GENOME EDITING COMPOSITIONS AND METHODS FOR TREATMENT OF WILSON'S DISEASE

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on Nov. 30, 2023, is named 59761-721.301 Sequence Listing.xml and is 8,280,972 bytes in size.

BACKGROUND

Wilson's disease is an autosomal recessive genetic copper storage disorder caused by mutations in the ATP7B gene (OMIM #606882). ATP7B is located in the human genome on 13q14.3 and contains 20 introns and 21 exons, for a total genomic length of about 80 kb. The ATP7B gene encodes ATPase copper transporting beta (ATP7B), a P-type transmembrane copper-transporting ATPase, which is mainly expressed in hepatic and neural tissues and functions in the transmembrane transport of copper. ATP7B deficiencies may lead to decreased hepatocellular excretion of copper into bile that may lead to systemic copper buildup primarily in the liver and subsequently in the neurologic system and other tissues, hepatic and neural toxicity, and early demise. The accumulation of copper can be manifested as neurological or psychiatric symptom. Over time without proper treatments, high copper levels can cause life-threatening organ damage. Failure to incorporate copper into ceruloplasmin is an additional consequence of the loss of functional ATP7B protein.

Current treatment approaches for Wilson's disease are daily oral therapy with chelating agents (such as penicillamine [Cuprimine] and trientine hydrochloride [Syprine]), zinc (to block enterocyte absorption of copper), and tetrathiomolybdate (TM), a copper chelator that forms complexes with albumin in the circulation; all of which require the affected individual to take medicines for their whole life. Furthermore, those treatments may cause side effects, such as drug induced lupus, myasthenia, paradoxical worsening, and do not restore normal copper metabolism. Liver transplantation is curative for Wilson's disease but transplant recipients are required to maintain a constant immune suppression regimen to prevent rejection. Therapeutic strategies, such as gene therapy, that can reverse the underlying metabolic defect would be greatly advantageous. However, the ATP7B gene is approximately 4.4 kb, nearing the adeno-associated virus (AAV) packaging size limit and making gene therapy approaches with the full-length gene difficult.

This disclosure provides Prime Editing methods and compositions for correcting mutations associated with Wilson's disease.

SUMMARY OF THE INVENTION

Provided herein, in some embodiments, are methods and compositions for prime editing of alterations in a target sequence in a target gene, for example, an ATP7B gene. The target ATP7B gene may comprise double stranded DNA. As exemplified in FIG. 1, in an embodiment, the target gene is edited by prime editing.

Without wishing to be bound by any particular theory, the prime editing process may search specific targets and edit endogenous sequences in a target gene, e.g., the ATP7B gene. As exemplified in FIG. 1, the spacer sequence of a PEgRNA recognizes and anneals with a search target sequence in a target strand of the target gene. A prime editing complex may generate a nick in the target gene on the edit strand which is the complementary strand of the target strand. The prime editing complex may then use a free 3′ end formed at the nick site of the edit strand to initiate DNA synthesis, where a primer binding site (PBS) of the PEgRNA complexes with the free 3′ end, and a single stranded DNA is synthesized using an editing template of the PEgRNA as a template. The editing template may comprise one or more nucleotide edits compared to the endogenous target ATP7B gene sequence. Accordingly, the newly-synthesized single stranded DNA also comprises the nucleotide edit(s) encoded by the editing template. Through removal of an editing target sequence on the edit strand of the target gene and DNA repair, the intended nucleotide edit(s) included in the newly synthesized single stranded DNA are incorporated into the target ATP7B gene.

One embodiment of the disclosure provides a prime editing guide RNA (PEgRNA) comprising: (a) a spacer that is complementary to a search target sequence on a first strand of an ATP7B gene, wherein the spacer comprises at its 3′ end nucleotides 5-20 of SEQ ID NO: 4425; (b) a gRNA core capable of binding to a Cas9 protein; (c) an extension arm comprising: (i) an editing template that comprises a region of complementarity to an editing target sequence on a second strand of the ATP7B gene, and (ii) a primer binding site that comprises at its 5′ end a sequence that is a reverse complement of nucleotides 15-17 of SEQ ID NO: 4425; wherein the first strand and second strand are complementary to each other and wherein the editing target sequence on the second strand is complementary to a portion of the ATP7B gene comprising a c.3207C>A substitution.

Another embodiment of the disclosure provides a prime editing guide RNA (PEgRNA) comprising: (a) a spacer comprising at its 3′ end nucleotides 5-20 of SEQ ID NO: 4425; (b) a gRNA core capable of binding to a Cas9 protein, and (c) an extension an comprising: (i) an editing template comprising at its 3′ end any one of SEQ ID NOs: 4437-4492, and (ii) a primer binding site (PBS) sequence comprising at its 5′ end any one of SEQ ID NOs: 2297, 4426, 4427, 4428, 4429, 4430, 4431, 4432, 4433, 4434, 4435, and 4436.

In some embodiments, the spacer of the PEgRNA is front 15 to 22 nucleotides in length. In some embodiments, the spacer of the PEgRNA comprises at its 3′ end nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 4425. In some embodiments, the spacer of the PEgRNA comprises at its 3′ end SEQ ID NO: 4425. In some embodiments, the spacer of the PEgRNA is 20 nucleotides in length. In some embodiments, the PEgRNA of the present disclosure, comprises from 5′ to 3′, the spacer, the gRNA core, the RTT, and the PBS. In some embodiments, the spacer, the gRNA core, the RTT, and the PBS form a contiguous sequence in a single molecule.

In some embodiments, the PEgRNA of the present disclosure comprises a pegRNA sequence selected from any one of SEQ ID NOs: 2445, 2446, 2447, 2448, 2449, 2450, 2451, 2452, 2453, 2454, 2455, 2456, 2457, 2458, 2459, 2460, 2461, 2462, 2463, 2464, 2465, 2466, 2467, 2468, 2469, 2470, 2471, 2472, 2473, 2474, 2475, 2476, 2477, 2478, 2479, 2480, 2481, 2482, 2483, 2484, 2485, 2486, 2487, 2488, 2489, 2490, 2491, 2492, 2493, 2494, 2495, 2496, 2497, 2498, 2499, 2500, 2501, 2502, 2503, 2504, 2505, 2506, 2507, 2508, 2509, 2510, 2511, 2512, 2513, 2514, 2515, 2516, 2517, 2518, 2519, 2520, 2521, 2522, 2523, 2524, 2525, 2526, 2527, 2528, 2529, 2530, 2531, 2532, 2533, 2534, 2535, 2538, 2539, 2540, 2541, 2542, 2543, 2544, 2545, 2546, 2547, 2548, 2549, 2550, 2551, 2553, 2554, 2555, 2556, 2557, 2558, 2559, 2560, 2561, 2562, 2563, 2564, 2565, 2566, 2567, 2568, 2569, 2570, 2571, 2572, 2573, 2574, 2575, 2576, 2577, 2578, 2580, 2582, 2584, 2585, 2586, 2587, 2588, 2589, 2590, 2591, 2592, 2593, 2594, 2595, 2596, 2597, 2598, 2600, 2601, 2602, 2603, 2605, 2606, 2607, 2608, 2609, 2610, 2611, 2612, 2613, 2614, 2615, 2616, 2617, 2618, 2619, 2620, 2621, 2623, 2624, 2628, 2629, 2630, 2631, 2632, 2633, 2634, 2635, 2636, 2637, 2638, 2643, 2645, 2646, 2647, 2648, 2649, 2650, 2651, 2652, 2653, 2654, 2655, 2656, 2657, 2658, 2659, 2660, 2663, 2664, 2665, 2667, 2668, 2669, 2670, 2671, 2672, 2674, 2675, 2676, 2677, 2678, 2680, 2681, 2683, 2685, 2687, 2688, 2689, 2690, 2692, 2694, 2695, 2696, 2697, 2699, 2701, 2702, 2704, 2706, 2708, 2711, 2713, 2715, 2716, 2717, 2720, 2721, 2722, 2723, 2725, 2726, 2727, 2728, 2729, 2730, 2733, 2734, 2735, 2744, 2747, 2748, 2749, 2752, 2753, 2757, 2758, 2759, 2760, 2761, 2762, 2764, 2765, 2768, 2769, 2770, 2772, 2773, 2774, 2777, 2786, 2788, 2789, 2790, 2791, 2792, 2793, 2794, 2795, 2796, 2797, 2798, 2799, 2800, 2801, 2802, 2807, 2810, 2811, 2812, 2814, 2816, 2824, 2825, 2826, 2828, 2829, 2830, 2832, 2833, 2834, 2841, 2842, 2843, 2844, 2846, 2847, 2854, 2855, 2856, 2857, 2862, 2864, 2866, 2867, 2868, 2869, 2870, 2871, 2885, 2886, 2887, 2888, 2889, 2890, 2891, 2893, 2894, 2896, 2898, 2899, 2901, 2902, 2909, 2910, 2914, 2916, 2918, 2919, 2920, 2926, 2927, 2932, 2933, 2937, 2938, 2939, 2941, 2942, 2945, 2953, 2954, 2956, 2957, 2960, 2962, 2963, 2964, 2965, 2967, 2972, 2973, 2977, 2979, 2980, 2982, 2983, 2988, 2991, 2993, 2994, 2995, 2997, 3006, 3008, 3012, 3013, 3015, 3023, 3024, 3027, 3028, 3029, 3030, 3031, 3032, 3033, 3034, 3035, 3036, 3037, 3038, 3039, 3043, 3044, 3045, 3046, 3048, 3049, 3050, 3051, 3052, 3053, 3054, 3055, 3059, 3064, 3065, 3071, 3072, 3075, 3076, 3080, 3082, 3084, 3093, 3096, 3098, 3099, 3101, 3119, 3121, 3122, 3123, 3124, 3126, 3128, 3130, 3133, 3142, 3144, 3148, 3159, 3161, 3162, 3163, 3164, 3165, 3166, 3168, 3169, 3170, 3176, 3182, 3188, 3190, 3191, 3195, 3200, 3202, 3203, 3210, 3212, 3216, 3218, 3226, 3227, 3228, 3229, 3230, 3231, 3232, 3234, 3235, 3238, 3239, 3241, 3243, 3244, 3245, 3246, 3247, 3248, 3249, 3250, 3260, 3262, 3263, 3271, 3273, 3275, 3281, 3282, 3283, 3287, 3288, 3289, 3300, 3301, 3302, 3303, 3304, 3305, 3307, 3310, 3311, 3312, 3313, 3314, 3315, 3316, 3317, 3318, 3322, 3324, 3325, 3328, 3330, 3346, 3347, 3348, 3349, 3350, 3358, 3359, 3362, 3364, 3365, 3366, 3367, 3368, 3372, 3373, 3382, 3385, 3387, 3388, 3389, 3390, 3391, 3392, 3393, 3400, 3403, 3404, 3405, 3407, 3408, 3409, 3412, 3414, 3420, 3423, 3425, 3426, 3427, 3428, 3429, 3430, 3431, 3434, 3438, 3441, 3442, 3446, 3449, 3450, 3451, 3452, 3453, 3454, 3455, 3463, 3466, 3469, 3470, 3471, 3472, 3473, 3474, 3477, 3478, 3480, 3481, 3482, 3487, 3490, 3494, 3498, 3499, 3502, 3503, 3505, 3506, 3508, 3509, 3510, 3511, 3513, 3520, 3522, 3523, 3526, 3529, 3533, 3535, 3536, 3542, 3543, 3546, 3547, 3549, 3550, 3553, 3554, 3555, 3557, 3560, 3561, 3563, 3564, 3567, 3568, 3569, 3571, 3574, 3575, 3576, 3578, 3579, 3580, 3581, 3583, 3584, 3585, 3592, 3594, 3595, 3596, 3597, 3603, 3612, 3613, 3617, 3622, 3625, 3626, 3627, 3628, 3630, 3631, 3632, 3633, 3635, 3636, 3638, 3639, 3640, 3641, 3642, 3646, 3647, 3648, 3654, 3657, 3659, 3660, 3661, 3664, 3668, 3669, 3673, 3674, 3678, 3679, 3680, 3681, 3684, 3685, 3687, 3688, 3697, 3699, 3702, 3703, 3704, 3705, 3706, 3708, 3710, 3711, 3712, 3714, 3715, 3721, 3722, 3724, 3725, 3728, 3729, 3730, 3731, 3732, 3733, 3734, 3735, 3736, 3737, 3739, 3740, 3741, 3743, 3744, 3746, 3748, 3755, 3761, 3770, 3771, 3773, 3774, 3776, 3778, 3779, 3781, 3782, 3784, 3785, 3792, 3793, 3794, 3795, 3796, 3797, 3798, 3799, 3800, 3801, 3802, 3803, 3804, 3805, 3806, 3807, 3808, 3809, 3810, 3811, 3812, 3814, 3815, 3816, 3820, 3829, 3839, 3841, 3842, 3843, 3844, 3845, 3851, 3852, 3853, 3854, 3855, 3856, 3857, 3858, 3859, 3860, 3861, 3862, 3863, 3864, 3865, 3868, 3869, 3871, 3874, 3875, 3876, 3877, 3878, 3879, 3880, 3882, 3883, 3884, 3885, 3887, 3895, 3899, 3904, 3907, 3908, 3909, 3910, 3911, 3912, 3913, 3914, 3915, 3916, 3917, 3921, 3924, 3927, 3928, 3929, 3931, 3932, 3935, 3936, 3937, 3938, 3939, 3940, 3941, 3942, 3943, 3945, 3946, 3956, 3957, 3961, 3962, 3965, 3971, 3977, 3978, 3979, 3980, 3981, 3982, 3983, 3985, 3988, 3989, 3990, 3991, 3992, 3993, 3994, 3995, 3997, 3998, 3999, 4001, 4002, 4003, 4004, 4009, 4011, 4012, 4013, 4015, 4016, 4017, 4020, 4021, 4023, 4025, 4026, 4028, 4029, 4031, 4032, 4034, 4035, 4036, 4037, 4038, 4040, 4052, 4055, 4056, 4060, 4061, 4066, 4067, 4070, 4077, 4078, 4080, 4081, 4082, 4083, 4084, 4085, 4086, 4087, 4088, 4089, 4090, 4102, 4105, 4106, 4108, 4109, 4110, 4114, 4115, 4117, 4118, 4119, 4128, 4129, 4132, 4136, 4137, 4142, 4147, 4159, 4163, 4168, 4170, 4171, 4172, 4173, 4175, 4182, 4183, 4186, 4188, 4192, 4194, 4199, 4208, 4225, 4226, 4227, 4228, 4232, 4239, 4240, and 4258.

In some embodiments, the PEgRNA of the present disclosure provides a pegRNA sequence selected from any one of SEQ ID NOs: 4588, 4657, 4719, 4589, 4624, 4500, 4618, 4649, and 4533.

Another embodiment of the disclosure provides a prime editing guide RNA (PEgRNA) comprising: (a) a spacer that is complementary to a search target sequence on a first strand of an ATP7B gene, wherein the spacer comprises at its 3′ end nucleotides 5-20 of SEQ ID NO: 2293: (b) a gRNA core capable of binding to a Cas9 protein; (c) an extension arm comprising: (i) an editing template that comprises a region of complementarity to an editing target sequence on a second strand of the ATP7B gene, and (ii) a primer binding site that comprises at its 5′ end a sequence that is a reverse complement of nucleotides 15-17 of SEQ ID NO: 2293; wherein the first strand and second strand are complementary to each other and wherein the editing target sequence on the second strand is complementary to a portion of the ATP7B gene comprising a c.3207C>A substitution.

Another embodiments of the disclosure provides a prime editing guide RNA (PEgRNA) comprising: (a) a spacer comprising at its 3′ end nucleotides 5-20 of SEQ ID NO: 2293; (b) a gRNA core capable of binding to a Cas9 protein, and (c) an extension arm comprising: (i) an editing template comprising at its 3′ end any one of SEQ ID NOs: 2305-2422, and (ii) a primer binding site (PBS) sequence comprising at its 5′ end any one of SEQ ID NOs: 2294-2304.

In some embodiments, the spacer of the PEgRNA is from 15 to 22 nucleotides in length. In some embodiments, the spacer of the PEgRNA comprises at its 3′ end nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 2293. In some embodiments, the spacer of the PEgRNA comprises at its 3′ end SEQ ID NO: 2293. In some embodiments, the spacer of the PEgRNA is 20 nucleotides in length. In some embodiments, the PEgRNA of the present disclosure comprises from 5′ to 3′, the spacer, the gRNA core, the RTT, and the PBS. In some embodiments, the spacer, the gRNA core, the RTT, and the PBS form a contiguous sequence in a single molecule.

In some embodiments, the PEgRNA of the present disclosure comprises a pegRNA sequence selected from any one of SEQ ID NOs: 2557, 2988, 2993, and 2585.

Another embodiment of the disclosure provides a prime editing system comprising: (a) the prime editing guide RNA (PEgRNA) of the present disclosure, or a nucleic acid encoding the PEgRNA; and (b) a nick guide RNA (ngRNA) comprising at its 3′ end nucleotides 5-20 of any one of SEQ ID NOs: 41, 60, 61, 62, 63, 64, 65, 66, 69, 71, 2045, 2046, 2047, 2048, 2049, 2050, 2051, 2052, 2053, 2054, 2055, 2056, 2057, 2058, 2059, 2060, 2061, 2062, 2063, 2064, 2065, 2066, 2067, 2068, 2069, 2070, 2071, 2072, 2073, 2074, 2075, 2076, 2077, 2078, 2079, 2080, 2081, 2082, 2083, 2084, 2085, 2086, 2087, 2088, 2089, 2090, 2091, 2092, 2093, 2094, 2095, 2096, 2423, 2424, 2425, 2426, 2427, 2428, 2429, 2430, 2431, 2432, 2433, 2434, 2435, 2436, 2437, 2438, 2439, 2440, 2441, 2442, 2443, and 2444 and a gRNA core capable of binding to a Cas9 protein, or a nucleic acid encoding the ngRNA.

In some embodiments, the spacer of the ngRNA is from 15 to 22 nucleotides in length. In some embodiments, the spacer of the ngRNA comprises at its 3′ end nucleotides 4-20, 3-20, 2-20, or 1-20 of any one of SEQ ID NOs: 41, 60, 61, 62, 63, 64, 65, 66, 69, 71, 2045, 2046, 2047, 2048, 2049, 2050, 2051, 2052, 2053, 2054, 2055, 2056, 2057, 2058, 2059, 2060, 2061, 2062, 2063, 2064, 2065, 2066, 2067, 2068, 2069, 2070, 2071, 2072, 2073, 2074, 2075, 2076, 2077, 2078, 2079, 2080, 2081, 2082, 2083, 2084, 2085, 2086, 2087, 2088, 2089, 2090, 2091, 2092, 2093, 2094, 2095, 2096, 2423, 2424, 2425, 2426, 2427, 2428, 2429, 2430, 2431, 2432, 2433, 2434, 2435, 2436, 2437, 2438, 2439, 2440, 2441, 2442, 2443, and 2444. In some embodiments, the spacer of the ngRNA comprises at its 3′ end of any one of SEQ ID NOs: 41, 60, 61, 62, 63, 64, 65, 66, 69, 71, 2045, 2046, 2047, 2048, 2049, 2050, 2051, 2052, 2053, 2054, 2055, 2056, 2057, 2058, 2059, 2060, 2061, 2062, 2063, 2064, 2065, 2066, 2067, 2068, 2069, 2070, 2071, 2072, 2073, 2074, 2075, 2076, 2077, 2078, 2079, 2080, 2081, 2082, 2083, 2084, 2085, 2086, 2087, 2088, 2089, 2090, 2091, 2092, 2093, 2094, 2095, 2096, 2423, 2424, 2425, 2426, 2427, 2428, 2429, 2430, 2431, 2432, 2433, 2434, 2435, 2436, 2437, 2438, 2439, 2440, 2441, 2442, 2443, and 2444.

In some embodiments, the spacer of the ngRNA is 20 nucleotides in length. In some embodiments, the ngRNA comprises SEQ ID NO: 2257, 2258, 2259, 2260, 2261, 2262, 2263, 2264, 2265, 2266, 2267, 2268, 2269, 2270, 2271, 2272, 2273, 2274, 2275, 2276, 2277, 2278, 2279, 2280, 2281, 2282, 2283, 2284, 2285, 2286, 2287, 2288, 2289, 4410, 4411, 4412, 4413, 4414, 4415, 4416, 4417, 4418, 4419, 4420, 4421, or 4422. In some embodiments, the ngRNA comprises SEQ ID NO: 2268, 2264, 4414, 4412, or 2265. In some embodiments, the prime editing system of present disclosure, further comprises: (c) a prime editor comprising a Cas9 nickase having a nuclease inactivating mutation in the HNH domain, or a nucleic acid encoding the Cas9 nickase, and a reverse transcriptase, or a nucleic acid encoding the reverse transcriptase.

In some embodiments, the Cas9 nickase comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 5786. In some embodiments, the reverse transcriptase comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 5842. In some embodiments, the sequence identities are determined by Needleman-Wunsch alignment of two protein sequences with Gap Costs set to Existence: 11 Extension: 1 where percent identity is calculated by dividing the number of identities by the length of the alignment. In some embodiments, the prime editor is a fusion protein.

Another embodiments of the disclosure provides an LNP comprising the prime editing system of the present disclosure. In some embodiments, the PEgRNA, the nucleic acid encoding the Cas9 nickase, and the nucleic acid encoding the reverse transcriptase. In some embodiments, the nucleic acid encoding the Cas9 nickase and the nucleic acid encoding the reverse transcriptase are mRNA. In some embodiments, the nucleic acid encoding the Cas9 nickase and the nucleic acid encoding the reverse transcriptase are the same molecule. In some embodiments, the LNP of the present disclosure, further comprises the ngRNA.

Another embodiment of the disclosure provides a method of correcting for editing an ATP7B gene, the method comprising contacting the ATP7B gene with: (a) the PEgRNA of the present disclosure and a prime editor comprising a Cas9 nickase having a nuclease inactivating mutation in the HNH domain and a reverse transcriptase, (b) the prime editing system of the present disclosure, or (c) the LNP of the present disclosure.

In some embodiments, the ATP7B gene is in a cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a primary cell. In some embodiments, the cell is a hepatocyte. In some embodiments, the cell is in a subject. In some embodiments, the subject is a human. In some embodiments, the cell is from a subject having Wilson's disease. In some embodiments, the method of the present disclosure, further comprises administering the cell to the subject after incorporation of the intended nucleotide edit.

Another embodiment of the disclosure provides a cell generated by the method of the present disclosure.

Another embodiment of the disclosure provides a population of cells generated by the method of the present disclosure.

Another embodiment of the disclosure provides a method for treating Wilson's disease in a subject in need thereof, the method comprising administering to the subject: (a) the PEgRNA of the present disclosure, (b) the prime editing system of the present disclosure, or (c) the LNP of the present disclosure.

In some embodiments, the method of the present disclosure, comprises administering to the subject the PEgRNA of the present disclosure and a prime editor comprising a Cas9 nickase having a nuclease inactivating mutation in the HNH domain and a reverse transcriptase or one or more nucleic acids encoding the prime editor or its components. In some embodiments, the prime editor is a fusion protein. Another embodiment of the disclosure provides a prime editing guide RNA (PEgRNA) comprising: (a) a spacer comprising at its 3′ end nucleotides 5-20 of a PEgRNA Spacer sequence selected from any one of Tables 6-32; (b) a gRNA core capable of binding to a Cas9 protein, and (c) an extension arm comprising: (i) an editing template comprising at its 3′ end an RTT sequence selected from the same Table as the PEgRNA Spacer sequence, and (ii) a primer binding site (PBS) comprising at its 5′ end a PBS sequence selected from the same Table as the PEgRNA Spacer sequence.

In some embodiments, the spacer of the PEgRNA is from 15 to 22 nucleotides in length. In some embodiments, the spacer of the PEgRNA comprises at its 3′ end nucleotides 4-20, 3-20, 2-20, or 1-20 of the PEgRNA Spacer sequence selected from any one of Tables 6-32. In some embodiments, the spacer of the PEgRNA comprises at its 3′ end the PEgRNA Spacer sequence selected from any one of Tables 6-32. In some embodiments, the spacer of the PEgRNA is 20 nucleotides in length. In some embodiments, the PEgRNA of the disclosure, comprises from 5′ to 3′, the spacer, the gRNA core, the editing template, and the PBS. In some embodiments, the spacer, the gRNA core, the editing template, and the PBS form a contiguous sequence in a single molecule. In some embodiments, the PEgRNA of the present disclosure, comprises a pegRNA sequence selected from the same Table as the PEgRNA Spacer sequence. Another embodiment of the disclosure provides a prime editing system comprising: (a) the prime editing guide RNA (PEgRNA) of the present disclosure, or a nucleic acid encoding the PEgRNA; and (b) a nick guide RNA (ngRNA) comprising a spacer comprising at its 3′ end nucleotides 5-20 of any ngRNA Spacer sequence selected from the same Table as the PEgRNA Spacer sequence and a gRNA core capable of binding to a Cas9 protein, or a nucleic acid encoding the ngRNA.

In some embodiments, the spacer of the ngRNA is from 15 to 22 nucleotides in length. In some embodiments, the spacer of the ngRNA comprises at its 3′ end nucleotides 4-20, 3-20, 2-20, or 1-20 of the ngRNA Spacer sequence selected from the same Table as the PEgRNA Spacer sequence. In some embodiments, the spacer of the ngRNA comprises at its 3′ end the ngRNA Spacer sequence selected from the same Table as the PEgRNA Spacer sequence. In some embodiments, the spacer of the ngRNA is 20 nucleotides in length. In some embodiments, the ngRNA comprises a ngRNA sequence selected from the same Table as the PEgRNA Spacer sequence. In some embodiments, the prime editing system of the present disclosure, further comprises: (c) a prime editor comprising a Cas9 nickase having a nuclease inactivating mutation in the HNH domain, or a nucleic acid encoding the Cas9 nickase, and a reverse transcriptase, or a nucleic acid encoding the reverse transcriptase.

In some embodiments, the Cas9 nickase comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 9%, 98% 99%, or 100% identity to SEQ ID NO: 5786. In some embodiments, the reverse transcriptase comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 5842. In some embodiments, the sequence identities are determined by Needleman-Wunsch alignment of two protein sequences with Gap Costs set to Existence: 11 Extension: 1 where percent identity is calculated by dividing the number of identities by the length of the alignment. In some embodiments, the prime editor is a fusion protein.

Another embodiment of the disclosure provides an LNP comprising the prime editing system of the present disclosure. In some embodiments, the LNP of the present disclosure, comprises the PEgRNA, the nucleic acid encoding the Cas9 nickase, and the nucleic acid encoding the reverse transcriptase. In some embodiments, the nucleic acid encoding the Cas9 nickase and the nucleic acid encoding the reverse transcriptase are mRNA. In some embodiments, the nucleic acid encoding the Cas9 nickase and the nucleic acid encoding the reverse transcriptase are the same molecule. In some embodiments, the LNP of the present disclosure, further comprises the ngRNA.

Another embodiment of the disclosure provides a method of correcting for editing an ATP7B gene, the method comprising contacting the ATP7B gene with: (A) the PEgRNA of the present disclosure and a prime editor comprising a Cas9 nickase having a nuclease inactivating mutation in the HNH domain and a reverse transcriptase, (B) the prime editing system of the present disclosure, or (C) the LNP of the present disclosure.

Another embodiment of the disclosure provides a cell generated by the method of the present disclosure.

Another embodiment of the disclosure provides a population of cells generated by the method of the present disclosure.

Another embodiment of the disclosure provides a method for treating Wilson's disease in a subject in need thereof, the method comprising administering to the subject: (a) the PEgRNA of the present disclosure, (B) the prime editing system of the present disclosure, or (C) the LNP of the present disclosure.

In some embodiments, the method, comprises administering to the subject the PEgRNA of the present disclosure and a prime editor comprising a Cas9 nickase having a nuclease inactivating mutation in the HNH domain and a reverse transcriptase or one or more nucleic acids encoding the prime editor or its components. In some embodiments, the prime editor is a fusion protein.

Another embodiment of the disclosure provides a prime editing guide RNA (PEgRNA) comprising: a spacer that is complementary to a search target sequence on a first strand of an ATP7B gene, an editing template that comprises a region of complementarity to an editing target sequence on a second strand of the ATP7B gene, and a gRNA core that associates with a prime editor comprising a DNA binding domain and a DNA polymerase domain, wherein the first strand and second strand are complementary to each other, and wherein the editing target sequence is in an exon selected from the group consisting of: exon 8, exon 13, exon 14, exon 15, and exon 17 of the ATP7B gene.

Another embodiment of the disclosure provides a prime editing guide RNA (PEgRNA) comprising: a spacer that that is complementary to a search target sequence on a first strand of an ATP7B gene, an editing template that comprises a region of complementarity to an editing target sequence on a second strand of the ATP7B gene, and a gRNA core that associates with a prime editor comprising a DNA binding domain and a DNA polymerase domain, wherein first strand and second strand are complementary to each other, and wherein if the editing target sequence is in exon 3 then the editing target sequence does not comprise a c.1288 duplication as compared to a wild type ATP7B gene.

Another embodiment of the disclosure provides a prime editing guide RNA (PEgRNA) comprising: a spacer that is complementary to a search target sequence on a first strand of an ATP7B gene, an editing template that comprises a region of complementarity to an editing target sequence on a second strand of the ATP7B gene, and a gRNA core that associates with a prime editor comprising a DNA binding domain and a DNA polymerase domain, wherein first strand and second strand are complementary to each other, and wherein the editing target sequence is between positions 51932669-51946368 and positions 51932370-52012130 of human chromosome 13.

Another embodiment of the disclosure provides a prime editing guide RNA (PEgRNA) comprising: a spacer that is complementary to a search target sequence on a first strand of a ATP7B gene, an editing template that comprises a region of complementarity to an editing target sequence on a second strand of the ATP7B gene, and a gRNA core that associates with a prime editor comprising a DNA binding domain and a DNA polymerase domain, wherein first strand and second strand are complementary to each other, wherein the editing target sequence comprises a mutation associated with Wilson's disease, and wherein the mutation does not encode the amino acid substitution p.Ser430fs.

In some embodiments, the PEgRNA comprises a primer binding site sequence (PBS) at least partially complementary to the spacer. In some embodiments, wherein the gRNA core is between the spacer and the editing template. In some embodiments, the editing template comprises an intended nucleotide edit compared to the ATP7B gene. In some embodiments, the PEgRNA guides the prime editor to incorporate the intended nucleotide edit into the ATP7B gene when contacted with the ATP7B gene. In some embodiments, the prime editor synthesizes a single stranded DNA encoded by the editing template, wherein the single stranded DNA replaces the editing target sequence and results in incorporation of the intended nucleotide edit into a region corresponding to the editing target in the ATP7B gene.

In some embodiments, the search target sequence is complementary to a protospacer sequence in the APT7B gene, and wherein the protospacer sequence is adjacent to a search target adjacent motif (PAM) in the ATP7B gene. In some embodiments, the PEgRNA results in incorporation of the intended nucleotide edit in the PAM when contacted with the ATP7B gene. In some embodiments, the PBS is about 2 to 20 base pairs in length. In some embodiments, the PBS is about 8 to 16 base pairs in length. In some embodiments, the editing template is about 4 to 30 base pairs in length. In some embodiments, the editing template is about 10 to 30 base pairs in length. In some embodiments, the PEgRNA results in incorporation of intended nucleotide edit about 0 to 27 base pairs downstream of the 5 end of the PAM when contacted with the ATP7B gene. In some embodiments, the intended nucleotide edit comprises a single nucleotide substitution compared to the region corresponding to the editing target in the ATP7B gene.

In some embodiments, the intended nucleotide edit comprise an insertion compared to the region corresponding to the editing target in the ATP7B gene. In some embodiments, the intended nucleotide edit comprises a deletion compared to the region corresponding to the editing target in the ATP7B gene. In some embodiments, the editing target sequence comprises a mutation associated with Wilson's disease. In some embodiments, the editing template comprises a wild type ATP7B gene sequence. In some embodiments, the PEgRNA results in correction of the mutation when contacted with the ATP7B gene. In some embodiments, the editing target sequence is between positions 51944045 and 51944245 of human chromosome 13.

In some embodiments, the intended nucleotide edit comprises an A>C nucleotide substitution at position 51944145 in human chromosome 13 as compared to the region corresponding to the editing target in the ATP7B gene. In some embodiments, the editing target sequence comprises a mutation that encodes an H1069Q amino acid substitution as compared to a wild type ATP7B protein as set forth in SEQ ID NO:5861. In some embodiments, the spacer comprises a sequence selected from the group consisting of SEQ ID Nos. 1, 182, 294, 483, 682, 1505, 2023, 2293, 4425, 5206, 5228, 5248, 5282, 5313, 5340, 5369, 5406, 5423, 5446, 5473, 5503, 5537, 5555, 5638, and 5706. In some embodiments, the editing template comprises a sequence selected from the group consisting of SEQ ID Nos.:13-17, 194-198, 306-336, 495-528, 694-735, 1517-1546, 2035-2044, 2305-2422, 4437-4492, 5218, 5240-5247, 5260-5279, 5294-5302, 5325-5338, 5352-5368, 5381-5401, 5418-5422, 5435-5445, 5458-5472, 5485-5502, 5515-5535, 5549-5554, 5567-5590, 5650-5668, and 5718-5738.

In some embodiments, the PBS comprises a sequence selected from the group consisting of SEQ ID Nos. 2-12, 183-193, 295-305, 484-494, 683-693, 1506-1516, 2024-2034, 2294-2304, 4426-4436, 5207-5217, 5229-5239, 5249-5259, 5283-5293, 5314-5324, 5341-5351, 5370-5380, 5407-5417, 5424-5434, 5447-5457, 5474-5484, 5504-5514, 5538-5548, 5556-5566, 5639-5649, and 5707-5717.

In some embodiments, the spacer comprises a sequence selected from the group consisting of SEQ ID Nos. 1, 182, 294, 483, 682, 1505, 2023, 2293, 4425, 5206, 5228, 5248, 5282, 5313, 5340, 5369, 5406, 5423, 5446, 5473, 5503, 5537, 5555, 5638, and 5706.

In some embodiments, the editing template comprises a sequence selected from the group consisting of SEQ ID Nos. 13-17, 194-198, 306-336, 495-528, 694-735, 1517-1546, 2035-2044, 2305-2422, 4437-4492, 5218, 5240-5247, 5260-5279, 5294-5302, 5325-5338, 5352-5368, 5381-5401, 5418-5422, 5435-5445, 5458-5472, 5485-5502, 5515-5535, 5549-5554, 5567-5590, 5650-5668, and 5718-5738.

In some embodiments, the PEgRNA comprises a PBS selected from the group consisting of SEQ ID Nos. 2-12, 183-193, 295-305, 484-494, 683-693, 1506-1516, 2024-2034, 2294-2304, 4426-4436, 5207-5217, 5229-5239, 5249-5259, 5283-5293, 5314-5324, 5341-5351, 5370-5380, 5407-5417, 5424-5434, 5447-5457, 5474-5484, 5504-5514, 5538-5548, 5556-5566, 5639-5649, and 5707-5717.

Another embodiment of the disclosure provides a PEgRNA comprising a sequence selected from the group consisting of SEQ ID Nos. 73-152, 210-289, 338-482, 530-680, 741-1500, 1547-2022, 2097-2256, 2445-4409, 4493-5205, 5591-5637, 5669-5705, and 5739-5779.

Another embodiment of the disclosure provides a PEgRNA system comprising the PEgRNA of the present disclosure and further comprising a nick guide RNA (ngRNA), wherein the ngRNA comprises an ng spacer that is complementary to a second search target sequence in the ATP7B gene.

In some embodiments, the second search target sequence is on the second strand of the ATP7B gene. In some embodiments, the ng spacer comprises a sequence selected from the group consisting of SEQ ID Nos. 18-72, 199-209, 337, 529, 736-740, 2045-2096, 2423-2444, 5219-5227, 5280-5281, 5303-5312, 5339, 5402-5405, and 5536. In some embodiments, the ng spacer comprises a sequence selected form SEQ ID Nos. 2052, 2053, 2059, 2438, and 2441. Another embodiment of the disclosure provides a PEgRNA system comprising a PEgRNA selected from the group consisting of SEQ ID Nos. 73-152, 210-289, 338-482, 530-680, 741-1500, 1547-2022, 2097-2256, 2445-4409, 4493-5205, 5591-5637, 5669-5705, or 5739-5779 and a ngRNA selected from the group consisting of SEQ ID Nos. 2290, 2291, 2292, 4423, and 4424.

Another embodiment of the disclosure provides a PEgRNA system comprising a PEgRNA selected from the group consisting of SEQ ID Nos. 2739, 2785, 3276, 3277, 4536, 4613, 4695, 4721, 4741, 4743, 4762, 4788, and 4824 and a ngRNA selected from the group consisting of SEQ ID Nos. 2290, 2291, 2292, 4423, and 4424.

Another embodiment of the disclosure provides a prime editing complex comprising: (i) the PEgRNA of the present disclosure or the PEgRNA system of the present disclosure; and (ii) a prime editor comprising a DNA binding domain and a DNA polymerase domain.

In some embodiments, the DNA binding domain is a CRISPR associated (Cas) protein domain. In some embodiments, the Cas protein domain has nickase activity. In some embodiments, the Cas protein domain is a Cas9. In some embodiments, the Cas9 comprises a mutation in an HNH domain. In some embodiments, the Cas9 comprises a H840A mutation in the HNH domain. In some embodiments, the Cas protein domain is a Cas12b. In some embodiments, the Cas protein domain is a Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas14a, Cas14b, Cas14c, Cas14d, Cas14e, Cas14f, Cas14g, Cas14h, Cas14u, or a Casφ. In some embodiments, the DNA polymerase domain is a reverse transcriptase. In some embodiments, the reverse transcriptase is a retrovirus reverse transcriptase. In some embodiments, the reverse transcriptase is a Moloney murine leukemia virus (M-MLV) reverse transcriptase.

In some embodiments, the DNA polymerase and the programmable DNA binding domain are fused or linked to form a fusion protein. In some embodiments, the fusion protein comprises the sequence of SEQ ID NO: 10740.

Another embodiment of the disclosure provides a lipid nanoparticle (LNP) or ribonucleoprotein (RNP) comprising the prime editing complex of the present disclosure, or a component thereof.

Another embodiment of the disclosure provides a polynucleotide encoding the PEgRNA of the present disclosure, the PEgRNA system of the present disclosure, or the fusion protein of the present disclosure.

In some embodiments, the polynucleotide is a mRNA. In some embodiments, the polynucleotide is operably linked to a regulatory element. In some embodiments, the regulatory element is an inducible regulatory element.

Another embodiment of the disclosure provides a vector comprising the polynucleotide of the present disclosure. In some embodiments, the vector is an AAV vector.

Another embodiment of the disclosure provides an isolated cell comprising the PEgRNA of the present disclosure, the PEgRNA system of the present disclosure, the prime editing complex of the present disclosure, the LNP or RNP of the present disclosure, the polynucleotide of the present disclosure, or the vector of the present disclosure.

In some embodiments, the cell is a human cell. In some embodiments, the cell is a hepatocyte.

Another embodiment of the disclosure provides a pharmaceutical composition comprising (i) the PEgRNA of the present disclosure, the PEgRNA system of the present disclosure, the prime editing complex of the present disclosure, the LNP or RNP of the present disclosure, the polynucleotide of the present disclosure, the vector of the present disclosure, or the cell of the present disclosure; and (ii) a pharmaceutically acceptable carrier.

Another embodiment of the disclosure provides a method for editing an ATP7B gene, the method comprising contacting the ATP7B gene with (i) the PEgRNA of the present disclosure or the PEgRNA system of the present disclosure and (ii) a prime editor comprising a DNA binding domain and a DNA polymerase domain, wherein the PEgRNA directs the prime editor to incorporate the intended nucleotide edit in the ATP7B gene, thereby editing the ATP7B gene.

Another embodiment of the disclosure provides a method for editing an ATP7B gene, the method comprising contacting the ATP7B gene with the prime editing complex of the present disclosure, wherein the PEgRNA directs the prime editor to incorporate the intended nucleotide edit in the ATP7B gene, thereby editing the ATP7B gene.

In some embodiments, the prime editor synthesizes a single stranded DNA encoded by the editing template, wherein the single stranded DNA replaces the editing target sequence and results in incorporation of the intended nucleotide edit into a region corresponding to the editing target in the ATP7B gene. In some embodiments, the ATP7B gene is in a cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, wherein the cell is a primary cell. In some embodiments, the cell is a hepatocyte. In some embodiments, the cell is in a subject. In some embodiments, the subject is a human. In some embodiments, the cell is from a subject having Wilson's disease. In some embodiments, the method further comprises administering the cell to the subject after incorporation of the intended nucleotide edit.

Another embodiment of the disclosure provides a cell generated by the method of the present disclosure.

Another embodiment of the disclosure provides a population of cells generated by the method of the present disclosure.

Another embodiment of the disclosure provides a method for treating Wilson's disease in a subject in need thereof, the method comprising administering to the subject (i) the PEgRNA of the present disclosure or the PEgRNA system of the present disclosure and (ii) a prime editor comprising a DNA binding domain and a DNA polymerase domain, wherein the PEgRNA directs the prime editor to incorporate the intended nucleotide edit in the ATP7B gene in the subject, thereby treating Wilson's disease in the subject.

Another embodiment of the disclosure provides a method for treating Wilson's disease in a subject in need thereof, the method comprising administering to the subject the prime editing complex of the present disclosure, the LNP or RNP of the present disclosure, or the pharmaceutical composition of the present disclosure, wherein the PEgRNA directs the prime editor to incorporate the intended nucleotide edit in the ATP7B gene in the subject, thereby treating Wilson's disease in the subject.

In some embodiments, the subject is a human. In some embodiment, the ATP7B gene in the subject comprises a mutation that encodes an H1069Q amino acid substitution as compared to a wild type ATP7B protein as set forth in SEQ ID NO:5861. In some embodiment, the ATP7B gene comprises a mutation that encodes an H1069Q amino acid substitution as compared to a wild type ATP7B protein as set forth in SEQ ID NO:5861.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

FIG. 1 depicts a schematic of a prime editing guide RNA (PEgRNA) binding to a double stranded target DNA sequence.

FIG. 2 depicts a PEgRNA architectural overview in an exemplary schematic of PEgRNA designed for a prime editor.

FIG. 3A depicts a 3′- to 5′ schematic (with the coding strand at the bottom) of an ATP7B H1069 locus with spacer sequences and an H1069Q mutation highlighted. FIG. 3B depicts a lentiviral screen design schematic.

FIG. 4 is a schematic showing the spacer and gRNA core part of an exemplary guide RNA, in two separate molecules. The rest of the PEgRNA structure is not shown.

DETAILED DESCRIPTION OF THE INVENTION

Provided herein, in some embodiments, are compositions and methods to edit the target gene ATP7B with prime editing. In certain embodiments, provided herein are compositions and methods for correction of mutations in the copper-transporting ATPase 2 (ATP7B) gene associated with Wilson's Disease. Compositions provided herein can comprise prime editors (PEs) that may use engineered guide polynucleotides, e.g., prime editing guide RNAs (PEgRNAs), that can direct PEs to specific DNA targets and can encode DNA edits on the target gene ATP7B that serve a variety of functions, including direct correction of disease-causing mutations.

The following description and examples illustrate embodiments of the present disclosure in detail. It is to be understood that this disclosure is not limited to the particular embodiments described herein and as such can vary. Those of skill in the art will recognize that there are numerous variations and modifications of this disclosure, which are encompassed within its scope. Although various features of the present disclosure can be described in the context of a single embodiment, the features can also be provided separately or in any suitable combination. Conversely, although the present disclosure can be described herein in the context of separate embodiments for clarity, the present disclosure can also be implemented in a single embodiment.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof as used herein mean “comprising”.

Unless otherwise specified, the words “comprising”, “comprise”, “comprises”, “having”, “have”, “has”, “including”, “includes”, “include”, “containing”, “contains” and “contain” are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

Reference to “some embodiments”, “an embodiment”, “one embodiment”, or “other embodiments” means that a particular feature or characteristic described in connection with the embodiments is included in at least one or more embodiments, but not necessarily all embodiments, of the present disclosure.

The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” meaning within an acceptable error range for the particular value should be assumed.

As used herein, a “cell” can generally refer to a biological cell. A cell can be the basic structural, functional and/or biological unit of a living organism. A cell can originate from any organism having one or more cells. Some non-limiting examples include: a prokaryotic cell, eukaryotic cell, a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a protozoa cell, a cell from a plant, an animal cell, a cell from an invertebrate animal (e.g. fruit fly, cnidarian, echinoderm, nematode, etc.), a cell from a vertebrate animal (e.g., fish, amphibian, reptile, bird, mammal), a cell from a mammal (e.g., a pig, a cow, a goat, a sheep, a rodent, a rat, a mouse, a non-human primate, a human, etc.), et cetera. Sometimes a cell may not originate from a natural organism (e.g., a cell can be synthetically made, sometimes termed an artificial cell).

In some embodiments, the cell is a human cell. A cell may be of or derived from different tissues, organs, and/or cell types. In some embodiments, the cell is a primary cell. As used herein, the term primary cell means a cell isolated from an organism, e.g., a mammal, which is grown in tissue culture (i.e., in vitro) for the first time before subdivision and transfer to a subculture. In some non-limiting examples, mammalian primary cells which can be transfected and further passaged include hepatocytes, fibroblasts, keratinocytes, epithelial cells (e.g., mammary epithelial cells, intestinal epithelial cells), endothelial cells, glial cells, neural cells, formed elements of the blood (e.g., lymphocytes, bone marrow cells), muscle cells and precursors of these somatic cell types. In some embodiments, the cell is a primary hepatocyte. In some embodiments, the cell is a primary human hepatocyte. In some embodiments, the cell is a primary human hepatocyte derived from an induced human pluripotent stem cell (iPSC). In some embodiments, the cell is a neuron. In some embodiments, the cell is a neuron from basal ganglia. In some embodiments, the cell is a neuron from basal ganglia of a subject.

In some embodiments, the cell comprises a prime editor or a prime editing composition. In some embodiments, the cell is from a human subject. In some embodiments, the human subject has a disease or condition associated with a mutation to be corrected by prime editing, for example, Wilsons's disease. In some embodiments, the cell is from a human subject, and comprises a prime editor or a prime editing composition for correction of the mutation. In some embodiments, the cell is from the human subject and the mutation has been edited or corrected by prime editing.

The term “substantially” as used herein may refer to a value approaching 100% of a given value. In some embodiments, the term may refer to an amount that may be at least about 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 99.99% of a total amount. In some embodiments, the term may refer to an amount that may be about 100% of a total amount.

The terms “protein” and “polypeptide” can be used interchangeably to refer to a polymer of two or more amino acids joined by covalent bonds (e.g., an amide bond) that can adopt a three-dimensional conformation. In some embodiments, a protein or polypeptide comprises at least 10 amino acids, 15 amino acids, 20 amino acids, 30 amino acids or 50 amino acids joined by covalent bonds (e.g., amide bonds). In some embodiments, a protein comprises at least two amide bonds. In some embodiments, a protein comprises multiple amide bonds. In some embodiments, a protein comprises an enzyme, enzyme precursor proteins, regulatory protein, structural protein, receptor, nucleic acid binding protein, a biomarker, a member of a specific binding pair (e.g., a ligand or aptamer), or an antibody. In some embodiments, a protein may be a full-length protein (e.g., a fully processed protein having certain biological function). In some embodiments, a protein may be a variant or a fragment of a full-length protein. For example, in some embodiments, a Cas9 protein domain comprises an H840A amino acid substitution compared to a naturally occurring S. pyogenes Cas9 protein. A variant of a protein or enzyme, for example a variant reverse transcriptase, comprises a polypeptide having an amino acid sequence that is about 60% identical, about 70% identical, about 80% identical, about 90% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, about 99% identical, about 99.5% identical, or about 99.9% identical to the amino acid sequence of a reference protein.

In some embodiments, a protein comprises one or more protein domains or subdomains. As used herein, the term “polypeptide domain”, “protein domain”, or “domain” when used in the context of a protein or polypeptide, refers to a polypeptide chain that has one or more biological functions, e.g., a catalytic function, a protein-protein binding function, or a protein-DNA function. In some embodiments, a protein comprises multiple protein domains. In some embodiments, a protein comprises multiple protein domains that are naturally occurring. In some embodiments, a protein comprises multiple protein domains from different naturally occurring proteins. For example, in some embodiments, a prime editor may be a fusion protein comprising a Cas9 protein domain of S. pyogenes and a reverse transcriptase protein domain of Moloney murine leukemia virus. A protein that comprises amino acid sequences from different origins or naturally occurring proteins may be referred to as a fusion, or chimeric protein.

In some embodiments, a protein comprises a functional variant or functional fragment of a full-length wild type protein. A “functional fragment” or “functional portion”, as used herein, refers to any portion of a reference protein (e.g., a wild type protein) that encompasses less than the entire amino acid sequence of the reference protein while retaining one or more of the functions, e.g., catalytic or binding functions. For example, a functional fragment of a reverse transcriptase may encompass less than the entire amino acid sequence of a wild type reverse transcriptase but retains the ability under at least one set of conditions to catalyze the polymerization of a polynucleotide. When the reference protein is a fusion of multiple functional domains, a functional fragment thereof may retain one or more of the functions of at least one of the functional domains. For example, a functional fragment of a Cas9 may encompass less than the entire amino acid sequence of a wild type Cas9 but retains its DNA binding ability and lacks its nuclease activity partially or completely.

A “functional variant” or “functional mutant”, as used herein, refers to any variant or mutant of a reference protein (e.g., a wild type protein) that encompasses one or more alterations to the amino acid sequence of the reference protein while retaining one or more of the functions, e.g., catalytic or binding functions. In some embodiments, the one or more alterations to the amino acid sequence comprises amino acid substitutions, insertions or deletions, or any combination thereof. In some embodiments, the one or more alterations to the amino acid sequence comprises amino acid substitutions. For example, a functional variant of a reverse transcriptase may comprise one or more amino acid substitutions compared to the amino acid sequence of a wild type reverse transcriptase but retains the ability under at least one set of conditions to catalyze the polymerization of a polynucleotide. When the reference protein is a fusion of multiple functional domains, a functional variant thereof may retain one or more of the functions of at least one of the functional domains. For example, in some embodiments, a functional fragment of a Cas9 may comprise one or more amino acid substitutions in a nuclease domain, e.g., an H840A amino acid substitution, compared to the amino acid sequence of a wild type Cas9, but retains the DNA binding ability and lacks the nuclease activity partially or completely.

The term “function” and its grammatical equivalents as used herein may refer to a capability of operating, having, or serving an intended purpose. Functional may comprise any percent from baseline to 100% of an intended purpose. For example, functional may comprise or comprise about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or up to about 100% of an intended purpose. In some embodiments, the term functional may mean over or over about 100% of normal function, for example, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 600%, 700% or up to about 1000% of an intended purpose.

In some embodiments, a protein or polypeptides includes naturally occurring amino acids (e.g., one of the twenty amino acids commonly found in peptides synthesized in nature, and known by the one letter abbreviations A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y and V). In some embodiments, a protein or polypeptides includes non-naturally occurring amino acids (e.g., amino acids which is not one of the twenty amino acids commonly found in peptides synthesized in nature, including synthetic amino acids, amino acid analogs, and amino acid mimetics). In some embodiments, a protein or polypeptide is modified.

In some embodiments, a protein comprises an isolated polypeptide. The term “isolated” means free or removed to varying degrees from components which normally accompany it as found in the natural state or environment. For example, a polypeptide naturally present in a living animal is not isolated, and the same polypeptide partially or completely separated from the coexisting materials of its natural state is isolated.

In some embodiments, a protein is present within a cell, a tissue, an organ, or a virus particle. In some embodiments, a protein is present within a cell or a part of a cell (e.g., a bacteria cell, a plant cell, or an animal cell). In some embodiments, the cell is in a tissue, in a subject, or in a cell culture. In some embodiments, the cell is a microorganism (e.g., a bacterium, fungus, protozoan, or virus). In some embodiments, a protein is present in a mixture of analytes (e.g., a lysate). In some embodiments, the protein is present in a lysate from a plurality of cells or from a lysate of a single cell.

The terms “homologous,” “homology,” or “percent homology” as used herein refer to the degree of sequence identity between an amino acid or polynucleotide sequence and a corresponding reference sequence. “Homology” can refer to polymeric sequences, e.g., polypeptide or DNA sequences that are similar. Homology can mean, for example, nucleic acid sequences with at least about: 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity. In other embodiments, a “homologous sequence” of nucleic acid sequences may exhibit 93%, 95% or 98% sequence identity to the reference nucleic acid sequence. For example, a “region of homology to a genomic region” can be a region of DNA that has a similar sequence to a given genomic region in the genome. A region of homology can be of any length that is sufficient to promote binding of a spacer or protospacer sequence to the genomic region. For example, the region of homology can comprise at least 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100 or more bases in length such that the region of homology has sufficient homology to undergo binding with the corresponding genomic region.

When a percentage of sequence homology or identity is specified, in the context of two nucleic acid sequences or two polypeptide sequences, the percentage of homology or identity generally refers to the alignment of two or more sequences across a portion of their length when compared and aligned for maximum correspondence. When a position in the compared sequence can be occupied by the same base or amino acid, then the molecules can be homologous at that position. Unless stated otherwise, sequence homology or identity is assessed over the specified length of the nucleic acid, polypeptide or portion thereof. In some embodiments, the homology or identity is assessed over a functional portion or specified portion of the length.

Alignment of sequences for assessment of sequence homology can be conducted by algorithms known in the art, such as the Basic Local Alignment Search Tool (BLAST) algorithm, which is described in Altschul et al, J. Mol. Biol. 215:403-410, 1990. A publicly available, internet interface, for performing BLAST analyses is accessible through the National Center for Biotechnology Information. Additional known algorithms include those published in: Smith & Waterman, “Comparison of Biosequences”, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, “A general method applicable to the search for similarities in the amino acid sequence of two proteins” J. Mol. Biol. 48:443, 1970; Pearson & Lipman “Improved tools for biological sequence comparison”, Proc. Natl. Acad. Sci. USA 85:2444, 1988; or by automated implementation of these or similar algorithms. Global alignment programs can also be used to align similar sequences of roughly equal size. Examples of global alignment programs include NEEDLE (available at www.ebi.ac.uk/Tools/psa/emboss_needle/) which is part of the EMBOSS package (Rice P et al., Trends Genet., 2000; 16: 276-277), and the GGSEARCH program https://fasta.bioch.virginia.edu/fasta_www2/, which is part of the FASTA package (Pearson W and Lipman D, 1988, Proc. Natl. Acad. Sci. USA, 85: 2444-2448). Both of these programs are based on the Needleman-Wunsch algorithm which is used to find the optimum alignment (including gaps) of two sequences along their entire length. A detailed discussion of sequence analysis can also be found in Unit 19.3 of Ausubel et al (“Current Protocols in Molecular Biology” John Wiley & Sons Inc, 1994-1998, Chapter 15, 1998). In some embodiments, alignment between a query sequence and a reference sequence is performed with Needleman-Wunsch alignment with Gap Costs set to Existence: 11 Extension: 1 where percent identity is calculated by dividing the number of identities by the length of the alignment, as further described in Altschul et al. (“Gapped BLAST and PSI-BLAST: a new generation of protein database search programs”, Nucleic Acids Res. 25:3389-3402, 1997) and Altschul et al, (“Protein database searches using compositionally adjusted substitution matrices”, FEBS J. 272:5101-5109, 2005).

A skilled person understands that amino acid (or nucleotide) positions may be determined in homologous sequences based on alignment, for example, “H840” in a reference Cas9 sequence may correspond to H839, or another position in a Cas9 homolog.

The term “polynucleotide” or “nucleic acid molecule” can be any polymeric form of nucleotides, including DNA, RNA, a hybridization thereof, or RNA-DNA chimeric molecules. In some embodiments, a polynucleotide comprises cDNA, genomic DNA, mRNA, tRNA, rRNA, or microRNA. In some embodiments, a polynucleotide is double stranded, e.g., a double-stranded DNA in a gene. In some embodiments, a polynucleotide is single-stranded or substantially single-stranded, e.g., single-stranded DNA or an mRNA. In some embodiments, a polynucleotide is a cell-free nucleic acid molecule. In some embodiments, a polynucleotide circulates in blood. In some embodiments, a polynucleotide is a cellular nucleic acid molecule. In some embodiments, a polynucleotide is a cellular nucleic acid molecule in a cell circulating in blood.

Polynucleotides can have any three-dimensional structure. The following are nonlimiting examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), an exon, an intron, intergenic DNA (including, without limitation, heterochromatic DNA), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), a ribozyme, cDNA, a recombinant polynucleotide, a branched polynucleotide, a plasmid, a vector, isolated DNA, isolated RNA, sgRNA, guide RNA, a nucleic acid probe, a primer, an snRNA, a long non-coding RNA, a snoRNA, a siRNA, a miRNA, a tRNA-derived small RNA (tsRNA), an antisense RNA, an shRNA, or a small rDNA-derived RNA (srRNA).

In some embodiments, a polynucleotide comprises deoxyribonucleotides, ribonucleotides or analogs thereof. In some embodiments, a polynucleotide comprises modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component.

In some embodiments, a polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA. In some embodiments, the polynucleotide may comprise one or more other nucleotide bases, such as inosine (I), which is read by the translation machinery as guanine (G).

In some embodiments, a polynucleotide may be modified. As used herein, the terms “modified” or “modification” refers to chemical modification with respect to the A, C, G, T and U nucleotides. In some embodiments, modifications may be on the nucleoside base and/or sugar portion of the nucleosides that comprise the polynucleotide. In some embodiments, the modification may be on the internucleoside linkage (e.g., phosphate backbone). In some embodiments, multiple modifications are included in the modified nucleic acid molecule. In some embodiments, a single modification is included in the modified nucleic acid molecule.

The term “complement”, “complementary”, or “complementarity” as used herein, refers to the ability of two polynucleotide molecules to base pair with each other. Complementary polynucleotides may base pair via hydrogen bonding, which may be Watson Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding. For example, an adenine on one polynucleotide molecule will base pair to a thymine or an uracil on a second polynucleotide molecule and a cytosine on one polynucleotide molecule will base pair to a guanine on a second polynucleotide molecule. Two polynucleotide molecules are complementary to each other when a first polynucleotide molecule comprising a first nucleotide sequence can base pair with a second polynucleotide molecule comprising a second nucleotide sequence. For instance, the two DNA molecules 5′-ATGC-3′ and 5′-GCAT-3′ are complementary, and the complement of the DNA molecule 5′-ATGC-3′ is 5′-GCAT-3′. A percentage of complementarity indicates the percentage of nucleotides in a polynucleotide molecule which can base pair with a second polynucleotide molecule (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary, respectively). “Perfectly complementary” means that all the contiguous nucleotides of a polynucleotide molecule will base pair with the same number of contiguous nucleotides in a second polynucleotide molecule. “Substantially complementary” as used herein refers to a degree of complementarity that can be 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% over all or a portion of two polynucleotide molecules. In some embodiments, the portion of complementarity may be a region of 10, 15, 20, 25, 30, 35, 40, 45, 50, or more nucleotides. “Substantial complementary” can also refer to a 100% complementarity over a portion of two polynucleotide molecules. In some embodiments, the portion of complementarity between the two polynucleotide molecules is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% of the length of at least one of the two polynucleotide molecules or a functional or defined portion thereof.

As used herein, “expression” refers to the process by which polynucleotides are transcribed into mRNA and/or the process by which polynucleotides, e.g., the transcribed mRNA, translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. In some embodiments, expression of a polynucleotide, e.g., a gene or a DNA encoding a protein, is determined by the amount of the protein encoded by the gene after transcription and translation of the gene. In some embodiments, expression of a polynucleotide, e.g., a gene or a DNA encoding a protein, is determined by the amount of a functional form of the protein encoded by the gene after transcription and translation of the gene. In some embodiments, expression of a gene is determined by the amount of the mRNA, or transcript, that is encoded by the gene after transcription the gene. In some embodiments, expression of a polynucleotide, e.g., an mRNA, is determined by the amount of the protein encoded by the mRNA after translation of the mRNA. In some embodiments, expression of a polynucleotide, e.g., a mRNA or coding RNA, is determined by the amount of a functional form of the protein encoded by the polypeptide after translation of the polynucleotide.

The term “sequencing” as used herein, may comprise capillary sequencing, bisulfite-free sequencing, bisulfite sequencing, TET-assisted bisulfite (TAB) sequencing, ACE-sequencing, high-throughput sequencing, Maxam-Gilbert sequencing, massively parallel signature sequencing, Polony sequencing, 454 pyrosequencing, Sanger sequencing, Illumina sequencing, SOLiD sequencing, Ion Torrent semiconductor sequencing, DNA nanoball sequencing, Heliscope single molecule sequencing, single molecule real time (SMRT) sequencing, nanopore sequencing, shot gun sequencing, RNA sequencing, or any combination thereof.

The terms “equivalent” or “biological equivalent” are used interchangeably when referring to a particular molecule, or biological or cellular material, and means a molecule having minimal homology to another molecule while still maintaining a desired structure or functionality.

The term “encode” as it is applied to polynucleotides refers to a polynucleotide which is said to “encode” another polynucleotide, a polypeptide, or an amino acid if, in its native state or when manipulated by methods well known to those skilled in the art, it can be used as polynucleotide synthesis template, e.g., transcribed into an RNA, reverse transcribed into a DNA or cDNA, and/or translated to produce an amino acid, or a polypeptide or fragment thereof. In some embodiments, a polynucleotide comprising three contiguous nucleotides form a codon that encodes a specific amino acid. In some embodiments, a polynucleotide comprises one or more codons that encode a polypeptide. In some embodiments, a polynucleotide comprising one or more codons comprises a mutation in a codon compared to a wild-type reference polynucleotide. In some embodiments, the mutation in the codon encodes an amino acid substitution in a polypeptide encoded by the polynucleotide as compared to a wild-type reference polypeptide.

The term “mutation” as used herein refers to a change and/or alteration in an amino acid sequence of a protein or nucleic acid sequence of a polynucleotide. Such changes and/or alterations may comprise the substitution, insertion, deletion and/or truncation of one or more amino acids, in the case of an amino acid sequence, and/or nucleotides, in the case of nucleic acid sequence, compared to a reference amino acid or nucleic acid sequence. In some embodiments, the reference sequence is a wild-type sequence. In some embodiments, a mutation in a nucleic acid sequence of a polynucleotide encodes a mutation in the amino acid sequence of a polypeptide. In some embodiments, the mutation in the amino acid sequence of the polypeptide or the mutation in the nucleic acid sequence of the polynucleotide is a mutation associated with a disease state.

The term “subject” and its grammatical equivalents as used herein may refer to a human or a non-human. A subject may be a mammal. A human subject may be male or female. A human subject may be of any age. A subject may be a human embryo. A human subject may be a newborn, an infant, a child, an adolescent, or an adult. A human subject may be up to about 100 years of age. A human subject may be in need of treatment for a genetic disease or disorder.

The terms “treatment” or “treating” and their grammatical equivalents may refer to the medical management of a subject with an intent to cure, ameliorate, or ameliorate a symptom of, a disease, condition, or disorder. Treatment may include active treatment, that is, treatment directed specifically toward the improvement of a disease, condition, or disorder. Treatment may include causal treatment, that is, treatment directed toward removal of the cause of the associated disease, condition, or disorder. In addition, this treatment may include palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, condition, or disorder. Treatment may include supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the disease, condition, or disorder. In some embodiments, a condition may be pathological. In some embodiments, a treatment may not completely cure or prevent a disease, condition, or disorder. In some embodiments, a treatment ameliorates, but does not completely cure or prevent a disease, condition, or disorder. In some embodiments, a subject may be treated for 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, indefinitely, or life of the subject.

The term “ameliorate” and its grammatical equivalents means to decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.

The terms “prevent” or “preventing” means delaying, forestalling, or avoiding the onset or development of a disease, condition, or disorder for a period of time. Prevent also means reducing risk of developing a disease, disorder, or condition. Prevention includes minimizing or partially or completely inhibiting the development of a disease, condition, or disorder. In some embodiments, a composition, e.g. a pharmaceutical composition, prevents a disorder by delaying the onset of the disorder for 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, indefinitely, or life of a subject.

The term “effective amount” or “therapeutically effective amount” may refer to a quantity of a composition, for example a composition comprising a construct, that can be sufficient to result in a desired activity upon introduction into a subject as disclosed herein. An effective amount of the prime editing compositions can be provided to the target gene or cell, whether the cell is ex vivo or in vivo. An effective amount can be the amount to induce, for example, at least about a 2-fold change (increase or decrease) or more in the amount of target nucleic acid modulation (e.g., expression of ATP7B gene to produce functional ATP7B protein) observed relative to a negative control. An effective amount or dose can induce, for example, about 2-fold increase, about 3-fold increase, about 4-fold increase, about 5-fold increase, about 6-fold increase, about 7-fold increase, about 8-fold increase, about 9-fold increase, about 10-fold increase, about 25-fold increase, about 50-fold increase, about 100-fold increase, about 200-fold increase, about 500-fold increase, about 700-fold increase, about 1000-fold increase, about 5000-fold increase, or about 10,000-fold increase in target gene modulation (e.g., expression of a target ATP7B gene to produce functional ATP7B protein). The amount of target gene modulation may be measured by any suitable method known in the art. In some embodiments, the “effective amount” or “therapeutically effective amount” is the amount of a composition that is required to ameliorate the symptoms of a disease relative to an untreated patient. In some embodiments, an effective amount is the amount of a composition sufficient to introduce an alteration in a gene of interest in a cell (e.g., a cell in vitro or in vivo).

As used herein, the terms “Wilson's disease,” “Wilsons disease,” and “Wilson disease” are used interchangeably. Wilson's disease is a monogenic autosomal-recessive disorder caused by pathogenic variants in ATP7B that decrease ATP7B function in hepatocytes and reduce excretion of excess copper into bile, leading to systemic copper buildup, hepatic and neural toxicity, and early demise. In some embodiments, mutations in the ATP7B gene are associated with diseases including Wilson's disease. The ATP7B gene codes for a copper transporter expressed in hepatic and neural tissues. The gene product is synthesized in the endoplasmic reticulum, then relocated to the trans Golgi network (TGN) within hepatocytes. ATP7B is most highly expressed in the liver, but is also found in the kidney, placenta, mammary glands, brain, and lung. Alternate names for ATP7B include: ATPase Copper Transporting Beta, Copper-Transporting ATPase, Copper Pump. ATPase, Cu++ Transporting. Beta Polypeptide, Wilson Disease-Associated Protein, PWD, WC1, WND, ATPase, Cu++ Transporting, Beta Polypeptide (Wilson Disease) 2. ATPase, Cu(2+)-Transporting, Beta Polypeptide, Copper-Transporting Protein ATP7B, Wilson Disease, EC 3.6.3.4, EC 7.2.2.8. EC 363, WD. In the human genome the ATP7B gene is located on 13q14.3 and contains 20 introns and 21 exons, for a total genomic length of 80 kb (chr13:51,930,436-52,012,130 (GRCh38/hg38)).

More than 600 pathogenic variants in ATP7B have been identified, with single-nucleotide missense and nonsense mutations being the most common, followed by insertions/deletions splice site mutations. A histidine-to-glutamate substitution at amino acid 1069 (p.H1069Q) (caused by c.3207C>A) in ATP7B maybe one of the most common cause of Wilson's disease, with a population allelic frequency of 10-40% (e.g., 30-70% among Caucasians. The p.H1069Q mutation occurs when histidine of the conserved SEHPL motif (SEQ ID NO: 5896) in the N-domain of ATP7B is replaced by glutamic acid, resulting in N-domain protein misfolding, abnormal phosphorylation in the P-domain, and decreased ATP binding affinity. This mutation may also lead to decreased heat stability and abnormal localization of the protein to the trans-Golgi network.

Prime Editing

The term “prime editing” refers to programmable editing of a target DNA using a prime editor complexed with a PEgRNA to incorporate an intended nucleotide edit into the target DNA through target-primed DNA synthesis. A target gene of prime editing may comprise a double stranded DNA molecule having two complementary strands: a first strand that may be referred to as a “target strand” or a “non-edit strand”, and a second strand that may be referred to as a “non-target strand,” or an “edit strand.” In some embodiments, in a prime editing guide RNA (PEgRNA), a spacer sequence is complementary or substantially complementary to a specific sequence on the target strand, which may be referred to as a “search target sequence”. In some embodiments, the spacer sequence anneals with the target strand at the search target sequence. The target strand may also be referred to as the “non-Protospacer Adjacent Motif (non-PAM strand).” In some embodiments, the non-target strand may also be referred to as the “PAM strand”. In some embodiments, the PAM strand comprises a protospacer sequence and optionally a PAM sequence. A protospacer sequence refers to a specific sequence in the PAM strand of the target gene that is complementary to the search target sequence. In a PEgRNA, a spacer sequence may have a substantially identical sequence as the protospacer sequence on the edit strand of a target gene, except that the spacer sequence may comprise Uracil (U) and the protospacer sequence may comprise Thymine (T).

In some embodiments, the double stranded target DNA comprises a nick site on the PAM strand (or non-target strand). As used herein, a “nick site” refers to a specific position in between two nucleotides or two base pairs of the double stranded target DNA. In some embodiments, the position of a nick site is determined relative to the position of a specific PAM sequence. In some embodiments, the nick site is the particular position where a nick will occur when the double stranded target DNA is contacted with a nickase, for example, a Cas nickase, that recognizes a specific PAM sequence. In some embodiments, the nick site is upstream of a specific PAM sequence on the PAM strand of the double stranded target DNA. In some embodiments, the nick site is downstream of a specific PAM sequence on the PAM strand of the double stranded target DNA. In some embodiments, the nick site is upstream of a PAM sequence recognized by a Cas9 nickase, wherein the Cas9 nickase comprises a nuclease active RuvC domain and a nuclease inactive NHN domain. In some embodiments, the nick site is 3 nucleotides upstream of the PAM sequence, and the PAM sequence is recognized by a Streptococcus pyogenes Cas9 nickase, a P. lavamentivorans Cas9 nickase, a C. diphtheriae Cas9 nickase, a N. cinerea Cas9, a S. aureus Cas9, or a N. lari Cas9 nickase that comprises a nuclease active RuvC domain and a nuclease inactive NHN domain. In some embodiments, the nick site is 2 nucleotides upstream of the PAM sequence, and the PAM sequence is recognized by a S. thermophilus Cas9 nickase that comprises a nuclease active RuvC domain and a nuclease inactive NHN domain.

A “primer binding site” (also referred to as PBS or primer binding site sequence) is a single-stranded portion of the PEgRNA that comprises a region of complementarity to the PAM strand (i.e., the non-target strand or the edit strand). The PBS is complementary or substantially complementary to a sequence on the PAM strand of the double stranded target DNA that is immediately upstream of the nick site. In some embodiments, in the process of prime editing, the PEgRNA complexes with and directs a prime editor to bind the search target sequence on the target strand of the double stranded target DNA, and generates a nick at the nick site on the non-target strand of the double stranded target DNA. In some embodiments, the PBS is complementary to or substantially complementary to, and can anneal to, a free 3′ end on the non-target strand of the double stranded target DNA at the nick site. In some embodiments, the PBS annealed to the free 3′ end on the non-target strand can initiate target-primed DNA synthesis.

An “editing template” of a PEgRNA is a single-stranded portion of the PEgRNA that is 5′ of the PBS and comprises a region of complementarity to the PAM strand (i.e. the non-target strand or the edit strand), and comprises one or more intended nucleotide edits compared to the endogenous sequence of the double stranded target DNA. In some embodiments, the editing template and the PBS are immediately adjacent to each other. Accordingly, in some embodiments, a PEgRNA in prime editing comprises a single-stranded portion that comprises the PBS and the editing template immediately adjacent to each other. In some embodiments, the single stranded portion of the PEgRNA comprising both the PBS and the editing template is complementary or substantially complementary to an endogenous sequence on the PAM strand (i.e., the non-target strand or the edit strand) of the double stranded target DNA except for one or more non-complementary nucleotides at the intended nucleotide edit positions. As used herein, regardless of relative 5′-3′ positioning in other context, the relative positions as between the PBS and the editing template, and the relative positions as among elements of a PEgRNA, are determined by the 5′ to 3′ order of the PEgRNA as a single molecule regardless of the position of sequences in the double stranded target DNA that may have complementarity or identity to elements of the PEgRNA. In some embodiments, the editing template is complementary or substantially complementary to a sequence on the PAM strand that is immediately downstream of the nick site, except for one or more non-complementary nucleotides at the intended nucleotide edit positions. The endogenous, e.g., genomic, sequence that is complementary or substantially complementary to the editing template, except for the one or more non-complementary nucleotides at the position corresponding to the intended nucleotide edit, may be referred to as an “editing target sequence”. In some embodiments, the editing template has identity or substantial identity to a sequence on the target strand that is complementary to, or having the same position in the genome as, the editing target sequence, except for one or more insertions, deletions, or substitutions at the intended nucleotide edit positions. In some embodiments, the editing template encodes a single stranded DNA, wherein the single stranded DNA has identity or substantial identity to the editing target sequence except for one or more insertions, deletions, or substitutions at the positions of the one or more intended nucleotide edits.

In some embodiments, a PEgRNA complexes with and directs a prime editor to bind to the search target sequence of the target gene. In some embodiments, the bound prime editor generates a nick on the edit strand (PAM strand) of the target gene. In some embodiments, a primer binding site (PBS) of the PEgRNA anneals with a free 3′ end formed at the nick site, and the prime editor initiates DNA synthesis from the nick site, using the free 3′ end as a primer. Subsequently, a single-stranded DNA encoded by the editing template of the PEgRNA is synthesized. In some embodiments, the newly synthesized single-stranded DNA comprises one or more intended nucleotide edits compared to the endogenous target gene sequence. Accordingly, in some embodiments, the editing template of a PEgRNA is complementary to a sequence in the edit strand except for one or more mismatches at the intended nucleotide edit positions in the editing template. The endogenous, e.g., genomic, sequence that is partially complementary to the editing template may be referred to as an “editing target sequence”.

In some embodiments, the newly synthesized single-stranded DNA equilibrates with the editing target on the edit strand of the target gene for pairing with the target strand of the target gene. In some embodiments, the editing target sequence of the target gene is excised by a flap endonuclease (FEN), for example, FEN1. In some embodiments, the FEN is an endogenous FEN, for example, in a cell comprising the target gene. In some embodiments, the FEN is provided as part of the prime editor, either linked to other components of the prime editor or provided in trans. In some embodiments, the newly synthesized single stranded DNA, which comprises the intended nucleotide edit, replaces the endogenous single stranded editing target sequence on the edit strand of the target gene. In some embodiments, the newly synthesized single stranded DNA and the endogenous DNA on the target strand form a heteroduplex DNA structure at the region corresponding to the editing target sequence of the target gene. In some embodiments, the newly synthesized single-stranded DNA comprising the nucleotide edit is paired in the heteroduplex with the target strand of the target DNA that does not comprise the nucleotide edit, thereby creating a mismatch. In some embodiments, the mismatch is recognized by DNA repair machinery, e.g., an endogenous DNA repair machinery. In some embodiments, through DNA repair, the intended nucleotide edit is incorporated into the target gene.

Prime Editor

The term “prime editor (PE)” refers to the polypeptide or polypeptide components involved in prime editing. In various embodiments, a prime editor includes a polypeptide domain having DNA binding activity and a polypeptide domain having DNA polymerase activity. In some embodiments, the prime editor further comprises a polypeptide domain having nuclease activity. In some embodiments, the polypeptide domain having DNA binding activity comprises a nuclease domain or nuclease activity. In some embodiments, the polypeptide domain having nuclease activity comprises a nickase, or a fully active nuclease. As used herein, the term “nickase” refers to a nuclease capable of cleaving only one strand of a double-stranded DNA target. In some embodiments, the prime editor comprises a polypeptide domain that is an inactive nuclease. In some embodiments, the polypeptide domain having programmable DNA binding activity comprises a nucleic acid guided DNA binding domain, for example, a CRISPR-Cas protein, for example, a Cas9 nickase, a Cpf1 nickase, or another CRISPR-Cas nuclease. In some embodiments, the polypeptide domain having DNA polymerase activity comprises a template-dependent DNA polymerase, for example, a DNA-dependent DNA polymerase or an RNA-dependent DNA polymerase. In some embodiments, the DNA polymerase is a reverse transcriptase. In some embodiments, the prime editor comprises additional polypeptides involved in prime editing, for example, a polypeptide domain having 5′ endonuclease activity, e.g., a 5′ endogenous DNA flap endonucleases (e.g., FEN1), for helping to drive the prime editing process towards the edited product formation. In some embodiments, the prime editor further comprises an RNA-protein recruitment polypeptide, for example, a MS2 coat protein.

A prime editor may be engineered. In some embodiments, the polypeptide components of a prime editor do not naturally occur in the same organism or cellular environment. In some embodiments, the polypeptide components of a prime editor may be of different origins or from different organisms. In some embodiments, a prime editor comprises a DNA binding domain and a DNA polymerase domain that are derived from different species. In some embodiments, a prime editor comprises a Cas polypeptide and a reverse transcriptase polypeptide that are derived from different species. For example, a prime editor may comprise a S. pyogenes Cas9 polypeptide and a Moloney murine leukemia virus (M-MLV) reverse transcriptase polypeptide.

In some embodiments, polypeptide domains of a prime editor may be fused or linked by a peptide linker to form a fusion protein. In other embodiments, a prime editor comprises one or more polypeptide domains provided in trans as separate proteins, which are capable of being associated to each other through non-peptide linkages or through aptamers or recruitment sequences. For example, a prime editor may comprise a DNA binding domain and a reverse transcriptase domain associated with each other by an RNA-protein recruitment aptamer, e.g. a MS2 aptamer, which may be linked to a PEgRNA. Prime editor polypeptide components may be encoded by one or more polynucleotides in whole or in part. In some embodiments, a single polynucleotide, construct, or vector encodes the prime editor fusion protein. In some embodiments, multiple polynucleotides, constructs, or vectors each encode a polypeptide domain or portion of a domain of a prime editor, or a portion of a prime editor fusion protein. For example, a prime editor fusion protein may comprise an N-terminal portion fused to an intein-N and a C-terminal portion fused to an intein-C, each of which is individually encoded by an AAV vector. In some embodiments, a prime editor comprises a nucleotide polymerase domain, e.g. a DNA polymerase domain. The DNA polymerase domain may be a wild-type DNA polymerase domain, a full-length DNA polymerase protein domain, or may be a functional mutant, a functional variant, or a functional fragment thereof. In some embodiments, the polymerase domain is a template dependent polymerase domain. For example, the DNA polymerase may rely on a template polynucleotide strand, e.g., the editing template sequence, for new strand DNA synthesis. In some embodiments, the prime editor comprises a DNA-dependent DNA polymerase. For example, a prime editor having a DNA-dependent DNA polymerase can synthesize a new single stranded DNA using a PEgRNA editing template that comprises a DNA sequence as a template. In such cases, the PEgRNA is a chimeric or hybrid PEgRNA, and comprising an extension arm comprising a DNA strand. The chimeric or hybrid PEgRNA may comprise an RNA portion (including the spacer and the gRNA core) and a DNA portion (the extension arm comprising the editing template that includes a strand of DNA). In some embodiments, the DNA polymerase is a bacteriophage polymerase, for example, a T4, T7, or phi29 DNA polymerase. In some embodiments, the DNA polymerase is an archaeal polymerase, for example, pol I type archaeal polymerase or a pol II type archaeal polymerase. In some embodiments, the DNA polymerase comprises a thermostable archaeal DNA polymerase. In some embodiments, the DNA polymerase comprises a eubacterial DNA polymerase, for example, Pol I, Pol II, or Pol III polymerase. In some embodiments, the DNA polymerase is a Pol I family DNA polymerase. In some embodiments, the DNA polymerase is a E. coli Pol I DNA polymerase. In some embodiments, the DNA polymerase is a Pol II family DNA polymerase. In some embodiments, the DNA polymerase is a Pyrococcusfuriosus (Pfu) Pol II DNA polymerase. In some embodiments, the DNA Polymerase is a Pol IV family DNA polymerase. In some embodiments, the DNA polymerase is a E. coli Pol IV DNA polymerase. In some embodiments, the DNA polymerase comprises a eukaryotic DNA polymerase. In some embodiments, the DNA polymerase is a Pol-beta DNA polymerase, a Pol-lamda DNA polymerase, a Pol-sigma DNA polymerase, or a Pol-mu DNA polymerase. In some embodiments, the DNA polymerase is a Pol-alpha DNA polymerase. In some embodiments, the DNA polymerase is a POLA1 DNA polymerase. In some embodiments, the DNA polymerase is a POLA2 DNA polymerase. In some embodiments, the DNA polymerase is a Pol-delta DNA polymerase. In some embodiments, the DNA polymerase is a POLD1 DNA polymerase. In some embodiments, the DNA polymerase is a POLD2 DNA polymerase. In some embodiments, the DNA polymerase is a human POLD1 DNA polymerase. In some embodiments, the DNA polymerase is a human POLD2 DNA polymerase. In some embodiments, the DNA polymerase is a POLD3 DNA polymerase. In some embodiments, the DNA polymerase is a POLD4 DNA polymerase. In some embodiments, the DNA polymerase is a Pol-epsilon DNA polymerase. In some embodiments, the DNA polymerase is a POLE1 DNA polymerase. In some embodiments, the DNA polymerase is a POLE2 DNA polymerase. In some embodiments, the DNA polymerase is a POLE3 DNA polymerase. In some embodiments, the DNA polymerase is a Pol-eta (POLH) DNA polymerase. In some embodiments, the DNA polymerase is a Pol-iota (POLI) DNA polymerase. In some embodiments, the DNA polymerase is a Pol-kappa (POLK) DNA polymerase. In some embodiments, the DNA polymerase is a Rev1 DNA polymerase. In some embodiments, the DNA polymerase is a human Rev1 DNA polymerase. In some embodiments, the DNA polymerase is a viral DNA-dependent DNA polymerase. In some embodiments, the DNA polymerase is a B family DNA polymerases. In some embodiments, the DNA polymerase is a herpes simplex virus (HSV) UL30 DNA polymerase. In some embodiments, the DNA polymerase is a cytomegalovirus (CMV) UL54 DNA polymerase.

In some embodiments, a prime editor comprises an RNA-dependent DNA polymerase domain, for example, a reverse transcriptase (RT). A RT or an RT domain may be a wild type RT domain, a full-length RT domain, or may be a functional mutant, a functional variant, or a functional fragment thereof. An RT or an RT domain of a prime editor may comprise a wild-type RT, or may be engineered or evolved to contain specific amino acid substitutions, truncations, or variants. An engineered RT may comprise sequences or amino acid changes different from a naturally occurring RT. In some embodiments, the engineered RT may have improved reverse transcription activity over a naturally occurring RT or RT domain. In some embodiments, the engineered RT may have improved features over a naturally occurring RT, for example, improved thermostability, reverse transcription efficiency, or target fidelity. In some embodiments, a prime editor comprising the engineered RT has improved prime editing efficiency over a prime editor having a reference naturally occurring RT.

In some embodiments, a prime editor comprises a virus RT, for example, a retrovirus RT. Non-limiting examples of virus RT include Moloney murine leukemia virus (M-MLV or MMLVRT); human T-cell leukemia virus type 1 (HTLV-1) RT; bovine leukemia virus (BLV) RT; Rous Sarcoma Virus (RSV) RT; human immunodeficiency virus (HIV) RT, M-MFV RT, Avian Sarcoma-Leukosis Virus (ASLV) RT, Rous Sarcoma Virus (RSV) RT, Avian Myeloblastosis Virus (AMV) RT, Avian Erythroblastosis Virus (AEV) Helper Virus MCAV RT, Avian Myelocytomatosis Virus MC29 Helper Virus MCAV RT, Avian Reticuloendotheliosis Virus (REV-T) Helper Virus REV-A RT, Avian Sarcoma Virus UR2 Helper Virus (UR2AV) RT, Avian Sarcoma Virus Y73 Helper Virus YAV RT, Rous Associated Virus (RAV) RT, and Myeloblastosis Associated Virus (MAV) RT, all of which may be suitably used in the methods and composition described herein.

In some embodiments, the prime editor comprises a wild-type M-MLV RT. An exemplary amino acid sequence of a wild-type M-MLV RT is provided in SEQ ID NO: 5780.

Exemplary wild type moloney murine leukemia virus reverse transcriptase:

(SEQ ID NO: 5780)

TLNIEDEHRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLII

PLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP

VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLD

LKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFD

EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNL

GYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQL

REFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQA

LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLD

PVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDR

WLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLQHNCLDILA

EAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAK

ALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRR

RGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNR

MADQAARKAAITETPDTSTLLIENSSP.

In some embodiments, the prime editor comprises a reference M-MLV RT. An exemplary amino acid sequence of a reference M-MLV RT is provided in SEQ ID NO: 5781.

Exemplary reference moloney murine leukemia virus reverse transcriptase:

(SEQ ID NO: 5781)

TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLII

PLKATSTPVSIKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPWNTPLLP

VKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLD

LKDAFFCLRLHPTSQPLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFD

EALHRDLADFRIQHPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNL

GYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQL

REFLGTAGFCRLWIPGFAEMAAPLYPLTKTGTLFNWGPDQQKAYQEIKQA

LLTAPALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLD

PVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPDR

WLSNARMTHYQALLLDTDRVQFGPVVALNPATLLPLPEEGLOHNCLDILA

EAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAK

ALPAGTSAQRAELIALTQALKMAEGKKLNVYTDSRYAFATAHIHGEIYRR

RGLLTSEGKEIKNKDEILALLKALFLPKRLSIIHCPGHQKGHSAEARGNR

MADQAARKAAITETPDTSTLLIENSSP.

In some embodiments, the prime editor comprises a M-MLV RT comprising one or more of amino acid substitutions P51X, S67X, E69X, L139X, T197X, D200X, H204X, F209X, E302X, T306X, F309X, W313X, T330X, L345X, L435X, N454X, D524X, E562X, D583X, H594X, L603X, E607X, or D653X as compared to the reference M-MLV RT as set forth in SEQ ID NO: 5781, where X is any amino acid other than the original amino acid in the reference M-MLV RT. In some embodiments, the prime editor comprises a M-MLV RT comprising one or more of amino acid substitutions P51L, S67K, E69K, L139P, T197A, D200N, H204R, F209N, E302K, E302R, T306K, F309N, W313F, T330P, L345G, L435G, N454K, D524G, E562Q, D583N, H594Q, L603W, E607K, and D653N as compared to the reference M-MLV RT as set forth in SEQ ID NO: 5781. In some embodiments, the prime editor comprises a M-MLV RT comprising one or more amino acid substitutions D200N, T330P, L603W, T306K, and W313F as compared to the reference M-MLV RT as set forth in SEQ ID NO: 5781. In some embodiments, the prime editor comprises a M-MLV RT comprising amino acid substitutions D200N, T330P, L603W, T306K, and W313F as compared to the reference M-MLV RT as set forth in SEQ ID NO: 5781. In some embodiments, a prime editor comprising the D200N, T330P, L603W, T306K, and W313F as compared to a reference M-MLV RT may be referred to as a “PE2” prime editor, and the corresponding prime editing system a PE2 prime editing system.

In some embodiments, a prime editor comprises a eukaryotic RT, for example, a yeast, drosophila, rodent, or primate RT. In some embodiments, the prime editor comprises a Group II intron RT, for example, a. Geobacillus stearothermophilus Group II Intron (GsI-IIC) RT or a Eubacterium rectale group II intron (Eu.re.I2) RT. In some embodiments, the prime editor comprises a retron RT.

In some embodiments, the DNA-binding domain of a prime editor is a programmable DNA binding domain. In some embodiments, the prime editors provided herein comprise a DNA binding domain comprising an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of the sequences set forth in SEQ ID NOs: 5783-5819. In some embodiments, the DNA binding domain comprises an amino acid sequence that has no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 differences e.g., mutations e.g., deletions, substitutions and/or insertions compared to any one of the amino acid sequences set forth in SEQ ID NOs: 5783-5819. A programmable DNA binding domain refers to a protein domain that is designed to bind a specific nucleic acid sequence, e.g., a target DNA or a target RNA. In some embodiments, the DNA binding domain is a polynucleotide programmable DNA-binding domain that can associate with a guide polynucleotide (e.g., a PEgRNA) that guides the DNA-binding domain to a specific DNA sequence, e.g., a search target sequence in a target gene. In some embodiments, the DNA binding domain comprises a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Associated (Cas) protein. A Cas protein may comprise any Cas protein described herein or a functional fragment or functional variant thereof. In some embodiments, a DNA binding domain may also comprise a zinc-finger protein domain. In other cases, a DNA binding domain comprises a transcription activator-like effector domain (TALE). In some embodiments, the DNA binding domain comprises a DNA nuclease. For example, the DNA binding domain of a prime editor may comprise an RNA-guided DNA endonuclease, e.g., a Cas protein. In some embodiments, the DNA binding domain comprises a zinc finger nuclease (ZFN) or a transcription activator like effector domain nuclease (TALEN), where one or more zinc finger motifs or TALE motifs are associated with one or more nucleases, e.g., a Fok I nuclease domain.

In some embodiments, the DNA binding domain comprises a nuclease activity. In some embodiments, the DNA binding domain of a prime editor comprises an endonuclease domain having single strand DNA cleavage activity. For example, the endonuclease domain may comprise a FokI nuclease domain. In some embodiments, the DNA binding domain of a prime editor comprises a nuclease having full nuclease activity. In some embodiments, the DNA-binding domain of a prime editor comprises a nuclease having modified or reduced nuclease activity as compared to a wild type endonuclease domain. For example, the endonuclease domain may comprise one or more amino acid substitutions as compared to a wild type endonuclease domain. In some embodiments, the DNA binding domain of a prime editor has a nickase activity. In some embodiments, the DNA binding domain of a prime editor comprises a Cas protein domain that is a nickase. In some embodiments, compared to a wild type Cas protein, the Cas nickase comprises one or more amino acid substitutions in a nuclease domain that reduces or abolishes its double strand nuclease activity but retains DNA binding activity. In some embodiments, the Cas nickase comprises an amino acid substitution in a HNH domain. In some embodiments, the Cas nickase comprises an amino acid substitution in a RuvC domain.

In some embodiments, the DNA-binding domain comprises a CRISPR associated protein (Cas protein) domain. A Cas protein may be a Class 1 or a Class 2 Cas protein. A Cas protein can be a type I, type II, type III, type IV, type V Cas protein, or a type VI Cas protein. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (e.g., Csn1 or Csx12), Cas10, Cas10d, Cas12a/Cpf1, Cas12b/C2c1, Cas12c/C2c3, Cas12d/CasY, Cas12e/CasX, Cas12g, Cas12h, Cas12i, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, Type II Cas effector proteins, Type V Cas effector proteins, Type VI Cas effector proteins, CARF, DinG, Cpf1, Cas12b/C2c1, Cas12c/C2c3, Cas12b/C2c1, Cas12c/C2c3, SpCas9 (K855A), eSpCas9 (1.1), SpCas9-HF1, hyper accurate Cas9 variant (HypaCas9), Cas Φ, and homologues, modified or engineered variants, mutants, and/or functional fragments thereof. A Cas protein can be a chimeric Cas protein that is fused to other proteins or polypeptides. A Cas protein can be a chimera of various Cas proteins, for example, comprising domains of Cas proteins from different organisms. A Cas protein, e.g., Cas9, can be from any suitable organism. In some aspects, the organism is Streptococcus pyogenes (S. pyogenes). In some aspects, the organism is Staphylococcus aureus (S. aureus). In some aspects, the organism is Streptococcus thermophilus (S. thermophilus). In some embodiments, the organism is Staphylococcus lugdunensis. Non-limiting examples of suitable organism include Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Nocardiopsis dassonvillei, Streptomyces pristinae spiralis, Streptomyces viridochromo genes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, AlicyclobacHlus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Pseudomonas aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, Acaryochloris marina, Leptotrichia shahii, and Francisella novicida. In some embodiments, the organism is Streptococcus pyogenes (S. pyogenes). In some embodiments, the organism is Staphylococcus aureus (S. aureus). In some embodiments, the organism is Streptococcus thermophilus (S. thermophilus). In some embodiments, the organism is Staphylococcus lugdunensis (S. lugdunensis).

In some embodiments, a Cas protein can be derived from a variety of bacterial species including, but not limited to, Veillonella atypical, Fusobacterium nucleatum, Filifactor alocis, Solobacterium moorei, Coprococcus catus, Treponema denticola, Peptoniphilus duerdenii, Catenibacterium mitsuokai, Streptococcus mutans, Listeria innocua, Staphylococcus pseudintermedius, Acidaminococcus intestine, Olsenella uli, Oenococcus kitaharae, Bifidobacterium bifidum, Lactobacillus rhamnosus, Lactobacillus gasseri, Finegoldia magna, Mycoplasma mobile, Mycoplasma gallisepticum, Mycoplasma ovipneumoniae, Mycoplasma canis, Mycoplasma synoviae, Eubacterium rectale, Streptococcus thermophilus, Eubacterium dolichum, Lactobacillus coryniformis subsp. Torquens, Ilyobacter polytropus, Ruminococcus albus, Akkermansia muciniphila, Acidothermus cellulolyticus, Bifidobacterium longum, Bifidobacterium dentium, Corynebacterium diphtheria, Elusimicrobium minutum, Nitratifractor salsuginis, Sphaerochaeta globus, Fibrobacter succinogenes subsp. Succinogenes, Bacteroides fragilis, Capnocytophaga ochracea, Rhodopseudomonas palustris, Prevotella micans, Prevotella ruminicola, Flavobacterium columnare, Aminomonas paucivorans, Rhodospirillum rubrum, Candidatus Puniceispirillum marinum, Verminephrobacter eiseniae, Ralstonia syzygii, Dinoroseobacter shibae, Azospirillum, Nitrobacter hamburgensis, Bradyrhizobium, Wolinella succinogenes, Campylobacter jejuni subsp. Jejuni, Helicobacter mustelae, Bacillus cereus, Acidovorax ebreus, Clostridium perfringens, Parvibaculum lavamentivorans, Roseburia intestinalis, Neisseria meningitidis, Pasteurella multocida subsp. Multocida, Sutterella wadsworthensis, proteobacterium, Legionella pneumophila, Parasutterella excrementihominis, Wolinella succinogenes, and Francisella novicida.

In some embodiments, a Cas protein, e.g., Cas9, can be a wild type or a modified form of a Cas protein. In some embodiments, a Cas protein, e.g., Cas9, can be a nuclease active variant, nuclease inactive variant, a nickase, or a functional variant or functional fragment of a wild type Cas protein. In some embodiments, a Cas protein, e.g., Cas9, can comprise an amino acid change such as a deletion, insertion, substitution, fusion, chimera, or any combination thereof relative to a corresponding wild-type version of the Cas protein. In some embodiments, a Cas protein can be a polypeptide with at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or sequence similarity to a wild type exemplary Cas protein.

A Cas protein, e.g., Cas9, may comprise one or more domains. Non-limiting examples of Cas domains include, guide nucleic acid recognition and/or binding domain, nuclease domains (e.g., DNase or RNase domains, RuvC, HNH), DNA binding domain, RNA binding domain, helicase domains, protein-protein interaction domains, and dimerization domains. In various embodiments, a Cas protein comprises a guide nucleic acid recognition and/or binding domain can interact with a guide nucleic acid, and one or more nuclease domains that comprise catalytic activity for nucleic acid cleavage.

In some embodiments, a Cas protein, e.g., Cas9, comprises one or more nuclease domains. A Cas protein can comprise an amino acid sequence having at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nuclease domain (e.g., RuvC domain, HNH domain) of a wild-type Cas protein. In some embodiments, a Cas protein comprises a single nuclease domain. For example, a Cpf1 may comprise a RuvC domain but lacks HNH domain. In some embodiments, a Cas protein comprises two nuclease domains, e.g., a Cas9 protein can comprise an HNH nuclease domain and a RuvC nuclease domain.

In some embodiments, a prime editor comprises a Cas protein, e.g., Cas9, wherein all nuclease domains of the Cas protein are active. In some embodiments, a prime editor comprises a Cas protein having one or more inactive nuclease domains. One or a plurality of the nuclease domains (e.g., RuvC, HNH) of a Cas protein can be deleted or mutated so that they are no longer functional or comprise reduced nuclease activity. In some embodiments, a Cas protein, e.g., Cas9, comprising mutations in a nuclease domain has reduced (e.g. nickase) or abolished nuclease activity while maintaining its ability to target a nucleic acid locus at a search target sequence when complexed with a guide nucleic acid, e.g. a PEgRNA.

In some embodiments, a prime editor comprises a Cas nickase that can bind to the target gene in a sequence-specific manner and generate a single-strand break at a protospacer within double-stranded DNA in the target gene, but not a double-strand break. For example, the Cas nickase can cleave the edit strand or the non-edit strand of the target gene, but may not cleave both. In some embodiments, a prime editor comprises a Cas nickase comprising two nuclease domains (e.g., Cas9), with one of the two nuclease domains modified to lack catalytic activity or deleted. In some embodiments, the Cas nickase of a prime editor comprises a nuclease inactive RuvC domain and a nuclease active HNH domain. In some embodiments, the Cas nickase of a prime editor comprises a nuclease inactive HNH domain and a nuclease active RuvC domain. In some embodiments, a prime editor comprises a Cas9 nickase having an amino acid substitution in the RuvC domain that reduces or abolishes nuclease activity of the RuvC domain. In some embodiments, the Cas9 nickase comprises a D10X amino acid substitution compared to a wild type S. pyogenes Cas9, wherein X is any amino acid other than D. In some embodiments, a prime editor comprises a Cas9 nickase having an amino acid substitution in the HNH domain that reduces or abolishes nuclease activity of the HNH domain. In some embodiments, the Cas9 nickase comprises a H840X amino acid substitution compared to a wild type S. pyogenes Cas9, wherein X is any amino acid other than H.

In some embodiments, a prime editor comprises a Cas protein that can bind to the target gene in a sequence-specific manner but lacks or has abolished nuclease activity and may not cleave either strand of a double stranded DNA in a target gene. Abolished activity or lacking activity can refer to an enzymatic activity less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, or less than 10% activity compared to a wild-type exemplary activity (e.g., wild-type Cas9 nuclease activity). In some embodiments, a Cas protein of a prime editor completely lacks nuclease activity. A nuclease, e.g., Cas9, that lacks nuclease activity may be referred to as nuclease inactive or “nuclease dead” (abbreviated by “d”). A nuclease dead Cas protein (e.g., dCas, dCas9) can bind to a target polynucleotide but may not cleave the target polynucleotide. In some embodiments, a dead Cas protein is a dead Cas9 protein. In some embodiments, a prime editor comprises a nuclease dead Cas protein wherein all of the nuclease domains (e.g., both RuvC and HNH nuclease domains in a Cas9 protein; RuvC nuclease domain in a Cpf1 protein) are mutated to lack catalytic activity, or are deleted.

A Cas protein can be modified. A Cas protein, e.g., Cas9, can be modified to increase or decrease nucleic acid binding affinity, nucleic acid binding specificity, and/or enzymatic activity. Cas proteins can also be modified to change any other activity or property of the protein, such as stability. For example, one or more nuclease domains of the Cas protein can be modified, deleted, or inactivated, or a Cas protein can be truncated to remove domains that are not essential for the function of the protein or to optimize (e.g., enhance or reduce) the activity of the Cas protein.

A Cas protein can be a fusion protein. For example, a Cas protein can be fused to a cleavage domain, an epigenetic modification domain, a transcriptional regulation domain, or a polymerase domain. A Cas protein can also be fused to a heterologous polypeptide providing increased or decreased stability. The fused domain or heterologous polypeptide can be located at the N-terminus, the C-terminus, or internally within the Cas protein.

In some embodiments, the Cas protein of a prime editor is a Class 2 Cas protein. In some embodiments, the Cas protein is a type II Cas protein. In some embodiments, the Cas protein is a Cas9 protein, a modified version of a Cas9 protein, a Cas9 protein homolog, mutant, variant, or a functional fragment thereof. As used herein, a Cas9, Cas9 protein, Cas9 polypeptide or a Cas9 nuclease refers to an RNA guided nuclease comprising one or more Cas9 nuclease domains and a Cas9 gRNA binding domain having the ability to bind a guide polynucleotide, e.g., a PEgRNA. A Cas9 protein may refer to a wild type Cas9 protein from any organism or a homolog, ortholog, or paralog from any organisms; any functional mutants or functional variants thereof; or any functional fragments or domains thereof. In some embodiments, a prime editor comprises a full-length Cas9 protein. In some embodiments, the Cas9 protein can generally comprises at least about 50%, 60%, 70%, 80%, 90%, 100% sequence identity to a wild type reference Cas9 protein (e.g., Cas9 from S. pyogenes). In some embodiments, the Cas9 comprises an amino acid change such as a deletion, insertion, substitution, fusion, chimera, or any combination thereof as compared to a wild type reference Cas9 protein.

In some embodiments, a Cas9 protein may comprise a Cas9 protein from Streptococcus pyogenes (Sp), Staphylococcus aureus (Sa), Streptococcus canis (Sc), Streptococcus thermophilus (St), Staphylococcus lugdunensis (Slu), Neisseria meningitidis (Nm), Campylobacter jejuni (Cj), Francisella novicida (Fn), or Treponema denticola (Td), or any Cas9 homolog or ortholog from an organism known in the art. In some embodiments, a Cas9 polypeptide is a SpCas9 polypeptide. In some embodiments, a Cas9 polypeptide is a SaCas9 polypeptide. In some embodiments, a Cas9 polypeptide is a ScCas9 polypeptide. In some embodiments, a Cas9 polypeptide is a StCas9 polypeptide. In some embodiments, a Cas9 polypeptide is a SluCas9 polypeptide. In some embodiments, a Cas9 polypeptide is a NmCas9 polypeptide. In some embodiments, a Cas9 polypeptide is a CjCas9 polypeptide. In some embodiments, a Cas9 polypeptide is a FnCas9 polypeptide. In some embodiments, a Cas9 polypeptide is a TdCas9 polypeptide. In some embodiments, a Cas9 polypeptide is a chimera comprising domains from two or more of the organisms described herein or those known in the art. In some embodiments, a Cas9 polypeptide is a Cas9 polypeptide from Streptococcus macacae. In some embodiments, a Cas9 polypeptide is a Cas9 polypeptide generated by replacing a PAM interaction domain of a SpCas9 with that of a Streptococcus macacae Cas9 (Spy-mac Cas9).

In some embodiments, a Cas9 protein comprises a Cas9 protein from Streptococcus pyogenes (Sp), e.g., as according to NC_002737.2:854751-858857 or the protein encoded by UniProt Q99ZW2, e.g., as according to SEQ ID NO: 5783. In some embodiments, the Cas9 protein is a SpCas9. In some embodiments, a SpCas9 can be a wild type SpCas9, a SpCas9 variant, or a nickase SpCas9. In some embodiments, the SpCas9 lacks the N-terminus methionine relative to a corresponding SpCas9 (e.g., wild type SpCas9, a SpCas9 variant or a nickase SpCas9). In some embodiments, a prime editor comprises a Cas9 protein, having an amino acid sequence as according to SEQ ID NO: 5783, not including the N-terminus methionine. In some embodiments, a wild type SpCas9 comprises an amino acid sequence set forth in SEQ ID NO: 5783 or SEQ ID NO: 5784. In some embodiments, a prime editor comprises a Cas9 protein, having an amino acid sequence as according to SEQ ID NO: 5783, not including the N-terminus methionine (e.g., as set forth in SEQ ID NO: 5784). In some embodiments, a prime editor comprises a Cas9 protein comprising one or more mutations (e.g., amino acid substitutions, insertions and/or deletions relative to a corresponding wild type Cas9 protein (e.g., wild type SpCas9). In some embodiments, the Cas9 protein comprising one or mutations relative to a wild type Cas9 protein comprises an amino acid sequence set forth in SEQ ID NO: 5785.

Exemplary Streptococcus pyogenes Cas9 (SpCas9) amino acid sequences useful in the prime editors disclosed herein are provided below in SEQ ID NOs: 5783-5789.

Exemplary wild type Streptococcus pyogenes Cas9 (SpCas9) amino acid sequence:

(SEQ ID NO: 5783)

MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA

LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR

LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD

LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP

INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP

NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI

LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI

FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR

KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY

YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK

NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD

LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI

IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ

LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD

SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV

MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP

VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD

SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL

TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI

REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK

YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI

TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV

QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE

KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK

YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPE

DNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK

PIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ

SITGLYETRIDLSQLGGD.

Exemplary wild type Streptococcus pyogenes Cas9 (SpCas9) amino acid sequence lacking the N-terminus methionine:

(SEQ ID NO: 5784)

DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL

LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL

EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADL

RLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPI

NASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPN

FKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL

LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIF

FDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK

QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY

VGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKN

LPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDL

LFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKII

KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQL

KRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDS

LTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM

GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV

ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDS

IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT

KAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIR

EVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKY

PKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT

LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQ

TGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEK

GKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY

SLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPED

NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP

IREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQS

ITGLYETRIDLSQLGGD.

Exemplary Streptococcus pyogenes Cas9 nickase (SpCas9 nickase) amino acid sequence:

(SEQ ID NO: 5785)

MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA

LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR

LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD

LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP

INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP

NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI

LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI

FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR

KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY

YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK

NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD

LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI

IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ

LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD

SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV

MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP

VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDD

SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL

TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI

REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK

YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI

TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV

QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVE

KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK

YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPE

DNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK

PIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQ

SITGLYETRIDLSQLGGD.

Exemplary Streptococcus pyogenes Cas9 nickase (SpCas9 nickase) amino acid sequence lacking the N-terminus methionine:

(SEQ ID NO: 5786)

DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL

LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL

EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADL

RLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPI

NASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPN

FKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL

LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIF

FDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK

QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY

VGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKN

LPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDL

LFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKII

KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQL

KRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDS

LTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM

GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV

ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDS

IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT

KAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIR

EVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKY

PKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT

LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQ

TGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEK

GKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY

SLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPED

NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP

IREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQS

ITGLYETRIDLSQLGGD.

Exemplary Streptococcus pyogenes Cas9 (SpCas9) variant; SpCas9 NG amino acid sequence

(SEQ ID NO: 5787)

MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA

LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR

LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD

LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP

INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP

NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI

LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI

FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR

KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY

YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK

NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD

LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI

IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ

LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD

SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV

MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP

VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD

SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL

TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI

REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK

YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI

TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV

QTGGFSKESIRPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVE

KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK

YSLFELENGRKRMLASARFLQKGNELALPSKYVNFLYLASHYEKLKGSPE

DNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK

PIREQAENIIHLFTLTNLGAPRAFKYFDTTIDRKVYRSTKEVLDATLIHQ

SITGLYETRIDLSQLGGD.

Exemplary Streptococcus pyogenes Cas9 (SpCas9) variant; SpCas9 NG amino acid sequence lacking the N-terminus methionine:

(SEQ ID NO: 5788)

DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL

LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL

EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADL

RLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPI

NASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPN

FKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL

LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIF

FDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK

QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY

VGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKN

LPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDL

LFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKII

KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQL

KRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDS

LTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM

GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV

ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDS

IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT

KAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIR

EVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKY

PKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT

LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQ

TGGFSKESIRPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEK

GKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY

SLFELENGRKRMLASARFLQKGNELALPSKYVNFLYLASHYEKLKGSPED

NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP

IREQAENIIHLFTLTNLGAPRAFKYFDTTIDRKVYRSTKEVLDATLIHQS

ITGLYETRIDLSQLGGD.

Exemplary Streptococcus pyogenes Cas9 (SpCas9) nickase; SpCas9 NG nickase amino acid sequence:

(SEQ ID NO: 5789)

MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA

LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR

LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD

LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP

INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP

NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI

LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI

FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR

KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY

YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK

NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD

LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI

IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ

LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD

SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV

MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP

VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDD

SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL

TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI

REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK

YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI

TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV

QTGGFSKESIRPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVE

KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK

YSLFELENGRKRMLASARFLQKGNELALPSKYVNFLYLASHYEKLKGSPE

DNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK

PIREQAENIIHLFTLTNLGAPRAFKYFDTTIDRKVYRSTKEVLDATLIHQ

SITGLYETRIDLSQLGGD.

Exemplary Streptococcus pyogenes Cas9 (SpCas9) nickase; SpCas9 NG nickase amino acid sequence lacking the N-terminus methionine:

(SEQ ID NO: 5790)

DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL

LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL

EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADL

RLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPI

NASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPN

FKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL

LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIF

FDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK

QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY

VGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKN

LPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDL

LFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKII

KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQL

KRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDS

LTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM

GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV

ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDS

IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT

KAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIR

EVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKY

PKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT

LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQ

TGGFSKESIRPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEK

GKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY

SLFELENGRKRMLASARFLQKGNELALPSKYVNFLYLASHYEKLKGSPED

NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP

IREQAENIIHLFTLTNLGAPRAFKYFDTTIDRKVYRSTKEVLDATLIHQS

ITGLYETRIDLSQLGGD.

Exemplary Streptococcus pyogenes Cas9 (SpCas9) variant; SpCas9 VRQR amino acid sequence:

(SEQ ID NO: 5791)

MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA

LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR

LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD

LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP

INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP

NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI

LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI

FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR

KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY

YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK

NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD

LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI

IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ

LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD

SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV

MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP

VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD

SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL

TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI

REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK

YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI

TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV

QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVE

KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK

YSLFELENGRKRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPE

DNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK

PIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRSTKEVLDATLIHQ

SITGLYETRIDLSQLGGD.

Exemplary Streptococcus pyogenes Cas9 (SpCas9) variant; SpCas9 VRQR amino acid sequence lacking the N-terminus methionine:

(SEQ ID NO: 5792)

DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL

LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL

EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADL

RLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPI

NASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPN

FKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL

LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIF

FDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK

QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY

VGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKN

LPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDL

LFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKII

KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQL

KRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDS

LTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM

GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV

ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDS

IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT

KAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIR

EVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKY

PKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT

LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQ

TGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEK

GKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY

SLFELENGRKRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPED

NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP

IREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRSTKEVLDATLIHQS

ITGLYETRIDLSQLGGD.

Exemplary Streptococcus pyogenes Cas9 (SpCas9) nickase; SpCas9 VRQR nickase amino acid sequence:

(SEQ ID NO: 5793)

MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA

LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR

LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD

LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP

INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP

NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI

LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI

FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR

KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY

YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK

NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD

LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI

IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ

LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD

SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV

MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP

VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDD

SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL

TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI

REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK

YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI

TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV

QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVE

KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK

YSLFELENGRKRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPE

DNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK

PIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRSTKEVLDATLIHQ

SITGLYETRIDLSQLGGD.

Exemplary Streptococcus pyogenes Cas9 (SpCas9) nickase; SpCas9 VRQR nickase amino acid sequence lacking the N-terminus methionine:

(SEQ ID NO: 5794)

DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL

LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL

EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADL

RLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPI

NASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPN

FKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL

LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIF

FDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK

QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY

VGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKN

LPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDL

LFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKII

KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQL

KRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDS

LTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM

GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV

ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDS

IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT

KAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIR

EVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKY

PKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT

LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQ

TGGFSKESILPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEK

GKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY

SLFELENGRKRMLASARELQKGNELALPSKYVNFLYLASHYEKLKGSPED

NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP

IREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRSTKEVLDATLIHQS

ITGLYETRIDLSQLGGD.

In some embodiments, a prime editor comprises a Cas9 protein as according to any of the SEQ ID NOS 5795-5798 or a variant thereof. In some embodiments, a prime editor comprises a Cas9 protein from Staphylococcus lugdunensis (Slu Cas9) e.g., as according to any of the SEQ ID NOS 5795-5798 or a variant thereof. In some embodiments, a sluCas9 lacks a N-terminal methionine relative to a corresponding sluCas9 (e.g., a wild type sluCas9, a sluCas9 variant, or a nickase sluCas9). In some embodiments, the Cas9 protein is a sluCas9. In some embodiments, a sluCas9 can be a wild type sluCas9, a sluCas9 variant or a nickase sluCas9. In some embodiments, a prime editor comprises a Cas9 protein, having an amino acid sequence as according to SEQ ID NO: 5795, not including the N-terminus methionine. In some embodiments, a wild type SluCas9 comprises an amino acid sequence set forth in SEQ ID NO: 5795 or SEQ ID NO: 5796. In some embodiments, a prime editor comprises a Cas9 protein, having an amino acid sequence as according to SEQ ID NO: 5795, not including the N-terminus methionine (e.g., as set forth in SEQ ID NO: 5796). In some embodiments, a prime editor comprises a Cas9 protein comprising one or more mutations (e.g., amino acid substitutions, insertions and/or deletions relative to a corresponding wild type Cas9 protein (e.g., wild type sluCas9). In some embodiments, the Cas9 protein comprising one or mutations relative to a wild type Cas9 protein comprises an amino acid sequence set forth in SEQ ID NO: 5797 or 5798.

Exemplary Staphylococcus lugdunensis Cas9 (SluCas9) amino acid sequences useful in the prime editors disclosed herein are provided below in SEQ ID NOs: 5795-5798.

Exemplary Staphylococcus lugdunensis amino acid sequence WP_002460848.1.

Exemplary wild type Staphylococcus lugdunensis Cas9 (SluCas9) amino acid sequence:

(SEQ ID NO: 5795)

MNQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENNEGRRSK

RGSRRLKRRRIHRLERVKKLLEDYNLLDQSQIPQSTNPYAIRVKGLSEAL

SKDELVIALLHIAKRRGIHKIDVIDSNDDVGNELSTKEQLNKNSKLLKDK

FVCQIQLERMNEGQVRGEKNRFKTADIIKEIIQLLNVQKNFHQLDENFIN

KYIELVEMRREYFEGPGKGSPYGWEGDPKAWYETLMGHCTYFPDELRSVK

YAYSADLFNALNDLNNLVIQRDGLSKLEYHEKYHIIENVFKQKKKPTLKQ

IANEINVNPEDIKGYRITKSGKPQFTEFKLYHDLKSVLFDQSILENEDVL

DQIAEILTIYQDKDSIKSKLTELDILLNEEDKENIAQLTGYTGTHRLSLK

CIRLVLEEQWYSSRNQMEIFTHLNIKPKKINLTAANKIPKAMIDEFILSP

VVKRTFGQAINLINKIIEKYGVPEDIIIELARENNSKDKQKFINEMQKKN

ENTRKRINEIIGKYGNQNAKRLVEKIRLHDEQEGKCLYSLESIPLEDLLN

NPNHYEVDHIIPRSVSFDNSYHNKVLVKQSENSKKSNLTPYQYFNSGKSK

LSYNQFKQHILNLSKSQDRISKKKKEYLLEERDINKFEVQKEFINRNLVD

TRYATRELTNYLKAYFSANNMNVKVKTINGSFTDYLRKVWKFKKERNHGY

KHHAEDALIIANADFLFKENKKLKAVNSVLEKPEIESKQLDIQVDSEDNY

SEMFIIPKQVQDIKDFRNFKYSHRVDKKPNRQLINDTLYSTRKKDNSTYI

VQTIKDIYAKDNTTLKKQFDKSPEKFLMYQHDPRTFEKLEVIMKQYANEK

NPLAKYHEETGEYLTKYSKKNNGPIVKSLKYIGNKLGSHLDVTHQFKSST

KKLVKLSIKPYRFDVYLTDKGYKFITISYLDVLKKDNYYYIPEQKYDKLK

LGKAIDKNAKFIASFYKNDLIKLDGEIYKIIGVNSDTRNMIELDLPDIRY

KEYCELNNIKGEPRIKKTIGKKVNSIEKLTTDVLGNVFTNTQYTKPQLLF

KRGN.

Exemplary wild type Staphylococcus lugdunensis Cas9 (SluCas9) amino acid sequence lacking N-terminus methionine:

(SEQ ID NO: 5796)

NQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENNEGRRSKR

GSRRLKRRRIHRLERVKKLLEDYNLLDQSQIPQSTNPYAIRVKGLSEALS

KDELVIALLHIAKRRGIHKIDVIDSNDDVGNELSTKEQLNKNSKLLKDKF

VCQIQLERMNEGQVRGEKNRFKTADIIKEIIQLLNVQKNFHOLDENFINK

YIELVEMRREYFEGPGKGSPYGWEGDPKAWYETLMGHCTYFPDELRSVKY

AYSADLFNALNDLNNLVIQRDGLSKLEYHEKYHIIENVFKQKKKPTLKQI

ANEINVNPEDIKGYRITKSGKPQFTEFKLYHDLKSVLFDQSILENEDVLD

QIAEILTIYQDKDSIKSKLTELDILLNEEDKENIAQLTGYTGTHRLSLKC

IRLVLEEQWYSSRNQMEIFTHLNIKPKKINLTAANKIPKAMIDEFILSPV

VKRTFGQAINLINKIIEKYGVPEDIIIELARENNSKDKQKFINEMQKKNE

NTRKRINEIIGKYGNQNAKRLVEKIRLHDEQEGKCLYSLESIPLEDLLNN

PNHYEVDHIIPRSVSFDNSYHNKVLVKQSENSKKSNLTPYQYFNSGKSKL

SYNQFKQHILNLSKSQDRISKKKKEYLLEERDINKFEVQKEFINRNLVDT

RYATRELTNYLKAYFSANNMNVKVKTINGSFTDYLRKVWKFKKERNHGYK

HHAEDALIIANADFLFKENKKLKAVNSVLEKPEIESKQLDIQVDSEDNYS

EMFIIPKQVQDIKDFRNFKYSHRVDKKPNRQLINDTLYSTRKKDNSTYIV

QTIKDIYAKDNTTLKKQFDKSPEKFLMYQHDPRTFEKLEVIMKQYANEKN

PLAKYHEETGEYLTKYSKKNNGPIVKSLKYIGNKLGSHLDVTHQFKSSTK

KLVKLSIKPYRFDVYLTDKGYKFITISYLDVLKKDNYYYIPEQKYDKLKL

GKAIDKNAKFIASFYKNDLIKLDGEIYKIIGVNSDTRNMIELDLPDIRYK

EYCELNNIKGEPRIKKTIGKKVNSIEKLTTDVLGNVFTNTQYTKPQLLFK

RGN.

Exemplary Staphylococcus lugdunensis Cas9 (SluCas9) nickase amino acid sequence:

(SEQ ID NO: 5797)

MNQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENNEGRRSK

RGSRRLKRRRIHRLERVKKLLEDYNLLDQSQIPQSTNPYAIRVKGLSEAL

SKDELVIALLHIAKRRGIHKIDVIDSNDDVGNELSTKEQLNKNSKLLKDK

FVCQIQLERMNEGQVRGEKNRFKTADIIKEIIQLLNVQKNFHQLDENFIN

KYIELVEMRREYFEGPGKGSPYGWEGDPKAWYETLMGHCTYFPDELRSVK

YAYSADLFNALNDLNNLVIQRDGLSKLEYHEKYHIIENVFKQKKKPTLKQ

IANEINVNPEDIKGYRITKSGKPQFTEFKLYHDLKSVLFDQSILENEDVL

DQIAEILTIYQDKDSIKSKLTELDILLNEEDKENIAQLTGYTGTHRLSLK

CIRLVLEEQWYSSRNQMEIFTHLNIKPKKINLTAANKIPKAMIDEFILSP

VVKRTFGQAINLINKIIEKYGVPEDIIIELARENNSKDKQKFINEMQKKN

ENTRKRINEIIGKYGNQNAKRLVEKIRLHDEQEGKCLYSLESIPLEDLLN

NPNHYEVDHIIPRSVSFDNSYHNKVLVKQSEASKKSNLTPYQYFNSGKSK

LSYNQFKQHILNLSKSQDRISKKKKEYLLEERDINKFEVQKEFINRNLVD

TRYATRELTNYLKAYFSANNMNVKVKTINGSFTDYLRKVWKFKKERNHGY

KHHAEDALIIANADFLFKENKKLKAVNSVLEKPEIESKQLDIQVDSEDNY

SEMFIIPKQVQDIKDFRNFKYSHRVDKKPNRQLINDTLYSTRKKDNSTYI

VQTIKDIYAKDNTTLKKQFDKSPEKFLMYQHDPRTFEKLEVIMKQYANEK

NPLAKYHEETGEYLTKYSKKNNGPIVKSLKYIGNKLGSHLDVTHQFKSST

KKLVKLSIKPYRFDVYLTDKGYKFITISYLDVLKKDNYYYIPEQKYDKLK

LGKAIDKNAKFIASFYKNDLIKLDGEIYKIIGVNSDTRNMIELDLPDIRY

KEYCELNNIKGEPRIKKTIGKKVNSIEKLTTDVLGNVFTNTQYTKPQLLF

KRGN.

Exemplary Staphylococcus lugdunensis Cas9 (SluCas9) nickase amino acid sequence lacking N-terminus methionine:

(SEQ ID NO: 5798)

NQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENNEGRRSKR

GSRRLKRRRIHRLERVKKLLEDYNLLDQSQIPQSTNPYAIRVKGLSEALS

KDELVIALLHIAKRRGIHKIDVIDSNDDVGNELSTKEQLNKNSKLLKDKF

VCQIQLERMNEGQVRGEKNRFKTADIIKEIIQLLNVQKNFHOLDENFINK

YIELVEMRREYFEGPGKGSPYGWEGDPKAWYETLMGHCTYFPDELRSVKY

AYSADLFNALNDLNNLVIQRDGLSKLEYHEKYHIIENVFKQKKKPTLKQI

ANEINVNPEDIKGYRITKSGKPQFTEFKLYHDLKSVLFDQSILENEDVLD

QIAEILTIYQDKDSIKSKLTELDILLNEEDKENIAQLTGYTGTHRLSLKC

IRLVLEEQWYSSRNQMEIFTHLNIKPKKINLTAANKIPKAMIDEFILSPV

VKRTFGQAINLINKIIEKYGVPEDIIIELARENNSKDKQKFINEMQKKNE

NTRKRINEIIGKYGNQNAKRLVEKIRLHDEQEGKCLYSLESIPLEDLLNN

PNHYEVDHIIPRSVSFDNSYHNKVLVKQSEASKKSNLTPYQYFNSGKSKL

SYNQFKQHILNLSKSQDRISKKKKEYLLEERDINKFEVQKEFINRNLVDT

RYATRELTNYLKAYFSANNMNVKVKTINGSFTDYLRKVWKFKKERNHGYK

HHAEDALIIANADFLFKENKKLKAVNSVLEKPEIESKQLDIQVDSEDNYS

EMFIIPKQVQDIKDFRNFKYSHRVDKKPNRQLINDTLYSTRKKDNSTYIV

QTIKDIYAKDNTTLKKQFDKSPEKFLMYQHDPRTFEKLEVIMKQYANEKN

PLAKYHEETGEYLTKYSKKNNGPIVKSLKYIGNKLGSHLDVTHQFKSSTK

KLVKLSIKPYRFDVYLTDKGYKFITISYLDVLKKDNYYYIPEQKYDKLKL

GKAIDKNAKFIASFYKNDLIKLDGEIYKIIGVNSDTRNMIELDLPDIRYK

EYCELNNIKGEPRIKKTIGKKVNSIEKLTTDVLGNVFTNTQYTKPQLLFK

RGN.

In some embodiments, a prime editor comprises a Cas9 protein from Staphylococcus aureus (SaCas9) e.g., as according to any of the SEQ ID NOS 5799-5800, 5802, 5803, or a variant thereof. In some embodiments, a SaCas9 may lack a N-terminal methionine. In some embodiments, a SaCas9 may comprise a mutation.

In some embodiments, a prime editor comprises a Cas9 protein as according to any of the SEQ ID NOS 5799-5800, 5802, 5803 or a variant thereof. In some embodiments, a SaCas9 lacks a N-terminal methionine relative to a corresponding SaCas9 (e.g., a wild type SaCas9, a SaCas9 variant, or a nickase SaCas9). In some embodiments, the Cas9 protein is a SaCas9. In some embodiments, a SaCas9 can be a wild type SaCas9, a SaCas9 variant or a nickase SaCas9. In some embodiments, a prime editor comprises a Cas9 protein, having an amino acid sequence as according to SEQ ID NO: 5799, not including the N-terminus methionine. In some embodiments, a wild type SaCas9 comprises an amino acid sequence set forth in SEQ ID NO: 5799 or SEQ ID NO: 5800. In some embodiments, a prime editor comprises a Cas9 protein, having an amino acid sequence as according to SEQ ID NO: 5799, not including the N-terminus methionine (e.g., as set forth in SEQ ID NO: 5800). In some embodiments, a prime editor comprises a Cas9 protein comprising one or more mutations (e.g., amino acid substitutions, insertions and/or deletions relative to a corresponding wild type Cas9 protein (e.g., wild type SaCas9). In some embodiments, the Cas9 protein comprising one or mutations relative to a wild type Cas9 protein comprises an amino acid sequence set forth in SEQ ID NO: 5802 or 5803. Exemplary SaCas9 amino acid sequences useful in the prime editors disclosed herein are provided below in SEQ ID NOs: 5799, 5800, 5802, 5803.

Exemplary wild type Staphylococcus aureus Cas9 (SaCas9) amino acid sequence:

(SEQ ID NO: 5799)

MKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSK

RGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKL

SEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYV

AELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDT

YIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYA

YNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIA

KEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQ

IAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI

NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVV

KRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQ

TNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNP

FNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKIS

YETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTR

YATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKH

HAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEY

KEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTL

IVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDE

KNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNS

RNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEA

KKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDIT

YREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQII

KKG.

Exemplary wild type Staphylococcus aureus Cas9 (SaCas9) amino acid sequence lacking N-terminus methionine:

(SEQ ID NO: 5800)

KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKR

GARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLS

EEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVA

ELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTY

IDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAY

NADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAK

EILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQI

AKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAIN

LILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVK

RSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQT

NERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPF

NYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISY

ETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRY

ATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHH

AEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYK

EIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLI

VNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEK

NPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSR

NKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAK

KLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITY

REYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIK

KG.

Exemplary Staphylococcus aureus Cas9 (SaCas9) nickase amino acid sequence:

(SEQ ID NO: 5802)

MKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSK

RGARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKL

SEEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYV

AELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDT

YIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYA

YNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIA

KEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQ

IAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAI

NLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVV

KRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQ

TNERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNP

FNYEVDHIIPRSVSFDNSFNNKVLVKQEEASKKGNRTPFQYLSSSDSKIS

YETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTR

YATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKH

HAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEY

KEIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTL

IVNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDE

KNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNS

RNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEA

KKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDIT

YREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQII

KKG.

Exemplary Staphylococcus aureus Cas9 (SaCas9) nickase amino acid sequence lacking N-terminus methionine:

(SEQ ID NO: 5803)

KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKR

GARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLS

EEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVA

ELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTY

IDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAY

NADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAK

EILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQI

AKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAIN

LILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVK

RSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQT

NERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPF

NYEVDHIIPRSVSFDNSFNNKVLVKQEEASKKGNRTPFQYLSSSDSKISY

ETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRY

ATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHH

AEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYK

EIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLI

VNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEK

NPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSR

NKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAK

KLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITY

REYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIK

KG.

In some embodiments, a prime editor comprises a Cas protein, e.g., Cas9 variant, containing modifications that allow altered PAM recognition. Exemplary Cas9 variants with altered PAM specificities that are useful in the Prime editors of the disclosure are provided below in SEQ ID NOs 5804-5819.

Exemplary Streptococcus pyogenes Cas9 variant (SpRY) amino acid sequence:

(SEQ ID NO: 5804)

KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKR

GARRLKRRRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLS

EEEFSAALLHLAKRRGVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVA

ELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQKAYHQLDQSFIDTY

IDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAY

NADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAK

EILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQI

AKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAIN

LILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVK

RSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQT

NERIEEIIRTTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPF

NYEVDHIIPRSVSFDNSFNNKVLVKQEEASKKGNRTPFQYLSSSDSKISY

ETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRY

ATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHH

AEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYK

EIFITPHQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLI

VNNLNGLYDKDNDKLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEK

NPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDDYPNSR

NKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAK

KLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITY

REYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIK

KG.

Exemplary Streptococcus pyogenes Cas9 variant (SpRY) amino acid sequence lacking N-terminus methionine:

(SEQ ID NO: 5805)

MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA

LLFDSGETAERTRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR

LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD

LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP

INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP

NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI

LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI

FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR

KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY

YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK

NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD

LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI

IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ

LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD

SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV

MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP

VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD

SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL

TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI

REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK

YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI

TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV

QTGGFSKESIRPKRNSDKLIARKKDWDPKKYGGFLWPTVAYSVLVVAKVE

KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK

YSLFELENGRKRMLASAKQLQKGNELALPSKYVNFLYLASHYEKLKGSPE

DNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK

PIREQAENIIHLFTLTRLGAPRAFKYFDTTIDPKQYRSTKEVLDATLIHQ

SITGLYETRIDLSQLGGD.

Exemplary Streptococcus pyogenes Cas9 variant nickase (SpRY nickase) amino acid sequence:

(SEQ ID NO: 5806)

MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA

LLFDSGETAERTRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR

LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD

LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP

INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP

NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI

LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI

FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR

KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY

YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK

NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD

LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI

IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ

LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD

SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV

MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP

VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDD

SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL

TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI

REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK

YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI

TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV

QTGGFSKESIRPKRNSDKLIARKKDWDPKKYGGFLWPTVAYSVLVVAKVE

KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK

YSLFELENGRKRMLASAKQLQKGNELALPSKYVNFLYLASHYEKLKGSPE

DNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK

PIREQAENIIHLFTLTRLGAPRAFKYFDTTIDPKQYRSTKEVLDATLIHQ

SITGLYETRIDLSQLGGD.

Exemplary Streptococcus pyogenes Cas9 variant nickase (SpRY nickase) amino acid sequence lacking N-terminus methionine:

(SEQ ID NO: 5807)

DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL

LFDSGETAERTRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL

EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADL

RLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPI

NASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPN

FKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL

LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIF

FDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK

QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY

VGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKN

LPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDL

LFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKII

KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQL

KRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDS

LTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM

GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV

ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDS

IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT

KAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIR

EVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKY

PKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT

LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQ

TGGFSKESIRPKRNSDKLIARKKDWDPKKYGGFLWPTVAYSVLVVAKVEK

GKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY

SLFELENGRKRMLASAKQLQKGNELALPSKYVNFLYLASHYEKLKGSPED

NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP

IREQAENIIHLFTLTRLGAPRAFKYFDTTIDPKQYRSTKEVLDATLIHQS

ITGLYETRIDLSQLGGD.

Exemplary Streptococcus pyogenes Cas9 variant (SpG) amino acid sequence:

(SEQ ID NO: 5816)

MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA

LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR

LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD

LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP

INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP

NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI

LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI

FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR

KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY

YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK

NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD

LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI

IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ

LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD

SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV

MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP

VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD

SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL

TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI

REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK

YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI

TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV

QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFLWPTVAYSVLVVAKVE

KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK

YSLFELENGRKRMLASAKQLQKGNELALPSKYVNFLYLASHYEKLKGSPE

DNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK

PIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRSTKEVLDATLIHQ

SITGLYETRIDLSQLGGD.

Exemplary Streptococcus pyogenes Cas9 variant (SpG) amino acid sequence lacking N-terminus methionine:

(SEQ ID NO: 5817)

DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL

LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL

EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADL

RLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPI

NASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPN

FKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL

LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIF

FDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK

QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY

VGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKN

LPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDL

LFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKII

KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQL

KRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDS

LTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM

GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV

ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDS

IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT

KAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIR

EVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKY

PKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT

LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQ

TGGFSKESILPKRNSDKLIARKKDWDPKKYGGFLWPTVAYSVLVVAKVEK

GKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY

SLFELENGRKRMLASAKQLQKGNELALPSKYVNFLYLASHYEKLKGSPED

NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP

IREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRSTKEVLDATLIHQS

ITGLYETRIDLSQLGGD.

Exemplary Streptococcus pyogenes Cas9 variant (SpG nickase) amino acid sequence:

(SEQ ID NO: 5818)

MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA

LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHR

LEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKAD

LRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENP

INASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP

NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAI

LLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEI

FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR

KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPY

YVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK

NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD

LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI

IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQ

LKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDD

SLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV

MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHP

VENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDD

SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNL

TKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLI

REVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKK

YPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEI

TLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEV

QTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFLWPTVAYSVLVVAKVE

KGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK

YSLFELENGRKRMLASAKQLQKGNELALPSKYVNFLYLASHYEKLKGSPE

DNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK

PIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRSTKEVLDATLIHQ

SITGLYETRIDLSQLGGD.

Exemplary Streptococcus pyogenes Cas9 variant (SpG nickase) amino acid sequence lacking N-terminus methionine:

(SEQ ID NO: 5819)

DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGAL

LFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRL

EESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADL

RLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPI

NASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPN

FKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAIL

LSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIF

FDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRK

QRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYY

VGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKN

LPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDL

LFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKII

KDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQL

KRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDS

LTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVM

GRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV

ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDS

IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLT

KAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIR

EVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKY

PKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEIT

LANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQ

TGGFSKESILPKRNSDKLIARKKDWDPKKYGGFLWPTVAYSVLVVAKVEK

GKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY

SLFELENGRKRMLASAKQLQKGNELALPSKYVNFLYLASHYEKLKGSPED

NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKP

IREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKQYRSTKEVLDATLIHQS

ITGLYETRIDLSQLGGD.

In some embodiments, a Cas9 is a chimeric Cas9, e.g., modified Cas9; e.g., synthetic RNA-guided nucleases (sRGNs), e.g., modified by DNA family shuffling, e.g., sRGN3.1, sRGN3.3. In some embodiments, the DNA family shuffling comprises, fragmentation and reassembly of parental Cas9 genes, e.g., one or more of Cas9s from Staphylococcus hyicus (Shy), Staphylococcus lugdunensis (Slu), Staphylococcus microti (Smi), and Staphylococcus pasteuri (Spa). In some embodiments, a modified sluCas9 shows increased editing efficiency and/or specificity relative to a sluCas9 that is not modified. In some embodiments, a modified Cas9, e.g., a sRGN shows at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000% increase in editing efficiency compared to a Cas9 that is not modified. In some embodiments, a Cas9, e.g., a sRGN shows at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000% increase in specificity compared to a Cas9 that is not modified. In some embodiments, a Cas9, e.g., a sRGN shows at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000% increase in cleavage activity compared to a Cas9 that is not modified. In some embodiments, a Cas9, e.g., a sRGN shows ability to cleave a 5′-NNGG-3′ PAM-containing target. In some embodiments, a prime editor may comprise a Cas9 (e.g., a chimeric Cas9), e.g., as according any of the sequences selected from 5808-5815 or a variant thereof. Exemplary amino acid sequences of sRGN useful in the prime editors disclosed herein are provided below in SEQ ID NOs: 5808-5815.

Exemplary sRGN3.1 amino acid sequence:

(SEQ ID NO: 5808)

MNQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENNEGRRSK

RGSRRLKRRRIHRLERVKLLLTEYDLINKEQIPTSNNPYQIRVKGLSEIL

SKDELAIALLHLAKRRGIHNVDVAADKEETASDSLSTKDQINKNAKFLES

RYVCELQKERLENEGHVRGVENRFLTKDIVREAKKIIDTQMQYYPEIDET

FKEKYISLVETRREYFEGPGQGSPFGWNGDLKKWYEMLMGHCTYFPQELR

SVKYAYSADLFNALNDLNNLIIQRDNSEKLEYHEKYHIIENVFKQKKKPT

LKQIAKEIGVNPEDIKGYRITKSGTPEFTSFKLFHDLKKVVKDHAILDDI

DLLNQIAEILTIYQDKDSIVAELGQLEYLMSEADKQSISELTGYTGTHSL

SLKCMNMIIDELWHSSMNQMEVFTYLNMRPKKYELKGYQRIPTDMIDDAI

LSPVVKRTFIQSINVINKVIEKYGIPEDIIIELARENNSDDRKKFINNLQ

KKNEATRKRINEIIGQTGNQNAKRIVEKIRLHDQQEGKCLYSLESIPLED

LLNNPNHYEVDHIIPRSVSFDNSYHNKVLVKQSENSKKSNLTPYQYFNSG

KSKLSYNQFKQHILNLSKSQDRISKKKKEYLLEERDINKFEVQKEFINRN

LVDTRYATRELTNYLKAYFSANNMNVKVKTINGSFTDYLRKVWKFKKERN

HGYKHHAEDALIIANADFLFKENKKLKAVNSVLEKPEIETKQLDIQVDSE

DNYSEMFIIPKQVQDIKDFRNFKYSHRVDKKPNRQLINDTLYSTRKKDNS

TYIVQTIKDIYAKDNTTLKKQFDKSPEKFLMYQHDPRTFEKLEVIMKQYA

NEKNPLAKYHEETGEYLTKYSKKNNGPIVKSLKYIGNKLGSHLDVTHQFK

SSTKKLVKLSIKNYRFDVYLTEKGYKFVTIAYLNVFKKDNYYYIPKDKYQ

ELKEKKKIKDTDQFIASFYKNDLIKLNGDLYKIIGVNSDDRNIIELDYYD

IKYKDYCEINNIKGEPRIKKTIGKKTESIEKFTTDVLGNLYLHSTEKAPQ

LIFKRGL.

Exemplary sRGN3.1 amino acid sequence lacking N-terminus methionine:

(SEQ ID NO: 5809)

NQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENNEGRRSKR

GSRRLKRRRIHRLERVKLLLTEYDLINKEQIPTSNNPYQIRVKGLSEILS

KDELAIALLHLAKRRGIHNVDVAADKEETASDSLSTKDQINKNAKFLESR

YVCELQKERLENEGHVRGVENRFLTKDIVREAKKIIDTQMQYYPEIDETF

KEKYISLVETRREYFEGPGQGSPFGWNGDLKKWYEMLMGHCTYFPQELRS

VKYAYSADLFNALNDLNNLIIQRDNSEKLEYHEKYHIIENVFKQKKKPTL

KQIAKEIGVNPEDIKGYRITKSGTPEFTSFKLFHDLKKVVKDHAILDDID

LLNQIAEILTIYQDKDSIVAELGQLEYLMSEADKQSISELTGYTGTHSLS

LKCMNMIIDELWHSSMNQMEVFTYLNMRPKKYELKGYQRIPTDMIDDAIL

SPVVKRTFIQSINVINKVIEKYGIPEDIIIELARENNSDDRKKFINNLQK

KNEATRKRINEIIGQTGNQNAKRIVEKIRLHDQQEGKCLYSLESIPLEDL

LNNPNHYEVDHIIPRSVSFDNSYHNKVLVKQSENSKKSNLTPYQYFNSGK

SKLSYNQFKQHILNLSKSQDRISKKKKEYLLEERDINKFEVQKEFINRNL

VDTRYATRELTNYLKAYFSANNMNVKVKTINGSFTDYLRKVWKFKKERNH

GYKHHAEDALIIANADFLFKENKKLKAVNSVLEKPEIETKQLDIQVDSED

NYSEMFIIPKQVQDIKDFRNFKYSHRVDKKPNRQLINDTLYSTRKKDNST

YIVQTIKDIYAKDNTTLKKQFDKSPEKFLMYQHDPRTFEKLEVIMKQYAN

EKNPLAKYHEETGEYLTKYSKKNNGPIVKSLKYIGNKLGSHLDVTHQFKS

STKKLVKLSIKNYRFDVYLTEKGYKFVTIAYLNVFKKDNYYYIPKDKYQE

LKEKKKIKDTDQFIASFYKNDLIKLNGDLYKIIGVNSDDRNIIELDYYDI

KYKDYCEINNIKGEPRIKKTIGKKTESIEKFTTDVLGNLYLHSTEKAPQL

IFKRGL.

Exemplary sRGN3.1 nickase amino acid sequence:

(SEQ ID NO: 5810)

MNQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENNEGRRSK

RGSRRLKRRRIHRLERVKLLLTEYDLINKEQIPTSNNPYQIRVKGLSEIL

SKDELAIALLHLAKRRGIHNVDVAADKEETASDSLSTKDQINKNAKFLES

RYVCELQKERLENEGHVRGVENRFLTKDIVREAKKIIDTQMQYYPEIDET

FKEKYISLVETRREYFEGPGQGSPFGWNGDLKKWYEMLMGHCTYFPQELR

SVKYAYSADLFNALNDLNNLIIQRDNSEKLEYHEKYHIIENVFKQKKKPT

LKQIAKEIGVNPEDIKGYRITKSGTPEFTSFKLFHDLKKVVKDHAILDDI

DLLNQIAEILTIYQDKDSIVAELGQLEYLMSEADKQSISELTGYTGTHSL

SLKCMNMIIDELWHSSMNQMEVFTYLNMRPKKYELKGYQRIPTDMIDDAI

LSPVVKRTFIQSINVINKVIEKYGIPEDIIIELARENNSDDRKKFINNLQ

KKNEATRKRINEIIGQTGNQNAKRIVEKIRLHDQQEGKCLYSLESIPLED

LLNNPNHYEVDHIIPRSVSFDNSYHNKVLVKQSEASKKSNLTPYQYFNSG

KSKLSYNQFKQHILNLSKSQDRISKKKKEYLLEERDINKFEVQKEFINRN

LVDTRYATRELTNYLKAYFSANNMNVKVKTINGSFTDYLRKVWKFKKERN

HGYKHHAEDALIIANADFLFKENKKLKAVNSVLEKPEIETKQLDIQVDSE

DNYSEMFIIPKQVQDIKDFRNFKYSHRVDKKPNRQLINDTLYSTRKKDNS

TYIVQTIKDIYAKDNTTLKKQFDKSPEKFLMYQHDPRTFEKLEVIMKQYA

NEKNPLAKYHEETGEYLTKYSKKNNGPIVKSLKYIGNKLGSHLDVTHQFK

SSTKKLVKLSIKNYRFDVYLTEKGYKFVTIAYLNVFKKDNYYYIPKDKYQ

ELKEKKKIKDTDQFIASFYKNDLIKLNGDLYKIIGVNSDDRNIIELDYYD

IKYKDYCEINNIKGEPRIKKTIGKKTESIEKFTTDVLGNLYLHSTEKAPQ

LIFKRGL.

Exemplary sRGN3.1 nickase amino acid sequence lacking N-terminus methionine:

(SEQ ID NO: 5811)

NQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENNEGRRSKR

GSRRLKRRRIHRLERVKLLLTEYDLINKEQIPTSNNPYQIRVKGLSEILS

KDELAIALLHLAKRRGIHNVDVAADKEETASDSLSTKDQINKNAKFLESR

YVCELQKERLENEGHVRGVENRFLTKDIVREAKKIIDTQMQYYPEIDETF

KEKYISLVETRREYFEGPGQGSPFGWNGDLKKWYEMLMGHCTYFPQELRS

VKYAYSADLFNALNDLNNLIIQRDNSEKLEYHEKYHIIENVFKQKKKPTL

KQIAKEIGVNPEDIKGYRITKSGTPEFTSFKLFHDLKKVVKDHAILDDID

LLNQIAEILTIYQDKDSIVAELGQLEYLMSEADKQSISELTGYTGTHSLS

LKCMNMIIDELWHSSMNQMEVFTYLNMRPKKYELKGYQRIPTDMIDDAIL

SPVVKRTFIQSINVINKVIEKYGIPEDIIIELARENNSDDRKKFINNLQK

KNEATRKRINEIIGQTGNQNAKRIVEKIRLHDQQEGKCLYSLESIPLEDL

LNNPNHYEVDHIIPRSVSFDNSYHNKVLVKQSEASKKSNLTPYQYFNSGK

SKLSYNQFKQHILNLSKSQDRISKKKKEYLLEERDINKFEVQKEFINRNL

VDTRYATRELTNYLKAYFSANNMNVKVKTINGSFTDYLRKVWKFKKERNH

GYKHHAEDALIIANADFLFKENKKLKAVNSVLEKPEIETKQLDIQVDSED

NYSEMFIIPKQVQDIKDFRNFKYSHRVDKKPNRQLINDTLYSTRKKDNST

YIVQTIKDIYAKDNTTLKKQFDKSPEKFLMYQHDPRTFEKLEVIMKQYAN

EKNPLAKYHEETGEYLTKYSKKNNGPIVKSLKYIGNKLGSHLDVTHQFKS

STKKLVKLSIKNYRFDVYLTEKGYKFVTIAYLNVFKKDNYYYIPKDKYQE

LKEKKKIKDTDQFIASFYKNDLIKLNGDLYKIIGVNSDDRNIIELDYYDI

KYKDYCEINNIKGEPRIKKTIGKKTESIEKFTTDVLGNLYLHSTEKAPQL

IFKRGL.

Exemplary sRGN3.3 amino acid sequence:

(SEQ ID NO: 5812)

MNQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENNEGRRSK

RGSRRLKRRRIHRLERVKLLLTEYDLINKEQIPTSNNPYQIRVKGLSEIL

SKDELAIALLHLAKRRGIHNVDVAADKEETASDSLSTKDQINKNAKFLES

RYVCELQKERLENEGHVRGVENRFLTKDIVREAKKIIDTQMQYYPEIDET

FKEKYISLVETRREYFEGPGQGSPFGWNGDLKKWYEMLMGHCTYFPQELR

SVKYAYSADLFNALNDLNNLIIQRDNSEKLEYHEKYHIIENVFKQKKKPT

LKQIAKEIGVNPEDIKGYRITKSGTPEFTSFKLFHDLKKVVKDHAILDDI

DLLNQIAEILTIYQDKDSIVAELGQLEYLMSEADKQSISELTGYTGTHSL

SLKCMNMIIDELWHSSMNQMEVFTYLNMRPKKYELKGYQRIPTDMIDDAI

LSPVVKRTFIQSINVINKVIEKYGIPEDIIIELARENNSDDRKKFINNLQ

KKNEATRKRINEIIGQTGNQNAKRIVEKIRLHDQQEGKCLYSLESIPLED

LLNNPNHYEVDHIIPRSVSFDNSYHNKVLVKQSENSKKSNLTPYQYFNSG

KSKLSYNQFKQHILNLSKSQDRISKKKKEYLLEERDINKFEVQKEFINRN

LVDTRYATRELTSYLKAYFSANNMDVKVKTINGSFTNHLRKVWRFDKYRN

HGYKHHAEDALIIANADFLFKENKKLQNTNKILEKPTIENNTKKVTVEKE

EDYNNVFETPKLVEDIKQYRDYKFSHRVDKKPNRQLINDTLYSTRMKDEH

DYIVQTITDIYGKDNTNLKKQFNKNPEKFLMYQNDPKTFEKLSIIMKQYS

DEKNPLAKYYEETGEYLTKYSKKNNGPIVKKIKLLGNKVGNHLDVTNKYE

NSTKKLVKLSIKNYRFDVYLTEKGYKFVTIAYLNVFKKDNYYYIPKDKYQ

ELKEKKKIKDTDQFIASFYKNDLIKLNGDLYKIIGVNSDDRNIIELDYYD

IKYKDYCEINNIKGEPRIKKTIGKKTESIEKFTTDVLGNLYLHSTEKAPQ

LIFKRGL.

Exemplary sRGN3.3 amino acid sequence lacking N-terminus methionine:

(SEQ ID NO: 5813)

NQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENNEGRRSKR

GSRRLKRRRIHRLERVKLLLTEYDLINKEQIPTSNNPYQIRVKGLSEILS

KDELAIALLHLAKRRGIHNVDVAADKEETASDSLSTKDQINKNAKFLESR

YVCELQKERLENEGHVRGVENRFLTKDIVREAKKIIDTQMQYYPEIDETF

KEKYISLVETRREYFEGPGQGSPFGWNGDLKKWYEMLMGHCTYFPQELRS

VKYAYSADLFNALNDLNNLIIQRDNSEKLEYHEKYHIIENVFKQKKKPTL

KQIAKEIGVNPEDIKGYRITKSGTPEFTSFKLFHDLKKVVKDHAILDDID

LLNQIAEILTIYQDKDSIVAELGQLEYLMSEADKQSISELTGYTGTHSLS

LKCMNMIIDELWHSSMNQMEVFTYLNMRPKKYELKGYQRIPTDMIDDAIL

SPVVKRTFIQSINVINKVIEKYGIPEDIIIELARENNSDDRKKFINNLQK

KNEATRKRINEIIGQTGNQNAKRIVEKIRLHDQQEGKCLYSLESIPLEDL

LNNPNHYEVDHIIPRSVSFDNSYHNKVLVKQSENSKKSNLTPYQYFNSGK

SKLSYNQFKQHILNLSKSQDRISKKKKEYLLEERDINKFEVQKEFINRNL

VDTRYATRELTSYLKAYFSANNMDVKVKTINGSFTNHLRKVWRFDKYRNH

GYKHHAEDALIIANADFLFKENKKLQNTNKILEKPTIENNTKKVTVEKEE

DYNNVFETPKLVEDIKQYRDYKFSHRVDKKPNRQLINDTLYSTRMKDEHD

YIVQTITDIYGKDNTNLKKQFNKNPEKFLMYQNDPKTFEKLSIIMKQYSD

EKNPLAKYYEETGEYLTKYSKKNNGPIVKKIKLLGNKVGNHLDVTNKYEN

STKKLVKLSIKNYRFDVYLTEKGYKFVTIAYLNVFKKDNYYYIPKDKYQE

LKEKKKIKDTDQFIASFYKNDLIKLNGDLYKIIGVNSDDRNIIELDYYDI

KYKDYCEINNIKGEPRIKKTIGKKTESIEKFTTDVLGNLYLHSTEKAPQL

IFKRGL.

Exemplary sRGN3.3 nickase amino acid sequence:

(SEQ ID NO: 5814)

MNQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENNEGRRSK

RGSRRLKRRRIHRLERVKLLLTEYDLINKEQIPTSNNPYQIRVKGLSEIL

SKDELAIALLHLAKRRGIHNVDVAADKEETASDSLSTKDQINKNAKFLES

RYVCELQKERLENEGHVRGVENRFLTKDIVREAKKIIDTQMQYYPEIDET

FKEKYISLVETRREYFEGPGQGSPFGWNGDLKKWYEMLMGHCTYFPQELR

SVKYAYSADLFNALNDLNNLIIQRDNSEKLEYHEKYHIIENVFKQKKKPT

LKQIAKEIGVNPEDIKGYRITKSGTPEFTSFKLFHDLKKVVKDHAILDDI

DLLNQIAEILTIYQDKDSIVAELGQLEYLMSEADKQSISELTGYTGTHSL

SLKCMNMIIDELWHSSMNQMEVFTYLNMRPKKYELKGYQRIPTDMIDDAI

LSPVVKRTFIQSINVINKVIEKYGIPEDIIIELARENNSDDRKKFINNLQ

KKNEATRKRINEIIGQTGNQNAKRIVEKIRLHDQQEGKCLYSLESIPLED

LLNNPNHYEVDHIIPRSVSFDNSYHNKVLVKQSEASKKSNLTPYQYFNSG

KSKLSYNQFKQHILNLSKSQDRISKKKKEYLLEERDINKFEVQKEFINRN

LVDTRYATRELTSYLKAYFSANNMDVKVKTINGSFTNHLRKVWRFDKYRN

HGYKHHAEDALIIANADFLFKENKKLQNTNKILEKPTIENNTKKVTVEKE

EDYNNVFETPKLVEDIKQYRDYKFSHRVDKKPNRQLINDTLYSTRMKDEH

DYIVQTITDIYGKDNTNLKKQFNKNPEKFLMYQNDPKTFEKLSIIMKQYS

DEKNPLAKYYEETGEYLTKYSKKNNGPIVKKIKLLGNKVGNHLDVTNKYE

NSTKKLVKLSIKNYRFDVYLTEKGYKFVTIAYLNVFKKDNYYYIPKDKYQ

ELKEKKKIKDTDQFIASFYKNDLIKLNGDLYKIIGVNSDDRNIIELDYYD

IKYKDYCEINNIKGEPRIKKTIGKKTESIEKFTTDVLGNLYLHSTEKAQP

LIFKRGL.

Exemplary sRGN3.3 nickase amino acid sequence lacking N-terminus methionine:

(SEQ ID NO: 5815)

NQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENNEGRRSKR

GSRRLKRRRIHRLERVKLLLTEYDLINKEQIPTSNNPYQIRVKGLSEILS

KDELAIALLHLAKRRGIHNVDVAADKEETASDSLSTKDQINKNAKFLESR

YVCELQKERLENEGHVRGVENRFLTKDIVREAKKIIDTQMQYYPEIDETF

KEKYISLVETRREYFEGPGQGSPFGWNGDLKKWYEMLMGHCTYFPQELRS

VKYAYSADLFNALNDLNNLIIQRDNSEKLEYHEKYHIIENVFKQKKKPTL

KQIAKEIGVNPEDIKGYRITKSGTPEFTSFKLFHDLKKVVKDHAILDDID

LLNQIAEILTIYQDKDSIVAELGQLEYLMSEADKQSISELTGYTGTHSLS

LKCMNMIIDELWHSSMNQMEVFTYLNMRPKKYELKGYQRIPTDMIDDAIL

SPVVKRTFIQSINVINKVIEKYGIPEDIIIELARENNSDDRKKFINNLQK

KNEATRKRINEIIGQTGNQNAKRIVEKIRLHDQQEGKCLYSLESIPLEDL

LNNPNHYEVDHIIPRSVSFDNSYHNKVLVKQSEASKKSNLTPYQYFNSGK

SKLSYNQFKQHILNLSKSQDRISKKKKEYLLEERDINKFEVQKEFINRNL

VDTRYATRELTSYLKAYFSANNMDVKVKTINGSFTNHLRKVWRFDKYRNH

GYKHHAEDALIIANADFLFKENKKLQNTNKILEKPTIENNTKKVTVEKEE

DYNNVFETPKLVEDIKQYRDYKFSHRVDKKPNRQLINDTLYSTRMKDEHD

YIVQTITDIYGKDNTNLKKQFNKNPEKFLMYQNDPKTFEKLSIIMKQYSD

EKNPLAKYYEETGEYLTKYSKKNNGPIVKKIKLLGNKVGNHLDVTNKYEN

STKKLVKLSIKNYRFDVYLTEKGYKFVTIAYLNVFKKDNYYYIPKDKYQE

LKEKKKIKDTDQFIASFYKNDLIKLNGDLYKIIGVNSDDRNIIELDYYDI

KYKDYCEINNIKGEPRIKKTIGKKTESIEKFTTDVLGNLYLHSTEKAPQL

IFKRGL.

In some embodiments, a Cas9 protein comprises a variant Cas9 protein containing one or more amino acid substitutions. In some embodiments, a wildtype Cas9 protein comprises a RuvC domain and an HNH domain. In some embodiments, a prime editor comprises a nuclease active Cas9 protein that may cleave both strands of a double stranded target DNA sequence. In some embodiments, the nuclease active Cas9 protein comprises a functional RuvC domain and a functional HNH domain. In some embodiments, a prime editor comprises a Cas9 nickase that can bind to a guide polynucleotide and recognize a target DNA, but can cleave only one strand of a double stranded target DNA. In some embodiments, the Cas9 nickase comprises only one functional RuvC domain or one functional HNH domain. In some embodiments, a prime editor comprises a Cas9 that has a non-functional HNH domain and a functional RuvC domain. In some embodiments, the prime editor can cleave the edit strand (i.e. the PAM strand), but not the non-edit strand of a double stranded target DNA sequence. In some embodiments, a prime editor comprises a Cas9 having a non-functional RuvC domain that can cleave the target strand (i.e. the non-PAM strand), but not the edit strand of a double stranded target DNA sequence. In some embodiments, a prime editor comprises a Cas9 that has neither a functional RuvC domain nor a functional HNH domain, which may not cleave any strand of a double stranded target DNA sequence.

In some embodiments, a prime editor comprises a Cas9 having a mutation in the RuvC domain that reduces or abolishes the nuclease activity of the RuvC domain. In some embodiments, the Cas9 comprise a mutation at amino acid D10 as compared to a wild type SpCas9 as set forth in SEQ ID NO:5783, or a corresponding mutation thereof. In some embodiments, the Cas9 comprise a D10A mutation as compared to a wild type SpCas9 as set forth in SEQ ID NO: 5783, or a corresponding mutation thereof. In some embodiments, the Cas9 polypeptide comprise a mutation at amino acid D10, G12, and/or G17 as compared to a wild type SpCas9 as set forth in SEQ ID NO: 5783, or a corresponding mutation thereof. In some embodiments, the Cas9 polypeptide comprise a D10A mutation, a G12A mutation, and/or a G17A mutation as compared to a wild type SpCas9 as set forth in SEQ ID NO: 5783, or a corresponding mutation thereof.

In some embodiments, a prime editor comprises a Cas9 polypeptide having a mutation in the HNH domain that reduces or abolishes the nuclease activity of the HNH domain. In some embodiments, the Cas9 polypeptide comprise a mutation at amino acid H840 as compared to a wild type SpCas9 as set forth in SEQ ID NO: 5783, or a corresponding mutation thereof. In some embodiments, the Cas9 polypeptide comprise a H840A mutation as compared to a wild type SpCas9 as set forth in SEQ ID NO: 5783, or a corresponding mutation thereof. In some embodiments, the Cas9 polypeptide comprise a mutation at amino acid E762, D839, H840, N854, N856, N863, H982, H983, A984, D986, and/or a A987 as compared to a wild type SpCas9 as set forth in SEQ ID NO: 5783, or a corresponding mutation thereof. In some embodiments, the Cas9 polypeptide comprise a E762A, D839A, H840A, N854A, N856A, N863A, H982A, H983A, A984A, and/or a D986A mutation as compared to a wild type SpCas9 as set forth in SEQ ID NO: 5783, or a corresponding mutation thereof.

In some embodiments, a prime editor comprises a Cas9 having one or more amino acid substitutions in both the HNH domain and the RuvC domain that reduce or abolish the nuclease activity of both the HNH domain and the RuvC domain. In some embodiments, the prime editor comprises a nuclease inactive Cas9, or a nuclease dead Cas9 (dCas9). In some embodiments, the dCas9 comprises a H840X substitution and a D10X mutation compared to a wild type SpCas9 as set forth in SEQ ID NO: 5783 or corresponding mutations thereof, wherein X is any amino acid other than H for the H840X substitution and any amino acid other than D for the D10X substitution. In some embodiments, the dead Cas9 comprises a H840A and a D10A mutation as compared to a wild type SpCas9 as set forth in SEQ ID NO: 5783, or corresponding mutations thereof.

In some embodiments, a prime editor comprises a Cas protein, e.g., Cas9, containing modifications that allow altered PAM recognition. In prime editing using a Cas-protein-based prime editor, a “protospacer adjacent motif (PAM)”, PAM sequence, or PAM-like motif, may be used to refer to a short DNA sequence immediately following the protospacer sequence on the PAM strand of the target gene. In some embodiments, the PAM is recognized by the Cas nuclease in the prime editor during prime editing. In certain embodiments, the PAM is required for target binding of the Cas protein. The specific PAM sequence required for Cas protein recognition may depend on the specific type of the Cas protein. A PAM can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides in length. In some embodiments, a PAM is between 2-6 nucleotides in length. In some embodiments, the PAM can be a 5′ PAM (i.e., located upstream of the 5′ end of the protospacer). In other embodiments, the PAM can be a 3′ PAM (i.e., located downstream of the 5′ end of the protospacer). In some embodiments, the Cas protein of a prime editor recognizes a canonical PAM, for example, a SpCas9 recognizes 5′-NGG-3′ PAM. In some embodiments, the Cas protein of a prime editor has altered or non-canonical PAM specificities. Exemplary PAM sequences and corresponding Cas variants are described in Table 1 below. It should be appreciated that for each of the variants provided, the Cas protein comprises one or more of the amino acid substitutions as indicated compared to a wild type Cas protein sequence, for example, the Cas9 as set forth in SEQ ID NO: 5783. The PAM motifs as shown in Table 1 below are in the order of 5′ to 3′. As used in PAM sequences in Table 1, “N” refers to any one of nucleotides A, G, C, and T, “R” refers to nucleotide A or G, and “Y” refers to nucleotide C or T.

TABLE 1

Cas protein variants and corresponding PAM

sequences

Variant
PAM

spCas9 (wild type)
NGG, NGA, NAG

spCas9-VRVRFRR
NG

R1335V/L1111R/D1135V/

G1218R/E1219F/A1322R/

T1337R

spCas9-VQR (D1135V/
NGA

R1335Q/T1337R)

spCas9-EQR (D1135E/
NGA

R1335Q/T1337R)

spCas9-VRER (D1135V/
NGCG

G1218R/R1335E/T1337R)

xCas9 (E480K, E543D,
NGN

E1219V, K294R, Q1256K,

A262T, S409I, M694I)

SluCa9
NNGG

saCas9
NNGRRT

(SEQ ID

NO: 5820),

NNGRRN

(SEQ ID

NO: 5821)

saCas9-KKH (E782K,
NNNRRT

N968K, R1015H)
(SEQ ID

NO: 5822)

spCas9-MQKSER (D1135M,
NGCG/NGCN

S1136Q, G1218K, E1219S,

R1335E, T1337R)

spCas9-LRKIQK (D1135L,
NGTN

S1136R, G1218K, E1219I,

R1335Q, T1337K)

spCas9-LRVSQK (D1135L,
NGTN

S1136R, G1218V, E1219S,

R1335Q, T1337K)

spCas9-LRVSQL(D1135L,
NGTN

S1136R, G1218V, E1219S,

R1335Q, T1337L)

Cpf1
TTTV

Spy-Mac
NAA

NmCas9 PAM
NNNNGATT

(SEQ ID

NO: 5823)

StCas9 PAM
NNAGAAW

(SEQ ID

NO: 5824)

TdCas9 PAM
NAAAAC

(SEQ ID

NO: 5825)

In some embodiments, a prime editor comprises a Cas9 polypeptide comprising one or mutations selected from the group consisting of: A61R, L111R, D1135V, R221K, A262T, R324L, N394K, S4091, S4091, E427G, E480K, M495V, N497A, Y515N, K526E, F539S, E543D), R654L, R661A, R661L, R691A, N692A, M694A, M6941, Q695A, H698A, R753G, M7631, K848A, K890N, Q926A, K1003A, R1060A, L111R, R1114G, D11135E, D11135L, D11135N, S1136W, V11139A, D11180G, G1218K, G1218R, G1218S, E1219Q, E1219V, E1219V, Q1221H, P1249S, E1253K, N1317R, A1320V, P1321S, A1322R, 11322V, D1332G, R1332N, A1332R, R1333K, R1333P, R1335L, R1335Q, R1335V, T1337N, T1337R, S1338T, H1349R, and any combinations thereof as compared to a wildtype SpCas9 polypeptide as set forth in SEQ ID NO: 5783.

In some embodiments, a prime editor comprises a SaCas9 polypeptide. In some embodiments, the SaCas9 polypeptide comprises one or more of mutations E782K, N968K, and R10115H as compared to a wild type SaCas9. In some embodiments, a prime editor comprises a FnCas9 polypeptide, for example, a wildtype FnCas9 polypeptide or a FnCas9 polypeptide comprising one or more of mutations E1369R, E1449H, or R1556A as compared to the wild type FnCas9. In some embodiments, a prime editor comprises a Sc Cas9, for example, a wild type ScCas9 or a ScCas9 polypeptide comprises one or more of mutations I367K, G368D, I369K, H371L, T375S, T376G, and T1227K as compared to the wild type ScCas9. In some embodiments, a prime editor comprises a St1 Cas9 polypeptide, a St3 Cas9 polypeptide, or a Slu Cas9 polypeptide.

In some embodiments, a prime editor comprises a Cas polypeptide that comprises a circular permutant Cas variant. For example, a Cas9 polypeptide of a prime editor may be engineered such that the N-terminus and the C-terminus of a Cas9 protein (e.g., a wild type Cas9 protein, or a Cas9 nickase) are topically rearranged to retain the ability to bind DNA when complexed with a guide RNA (gRNA). An exemplary circular permutant configuration may be N-terminus-[original C-terminus]-[original N-terminus]-C-terminus. Any of the Cas9 proteins described herein, including any variant, ortholog, or naturally occurring Cas9 or equivalent thereof, may be reconfigured as a circular permutant variant.

Prime editors described herein may also comprise Cas proteins other than Cas9. For example, a prime editor as described herein may comprise a Cas12a (Cpf1) polypeptide or functional variants thereof. In some embodiments, the Cas12a polypeptide comprises a mutation that reduces or abolishes the endonuclease domain of the Cas12a polypeptide. In some embodiments, the Cas12a polypeptide is a Cas12a nickase. In some embodiments, the Cas protein comprises an amino acid sequence that comprises at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a naturally occurring Cas12a polypeptide.

In some embodiments, a prime editor comprises a Cas protein that is a Cas12b (C2c1) or a Cas12c (C2c3) polypeptide. In some embodiments, the Cas protein comprises an amino acid sequence that comprises at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a naturally occurring Cas12b (C2c1) or Cas12c (C2c3) protein. In some embodiments, the Cas protein is a Cas12b nickase or a Cas12c nickase. In some embodiments, the Cas protein is a Cas12e, a Cas12d, a Cas13, Cas14a, Cas14b, Cas14c, Cas14d, Cas14e, Cas14f, Cas14g, Cas14h, Cas14u, or a CasΦ polypeptide. In some embodiments, the Cas protein comprises an amino acid sequence that comprises at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a naturally-occurring Cas12e, Cas12d, Cas13, Cas14a, Cas14b, Cas14c, Cas14d, Cas14e, Cas14f, Cas14g, Cas14h, Cas14u, or CasΦ protein. In some embodiments, the Cas protein is a Cas12e, Cas12d, Cas13, or Cas Φ nickase.

In some embodiments, a prime editor further comprises additional polypeptide components, for example, a flap endonuclease (FEN, e.g., FEN1). In some embodiments, the flap endonuclease excises the 5′ single stranded DNA of the edit strand of the target gene and assists incorporation of the intended nucleotide edit into the target gene. In some embodiments, the FEN is linked or fused to another component. In some embodiments, the FEN is provided in trans, for example, as a separate polypeptide or polynucleotide encoding the FEN.

In some embodiments, a prime editor further comprises one or more nuclear localization sequence (NLS). In some embodiments, the NLS helps promote translocation of a protein into the cell nucleus. In some embodiments, a prime editor comprises a fusion protein, e.g., a fusion protein comprising a DNA binding domain and a DNA polymerase, that comprises one or more NLSs. In some embodiments, one or more polypeptides of the prime editor are fused to or linked to one or more NLSs. In some embodiments, the prime editor comprises a DNA binding domain and a DNA polymerase domain that are provided in trans, wherein the DNA binding domain and/or the DNA polymerase domain is fused or linked to one or more NLSs.

In certain embodiments, a prime editor or prime editing complex comprises at least one NLS. In some embodiments, a prime editor or prime editing complex comprises at least two NLSs. In embodiments with at least two NLSs, the NLSs can be the same NLS, or they can be different NLSs.

Any NLSs that are known in the art are also contemplated herein. The NLSs may be any naturally occurring NLS, or any non-naturally occurring NLS (e.g., an NLS with one or more mutations relative to a wild-type NLS). In some embodiments, the one or more NLSs of a prime editor comprise bipartite NLSs. In some embodiments, the one or more NLSs of a prime editor are rich in lysine and arginine residues. In some embodiments, the one or more NLSs of a prime editor comprise proline residues. Non-limiting examples of NLS sequences are provided in Table 2 below.

TABLE 2

Exemplary nuclear localization sequences

SEQ

ID

Description
Sequence
NO:

NLS of SV40
PKKKRKV
5826

Large T-AG

NLS
MKRTADGSEFESPKKKRKV
5827

NLS
MDSLLMNRRKFLYQFKNVRWAKGRRETYLC
5828

NLS of
AVKRPAATKKAGQAKKKKLD
5829

Nucleoplasmin

NLS of EGL-13
MSRRRKANPTKLSENAKKLAKEVEN
5830

NLS of C-Myc
PAAKRVKLD
5831

NLS of Tus-
KLKIKRPVK
5832

protein

NLS of polyoma
VSRKRPRP
5833

large T-AG

NLS of
EGAPPAKRAR
5834

Hepatitis

D virus

antigen

NLS of
PPQPKKKPLDGE
5835

murine p53

NLS of PE1
SGGSKRTADGSEFEPKKKRKV
5836

and PE2

In some embodiments, a prime editing complex comprises a fusion protein comprising a DNA binding domain (e.g., Cas9 (H840A)) and a reverse transcriptase (e.g., a variant MMLV RT) having the following structure: [NLS]-[Cas9(H840A)]-[linker]-[MMLV_RT(D200N)(T330P)(L603W)(T306K)(W313F)], and a desired PEgRNA. In some embodiments, the prime editing complex comprises a prime editor fusion protein that has the amino acid sequence of SEQ ID NO: 5837 or 5838. The sequences and components of these exemplary prime editor fusion proteins which are shown as follows:

TABLE 34A

Exemplary prime editor fusion protein and

component sequences.

SEQ

ID
DESCRIP-

NO
TION
SEQUENCE

5837
Prime

MKRTADGSEFESPKKKRKV
DKKYSIGLD

Editor

IGTNSVGWAVITDEYKVPSKKFKVLGNT

Structure:

DRHSIKKNLIGALLFDSGETAEATRLKR

[N-

TARRRYTRRKNRICYLQEIFSNEMAKVD

terminal

DSFFHRLEESFLVEEDKKHERHPIFGNI

NLS]-

VDEVAYHEKYPTIYHLRKKLVDSTDKAD

[Cas9

LRLIYLALAHMIKFRGHFLIEGDLNPDN

(H840A)]-

SDVDKLFIQLVQTYNQLFEENPINASGV

[LINKER]-

DAKAILSARLSKSRRLENLIAQLPGEKK

[MMLV_RT

NGLFGNLIALSLGLTPNFKSNFDLAEDA

(D200N)

KLQLSKDTYDDDLDNLLAQIGDQYADLF

(T330P)

LAAKNLSDAILLSDILRVNTEITKAPLS

(L603W)

ASMIKRYDEHHQDLTLLKALVRQQLPEK

(T306K)

YKEIFFDQSKNGYAGYIDGGASQEEFYK

(W313F)]-

FIKPILEKMDGTEELLVKLNREDLLRKQ

[C-

RTFDNGSIPHQIHLGELHAILRRQEDFY

terminal

PFLKDNREKIEKILTFRIPYYVGPLARG

linker

NSRFAWMTRKSEETITPWNFEEVVDKGA

and NLS]

SAQSFIERMTNFDKNLPNEKVLPKHSLL

YEYFTVYNELTKVKYVTEGMRKPAFLSG

EQKKAIVDLLFKTNRKVTVKQLKEDYFK

KIECFDSVEISGVEDRFNASLGTYHDLL

KIIKDKDFLDNEENEDILEDIVLTLTLF

EDREMIEERLKTYAHLFDDKVMKQLKRR

RYTGWGRLSRKLINGIRDKQSGKTILDF

LKSDGFANRNFMQLIHDDSLTFKEDIQK

AQVSGQGDSLHEHIANLAGSPAIKKGIL

QTVKVVDELVKVMGRHKPENIVIEMARE

NQTTQKGQKNSRERMKRIEEGIKELGSQ

ILKEHPVENTQLQNEKLYLYYLQNGRDM

YVDQELDINRLSDYDVDAIVPQSFLKDD

SIDNKVLTRSDKNRGKSDNVPSEEVVKK

MKNYWRQLLNAKLITQRKFDNLTKAERG

GLSELDKAGFIKRQLVETRQITKHVAQI

LDSRMNTKYDENDKLIREVKVITLKSKL

VSDFRKDFQFYKVREINNYHHAHDAYLN

AVVGTALIKKYPKLESEFVYGDYKVYDV

RKMIAKSEQEIGKATAKYFFYSNIMNFF

KTEITLANGEIRKRPLIETNGETGEIVW

DKGRDFATVRKVLSMPQVNIVKKTEVQT

GGFSKESILPKRNSDKLIARKKDWDPKK

YGGFDSPTVAYSVLVVAKVEKGKSKKLK

SVKELLGITIMERSSFEKNPIDFLEAKG

YKEVKKDLIIKLPKYSLFELENGRKRML

ASAGELQKGNELALPSKYVNFLYLASHY

EKLKGSPEDNEQKQLFVEQHKHYLDEII

EQISEFSKRVILADANLDKVLSAYNKHR

DKPIREQAENIIHLFTLTNLGAPAAFKY

FDTTIDRKRYTSTKEVLDATLIHQSITG

LYETRIDLSQLGGD

custom-character

TLNIEDEYRLHETSKEPDVSLGSTWLSD

FPQAWAETGGMGLAVRQAPLIIPLKATS

TPVSIKQYPMSQEARLGIKPHIQRLLDQ

GILVPCQSPWNTPLLPVKKPGTNDYRPV

QDLREVNKRVEDIHPTVPNPYNLLSGLP

PSHQWYTVLDLKDAFFCLRLHPTSQPLF

AFEWRDPEMGISGQLTWTRLPQGFKNSP

TLFNEALHRDLADFRIQHPDLILLQYVD

DLLLAATSELDCQQGTRALLQTLGNLGY

RASAKKAQICQKQVKYLGYLLKEGQRWL

TEARKETVMGQPTPKTPRQLREFLGKAG

FCRLFIPGFAEMAAPLYPLTKPGTLFNW

GPDQQKAYQEIKQALLTAPALGLPDLTK

PFELFVDEKQGYAKGVLTQKLGPWRRPV

AYLSKKLDPVAAGWPPCLRMVAAIAVLT

KDAGKLTMGQPLVILAPHAVEALVKQPP

DRWLSNARMTHYQALLLDTDRVQFGPVV

ALNPATLLPLPEEGLQHNCLDILAEAHG

TRPDLTDQPLPDADHTWYTDGSSLLQEG

QRKAGAAVTTETEVIWAKALPAGTSAQR

AELIALTQALKMAEGKKLNVYTDSRYAF

ATAHIHGEIYRRRGWLTSEGKEIKNKDE

ILALLKALFLPKRLSIIHCPGHQKGHSA

EARGNRMADQAARKAAITETPDTSTLLI

ENSSP

SGGSKRTADGSEFEPKKKRKV

5827
N
MKRTADGSEFESPKKKRKV

terminal

NLS

5786
SpCas9
DKKYSIGLDIGTNSVGWAVITDEYKVPS

(H840A)
KKFKVLGNTDRHSIKKNLIGALLFDSGE

TAEATRLKRTARRRYTRRKNRICYLQEI

FSNEMAKVDDSFFHRLEESFLVEEDKKH

ERHPIFGNIVDEVAYHEKYPTIYHLRKK

LVDSTDKADLRLIYLALAHMIKFRGHFL

IEGDLNPDNSDVDKLFIQLVQTYNQLFE

ENPINASGVDAKAILSARLSKSRRLENL

IAQLPGEKKNGLFGNLIALSLGLTPNFK

SNFDLAEDAKLQLSKDTYDDDLDNLLAQ

IGDQYADLFLAAKNLSDAILLSDILRVN

TEITKAPLSASMIKRYDEHHQDLTLLKA

LVRQQLPEKYKEIFFDQSKNGYAGYIDG

GASQEEFYKFIKPILEKMDGTEELLVKL

NREDLLRKQRTFDNGSIPHQIHLGELHA

ILRRQEDFYPFLKDNREKIEKILTFRIP

YYVGPLARGNSRFAWMTRKSEETITPWN

FEEVVDKGASAQSFIERMTNFDKNLPNE

KVLPKHSLLYEYFTVYNELTKVKYVTEG

MRKPAFLSGEQKKAIVDLLFKTNRKVTV

KQLKEDYFKKIECFDSVEISGVEDRFNA

SLGTYHDLLKIIKDKDFLDNEENEDILE

DIVLTLTLFEDREMIEERLKTYAHLFDD

KVMKQLKRRRYTGWGRLSRKLINGIRDK

QSGKTILDFLKSDGFANRNFMQLIHDDS

LTFKEDIQKAQVSGQGDSLHEHIANLAG

SPAIKKGILQTVKVVDELVKVMGRHKPE

NIVIEMARENQTTQKGQKNSRERMKRIE

EGIKELGSQILKEHPVENTQLQNEKLYL

YYLQNGRDMYVDQELDINRLSDYDVDAI

VPQSFLKDDSIDNKVLTRSDKNRGKSDN

VPSEEVVKKMKNYWRQLLNAKLITQRKF

DNLTKAERGGLSELDKAGFIKRQLVETR

QITKHVAQILDSRMNTKYDENDKLIREV

KVITLKSKLVSDFRKDFQFYKVREINNY

HHAHDAYLNAVVGTALIKKYPKLESEFV

YGDYKVYDVRKMIAKSEQEIGKATAKYF

FYSNIMNFFKTEITLANGEIRKRPLIET

NGETGEIVWDKGRDFATVRKVLSMPQVN

IVKKTEVQTGGFSKESILPKRNSDKLIA

RKKDWDPKKYGGFDSPTVAYSVLVVAKV

EKGKSKKLKSVKELLGITIMERSSFEKN

PIDFLEAKGYKEVKKDLIIKLPKYSLFE

LENGRKRMLASAGELQKGNELALPSKYV

NFLYLASHYEKLKGSPEDNEQKQLFVEQ

HKHYLDEIIEQISEFSKRVILADANLDK

VLSAYNKHRDKPIREQAENIIHLFTLTN

LGAPAAFKYFDTTIDRKRYTSTKEVLDA

TLIHQSITGLYETRIDLSQLGGD

5852
LINKER
SGGSSGGSSGSETPGTSESATPESSGGS

SGGSS

5842
MMLV_
TLNIEDEYRLHETSKEPDVSLGSTWLSD

RT
FPQAWAETGGMGLAVRQAPLIIPLKATS

(D200N)
TPVSIKQYPMSQEARLGIKPHIQRLLDQ

(T330P)
GILVPCQSPWNTPLLPVKKPGTNDYRPV

(L603W)
QDLREVNKRVEDIHPTVPNPYNLLSGLP

(T306K)
PSHQWYTVLDLKDAFFCLRLHPTSQPLF

(W313F)
AFEWRDPEMGISGQLTWTRLPQGFKNSP

TLFNEALHRDLADFRIQHPDLILLQYVD

DLLLAATSELDCQQGTRALLQTLGNLGY

RASAKKAQICQKQVKYLGYLLKEGQRWL

TEARKETVMGQPTPKTPRQLREFLGKAG

FCRLFIPGFAEMAAPLYPLTKPGTLFNW

GPDQQKAYQEIKQALLTAPALGLPDLTK

PFELFVDEKQGYAKGVLTQKLGPWRRPV

AYLSKKLDPVAAGWPPCLRMVAAIAVLT

KDAGKLTMGQPLVILAPHAVEALVKQPP

DRWLSNARMTHYQALLLDTDRVQFGPVV

ALNPATLLPLPEEGLQHNCLDILAEAHG

TRPDLTDQPLPDADHTWYTDGSSLLQEG

QRKAGAAVTTETEVIWAKALPAGTSAQR

AELIALTQALKMAEGKKLNVYTDSRYAF

ATAHIHGEIYRRRGWLTSEGKEIKNKDE

ILALLKALFLPKRLSIIHCPGHQKGHSA

EARGNRMADQAARKAAITETPDTSTLLI

ENSSP

5836
C-
SGGSKRTADGSEFEPKKKRKV

Terminal

linker

and NLS

TABLE 34B

Exemplary prime editor fusion protein and

component sequences.

SEQ

ID
DESCRIP-

NO
TION
SEQUENCE

5838
PEmax

MKRTADGSEFESPKKKRKV

Prime

DKKYSIGLDIGTNSVGWAVITDEYKVP

Editor

SKKFKVLGNTDRHSIKKNLIGALLFDS

structure:

GETAEATRLKRTARRRYTRRKNRICYL

[N-

QEIFSNEMAKVDDSFFHRLEESFLVEE

terminal

DKKHERHPIFGNIVDEVAYHEKYPTIY

NLS]-

HLRKKLVDSTDKADLRLIYLALAHMIK

[Cas9

FRGHFLIEGDLNPDNSDVDKLFIQLVQ

((R221K

TYNQLFEENPINASGVDAKAILSARLS

N394K

KSRKLENLIAQLPGEKKNGLFGNLIAL

H840A)]-

SLGLTPNFKSNFDLAEDAKLQLSKDTY

[SGGSx2-

DDDLDNLLAQIGDQYADLFLAAKNLSD

bpSV40NLS-

AILLSDILRVNTEITKAPLSASMIKRY

SGGSx2]-

DEHHQDLTLLKALVRQQLPEKYKEIFF

[MMLV_RT

DQSKNGYAGYIDGGASQEEFYKFIKPI

(D200N)

LEKMDGTEELLVKLKREDLLRKQRTFD

(T330P)

NGSIPHQIHLGELHAILRRQEDFYPFL

(L603W)

KDNREKIEKILTFRIPYYVGPLARGNS

(T306K)

RFAWMTRKSEETITPWNFEEVVDKGAS

(W313F)]-

AQSFIERMTNFDKNLPNEKVLPKHSLL

[C-

YEYFTVYNELTKVKYVTEGMRKPAFLS

terminal

GEQKKAIVDLLFKTNRKVTVKQLKEDY

linker

FKKIECFDSVEISGVEDRFNASLGTYH

and NLS-

DLLKIIKDKDFLDNEENEDILEDIVLT

linker-

LTLFEDREMIEERLKTYAHLFDDKVMK

NLS2]

QLKRRRYTGWGRLSRKLINGIRDKQSG

KTILDFLKSDGFANRNFMQLIHDDSLT

FKEDIQKAQVSGQGDSLHEHIANLAGS

PAIKKGILQTVKVVDELVKVMGRHKPE

NIVIEMARENQTTQKGQKNSRERMKRI

EEGIKELGSQILKEHPVENTQLQNEKL

YLYYLQNGRDMYVDQELDINRLSDYDV

DAIVPQSFLKDDSIDNKVLTRSDKNRG

KSDNVPSEEVVKKMKNYWRQLLNAKLI

TQRKFDNLTKAERGGLSELDKAGFIKR

QLVETRQITKHVAQILDSRMNTKYDEN

DKLIREVKVITLKSKLVSDFRKDFQFY

KVREINNYHHAHDAYLNAVVGTALIKK

YPKLESEFVYGDYKVYDVRKMIAKSEQ

EIGKATAKYFFYSNIMNFFKTEITLAN

GEIRKRPLIETNGETGEIVWDKGRDFA

TVRKVLSMPQVNIVKKTEVQTGGFSKE

SILPKRNSDKLIARKKDWDPKKYGGFD

SPTVAYSVLVVAKVEKGKSKKLKSVKE

LLGITIMERSSFEKNPIDFLEAKGYKE

VKKDLIIKLPKYSLFELENGRKRMLAS

AGELQKGNELALPSKYVNFLYLASHYE

KLKGSPEDNEQKQLFVEQHKHYLDEII

EQISEFSKRVILADANLDKVLSAYNKH

RDKPIREQAENIIHLFTLTNLGAPAAF

KYFDTTIDRKRYTSTKEVLDATLIHQS

ITGLYETRIDLSQLGGD

custom-character

TLNIEDEYRLHETSKEPDVSLGSTWLS

DFPQAWAETGGMGLAVRQAPLIIPLKA

TSTPVSIKQYPMSQEARLGIKPHIQRL

LDQGILVPCQSPWNTPLLPVKKPGTND

YRPVQDLREVNKRVEDIHPTVPNPYNL

LSGLPPSHQWYTVLDLKDAFFCLRLHP

TSQPLFAFEWRDPEMGISGQLTWTRLP

QGFKNSPTLFNEALHRDLADFRIQHPD

LILLQYVDDLLLAATSELDCQQGTRAL

LQTLGNLGYRASAKKAQICQKQVKYLG

YLLKEGQRWLTEARKETVMGQPTPKTP

RQLREFLGKAGFCRLFIPGFAEMAAPL

YPLTKPGTLFNWGPDQQKAYQEIKQAL

LTAPALGLPDLTKPFELFVDEKQGYAK

GVLTQKLGPWRRPVAYLSKKLDPVAAG

WPPCLRMVAAIAVLTKDAGKLTMGQPL

VILAPHAVEALVKQPPDRWLSNARMTH

YQALLLDTDRVQFGPVVALNPATLLPL

PEEGLQHNCLDILAEAHGTRPDLTDQP

LPDADHTWYTDGSSLLQEGQRKAGAAV

TTETEVIWAKALPAGTSAQRAELIALT

QALKMAEGKKLNVYTDSRYAFATAHIH

GEIYRRRGWLTSEGKEIKNKDEILALL

KALFLPKRLSIIHCPGHQKGHSAEARG

NRMADQAARKAAITETPDTSTLLIENS

SP

SGGSKRTADGSEFESPKKKRKVGS

GPAAKRVKLD

5839
PEmax-
MKRTADGSEFESPKKKRKV

N-

terminal

bpSV40NLS

5840
PEmax-
DKKYSIGLDIGTNSVGWAVITDEYKVP

CAS9
SKKFKVLGNTDRHSIKKNLIGALLFDS

(R221K
GETAEATRLKRTARRRYTRRKNRICYL

N394K
QEIFSNEMAKVDDSFFHRLEESFLVEE

H840A)
DKKHERHPIFGNIVDEVAYHEKYPTIY

(not
HLRKKLVDSTDKADLRLIYLALAHMIK

including
FRGHFLIEGDLNPDNSDVDKLFIQLVQ

N-
TYNQLFEENPINASGVDAKAILSARLS

terminal
KSRKLENLIAQLPGEKKNGLFGNLIAL

Met in
SLGLTPNFKSNFDLAEDAKLQLSKDTY

Cas9)
DDDLDNLLAQIGDQYADLFLAAKNLSD

AILLSDILRVNTEITKAPLSASMIKRY

DEHHQDLTLLKALVRQQLPEKYKEIFF

DQSKNGYAGYIDGGASQEEFYKFIKPI

LEKMDGTEELLVKLKREDLLRKQRTFD

NGSIPHQIHLGELHAILRRQEDFYPFL

KDNREKIEKILTFRIPYYVGPLARGNS

RFAWMTRKSEETITPWNFEEVVDKGAS

AQSFIERMTNFDKNLPNEKVLPKHSLL

YEYFTVYNELTKVKYVTEGMRKPAFLS

GEQKKAIVDLLFKTNRKVTVKQLKEDY

FKKIECFDSVEISGVEDRFNASLGTYH

DLLKIIKDKDFLDNEENEDILEDIVLT

LTLFEDREMIEERLKTYAHLFDDKVMK

QLKRRRYTGWGRLSRKLINGIRDKQSG

KTILDFLKSDGFANRNFMQLIHDDSLT

FKEDIQKAQVSGQGDSLHEHIANLAGS

PAIKKGILQTVKVVDELVKVMGRHKPE

NIVIEMARENQTTQKGQKNSRERMKRI

EEGIKELGSQILKEHPVENTQLQNEKL

YLYYLQNGRDMYVDQELDINRLSDYDV

DAIVPQSFLKDDSIDNKVLTRSDKNRG

KSDNVPSEEVVKKMKNYWRQLLNAKLI

TQRKFDNLTKAERGGLSELDKAGFIKR

QLVETRQITKHVAQILDSRMNTKYDEN

DKLIREVKVITLKSKLVSDFRKDFQFY

KVREINNYHHAHDAYLNAVVGTALIKK

YPKLESEFVYGDYKVYDVRKMIAKSEQ

EIGKATAKYFFYSNIMNFFKTEITLAN

GEIRKRPLIETNGETGEIVWDKGRDFA

TVRKVLSMPQVNIVKKTEVQTGGFSKE

SILPKRNSDKLIARKKDWDPKKYGGFD

SPTVAYSVLVVAKVEKGKSKKLKSVKE

LLGITIMERSSFEKNPIDFLEAKGYKE

VKKDLIIKLPKYSLFELENGRKRMLAS

AGELQKGNELALPSKYVNFLYLASHYE

KLKGSPEDNEQKQLFVEQHKHYLDEII

EQISEFSKRVILADANLDKVLSAYNKH

RDKPIREQAENIIHLFTLTNLGAPAAF

KYFDTTIDRKRYTSTKEVLDATLIHQS

ITGLYETRIDLSQLGGD

5841
PEmax-
SGGSSGGSKRTADGSEFESPKKKRKVS

SGGSx2-
GGSSGGS

bpSV40NLS-

SGGSx2

linker

5842
PEmax-
TLNIEDEYRLHETSKEPDVSLGSTWLS

MMLV RT
DFPQAWAETGGMGLAVRQAPLIIPLKA

D200N
TSTPVSIKQYPMSQEARLGIKPHIQRL

T330P
LDQGILVPCQSPWNTPLLPVKKPGTND

L603W
YRPVQDLREVNKRVEDIHPTVPNPYNL

T306K
LSGLPPSHQWYTVLDLKDAFFCLRLHP

W313F
TSQPLFAFEWRDPEMGISGQLTWTRLP

QGFKNSPTLFNEALHRDLADFRIQHPD

LILLQYVDDLLLAATSELDCQQGTRAL

LQTLGNLGYRASAKKAQICQKQVKYLG

YLLKEGQRWLTEARKETVMGQPTPKTP

RQLREFLGKAGFCRLFIPGFAEMAAPL

YPLTKPGTLFNWGPDQQKAYQEIKQAL

LTAPALGLPDLTKPFELFVDEKQGYAK

GVLTQKLGPWRRPVAYLSKKLDPVAAG

WPPCLRMVAAIAVLTKDAGKLTMGQPL

VILAPHAVEALVKQPPDRWLSNARMTH

YQALLLDTDRVQFGPVVALNPATLLPL

PEEGLQHNCLDILAEAHGTRPDLTDQP

LPDADHTWYTDGSSLLQEGQRKAGAAV

TTETEVIWAKALPAGTSAQRAELIALT

QALKMAEGKKLNVYTDSRYAFATAHIH

GEIYRRRGWLTSEGKEIKNKDEILALL

KALFLPKRLSIIHCPGHQKGHSAEARG

NRMADQAARKAAITETPDTSTLLIENS

SP

5843
PEmax-
SGGSKRTADGSEFESPKKKRKV

C-

terminal

linker-

NLS

5844
PEmax-
GSGPAAKRVKLD

C-

terminal

linker-

NLS2

Polypeptides comprising components of a prime editor may be fused via peptide linkers, or may be provided in trans relevant to each other. For example, a reverse transcriptase may be expressed, delivered, or otherwise provided as an individual component rather than as a part of a fusion protein with the DNA binding domain. In such cases, components of the prime editor may be associated through non-peptide linkages or co-localization functions. In some embodiments, a prime editor further comprises additional components capable of interacting with, associating with, or capable of recruiting other components of the prime editor or the prime editing system. For example, a prime editor may comprise an RNA-protein recruitment polypeptide that can associate with an RNA-protein recruitment RNA aptamer. In some embodiments, an RNA-protein recruitment polypeptide can recruit, or be recruited by, a specific RNA sequence. Non limiting examples of RNA-protein recruitment polypeptide and RNA aptamer pairs include a MS2 coat protein and a MS2 RNA hairpin, a PCP polypeptide and a PP7 RNA hairpin, a Com polypeptide and a Com RNA hairpin, a Ku protein and a telomerase Ku binding RNA motif, and a Sm7 protein and a telomerase Sm7 binding RNA motif. In some embodiments, the prime editor comprises a DNA binding domain fused or linked to an RNA-protein recruitment polypeptide. In some embodiments, the prime editor comprises a DNA polymerase domain fused or linked to an RNA-protein recruitment polypeptide. In some embodiments, the DNA binding domain and the DNA polymerase domain fused to the RNA-protein recruitment polypeptide, or the DNA binding domain fused to the RNA-protein recruitment polypeptide and the DNA polymerase domain are co-localized by the corresponding RNA-protein recruitment RNA aptamer of the RNA-protein recruitment polypeptide. In some embodiments, the corresponding RNA-protein recruitment RNA aptamer fused or linked to a portion of the PEgRNA or ngRNA. For example, an MS2 coat protein fused or linked to the DNA polymerase and a MS2 hairpin installed on the PEgRNA for co-localization of the DNA polymerase and the RNA-guided DNA binding domain (e.g., a Cas9 nickase).

In certain embodiments, components of a prime editor are directly fused to each other. In certain embodiments, components of a prime editor are associated to each other via a linker.

As used herein, a linker can be any chemical group or a molecule linking two molecules or moieties, e.g., a DNA binding domain and a polymerase domain of a prime editor. In some embodiments, a linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker comprises a non-peptide moiety. The linker may be as simple as a covalent bond, or it may be a polymeric linker many atoms in length, for example, a polynucleotide sequence. In certain embodiments, the linker is a covalent bond (e.g., a carbon-carbon bond, disulfide bond, carbon-heteroatom bond, etc.).

In certain embodiments, two or more components of a prime editor are linked to each other by a peptide linker. In some embodiments, a peptide linker is 5-100 amino acids in length, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids in length. In some embodiments, the peptide linker is 16 amino acids in length, 24 amino acids in length, 64 amino acids in length, or 96 amino acids in length.

In some embodiments, the linker comprises the amino acid sequence (GGGGS)n (SEQ ID NO: 5845), (G)n (SEQ ID NO: 5846), (EAAAK)n (SEQ ID NO: 5847), (GGS)n (SEQ ID NO: 5848), (SGGS)n (SEQ ID NO: 5849), (XP)n (SEQ ID NO: 5850), or any combination thereof, wherein n is independently an integer between 1 and 30, and wherein X is any amino acid. In some embodiments, the linker comprises the amino acid sequence (GGS)n (SEQ ID NO: 5903), wherein n is 1, 3, or 7. In some embodiments, the linker comprises the amino acid sequence SGSETPGTSESATPES (SEQ ID NO: 5851). In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 5852). In some embodiments, the linker comprises the amino acid sequence SGGSGGSGGS (SEQ ID NO: 5854). In some embodiments, the linker comprises the amino acid sequence SGGS (SEQ ID NO: 5855). In other embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESAGSYPYDVPDYAGSAAPAAKKKKLDGSGSGGSSGGS (SEQ ID NO: 5856).

In certain embodiments, two or more components of a prime editor are linked to each other by a non-peptide linker. In some embodiments, the linker is a carbon-nitrogen bond of an amide linkage. In certain embodiments, the linker is a cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic or heteroaliphatic linker. In certain embodiments, the linker is polymeric (e.g., polyethylene, polyethylene glycol, polyamide, polyester, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of aminoalkanoic acid. In certain embodiments, the linker comprises an aminoalkanoic acid (e.g., glycine, ethanoic acid, alanine, beta-alanine, 3-aminopropanoic acid, 4-aminobutanoic acid, 5-pentanoic acid, etc.). In certain embodiments, the linker comprises a monomer, dimer, or polymer of aminohexanoic acid (Ahx). In certain embodiments, the linker is based on a carbocyclic moiety (e.g., cyclopentane, cyclohexane). In other embodiments, the linker comprises a polyethylene glycol moiety (PEG). In certain embodiments, the linker comprises an aryl or heteroaryl moiety. In certain embodiments, the linker is based on a phenyl ring. The linker may include functionalized moieties to facilitate attachment of a nucleophile (e.g., thiol, amino) from the peptide to the linker. Any electrophile may be used as part of the linker. Exemplary electrophiles include, but are not limited to, activated esters, activated amides, Michael acceptors, alkyl halides, aryl halides, acyl halides, and isothiocyanates.

Components of a prime editor may be connected to each other in any order. In some embodiments, the DNA binding domain and the DNA polymerase domain of a prime editor may be fused to form a fusion protein, or may be joined by a peptide or protein linker, in any order from the N terminus to the C terminus. In some embodiments, a prime editor comprises a DNA binding domain fused or linked to the C-terminal end of a DNA polymerase domain. In some embodiments, a prime editor comprises a DNA binding domain fused or linked to the N-terminal end of a DNA polymerase domain. In some embodiments, the prime editor comprises a fusion protein comprising the structure NH2-[DNA binding domain]-[polymerase]-COOH; or NH2-[polymerase]-[DNA binding domain]-COOH, wherein each instance of “]-[” indicates the presence of an optional linker sequence. In some embodiments, a prime editor comprises a fusion protein and a DNA polymerase domain provided in trans, wherein the fusion protein comprises the structure NH2-[DNA binding domain]-[RNA-protein recruitment polypeptide]-COOH. In some embodiments, a prime editor comprises a fusion protein and a DNA binding domain provided in trans, wherein the fusion protein comprises the structure NH2-[DNA polymerase domain]-[RNA-protein recruitment polypeptide]-COOH.

In addition, the NLSs may be expressed as part of a prime editor complex. The location of the NLS fusion can be at the N-terminus, the C-terminus, or positioned anywhere within a sequence of a prime editor or a component thereof (e.g., inserted between the DNA-binding domain and the DNA polymerase domain of a prime editor fusion protein, between the DNA binding domain and a linker sequence, between a DNA polymerase and a linker sequence, between two linker sequences of a prime editor fusion protein or a component thereof, in either N-terminus to C-terminus or C-terminus to N-terminus order). In some embodiments, a prime editor is fusion protein that comprises an NLS at the N terminus. In some embodiments, a prime editor is fusion protein that comprises an NLS at the C terminus. In some embodiments, a prime editor is fusion protein that comprises at least one NLS at both the N terminus and the C terminus. In some embodiments, the prime editor is a fusion protein that comprises two NLSs at the N terminus and/or the C terminus.

In some embodiments, a prime editor fusion protein, a polypeptide component of a prime editor, or a polynucleotide encoding the prime editor fusion protein or polypeptide component, may be split into an N-terminal half and a C-terminal half or polypeptides that encode the N-terminal half and the C terminal half, and provided to a target DNA in a cell separately. For example, in certain embodiments, a prime editor fusion protein may be split into a N-terminal and a C-terminal half for separate delivery in AAV vectors, and subsequently translated and colocalized in a target cell to reform the complete polypeptide or prime editor protein. In such cases, separate halves of a protein or a fusion protein may each comprise a split-intein to facilitate colocalization and reformation of the complete protein or fusion protein by the mechanism of intein facilitated trans splicing. In some embodiments, a prime editor comprises a N-terminal half fused to an intein-N, and a C-terminal half fused to an intein-C, or polynucleotides or vectors (e.g. AAV vectors) encoding each thereof. When delivered and/or expressed in a target cell, the intein-N and the intein-C can be excised via protein trans-splicing, resulting in a complete prime editor fusion protein in the target cell.

PEgRNA for Editing of ATP7B Gene

The term “prime editing guide RNA”, or “PEgRNA”, refers to a guide polynucleotide that comprises one or more intended nucleotide edits for incorporation into the target DNA. In some embodiments, the PEgRNA associates with and directs a prime editor to incorporate the one or more intended nucleotide edits into the target gene via prime editing. “Nucleotide edit” or “intended nucleotide edit” refers to a specified deletion of one or more nucleotides at one specific position, insertion of one or more nucleotides at one specific position, substitution of a single nucleotide, or other alterations at one specific position to be incorporated into the sequence of the target gene. Intended nucleotide edit may refer to the edit on the editing template as compared to the sequence on the target strand of the target gene or may refer to the edit encoded by the editing template on the newly synthesized single stranded DNA that replaces the editing target sequence, as compared to the editing target sequence. In some embodiments, a PEgRNA comprises a spacer sequence that is complementary or substantially complementary to a search target sequence on a target strand of the target gene. In some embodiments, the PEgRNA comprises a gRNA core that associates with a DNA binding domain, e.g., a CRISPR-Cas protein domain, of a prime editor. In some embodiments, the PEgRNA further comprises an extended nucleotide sequence comprising one or more intended nucleotide edits compared to the endogenous sequence of the target gene, wherein the extended nucleotide sequence may be referred to as an extension arm.

In certain embodiments, the extension arm comprises a primer binding site sequence (PBS) that can initiate target-primed DNA synthesis. In some embodiments, the PBS is complementary or substantially complementary to a free 3′ end on the edit strand of the target gene at a nick site generated by the prime editor. In some embodiments, the extension arm further comprises an editing template that comprises one or more intended nucleotide edits to be incorporated in the target gene by prime editing. In some embodiments, the editing template is a template for an RNA-dependent DNA polymerase domain or polypeptide of the prime editor, for example, a reverse transcriptase domain. The reverse transcriptase editing template may also be referred to herein as an RT template, or RTT. In some embodiments, the editing template comprises partial complementarity to an editing target sequence in the target gene. e.g., an ATP7B gene. In some embodiments, the editing template comprises substantial or partial complementarity to the editing target sequence except at the position of the intended nucleotide edits to be incorporated into the target gene. An exemplary architecture of a PEgRNA including its components is as demonstrated in FIG. 2.

In some embodiments, a PEgRNA includes only RNA nucleotides and forms an RNA polynucleotide. In some embodiments, a PEgRNA is a chimeric polynucleotide that includes both RNA and DNA nucleotides. For example, a PEgRNA can include DNA in the spacer sequence, the gRNA core, or the extension arm. In some embodiments, a PEgRNA comprises DNA in the spacer sequence. In some embodiments, the entire spacer sequence of a PEgRNA is a DNA sequence. In some embodiments, the PEgRNA comprises DNA in the gRNA core, for example, in a stem region of the gRNA core. In some embodiments, the PEgRNA comprises DNA in the extension arm, for example, in the editing template. An editing template that comprises a DNA sequence may serve as a DNA synthesis template for a DNA polymerase in a prime editor, for example, a DNA-dependent DNA polymerase. Accordingly, the PEgRNA may be a chimeric polynucleotide that comprises RNA in the spacer, gRNA core, and/or the PBS sequences and DNA in the editing template.

Components of a PEgRNA may be arranged in a modular fashion. In some embodiments, the spacer and the extension arm comprising a primer binding site sequence (PBS) and an editing template, e.g., a reverse transcriptase template (RTT), can be interchangeably located in the 5′ portion of the PEgRNA, the 3′ portion of the PEgRNA, or in the middle of the gRNA core. In some embodiments, a PEgRNA comprises a PBS and an editing template sequence in 5′ to 3′ order. In some embodiments, the gRNA core of a PEgRNA of this disclosure may be located in between a spacer and an extension arm of the PEgRNA. In some embodiments, the gRNA core of a PEgRNA may be located at the 3′ end of a spacer. In some embodiments, the gRNA core of a PEgRNA may be located at the 5′ end of a spacer. In some embodiments, the gRNA core of a PEgRNA may be located at the 3′ end of an extension arm. In some embodiments, the gRNA core of a PEgRNA may be located at the 5′ end of an extension arm. In some embodiments, the PEgRNA comprises, from 5′ to 3′: a spacer, a gRNA core, and an extension arm. In some embodiments, the PEgRNA comprises, from 5′ to 3′: a spacer, a gRNA core, an editing template, and a PBS. In some embodiments, the PEgRNA comprises, from 5′ to 3′: an extension arm, a spacer, and a gRNA core. In some embodiments, the PEgRNA comprises, from 5′ to 3′ an editing template, a PBS, a spacer, and a gRNA core.

In some embodiments, a PEgRNA comprises a single polynucleotide molecule that comprises the spacer sequence, the gRNA core, and the extension arm. In some embodiments, a PEgRNA comprises multiple polynucleotide molecules, for example, two polynucleotide molecules. In some embodiments, a PEgRNA comprise a first polynucleotide molecule that comprises the spacer and a portion of the gRNA core, and a second polynucleotide molecule that comprises the rest of the gRNA core and the extension arm. In some embodiments, the gRNA core portion in the first polynucleotide molecule and the gRNA core portion in the second polynucleotide molecule are at least partly complementary to each other. In some embodiments, the PEgRNA may comprise a first polynucleotide comprising the spacer and a first portion of a gRNA core comprising, which may be also be referred to as a crRNA. In some embodiments, the PEgRNA comprise a second polynucleotide comprising a second portion of the gRNA core and the extension arm, wherein the second portion of the gRNA core may also be referred to as a trans-activating crRNA, or tracr RNA. In some embodiments, the crRNA portion and the tracr RNA portion of the gRNA core are at least partially complementary to each other. In some embodiments, the partially complementary portions of the crRNA and the tracr RNA form a lower stem, a bulge, and an upper stem, as exemplified in FIG. 4.

In some embodiments, a spacer sequence comprises a region that has substantial complementarity to a search target sequence on the target strand of a double stranded target DNA, e.g., an ATP7B gene. In some embodiments, the spacer sequence of a PEgRNA is identical or substantially identical to a protospacer sequence on the edit strand of the target gene (except that the protospacer sequence comprises thymine and the spacer sequence may comprise uracil). In some embodiments, the spacer sequence is at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to a search target sequence in the target gene. In some embodiments, the spacer comprises is substantially complementary to the search target sequence.

In some embodiments, the length of the spacer varies from about 10 to about 100 nucleotides. In some embodiments, the spacer is 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, or 25 nucleotides in length. In some embodiments, the spacer is from 15 nucleotides to 30 nucleotides in length, 15 to 25 nucleotides in length, 18 to 22 nucleotides in length, 10 to 20 nucleotides in length, or 20 to 30 nucleotides in length. In some embodiments, the spacer is 16 to 22 nucleotides in length. In some embodiments, the spacer is 16 to 20 nucleotides in length. In some embodiments, the spacer is 17 to 18 nucleotides in length. In some embodiments, the spacer is 20 nucleotides in length.

As used herein in a PEgRNA or a nick guide RNA sequence, or fragments thereof such as a spacer, PBS, or RTT sequence, unless indicated otherwise, it should be appreciated that the letter “T” or “thymine” indicates a nucleobase in a DNA sequence that encodes the PEgRNA or guide RNA sequence, and is intended to refer to a uracil (U) nucleobase of the PEgRNA or guide RNA or any chemically modified uracil nucleobase known in the art, such as 5-methoxyuracil.

The extension arm of a PEgRNA may comprise a primer binding site (PBS) and an editing template (e.g., an RTT). The extension arm may be partially complementary to the spacer. In some embodiments, the editing template (e.g., RTT) is partially complementary to the spacer. In some embodiments, the editing template (e.g., RTT) and the primer binding site (PBS) are each partially complementary to the spacer.

An extension arm of a PEgRNA may comprise a primer binding site sequence (PBS, or PBS sequence) that comprises complementarity to and can hybridize with a free 3′ end of a single stranded DNA in the target gene (e.g. the ATP7B gene) generated by nicking with a prime editor at the nick site on the PAM strand. The length of the PBS sequence may vary depending on, e.g., the prime editor components, the search target sequence and other components of the PEgRNA. In some embodiments, the PBS is about 3 to 19 nucleotides in length. in some embodiments, the PBS is about 3 to 17 nucleotides in length. In some embodiments, the PBS is about 4 to 16 nucleotides, about 6 to 16 nucleotides, about 6 to 18 nucleotides, about 6 to 20 nucleotides, about 8 to 20 nucleotides, about 10 to 20 nucleotides, about 12 to 20 nucleotides, about 14 to 20 nucleotides, about 16 to 20 nucleotides, or about 18 to 20 nucleotides in length. In some embodiments, the PBS is 8 to 17 nucleotides in length. In some embodiments, the PBS is 8 to 16 nucleotides in length. In some embodiments, the PBS is 8 to 15 nucleotides in length. In some embodiments, the PBS is 8 to 14 nucleotides in length. In some embodiments, the PBS is 8 to 13 nucleotides in length. In some embodiments, the PBS is 8 to 12 nucleotides in length. In some embodiments, the PBS is 8 to 11 nucleotides in length. In some embodiments, the PBS is 8 to 10 nucleotides in length. In some embodiments, the PBS is 8 or 9 nucleotides in length. In some embodiments, the PBS is 16 or 17 nucleotides in length. In some embodiments, the PBS is 15 to 17 nucleotides in length. In some embodiments, the PBS is 14 to 17 nucleotides in length. In some embodiments, the PBS is 13 to 17 nucleotides in length. In some embodiments, the PBS is 12 to 17 nucleotides in length. In some embodiments, the PBS is 11 to 17 nucleotides in length. In some embodiments, the PBS is 10 to 17 nucleotides in length. In some embodiments, the PBS is 9 to 17 nucleotides in length. In some embodiments, the PBS is about 7 to 15 nucleotides in length. In some embodiments, the PBS is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the PBS is 8, 9, 10, 11, 12, 13, or 14 nucleotides in length.

The PBS may be complementary or substantially complementary to a DNA sequence in the edit strand of the target gene. By annealing with the edit strand at a free hydroxy group, e.g. a free 3′ end generated by prime editor nicking, the PBS may initiate synthesis of a new single stranded DNA encoded by the editing template at the nick site. In some embodiments, the PBS is at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to a region of the edit strand of the target gene (e.g., the ATP7B gene). In some embodiments, the PBS is perfectly complementary, or 100% complementary, to a region of the edit strand of the target gene (e.g., the ATP7B gene).

An extension arm of a PEgRNA may comprise an editing template that serves as a DNA synthesis template for the DNA polymerase in a prime editor during prime editing.

The length of an editing template may vary depending on, e.g., the prime editor components, the search target sequence and other components of the PEgRNA. In some embodiments, the editing template serves as a DNA synthesis template for a reverse transcriptase, and the editing template is referred to as a reverse transcription editing template (RTT).

The editing template (e.g., RTT), in some embodiments, is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the RTT is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the RTT is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length. In some embodiments, the RTT is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides in length.

In some embodiments, the editing template (e.g., RTT) sequence is about 70%, 75%, 80%, 85%, 90%, 95%, or 99% complementary to the editing target sequence on the edit strand of the target gene. In some embodiments, the editing template sequence (e.g., RTT) is substantially complementary to the editing target sequence. In some embodiments, the editing template sequence (e.g., RTT) is complementary to the editing target sequence except at positions of the intended nucleotide edits to be incorporated in the target gene. In some embodiments, the editing template comprises a nucleotide sequence comprising about 85% to about 95% complementarity to an editing target sequence in the edit strand in the target gene (e.g., the ATP7B gene). In some embodiments, the editing template comprises about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementarity to an editing target sequence in the edit strand of the target gene (e.g., the ATP7B gene).

An intended nucleotide edit in an editing template of a PEgRNA may comprise various types of alterations as compared to the target gene sequence. In some embodiments, the nucleotide edit is a single nucleotide substitution as compared to the target gene sequence. In some embodiments, the nucleotide edit is a deletion as compared to the target gene sequence. In some embodiments, the nucleotide edit is an insertion as compared to the target gene sequence. In some embodiments, the editing template comprises one to ten intended nucleotide edits as compared to the target gene sequence. In some embodiments, the editing template comprises one or more intended nucleotide edits as compared to the target gene sequence. In some embodiments, the editing template comprises two or more intended nucleotide edits as compared to the target gene sequence. In some embodiments, the editing template comprises three or more intended nucleotide edits as compared to the target gene sequence. In some embodiments, the editing template comprises four or more, five or more, or six or more intended nucleotide edits as compared to the target gene sequence. In some embodiments, the editing template comprises two single nucleotide substitutions, insertions, deletions, or any combination thereof, as compared to the target gene sequence. In some embodiments, the editing template comprises three single nucleotide substitutions, insertions, deletions, or any combination thereof, as compared to the target gene sequence. In some embodiments, the editing template comprises four, five, or six single nucleotide substitutions, insertions, deletions, or any combination thereof, as compared to the target gene sequence. In some embodiments, a nucleotide substitution comprises an adenine (A)-to-thymine (T) substitution. In some embodiments, a nucleotide substitution comprises an A-to-guanine (G) substitution. In some embodiments, a nucleotide substitution comprises an A-to-cytosine (C) substitution. In some embodiments, a nucleotide substitution comprises a T-A substitution. In some embodiments, a nucleotide substitution comprises a T-G substitution. In some embodiments, a nucleotide substitution comprises a T-C substitution. In some embodiments, a nucleotide substitution comprises a G-to-A substitution. In some embodiments, a nucleotide substitution comprises a G-to-T substitution. In some embodiments, a nucleotide substitution comprises a G-to-C substitution. In some embodiments, a nucleotide substitution comprises a C-to-A substitution. In some embodiments, a nucleotide substitution comprises a C-to-T substitution. In some embodiments, a nucleotide substitution comprises a C-to-G substitution.

In some embodiments, a nucleotide insertion is at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, or at least 20 nucleotides in length. In some embodiments, a nucleotide insertion is from 1 to 2 nucleotides, from 1 to 3 nucleotides, from 1 to 4 nucleotides, from 1 to 5 nucleotides, form 2 to 5 nucleotides, from 3 to 5 nucleotides, from 3 to 6 nucleotides, from 3 to 8 nucleotides, from 4 to 9 nucleotides, from 5 to 10 nucleotides, from 6 to 11 nucleotides, from 7 to 12 nucleotides, from 8 to 13 nucleotides, from 9 to 14 nucleotides, from 10 to 15 nucleotides, from 11 to 16 nucleotides, from 12 to 17 nucleotides, from 13 to 18 nucleotides, from 14 to 19 nucleotides, from 15 to 20 nucleotides in length. In some embodiments, a nucleotide insertion is a single nucleotide insertion. In some embodiments, a nucleotide insertion comprises insertion of two nucleotides.

The editing template of a PEgRNA may comprise one or more intended nucleotide edits, compared to the ATP7B gene to be edited. Position of the intended nucleotide edit(s) relevant to other components of the PEgRNA, or to particular nucleotides (e.g., mutations) in the ATP7B target gene may vary. In some embodiments, the nucleotide edit is in a region of the PEgRNA corresponding to or homologous to the protospacer sequence. In some embodiments, the nucleotide edit is in a region of the PEgRNA corresponding to a region of the ATP7B gene outside of the protospacer sequence.

In some embodiments, the position of a nucleotide edit incorporation in the target gene may be determined based on position of the protospacer adjacent motif (PAM). For instance, the intended nucleotide edit may be installed in a sequence corresponding to the protospacer adjacent motif (PAM) sequence. In some embodiments, a nucleotide edit in the editing template is at a position corresponding to the 5′ most nucleotide of the PAM sequence. In some embodiments, a nucleotide edit in the editing template is at a position corresponding to the 3′ most nucleotide of the PAM sequence. In some embodiments, position of an intended nucleotide edit in the editing template may be referred to by aligning the editing template with the partially complementary edit strand of the target gene, and referring to nucleotide positions on the editing strand where the intended nucleotide edit is incorporated. In some embodiments, a nucleotide edit is incorporated at a position corresponding to about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides upstream of the 5′ most nucleotide of the PAM sequence in the edit strand of the target gene. By 0 base pair upstream or downstream of a reference position, it is meant that the intended nucleotide is immediately upstream or downstream of the reference position. In some embodiments, a nucleotide edit is incorporated at a position corresponding to about 0 to 2 nucleotides, 0 to 4 nucleotides, 0 to 6 nucleotides, 0 to 8 nucleotides, 0 to 10 nucleotides, 2 to 4 nucleotides, 2 to 6 nucleotides, 2 to 8 nucleotides, 2 to 10 nucleotides, 2 to 12 nucleotides, 4 to 6 nucleotides, 4 to 8 nucleotides, 4 to 10 nucleotides, 4 to 12 nucleotides, 4 to 14 nucleotides, 6 to 8 nucleotides, 6 to 10 nucleotides, 6 to 12 nucleotides, 6 to 14 nucleotides, 6 to 16 nucleotides, 8 to 10 nucleotides, 8 to 12 nucleotides, 8 to 14 nucleotides, 8 to 16 nucleotides, 8 to 18 nucleotides, 10 to 12 nucleotides, 10 to 14 nucleotides, 10 to 16 nucleotides, 10 to 18 nucleotides, 10 to 20 nucleotides, 12 to 14 nucleotides, 12 to 16 nucleotides, 12 to 18 nucleotides, 12 to 20 nucleotides, 12 to 22 nucleotides, 14 to 16 nucleotides, 14 to 18 nucleotides, 14 to 20 nucleotides, 14 to 22 nucleotides, 14 to 24 nucleotides, 16 to 18 nucleotides, 16 to 20 nucleotides, 16 to 22 nucleotides, 16 to 24 nucleotides, 16 to 26 nucleotides, 18 to 20 nucleotides, 18 to 22 nucleotides, 18 to 24 nucleotides, 18 to 26 nucleotides, 18 to 28 nucleotides, 20 to 22 nucleotides, 20 to 24 nucleotides, 20 to 26 nucleotides, 20 to 28 nucleotides, or 20 to 30 nucleotides upstream of the 5′ most nucleotide of the PAM sequence. In some embodiments, the nucleotide edit is incorporated at a position corresponding to 3 nucleotides upstream of the 5′ most nucleotide of the PAM sequence. In some embodiments, the nucleotide edit in is incorporated at a position corresponding to 4 nucleotides upstream of the 5′ most nucleotide of the PAM sequence. In some embodiments, the nucleotide edit is incorporated at a position corresponding to 5 nucleotides upstream of the 5′ most nucleotide of the PAM sequence. In some embodiments, the nucleotide edit in the editing template is at a position corresponding to 6 nucleotides upstream of the 5′ most nucleotide of the PAM sequence.

In some embodiments, an intended nucleotide edit is incorporated at a position corresponding to about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides downstream of the 5′ most nucleotide of the PAM sequence in the edit strand of the target gene. In some embodiments, a nucleotide edit is incorporated at a position corresponding to about 0 to 2 nucleotides, 0 to 4 nucleotides, 0 to 6 nucleotides, 0 to 8 nucleotides, 0 to 10 nucleotides, 2 to 4 nucleotides, 2 to 6 nucleotides, 2 to 8 nucleotides, 2 to 10 nucleotides, 2 to 12 nucleotides, 4 to 6 nucleotides, 4 to 8 nucleotides, 4 to 10 nucleotides, 4 to 12 nucleotides, 4 to 14 nucleotides, 6 to 8 nucleotides, 6 to 10 nucleotides, 6 to 12 nucleotides, 6 to 14 nucleotides, 6 to 16 nucleotides, 8 to 10 nucleotides, 8 to 12 nucleotides, 8 to 14 nucleotides, 8 to 16 nucleotides, 8 to 18 nucleotides, 10 to 12 nucleotides, 10 to 14 nucleotides, 10 to 16 nucleotides, 10 to 18 nucleotides, 10 to 20 nucleotides, 12 to 14 nucleotides, 12 to 16 nucleotides, 12 to 18 nucleotides, 12 to 20 nucleotides, 12 to 22 nucleotides, 14 to 16 nucleotides, 14 to 18 nucleotides, 14 to 20 nucleotides, 14 to 22 nucleotides, 14 to 24 nucleotides, 16 to 18 nucleotides, 16 to 20 nucleotides, 16 to 22 nucleotides, 16 to 24 nucleotides, 16 to 26 nucleotides, 18 to 20 nucleotides, 18 to 22 nucleotides, 18 to 24 nucleotides, 18 to 26 nucleotides, 18 to 28 nucleotides, 20 to 22 nucleotides, 20 to 24 nucleotides, 20 to 26 nucleotides, 20 to 28 nucleotides, or 20 to 30 nucleotides downstream of the 5′ most nucleotide of the PAM sequence. In some embodiments, a nucleotide edit is incorporated at a position corresponding to 3 nucleotides downstream of the 5′ most nucleotide of the PAM sequence. In some embodiments, a nucleotide edit is incorporated at a position corresponding to 4 nucleotides downstream of the 5′ most nucleotide of the PAM sequence. In some embodiments, a nucleotide edit is incorporated at a position corresponding to 5 nucleotides downstream of the 5′ most nucleotide of the PAM sequence. In some embodiments, a nucleotide edit is incorporated at a position corresponding to 6 nucleotides downstream of the 5′ most nucleotide of the PAM sequence. By “upstream” and “downstream” it is intended to define relevant positions at least two regions or sequences in a nucleic acid molecule orientated in a 5′-to-3′ direction. For example, a first sequence is upstream of a second sequence in a DNA molecule where the first sequence is positioned 5′ to the second sequence. Accordingly, the second sequence is downstream of the first sequence.

When referred to within the PEgRNA, positions of the one or more intended nucleotide edits may be referred to relevant to components of the PEgRNA. For example, an intended nucleotide edit may be 5′ or 3′ to the PBS. In some embodiments, a PEgRNA comprises the structure, from 5′ to 3′: a spacer, a gRNA core, an editing template, and a PBS. In some embodiments, the intended nucleotide edit is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides upstream to the 5′ most nucleotide of the PBS. In some embodiments, the intended nucleotide edit is 0 to 2 nucleotides, 0 to 4 nucleotides, 0 to 6 nucleotides, 0 to 8 nucleotides, 0 to 10 nucleotides, 2 to 4 nucleotides, 2 to 6 nucleotides, 2 to 8 nucleotides, 2 to 10 nucleotides, 2 to 12 nucleotides, 4 to 6 nucleotides, 4 to 8 nucleotides, 4 to 10 nucleotides, 4 to 12 nucleotides, 4 to 14 nucleotides, 6 to 8 nucleotides, 6 to 10 nucleotides, 6 to 12 nucleotides, 6 to 14 nucleotides, 6 to 16 nucleotides, 8 to 10 nucleotides, 8 to 12 nucleotides, 8 to 14 nucleotides, 8 to 16 nucleotides, 8 to 18 nucleotides, 10 to 12 nucleotides, 10 to 14 nucleotides, 10 to 16 nucleotides, 10 to 18 nucleotides, 10 to 20 nucleotides, 12 to 14 nucleotides, 12 to 16 nucleotides, 12 to 18 nucleotides, 12 to 20 nucleotides, 12 to 22 nucleotides, 14 to 16 nucleotides, 14 to 18 nucleotides, 14 to 20 nucleotides, 14 to 22 nucleotides, 14 to 24 nucleotides, 16 to 18 nucleotides, 16 to 20 nucleotides, 16 to 22 nucleotides, 16 to 24 nucleotides, 16 to 26 nucleotides, 18 to 20 nucleotides, 18 to 22 nucleotides, 18 to 24 nucleotides, 18 to 26 nucleotides, 18 to 28 nucleotides, 20 to 22 nucleotides, 20 to 24 nucleotides, 20 to 26 nucleotides, 20 to 28 nucleotides, or 20 to 30 nucleotides upstream to the 5′ most nucleotide of the PBS.

The corresponding positions of the intended nucleotide edit incorporated in the target gene may also be referred to based on the nicking position (i.e. the nick site) generated by a prime editor based on sequence homology and complementarity. For example, in embodiments, the distance between the intended nucleotide edit to be incorporated into the target ATP7B gene and the nick site (also referred to as the “nick to edit distance”) may be determined by the position of the nick site and the position of the nucleotide(s) corresponding to the intended nucleotide edit(s), for example, by identifying sequence complementarity between the spacer and the search target sequence and sequence complementarity between the editing template and the editing target sequence. In certain embodiments, the position of the nucleotide edit can be in any position downstream of the nick site on the edit strand (or the PAM strand) generated by the prime editor, such that the distance between the nick site and the intended nucleotide edit is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, the position of the nucleotide edit is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides upstream of the nick site on the edit strand. In some embodiments, the position of the nucleotide edit is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides downstream of the nick site on the edit strand. In some embodiments, the position of the nucleotide edit is 0 base pair from the nick site on the edit strand, that is, the editing position is at the same position as the nick site. As used herein, the distance between the nick site and the nucleotide edit, for example, where the nucleotide edit comprises an insertion or deletion, refers to the 5′ most position of the nucleotide edit for a nick that creates a 3′ free end on the edit strand (i.e., the “near position” of the nucleotide edit to the nick site). Similarly, as used herein, the distance between the nick site and a PAM position edit, for example, where the nucleotide edit comprises an insertion, deletion, or substitution of two or more contiguous nucleotides, refers to the 5′ most position of the nucleotide edit and the 5′ most position of the PAM sequence.

In some embodiments, the editing template extends beyond a nucleotide edit to be incorporated to the target ATP7B gene sequence. For example, in some embodiments, the editing template comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence.

In some embodiments, the editing template comprises 1 to 2 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 3 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 4 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 5 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 6 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 7 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 8 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 9 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 10 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 11 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 12 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 13 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 14 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 15 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 16 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 17 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 18 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 19 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 20 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 21 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 22 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 23 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 24 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 25 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 26 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 27 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 28 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 29 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 30 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 31 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 32 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 33 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 34 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 35 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 36 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 37 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 38 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 39 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 40 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 41 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 42 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 43 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 44 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 45 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 46 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 47 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 48 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 49 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 50 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 51 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 52 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 53 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 54 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 55 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 56 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 57 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 58 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 59 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 60 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 61 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 62 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 63 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 64 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 65 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 66 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 67 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 68 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 69 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 70 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 71 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 72 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 73 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 74 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 75 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 76 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 77 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 1 to 78 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 2 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 3 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 4 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 5 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 6 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 7 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 8 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 9 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 10 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 11 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 12 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 13 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 14 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 15 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 16 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 17 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 18 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 19 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 20 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 21 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 22 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 23 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 24 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 25 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 26 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 27 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 28 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 29 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 30 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 31 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 32 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 33 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 34 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 35 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 36 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 37 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 38 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 39 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 40 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 41 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 42 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 43 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 44 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 45 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 46 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 47 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 48 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 49 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 50 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 51 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 52 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 53 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 54 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 55 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 56 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 57 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 58 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 59 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 60 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 61 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 62 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 63 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 64 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 65 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 66 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 67 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 68 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 69 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 70 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 71 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 72 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 73 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 74 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 75 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 76 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 77 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 78 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 79 to 80 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence.

In some embodiments, the editing template comprises 2 to 40 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 2 to 38 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 2 to 36 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 2 to 34 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 2 to 32 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 4 to 30 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 2 to 25 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 2 to 20 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 2 to 15 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 2 to 10 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 2 to 5 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 4 to 25 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 4 to 20 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 4 to 25 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 4 to 15 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 4 to 10 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 10 to 15 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 10 to 20 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 10 to 30 nucleotides 3′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 4 to 30 nucleotides 5′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 4 to 25 nucleotides 5′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence. In some embodiments, the editing template comprises 4 to 20 nucleotides 5′ to the nucleotide edit to be incorporated to the target ATP7B gene sequence.

In some embodiments, the length of the editing template is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides longer than the nick to edit distance. In some embodiments, for example, the nick to edit distance is 8 nucleotides, and the editing template is 10 to 15, 10 to 20, 10 to 25, 10 to 30, 10 to 35, 10 to 40, 10 to 45, 10 to 50, 10 to 55, 10 to 60, 10 to 65, 10 to 70, 10 to 75, or 10 to 80 nucleotides in length. In some embodiments, the nick to edit distance is 22 nucleotides, and the editing template is 24 to 28, 24 to 30, 24 to 32, 24 to 34, 24 to 36, 24 to 37, 24 to 38, 24 to 40, 24 to 45, 24 to 50, 24 to 55, 24 to 60, 24 to 65, 24 to 70, 24 to 75, 24 to 80, 24 to 85, 24 to 90, 24 to 95, 24 to 100, 24 to 105, 24 to 100, 24 to 105, or 24 to 110 nucleotides in length.

In some embodiments, the editing template comprises an adenine at the first nucleobase position (e.g., for a PEgRNA following 5′-spacer-gRNA core-RTT-PBS-3′ orientation, the 5′ most nucleobase is the “first base”). In some embodiments, the editing template comprises a guanine at the first nucleobase position (e.g., for a PEgRNA following 5′-spacer-gRNA core-RTT-PBS-3′ orientation, the 5′ most nucleobase is the “first base”). In some embodiments, the editing template comprises an uracil at the first nucleobase position (e.g., for a PEgRNA following 5′-spacer-gRNA core-RTT-PBS-3′ orientation, the 5′ most nucleobase is the “first base”). In some embodiments, the editing template comprises a cytosine at the first nucleobase position (e.g., for a PEgRNA following 5′-spacer-gRNA core-RTT-PBS-3′ orientation, the 5′ most nucleobase is the “first base”). In some embodiments, the editing template does not comprise a cytosine at the first nucleobase position (e.g., for a PEgRNA following 5′-spacer-gRNA core-RT T-PBS-3′ orientation, the 5′ most nucleobase is the “first base”).

The editing template of a PEgRNA may encode a new single stranded DNA (e.g. by reverse transcription) to replace a editing target sequence in the target gene. In some embodiments, the editing target sequence in the edit strand of the target gene is replaced by the newly synthesized strand, and the nucleotide edit(s) are incorporated in the region of the target gene. In some embodiments, the target gene is an ATP7B gene. In some embodiments, the editing template of the PEgRNA encodes a newly synthesized single stranded DNA that comprises a wild type APT7B gene sequence. In some embodiments, the newly synthesized DNA strand replaces the editing target sequence in the target ATP7B gene, wherein the editing target sequence (or the endogenous sequence complementary to the editing target sequence on the target strand of the ATP7B gene) comprises a mutation compared to a wild type ATP7B gene. In some embodiments, the mutation is associated with Wilson's disease.

In some embodiments, the editing target sequence comprises a mutation in exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, or exon 21 of the ATP7B gene as compared to a wild type ATP7B gene. In some embodiments, the editing target sequence comprises a mutation in exon 8, exon 13, exon 14, exon 15, or exon 17 of the ATP7B gene as compared to a wild type ATP7B gene. In some embodiments, the editing target sequence comprises a mutation in exon 14 of the ATP7B gene as compared to a wild type ATP7B gene. In some embodiments, the editing target sequence comprises a mutation in exon 3 of the ATP7B gene as compared to a wild type ATP7B gene. In some embodiments, the editing target sequence comprises a mutation that is located in exon 8 of the ATP7B gene as compared to a wild type ATP7B gene. In some embodiments, the mutation is not a c.1288dup duplication. In some embodiments, the editing target sequence comprises a mutation that is located between positions 51932669 and 52012130 of human chromosome 13 as set forth in human genome assembly consortium Human build 38 (GRCh38), GenBank accession GCA_000001405.15. In some embodiments, the editing target sequence comprises a mutation that is located between positions 51944045 and 51944245 of human chromosome 13 as set forth in human genome assembly consortium Human build 38 (GRCh38), GenBank accession GCA_000001405.15. In some embodiments, the editing target sequence comprises a mutation that encodes an amino acid substitution H1069Q relative to a wild type ATP7B polypeptide set forth in SEQ ID NO: 5861. In some embodiments, the editing target sequence comprises a C>A mutation at position 51944145 in human chromosome 13 as set forth in human genome assembly consortium Human build 38 (GRCh38), GenBank accession GCA_000001405.15. As used herein, unless otherwise noted, reference to positions in human genome is as set forth in human genome assembly consortium Human build 38 (GRCh38), GenBank accession GCA_000001405.15.

A guide RNA core (also referred to herein as the gRNA core, gRNA scaffold, or gRNA backbone sequence) of a PEgRNA may contain a polynucleotide sequence that binds to a DNA binding domain (e.g., Cas9) of a prime editor. The gRNA core may interact with a prime editor as described herein, for example, by association with a DNA binding domain, such as a DNA nickase of the prime editor.

One of skill in the art will recognize that different prime editors having different DNA binding domains from different DNA binding proteins may require different gRNA core sequences specific to the DNA binding protein. In some embodiments, the gRNA core is capable of binding to a Cas9-based prime editor. In some embodiments, the gRNA core is capable of binding to a Cpf1-based prime editor. In some embodiments, the gRNA core is capable of binding to a Cas12b-based prime editor.

In some embodiments, the gRNA core comprises regions and secondary structures involved in binding with specific CRISPR Cas proteins. For example, in a Cas9 based prime editing system, the gRNA core of a PEgRNA may comprise one or more regions of a base paired “lower stem” adjacent to the spacer sequence and a base paired “upper stem” following the lower stem, where the lower stem and upper stem may be connected by a “bulge” comprising unpaired RNAs. The gRNA core may further comprise a “nexus” distal from the spacer sequence, followed by a hairpin structure, e.g., at the 3′ end, as exemplified in FIG. 4. In some embodiments, the gRNA core comprises modified nucleotides as compared to a wild type gRNA core in the lower stem, upper stem, and/or the hairpin. For example, nucleotides in the lower stem, upper stem, an/or the hairpin regions may be modified, deleted, or replaced. In some embodiments. RNA nucleotides in the lower stem, upper stem, an/or the hairpin regions may be replaced with one or more DNA sequences. In some embodiments, the gRNA core comprises unmodified or wild type RNA sequences in the nexus and/or the bulge regions. In some embodiments, the gRNA core does not include long stretches of A-T pairs, for example, a GUUUU-AAAAC pairing element.

In some embodiments, a prime editing system comprises a prime editor and a PEgRNA, wherein the prime editor comprises a SpCas9 nickase or a variant thereof, and the gRNA core of the PEgRNA comprises the sequence:

(SEQ ID NO: 5857)

GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC

UUGAAAAAGUGGCACCGAGUCGGUGC;

(SEQ ID NO: 5858)

GUUUGAGAGCUAGAAAUAGCAAGUUUAAAUAAGGCUAGUCCGUUAUCAAC

UUGAAAAAGUGGGACCGAGUCGGUCC,

or

(SEQ ID NO: 5859)

GUUUAAGAGCUAUGCUGGAAACAGCAUAGCAAGUUUAAAUAAGGCUAGUC

CGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC.

In some embodiments, the gRNA core comprises the sequence

(SEQ ID NO: 5857)

GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC

UUGAAAAAGUGGCACCGAGUCGGUGC.

Any gRNA core sequences known in the art are also contemplated in the prime editing compositions described herein.

A PEgRNA may also comprise optional modifiers, e.g., 3′ end modifier region and/or an 5′ end modifier region. In some embodiments, a PEgRNA comprises at least one nucleotide that is not part of a spacer, a gRNA core, or an extension arm. The optional sequence modifiers could be positioned within or between any of the other regions shown, and not limited to being located at the 3′ and 5′ ends. In certain embodiments, the PEgRNA comprises secondary RNA structure, such as, but not limited to, aptamers, hairpins, stem/loops, toeloops, and/or RNA-binding protein recruitment domains (e.g., the MS2 aptamer which recruits and binds to the MS2cp protein). In some embodiments, a PEgRNA comprises a short stretch of uracil at the 5′ end or the 3′ end. For example, in some embodiments, a PEgRNA comprising a 3′ extension arm comprises a “UUU” sequence at the 3′ end of the extension arm. In some embodiments, a PEgRNA comprises a toeloop sequence at the 3′ end. In some embodiments, the PEgRNA comprises a 3′ extension arm and a toeloop sequence at the 3′ end of the extension arm. In some embodiments, the PEgRNA comprises a 5′ extension arm and a toeloop sequence at the 5′ end of the extension arm. In some embodiments, the PEgRNA comprises a toeloop element having the sequence 5′-GAAANNNNN-3′, wherein N is any nucleobase. In some embodiments, the secondary RNA structure is positioned within the spacer. In some embodiments, the secondary structure is positioned within the extension arm. In some embodiments, the secondary structure is positioned within the gRNA core. In some embodiments, the secondary structure is positioned between the spacer and the gRNA core, between the gRNA core and the extension arm, or between the spacer and the extension arm. In some embodiments, the secondary structure is positioned between the PBS and the editing template. In some embodiments the secondary structure is positioned at the 3′ end or at the 5′ end of the PEgRNA. In some embodiments, the PEgRNA comprises a transcriptional termination signal at the 3′ end of the PEgRNA. In addition to secondary RNA structures, the PEgRNA may comprise a chemical linker or a poly(N) linker or tail, where “N” can be any nucleobase. In some embodiments, the chemical linker may function to prevent reverse transcription of the gRNA core.

In some embodiments, a prime editing system or composition further comprises a nick guide polynucleotide, such as a nick guide RNA (ngRNA). Without wishing to be bound by any particular theory, the non-edit strand of a double stranded target DNA in the target gene may be nicked by a CRISPR-Cas nickase directed by an ngRNA. In some embodiments, the nick on the non-edit strand directs endogenous DNA repair machinery to use the edit strand as a template for repair of the non-edit strand, which may increase efficiency of prime editing. In some embodiments, the non-edit strand is nicked by a prime editor localized to the non-edit strand by the ngRNA. Accordingly, also provided herein are PEgRNA systems comprising at least one PEgRNA and at least one ngRNA.

In some embodiments, the ngRNA is a guide RNA which contains a variable spacer sequence and a guide RNA scaffold or core region that interacts with the DNA binding domain, e.g. Cas9 of the prime editor. In some embodiments, the ngRNA comprises a spacer sequence (referred to herein as an ng spacer, or a second spacer) that is substantially complementary to a second search target sequence (or ng search target sequence), which is located on the edit strand, or the non-target strand. Thus, in some embodiments, the ng search target sequence recognized by the ng spacer and the search target sequence recognized by the spacer sequence of the PEgRNA are on opposite strands of the double stranded target DNA of target gene, e.g., the ATP7B gene. A prime editing system or complex comprising a ngRNA may be referred to as a “PE3” prime editing system or PE3 prime editing complex.

In some embodiments, the ng search target sequence is located on the non-target strand, within 10 base pairs to 100 base pairs of an intended nucleotide edit incorporated by the PEgRNA on the edit strand. In some embodiments, the ng target search target sequence is within 10 bp, 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 91 bp, 92 bp, 93 bp, 94 bp, 95 bp, 96 bp, 97 bp, 98 bp, 99 bp, or 100 bp of an intended nucleotide edit incorporated by the PEgRNA on the edit strand. In some embodiments, the 5′ ends of the ng search target sequence and the PEgRNA search target sequence are within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bp apart from each other. In some embodiments, the 5′ ends of the ng search target sequence and the PEgRNA search target sequence are within 10 bp, 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 91 bp, 92 bp, 93 bp, 94 bp, 95 bp, 96 bp, 97 bp, 98 bp, 99 bp, or 100 bp apart from each other.

In some embodiments, an ng spacer sequence is complementary to, and may hybridize with the second search target sequence only after an intended nucleotide edit has been incorporated on the edit strand, by the editing template of a PEgRNA. Such a prime editing system maybe referred to as a “PE3b” prime editing system or composition. In some embodiments, the ngRNA comprises a spacer sequence that matches only the edit strand after incorporation of the nucleotide edits, but not the endogenous target gene sequence on the edit strand. Accordingly, in some embodiments, an intended nucleotide edit is incorporated within the ng search target sequence. In some embodiments, the intended nucleotide edit is incorporated within about 1-10 nucleotides of the position corresponding to the PAM of the ng search target sequence.

Exemplary combinations of PEgRNA components, e.g., spacer, PBS, and RTT, as well as combinations of each PEgRNA and corresponding ngRNA(s) are provided in Tables 6-12 and 15-32. Tables 6-12, 15-32 each contains two columns, the left column lists the respective PEgRNA components, and the right column is the corresponding sequence identifiers. Each of the PEgRNA components in Tables 6-12, 15-32 is listed consecutively and should be read from left to right, continuously.

TABLE 6

Sequence region
SEQ ID

pegRNA Spacer
1

PBS
2

PBS
3

PBS
4

PBS
5

PBS
6

PBS
7

PBS
8

PBS
9

PBS
10

PBS
11

PBS
12

RTT
13

RTT
14

RTT
15

RTT
16

RTT
17

ngRNA Spacer
18

ngRNA Spacer
19

ngRNA Spacer
20

ngRNA Spacer
21

ngRNA Spacer
22

ngRNA Spacer
23

ngRNA Spacer
24

ngRNA Spacer
25

ngRNA Spacer
26

ngRNA Spacer
27

ngRNA Spacer
28

ngRNA Spacer
29

ngRNA Spacer
30

ngRNA Spacer
31

ngRNA Spacer
32

ngRNA Spacer
33

ngRNA Spacer
34

ngRNA Spacer
35

ngRNA Spacer
36

ngRNA Spacer
37

ngRNA Spacer
38

ngRNA Spacer
39

ngRNA Spacer
40

ngRNA Spacer
41

ngRNA Spacer
42

ngRNA Spacer
43

ngRNA Spacer
44

ngRNA Spacer
45

ngRNA Spacer
46

ngRNA Spacer
47

ngRNA Spacer
48

ngRNA Spacer
49

ngRNA Spacer
50

ngRNA Spacer
51

ngRNA Spacer
52

ngRNA Spacer
53

ngRNA Spacer
54

ngRNA Spacer
55

ngRNA Spacer
56

ngRNA Spacer
57

ngRNA Spacer
58

ngRNA Spacer
59

ngRNA Spacer
60

ngRNA Spacer
61

ngRNA Spacer
62

ngRNA Spacer
63

ngRNA Spacer
64

ngRNA Spacer
65

ngRNA Spacer
66

ngRNA Spacer
67

ngRNA Spacer
68

ngRNA Spacer
69

ngRNA Spacer
70

ngRNA Spacer
71

ngRNA Spacer
72

pegRNA
73

pegRNA
74

pegRNA
75

pegRNA
76

pegRNA
77

pegRNA
78

pegRNA
79

pegRNA
80

pegRNA
81

pegRNA
82

pegRNA
83

pegRNA
84

pegRNA
85

pegRNA
86

pegRNA
87

pegRNA
88

pegRNA
89

pegRNA
90

pegRNA
91

pegRNA
92

pegRNA
93

pegRNA
94

pegRNA
95

pegRNA
96

pegRNA
97

pegRNA
98

pegRNA
99

pegRNA
100

pegRNA
101

pegRNA
102

pegRNA
103

pegRNA
104

pegRNA
105

pegRNA
106

pegRNA
107

pegRNA
108

pegRNA
109

pegRNA
110

pegRNA
111

pegRNA
112

pegRNA
113

pegRNA
114

pegRNA
115

pegRNA
116

pegRNA
117

pegRNA
118

pegRNA
119

pegRNA
120

pegRNA
121

pegRNA
122

pegRNA
123

pegRNA
124

pegRNA
125

pegRNA
126

pegRNA
127

pegRNA
128

pegRNA
129

pegRNA
130

pegRNA
131

pegRNA
132

pegRNA
133

pegRNA
134

pegRNA
135

pegRNA
136

pegRNA
137

pegRNA
138

pegRNA
139

pegRNA
140

pegRNA
141

pegRNA
142

pegRNA
143

pegRNA
144

pegRNA
145

pegRNA
146

pegRNA
147

pegRNA
148

pegRNA
149

pegRNA
150

pegRNA
151

pegRNA
152

ngRNA
153

ngRNA
154

ngRNA
155

ngRNA
156

ngRNA
157

ngRNA
158

ngRNA
159

ngRNA
160

ngRNA
161

ngRNA
162

ngRNA
163

ngRNA
164

ngRNA
165

ngRNA
166

ngRNA
167

ngRNA
168

ngRNA
169

ngRNA
170

ngRNA
171

ngRNA
172

ngRNA
173

ngRNA
174

ngRNA
175

ngRNA
176

ngRNA
177

ngRNA
178

ngRNA
179

ngRNA
180

ngRNA
181

TABLE 7

Sequence region
SEQ ID

pegRNA Spacer
182

PBS
183

PBS
184

PBS
185

PBS
186

PBS
187

PBS
188

PBS
189

PBS
190

PBS
191

PBS
192

PBS
193

RTT
194

RTT
195

RTT
196

RTT
197

RTT
198

ngRNA Spacer
42

ngRNA Spacer
22

ngRNA Spacer
55

ngRNA Spacer
54

ngRNA Spacer
199

ngRNA Spacer
62

ngRNA Spacer
34

ngRNA Spacer
72

ngRNA Spacer
63

ngRNA Spacer
43

ngRNA Spacer
66

ngRNA Spacer
200

ngRNA Spacer
28

ngRNA Spacer
30

ngRNA Spacer
20

ngRNA Spacer
50

ngRNA Spacer
35

ngRNA Spacer
27

ngRNA Spacer
31

ngRNA Spacer
48

ngRNA Spacer
61

ngRNA Spacer
69

ngRNA Spacer
26

ngRNA Spacer
45

ngRNA Spacer
37

ngRNA Spacer
33

ngRNA Spacer
60

ngRNA Spacer
58

ngRNA Spacer
68

ngRNA Spacer
70

ngRNA Spacer
25

ngRNA Spacer
46

ngRNA Spacer
24

ngRNA Spacer
38

ngRNA Spacer
56

ngRNA Spacer
201

ngRNA Spacer
36

ngRNA Spacer
44

ngRNA Spacer
57

ngRNA Spacer
23

ngRNA Spacer
21

ngRNA Spacer
202

ngRNA Spacer
41

ngRNA Spacer
29

ngRNA Spacer
203

ngRNA Spacer
71

ngRNA Spacer
32

ngRNA Spacer
67

ngRNA Spacer
19

ngRNA Spacer
39

ngRNA Spacer
51

ngRNA Spacer
47

ngRNA Spacer
40

ngRNA Spacer
204

ngRNA Spacer
64

ngRNA Spacer
205

ngRNA Spacer
53

ngRNA Spacer
206

ngRNA Spacer
65

ngRNA Spacer
207

ngRNA Spacer
18

ngRNA Spacer
52

ngRNA Spacer
208

ngRNA Spacer
59

ngRNA Spacer
49

ngRNA Spacer
209

pegRNA
210

pegRNA
211

pegRNA
212

pegRNA
213

pegRNA
214

pegRNA
215

pegRNA
216

pegRNA
217

pegRNA
218

pegRNA
219

pegRNA
220

pegRNA
221

pegRNA
222

pegRNA
223

pegRNA
224

pegRNA
225

pegRNA
226

pegRNA
227

pegRNA
228

pegRNA
229

pegRNA
230

pegRNA
231

pegRNA
232

pegRNA
233

pegRNA
234

pegRNA
235

pegRNA
236

pegRNA
237

pegRNA
238

pegRNA
239

pegRNA
240

pegRNA
241

pegRNA
242

pegRNA
243

pegRNA
244

pegRNA
245

pegRNA
246

pegRNA
247

pegRNA
248

pegRNA
249

pegRNA
250

pegRNA
251

pegRNA
252

pegRNA
253

pegRNA
254

pegRNA
255

pegRNA
256

pegRNA
257

pegRNA
258

pegRNA
259

pegRNA
260

pegRNA
261

pegRNA
262

pegRNA
263

pegRNA
265

pegRNA
266

pegRNA
267

pegRNA
268

pegRNA
269

pegRNA
270

pegRNA
271

pegRNA
272

pegRNA
273

pegRNA
274

pegRNA
275

pegRNA
276

pegRNA
277

pegRNA
278

pegRNA
279

pegRNA
280

pegRNA
281

pegRNA
282

pegRNA
283

pegRNA
284

pegRNA
285

pegRNA
286

pegRNA
287

pegRNA
288

pegRNA
289

ngRNA
153

ngRNA
156

ngRNA
163

ngRNA
162

ngRNA
165

ngRNA
169

ngRNA
158

ngRNA
164

ngRNA
290

ngRNA
159

ngRNA
155

ngRNA
161

ngRNA
168

ngRNA
167

ngRNA
157

ngRNA
291

ngRNA
166

ngRNA
154

ngRNA
160

ngRNA
173

ngRNA
175

ngRNA
177

ngRNA
170

ngRNA
292

ngRNA
176

ngRNA
172

ngRNA
179

ngRNA
293

ngRNA
178

ngRNA
180

ngRNA
171

ngRNA
181

ngRNA
174

TABLE 8

Sequence region
SEQ ID

pegRNA Spacer
294

PBS
295

PBS
296

PBS
297

PBS
298

PBS
299

PBS
300

PBS
301

PBS
302

PBS
303

PBS
304

PBS
305

RTT
306

RTT
307

RTT
308

RTT
309

RTT
310

RTT
311

RTT
312

RTT
313

RTT
314

RTT
315

RTT
316

RTT
317

RTT
318

RTT
319

RTT
320

RTT
321

RTT
322

RTT
323

RTT
324

RTT
325

RTT
326

RTT
327

RTT
328

RTT
329

RTT
330

RTT
331

RTT
332

RTT
333

RTT
334

RTT
335

RTT
336

ngRNA Spacer
69

ngRNA Spacer
63

ngRNA Spacer
62

ngRNA Spacer
66

ngRNA Spacer
61

ngRNA Spacer
71

ngRNA Spacer
67

ngRNA Spacer
53

ngRNA Spacer
59

ngRNA Spacer
24

ngRNA Spacer
52

ngRNA Spacer
39

ngRNA Spacer
40

ngRNA Spacer
20

ngRNA Spacer
200

ngRNA Spacer
46

ngRNA Spacer
35

ngRNA Spacer
201

ngRNA Spacer
206

ngRNA Spacer
36

ngRNA Spacer
70

ngRNA Spacer
28

ngRNA Spacer
45

ngRNA Spacer
47

ngRNA Spacer
27

ngRNA Spacer
29

ngRNA Spacer
44

ngRNA Spacer
204

ngRNA Spacer
19

ngRNA Spacer
202

ngRNA Spacer
65

ngRNA Spacer
207

ngRNA Spacer
64

ngRNA Spacer
37

ngRNA Spacer
60

ngRNA Spacer
205

ngRNA Spacer
57

ngRNA Spacer
209

ngRNA Spacer
18

ngRNA Spacer
51

ngRNA Spacer
49

ngRNA Spacer
42

ngRNA Spacer
56

ngRNA Spacer
208

ngRNA Spacer
33

ngRNA Spacer
199

ngRNA Spacer
54

ngRNA Spacer
43

ngRNA Spacer
55

ngRNA Spacer
58

ngRNA Spacer
41

ngRNA Spacer
25

ngRNA Spacer
26

ngRNA Spacer
34

ngRNA Spacer
38

ngRNA Spacer
68

ngRNA Spacer
72

ngRNA Spacer
23

ngRNA Spacer
203

ngRNA Spacer
32

ngRNA Spacer
50

ngRNA Spacer
337

ngRNA Spacer
22

ngRNA Spacer
48

ngRNA Spacer
31

ngRNA Spacer
21

pegRNA
338

pegRNA
339

pegRNA
340

pegRNA
341

pegRNA
342

pegRNA
343

pegRNA
344

pegRNA
345

pegRNA
346

pegRNA
347

pegRNA
348

pegRNA
349

pegRNA
349

pegRNA
351

pegRNA
352

pegRNA
353

pegRNA
354

pegRNA
355

pegRNA
356

pegRNA
357

pegRNA
356

pegRNA
358

pegRNA
359

pegRNA
360

pegRNA
361

pegRNA
362

pegRNA
363

pegRNA
364

pegRNA
365

pegRNA
366

pegRNA
366

pegRNA
365

pegRNA
367

pegRNA
368

pegRNA
369

pegRNA
370

pegRNA
371

pegRNA
372

pegRNA
373

pegRNA
374

pegRNA
375

pegRNA
375

pegRNA
376

pegRNA
377

pegRNA
378

pegRNA
379

pegRNA
380

pegRNA
381

pegRNA
382

pegRNA
383

pegRNA
384

pegRNA
385

pegRNA
386

pegRNA
387

pegRNA
388

pegRNA
389

pegRNA
389

pegRNA
390

pegRNA
390

pegRNA
391

pegRNA
392

pegRNA
393

pegRNA
394

pegRNA
395

pegRNA
396

pegRNA
397

pegRNA
398

pegRNA
399

pegRNA
400

pegRNA
401

pegRNA
402

pegRNA
403

pegRNA
404

pegRNA
405

pegRNA
406

pegRNA
407

pegRNA
408

pegRNA
409

pegRNA
410

pegRNA
411

pegRNA
412

pegRNA
413

pegRNA
414

pegRNA
415

pegRNA
416

pegRNA
417

pegRNA
417

pegRNA
418

pegRNA
419

pegRNA
420

pegRNA
421

pegRNA
422

pegRNA
423

pegRNA
424

pegRNA
425

pegRNA
426

pegRNA
427

pegRNA
428

pegRNA
429

pegRNA
430

pegRNA
431

pegRNA
432

pegRNA
433

pegRNA
434

pegRNA
435

pegRNA
436

pegRNA
437

pegRNA
438

pegRNA
439

pegRNA
440

pegRNA
441

pegRNA
442

pegRNA
443

pegRNA
444

pegRNA
445

pegRNA
446

pegRNA
447

pegRNA
448

pegRNA
449

pegRNA
450

pegRNA
451

pegRNA
452

pegRNA
453

pegRNA
454

pegRNA
455

pegRNA
456

pegRNA
457

pegRNA
458

pegRNA
459

pegRNA
460

pegRNA
461

pegRNA
462

pegRNA
463

pegRNA
464

pegRNA
465

pegRNA
466

pegRNA
467

pegRNA
468

pegRNA
469

pegRNA
470

pegRNA
471

pegRNA
472

pegRNA
473

pegRNA
474

pegRNA
475

pegRNA
476

pegRNA
477

pegRNA
478

pegRNA
479

pegRNA
480

pegRNA
481

pegRNA
482

ngRNA
162

ngRNA
154

ngRNA
291

ngRNA
153

ngRNA
161

ngRNA
164

ngRNA
157

ngRNA
155

ngRNA
159

ngRNA
160

ngRNA
290

ngRNA
156

ngRNA
166

ngRNA
158

ngRNA
168

ngRNA
167

ngRNA
163

ngRNA
165

ngRNA
169

ngRNA
173

ngRNA
293

ngRNA
171

ngRNA
176

ngRNA
179

ngRNA
181

ngRNA
178

ngRNA
172

ngRNA
180

ngRNA
175

ngRNA
170

ngRNA
174

ngRNA
292

ngRNA
177

TABLE 9

Sequence region
SEQ ID

pegRNA Spacer
483

PBS
484

PBS
485

PBS
486

PBS
487

PBS
488

PBS
489

PBS
490

PBS
491

PBS
492

PBS
493

PBS
494

RTT
495

RTT
496

RTT
497

RTT
498

RTT
499

RTT
500

RTT
501

RTT
502

RTT
503

RTT
504

RTT
505

RTT
506

RTT
507

RTT
508

RTT
509

RTT
510

RTT
511

RTT
512

RTT
513

RTT
514

RTT
515

RTT
516

RTT
517

RTT
518

RTT
519

RTT
520

RTT
521

RTT
522

RTT
523

RTT
524

RTT
525

RTT
526

RTT
527

RTT
528

ngRNA Spacer
68

ngRNA Spacer
66

ngRNA Spacer
61

ngRNA Spacer
71

ngRNA Spacer
67

ngRNA Spacer
46

ngRNA Spacer
38

ngRNA Spacer
58

ngRNA Spacer
36

ngRNA Spacer
30

ngRNA Spacer
37

ngRNA Spacer
29

ngRNA Spacer
203

ngRNA Spacer
54

ngRNA Spacer
59

ngRNA Spacer
24

ngRNA Spacer
31

ngRNA Spacer
53

ngRNA Spacer
23

ngRNA Spacer
72

ngRNA Spacer
44

ngRNA Spacer
20

ngRNA Spacer
26

ngRNA Spacer
22

ngRNA Spacer
40

ngRNA Spacer
21

ngRNA Spacer
60

ngRNA Spacer
62

ngRNA Spacer
63

ngRNA Spacer
19

ngRNA Spacer
65

ngRNA Spacer
42

ngRNA Spacer
64

ngRNA Spacer
28

ngRNA Spacer
25

ngRNA Spacer
18

ngRNA Spacer
32

ngRNA Spacer
34

ngRNA Spacer
50

ngRNA Spacer
33

ngRNA Spacer
48

ngRNA Spacer
39

ngRNA Spacer
27

ngRNA Spacer
41

ngRNA Spacer
529

ngRNA Spacer
199

ngRNA Spacer
43

ngRNA Spacer
70

ngRNA Spacer
45

ngRNA Spacer
35

ngRNA Spacer
200

ngRNA Spacer
52

ngRNA Spacer
56

ngRNA Spacer
55

ngRNA Spacer
51

ngRNA Spacer
57

ngRNA Spacer
49

ngRNA Spacer
47

ngRNA Spacer
69

pegRNA
530

pegRNA
531

pegRNA
532

pegRNA
533

pegRNA
534

pegRNA
535

pegRNA
536

pegRNA
537

pegRNA
538

pegRNA
539

pegRNA
540

pegRNA
541

pegRNA
542

pegRNA
543

pegRNA
544

pegRNA
545

pegRNA
546

pegRNA
547

pegRNA
545

pegRNA
548

pegRNA
549

pegRNA
550

pegRNA
551

pegRNA
552

pegRNA
553

pegRNA
554

pegRNA
555

pegRNA
556

pegRNA
557

pegRNA
558

pegRNA
553

pegRNA
559

pegRNA
560

pegRNA
561

pegRNA
562

pegRNA
563

pegRNA
564

pegRNA
564

pegRNA
565

pegRNA
566

pegRNA
567

pegRNA
568

pegRNA
569

pegRNA
570

pegRNA
571

pegRNA
572

pegRNA
573

pegRNA
574

pegRNA
575

pegRNA
576

pegRNA
577

pegRNA
578

pegRNA
579

pegRNA
580

pegRNA
581

pegRNA
582

pegRNA
578

pegRNA
583

pegRNA
584

pegRNA
585

pegRNA
586

pegRNA
587

pegRNA
588

pegRNA
589

pegRNA
590

pegRNA
591

pegRNA
592

pegRNA
593

pegRNA
594

pegRNA
595

pegRNA
596

pegRNA
597

pegRNA
598

pegRNA
599

pegRNA
600

pegRNA
601

pegRNA
602

pegRNA
603

pegRNA
604

pegRNA
605

pegRNA
606

pegRNA
607

pegRNA
608

pegRNA
609

pegRNA
610

pegRNA
611

pegRNA
612

pegRNA
613

pegRNA
614

pegRNA
615

pegRNA
616

pegRNA
617

pegRNA
618

pegRNA
619

pegRNA
620

pegRNA
621

pegRNA
622

pegRNA
623

pegRNA
624

pegRNA
625

pegRNA
626

pegRNA
627

pegRNA
628

pegRNA
629

pegRNA
630

pegRNA
631

pegRNA
632

pegRNA
633

pegRNA
634

pegRNA
635

pegRNA
636

pegRNA
637

pegRNA
638

pegRNA
639

pegRNA
640

pegRNA
641

pegRNA
642

pegRNA
643

pegRNA
644

pegRNA
645

pegRNA
646

pegRNA
647

pegRNA
648

pegRNA
649

pegRNA
650

pegRNA
651

pegRNA
652

pegRNA
653

pegRNA
654

pegRNA
655

pegRNA
656

pegRNA
657

pegRNA
658

pegRNA
659

pegRNA
660

pegRNA
661

pegRNA
662

pegRNA
663

pegRNA
664

pegRNA
665

pegRNA
666

pegRNA
667

pegRNA
668

pegRNA
669

pegRNA
670

pegRNA
671

pegRNA
672

pegRNA
673

pegRNA
674

pegRNA
675

pegRNA
676

pegRNA
677

pegRNA
678

pegRNA
679

pegRNA
680

ngRNA
161

ngRNA
154

ngRNA
166

ngRNA
155

ngRNA
162

ngRNA
159

ngRNA
168

ngRNA
157

ngRNA
160

ngRNA
291

ngRNA
169

ngRNA
153

ngRNA
290

ngRNA
167

ngRNA
165

ngRNA
158

ngRNA
681

ngRNA
156

ngRNA
163

ngRNA
164

ngRNA
293

ngRNA
180

ngRNA
173

ngRNA
176

ngRNA
181

ngRNA
178

ngRNA
292

ngRNA
175

ngRNA
177

ngRNA
172

ngRNA
171

ngRNA
170

ngRNA
174

ngRNA
179

TABLE 10

Sequence region
SEQ ID

pegRNA Spacer
682

PBS
683

PBS
684

PBS
685

PBS
686

PBS
687

PBS
688

PBS
689

PBS
690

PBS
691

PBS
692

PBS
693

RTT
694

RTT
695

RTT
696

RTT
697

RTT
698

RTT
699

RTT
700

RTT
701

RTT
702

RTT
703

RTT
704

RTT
705

RTT
706

RTT
707

RTT
708

RTT
709

RTT
710

RTT
711

RTT
712

RTT
713

RTT
714

RTT
715

RTT
716

RTT
717

RTT
718

RTT
719

RTT
720

RTT
721

RTT
722

RTT
723

RTT
724

RTT
725

RTT
726

RTT
727

RTT
728

RTT
729

RTT
730

RTT
731

RTT
732

RTT
733

RTT
734

RTT
735

ngRNA Spacer
67

ngRNA Spacer
72

ngRNA Spacer
34

ngRNA Spacer
50

ngRNA Spacer
27

ngRNA Spacer
36

ngRNA Spacer
31

ngRNA Spacer
53

ngRNA Spacer
57

ngRNA Spacer
22

ngRNA Spacer
49

ngRNA Spacer
43

ngRNA Spacer
54

ngRNA Spacer
59

ngRNA Spacer
56

ngRNA Spacer
70

ngRNA Spacer
55

ngRNA Spacer
44

ngRNA Spacer
30

ngRNA Spacer
200

ngRNA Spacer
203

ngRNA Spacer
52

ngRNA Spacer
41

ngRNA Spacer
51

ngRNA Spacer
736

ngRNA Spacer
737

ngRNA Spacer
32

ngRNA Spacer
738

ngRNA Spacer
45

ngRNA Spacer
60

ngRNA Spacer
199

ngRNA Spacer
37

ngRNA Spacer
18

ngRNA Spacer
23

ngRNA Spacer
42

ngRNA Spacer
21

ngRNA Spacer
38

ngRNA Spacer
58

ngRNA Spacer
33

ngRNA Spacer
68

ngRNA Spacer
47

ngRNA Spacer
40

ngRNA Spacer
529

ngRNA Spacer
739

ngRNA Spacer
46

ngRNA Spacer
19

ngRNA Spacer
20

ngRNA Spacer
26

ngRNA Spacer
48

ngRNA Spacer
24

ngRNA Spacer
35

ngRNA Spacer
28

ngRNA Spacer
61

ngRNA Spacer
66

ngRNA Spacer
71

ngRNA Spacer
63

ngRNA Spacer
64

ngRNA Spacer
62

ngRNA Spacer
69

ngRNA Spacer
740

ngRNA Spacer
39

ngRNA Spacer
29

ngRNA Spacer
25

ngRNA Spacer
65

pegRNA
741

pegRNA
742

pegRNA
743

pegRNA
744

pegRNA
745

pegRNA
746

pegRNA
747

pegRNA
748

pegRNA
749

pegRNA
750

pegRNA
751

pegRNA
752

pegRNA
753

pegRNA
754

pegRNA
755

pegRNA
756

pegRNA
757

pegRNA
758

pegRNA
760

pegRNA
761

pegRNA
762

pegRNA
763

pegRNA
764

pegRNA
765

pegRNA
766

pegRNA
767

pegRNA
768

pegRNA
769

pegRNA
770

pegRNA
771

pegRNA
772

pegRNA
773

pegRNA
774

pegRNA
775

pegRNA
776

pegRNA
777

pegRNA
778

pegRNA
779

pegRNA
780

pegRNA
781

pegRNA
782

pegRNA
783

pegRNA
784

pegRNA
785

pegRNA
786

pegRNA
787

pegRNA
788

pegRNA
789

pegRNA
790

pegRNA
791

pegRNA
792

pegRNA
793

pegRNA
794

pegRNA
795

pegRNA
796

pegRNA
797

pegRNA
798

pegRNA
799

pegRNA
800

pegRNA
801

pegRNA
802

pegRNA
803

pegRNA
804

pegRNA
806

pegRNA
807

pegRNA
808

pegRNA
809

pegRNA
810

pegRNA
811

pegRNA
812

pegRNA
813

pegRNA
814

pegRNA
815

pegRNA
816

pegRNA
817

pegRNA
818

pegRNA
819

pegRNA
820

pegRNA
821

pegRNA
822

pegRNA
823

pegRNA
824

pegRNA
825

pegRNA
826

pegRNA
827

pegRNA
828

pegRNA
829

pegRNA
830

pegRNA
831

pegRNA
832

pegRNA
833

pegRNA
834

pegRNA
835

pegRNA
836

pegRNA
837

pegRNA
838

pegRNA
839

pegRNA
840

pegRNA
841

pegRNA
842

pegRNA
843

pegRNA
844

pegRNA
845

pegRNA
846

pegRNA
847

pegRNA
848

pegRNA
849

pegRNA
850

pegRNA
851

pegRNA
852

pegRNA
853

pegRNA
854

pegRNA
855

pegRNA
856

pegRNA
857

pegRNA
858

pegRNA
859

pegRNA
860

pegRNA
861

pegRNA
862

pegRNA
863

pegRNA
864

pegRNA
865

pegRNA
866

pegRNA
867

pegRNA
868

pegRNA
869

pegRNA
870

pegRNA
871

pegRNA
872

pegRNA
873

pegRNA
874

pegRNA
875

pegRNA
876

pegRNA
877

pegRNA
878

pegRNA
879

pegRNA
880

pegRNA
881

pegRNA
882

pegRNA
883

pegRNA
884

pegRNA
885

pegRNA
886

pegRNA
887

pegRNA
888

pegRNA
889

pegRNA
890

pegRNA
891

pegRNA
892

pegRNA
893

pegRNA
894

pegRNA
895

pegRNA
896

pegRNA
897

pegRNA
898

pegRNA
899

pegRNA
900

pegRNA
901

pegRNA
902

pegRNA
903

pegRNA
904

pegRNA
905

pegRNA
906

pegRNA
907

pegRNA
908

pegRNA
909

pegRNA
910

pegRNA
911

pegRNA
912

pegRNA
913

pegRNA
914

pegRNA
915

pegRNA
916

pegRNA
917

pegRNA
918

pegRNA
919

pegRNA
920

pegRNA
921

pegRNA
922

pegRNA
923

pegRNA
924

pegRNA
925

pegRNA
926

pegRNA
927

pegRNA
928

pegRNA
929

pegRNA
930

pegRNA
931

pegRNA
932

pegRNA
933

pegRNA
934

pegRNA
935

pegRNA
936

pegRNA
937

pegRNA
938

pegRNA
939

pegRNA
940

pegRNA
941

pegRNA
942

pegRNA
943

pegRNA
944

pegRNA
945

pegRNA
946

pegRNA
947

pegRNA
948

pegRNA
949

pegRNA
950

pegRNA
951

pegRNA
952

pegRNA
953

pegRNA
954

pegRNA
955

pegRNA
956

pegRNA
957

pegRNA
958

pegRNA
959

pegRNA
960

pegRNA
961

pegRNA
962

pegRNA
963

pegRNA
964

pegRNA
965

pegRNA
966

pegRNA
967

pegRNA
968

pegRNA
969

pegRNA
970

pegRNA
971

pegRNA
972

pegRNA
973

pegRNA
974

pegRNA
975

pegRNA
976

pegRNA
977

pegRNA
978

pegRNA
979

pegRNA
980

pegRNA
981

pegRNA
982

pegRNA
983

pegRNA
984

pegRNA
985

pegRNA
986

pegRNA
987

pegRNA
988

pegRNA
989

pegRNA
990

pegRNA
991

pegRNA
992

pegRNA
993

pegRNA
994

pegRNA
995

pegRNA
996

pegRNA
997

pegRNA
998

pegRNA
999

pegRNA
1000

pegRNA
1001

pegRNA
1002

pegRNA
1003

pegRNA
1004

pegRNA
1005

pegRNA
1006

pegRNA
1007

pegRNA
1008

pegRNA
1009

pegRNA
1010

pegRNA
1011

pegRNA
1012

pegRNA
1013

pegRNA
1014

pegRNA
1015

pegRNA
1016

pegRNA
1017

pegRNA
1018

pegRNA
1019

pegRNA
1020

pegRNA
1021

pegRNA
1022

pegRNA
1023

pegRNA
1024

pegRNA
1025

pegRNA
1026

pegRNA
1027

pegRNA
1028

pegRNA
1029

pegRNA
1030

pegRNA
1031

pegRNA
1032

pegRNA
1033

pegRNA
1034

pegRNA
1035

pegRNA
1036

pegRNA
1037

pegRNA
1038

pegRNA
1039

pegRNA
1040

pegRNA
1041

pegRNA
1042

pegRNA
1043

pegRNA
1044

pegRNA
1045

pegRNA
1046

pegRNA
1047

pegRNA
1048

pegRNA
1049

pegRNA
1050

pegRNA
1051

pegRNA
1052

pegRNA
1053

pegRNA
1054

pegRNA
1055

pegRNA
1056

pegRNA
1057

pegRNA
1058

pegRNA
1059

pegRNA
1060

pegRNA
1061

pegRNA
1062

pegRNA
1063

pegRNA
1064

pegRNA
1065

pegRNA
1066

pegRNA
1067

pegRNA
1068

pegRNA
1069

pegRNA
1070

pegRNA
1071

pegRNA
1072

pegRNA
1073

pegRNA
1074

pegRNA
1075

pegRNA
1076

pegRNA
1077

pegRNA
1078

pegRNA
1079

pegRNA
1080

pegRNA
1081

pegRNA
1082

pegRNA
1083

pegRNA
1084

pegRNA
1085

pegRNA
1086

pegRNA
1087

pegRNA
1088

pegRNA
1089

pegRNA
1090

pegRNA
1091

pegRNA
1092

pegRNA
1093

pegRNA
1094

pegRNA
1095

pegRNA
1096

pegRNA
1097

pegRNA
1098

pegRNA
1099

pegRNA
1100

pegRNA
1101

pegRNA
1102

pegRNA
1103

pegRNA
1104

pegRNA
1105

pegRNA
1106

pegRNA
1107

pegRNA
1108

pegRNA
1109

pegRNA
1110

pegRNA
1111

pegRNA
1112

pegRNA
1113

pegRNA
1114

pegRNA
1115

pegRNA
1116

pegRNA
1117

pegRNA
1118

pegRNA
1119

pegRNA
1120

pegRNA
1121

pegRNA
1122

pegRNA
1123

pegRNA
1124

pegRNA
1125

pegRNA
1126

pegRNA
1127

pegRNA
1128

pegRNA
1129

pegRNA
1130

pegRNA
1131

pegRNA
1132

pegRNA
1133

pegRNA
1134

pegRNA
1135

pegRNA
1136

pegRNA
1137

pegRNA
1138

pegRNA
1139

pegRNA
1140

pegRNA
1141

pegRNA
1142

pegRNA
1143

pegRNA
1144

pegRNA
1145

pegRNA
1146

pegRNA
1147

pegRNA
1148

pegRNA
1149

pegRNA
1150

pegRNA
1151

pegRNA
1152

pegRNA
1153

pegRNA
1154

pegRNA
1155

pegRNA
1156

pegRNA
1157

pegRNA
1158

pegRNA
1159

pegRNA
1160

pegRNA
1161

pegRNA
1162

pegRNA
1163

pegRNA
1164

pegRNA
1165

pegRNA
1166

pegRNA
1167

pegRNA
1168

pegRNA
1169

pegRNA
1170

pegRNA
1171

pegRNA
1172

pegRNA
1173

pegRNA
1174

pegRNA
1175

pegRNA
1176

pegRNA
1177

pegRNA
1178

pegRNA
1179

pegRNA
1180

pegRNA
1181

pegRNA
1182

pegRNA
1183

pegRNA
1184

pegRNA
1185

pegRNA
1186

pegRNA
1187

pegRNA
1188

pegRNA
1189

pegRNA
1190

pegRNA
1191

pegRNA
1192

pegRNA
1193

pegRNA
1194

pegRNA
1195

pegRNA
1196

pegRNA
1197

pegRNA
1198

pegRNA
1199

pegRNA
1200

pegRNA
1201

pegRNA
1202

pegRNA
1203

pegRNA
1204

pegRNA
1205

pegRNA
1206

pegRNA
1207

pegRNA
1208

pegRNA
1209

pegRNA
1210

pegRNA
1211

pegRNA
1212

pegRNA
1213

pegRNA
1214

pegRNA
1215

pegRNA
1216

pegRNA
1217

pegRNA
1218

pegRNA
1219

pegRNA
1220

pegRNA
1221

pegRNA
1222

pegRNA
1223

pegRNA
1224

pegRNA
1225

pegRNA
1226

pegRNA
1227

pegRNA
1228

pegRNA
1229

pegRNA
1230

pegRNA
1231

pegRNA
1232

pegRNA
1233

pegRNA
1234

pegRNA
1235

pegRNA
1236

pegRNA
1237

pegRNA
1238

pegRNA
1239

pegRNA
1240

pegRNA
1241

pegRNA
1242

pegRNA
1243

pegRNA
1244

pegRNA
1245

pegRNA
1246

pegRNA
1247

pegRNA
1248

pegRNA
1249

pegRNA
1250

pegRNA
1251

pegRNA
1252

pegRNA
1253

pegRNA
1254

pegRNA
1255

pegRNA
1256

pegRNA
1257

pegRNA
1258

pegRNA
1259

pegRNA
1260

pegRNA
1261

pegRNA
1262

pegRNA
1263

pegRNA
1264

pegRNA
1265

pegRNA
1266

pegRNA
1267

pegRNA
1268

pegRNA
1269

pegRNA
1270

pegRNA
1271

pegRNA
1272

pegRNA
1273

pegRNA
1274

pegRNA
1275

pegRNA
1276

pegRNA
1277

pegRNA
1278

pegRNA
1279

pegRNA
1280

pegRNA
1281

pegRNA
1282

pegRNA
1283

pegRNA
1284

pegRNA
1285

pegRNA
1286

pegRNA
1287

pegRNA
1288

pegRNA
1289

pegRNA
1290

pegRNA
1291

pegRNA
1292

pegRNA
1293

pegRNA
1294

pegRNA
1295

pegRNA
1296

pegRNA
1297

pegRNA
1298

pegRNA
1299

pegRNA
1300

pegRNA
1301

pegRNA
1302

pegRNA
1303

pegRNA
1304

pegRNA
1305

pegRNA
1306

pegRNA
1307

pegRNA
1308

pegRNA
1309

pegRNA
1310

pegRNA
1311

pegRNA
1312

pegRNA
1313

pegRNA
1314

pegRNA
1315

pegRNA
1316

pegRNA
1317

pegRNA
1318

pegRNA
1319

pegRNA
1320

pegRNA
1321

pegRNA
1322

pegRNA
1323

pegRNA
1324

pegRNA
1325

pegRNA
1326

pegRNA
1327

pegRNA
1328

pegRNA
1329

pegRNA
1330

pegRNA
1331

pegRNA
1332

pegRNA
1333

pegRNA
1334

pegRNA
1335

pegRNA
1336

pegRNA
1337

pegRNA
1338

pegRNA
1339

pegRNA
1340

pegRNA
1341

pegRNA
1342

pegRNA
1343

pegRNA
1344

pegRNA
1345

pegRNA
1346

pegRNA
1347

pegRNA
1348

pegRNA
1349

pegRNA
1350

pegRNA
1351

pegRNA
1352

pegRNA
1353

pegRNA
1354

pegRNA
1355

pegRNA
1356

pegRNA
1357

pegRNA
1358

pegRNA
1359

pegRNA
1360

pegRNA
1361

pegRNA
1362

pegRNA
1363

pegRNA
1364

pegRNA
1365

pegRNA
1366

pegRNA
1367

pegRNA
1368

pegRNA
1369

pegRNA
1370

pegRNA
1371

pegRNA
1372

pegRNA
1373

pegRNA
1374

pegRNA
1375

pegRNA
1376

pegRNA
1377

pegRNA
1378

pegRNA
1379

pegRNA
1380

pegRNA
1381

pegRNA
1382

pegRNA
1383

pegRNA
1384

pegRNA
1385

pegRNA
1386

pegRNA
1387

pegRNA
1388

pegRNA
1389

pegRNA
1390

pegRNA
1391

pegRNA
1392

pegRNA
1393

pegRNA
1394

pegRNA
1395

pegRNA
1396

pegRNA
1397

pegRNA
1398

pegRNA
1399

pegRNA
1400

pegRNA
1401

pegRNA
1402

pegRNA
1403

pegRNA
1404

pegRNA
1405

pegRNA
1406

pegRNA
1407

pegRNA
1408

pegRNA
1409

pegRNA
1410

pegRNA
1411

pegRNA
1412

pegRNA
1413

pegRNA
1414

pegRNA
1415

pegRNA
1416

pegRNA
1417

pegRNA
1418

pegRNA
1419

pegRNA
1420

pegRNA
1421

pegRNA
1422

pegRNA
1423

pegRNA
1424

pegRNA
1425

pegRNA
1426

pegRNA
1427

pegRNA
1428

pegRNA
1429

pegRNA
1430

pegRNA
1431

pegRNA
1432

pegRNA
1433

pegRNA
1434

pegRNA
1435

pegRNA
1436

pegRNA
1437

pegRNA
1438

pegRNA
1439

pegRNA
1440

pegRNA
1441

pegRNA
1442

pegRNA
1443

pegRNA
1444

pegRNA
1445

pegRNA
1446

pegRNA
1447

pegRNA
1448

pegRNA
1450

pegRNA
1451

pegRNA
1452

pegRNA
1453

pegRNA
1454

pegRNA
1455

pegRNA
1456

pegRNA
1457

pegRNA
1458

pegRNA
1459

pegRNA
1460

pegRNA
1461

pegRNA
1462

pegRNA
1463

pegRNA
1464

pegRNA
1465

pegRNA
1466

pegRNA
1467

pegRNA
1468

pegRNA
1469

pegRNA
1470

pegRNA
1471

pegRNA
1472

pegRNA
1473

pegRNA
1474

pegRNA
1475

pegRNA
1476

pegRNA
1477

pegRNA
1478

pegRNA
1479

pegRNA
1480

pegRNA
1481

pegRNA
1482

pegRNA
1483

pegRNA
1484

pegRNA
1485

pegRNA
1486

pegRNA
1487

pegRNA
1488

pegRNA
1489

pegRNA
1490

pegRNA
1491

pegRNA
1492

pegRNA
1493

pegRNA
1494

pegRNA
1495

pegRNA
1496

pegRNA
1497

pegRNA
1498

pegRNA
1499

pegRNA
1500

ngRNA
291

ngRNA
164

ngRNA
681

ngRNA
1501

ngRNA
166

ngRNA
158

ngRNA
161

ngRNA
157

ngRNA
154

ngRNA
153

ngRNA
167

ngRNA
168

ngRNA
169

ngRNA
155

ngRNA
1502

ngRNA
160

ngRNA
1503

ngRNA
156

ngRNA
159

ngRNA
162

ngRNA
165

ngRNA
163

ngRNA
290

ngRNA
1504

ngRNA
177

ngRNA
178

ngRNA
171

ngRNA
175

ngRNA
173

ngRNA
180

ngRNA
293

ngRNA
176

ngRNA
181

ngRNA
292

ngRNA
174

ngRNA
170

ngRNA
172

ngRNA
179

TABLE 11

Sequence region
SEQ ID

pegRNA Spacer
1505

PBS
1506

PBS
1507

PBS
1508

PBS
1509

PBS
1510

PBS
1511

PBS
1512

PBS
1513

PBS
1514

PBS
1515

PBS
1516

RTT
1517

RTT
1518

RTT
1519

RTT
1520

RTT
1521

RTT
1522

RTT
1523

RTT
1524

RTT
1525

RTT
1526

RTT
1527

RTT
1528

RTT
1529

RTT
1530

RTT
1531

RTT
1532

RTT
1533

RTT
1534

RTT
1535

RTT
1536

RTT
1537

RTT
1538

RTT
1539

RTT
1540

RTT
1541

RTT
1542

RTT
1543

RTT
1544

RTT
1545

RTT
1546

ngRNA Spacer
65

ngRNA Spacer
63

ngRNA Spacer
62

ngRNA Spacer
66

ngRNA Spacer
61

ngRNA Spacer
71

ngRNA Spacer
53

ngRNA Spacer
37

ngRNA Spacer
44

ngRNA Spacer
529

ngRNA Spacer
42

ngRNA Spacer
58

ngRNA Spacer
50

ngRNA Spacer
31

ngRNA Spacer
19

ngRNA Spacer
737

ngRNA Spacer
738

ngRNA Spacer
36

ngRNA Spacer
739

ngRNA Spacer
34

ngRNA Spacer
203

ngRNA Spacer
49

ngRNA Spacer
54

ngRNA Spacer
52

ngRNA Spacer
18

ngRNA Spacer
43

ngRNA Spacer
57

ngRNA Spacer
56

ngRNA Spacer
64

ngRNA Spacer
24

ngRNA Spacer
23

ngRNA Spacer
72

ngRNA Spacer
51

ngRNA Spacer
70

ngRNA Spacer
48

ngRNA Spacer
60

ngRNA Spacer
21

ngRNA Spacer
20

ngRNA Spacer
25

ngRNA Spacer
40

ngRNA Spacer
22

ngRNA Spacer
38

ngRNA Spacer
59

ngRNA Spacer
39

ngRNA Spacer
46

ngRNA Spacer
26

ngRNA Spacer
29

ngRNA Spacer
67

ngRNA Spacer
28

ngRNA Spacer
69

ngRNA Spacer
30

ngRNA Spacer
41

ngRNA Spacer
740

ngRNA Spacer
68

ngRNA Spacer
32

ngRNA Spacer
35

ngRNA Spacer
736

ngRNA Spacer
47

ngRNA Spacer
199

ngRNA Spacer
33

ngRNA Spacer
55

ngRNA Spacer
45

ngRNA Spacer
27

ngRNA Spacer
200

pegRNA
1547

pegRNA
1548

pegRNA
1549

pegRNA
1550

pegRNA
1551

pegRNA
1552

pegRNA
1553

pegRNA
1554

pegRNA
1555

pegRNA
1556

pegRNA
1557

pegRNA
1558

pegRNA
1559

pegRNA
1560

pegRNA
1561

pegRNA
1562

pegRNA
1563

pegRNA
1564

pegRNA
1565

pegRNA
1566

pegRNA
1567

pegRNA
1568

pegRNA
1569

pegRNA
1570

pegRNA
1571

pegRNA
1572

pegRNA
1573

pegRNA
1574

pegRNA
1575

pegRNA
1576

pegRNA
1577

pegRNA
1578

pegRNA
1579

pegRNA
1580

pegRNA
1581

pegRNA
1582

pegRNA
1583

pegRNA
1584

pegRNA
1585

pegRNA
1586

pegRNA
1587

pegRNA
1588

pegRNA
1589

pegRNA
1590

pegRNA
1591

pegRNA
1592

pegRNA
1593

pegRNA
1594

pegRNA
1595

pegRNA
1596

pegRNA
1597

pegRNA
1598

pegRNA
1599

pegRNA
1600

pegRNA
1601

pegRNA
1602

pegRNA
1603

pegRNA
1604

pegRNA
1605

pegRNA
1606

pegRNA
1607

pegRNA
1608

pegRNA
1609

pegRNA
1610

pegRNA
1611

pegRNA
1612

pegRNA
1613

pegRNA
1614

pegRNA
1615

pegRNA
1616

pegRNA
1617

pegRNA
1618

pegRNA
1619

pegRNA
1620

pegRNA
1621

pegRNA
1622

pegRNA
1623

pegRNA
1624

pegRNA
1625

pegRNA
1626

pegRNA
1627

pegRNA
1628

pegRNA
1629

pegRNA
1630

pegRNA
1631

pegRNA
1632

pegRNA
1633

pegRNA
1634

pegRNA
1635

pegRNA
1636

pegRNA
1637

pegRNA
1638

pegRNA
1639

pegRNA
1640

pegRNA
1641

pegRNA
1642

pegRNA
1643

pegRNA
1644

pegRNA
1645

pegRNA
1646

pegRNA
1647

pegRNA
1648

pegRNA
1649

pegRNA
1650

pegRNA
1651

pegRNA
1652

pegRNA
1653

pegRNA
1654

pegRNA
1655

pegRNA
1656

pegRNA
1657

pegRNA
1658

pegRNA
1659

pegRNA
1660

pegRNA
1661

pegRNA
1662

pegRNA
1663

pegRNA
1664

pegRNA
1665

pegRNA
1666

pegRNA
1667

pegRNA
1668

pegRNA
1669

pegRNA
1670

pegRNA
1671

pegRNA
1672

pegRNA
1673

pegRNA
1674

pegRNA
1675

pegRNA
1676

pegRNA
1677

pegRNA
1678

pegRNA
1679

pegRNA
1680

pegRNA
1681

pegRNA
1682

pegRNA
1683

pegRNA
1684

pegRNA
1685

pegRNA
1686

pegRNA
1687

pegRNA
1688

pegRNA
1689

pegRNA
1690

pegRNA
1691

pegRNA
1692

pegRNA
1693

pegRNA
1694

pegRNA
1695

pegRNA
1696

pegRNA
1697

pegRNA
1698

pegRNA
1699

pegRNA
1700

pegRNA
1701

pegRNA
1702

pegRNA
1703

pegRNA
1704

pegRNA
1705

pegRNA
1706

pegRNA
1707

pegRNA
1708

pegRNA
1709

pegRNA
1710

pegRNA
1711

pegRNA
1712

pegRNA
1713

pegRNA
1714

pegRNA
1715

pegRNA
1716

pegRNA
1717

pegRNA
1718

pegRNA
1719

pegRNA
1720

pegRNA
1721

pegRNA
1722

pegRNA
1723

pegRNA
1724

pegRNA
1725

pegRNA
1726

pegRNA
1727

pegRNA
1728

pegRNA
1729

pegRNA
1730

pegRNA
1731

pegRNA
1732

pegRNA
1733

pegRNA
1734

pegRNA
1735

pegRNA
1736

pegRNA
1737

pegRNA
1738

pegRNA
1739

pegRNA
1740

pegRNA
1741

pegRNA
1742

pegRNA
1743

pegRNA
1744

pegRNA
1745

pegRNA
1746

pegRNA
1747

pegRNA
1748

pegRNA
1749

pegRNA
1750

pegRNA
1751

pegRNA
1752

pegRNA
1753

pegRNA
1754

pegRNA
1755

pegRNA
1756

pegRNA
1757

pegRNA
1758

pegRNA
1759

pegRNA
1760

pegRNA
1761

pegRNA
1762

pegRNA
1763

pegRNA
1764

pegRNA
1765

pegRNA
1766

pegRNA
1767

pegRNA
1768

pegRNA
1769

pegRNA
1770

pegRNA
1771

pegRNA
1772

pegRNA
1773

pegRNA
1774

pegRNA
1775

pegRNA
1776

pegRNA
1777

pegRNA
1778

pegRNA
1779

pegRNA
1780

pegRNA
1781

pegRNA
1782

pegRNA
1783

pegRNA
1784

pegRNA
1785

pegRNA
1786

pegRNA
1787

pegRNA
1788

pegRNA
1789

pegRNA
1790

pegRNA
1791

pegRNA
1792

pegRNA
1793

pegRNA
1794

pegRNA
1795

pegRNA
1796

pegRNA
1797

pegRNA
1798

pegRNA
1799

pegRNA
1800

pegRNA
1801

pegRNA
1802

pegRNA
1803

pegRNA
1804

pegRNA
1805

pegRNA
1806

pegRNA
1807

pegRNA
1808

pegRNA
1809

pegRNA
1810

pegRNA
1811

pegRNA
1812

pegRNA
1813

pegRNA
1814

pegRNA
1815

pegRNA
1816

pegRNA
1817

pegRNA
1818

pegRNA
1819

pegRNA
1820

pegRNA
1821

pegRNA
1822

pegRNA
1823

pegRNA
1824

pegRNA
1825

pegRNA
1826

pegRNA
1827

pegRNA
1828

pegRNA
1829

pegRNA
1830

pegRNA
1831

pegRNA
1832

pegRNA
1833

pegRNA
1834

pegRNA
1835

pegRNA
1836

pegRNA
1837

pegRNA
1838

pegRNA
1839

pegRNA
1840

pegRNA
1841

pegRNA
1842

pegRNA
1843

pegRNA
1844

pegRNA
1845

pegRNA
1846

pegRNA
1847

pegRNA
1848

pegRNA
1849

pegRNA
1850

pegRNA
1851

pegRNA
1852

pegRNA
1853

pegRNA
1854

pegRNA
1855

pegRNA
1856

pegRNA
1857

pegRNA
1858

pegRNA
1859

pegRNA
1860

pegRNA
1861

pegRNA
1862

pegRNA
1863

pegRNA
1864

pegRNA
1865

pegRNA
1866

pegRNA
1867

pegRNA
1868

pegRNA
1869

pegRNA
1870

pegRNA
1871

pegRNA
1872

pegRNA
1873

pegRNA
1874

pegRNA
1875

pegRNA
1876

pegRNA
1877

pegRNA
1878

pegRNA
1879

pegRNA
1880

pegRNA
1881

pegRNA
1882

pegRNA
1883

pegRNA
1884

pegRNA
1885

pegRNA
1886

pegRNA
1887

pegRNA
1888

pegRNA
1889

pegRNA
1890

pegRNA
1891

pegRNA
1892

pegRNA
1893

pegRNA
1894

pegRNA
1895

pegRNA
1896

pegRNA
1897

pegRNA
1898

pegRNA
1899

pegRNA
1900

pegRNA
1901

pegRNA
1902

pegRNA
1903

pegRNA
1904

pegRNA
1905

pegRNA
1906

pegRNA
1907

pegRNA
1908

pegRNA
1909

pegRNA
1910

pegRNA
1911

pegRNA
1912

pegRNA
1913

pegRNA
1914

pegRNA
1915

pegRNA
1916

pegRNA
1917

pegRNA
1918

pegRNA
1919

pegRNA
1920

pegRNA
1921

pegRNA
1922

pegRNA
1923

pegRNA
1924

pegRNA
1925

pegRNA
1926

pegRNA
1927

pegRNA
1928

pegRNA
1929

pegRNA
1930

pegRNA
1931

pegRNA
1932

pegRNA
1933

pegRNA
1934

pegRNA
1935

pegRNA
1936

pegRNA
1937

pegRNA
1938

pegRNA
1939

pegRNA
1940

pegRNA
1941

pegRNA
1942

pegRNA
1943

pegRNA
1944

pegRNA
1945

pegRNA
1946

pegRNA
1947

pegRNA
1948

pegRNA
1949

pegRNA
1950

pegRNA
1951

pegRNA
1952

pegRNA
1953

pegRNA
1954

pegRNA
1955

pegRNA
1956

pegRNA
1957

pegRNA
1958

pegRNA
1959

pegRNA
1960

pegRNA
1961

pegRNA
1962

pegRNA
1963

pegRNA
1964

pegRNA
1965

pegRNA
1966

pegRNA
1967

pegRNA
1968

pegRNA
1969

pegRNA
1970

pegRNA
1971

pegRNA
1972

pegRNA
1973

pegRNA
1974

pegRNA
1975

pegRNA
1976

pegRNA
1977

pegRNA
1978

pegRNA
1979

pegRNA
1980

pegRNA
1981

pegRNA
1982

pegRNA
1983

pegRNA
1984

pegRNA
1985

pegRNA
1986

pegRNA
1987

pegRNA
1988

pegRNA
1989

pegRNA
1990

pegRNA
1991

pegRNA
1992

pegRNA
1993

pegRNA
1994

pegRNA
1995

pegRNA
1996

pegRNA
1997

pegRNA
1998

pegRNA
1999

pegRNA
2000

pegRNA
2001

pegRNA
2002

pegRNA
2003

pegRNA
2004

pegRNA
2005

pegRNA
2006

pegRNA
2007

pegRNA
2008

pegRNA
2009

pegRNA
2010

pegRNA
2011

pegRNA
2012

pegRNA
2013

pegRNA
2014

pegRNA
2015

pegRNA
2016

pegRNA
2017

pegRNA
2018

pegRNA
2019

pegRNA
2020

pegRNA
2021

pegRNA
2022

ngRNA
169

ngRNA
153

ngRNA
291

ngRNA
1503

ngRNA
159

ngRNA
155

ngRNA
1502

ngRNA
160

ngRNA
681

ngRNA
158

ngRNA
161

ngRNA
168

ngRNA
157

ngRNA
166

ngRNA
167

ngRNA
164

ngRNA
162

ngRNA
154

ngRNA
165

ngRNA
290

ngRNA
163

ngRNA
156

ngRNA
1501

ngRNA
181

ngRNA
173

ngRNA
170

ngRNA
175

ngRNA
172

ngRNA
176

ngRNA
292

ngRNA
171

ngRNA
180

ngRNA
177

ngRNA
1504

ngRNA
179

ngRNA
174

ngRNA
178

ngRNA
293

TABLE 12

Sequence region
SEQ ID

pegRNA Spacer
2023

PBS
2024

PBS
2025

PBS
2026

PBS
2027

PBS
2028

PBS
2029

PBS
2030

PBS
2031

PBS
2032

PBS
2033

PBS
2034

RTT
2035

RTT
2036

RTT
2037

RTT
2038

RTT
2039

RTT
2040

RTT
2041

RTT
2042

RTT
2043

RTT
2044

ngRNA Spacer
2045

ngRNA Spacer
2046

ngRNA Spacer
2047

ngRNA Spacer
2048

ngRNA Spacer
2049

ngRNA Spacer
2050

ngRNA Spacer
2051

ngRNA Spacer
2052

ngRNA Spacer
2053

ngRNA Spacer
2054

ngRNA Spacer
2055

ngRNA Spacer
2056

ngRNA Spacer
2057

ngRNA Spacer
2058

ngRNA Spacer
2059

ngRNA Spacer
2060

ngRNA Spacer
2061

ngRNA Spacer
2062

ngRNA Spacer
2063

ngRNA Spacer
2064

ngRNA Spacer
2065

ngRNA Spacer
2066

ngRNA Spacer
2067

ngRNA Spacer
2068

ngRNA Spacer
41

ngRNA Spacer
2069

ngRNA Spacer
2070

ngRNA Spacer
2071

ngRNA Spacer
2072

ngRNA Spacer
2073

ngRNA Spacer
2074

ngRNA Spacer
2075

ngRNA Spacer
2076

ngRNA Spacer
2077

ngRNA Spacer
2078

ngRNA Spacer
2079

ngRNA Spacer
2080

ngRNA Spacer
2081

ngRNA Spacer
2082

ngRNA Spacer
2083

ngRNA Spacer
2084

ngRNA Spacer
2085

ngRNA Spacer
2086

ngRNA Spacer
2087

ngRNA Spacer
2088

ngRNA Spacer
2089

ngRNA Spacer
2090

ngRNA Spacer
2091

ngRNA Spacer
2092

ngRNA Spacer
2093

ngRNA Spacer
2094

ngRNA Spacer
2095

ngRNA Spacer
60

ngRNA Spacer
69

ngRNA Spacer
2096

ngRNA Spacer
65

ngRNA Spacer
64

ngRNA Spacer
63

ngRNA Spacer
62

ngRNA Spacer
66

ngRNA Spacer
61

ngRNA Spacer
71

pegRNA
2097

pegRNA
2098

pegRNA
2099

pegRNA
2100

pegRNA
2101

pegRNA
2102

pegRNA
2103

pegRNA
2104

pegRNA
2105

pegRNA
2106

pegRNA
2107

pegRNA
2108

pegRNA
2109

pegRNA
2110

pegRNA
2111

pegRNA
2112

pegRNA
2113

pegRNA
2114

pegRNA
2115

pegRNA
2116

pegRNA
2117

pegRNA
2118

pegRNA
2119

pegRNA
2120

pegRNA
2121

pegRNA
2122

pegRNA
2123

pegRNA
2124

pegRNA
2125

pegRNA
2127

pegRNA
2128

pegRNA
2129

pegRNA
2130

pegRNA
2131

pegRNA
2132

pegRNA
2133

pegRNA
2134

pegRNA
2135

pegRNA
2136

pegRNA
2137

pegRNA
2138

pegRNA
2139

pegRNA
2140

pegRNA
2141

pegRNA
2142

pegRNA
2143

pegRNA
2144

pegRNA
2145

pegRNA
2146

pegRNA
2147

pegRNA
2148

pegRNA
2149

pegRNA
2150

pegRNA
2151

pegRNA
2152

pegRNA
2153

pegRNA
2154

pegRNA
2155

pegRNA
2156

pegRNA
2157

pegRNA
2158

pegRNA
2159

pegRNA
2160

pegRNA
2161

pegRNA
2162

pegRNA
2163

pegRNA
2164

pegRNA
2165

pegRNA
2166

pegRNA
2167

pegRNA
2168

pegRNA
2169

pegRNA
2170

pegRNA
2171

pegRNA
2173

pegRNA
2174

pegRNA
2175

pegRNA
2176

pegRNA
2177

pegRNA
2178

pegRNA
2179

pegRNA
2180

pegRNA
2181

pegRNA
2182

pegRNA
2183

pegRNA
2184

pegRNA
2185

pegRNA
2186

pegRNA
2187

pegRNA
2188

pegRNA
2189

pegRNA
2190

pegRNA
2191

pegRNA
2192

pegRNA
2193

pegRNA
2194

pegRNA
2195

pegRNA
2196

pegRNA
2197

pegRNA
2198

pegRNA
2199

pegRNA
2200

pegRNA
2201

pegRNA
2202

pegRNA
2203

pegRNA
2204

pegRNA
2205

pegRNA
2206

pegRNA
2207

pegRNA
2208

pegRNA
2209

pegRNA
2210

pegRNA
2211

pegRNA
2212

pegRNA
2213

pegRNA
2214

pegRNA
2215

pegRNA
2216

pegRNA
2217

pegRNA
2218

pegRNA
2219

pegRNA
2220

pegRNA
2221

pegRNA
2222

pegRNA
2223

pegRNA
2224

pegRNA
2225

pegRNA
2226

pegRNA
2227

pegRNA
2228

pegRNA
2229

pegRNA
2230

pegRNA
2231

pegRNA
2232

pegRNA
2233

pegRNA
2234

pegRNA
2235

pegRNA
2236

pegRNA
2237

pegRNA
2238

pegRNA
2239

pegRNA
2240

pegRNA
2241

pegRNA
2242

pegRNA
2243

pegRNA
2244

pegRNA
2245

pegRNA
2246

pegRNA
2247

pegRNA
2248

pegRNA
2249

pegRNA
2250

pegRNA
2251

pegRNA
2252

pegRNA
2253

pegRNA
2254

pegRNA
2255

pegRNA
2256

ngRNA
2257

ngRNA
2258

ngRNA
2259

ngRNA
2260

ngRNA
2261

ngRNA
2262

ngRNA
2263

ngRNA
2264

ngRNA
2265

ngRNA
2266

ngRNA
2267

ngRNA
2268

ngRNA
2269

ngRNA
2270

ngRNA
2271

ngRNA
2272

ngRNA
2273

ngRNA
2274

ngRNA
2275

ngRNA
2276

ngRNA
2277

ngRNA
2278

ngRNA
2279

ngRNA
2280

ngRNA
2281

ngRNA
2282

ngRNA
2283

ngRNA
2284

ngRNA
2285

ngRNA
2286

ngRNA
2287

ngRNA
2288

ngRNA
2289

ngRNA
2290

ngRNA
2291

ngRNA
2292

TABLE 15

Sequence region
SEQ ID

pegRNA Spacer
2293

PBS
2294

PBS
2295

PBS
2296

PBS
2297

PBS
2298

PBS
2299

PBS
2300

PBS
2301

PBS
2302

PBS
2303

PBS
2304

RTT
2305

RTT
2306

RTT
2353

RTT
2307

RTT
2308

RTT
2309

RTT
2310

RTT
2311

RTT
2312

RTT
2313

RTT
2314

RTT
2315

RTT
2316

RTT
2317

RTT
2318

RTT
2319

RTT
2320

RTT
2321

RTT
2322

RTT
2323

RTT
2324

RTT
2325

RTT
2326

RTT
2327

RTT
2328

RTT
2329

RTT
2330

RTT
2331

RTT
2332

RTT
2333

RTT
2334

RTT
2335

RTT
2336

RTT
2337

RTT
2338

RTT
2339

RTT
2340

RTT
2341

RTT
2342

RTT
2343

RTT
2344

RTT
2345

RTT
2346

RTT
2347

RTT
2348

RTT
2349

RTT
2350

RTT
2351

RTT
2352

RTT
2354

RTT
2355

RTT
2356

RTT
2357

RTT
2358

RTT
2359

RTT
2360

RTT
2361

RTT
2362

RTT
2363

RTT
2364

RTT
2365

RTT
2366

RTT
2367

RTT
2368

RTT
2369

RTT
2370

RTT
2371

RTT
2372

RTT
2373

RTT
2374

RTT
2375

RTT
2376

RTT
2377

RTT
2378

RTT
2379

RTT
2380

RTT
2381

RTT
2382

RTT
2383

RTT
2384

RTT
2385

RTT
2386

RTT
2387

RTT
2388

RTT
2389

RTT
2390

RTT
2391

RTT
2392

RTT
2393

RTT
2394

RTT
2395

RTT
2396

RTT
2397

RTT
2398

RTT
2400

RTT
2401

RTT
2402

RTT
2403

RTT
2404

RTT
2405

RTT
2406

RTT
2407

RTT
2408

RTT
2409

RTT
2410

RTT
2411

RTT
2412

RTT
2413

RTT
2414

RTT
2415

RTT
2416

RTT
2417

RTT
2418

RTT
2419

RTT
2420

RTT
2421

RTT
2422

ngRNA Spacer
2064

ngRNA Spacer
2423

ngRNA Spacer
2424

ngRNA Spacer
2425

ngRNA Spacer
2426

ngRNA Spacer
2427

ngRNA Spacer
2428

ngRNA Spacer
2429

ngRNA Spacer
2430

ngRNA Spacer
2431

ngRNA Spacer
2432

ngRNA Spacer
2433

ngRNA Spacer
2434

ngRNA Spacer
2435

ngRNA Spacer
2091

ngRNA Spacer
2060

ngRNA Spacer
2436

ngRNA Spacer
2437

ngRNA Spacer
2054

ngRNA Spacer
2070

ngRNA Spacer
2078

ngRNA Spacer
2046

ngRNA Spacer
2047

ngRNA Spacer
2050

ngRNA Spacer
2051

ngRNA Spacer
2057

ngRNA Spacer
2058

ngRNA Spacer
2061

ngRNA Spacer
2438

ngRNA Spacer
2080

ngRNA Spacer
71

ngRNA Spacer
61

ngRNA Spacer
66

ngRNA Spacer
2074

ngRNA Spacer
62

ngRNA Spacer
64

ngRNA Spacer
65

ngRNA Spacer
69

ngRNA Spacer
60

ngRNA Spacer
2083

ngRNA Spacer
2066

ngRNA Spacer
2056

ngRNA Spacer
2062

ngRNA Spacer
2089

ngRNA Spacer
2439

ngRNA Spacer
2090

ngRNA Spacer
2059

ngRNA Spacer
2081

ngRNA Spacer
2069

ngRNA Spacer
63

ngRNA Spacer
2088

ngRNA Spacer
2068

ngRNA Spacer
2440

ngRNA Spacer
2072

ngRNA Spacer
2079

ngRNA Spacer
2073

ngRNA Spacer
2077

ngRNA Spacer
2093

ngRNA Spacer
2071

ngRNA Spacer
2048

ngRNA Spacer
2049

ngRNA Spacer
41

ngRNA Spacer
2085

ngRNA Spacer
2087

ngRNA Spacer
2441

ngRNA Spacer
2045

ngRNA Spacer
2063

ngRNA Spacer
2084

ngRNA Spacer
2076

ngRNA Spacer
2095

ngRNA Spacer
2075

ngRNA Spacer
2442

ngRNA Spacer
2443

ngRNA Spacer
2092

ngRNA Spacer
2055

ngRNA Spacer
2052

ngRNA Spacer
2086

ngRNA Spacer
2444

ngRNA Spacer
2082

ngRNA Spacer
2067

ngRNA Spacer
2096

ngRNA Spacer
2094

ngRNA Spacer
2053

pegRNA
2445

pegRNA
2446

pegRNA
2447

pegRNA
2448

pegRNA
2449

pegRNA
2450

pegRNA
2451

pegRNA
2452

pegRNA
2453

pegRNA
2454

pegRNA
2455

pegRNA
2456

pegRNA
2457

pegRNA
2458

pegRNA
2459

pegRNA
2460

pegRNA
2461

pegRNA
2462

pegRNA
2463

pegRNA
2464

pegRNA
2465

pegRNA
2466

pegRNA
2467

pegRNA
2468

pegRNA
2469

pegRNA
2470

pegRNA
2471

pegRNA
2472

pegRNA
2473

pegRNA
2474

pegRNA
2475

pegRNA
2476

pegRNA
2477

pegRNA
2478

pegRNA
2479

pegRNA
2480

pegRNA
2481

pegRNA
2482

pegRNA
2483

pegRNA
2484

pegRNA
2485

pegRNA
2486

pegRNA
2487

pegRNA
2488

pegRNA
2489

pegRNA
2490

pegRNA
2491

pegRNA
2492

pegRNA
2493

pegRNA
2494

pegRNA
2495

pegRNA
2496

pegRNA
2497

pegRNA
2498

pegRNA
2499

pegRNA
2500

pegRNA
2501

pegRNA
2502

pegRNA
2503

pegRNA
2504

pegRNA
2505

pegRNA
2506

pegRNA
2507

pegRNA
2508

pegRNA
2509

pegRNA
2510

pegRNA
2511

pegRNA
2512

pegRNA
2513

pegRNA
2514

pegRNA
2515

pegRNA
2516

pegRNA
2517

pegRNA
2518

pegRNA
2519

pegRNA
2520

pegRNA
2521

pegRNA
2522

pegRNA
2523

pegRNA
2524

pegRNA
2525

pegRNA
2526

pegRNA
2527

pegRNA
2528

pegRNA
2529

pegRNA
2530

pegRNA
2531

pegRNA
2532

pegRNA
2533

pegRNA
2534

pegRNA
2535

pegRNA
2536

pegRNA
2537

pegRNA
2538

pegRNA
2539

pegRNA
2540

pegRNA
2541

pegRNA
2542

pegRNA
2543

pegRNA
2544

pegRNA
2545

pegRNA
2546

pegRNA
2547

pegRNA
2548

pegRNA
2549

pegRNA
2550

pegRNA
2551

pegRNA
2552

pegRNA
2553

pegRNA
2554

pegRNA
2555

pegRNA
2556

pegRNA
2557

pegRNA
2558

pegRNA
2559

pegRNA
2560

pegRNA
2561

pegRNA
2562

pegRNA
2563

pegRNA
2564

pegRNA
2565

pegRNA
2566

pegRNA
2567

pegRNA
2568

pegRNA
2569

pegRNA
2570

pegRNA
2571

pegRNA
2572

pegRNA
2573

pegRNA
2574

pegRNA
2575

pegRNA
2576

pegRNA
2577

pegRNA
2578

pegRNA
2579

pegRNA
2580

pegRNA
2581

pegRNA
2582

pegRNA
2583

pegRNA
2584

pegRNA
2585

pegRNA
2586

pegRNA
2587

pegRNA
2588

pegRNA
2589

pegRNA
2590

pegRNA
2591

pegRNA
2592

pegRNA
2593

pegRNA
2594

pegRNA
2595

pegRNA
2596

pegRNA
2597

pegRNA
2598

pegRNA
2599

pegRNA
2600

pegRNA
2601

pegRNA
2602

pegRNA
2603

pegRNA
2604

pegRNA
2605

pegRNA
2606

pegRNA
2607

pegRNA
2608

pegRNA
2609

pegRNA
2610

pegRNA
2611

pegRNA
2612

pegRNA
2613

pegRNA
2614

pegRNA
2615

pegRNA
2616

pegRNA
2617

pegRNA
2618

pegRNA
2619

pegRNA
2620

pegRNA
2621

pegRNA
2622

pegRNA
2623

pegRNA
2624

pegRNA
2625

pegRNA
2626

pegRNA
2627

pegRNA
2628

pegRNA
2629

pegRNA
2630

pegRNA
2631

pegRNA
2632

pegRNA
2633

pegRNA
2634

pegRNA
2635

pegRNA
2636

pegRNA
2637

pegRNA
2638

pegRNA
2639

pegRNA
2640

pegRNA
2641

pegRNA
2642

pegRNA
2643

pegRNA
2644

pegRNA
2645

pegRNA
2646

pegRNA
2647

pegRNA
2648

pegRNA
2649

pegRNA
2650

pegRNA
2651

pegRNA
2652

pegRNA
2653

pegRNA
2654

pegRNA
2655

pegRNA
2656

pegRNA
2657

pegRNA
2658

pegRNA
2659

pegRNA
2660

pegRNA
2661

pegRNA
2662

pegRNA
2663

pegRNA
2664

pegRNA
2665

pegRNA
2666

pegRNA
2667

pegRNA
2668

pegRNA
2669

pegRNA
2670

pegRNA
2671

pegRNA
2672

pegRNA
2673

pegRNA
2674

pegRNA
2675

pegRNA
2676

pegRNA
2677

pegRNA
2678

pegRNA
2679

pegRNA
2680

pegRNA
2681

pegRNA
2682

pegRNA
2683

pegRNA
2684

pegRNA
2685

pegRNA
2686

pegRNA
2687

pegRNA
2688

pegRNA
2689

pegRNA
2690

pegRNA
2691

pegRNA
2692

pegRNA
2693

pegRNA
2694

pegRNA
2695

pegRNA
2696

pegRNA
2697

pegRNA
2698

pegRNA
2699

pegRNA
2700

pegRNA
2701

pegRNA
2702

pegRNA
2703

pegRNA
2704

pegRNA
2705

pegRNA
2706

pegRNA
2707

pegRNA
2708

pegRNA
2709

pegRNA
2710

pegRNA
2711

pegRNA
2712

pegRNA
2713

pegRNA
2714

pegRNA
2715

pegRNA
2716

pegRNA
2717

pegRNA
2718

pegRNA
2719

pegRNA
2720

pegRNA
2721

pegRNA
2722

pegRNA
2723

pegRNA
2724

pegRNA
2725

pegRNA
2726

pegRNA
2727

pegRNA
2728

pegRNA
2729

pegRNA
2730

pegRNA
2731

pegRNA
2732

pegRNA
2733

pegRNA
2734

pegRNA
2735

pegRNA
2736

pegRNA
2737

pegRNA
2738

pegRNA
2739

pegRNA
2740

pegRNA
2741

pegRNA
2742

pegRNA
2743

pegRNA
2744

pegRNA
2745

pegRNA
2746

pegRNA
2747

pegRNA
2748

pegRNA
2749

pegRNA
2750

pegRNA
2751

pegRNA
2752

pegRNA
2753

pegRNA
2754

pegRNA
2755

pegRNA
2756

pegRNA
2757

pegRNA
2758

pegRNA
2759

pegRNA
2760

pegRNA
2761

pegRNA
2762

pegRNA
2763

pegRNA
2764

pegRNA
2765

pegRNA
2766

pegRNA
2767

pegRNA
2768

pegRNA
2769

pegRNA
2770

pegRNA
2771

pegRNA
2772

pegRNA
2773

pegRNA
2774

pegRNA
2775

pegRNA
2776

pegRNA
2777

pegRNA
2778

pegRNA
2779

pegRNA
2780

pegRNA
2781

pegRNA
2782

pegRNA
2783

pegRNA
2784

pegRNA
2785

pegRNA
2786

pegRNA
2787

pegRNA
2788

pegRNA
2789

pegRNA
2790

pegRNA
2791

pegRNA
2792

pegRNA
2793

pegRNA
2794

pegRNA
2795

pegRNA
2796

pegRNA
2797

pegRNA
2798

pegRNA
2799

pegRNA
2800

pegRNA
2801

pegRNA
2802

pegRNA
2803

pegRNA
2804

pegRNA
2805

pegRNA
2806

pegRNA
2807

pegRNA
2808

pegRNA
2809

pegRNA
2810

pegRNA
2811

pegRNA
2812

pegRNA
2813

pegRNA
2814

pegRNA
2815

pegRNA
2816

pegRNA
2817

pegRNA
2818

pegRNA
2819

pegRNA
2820

pegRNA
2821

pegRNA
2822

pegRNA
2823

pegRNA
2824

pegRNA
2825

pegRNA
2826

pegRNA
2827

pegRNA
2828

pegRNA
2829

pegRNA
2830

pegRNA
2831

pegRNA
2832

pegRNA
2833

pegRNA
2834

pegRNA
2835

pegRNA
2836

pegRNA
2837

pegRNA
2838

pegRNA
2839

pegRNA
2840

pegRNA
2841

pegRNA
2842

pegRNA
2843

pegRNA
2844

pegRNA
2845

pegRNA
2846

pegRNA
2847

pegRNA
2848

pegRNA
2849

pegRNA
2850

pegRNA
2851

pegRNA
2852

pegRNA
2853

pegRNA
2854

pegRNA
2855

pegRNA
2856

pegRNA
2857

pegRNA
2858

pegRNA
2859

pegRNA
2860

pegRNA
2861

pegRNA
2862

pegRNA
2863

pegRNA
2864

pegRNA
2865

pegRNA
2866

pegRNA
2867

pegRNA
2868

pegRNA
2869

pegRNA
2870

pegRNA
2871

pegRNA
2872

pegRNA
2873

pegRNA
2874

pegRNA
2875

pegRNA
2876

pegRNA
2877

pegRNA
2878

pegRNA
2879

pegRNA
2880

pegRNA
2881

pegRNA
2882

pegRNA
2883

pegRNA
2884

pegRNA
2885

pegRNA
2886

pegRNA
2887

pegRNA
2888

pegRNA
2889

pegRNA
2890

pegRNA
2891

pegRNA
2892

pegRNA
2893

pegRNA
2894

pegRNA
2895

pegRNA
2896

pegRNA
2897

pegRNA
2898

pegRNA
2899

pegRNA
2900

pegRNA
2901

pegRNA
2902

pegRNA
2903

pegRNA
2904

pegRNA
2905

pegRNA
2906

pegRNA
2907

pegRNA
2908

pegRNA
2909

pegRNA
2910

pegRNA
2911

pegRNA
2912

pegRNA
2913

pegRNA
2914

pegRNA
2915

pegRNA
2916

pegRNA
2917

pegRNA
2918

pegRNA
2919

pegRNA
2920

pegRNA
2921

pegRNA
2922

pegRNA
2923

pegRNA
2924

pegRNA
2925

pegRNA
2926

pegRNA
2927

pegRNA
2928

pegRNA
2929

pegRNA
2930

pegRNA
2931

pegRNA
2932

pegRNA
2933

pegRNA
2934

pegRNA
2935

pegRNA
2936

pegRNA
2937

pegRNA
2938

pegRNA
2939

pegRNA
2940

pegRNA
2941

pegRNA
2942

pegRNA
2943

pegRNA
2944

pegRNA
2945

pegRNA
2946

pegRNA
2947

pegRNA
2948

pegRNA
2949

pegRNA
2950

pegRNA
2951

pegRNA
2952

pegRNA
2953

pegRNA
2954

pegRNA
2955

pegRNA
2956

pegRNA
2957

pegRNA
2958

pegRNA
2959

pegRNA
2960

pegRNA
2961

pegRNA
2962

pegRNA
2963

pegRNA
2964

pegRNA
2965

pegRNA
2966

pegRNA
2967

pegRNA
2968

pegRNA
2969

pegRNA
2970

pegRNA
2971

pegRNA
2972

pegRNA
2973

pegRNA
2974

pegRNA
2975

pegRNA
2976

pegRNA
2977

pegRNA
2978

pegRNA
2979

pegRNA
2980

pegRNA
2981

pegRNA
2982

pegRNA
2983

pegRNA
2984

pegRNA
2985

pegRNA
2986

pegRNA
2987

pegRNA
2988

pegRNA
2989

pegRNA
2990

pegRNA
2991

pegRNA
2992

pegRNA
2993

pegRNA
2994

pegRNA
2995

pegRNA
2996

pegRNA
2997

pegRNA
2998

pegRNA
2999

pegRNA
3000

pegRNA
3001

pegRNA
3002

pegRNA
3003

pegRNA
3004

pegRNA
3005

pegRNA
3006

pegRNA
3007

pegRNA
3008

pegRNA
3009

pegRNA
3010

pegRNA
3011

pegRNA
3012

pegRNA
3013

pegRNA
3014

pegRNA
3015

pegRNA
3016

pegRNA
3017

pegRNA
3018

pegRNA
3019

pegRNA
3020

pegRNA
3021

pegRNA
3022

pegRNA
3023

pegRNA
3024

pegRNA
3025

pegRNA
3026

pegRNA
3027

pegRNA
3028

pegRNA
3029

pegRNA
3030

pegRNA
3031

pegRNA
3032

pegRNA
3033

pegRNA
3034

pegRNA
3035

pegRNA
3036

pegRNA
3037

pegRNA
3038

pegRNA
3039

pegRNA
3040

pegRNA
3041

pegRNA
3042

pegRNA
3043

pegRNA
3044

pegRNA
3045

pegRNA
3046

pegRNA
3047

pegRNA
3048

pegRNA
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4071

pegRNA
4072

pegRNA
4073

pegRNA
4074

pegRNA
4075

pegRNA
4076

pegRNA
4077

pegRNA
4078

pegRNA
4079

pegRNA
4080

pegRNA
4081

pegRNA
4082

pegRNA
4083

pegRNA
4084

pegRNA
4085

pegRNA
4086

pegRNA
4087

pegRNA
4088

pegRNA
4089

pegRNA
4090

pegRNA
4091

pegRNA
4092

pegRNA
4093

pegRNA
4094

pegRNA
4095

pegRNA
4096

pegRNA
4097

pegRNA
4098

pegRNA
4099

pegRNA
4100

pegRNA
4101

pegRNA
4102

pegRNA
4103

pegRNA
4104

pegRNA
4105

pegRNA
4106

pegRNA
4107

pegRNA
4108

pegRNA
4109

pegRNA
4110

pegRNA
4111

pegRNA
4112

pegRNA
4113

pegRNA
4114

pegRNA
4115

pegRNA
4116

pegRNA
4117

pegRNA
4118

pegRNA
4119

pegRNA
4120

pegRNA
4121

pegRNA
4122

pegRNA
4123

pegRNA
4124

pegRNA
4125

pegRNA
4126

pegRNA
4127

pegRNA
4128

pegRNA
4129

pegRNA
4130

pegRNA
4131

pegRNA
4132

pegRNA
4133

pegRNA
4134

pegRNA
4135

pegRNA
4136

pegRNA
4137

pegRNA
4138

pegRNA
4139

pegRNA
4140

pegRNA
4141

pegRNA
4142

pegRNA
4143

pegRNA
4144

pegRNA
4145

pegRNA
4146

pegRNA
4147

pegRNA
4148

pegRNA
4149

pegRNA
4150

pegRNA
4151

pegRNA
4152

pegRNA
4153

pegRNA
4154

pegRNA
4155

pegRNA
4156

pegRNA
4157

pegRNA
4158

pegRNA
4159

pegRNA
4160

pegRNA
4161

pegRNA
4162

pegRNA
4163

pegRNA
4164

pegRNA
4165

pegRNA
4166

pegRNA
4167

pegRNA
4168

pegRNA
4169

pegRNA
4170

pegRNA
4171

pegRNA
4172

pegRNA
4173

pegRNA
4174

pegRNA
4175

pegRNA
4176

pegRNA
4177

pegRNA
4178

pegRNA
4179

pegRNA
4180

pegRNA
4181

pegRNA
4182

pegRNA
4183

pegRNA
4184

pegRNA
4185

pegRNA
4186

pegRNA
4187

pegRNA
4188

pegRNA
4189

pegRNA
4190

pegRNA
4191

pegRNA
4192

pegRNA
4193

pegRNA
4194

pegRNA
4195

pegRNA
4196

pegRNA
4197

pegRNA
4198

pegRNA
4199

pegRNA
4200

pegRNA
4201

pegRNA
4202

pegRNA
4203

pegRNA
4204

pegRNA
4205

pegRNA
4206

pegRNA
4207

pegRNA
4208

pegRNA
4209

pegRNA
4210

pegRNA
4211

pegRNA
4212

pegRNA
4213

pegRNA
4214

pegRNA
4215

pegRNA
4216

pegRNA
4217

pegRNA
4218

pegRNA
4219

pegRNA
4220

pegRNA
4221

pegRNA
4222

pegRNA
4223

pegRNA
4224

pegRNA
4225

pegRNA
4226

pegRNA
4227

pegRNA
4228

pegRNA
4229

pegRNA
4230

pegRNA
4231

pegRNA
4232

pegRNA
4233

pegRNA
4234

pegRNA
4235

pegRNA
4236

pegRNA
4237

pegRNA
4238

pegRNA
4239

pegRNA
4240

pegRNA
4241

pegRNA
4242

pegRNA
4243

pegRNA
4244

pegRNA
4245

pegRNA
4246

pegRNA
4247

pegRNA
4248

pegRNA
4249

pegRNA
4250

pegRNA
4251

pegRNA
4252

pegRNA
4253

pegRNA
4254

pegRNA
4255

pegRNA
4256

pegRNA
4257

pegRNA
4258

pegRNA
4259

pegRNA
4260

pegRNA
4261

pegRNA
4262

pegRNA
4263

pegRNA
4264

pegRNA
4265

pegRNA
4266

pegRNA
4267

pegRNA
4268

pegRNA
4269

pegRNA
4270

pegRNA
4271

pegRNA
4272

pegRNA
4273

pegRNA
4274

pegRNA
4275

pegRNA
4276

pegRNA
4277

pegRNA
4278

pegRNA
4279

pegRNA
4280

pegRNA
4281

pegRNA
4282

pegRNA
4283

pegRNA
4284

pegRNA
4285

pegRNA
4286

pegRNA
4287

pegRNA
4288

pegRNA
4289

pegRNA
4290

pegRNA
4291

pegRNA
4292

pegRNA
4293

pegRNA
4294

pegRNA
4295

pegRNA
4296

pegRNA
4297

pegRNA
4298

pegRNA
4299

pegRNA
4300

pegRNA
4301

pegRNA
4302

pegRNA
4303

pegRNA
4304

pegRNA
4305

pegRNA
4306

pegRNA
4307

pegRNA
4308

pegRNA
4309

pegRNA
4310

pegRNA
4311

pegRNA
4312

pegRNA
4313

pegRNA
4314

pegRNA
4315

pegRNA
4316

pegRNA
4317

pegRNA
4318

pegRNA
4319

pegRNA
4320

pegRNA
4321

pegRNA
4322

pegRNA
4323

pegRNA
4324

pegRNA
4325

pegRNA
4326

pegRNA
4327

pegRNA
4328

pegRNA
4329

pegRNA
4330

pegRNA
4331

pegRNA
4332

pegRNA
4333

pegRNA
4334

pegRNA
4335

pegRNA
4336

pegRNA
4337

pegRNA
4338

pegRNA
4339

pegRNA
4340

pegRNA
4341

pegRNA
4342

pegRNA
4343

pegRNA
4344

pegRNA
4345

pegRNA
4346

pegRNA
4347

pegRNA
4348

pegRNA
4349

pegRNA
4350

pegRNA
4351

pegRNA
4352

pegRNA
4353

pegRNA
4354

pegRNA
4355

pegRNA
4356

pegRNA
4357

pegRNA
4358

pegRNA
4359

pegRNA
4360

pegRNA
4361

pegRNA
4362

pegRNA
4363

pegRNA
4364

pegRNA
4365

pegRNA
4366

pegRNA
4367

pegRNA
4368

pegRNA
4369

pegRNA
4370

pegRNA
4371

pegRNA
4372

pegRNA
4373

pegRNA
4374

pegRNA
4375

pegRNA
4376

pegRNA
4377

pegRNA
4378

pegRNA
4379

pegRNA
4380

pegRNA
4381

pegRNA
4382

pegRNA
4383

pegRNA
4384

pegRNA
4385

pegRNA
4386

pegRNA
4387

pegRNA
4388

pegRNA
4389

pegRNA
4390

pegRNA
4391

pegRNA
4392

pegRNA
4393

pegRNA
4394

pegRNA
4395

pegRNA
4396

pegRNA
4397

pegRNA
4398

pegRNA
4399

pegRNA
4400

pegRNA
4401

pegRNA
4402

pegRNA
4403

pegRNA
4404

pegRNA
4405

pegRNA
4406

pegRNA
4407

pegRNA
4408

pegRNA
4409

ngRNA
2268

ngRNA
2257

ngRNA
4410

ngRNA
4411

ngRNA
4412

ngRNA
2275

ngRNA
2267

ngRNA
2266

ngRNA
4413

ngRNA
2272

ngRNA
2271

ngRNA
4414

ngRNA
2270

ngRNA
2263

ngRNA
2265

ngRNA
2261

ngRNA
2264

ngRNA
4415

ngRNA
2258

ngRNA
2276

ngRNA
2262

ngRNA
2273

ngRNA
2274

ngRNA
2260

ngRNA
4416

ngRNA
2269

ngRNA
4417

ngRNA
2277

ngRNA
4418

ngRNA
2259

ngRNA
4419

ngRNA
2280

ngRNA
4420

ngRNA
4421

ngRNA
2286

ngRNA
4422

ngRNA
2288

ngRNA
2289

ngRNA
2279

ngRNA
2281

ngRNA
2282

ngRNA
2285

ngRNA
2283

ngRNA
2287

ngRNA
2278

ngRNA
2284

ngRNA
4423

ngRNA
2291

ngRNA
4424

ngRNA
2290

ngRNA
2292

TABLE 16

Sequence region
SEQ ID

pegRNA Spacer
4425

PBS
4426

PBS
4427

PBS
4428

PBS
2297

PBS
4429

PBS
4430

PBS
4431

PBS
4432

PBS
4433

PBS
4434

PBS
4435

PBS
4436

RTT
4437

RTT
4438

RTT
4439

RTT
4440

RTT
4441

RTT
4442

RTT
4443

RTT
4444

RTT
4445

RTT
4446

RTT
4447

RTT
4448

RTT
4449

RTT
4450

RTT
4451

RTT
4452

RTT
4453

RTT
4454

RTT
4455

RTT
4456

RTT
4457

RTT
4458

RTT
4459

RTT
4460

RTT
4461

RTT
4462

RTT
4463

RTT
4464

RTT
4465

RTT
4466

RTT
4467

RTT
4468

RTT
4469

RTT
4470

RTT
4471

RTT
4472

RTT
4473

RTT
4474

RTT
4475

RTT
4476

RTT
4477

RTT
4478

RTT
4479

RTT
4480

RTT
4481

RTT
4482

RTT
4483

RTT
4484

RTT
4485

RTT
4486

RTT
4487

RTT
4488

RTT
4489

RTT
4490

RTT
4491

RTT
4492

ngRNA Spacer
2083

ngRNA Spacer
2080

ngRNA Spacer
2091

ngRNA Spacer
2442

ngRNA Spacer
2066

ngRNA Spacer
2067

ngRNA Spacer
2075

ngRNA Spacer
2057

ngRNA Spacer
2050

ngRNA Spacer
2058

ngRNA Spacer
2072

ngRNA Spacer
2076

ngRNA Spacer
2079

ngRNA Spacer
2078

ngRNA Spacer
2051

ngRNA Spacer
2046

ngRNA Spacer
2065

ngRNA Spacer
2085

ngRNA Spacer
2049

ngRNA Spacer
2047

ngRNA Spacer
2061

ngRNA Spacer
2064

ngRNA Spacer
2068

ngRNA Spacer
2082

ngRNA Spacer
2054

ngRNA Spacer
2062

ngRNA Spacer
2056

ngRNA Spacer
2094

ngRNA Spacer
2045

ngRNA Spacer
2090

ngRNA Spacer
2086

ngRNA Spacer
2074

ngRNA Spacer
2095

ngRNA Spacer
2084

ngRNA Spacer
65

ngRNA Spacer
2059

ngRNA Spacer
71

ngRNA Spacer
66

ngRNA Spacer
62

ngRNA Spacer
63

ngRNA Spacer
64

ngRNA Spacer
69

ngRNA Spacer
2088

ngRNA Spacer
2070

ngRNA Spacer
60

ngRNA Spacer
61

ngRNA Spacer
2089

ngRNA Spacer
2063

ngRNA Spacer
2093

ngRNA Spacer
2073

ngRNA Spacer
2081

ngRNA Spacer
2071

ngRNA Spacer
2055

ngRNA Spacer
2087

ngRNA Spacer
2069

ngRNA Spacer
2048

ngRNA Spacer
2437

ngRNA Spacer
41

ngRNA Spacer
2077

ngRNA Spacer
2439

ngRNA Spacer
2092

ngRNA Spacer
2060

ngRNA Spacer
2052

ngRNA Spacer
2443

ngRNA Spacer
2053

ngRNA Spacer
2096

ngRNA Spacer
2444

pegRNA
4493

pegRNA
4494

pegRNA
4495

pegRNA
4496

pegRNA
4497

pegRNA
4498

pegRNA
4499

pegRNA
4500

pegRNA
4501

pegRNA
4502

pegRNA
4503

pegRNA
4504

pegRNA
4505

pegRNA
4504

pegRNA
4506

pegRNA
4507

pegRNA
4508

pegRNA
4509

pegRNA
4510

pegRNA
4511

pegRNA
4512

pegRNA
4513

pegRNA
4514

pegRNA
4515

pegRNA
4516

pegRNA
4517

pegRNA
4518

pegRNA
4519

pegRNA
4520

pegRNA
4521

pegRNA
4522

pegRNA
4523

pegRNA
4524

pegRNA
4525

pegRNA
4526

pegRNA
4527

pegRNA
4528

pegRNA
4529

pegRNA
4522

pegRNA
4530

pegRNA
4531

pegRNA
4532

pegRNA
4533

pegRNA
4534

pegRNA
4535

pegRNA
4536

pegRNA
4537

pegRNA
4538

pegRNA
4539

pegRNA
4540

pegRNA
4541

pegRNA
4542

pegRNA
4543

pegRNA
4544

pegRNA
4545

pegRNA
4546

pegRNA
4547

pegRNA
4548

pegRNA
4549

pegRNA
4550

pegRNA
4551

pegRNA
4552

pegRNA
4553

pegRNA
4554

pegRNA
4555

pegRNA
4556

pegRNA
4557

pegRNA
4558

pegRNA
4559

pegRNA
4560

pegRNA
4559

pegRNA
4561

pegRNA
4562

pegRNA
4563

pegRNA
4564

pegRNA
4565

pegRNA
4566

pegRNA
4567

pegRNA
4568

pegRNA
4569

pegRNA
4570

pegRNA
4571

pegRNA
4572

pegRNA
4573

pegRNA
4574

pegRNA
4575

pegRNA
4576

pegRNA
4577

pegRNA
4578

pegRNA
4579

pegRNA
4580

pegRNA
4581

pegRNA
4582

pegRNA
4583

pegRNA
4584

pegRNA
4585

pegRNA
4586

pegRNA
4587

pegRNA
4588

pegRNA
4589

pegRNA
4590

pegRNA
4591

pegRNA
4592

pegRNA
4593

pegRNA
4594

pegRNA
4595

pegRNA
4588

pegRNA
4596

pegRNA
4591

pegRNA
4597

pegRNA
4598

pegRNA
4599

pegRNA
4600

pegRNA
4601

pegRNA
4602

pegRNA
4603

pegRNA
4604

pegRNA
4605

pegRNA
4606

pegRNA
4607

pegRNA
4608

pegRNA
4609

pegRNA
4610

pegRNA
4611

pegRNA
4612

pegRNA
4613

pegRNA
4614

pegRNA
4615

pegRNA
4616

pegRNA
4617

pegRNA
4618

pegRNA
4619

pegRNA
4620

pegRNA
4621

pegRNA
4622

pegRNA
4623

pegRNA
4624

pegRNA
4625

pegRNA
4626

pegRNA
4627

pegRNA
4628

pegRNA
4629

pegRNA
4630

pegRNA
4631

pegRNA
4632

pegRNA
4633

pegRNA
4634

pegRNA
4635

pegRNA
4636

pegRNA
4637

pegRNA
4638

pegRNA
4639

pegRNA
4640

pegRNA
4641

pegRNA
4642

pegRNA
4643

pegRNA
4644

pegRNA
4645

pegRNA
4646

pegRNA
4647

pegRNA
4648

pegRNA
4649

pegRNA
4650

pegRNA
4651

pegRNA
4652

pegRNA
4653

pegRNA
4654

pegRNA
4655

pegRNA
4656

pegRNA
4657

pegRNA
4658

pegRNA
4659

pegRNA
4660

pegRNA
4661

pegRNA
4662

pegRNA
4663

pegRNA
4664

pegRNA
4665

pegRNA
4666

pegRNA
4667

pegRNA
4668

pegRNA
4669

pegRNA
4670

pegRNA
4671

pegRNA
4672

pegRNA
4673

pegRNA
4674

pegRNA
4675

pegRNA
4676

pegRNA
4677

pegRNA
4678

pegRNA
4679

pegRNA
4680

pegRNA
4681

pegRNA
4682

pegRNA
4683

pegRNA
4684

pegRNA
4685

pegRNA
4686

pegRNA
4687

pegRNA
4688

pegRNA
4689

pegRNA
4690

pegRNA
4691

pegRNA
4692

pegRNA
4693

pegRNA
4694

pegRNA
4695

pegRNA
4696

pegRNA
4697

pegRNA
4698

pegRNA
4699

pegRNA
4700

pegRNA
4701

pegRNA
4702

pegRNA
4703

pegRNA
4697

pegRNA
4704

pegRNA
4700

pegRNA
4705

pegRNA
4706

pegRNA
4707

pegRNA
4708

pegRNA
4709

pegRNA
4710

pegRNA
4711

pegRNA
4712

pegRNA
4713

pegRNA
4714

pegRNA
4715

pegRNA
4716

pegRNA
4717

pegRNA
4718

pegRNA
4719

pegRNA
4720

pegRNA
4721

pegRNA
4722

pegRNA
4723

pegRNA
4724

pegRNA
4725

pegRNA
4726

pegRNA
4727

pegRNA
4728

pegRNA
4729

pegRNA
4730

pegRNA
4731

pegRNA
4732

pegRNA
4733

pegRNA
4734

pegRNA
4735

pegRNA
4736

pegRNA
4737

pegRNA
4738

pegRNA
4739

pegRNA
4740

pegRNA
4741

pegRNA
4742

pegRNA
4743

pegRNA
4744

pegRNA
4745

pegRNA
4746

pegRNA
4747

pegRNA
4748

pegRNA
4749

pegRNA
4750

pegRNA
4751

pegRNA
4752

pegRNA
4753

pegRNA
4754

pegRNA
4755

pegRNA
4756

pegRNA
4757

pegRNA
4758

pegRNA
4759

pegRNA
4760

pegRNA
4761

pegRNA
4762

pegRNA
4763

pegRNA
4764

pegRNA
4765

pegRNA
4766

pegRNA
4767

pegRNA
4768

pegRNA
4769

pegRNA
4770

pegRNA
4771

pegRNA
4772

pegRNA
4773

pegRNA
4774

pegRNA
4775

pegRNA
4776

pegRNA
4777

pegRNA
4778

pegRNA
4779

pegRNA
4780

pegRNA
4781

pegRNA
4782

pegRNA
4783

pegRNA
4784

pegRNA
4785

pegRNA
4786

pegRNA
4763

pegRNA
4787

pegRNA
4788

pegRNA
4789

pegRNA
4790

pegRNA
4791

pegRNA
4792

pegRNA
4793

pegRNA
4794

pegRNA
4795

pegRNA
4796

pegRNA
4797

pegRNA
4798

pegRNA
4799

pegRNA
4800

pegRNA
4801

pegRNA
4802

pegRNA
4803

pegRNA
4804

pegRNA
4805

pegRNA
4806

pegRNA
4807

pegRNA
4808

pegRNA
4809

pegRNA
4810

pegRNA
4811

pegRNA
4812

pegRNA
4813

pegRNA
4814

pegRNA
4815

pegRNA
4816

pegRNA
4817

pegRNA
4818

pegRNA
4819

pegRNA
4820

pegRNA
4821

pegRNA
4822

pegRNA
4823

pegRNA
4824

pegRNA
4825

pegRNA
4826

pegRNA
4827

pegRNA
4828

pegRNA
4829

pegRNA
4830

pegRNA
4831

pegRNA
4832

pegRNA
4833

pegRNA
4834

pegRNA
4835

pegRNA
4836

pegRNA
4837

pegRNA
4838

pegRNA
4839

pegRNA
4840

pegRNA
4841

pegRNA
4826

pegRNA
4842

pegRNA
4843

pegRNA
4844

pegRNA
4845

pegRNA
4846

pegRNA
4847

pegRNA
4848

pegRNA
4849

pegRNA
4850

pegRNA
4851

pegRNA
4852

pegRNA
4853

pegRNA
4854

pegRNA
4855

pegRNA
4856

pegRNA
4857

pegRNA
4858

pegRNA
4859

pegRNA
4860

pegRNA
4861

pegRNA
4862

pegRNA
4863

pegRNA
4864

pegRNA
4865

pegRNA
4866

pegRNA
4867

pegRNA
4868

pegRNA
4869

pegRNA
4870

pegRNA
4871

pegRNA
4872

pegRNA
4873

pegRNA
4874

pegRNA
4875

pegRNA
4873

pegRNA
4876

pegRNA
4877

pegRNA
4878

pegRNA
4879

pegRNA
4871

pegRNA
4880

pegRNA
4881

pegRNA
4882

pegRNA
4883

pegRNA
4884

pegRNA
4885

pegRNA
4886

pegRNA
4887

pegRNA
4888

pegRNA
4889

pegRNA
4890

pegRNA
4891

pegRNA
4892

pegRNA
4893

pegRNA
4894

pegRNA
4895

pegRNA
4896

pegRNA
4897

pegRNA
4898

pegRNA
4899

pegRNA
4900

pegRNA
4901

pegRNA
4902

pegRNA
4903

pegRNA
4904

pegRNA
4905

pegRNA
4906

pegRNA
4907

pegRNA
4908

pegRNA
4907

pegRNA
4909

pegRNA
4910

pegRNA
4911

pegRNA
4912

pegRNA
4913

pegRNA
4914

pegRNA
4915

pegRNA
4916

pegRNA
4917

pegRNA
4918

pegRNA
4919

pegRNA
4920

pegRNA
4921

pegRNA
4922

pegRNA
4923

pegRNA
4924

pegRNA
4925

pegRNA
4926

pegRNA
4927

pegRNA
4928

pegRNA
4929

pegRNA
4930

pegRNA
4931

pegRNA
4932

pegRNA
4933

pegRNA
4934

pegRNA
4935

pegRNA
4936

pegRNA
4937

pegRNA
4938

pegRNA
4939

pegRNA
4940

pegRNA
4941

pegRNA
4942

pegRNA
4943

pegRNA
4944

pegRNA
4945

pegRNA
4946

pegRNA
4947

pegRNA
4948

pegRNA
4949

pegRNA
4950

pegRNA
4951

pegRNA
4952

pegRNA
4949

pegRNA
4953

pegRNA
4951

pegRNA
4954

pegRNA
4955

pegRNA
4956

pegRNA
4957

pegRNA
4958

pegRNA
4959

pegRNA
4960

pegRNA
4961

pegRNA
4962

pegRNA
4963

pegRNA
4964

pegRNA
4965

pegRNA
4966

pegRNA
4967

pegRNA
4968

pegRNA
4969

pegRNA
4970

pegRNA
4971

pegRNA
4972

pegRNA
4973

pegRNA
4974

pegRNA
4970

pegRNA
4975

pegRNA
4976

pegRNA
4977

pegRNA
4978

pegRNA
4979

pegRNA
4971

pegRNA
4980

pegRNA
4981

pegRNA
4982

pegRNA
4983

pegRNA
4984

pegRNA
4985

pegRNA
4986

pegRNA
4987

pegRNA
4988

pegRNA
4989

pegRNA
4990

pegRNA
4991

pegRNA
4992

pegRNA
4993

pegRNA
4994

pegRNA
4995

pegRNA
4996

pegRNA
4997

pegRNA
4998

pegRNA
4999

pegRNA
5000

pegRNA
5001

pegRNA
5002

pegRNA
5003

pegRNA
5004

pegRNA
5005

pegRNA
5006

pegRNA
5007

pegRNA
5008

pegRNA
5009

pegRNA
5010

pegRNA
5004

pegRNA
5005

pegRNA
5011

pegRNA
5012

pegRNA
5013

pegRNA
5014

pegRNA
5015

pegRNA
5016

pegRNA
5017

pegRNA
5018

pegRNA
5019

pegRNA
5020

pegRNA
5021

pegRNA
5022

pegRNA
5023

pegRNA
5024

pegRNA
5025

pegRNA
5026

pegRNA
5027

pegRNA
5028

pegRNA
5029

pegRNA
5030

pegRNA
5031

pegRNA
5032

pegRNA
5033

pegRNA
5034

pegRNA
5033

pegRNA
5035

pegRNA
5036

pegRNA
5037

pegRNA
5038

pegRNA
5039

pegRNA
5039

pegRNA
5040

pegRNA
5041

pegRNA
5042

pegRNA
5043

pegRNA
5044

pegRNA
5045

pegRNA
5046

pegRNA
5047

pegRNA
5048

pegRNA
5049

pegRNA
5050

pegRNA
5051

pegRNA
5052

pegRNA
5053

pegRNA
5054

pegRNA
5055

pegRNA
5056

pegRNA
5057

pegRNA
5058

pegRNA
5059

pegRNA
5060

pegRNA
5058

pegRNA
5061

pegRNA
5062

pegRNA
5063

pegRNA
5064

pegRNA
5065

pegRNA
5066

pegRNA
5067

pegRNA
5063

pegRNA
5068

pegRNA
5069

pegRNA
5070

pegRNA
5071

pegRNA
5072

pegRNA
5073

pegRNA
5074

pegRNA
5075

pegRNA
5076

pegRNA
5077

pegRNA
5078

pegRNA
5079

pegRNA
5080

pegRNA
5081

pegRNA
5082

pegRNA
5083

pegRNA
5084

pegRNA
5085

pegRNA
5086

pegRNA
5084

pegRNA
5087

pegRNA
5088

pegRNA
5089

pegRNA
5090

pegRNA
5091

pegRNA
5092

pegRNA
5093

pegRNA
5094

pegRNA
5095

pegRNA
5096

pegRNA
5097

pegRNA
5098

pegRNA
5099

pegRNA
5100

pegRNA
5101

pegRNA
5102

pegRNA
5103

pegRNA
5104

pegRNA
5105

pegRNA
5106

pegRNA
5107

pegRNA
5108

pegRNA
5109

pegRNA
5110

pegRNA
5111

pegRNA
5112

pegRNA
5113

pegRNA
5114

pegRNA
5115

pegRNA
5116

pegRNA
5117

pegRNA
5115

pegRNA
5118

pegRNA
5119

pegRNA
5120

pegRNA
5121

pegRNA
5122

pegRNA
5123

pegRNA
5124

pegRNA
5125

pegRNA
5126

pegRNA
5127

pegRNA
5128

pegRNA
5129

pegRNA
5130

pegRNA
5131

pegRNA
5132

pegRNA
5133

pegRNA
5134

pegRNA
5135

pegRNA
5136

pegRNA
5137

pegRNA
5138

pegRNA
5139

pegRNA
5140

pegRNA
5141

pegRNA
5142

pegRNA
5143

pegRNA
5144

pegRNA
5145

pegRNA
5146

pegRNA
5147

pegRNA
5148

pegRNA
5149

pegRNA
5150

pegRNA
5151

pegRNA
5152

pegRNA
5153

pegRNA
5154

pegRNA
5155

pegRNA
5156

pegRNA
5157

pegRNA
5158

pegRNA
5159

pegRNA
5160

pegRNA
5161

pegRNA
5162

pegRNA
5163

pegRNA
5164

pegRNA
5165

pegRNA
5166

pegRNA
5167

pegRNA
5168

pegRNA
5169

pegRNA
5170

pegRNA
5171

pegRNA
5172

pegRNA
5173

pegRNA
5174

pegRNA
5175

pegRNA
5176

pegRNA
5177

pegRNA
5178

pegRNA
5179

pegRNA
5180

pegRNA
5181

pegRNA
5182

pegRNA
5183

pegRNA
5184

pegRNA
5185

pegRNA
5186

pegRNA
5187

pegRNA
5188

pegRNA
5189

pegRNA
5190

pegRNA
5191

pegRNA
5192

pegRNA
5193

pegRNA
5194

pegRNA
5195

pegRNA
5196

pegRNA
5197

pegRNA
5198

pegRNA
5199

pegRNA
5200

pegRNA
5201

pegRNA
5202

pegRNA
5203

pegRNA
5204

pegRNA
5205

ngRNA
2267

ngRNA
2257

ngRNA
2277

ngRNA
2276

ngRNA
2262

ngRNA
4418

ngRNA
2270

ngRNA
2274

ngRNA
2264

ngRNA
2268

ngRNA
2275

ngRNA
2272

ngRNA
2271

ngRNA
2265

ngRNA
2263

ngRNA
2266

ngRNA
2273

ngRNA
4411

ngRNA
2269

ngRNA
4416

ngRNA
2258

ngRNA
2261

ngRNA
2260

ngRNA
2259

ngRNA
2278

ngRNA
2284

ngRNA
2282

ngRNA
2288

ngRNA
4420

ngRNA
2285

ngRNA
2283

ngRNA
2280

ngRNA
4422

ngRNA
2281

ngRNA
2287

ngRNA
2279

ngRNA
2286

ngRNA
2289

ngRNA
2291

ngRNA
2290

ngRNA
2292

TABLE 17

Sequence region
SEQ ID

pegRNA Spacer
5206

PBS
5207

PBS
5208

PBS
5209

PBS
5210

PBS
5211

PBS
5212

PBS
5213

PBS
5214

PBS
5215

PBS
5216

PBS
5217

RTT
5218

ngRNA Spacer
199

ngRNA Spacer
24

ngRNA Spacer
20

ngRNA Spacer
200

ngRNA Spacer
52

ngRNA Spacer
21

ngRNA Spacer
56

ngRNA Spacer
57

ngRNA Spacer
51

ngRNA Spacer
45

ngRNA Spacer
5219

ngRNA Spacer
27

ngRNA Spacer
55

ngRNA Spacer
737

ngRNA Spacer
18

ngRNA Spacer
32

ngRNA Spacer
72

ngRNA Spacer
529

ngRNA Spacer
43

ngRNA Spacer
23

ngRNA Spacer
31

ngRNA Spacer
22

ngRNA Spacer
34

ngRNA Spacer
30

ngRNA Spacer
35

ngRNA Spacer
46

ngRNA Spacer
40

ngRNA Spacer
33

ngRNA Spacer
39

ngRNA Spacer
58

ngRNA Spacer
25

ngRNA Spacer
28

ngRNA Spacer
29

ngRNA Spacer
47

ngRNA Spacer
5220

ngRNA Spacer
68

ngRNA Spacer
37

ngRNA Spacer
67

ngRNA Spacer
44

ngRNA Spacer
19

ngRNA Spacer
38

ngRNA Spacer
70

ngRNA Spacer
42

ngRNA Spacer
59

ngRNA Spacer
48

ngRNA Spacer
36

ngRNA Spacer
49

ngRNA Spacer
54

ngRNA Spacer
5221

ngRNA Spacer
5222

ngRNA Spacer
5223

ngRNA Spacer
5224

ngRNA Spacer
5225

ngRNA Spacer
5226

ngRNA Spacer
5227

ngRNA Spacer
26

ngRNA Spacer
53

ngRNA Spacer
203

ngRNA Spacer
50

ngRNA
155

ngRNA
163

ngRNA
156

ngRNA
154

ngRNA
167

ngRNA
153

ngRNA
157

ngRNA
169

ngRNA
168

ngRNA
290

ngRNA
159

ngRNA
160

ngRNA
291

ngRNA
1501

ngRNA
161

ngRNA
681

ngRNA
164

ngRNA
162

ngRNA
166

ngRNA
165

ngRNA
158

ngRNA
171

ngRNA
170

ngRNA
173

ngRNA
293

ngRNA
179

ngRNA
1504

ngRNA
180

ngRNA
176

ngRNA
175

ngRNA
181

ngRNA
178

ngRNA
292

ngRNA
177

ngRNA
172

ngRNA
174

TABLE 18

Sequence region
SEQ ID

pegRNA Spacer
5228

PBS
5229

PBS
5230

PBS
5231

PBS
5232

PBS
5233

PBS
5234

PBS
5235

PBS
5236

PBS
5237

PBS
5238

PBS
5239

RTT
5240

RTT
5241

RTT
5242

RTT
5243

RTT
5244

RTT
5245

RTT
5246

RTT
5247

ngRNA Spacer
28

ngRNA Spacer
55

ngRNA Spacer
35

ngRNA Spacer
737

ngRNA Spacer
736

ngRNA Spacer
529

ngRNA Spacer
43

ngRNA Spacer
200

ngRNA Spacer
203

ngRNA Spacer
199

ngRNA Spacer
34

ngRNA Spacer
5219

ngRNA Spacer
740

ngRNA Spacer
33

ngRNA Spacer
37

ngRNA Spacer
48

ngRNA Spacer
20

ngRNA Spacer
19

ngRNA Spacer
38

ngRNA Spacer
46

ngRNA Spacer
738

ngRNA Spacer
30

ngRNA Spacer
50

ngRNA Spacer
24

ngRNA Spacer
23

ngRNA Spacer
47

ngRNA Spacer
22

ngRNA Spacer
29

ngRNA Spacer
70

ngRNA Spacer
58

ngRNA Spacer
52

ngRNA Spacer
51

ngRNA Spacer
49

ngRNA Spacer
45

ngRNA Spacer
68

ngRNA Spacer
59

ngRNA Spacer
31

ngRNA Spacer
18

ngRNA Spacer
67

ngRNA Spacer
53

ngRNA Spacer
5220

ngRNA Spacer
21

ngRNA Spacer
26

ngRNA Spacer
36

ngRNA Spacer
56

ngRNA Spacer
32

ngRNA Spacer
44

ngRNA Spacer
5221

ngRNA Spacer
739

ngRNA Spacer
39

ngRNA Spacer
40

ngRNA Spacer
42

ngRNA Spacer
72

ngRNA Spacer
54

ngRNA Spacer
57

ngRNA Spacer
5227

ngRNA Spacer
27

ngRNA Spacer
5225

ngRNA Spacer
5224

ngRNA Spacer
5226

ngRNA Spacer
5223

ngRNA Spacer
5222

ngRNA Spacer
25

ngRNA
162

ngRNA
164

ngRNA
160

ngRNA
1503

ngRNA
681

ngRNA
154

ngRNA
155

ngRNA
156

ngRNA
290

ngRNA
153

ngRNA
166

ngRNA
1501

ngRNA
291

ngRNA
158

ngRNA
163

ngRNA
157

ngRNA
169

ngRNA
167

ngRNA
168

ngRNA
159

ngRNA
161

ngRNA
1502

ngRNA
165

ngRNA
1504

ngRNA
180

ngRNA
292

ngRNA
178

ngRNA
179

ngRNA
170

ngRNA
172

ngRNA
173

ngRNA
177

ngRNA
293

ngRNA
176

ngRNA
174

ngRNA
175

ngRNA
181

ngRNA
171

TABLE 19

Sequence region
SEQ ID

pegRNA Spacer
5248

PBS
5249

PBS
5250

PBS
5251

PBS
5252

PBS
5253

PBS
5254

PBS
5255

PBS
5256

PBS
5257

PBS
5258

PBS
5259

RTT
5260

RTT
5261

RTT
5262

RTT
5263

RTT
5264

RTT
5265

RTT
5266

RTT
5267

RTT
5268

RTT
5269

RTT
5270

RTT
5271

RTT
5272

RTT
5273

RTT
5274

RTT
5275

RTT
5276

RTT
5277

RTT
5278

RTT
5279

ngRNA Spacer
40

ngRNA Spacer
738

ngRNA Spacer
529

ngRNA Spacer
44

ngRNA Spacer
29

ngRNA Spacer
26

ngRNA Spacer
42

ngRNA Spacer
5280

ngRNA Spacer
30

ngRNA Spacer
50

ngRNA Spacer
46

ngRNA Spacer
5219

ngRNA Spacer
25

ngRNA Spacer
52

ngRNA Spacer
737

ngRNA Spacer
55

ngRNA Spacer
49

ngRNA Spacer
48

ngRNA Spacer
5281

ngRNA Spacer
740

ngRNA Spacer
739

ngRNA Spacer
57

ngRNA Spacer
203

ngRNA Spacer
43

ngRNA Spacer
200

ngRNA Spacer
33

ngRNA Spacer
24

ngRNA Spacer
54

ngRNA Spacer
45

ngRNA Spacer
21

ngRNA Spacer
47

ngRNA Spacer
37

ngRNA Spacer
19

ngRNA Spacer
58

ngRNA Spacer
32

ngRNA Spacer
59

ngRNA Spacer
5220

ngRNA Spacer
56

ngRNA Spacer
736

ngRNA Spacer
67

ngRNA Spacer
28

ngRNA Spacer
39

ngRNA Spacer
20

ngRNA Spacer
70

ngRNA Spacer
199

ngRNA Spacer
22

ngRNA Spacer
68

ngRNA Spacer
5221

ngRNA Spacer
18

ngRNA Spacer
36

ngRNA Spacer
27

ngRNA Spacer
31

ngRNA Spacer
23

ngRNA Spacer
53

ngRNA Spacer
35

ngRNA Spacer
38

ngRNA Spacer
34

ngRNA Spacer
72

ngRNA Spacer
5227

ngRNA Spacer
5225

ngRNA Spacer
5224

ngRNA Spacer
5226

ngRNA Spacer
5223

ngRNA Spacer
51

ngRNA Spacer
5222

ngRNA
164

ngRNA
162

ngRNA
160

ngRNA
1503

ngRNA
161

ngRNA
153

ngRNA
1502

ngRNA
290

ngRNA
168

ngRNA
167

ngRNA
169

ngRNA
157

ngRNA
165

ngRNA
158

ngRNA
166

ngRNA
291

ngRNA
154

ngRNA
156

ngRNA
163

ngRNA
159

ngRNA
681

ngRNA
155

ngRNA
1501

ngRNA
174

ngRNA
176

ngRNA
181

ngRNA
179

ngRNA
175

ngRNA
292

ngRNA
1504

ngRNA
172

ngRNA
171

ngRNA
293

ngRNA
170

ngRNA
173

ngRNA
177

ngRNA
178

ngRNA
180

TABLE 20

Sequence region
SEQ ID

pegRNA Spacer
5282

PBS
5283

PBS
5284

PBS
5285

PBS
5286

PBS
5287

PBS
5288

PBS
5289

PBS
5290

PBS
5291

PBS
5292

PBS
5293

RTT
5294

RTT
5295

RTT
5296

RTT
5297

RTT
5298

RTT
5299

RTT
5300

RTT
5301

RTT
5302

ngRNA Spacer
202

ngRNA Spacer
5303

ngRNA Spacer
337

ngRNA Spacer
5304

ngRNA Spacer
5305

ngRNA Spacer
209

ngRNA Spacer
205

ngRNA Spacer
5306

ngRNA Spacer
204

ngRNA Spacer
740

ngRNA Spacer
70

ngRNA Spacer
33

ngRNA Spacer
35

ngRNA Spacer
206

ngRNA Spacer
50

ngRNA Spacer
5307

ngRNA Spacer
201

ngRNA Spacer
27

ngRNA Spacer
68

ngRNA Spacer
5308

ngRNA Spacer
30

ngRNA Spacer
5309

ngRNA Spacer
5310

ngRNA Spacer
200

ngRNA Spacer
736

ngRNA Spacer
207

ngRNA Spacer
208

ngRNA Spacer
5311

ngRNA Spacer
5312

ngRNA
1503

ngRNA
153

ngRNA
291

ngRNA
154

ngRNA
155

ngRNA
167

ngRNA
157

ngRNA
165

ngRNA
163

ngRNA
166

ngRNA
1501

ngRNA
160

ngRNA
162

ngRNA
173

ngRNA
172

ngRNA
171

ngRNA
170

ngRNA
178

ngRNA
1504

ngRNA
177

ngRNA
180

ngRNA
292

ngRNA
181

TABLE 21

Sequence region
SEQ ID

pegRNA Spacer
5313

PBS
5314

PBS
5315

PBS
5316

PBS
5317

PBS
5318

PBS
5319

PBS
5320

PBS
5321

PBS
5322

PBS
5323

PBS
5324

RTT
5325

RTT
5326

RTT
5327

RTT
5328

RTT
5329

RTT
5330

RTT
5331

RTT
5332

RTT
5333

RTT
5334

RTT
5335

RTT
5336

RTT
5337

RTT
5338

ngRNA Spacer
5312

ngRNA Spacer
5310

ngRNA Spacer
5308

ngRNA Spacer
207

ngRNA Spacer
5309

ngRNA Spacer
208

ngRNA Spacer
5303

ngRNA Spacer
5305

ngRNA Spacer
5311

ngRNA Spacer
201

ngRNA Spacer
5339

ngRNA Spacer
206

ngRNA Spacer
337

ngRNA Spacer
5307

ngRNA
160

ngRNA
165

ngRNA
166

ngRNA
155

ngRNA
291

ngRNA
171

ngRNA
177

ngRNA
172

ngRNA
292

TABLE 22

Sequence region
SEQ ID

pegRNA Spacer
5340

PBS
5341

PBS
5342

PBS
5343

PBS
5344

PBS
5345

PBS
5346

PBS
5347

PBS
5348

PBS
5349

PBS
5350

PBS
5351

RTT
5352

RTT
5353

RTT
5354

RTT
5355

RTT
5356

RTT
5357

RTT
5358

RTT
5359

RTT
5360

RTT
5361

RTT
5362

RTT
5363

RTT
5364

RTT
5365

RTT
5366

RTT
5367

RTT
5368

ngRNA Spacer
337

ngRNA Spacer
207

ngRNA Spacer
5305

ngRNA Spacer
5303

ngRNA Spacer
208

ngRNA Spacer
5307

ngRNA Spacer
5312

ngRNA Spacer
5339

ngRNA Spacer
206

ngRNA Spacer
5308

ngRNA Spacer
201

ngRNA Spacer
5311

ngRNA Spacer
5310

ngRNA Spacer
5309

ngRNA
160

ngRNA
165

ngRNA
166

ngRNA
155

ngRNA
291

ngRNA
171

ngRNA
292

ngRNA
172

ngRNA
177

TABLE 23

Sequence region
SEQ ID

pegRNA Spacer
5369

PBS
5370

PBS
5371

PBS
5372

PBS
5373

PBS
5374

PBS
5375

PBS
5376

PBS
5377

PBS
5378

PBS
5379

PBS
5380

RTT
5381

RTT
5382

RTT
5383

RTT
5384

RTT
5385

RTT
5386

RTT
5387

RTT
5388

RTT
5389

RTT
5390

RTT
5391

RTT
5392

RTT
5393

RTT
5394

RTT
5395

RTT
5396

RTT
5397

RTT
5398

RTT
5399

RTT
5400

RTT
5401

ngRNA Spacer
2434

ngRNA Spacer
2080

ngRNA Spacer
5402

ngRNA Spacer
2430

ngRNA Spacer
2436

ngRNA Spacer
5403

ngRNA Spacer
5404

ngRNA Spacer
2425

ngRNA Spacer
2052

ngRNA Spacer
2424

ngRNA Spacer
2088

ngRNA Spacer
5405

ngRNA Spacer
2426

ngRNA Spacer
2443

ngRNA Spacer
2427

ngRNA Spacer
2440

ngRNA Spacer
2048

ngRNA Spacer
2089

ngRNA Spacer
2439

ngRNA Spacer
2429

ngRNA Spacer
2435

ngRNA Spacer
2428

ngRNA Spacer
2444

ngRNA Spacer
2433

ngRNA Spacer
2070

ngRNA Spacer
2431

ngRNA Spacer
2086

ngRNA Spacer
2438

ngRNA Spacer
2432

ngRNA Spacer
2085

ngRNA Spacer
2423

ngRNA Spacer
2091

ngRNA
4412

ngRNA
4416

ngRNA
2270

ngRNA
2274

ngRNA
2262

ngRNA
2275

ngRNA
2272

ngRNA
4413

ngRNA
4415

ngRNA
2276

ngRNA
4417

ngRNA
2259

ngRNA
2260

ngRNA
2261

ngRNA
2257

ngRNA
4411

ngRNA
2264

ngRNA
2278

ngRNA
2282

ngRNA
2285

ngRNA
2283

ngRNA
2284

ngRNA
2288

ngRNA
4421

ngRNA
4419

ngRNA
2280

ngRNA
4420

ngRNA
2292

ngRNA
4424

TABLE 24

Sequence region
SEQ ID

pegRNA Spacer
5406

PBS
5407

PBS
5408

PBS
5409

PBS
5410

PBS
5411

PBS
5412

PBS
5413

PBS
5414

PBS
5415

PBS
5416

PBS
5417

RTT
5418

RTT
5419

RTT
5420

RTT
5421

RTT
5422

ngRNA Spacer
736

ngRNA Spacer
55

ngRNA Spacer
737

ngRNA Spacer
51

ngRNA Spacer
28

ngRNA Spacer
199

ngRNA Spacer
529

ngRNA Spacer
70

ngRNA Spacer
68

ngRNA Spacer
38

ngRNA Spacer
740

ngRNA Spacer
37

ngRNA Spacer
18

ngRNA Spacer
57

ngRNA Spacer
20

ngRNA Spacer
58

ngRNA Spacer
54

ngRNA Spacer
56

ngRNA Spacer
29

ngRNA Spacer
67

ngRNA Spacer
31

ngRNA Spacer
35

ngRNA Spacer
33

ngRNA Spacer
25

ngRNA Spacer
44

ngRNA Spacer
21

ngRNA Spacer
52

ngRNA Spacer
39

ngRNA Spacer
34

ngRNA Spacer
36

ngRNA Spacer
27

ngRNA Spacer
32

ngRNA Spacer
203

ngRNA Spacer
23

ngRNA Spacer
48

ngRNA Spacer
22

ngRNA Spacer
47

ngRNA Spacer
40

ngRNA Spacer
45

ngRNA Spacer
43

ngRNA Spacer
42

ngRNA Spacer
49

ngRNA Spacer
24

ngRNA Spacer
26

ngRNA Spacer
72

ngRNA Spacer
19

ngRNA Spacer
50

ngRNA Spacer
53

ngRNA Spacer
739

ngRNA Spacer
200

ngRNA Spacer
59

ngRNA Spacer
30

ngRNA Spacer
46

ngRNA
162

ngRNA
681

ngRNA
154

ngRNA
161

ngRNA
290

ngRNA
156

ngRNA
155

ngRNA
160

ngRNA
159

ngRNA
168

ngRNA
167

ngRNA
169

ngRNA
1503

ngRNA
165

ngRNA
158

ngRNA
166

ngRNA
1501

ngRNA
291

ngRNA
153

ngRNA
157

ngRNA
164

ngRNA
163

ngRNA
176

ngRNA
170

ngRNA
171

ngRNA
175

ngRNA
181

ngRNA
172

ngRNA
179

ngRNA
173

ngRNA
292

ngRNA
178

ngRNA
180

ngRNA
1504

ngRNA
293

ngRNA
177

ngRNA
174

TABLE 25

Sequence region
SEQ ID

pegRNA Spacer
5423

PBS
5424

PBS
5425

PBS
5426

PBS
5427

PBS
5428

PBS
5429

PBS
5430

PBS
5431

PBS
5432

PBS
5433

PBS
5434

RTT
5435

RTT
5436

RTT
5437

RTT
5438

RTT
5439

RTT
5440

RTT
5441

RTT
5442

RTT
5443

RTT
5444

RTT
5445

ngRNA Spacer
200

ngRNA Spacer
53

ngRNA Spacer
736

ngRNA Spacer
49

ngRNA Spacer
23

ngRNA Spacer
740

ngRNA Spacer
30

ngRNA Spacer
203

ngRNA Spacer
32

ngRNA Spacer
5280

ngRNA Spacer
22

ngRNA Spacer
35

ngRNA Spacer
45

ngRNA Spacer
26

ngRNA Spacer
70

ngRNA Spacer
737

ngRNA Spacer
59

ngRNA Spacer
44

ngRNA Spacer
46

ngRNA Spacer
37

ngRNA Spacer
56

ngRNA Spacer
58

ngRNA Spacer
20

ngRNA Spacer
21

ngRNA Spacer
67

ngRNA Spacer
38

ngRNA Spacer
738

ngRNA Spacer
47

ngRNA Spacer
34

ngRNA Spacer
68

ngRNA Spacer
52

ngRNA Spacer
54

ngRNA Spacer
51

ngRNA Spacer
739

ngRNA Spacer
43

ngRNA Spacer
18

ngRNA Spacer
42

ngRNA Spacer
39

ngRNA Spacer
72

ngRNA Spacer
57

ngRNA Spacer
55

ngRNA Spacer
25

ngRNA Spacer
27

ngRNA Spacer
33

ngRNA Spacer
29

ngRNA Spacer
31

ngRNA Spacer
40

ngRNA Spacer
50

ngRNA Spacer
19

ngRNA Spacer
24

ngRNA Spacer
529

ngRNA Spacer
28

ngRNA Spacer
36

ngRNA Spacer
48

ngRNA Spacer
199

ngRNA
681

ngRNA
154

ngRNA
158

ngRNA
1503

ngRNA
161

ngRNA
159

ngRNA
156

ngRNA
164

ngRNA
160

ngRNA
290

ngRNA
168

ngRNA
167

ngRNA
155

ngRNA
162

ngRNA
169

ngRNA
163

ngRNA
166

ngRNA
153

ngRNA
1501

ngRNA
291

ngRNA
157

ngRNA
1502

ngRNA
165

ngRNA
181

ngRNA
170

ngRNA
171

ngRNA
174

ngRNA
179

ngRNA
172

ngRNA
175

ngRNA
173

ngRNA
292

ngRNA
178

ngRNA
180

ngRNA
176

ngRNA
293

ngRNA
1504

ngRNA
177

TABLE 26

Sequence region
SEQ ID

pegRNA Spacer
5446

PBS
5447

PBS
5448

PBS
5449

PBS
5450

PBS
5451

PBS
5452

PBS
5453

PBS
5454

PBS
5455

PBS
5456

PBS
5457

RTT
5458

RTT
5459

RTT
5460

RTT
5461

RTT
5462

RTT
5463

RTT
5464

RTT
5465

RTT
5466

RTT
5467

RTT
5468

RTT
5469

RTT
5470

RTT
5471

RTT
5472

ngRNA Spacer
59

ngRNA Spacer
25

ngRNA Spacer
740

ngRNA Spacer
28

ngRNA Spacer
22

ngRNA Spacer
54

ngRNA Spacer
21

ngRNA Spacer
5280

ngRNA Spacer
5281

ngRNA Spacer
56

ngRNA Spacer
52

ngRNA Spacer
738

ngRNA Spacer
34

ngRNA Spacer
38

ngRNA Spacer
24

ngRNA Spacer
49

ngRNA Spacer
739

ngRNA Spacer
55

ngRNA Spacer
43

ngRNA Spacer
58

ngRNA Spacer
46

ngRNA Spacer
737

ngRNA Spacer
37

ngRNA Spacer
40

ngRNA Spacer
31

ngRNA Spacer
70

ngRNA Spacer
47

ngRNA Spacer
33

ngRNA Spacer
736

ngRNA Spacer
203

ngRNA Spacer
51

ngRNA Spacer
68

ngRNA Spacer
50

ngRNA Spacer
72

ngRNA Spacer
29

ngRNA Spacer
20

ngRNA Spacer
36

ngRNA Spacer
30

ngRNA Spacer
529

ngRNA Spacer
18

ngRNA Spacer
27

ngRNA Spacer
48

ngRNA Spacer
199

ngRNA Spacer
35

ngRNA Spacer
57

ngRNA Spacer
200

ngRNA Spacer
53

ngRNA Spacer
39

ngRNA Spacer
23

ngRNA Spacer
201

ngRNA Spacer
19

ngRNA Spacer
44

ngRNA Spacer
32

ngRNA Spacer
26

ngRNA Spacer
67

ngRNA Spacer
45

ngRNA Spacer
42

ngRNA
158

ngRNA
154

ngRNA
164

ngRNA
1503

ngRNA
161

ngRNA
155

ngRNA
156

ngRNA
1502

ngRNA
160

ngRNA
159

ngRNA
290

ngRNA
168

ngRNA
167

ngRNA
162

ngRNA
163

ngRNA
165

ngRNA
166

ngRNA
1501

ngRNA
153

ngRNA
29

ngRNA
169

ngRNA
681

ngRNA
157

ngRNA
181

ngRNA
170

ngRNA
177

ngRNA
174

ngRNA
179

ngRNA
180

ngRNA
175

ngRNA
173

ngRNA
292

ngRNA
178

ngRNA
172

ngRNA
176

ngRNA
293

ngRNA
1504

ngRNA
171

TABLE 27

Sequence region
SEQ ID

pegRNA Spacer
5473

PBS
5474

PBS
5475

PBS
5476

PBS
5477

PBS
5478

PBS
5479

PBS
5480

PBS
5481

PBS
5482

PBS
5483

PBS
5484

RTT
5485

RTT
5486

RTT
5487

RTT
5488

RTT
5489

RTT
5490

RTT
5491

RTT
5492

RTT
5493

RTT
5494

RTT
5495

RTT
5496

RTT
5497

RTT
5498

RTT
5499

RTT
5500

RTT
5501

RTT
5502

ngRNA Spacer
200

ngRNA Spacer
737

ngRNA Spacer
28

ngRNA Spacer
739

ngRNA Spacer
45

ngRNA Spacer
46

ngRNA Spacer
30

ngRNA Spacer
50

ngRNA Spacer
29

ngRNA Spacer
22

ngRNA Spacer
47

ngRNA Spacer
736

ngRNA Spacer
26

ngRNA Spacer
58

ngRNA Spacer
203

ngRNA Spacer
18

ngRNA Spacer
59

ngRNA Spacer
39

ngRNA Spacer
5280

ngRNA Spacer
68

ngRNA Spacer
43

ngRNA Spacer
54

ngRNA Spacer
33

ngRNA Spacer
740

ngRNA Spacer
34

ngRNA Spacer
55

ngRNA Spacer
49

ngRNA Spacer
51

ngRNA Spacer
19

ngRNA Spacer
42

ngRNA Spacer
67

ngRNA Spacer
57

ngRNA Spacer
36

ngRNA Spacer
23

ngRNA Spacer
21

ngRNA Spacer
738

ngRNA Spacer
38

ngRNA Spacer
529

ngRNA Spacer
31

ngRNA Spacer
48

ngRNA Spacer
53

ngRNA Spacer
35

ngRNA Spacer
5281

ngRNA Spacer
25

ngRNA Spacer
52

ngRNA Spacer
27

ngRNA Spacer
70

ngRNA Spacer
40

ngRNA Spacer
32

ngRNA Spacer
20

ngRNA Spacer
24

ngRNA Spacer
37

ngRNA Spacer
199

ngRNA Spacer
56

ngRNA Spacer
72

ngRNA Spacer
44

ngRNA
162

ngRNA
161

ngRNA
154

ngRNA
164

ngRNA
1503

ngRNA
155

ngRNA
681

ngRNA
158

ngRNA
1502

ngRNA
160

ngRNA
159

ngRNA
290

ngRNA
168

ngRNA
156

ngRNA
167

ngRNA
157

ngRNA
163

ngRNA
166

ngRNA
153

ngRNA
1501

ngRNA
291

ngRNA
169

ngRNA
165

ngRNA
175

ngRNA
1504

ngRNA
176

ngRNA
179

ngRNA
177

ngRNA
170

ngRNA
180

ngRNA
171

ngRNA
173

ngRNA
293

ngRNA
172

ngRNA
174

ngRNA
178

ngRNA
292

ngRNA
181

TABLE 28

Sequence region
SEQ ID

pegRNA Spacer
5503

PBS
5504

PBS
5505

PBS
5506

PBS
5507

PBS
5508

PBS
5509

PBS
5510

PBS
5511

PBS
5512

PBS
5513

PBS
5514

RTT
5515

RTT
5516

RTT
5517

RTT
5518

RTT
5519

RTT
5520

RTT
5521

RTT
5522

RTT
5523

RTT
5524

RTT
5525

RTT
5526

RTT
5527

RTT
5528

RTT
5529

RTT
5530

RTT
5531

RTT
5532

RTT
5533

RTT
5534

RTT
5535

ngRNA Spacer
2052

ngRNA Spacer
2437

ngRNA Spacer
2096

ngRNA Spacer
2074

ngRNA Spacer
2066

ngRNA Spacer
2065

ngRNA Spacer
2094

ngRNA Spacer
2090

ngRNA Spacer
2444

ngRNA Spacer
2073

ngRNA Spacer
2077

ngRNA Spacer
2063

ngRNA Spacer
2046

ngRNA Spacer
2064

ngRNA Spacer
2089

ngRNA Spacer
2060

ngRNA Spacer
2091

ngRNA Spacer
2086

ngRNA Spacer
2093

ngRNA Spacer
2079

ngRNA Spacer
2054

ngRNA Spacer
2049

ngRNA Spacer
2443

ngRNA Spacer
2057

ngRNA Spacer
2069

ngRNA Spacer
2085

ngRNA Spacer
2059

ngRNA Spacer
2047

ngRNA Spacer
2071

ngRNA Spacer
2081

ngRNA Spacer
2072

ngRNA Spacer
2048

ngRNA Spacer
2068

ngRNA Spacer
2045

ngRNA Spacer
2078

ngRNA Spacer
5536

ngRNA Spacer
2088

ngRNA Spacer
2055

ngRNA Spacer
2084

ngRNA Spacer
2050

ngRNA Spacer
2092

ngRNA Spacer
2062

ngRNA Spacer
2087

ngRNA Spacer
2082

ngRNA Spacer
2061

ngRNA Spacer
2080

ngRNA Spacer
2095

ngRNA Spacer
2056

ngRNA Spacer
2083

ngRNA Spacer
2442

ngRNA Spacer
2075

ngRNA Spacer
2058

ngRNA Spacer
2067

ngRNA Spacer
2439

ngRNA Spacer
2051

ngRNA Spacer
2070

ngRNA Spacer
2076

ngRNA Spacer
2053

ngRNA
2270

ngRNA
2267

ngRNA
4416

ngRNA
2268

ngRNA
2263

ngRNA
2262

ngRNA
2265

ngRNA
2272

ngRNA
2275

ngRNA
4411

ngRNA
2276

ngRNA
2266

ngRNA
2258

ngRNA
2271

ngRNA
2261

ngRNA
2259

ngRNA
2274

ngRNA
2260

ngRNA
2257

ngRNA
2277

ngRNA
4418

ngRNA
2273

ngRNA
2264

ngRNA
2269

ngRNA
2279

ngRNA
2281

ngRNA
2285

ngRNA
2283

ngRNA
4420

ngRNA
2280

ngRNA
2286

ngRNA
2282

ngRNA
2284

ngRNA
2278

ngRNA
2288

ngRNA
2287

ngRNA
4422

ngRNA
2289

ngRNA
2291

ngRNA
2290

ngRNA
2292

TABLE 29

Sequence region
SEQ ID

pegRNA Spacer
5537

PBS
5538

PBS
5539

PBS
5540

PBS
5541

PBS
5542

PBS
5543

PBS
5544

PBS
5545

PBS
5546

PBS
5547

PBS
5548

RTT
5549

RTT
5550

RTT
5551

RTT
5552

RTT
5553

RTT
5554

ngRNA Spacer
70

ngRNA Spacer
30

ngRNA Spacer
204

ngRNA Spacer
68

ngRNA Spacer
209

ngRNA Spacer
206

ngRNA Spacer
208

ngRNA Spacer
201

ngRNA Spacer
35

ngRNA Spacer
337

ngRNA Spacer
27

ngRNA Spacer
202

ngRNA Spacer
200

ngRNA Spacer
736

ngRNA Spacer
33

ngRNA Spacer
5306

ngRNA Spacer
50

ngRNA Spacer
205

ngRNA Spacer
207

ngRNA Spacer
740

ngRNA
1503

ngRNA
153

ngRNA
157

ngRNA
162

ngRNA
167

ngRNA
165

ngRNA
291

ngRNA
163

ngRNA
166

ngRNA
1501

ngRNA
160

ngRNA
154

ngRNA
181

ngRNA
171

ngRNA
1504

ngRNA
292

ngRNA
173

ngRNA
170

ngRNA
172

ngRNA
180

ngRNA
178

ngRNA
177

TABLE 30

Sequence region
SEQ ID

pegRNA Spacer
5555

PBS
5556

PBS
5557

PBS
5558

PBS
5559

PBS
5560

PBS
5561

PBS
5562

PBS
5563

PBS
5564

PBS
5565

PBS
5566

RTT
5567

RTT
5568

RTT
5569

RTT
5570

RTT
5571

RTT
5572

RTT
5573

RTT
5574

RTT
5575

RTT
5576

RTT
5577

RTT
5578

RTT
5579

RTT
5580

RTT
5581

RTT
5582

RTT
5583

RTT
5584

RTT
5585

RTT
5586

RTT
5587

RTT
5588

RTT
5589

RTT
5590

ngRNA Spacer
65

ngRNA Spacer
64

ngRNA Spacer
63

ngRNA Spacer
61

ngRNA Spacer
62

ngRNA Spacer
69

ngRNA Spacer
66

ngRNA Spacer
60

ngRNA Spacer
41

ngRNA Spacer
71

pegRNA
5591

pegRNA
5592

pegRNA
5593

pegRNA
5594

pegRNA
5595

pegRNA
5596

pegRNA
5597

pegRNA
5598

pegRNA
5599

pegRNA
5600

pegRNA
5601

pegRNA
5602

pegRNA
5603

pegRNA
5604

pegRNA
5605

pegRNA
5606

pegRNA
5607

pegRNA
5608

pegRNA
5609

pegRNA
5610

pegRNA
5611

pegRNA
5612

pegRNA
5613

pegRNA
5614

pegRNA
5615

pegRNA
5616

pegRNA
5617

pegRNA
5618

pegRNA
5619

pegRNA
5620

pegRNA
5621

pegRNA
5622

pegRNA
5623

pegRNA
5624

pegRNA
5625

pegRNA
5626

pegRNA
5627

pegRNA
5628

pegRNA
5629

pegRNA
5630

pegRNA
5631

pegRNA
5632

pegRNA
5633

pegRNA
5634

pegRNA
5635

pegRNA
5636

pegRNA
5637

TABLE 31

Sequence region
SEQ ID

pegRNA Spacer
5638

PBS
5639

PBS
5640

PBS
5641

PBS
5642

PBS
5643

PBS
5644

PBS
5645

PBS
5646

PBS
5647

PBS
5648

PBS
5649

RTT
5650

RTT
5651

RTT
5652

RTT
5653

RTT
5654

RTT
5655

RTT
5656

RTT
5657

RTT
5658

RTT
5659

RTT
5660

RTT
5661

RTT
5662

RTT
5663

RTT
5664

RTT
5665

RTT
5666

RTT
5667

RTT
5668

ngRNA Spacer
65

ngRNA Spacer
64

ngRNA Spacer
62

ngRNA Spacer
61

ngRNA Spacer
63

ngRNA Spacer
69

ngRNA Spacer
66

ngRNA Spacer
60

ngRNA Spacer
41

ngRNA Spacer
71

pegRNA
5669

pegRNA
5670

pegRNA
5671

pegRNA
5672

pegRNA
5673

pegRNA
5674

pegRNA
5675

pegRNA
5676

pegRNA
5677

pegRNA
5678

pegRNA
5679

pegRNA
5680

pegRNA
5681

pegRNA
5682

pegRNA
5683

pegRNA
5684

pegRNA
5685

pegRNA
5686

pegRNA
5687

pegRNA
5688

pegRNA
5689

pegRNA
5690

pegRNA
5691

pegRNA
5692

pegRNA
5693

pegRNA
5694

pegRNA
5695

pegRNA
5696

pegRNA
5697

pegRNA
5698

pegRNA
5699

pegRNA
5700

pegRNA
5701

pegRNA
5702

pegRNA
5703

pegRNA
5704

pegRNA
5705

TABLE 32

Sequence region
SEQ ID

pegRNA Spacer
5706

PBS
5707

PBS
5708

PBS
5709

PBS
5710

PBS
5711

PBS
5712

PBS
5713

PBS
5714

PBS
5715

PBS
5716

PBS
5717

RTT
5718

RTT
5719

RTT
5720

RTT
5721

RTT
5722

RTT
5723

RTT
5724

RTT
5725

RTT
5726

RTT
5727

RTT
5728

RTT
5729

RTT
5730

RTT
5731

RTT
5732

RTT
5733

RTT
5734

RTT
5735

RTT
5736

RTT
5737

RTT
5738

ngRNA Spacer
65

ngRNA Spacer
64

ngRNA Spacer
62

ngRNA Spacer
61

ngRNA Spacer
63

ngRNA Spacer
69

ngRNA Spacer
66

ngRNA Spacer
60

ngRNA Spacer
41

ngRNA Spacer
71

pegRNA
5739

pegRNA
5740

pegRNA
5741

pegRNA
5742

pegRNA
5743

pegRNA
5744

pegRNA
5745

pegRNA
5746

pegRNA
5747

pegRNA
5748

pegRNA
5749

pegRNA
5750

pegRNA
5751

pegRNA
5752

pegRNA
5753

pegRNA
5754

pegRNA
5755

pegRNA
5756

pegRNA
5757

pegRNA
5758

pegRNA
5759

pegRNA
5760

pegRNA
5761

pegRNA
5762

pegRNA
5763

pegRNA
5764

pegRNA
5765

pegRNA
5766

pegRNA
5767

pegRNA
5768

pegRNA
5769

pegRNA
5770

pegRNA
5771

pegRNA
5772

pegRNA
5773

pegRNA
5774

pegRNA
5775

pegRNA
5776

pegRNA
5777

pegRNA
5778

pegRNA
5779

In some embodiments, a PEgRNA as described herein comprises a spacer comprising a PEgRNA spacer sequence as provided in Table x, a PBS comprising a PBS sequence as provided in Table x, and an editing template comprising an RTT sequence as provided in Table x, wherein for each PEgRNA, x is the same integer for the spacer, the PBS, and the editing template, and wherein x is an integer selected from 6, 7, 8, 9, 10, 11, 12, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, and 32. In some embodiments, the PEgRNA is a part of a prime editing system, wherein the PEgRNA comprises a spacer comprising a PEgRNA spacer sequence as provided in Table x, a PBS comprising a PBS sequence as provided in Table x, and an editing template comprising an RTT sequence as provided in Table x, and wherein the prime editing system further comprises an ngRNA, wherein the ngRNA comprises a ngRNA spacer sequence as provided in Table x, wherein x is the same integer for the spacer, PBS, and editing template selection and for the ngRNA spacer selection, and wherein x is an integer selected from 6, 7, 8, 9, 10, 11, 12, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 and 32.

In some embodiments, the PEgRNA and/or the ngRNA comprises a gRNA core, wherein the gRNA core comprises a sequence selected from SEQ ID Nos 5857-5859.

Table 6 provides Prime Editing guide RNAs (PEgRNAs), which can be used in Prime Editing systems disclosed herein. Such Prime Editing systems can comprise a Cas9 protein capable of recognizing an NGG or an NG PAM sequence (e.g., TGG or TG), and a reverse transcriptase. The Prime Editing systems (e.g., PE3 or PE3b systems) can further comprise a nick guide RNA (ngRNA). Such PEgRNAs and Prime Editing systems can be used, for example, to correct an H1069Q mutation in ATP7B.

The PEgRNAs of Table 6 comprise: (a) a spacer comprising nucleotides 5-20 of SEQ ID NO: 1, (b) a gRNA core capable of binding to a Cas9 protein, and (c) an extension arm comprising: (i) an editing template comprising any one of SEQ ID NOs: 13-17, and (ii) a primer binding site (PBS) comprising any one of SEQ ID NOs: 2-12. The spacer of the PEgRNA can comprise, for example, nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 1. The spacer of the PEgRNA can comprise SEQ ID NO: 1. The RTT and the PBS can comprise respectively SEQ ID NOs: 13 and 2, 13 and 3, 13 and 4, 13 and 5, 13 and 6, 13 and 7, 13 and 8, 13 and 9, 13 and 10, 13 and 11, 13 and 12, 14 and 2, 14 and 3, 14 and 4, 14 and 5, 14 and 6, 14 and 7, 14 and 8, 14 and 9, 14 and 10, 14 and 11, 14 and 12, 15 and 2, 15 and 3, 15 and 4, 15 and 5, 15 and 6, 15 and 7, 15 and 8, 15 and 9, 15 and 10, 15 and 11, 15 and 12, 16 and 2, 16 and 3, 16 and 4, 16 and 5, 16 and 6, 16 and 7, 16 and 8, 16 and 9, 16 and 10, 16 and 11, 16 and 12, 17 and 2, 17 and 3, 17 and 4, 17 and 5, 17 and 6, 17 and 7, 17 and 8, 17 and 9, 17 and 10, 17 and 11, or 17 and 12. The gRNA core of the PEgRNA can comprise SEQ ID NO: 5857-5859. Exemplary PEgRNAs provided in Table 6 can comprise SEQ ID NOs: 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 106, 107, 108, 109, 111, 116, 117, or 120. Any PEgRNA sequence disclosed in Table 6 may further comprise a 3′ motif at the 3′ end of the extension arm, for example, a hairpin-forming motif or a series of 3, 4 or more U nucleotides. Without being bound by theory, such 3′ motifs are believed to increase PEgRNA stability. Such PEgRNA sequences may alternatively or additionally be adapted for transcription from a DNA template, for example, by including a 5′ terminal G if the spacer of the PEgRNA begins with another nucleotide, by including 6 or 7 U nucleotides at the 3′ end of the extension arm, or both. Exemplary transcription-adapted sequences include SEQ ID NOs: 105, 110, 112, 113, 114, 115, 118, 119, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, and 152. Such plasmid adapted sequences may further comprise a hairpin-forming motif between the PBS and the 3′ terminal U series.

Any of the PEgRNAs of Table 6 can be used in a Prime Editing system further comprising a nick guide RNA (ngRNA). Such ngRNA can comprise a spacer comprising nucleotides 5-20 of any one of SEQ ID NOs: 18-72 and a gRNA core capable of binding to a Cas9 protein. The spacer of the ngRNA can comprise nucleotides 4-20, 3-20, 2-20, or 1-20 of any one of SEQ ID NO: 18-72. The spacer of the ngRNA can comprise any one of SEQ ID NO: 18-72. The gRNA core of the ngRNA can comprise SEQ ID NO: 5857-5859. Exemplary ngRNA provided in Table 6 can comprise any one of SEQ ID NOs: 153-181. Such ngRNA sequences may further comprise a 3′ motif at the 3′ end of the gRNA core, for example, a series of 3, 4 or more U nucleotides. Without being bound by theory, such 3′ motifs are believed to increase ngRNA stability.

Table 7 provides Prime Editing guide RNAs (PEgRNAs), which can be used in Prime Editing systems disclosed herein. Such Prime Editing systems can comprise a Cas9 protein capable of recognizing an NGG, NG, or NNGG PAM sequence (e.g., TGG, TG, or TGGG), and a reverse transcriptase. The Prime Editing systems (e.g., PE3 or PE3b systems) can further comprise a nick guide RNA (ngRNA). Such PEgRNAs and Prime Editing systems can be used, for example, to correct an H1069Q mutation in ATP7B.

The PEgRNAs of Table 7 comprise: (a) a spacer comprising nucleotides 5-20 of SEQ ID NO: 182, (b) a gRNA core capable of binding to a Cas9 protein, and (c) an extension arm comprising: (i) an editing template comprising any one of SEQ ID NOs: 194-198, and (ii) a primer binding site (PBS) comprising any one of SEQ ID NOs: 183-193. The spacer of the PEgRNA can comprise, for example, nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 182. The spacer of the PEgRNA can comprise SEQ ID NO: 182. The RTT and the PBS can comprise respectively SEQ ID NOs: 194 and 183, 194 and 184, 194 and 185, 194 and 186, 194 and 187, 194 and 188, 194 and 189, 194 and 190, 194 and 191, 194 and 192, 194 and 193, 195 and 183, 195 and 184, 195 and 185, 195 and 186, 195 and 187, 195 and 188, 195 and 189, 195 and 190, 195 and 191, 195 and 192, 195 and 193, 196 and 183, 196 and 184, 196 and 185, 196 and 186, 196 and 187, 196 and 188, 196 and 189, 196 and 190, 196 and 191, 196 and 192, 196 and 193, 197 and 183, 197 and 184, 197 and 185, 197 and 186, 197 and 187, 197 and 188, 197 and 189, 197 and 190, 197 and 191, 197 and 192, 197 and 193, 198 and 183, 198 and 184, 198 and 185, 198 and 186, 198 and 187, 198 and 188, 198 and 189, 198 and 190, 198 and 191, 198 and 192, or 198 and 193. The gRNA core of the PEgRNA can comprise SEQ ID NO: 5857-5859. Exemplary PEgRNAs provided in Table 7 can comprise SEQ ID NOs. 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226,227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 245, 247, 248, 250, 251, or 255. Such PEgRNA sequences may further comprise a 3′ motif at the 3′ end of the extension arm, for example, a hairpin-forming motif or a series of 3, 4 or more U nucleotides. Without being bound by theory, such 3′ motifs are believed to increase PEgRNA stability. Such PEgRNA sequences may alternatively or additionally be adapted for transcription from a DNA template, for example, by including a 5′ terminal G if the spacer of the PEgRNA begins with another nucleotide, by including 6 or 7 U nucleotides at the 3′ end of the extension arm, or both. Exemplary transcription-adapted sequences include SEQ ID NOs: 244, 246, 249, 252, 253, 254, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, and 289. Such plasmid adapted sequences may further comprise a hairpin-forming motif between the PBS and the 3′ terminal U series.

Any of the PEgRNAs of Table 7 can be used in a Prime Editing system further comprising a nick guide RNA (ngRNA). Such ngRNA can comprise a spacer comprising nucleotides 5-20 of SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, or 209 and a gRNA core capable of binding to a Cas9 protein. The spacer of the ngRNA can comprise nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, or 209. The spacer of the ngRNA can comprise SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, or 209. The gRNA core of the ngRNA can comprise SEQ ID NO: 5857-5859. Exemplary ngRNA provided in Table 7 can comprise SEQ ID NOs: 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 290, 291, 292, or 293. Such ngRNA sequences may further comprise a 3′ motif at the 3′ end of the gRNA core, for example, a series of 3, 4 or more U nucleotides. Without being bound by theory, such 3′ motifs are believed to increase ngRNA stability.

Table 8 provides Prime Editing guide RNAs (PEgRNAs), which can be used in Prime Editing systems disclosed herein. Such Prime Editing systems can comprise a Cas9 protein capable of recognizing an NGG, NG, or NNGG PAM sequence (e.g., GGG, GG, GGGG), and a reverse transcriptase. The Prime Editing systems (e.g., PE3 or PE3b systems) can further comprise a nick guide RNA (ngRNA). Such PEgRNAs and Prime Editing systems can be used, for example, to correct an H1069Q mutation in ATP7B.

The PEgRNAs of Table 8 comprise: (a) a spacer comprising nucleotides 5-20 of SEQ ID NO: 294, (b) a gRNA core capable of binding to a Cas9 protein, and (c) an extension arm comprising: (i) an editing template comprising any one of SEQ ID NOs: 306-336, and (ii) a primer binding site (PBS) comprising any one of SEQ ID NOs: 295-305. The spacer of the PEgRNA can comprise, for example, nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 294. The spacer of the PEgRNA can comprise SEQ ID NO: 294. The RTT and the PBS can comprise respectively SEQ ID NOs: 306 and 295, 306 and 296, 306 and 297, 306 and 298, 306 and 299, 306 and 300, 306 and 301, 306 and 302, 306 and 303, 306 and 304, 306 and 305, 307 and 295, 307 and 296, 307 and 297, 307 and 298, 307 and 299, 307 and 300, 307 and 301, 307 and 302, 307 and 303, 307 and 304, 307 and 305, 308 and 295, 308 and 296, 308 and 297, 308 and 298, 308 and 299, 308 and 300, 308 and 301, 308 and 302, 308 and 303, 308 and 304, 308 and 305, 309 and 295, 309 and 296, 309 and 297, 309 and 298, 309 and 299, 309 and 300, 309 and 301, 309 and 302, 309 and 303, 309 and 304, 309 and 305, 310 and 295, 310 and 296, 310 and 297, 310 and 298, 310 and 299, 310 and 300, 310 and 301, 310 and 302, 310 and 303, 310 and 304, 310 and 305, 311 and 295, 311 and 296, 311 and 297.311 and 298, 311 and 299, 311 and 300, 311 and 301, 311 and 302, 311 and 303, 311 and 304, 311 and 305, 312 and 295, 312 and 296, 312 and 297, 312 and 298, 312 and 299, 312 and 300, 312 and 301, 312 and 302, 312 and 303, 312 and 304, 312 and 305, 313 and 295, 313 and 296, 313 and 297.313 and 298, 313 and 299, 313 and 300, 313 and 301, 313 and 302, 313 and 303, 313 and 304, 313 and 305, 314 and 295, 314 and 296, 314 and 297, 314 and 298, 314 and 299, 314 and 300, 314 and 301, 314 and 302, 314 and 303, 314 and 304, 314 and 305, 315 and 295, 315 and 296, 315 and 297, 315 and 298, 315 and 299, 315 and 300, 315 and 301, 315 and 302, 315 and 303, 315 and 304, 315 and 305, 316 and 295, 316 and 296, 316 and 297, 316 and 298, 316 and 299, 316 and 304, 316 and 301, 316 and 302, 316 and 303, 316 and 304, 316 and 305, 317 and 295, 317 and 296, 317 and 297, 317 and 298, 317 and 299, 317 and 300, 317 and 301, 317 and 302, 317 and 303, 317 and 304, 317 and 305, 318 and 295, 318 and 296, 318 and 297, 318 and 298, 318 and 299, 318 and 300, 318 and 301, 318 and 302, 318 and 303, 318 and 304, 318 and 305, 319 and 295, 319 and 296, 319 and 297, 319 and 298, 319 and 299, 319 and 300, 319 and 301, 319 and 302, 319 and 303, 319 and 304, 319 and 305, 320 and 295, 320 and 296, 320 and 297, 320 and 298, 320 and 299, 320 and 300, 320 and 301, 320 and 302, 320 and 303, 320 and 304, 320 and 305, 321 and 295, 321 and 296, 321 and 297, 321 and 298, 321 and 299, 321 and 300, 321 and 301, 321 and 302, 321 and 303, 321 and 304, 321 and 305, 322 and 295, 322 and 296, 322 and 297, 322 and 298, 322 and 299, 322 and 300, 322 and 301, 322 and 302, 322 and 303, 322 and 304, 322 and 305, 323 and 295, 323 and 296, 323 and 297, 323 and 298, 323 and 299, 323 and 300, 323 and 301, 323 and 302, 323 and 303, 323 and 304, 323 and 305, 324 and 295, 324 and 296, 324 and 297, 324 and 298, 324 and 299, 324 and 300, 324 and 301, 324 and 302, 324 and 303, 324 and 304, 324 and 305, 325 and 295, 325 and 296, 325 and 297, 325 and 298, 325 and 299, 325 and 300, 325 and 301, 325 and 302, 325 and 303, 325 and 304, 325 and 305, 326 and 295, 326 and 296, 326 and 297, 326 and 298, 326 and 299, 326 and 300, 326 and 301, 326 and 302, 326 and 303, 326 and 304, 326 and 305, 327 and 295, 327 and 296, 327 and 297, 327 and 298, 327 and 299, 327 and 300, 327 and 301, 327 and 302, 327 and 303, 327 and 304, 327 and 305, 328 and 295, 328 and 296, 328 and 297, 328 and 298, 328 and 299, 328 and 300, 328 and 301, 328 and 302, 328 and 303, 328 and 304, 328 and 305, 329 and 295, 329 and 296, 329 and 297, 329 and 298, 329 and 299, 329 and 300, 329 and 301, 329 and 302, 329 and 303, 329 and 304, 329 and 305, 330 and 295, 330 and 296, 330 and 297, 330 and 298, 330 and 299, 330 and 300, 330 and 301, 330 and 302, 330 and 303, 330 and 304, 330 and 305, 331 and 295, 331 and 296, 331 and 297, 331 and 298, 331 and 299, 331 and 300, 331 and 301, 331 and 302, 331 and 303, 331 and 304, 331 and 305, 332 and 295, 332 and 296, 332 and 297, 332 and 298, 332 and 299, 332 and 300, 332 and 301, 332 and 302, 332 and 303, 332 and 304, 332 and 305, 333 and 295, 333 and 296, 333 and 297, 333 and 298, 333 and 299, 333 and 300, 333 and 301, 333 and 302, 333 and 303, 333 and 304, 333 and 305, 334 and 295, 334 and 296, 334 and 297, 334 and 298, 334 and 299, 334 and 300, 334 and 301, 334 and 302, 334 and 303, 334 and 304, 334 and 305, 335 and 295, 335 and 296, 335 and 297, 335 and 298, 335 and 299, 335 and 300, 335 and 301, 335 and 302, 335 and 303, 335 and 304, 335 and 305, 336 and 295, 336 and 296, 336 and 297, 336 and 298, 336 and 299, 336 and 300, 336 and 301, 336 and 302, 336 and 303, 336 and 304, or 336 and 305. The gRNA core of the PEgRNA can comprise SEQ ID NOs. any one of 5857-5859. Exemplary PEgRNAs provided in Table 8 can comprise SEQ ID NOs. 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 386, 389, 390, 391, 392, 394, 397, 398, 400, 401, 404, 406, 408, 415, 417, 425, 427, 429, 433, 442, 444, 448, 449, 453, 454, 457, 458, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, or 482. Such PEgRNA sequences may further comprise a 3′ motif at the 3′ end of the extension arm, for example, a hairpin-forming motif or a series of 3, 4 or more U nucleotides. Without being bound by theory, such 3′ motifs are believed to increase PEgRNA stability. Such PEgRNA sequences may alternatively or additionally be adapted for transcription from a DNA template, for example, by including a 5′ terminal G if the spacer of the PEgRNA begins with another nucleotide, by including 6 or 7 U nucleotides at the 3′ end of the extension arm, or both. Exemplary transcription-adapted sequences include SEQ ID NOs: 385, 387, 388, 393, 395, 396, 399, 402, 403, 405, 407, 409, 410, 411, 412, 413, 414, 416, 418, 419, 420, 421, 422, 423, 424, 426, 428, 430, 431, 432, 434, 435, 436, 437, 438, 439, 440, 441, 443, 445, 446, 447, 450, 451, 452, 455, 456, and 459. Such plasmid adapted sequences may further comprise a hairpin-forming motif between the PBS and the 3′ terminal U series.

Any of the PEgRNAs of Table 8 can be used in a Prime Editing system further comprising a nick guide RNA (ngRNA). Such ngRNA can comprise a spacer comprising nucleotides 5-20 of SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, or 337 and a gRNA core capable of binding to a Cas9 protein. The spacer of the ngRNA can comprise nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, or 337. The spacer of the ngRNA can comprise SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, or 337. The gRNA core of the ngRNA can comprise SEQ ID NO. 5857-5859. Exemplary ngRNA provided in Table 8 can comprise SEQ ID NO: 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 290, 291, 292, or 293. Such ngRNA sequences may further comprise a 3′ motif at the 3′ end of the gRNA core, for example, a series of 3, 4 or more U nucleotides. Without being bound by theory, such 3′ motifs are believed to increase ngRNA stability.

Table 9 provides Prime Editing guide RNAs (PEgRNAs), which can be used in Prime Editing systems disclosed herein. Such Prime Editing systems can comprise a Cas9 protein capable of recognizing an NGG or NG PAM sequence (e.g., GGG or GG), and a reverse transcriptase. The Prime Editing systems (e.g., PE3 or PE3b systems) can further comprise a nick guide RNA (ngRNA). Such PEgRNAs and Prime Editing systems can be used, for example, to correct an H1069Q mutation in ATP7B.

The PEgRNAs of Table 9 comprise: (a) a spacer comprising nucleotides 5-20 of SEQ ID NO: 483, (b) a gRNA core capable of binding to a Cas9 protein, and (c) an extension arm comprising: (i) an editing template comprising any one of SEQ ID NOs: 495-528, and (ii) a primer binding site (PBS) comprising any one of SEQ ID NOs: 484-494. The spacer of the PEgRNA can comprise, for example, nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 483. The spacer of the PEgRNA can comprise SEQ ID NO: 483. The RTT and the PBS can comprise respectively SEQ ID NOs: 495 and 484, 495 and 485, 495 and 486, 495 and 487, 495 and 488, 495 and 489, 495 and 490, 495 and 491, 495 and 492, 495 and 493, 495 and 494, 496 and 484, 496 and 485, 496 and 486, 496 and 487, 496 and 488, 496 and 489, 496 and 490, 496 and 491, 496 and 492, 496 and 493, 496 and 494, 497 and 484, 497 and 485, 497 and 486, 497 and 487, 497 and 488, 497 and 489, 497 and 490, 497 and 491, 497 and 492, 497 and 493, 497 and 494, 498 and 484, 498 and 485, 498 and 486, 498 and 487, 498 and 488, 498 and 489, 498 and 490, 498 and 491, 498 and 492, 498 and 493, 498 and 494, 499 and 484, 499 and 485, 499 and 486, 499 and 487, 499 and 488, 499 and 489, 499 and 490, 499 and 491, 499 and 492, 499 and 493, 499 and 494, 500 and 484, 500 and 485, 500 and 486, 500 and 487, 500 and 488, 500 and 489, 500 and 490, 500 and 491, 500 and 492, 500 and 493, 500 and 494, 501 and 484, 501 and 485, 501 and 486, 501 and 487, 501 and 488, 501 and 489, 501 and 490, 501 and 491, 501 and 492, 501 and 493, 501 and 494, 502 and 484, 502 and 485, 502 and 486, 502 and 487, 502 and 488, 502 and 489, 502 and 490, 502 and 491, 502 and 492, 502 and 493, 502 and 494, 503 and 484, 503 and 485, 503 and 486, 503 and 487, 503 and 488, 503 and 489, 503 and 490, 503 and 491, 503 and 492, 503 and 493, 503 and 494, 504 and 484, 504 and 485, 504 and 486, 504 and 487, 504 and 488, 504 and 489, 504 and 490, 504 and 491, 504 and 492, 504 and 493, 504 and 494, 505 and 484, 505 and 485, 505 and 486, 505 and 487, 505 and 488, 505 and 489, 505 and 490, 505 and 491, 505 and 492, 505 and 493, 505 and 494, 506 and 484, 506 and 485, 506 and 486, 506 and 487, 506 and 488, 506 and 489, 506 and 490, 506 and 491, 506 and 492, 506 and 493, 506 and 494, 507 and 484, 507 and 485, 507 and 486, 507 and 487, 507 and 488, 507 and 489, 507 and 490, 507 and 491, 507 and 492, 507 and 493, 507 and 494, 508 and 484, 508 and 485, 508 and 486, 508 and 487, 508 and 488, 508 and 489, 508 and 490, 508 and 491, 508 and 492, 508 and 493, 508 and 494, 509 and 484, 509 and 485, 509 and 486, 509 and 487, 509 and 488, 509 and 489, 509 and 490, 509 and 491, 509 and 492, 509 and 493, 509 and 494, 510 and 484, 510 and 485, 510 and 486, 510 and 487, 510 and 488, 510 and 489, 510 and 490, 510 and 491, 510 and 492, 510 and 493, 510 and 494, 511 and 484, 511 and 485, 511 and 486, 511 and 487, 511 and 488, 511 and 489, 511 and 490, 511 and 491, 511 and 492, 511 and 493, 511 and 494, 512 and 484, 512 and 485, 512 and 486, 512 and 487, 512 and 488, 512 and 489, 512 and 490, 512 and 491, 512 and 492, 512 and 493, 512 and 494, 513 and 484, 513 and 485, 513 and 486, 513 and 487, 513 and 488, 513 and 489, 513 and 490, 513 and 491, 513 and 492, 513 and 493, 513 and 494, 514 and 484, 514 and 485, 514 and 486, 514 and 487, 514 and 488, 514 and 489, 514 and 490, 514 and 491, 514 and 492, 514 and 493, 514 and 494, 515 and 484, 515 and 485, 515 and 486, 515 and 487, 515 and 488, 515 and 489, 515 and 490, 515 and 491, 515 and 492, 515 and 493, 515 and 494, 516 and 484, 516 and 485, 516 and 486, 516 and 487, 516 and 488, 516 and 489, 516 and 490, 516 and 491, 516 and 492, 516 and 493, 516 and 494, 517 and 484, 517 and 485, 517 and 486, 517 and 487, 517 and 488, 517 and 489, 517 and 490, 517 and 491, 517 and 492, 517 and 493, 517 and 494, 518 and 484, 518 and 485, 518 and 486, 518 and 487, 518 and 488, 518 and 489, 518 and 490, 518 and 491, 518 and 492, 518 and 493, 518 and 494, 519 and 484, 519 and 485, 519 and 486, 519 and 487, 519 and 488, 519 and 489, 519 and 490, 519 and 491, 519 and 492, 519 and 493, 519 and 494, 520 and 484, 520 and 485, 520 and 486, 520 and 487, 520 and 488, 520 and 489, 520 and 490, 520 and 491, 520 and 492, 520 and 493, 520 and 494, 521 and 484, 521 and 485, 521 and 486, 521 and 487, 521 and 488, 521 and 489, 521 and 490, 521 and 491, 521 and 492, 521 and 493, 521 and 494, 522 and 484, 522 and 485, 522 and 486, 522 and 487, 522 and 488, 522 and 489, 522 and 490, 522 and 491, 522 and 492, 522 and 493, 522 and 494, 523 and 484, 523 and 485, 523 and 486, 523 and 487, 523 and 488, 523 and 489, 523 and 490, 523 and 491, 523 and 492, 523 and 493, 523 and 494, 524 and 484, 524 and 485, 524 and 486, 524 and 487, 524 and 488, 524 and 489, 524 and 490, 524 and 491, 524 and 492, 524 and 493, 524 and 494, 525 and 484, 525 and 485, 525 and 486, 525 and 487, 525 and 488, 525 and 489, 525 and 490, 525 and 491, 525 and 492, 525 and 493, 525 and 494, 526 and 484, 526 and 485, 526 and 486, 526 and 487, 526 and 488, 526 and 489, 526 and 490, 526 and 491, 526 and 492, 526 and 493, 526 and 494, 527 and 484, 527 and 485, 527 and 486, 527 and 487, 527 and 488, 527 and 489, 527 and 490, 527 and 491, 527 and 492, 527 and 493, 527 and 494, 528 and 484, 528 and 485, 528 and 486, 528 and 487, 528 and 488, 528 and 489, 528 and 490, 528 and 491, 528 and 492, 528 and 493, or 528 and 494. The gRNA core of the PEgRNA can comprise SEQ ID NO. 5857-5859. Exemplary PEgRNAs provided in Table 9 can comprise SEQ ID NO. 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 574, 575, 577, 578, 580, 581, 582, 583, 584, 585, 586, 588, 592, 593, 594, 597, 598, 600, 601, 608, 609, 612, 620, 621, 627, 628, 631, 639, 640, 644, 647, 649, 650, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, or 680. Such PEgRNA sequences may further comprise a 3′ motif at the 3′ end of the extension arm, for example, a hairpin-forming motif or a series of 3, 4 or more U nucleotides. Without being bound by theory, such 3′ motifs are believed to increase PEgRNA stability. Such PEgRNA sequences may alternatively or additionally be adapted for transcription from a DNA template, for example, by including a 5′ terminal G if the spacer of the PEgRNA begins with another nucleotide, by including 6 or 7 U nucleotides at the 3′ end of the extension arm, or both. Exemplary transcription-adapted sequences include SEQ ID NOs: 573, 576, 579, 587, 589, 590, 591, 595, 596, 599, 602, 603, 604, 605, 606, 607, 610, 611, 613, 614, 615, 616, 617,618, 619, 622, 623, 624, 625, 626, 629, 630, 632, 633, 634, 635, 636, 637, 638, 641, 642, 643, 645, 646, 648, 651, 652, and 653. Such plasm id adapted sequences may further comprise a hairpin-forming motif between the PBS and the 3′ terminal U series.

Any of the PEgRNAs of Table 9 can be used in a Prime Editing system further comprising a nick guide RNA (ngRNA). Such ngRNA can comprise a spacer comprising nucleotides 5-20 of SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 199, 200, 203, or 529 and a gRNA core capable of binding to a Cas9 protein. The spacer of the ngRNA can comprise nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 199, 200, 203, or 529. The spacer of the ngRNA can comprise SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 199, 200, 203, or 529. The gRNA core of the ngRNA can comprise SEQ ID NO. 5857-5859. Exemplary ngRNA provided in Table 9 can comprise SEQ ID NO: 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 290, 291, 292, 293, or 681. Such ngRNA sequences may further comprise a 3′ motif at the 3′ end of the gRNA core, for example, a series of 3, 4 or more U nucleotides. Without being bound by theory, such 3′ motifs are believed to increase ngRNA stability.

Table 10 provides Prime Editing guide RNAs (PEgRNAs), which can be used in Prime Editing systems disclosed herein. Such Prime Editing systems can comprise a Cas9 protein capable of recognizing an NOG or NO PAM sequence (e.g., CGG or CG), and a reverse transcriptase. The Prime Editing systems (e.g., PE3 or PE3b systems) can further comprise a nick guide RNA (ngRNA). Such PEgRNAs and Prime Editing systems can be used, for example, to correct an 11069Q mutation in ATP7B.

The PEgRNAs of Table 10 comprise: (a) a spacer comprising nucleotides 5-20 of SEQ ID NO: 682, (b) a gRNA core capable of binding to a Cas9 protein, and (c) an extension arm comprising: (i) an editing template comprising any one of SEQ ID NOs: 694-735, and (ii) a primer binding site (PBS) comprising any one of SEQ ID NOs: 683-693. The spacer of the PEgRNA can comprise, for example, nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 682. The spacer of the PEgRNA can comprise SEQ ID NO: 682. The RTT and the PBS can comprise respectively SEQ ID NOs: 694 and 683, 694 and 684, 694 and 685, 694 and 686, 694 and 687, 694 and 688, 694 and 689, 694 and 690, 694 and 691, 694 and 692, 694 and 693, 695 and 683, 695 and 684, 695 and 685, 695 and 686, 695 and 687, 695 and 688, 695 and 689, 695 and 690, 695 and 691, 695 and 692, 695 and 693, 696 and 683, 696 and 684, 696 and 685, 696 and 686, 696 and 687, 696 and 688, 696 and 689, 696 and 690, 696 and 691, 696 and 692, 696 and 693, 697 and 683, 697 and 684, 697 and 685, 697 and 686, 697 and 687, 697 and 688, 697 and 689, 697 and 690, 697 and 691, 697 and 692, 697 and 693, 698 and 683, 698 and 684, 698 and 685, 698 and 686, 698 and 687, 698 and 688, 698 and 689, 698 and 690, 698 and 691, 698 and 692, 698 and 693, 699 and 683, 699 and 684, 699 and 685, 699 and 686, 699 and 687, 699 and 688, 699 and 689, 699 and 690, 699 and 691, 699 and 692, 699 and 693, 700 and 683, 700 and 684, 700 and 685, 700 and 686, 700 and 687, 700 and 688, 700 and 689, 700 and 690, 700 and 691, 700 and 692, 700 and 693, 701 and 683, 701 and 684, 701 and 685, 701 and 686, 701 and 687, 701 and 688, 701 and 689, 701 and 690, 701 and 691, 701 and 692, 701 and 693, 702 and 683, 702 and 684, 702 and 685, 702 and 686, 702 and 687, 702 and 688, 702 and 689, 702 and 690, 702 and 691, 702 and 692, 702 and 693, 703 and 683, 703 and 684, 703 and 685, 703 and 686, 703 and 687, 703 and 688, 703 and 689, 703 and 690, 703 and 691, 703 and 692, 703 and 693, 704 and 683, 704 and 684, 704 and 685, 704 and 686, 704 and 687, 704 and 688, 704 and 689, 704 and 690, 704 and 691, 704 and 692, 704 and 693, 705 and 683, 705 and 684, 705 and 685, 705 and 686, 705 and 687, 705 and 688, 705 and 689, 705 and 690, 705 and 691, 705 and 692, 705 and 693, 706 and 683, 706 and 684, 706 and 685, 706 and 686, 706 and 687, 706 and 688, 706 and 689, 706 and 690, 706 and 691, 706 and 692, 706 and 693, 707 and 683, 707 and 684, 707 and 685, 707 and 686, 707 and 687, 707 and 688, 707 and 689, 707 and 690, 707 and 691, 707 and 692, 707 and 693, 708 and 683, 708 and 684, 708 and 685, 708 and 686, 708 and 687, 708 and 688, 708 and 689, 708 and 690, 708 and 691, 708 and 692, 708 and 693, 709 and 683, 709 and 684, 709 and 685, 709 and 686, 709 and 687, 709 and 688, 709 and 689, 709 and 690, 709 and 691, 709 and 692, 709 and 693, 710 and 683, 710 and 684, 710 and 685, 710 and 686, 710 and 687, 710 and 688, 710 and 689, 710 and 690, 710 and 691, 710 and 692, 710 and 693, 711 and 683, 711 and 684, 711 and 685, 711 and 686, 711 and 687, 711 and 688, 711 and 689, 711 and 690, 711 and 691, 711 and 692, 711 and 693, 712 and 683, 712 and 684, 712 and 685, 712 and 686, 712 and 687, 712 and 688, 712 and 689, 712 and 690, 712 and 691, 712 and 692, 712 and 693, 713 and 683, 713 and 684, 713 and 685, 713 and 686, 713 and 687, 713 and 688, 713 and 689, 713 and 690, 713 and 691, 713 and 692, 713 and 693, 714 and 683, 714 and 684, 714 and 685, 714 and 686, 714 and 687, 714 and 688, 714 and 689, 714 and 690, 714 and 691, 714 and 692, 714 and 693, 715 and 683, 715 and 684, 715 and 685, 715 and 686, 715 and 687, 715 and 688, 715 and 689, 715 and 690, 715 and 691, 715 and 692, 715 and 693, 716 and 683, 716 and 684, 716 and 685, 716 and 686, 716 and 687, 716 and 688, 716 and 689, 716 and 690, 716 and 691, 716 and 692, 716 and 693, 717 and 683, 717 and 684, 717 and 685, 717 and 686, 717 and 687, 717 and 688, 717 and 689, 717 and 690, 717 and 691, 717 and 692, 717 and 693, 718 and 683, 718 and 684, 718 and 685, 718 and 686, 718 and 687, 718 and 688, 718 and 689, 718 and 690, 718 and 691, 718 and 692, 718 and 693, 719 and 683, 719 and 684, 719 and 685, 719 and 686, 719 and 687, 719 and 688, 719 and 689, 719 and 690, 719 and 691, 719 and 692, 719 and 693, 720 and 683, 720 and 684, 720 and 685, 720 and 686, 720 and 687, 720 and 688, 720 and 689, 720 and 690, 720 and 691, 720 and 692, 720 and 693, 721 and 683, 721 and 684, 721 and 685, 721 and 686, 721 and 687, 721 and 688, 721 and 689, 721 and 690, 721 and 691, 721 and 692, 721 and 693, 722 and 683, 722 and 684, 722 and 685, 722 and 686, 722 and 687, 722 and 688, 722 and 689, 722 and 690, 722 and 691, 722 and 692, 722 and 693, 723 and 683, 723 and 684, 723 and 685, 723 and 686, 723 and 687, 723 and 688, 723 and 689, 723 and 690, 723 and 691, 723 and 692, 723 and 693, 724 and 683, 724 and 684, 724 and 685, 724 and 686, 724 and 687, 724 and 688, 724 and 689, 724 and 690, 724 and 691, 724 and 692, 724 and 693, 725 and 683, 725 and 684, 725 and 685, 725 and 686, 725 and 687, 725 and 688, 725 and 689, 725 and 690, 725 and 691, 725 and 692, 725 and 693, 726 and 683, 726 and 684, 726 and 685, 726 and 686, 726 and 687, 726 and 688, 726 and 689, 726 and 690, 726 and 691, 726 and 692, 726 and 693, 727 and 683, 727 and 684, 727 and 685, 727 and 686, 727 and 687, 727 and 688, 727 and 689, 727 and 690, 727 and 691, 727 and 692, 727 and 693, 728 and 683, 728 and 684, 728 and 685, 728 and 686, 728 and 687, 728 and 688, 728 and 689, 728 and 690, 728 and 691, 728 and 692, 728 and 693, 729 and 683, 729 and 684, 729 and 685, 729 and 686, 729 and 687, 729 and 688, 729 and 689, 729 and 690, 729 and 691, 729 and 692, 729 and 693, 730 and 683, 730 and 684, 730 and 685, 730 and 686, 730 and 687, 730 and 688, 730 and 689, 730 and 690, 730 and 691, 730 and 692, 730 and 693, 731 and 683, 731 and 684, 731 and 685, 731 and 686, 731 and 687, 731 and 688, 731 and 689, 731 and 690, 731 and 691, 731 and 692, 731 and 693, 732 and 683, 732 and 684, 732 and 685, 732 and 686, 732 and 687, 732 and 688, 732 and 689, 732 and 690, 732 and 691, 732 and 692, 732 and 693, 733 and 683, 733 and 684, 733 and 685, 733 and 686, 733 and 687, 733 and 688, 733 and 689, 733 and 690, 733 and 691, 733 and 692, 733 and 693, 734 and 683, 734 and 684, 734 and 685, 734 and 686, 734 and 687, 734 and 688, 734 and 689, 734 and 690, 734 and 691, 734 and 692, 734 and 693, 735 and 683, 735 and 684, 735 and 685, 735 and 686, 735 and 687, 735 and 688, 735 and 689, 735 and 690, 735 and 691, 735 and 692, or 735 and 693, The gRNA core of the PEgRNA can comprise SEQ ID NO. 5857-5859. Exemplary PEgRNAs provided in Table 10 can comprise SEQ ID NO. 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 865, 866, 869, 870, 871, 872, 873, 874, 878, 879, 880, 881, 882, 883, 885, 887, 888, 890, 893, 894, 895, 896, 897, 898, 900, 902, 905, 906, 908, 909, 910, 911, 912, 914, 916, 918, 920, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 959, 960, 961, 962, 963, 965, 966, 967, 970, 971, 977, 979, 983, 987, 989, 992, 997, 1000, 1018, 1019, 1020, 1021, 1022, 1023, 1024, 1025, 1037, 1041, 1043, 1044, 1045, 1046, 1050, 1057, 1068, 1074, 1076, 1077, 1078, 1079, 1080, 1081, 1082, 1084, 1085, 1087, 1101, 1103, 1104, 1105, 1106, 1107, 1108, 1109, 1110, 1111, 1112, 1113, 1114, 1115, 1116, 1124, 1139, 1140, 1141, 1144, 1147, 1151, 1155, 1159, 1160, 1163, 1166, 1167, 1168, 1169, 1171, 1172, 1185, 1187, 1190, 1191, 1197, 1199, 1203, 1208, 1209, 1210, 1218, 1219, 1220, 1221, 1222, 1223, 1229, 1230, 1233, 1235, 1236, 1237, 1240, 1244, 1248, 1261, 1263, 1264, 1265, 1266, 1267, 1268, 1269, 1270, 1272, 1273, 1278, 1282, 1285, 1289, 1292, 1294, 1297, 1299, 1305, 1309, 1310, 1311, 1313, 1314, 1315, 1316, 1317, 1318, 1319, 1329, 1333, 1334, 1338, 1340, 1342, 1344, 1345, 1346, 1347, 1348, 1353, 1355, 1357, 1362, 1363, 1364, 1368, 1369, 1370, 1376, 1383, 1388, 1395, 1396, 1413, 1414, 1415, or 1416. Such PEgRNA sequences may further comprise a 3′ motif at the 3′ end of the extension arm, for example, a hairpin-forming motif or a series of 3, 4 or more U nucleotides. Without being bound by theory, such 3′ motifs are believed to increase PEgRNA stability. Such PEgRNA sequences may alternatively or additionally be adapted for transcription from a DNA template, for example, by including a 5′ terminal G if the spacer of the PEgRNA begins with another nucleotide, by including 6 or 7 U nucleotides at the 3′ end of the extension arm, or both. Exemplary transcription-adapted sequences include SEQ ID NOs: 864, 867, 868, 875, 876, 877, 884, 886, 889, 891, 892, 899, 901, 903, 904, 907, 913, 915, 917, 919, 921, 940, 941, 942, 943, 944, 945, 946, 947, 948,949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 964, 968, 969, 972, 973, 974, 975, 976, 978, 980, 981, 982, 984, 985, 986, 988, 990, 991, 993, 994, 995, 996, 998, 999, 1001, 1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1010, 1011, 1012, 1013, 1014, 1015, 1016, 1017, 1026, 1027, 1028, 1029, 1030, 1031, 1032, 1033, 1034, 1035, 1036, 1038, 1039, 1040, 1042, 1047, 1048, 1049, 1051, 1052, 1053, 1054, 1055, 1056, 1058, 1059, 1060, 1061, 1062, 1063, 1064, 1065, 1066, 1067, 1069, 1070, 1071, 1072, 1073, 1075, 1083, 1086, 1088, 1089, 1090, 1091, 1092, 1093, 1094, 1095, 1096, 1097, 1098, 1099, 1100, 1102, 1117, 1118, 1119, 1120, 1121, 1122, 1123, 1125, 1126, 1127, 1128, 1129, 1130, 1131, 1132, 1133, 1134, 1135, 1136, 1137, 1138, 1142, 1143, 1145, 1146, 1148, 1149, 1150, 1152, 1153, 1154, 1156, 1157, 1158, 1161, 1162, 1164, 1165, 1170, 1173, 1174, 1175, 1176, 1177, 1178, 1179, 1180, 1181, 1182, 1183, 1184, 1186, 1188, 1189, 1192, 1193, 1194, 1195, 1196, 1198, 1200, 1201, 1202, 1204, 1205, 1206, 1207, 1211, 1212, 1213, 1214, 1215, 1216, 1217, 1224, 1225, 1226, 1227, 1228, 1231, 1232, 1234, 1238, 1239, 1241, 1242, 1243, 1245, 1246, 1247, 1249, 1250, 1251, 1252, 1253, 1254, 1255, 1256, 1257, 1258, 1259, 1260, 1262, 1271, 1274, 1275, 1276, 1277, 1279, 1280, 1281, 1283, 1284, 1286, 1287, 1288, 1290, 1291, 1293, 1295, 1296, 1298, 1300, 1301, 1302, 1303, 1304, 1306, 1307, 1308, 1312, 1320, 1321, 1322, 1323, 1324, 1325, 1326, 1327, 1328, 1330, 1331, 1332, 1335, 1336, 1337, 1339, 1341, 1343, 1349, 1350, 1351, 1352, 1354, 1356, 1358, 1359, 1360, 1361, 1365, 1366, 1367, 1371, 1372, 1373, 1374, 1375, 1377, 1378, 1379, 1380, 1381, 1382, 1384, 1385, 1386, 1387, 1389, 1390, 1391, 1392, 1393, 1394, 1397, 1398, 1399, 1400, 1401, 1402, 1403, 1404, 1405, 1406, 1407, 1408, 1409, 1410, 1411, 1412, 1417, 1418, 1419, 1420, 1421, 1422, 1423, 1424, 1425, 1426, 1427, 1428, 1429, 1430, 1431, 1432, 1433, 1434, 1435, 1436, 1437, 1438, 1439, 1440, 1441, 1442, 1443, 1444, 1445, 1446, 1447, 1448, 1449, 1450, 1451, 1452, 1453, 1454, 1455, 1456, 1457, 1458, 1459, 1460, 1461, 1462, 1463, 1464, 1465, 1466, 1467, 1468, 1469, 1470, 1471, 1472, 1473, 1474, 1475, 1476, 1477, 1478, 1479, 1480, 1481, 1482, 1483, 1484, 1485, 1486, 1487, 1488, 1489, 1490, 1491, 1492, 1493, 1494, 1495, 1496, 1497, 1498, 1499, and 1500. Such plasmid adapted sequences may further comprise a hairpin-forming motif between the PBS and the 3′ terminal U series.

Any of the PEgRNAs of Table 10 can be used in a Prime Editing system further comprising a nick guide RNA (ngRNA). Such ngRNA can comprise a spacer comprising nucleotides 5-20 of SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 199, 200, 203, 529, 736, 737, 738, 739, or 740 and a gRNA core capable of binding to a Cas9 protein. The spacer of the ngRNA can comprise nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 199, 200, 203, 529, 736, 737, 738, 739, or 740. The spacer of the ngRNA can comprise SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 199, 200, 203, 529, 736, 737, 738, 739, or 740. The gRNA core of the ngRNA can comprise SEQ ID NO. 5857-5859. Exemplary ngRNA provided in Table 10 can comprise SEQ ID NO: 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 290, 291, 292, 293, 681, 1501, 1502, 1503, or 1504. Such ngRNA sequences may further comprise a 3′ motif at the 3′ end of the gRNA core, for example, a series of 3, 4 or more U nucleotides. Without being bound by theory, such 3′ motifs are believed to increase ngRNA stability.

Table 11 provides Prime Editing guide RNAs (PEgRNAs), which can be used in Prime Editing systems disclosed herein. Such Prime Editing systems can comprise a Cas9 protein capable of recognizing an NGG or NG PAM sequence (e.g., AGG or AG), and a reverse transcriptase. The Prime Editing systems (e.g., PE3 or PE3b systems) can further comprise a nick guide RNA (ngRNA). Such PEgRNAs and Prime Editing systems can be used, for example, to correct an 1H1069Q mutation in ATP7B.

The PEgRNAs of Table 11 comprise: (a) a spacer comprising nucleotides 5-20 of SEQ ID NO: 1505, (b) a gRNA core capable of binding to a Cas9 protein, and (c) an extension arm comprising: (i) an editing template comprising any one of SEQ ID NOs: 1517-1546, and (ii) a primer binding site (PBS) comprising any one of SEQ ID NOs: 1506-1516. The spacer of the PEgRNA can comprise, for example, nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 1505. The spacer of the PEgRNA can comprise SEQ ID NO: 1505. The RTT and the PBS can comprise respectively SEQ ID NOs: 1517 and 1506, 1517 and 1507, 1517 and 1508, 1517 and 1509, 1517 and 1510, 1517 and 1511, 1517 and 1512, 1517 and 1513, 1517 and 1514, 1517 and 1515, 1517 and 1516, 1518 and 1506, 1518 and 1507, 1518 and 1508, 1518 and 1509, 1518 and 1510, 1518 and 1511, 1518 and 1512, 1518 and 1513, 1518 and 1514, 1518 and 1515, 1518 and 1516, 1519 and 1506, 1519 and 1507, 1519 and 1508, 1519 and 1509, 1519 and 1510, 1519 and 1511, 1519 and 1512, 1519 and 1513, 1519 and 1514, 1519 and 1515, 1519 and 1516, 1520 and 1506, 1520 and 1507, 1520 and 1508, 1520 and 1509, 1520 and 1510, 1520 and 1511, 1520 and 1512, 1520 and 1513, 1520 and 1514, 1520 and 1515, 1520 and 1516, 1521 and 1506, 1521 and 1507, 1521 and 1508, 1521 and 1509, 1521 and 1510, 1521 and 1511, 1521 and 1512, 1521 and 1513, 1521 and 1514, 1521 and 1515, 1521 and 1516, 1522 and 1506, 1522 and 1507, 1522 and 1508, 1522 and 1509, 1522 and 1510, 1522 and 1511, 1522 and 1512, 1522 and 1513, 1522 and 1514, 1522 and 1515, 1522 and 1516, 1523 and 1506, 1523 and 1507, 1523 and 1508, 1523 and 1509, 1523 and 1510, 1523 and 1511, 1523 and 1512, 1523 and 1513, 1523 and 1514, 1523 and 1515, 1523 and 1516, 1524 and 1506, 1524 and 1507, 1524 and 1508, 1524 and 1509, 1524 and 1510, 1524 and 1511, 1524 and 1512, 1524 and 1513, 1524 and 1514, 1524 and 1515, 1524 and 1516, 1525 and 1506, 1525 and 1507, 1525 and 1508, 1525 and 1509, 1525 and 1510, 1525 and 1511, 1525 and 1512, 1525 and 1513, 1525 and 1514, 1525 and 1515, 1525 and 1516, 1526 and 1506, 1526 and 1507, 1526 and 1508, 1526 and 1509, 1526 and 1510, 1526 and 1511, 1526 and 1512, 1526 and 1513, 1526 and 1514, 1526 and 1515, 1526 and 1516, 1527 and 1506, 1527 and 1507, 1527 and 1508, 1527 and 1509, 1527 and 1510, 1527 and 1511, 1527 and 1512, 1527 and 1513, 1527 and 1514, 1527 and 1515, 1527 and 1516, 1528 and 1506, 1528 and 1507, 1528 and 1508, 1528 and 1509, 1528 and 1510, 1528 and 1511, 1528 and 1512, 1528 and 1513, 1528 and 1514, 1528 and 1515, 1528 and 1516, 1529 and 1506, 1529 and 1507, 1529 and 1508, 1529 and 1509, 1529 and 1510, 1529 and 1511, 1529 and 1512, 1529 and 1513, 1529 and 1514, 1529 and 1515, 1529 and 1516, 1530 and 1506, 1530 and 1507, 1530 and 1508, 1530 and 1509, 1530 and 1510, 1530 and 1511, 1530 and 1512, 1530 and 1513, 1530 and 1514, 1530 and 1515, 1530 and 1516, 1531 and 1506, 1531 and 1507, 1531 and 1508, 1531 and 1509, 1531 and 1510, 1531 and 1511, 1531 and 1512, 1531 and 1513, 1531 and 1514, 1531 and 1515, 1531 and 1516, 1532 and 1506, 1532 and 1507, 1532 and 1508, 1532 and 1509, 1532 and 1510, 1532 and 1511, 1532 and 1512, 1532 and 1513, 1532 and 1514, 1532 and 1515, 1532 and 1516, 1533 and 1506, 1533 and 1507, 1533 and 1508, 1533 and 1509, 1533 and 1510, 1533 and 1511, 1533 and 1512, 1533 and 1513, 1533 and 1514, 1533 and 1515, 1533 and 1516, 1534 and 1506, 1534 and 1507, 1534 and 1508, 1534 and 1509, 1534 and 1510, 1534 and 1511, 1534 and 1512, 1534 and 1513, 1534 and 1514, 1534 and 1515, 1534 and 1516, 1535 and 1506, 1535 and 1507, 1535 and 1508, 1535 and 1509, 1535 and 1510, 1535 and 1511, 1535 and 1512, 1535 and 1513, 1535 and 1514, 1535 and 1515, 1535 and 1516, 1536 and 1506, 1536 and 1507, 1536 and 1508, 1536 and 1509, 1536 and 1510, 1536 and 1511, 1536 and 1512, 1536 and 1513, 1536 and 1514, 1536 and 1515, 1536 and 1516, 1537 and 1506, 1537 and 1507, 1537 and 1508, 1537 and 1509, 1537 and 1510, 1537 and 1511, 1537 and 1512, 1537 and 1513, 1537 and 1514, 1537 and 1515, 1537 and 1516, 1538 and 1506, 1538 and 1507, 1538 and 1508, 1538 and 1509, 1538 and 1510, 1538 and 1511, 1538 and 1512, 1538 and 1513, 1538 and 1514, 1538 and 1515, 1538 and 1516, 1539 and 1506, 1539 and 1507, 1539 and 1508, 1539 and 1509, 1539 and 1510, 1539 and 1511, 1539 and 1512, 1539 and 1513, 1539 and 1514, 1539 and 1515, 1539 and 1516, 1540 and 1506, 1540 and 1507, 1540 and 1508, 1540 and 1509, 1540 and 1510, 1540 and 1511, 1540 and 1512, 1540 and 1513, 1540 and 1514, 1540 and 1515, 1540 and 1516, 1541 and 1506, 1541 and 1507, 1541 and 1508, 1541 and 1509, 1541 and 1510, 1541 and 1511, 1541 and 1512, 1541 and 1513, 1541 and 1514, 1541 and 1515, 1541 and 1516, 1542 and 1506, 1542 and 1507, 1542 and 1508, 1542 and 1509, 1542 and 1510, 1542 and 1511, 1542 and 1512, 1542 and 1513, 1542 and 1514, 1542 and 1515, 1542 and 1516, 1543 and 1506, 1543 and 1507, 1543 and 1508, 1543 and 1509, 1543 and 1510, 1543 and 1511, 1543 and 1512, 1543 and 1513, 1543 and 1514, 1543 and 1515, 1543 and 1516, 1544 and 1506, 1544 and 1507, 1544 and 1508, 1544 and 1509, 1544 and 1510, 1544 and 1511, 1544 and 1512, 1544 and 1513, 1544 and 1514, 1544 and 1515, 1544 and 1516, 1545 and 1506, 1545 and 1507, 1545 and 1508, 1545 and 1509, 1545 and 1510, 1545 and 1511, 1545 and 1512, 1545 and 1513, 1545 and 1514, 1545 and 1515, 1545 and 1516, 1546 and 1506, 1546 and 1507, 1546 and 1508, 1546 and 1509, 1546 and 1510, 1546 and 1511, 1546 and 1512, 1546 and 1513, 1546 and 1514, 1546 and 1515, or 1546 and 1516. The gRNA core of the PEgRNA can comprise SEQ ID NO. 5857-5859. Exemplary PEgRNAs provided in Table 11 can comprise SEQ ID NO. 1547, 1548, 1549, 1550, 1551, 1552, 1553, 1554, 1555, 1556, 1557, 1558, 1559, 1560, 1561, 1562, 1563, 1564, 1565, 1566, 1567, 1568, 1569, 1570, 1571, 1572, 1573, 1574, 1575, 1576, 1577, 1578, 1579, 1580, 1581, 1582, 1583, 1584, 1585, 1586, 1587, 1588, 1589, 1590, 1591, 1592, 1593, 1594, 1595, 1596, 1598, 1600, 1601, 1602, 1604, 1606, 1607, 1608, 1609, 1610, 1614, 1615, 1616, 1619, 1620, 1621, 1622, 1623, 1624, 1626, 1628, 1632, 1633, 1634, 1635, 1637, 1640, 1643, 1645, 1646, 1647, 1648, 1649, 1650, 1651, 1653, 1654, 1663, 1664, 1668, 1671, 1678, 1679, 1684, 1686, 1687, 1688, 1691, 1694, 1696, 1697, 1699, 1702, 1708, 1721, 1725, 1728, 1729, 1730, 1731, 1732, 1733, 1737, 1738, 1741, 1742, 1743, 1744, 1745, 1756, 1760, 1761, 1766, 1769, 1770, 1771, 1773, 1777, 1785, 1786, 1788, 1789, 1792, 1796, 1798, 1801, 1803, 1804, 1805, 1806, 1807, 1810, 1813, 1815, 1819, 1824, 1825, 1826, 1830, 1832, 1833, 1835, 1837, 1840, 1841, 1842, 1845, 1846, 1847, 1848, 1850, 1852, 1855, 1856, 1857, 1858, 1859, 1860, 1861, 1862, 1863, 1864, 1865, 1866, 1868, 1870, 1872, 1874, 1876, 1877, 1880, 1882, 1883, 1884, 1888, 1890, 1892, 1893, 1895, 1896, 1902, 1905, 1907, 1908, 1909, 1910, 1911, 1912, 1915, 1918, 1919, 1920, 1921, 1923, 1924, 1928, 1930, 1933, 1935, 1940, 1941, 1943, 1945, 1949, 1954, 1958, 1966, or 1967. Such PEgRNA sequences may further comprise a 3′ motif at the 3′ end of the extension arm, for example, a hairpin-forming motif or a series of 3, 4 or more U nucleotides. Without being bound by theory, such 3′ motifs are believed to increase PEgRNA stability. Such PEgRNA sequences ma alternatively or additionally be adapted for transcription from a DNA template, for example, by including a 5′ terminal G if the spacer of the PEgRNA begins with another nucleotide, by including 6 or 7 U nucleotides at the 3′ end of the extension arm, or both. Exemplary transcription-adapted sequences include SEQ ID NOs: 1597, 1599, 1603, 1605, 1611, 1612, 1613, 1617, 1618, 1625, 1627, 1629, 1630, 1631, 1636, 1638, 1639, 1641, 1642, 1644, 1652, 1655, 1656, 1657, 1658, 1659, 1660, 1661, 1662, 1665, 1666, 1667, 1669, 1670, 1672, 1673, 1674, 1675, 1676, 1677, 1680, 1681, 1682, 1683, 1685, 1689, 1690, 1692, 1693, 1695, 1698, 1700, 1701, 1703, 1704, 1705, 1706, 1707, 1709, 1710, 1711, 1712, 1713, 1714, 1715, 1716, 1717, 1718, 1719, 1720, 1722, 1723, 1724, 1726, 1727, 1734, 1735, 1736, 1739, 1740, 1746, 1747, 1748, 1749, 1750, 1751, 1752, 1753, 1754, 1755, 1757, 1758, 1759, 1762, 1763, 1764, 1765, 1767, 1768, 1772, 1774, 1775, 1776, 1778, 1779, 1780, 1781, 1782, 1783, 1784, 1787, 1790, 1791, 1793, 1794, 1795, 1797, 1799, 1800, 1802, 1808, 1809, 1811, 1812, 1814, 1816, 1817, 1818, 1820, 1821, 1822, 1823, 1827, 1828, 1829, 1831, 1834, 1836, 1838, 1839, 1843, 1844, 1849, 1851, 1853, 1854, 1867, 1869, 1871, 1873, 1875, 1878, 1879, 1881, 1885, 1886, 1887, 1889, 1891, 1894, 1897, 1898, 1899, 1900, 1901, 1903, 1904, 1906, 1913, 1914, 1916, 1917, 1922, 1925, 1926, 1927, 1929, 1931, 1932, 1934, 1936, 1937, 1938, 1939, 1942, 1944, 1946, 1947, 1948, 1950, 1951, 1952, 1953, 1955, 1956, 1957, 1959, 1960, 1961, 1962, 1963, 1964, 1965, 1968, 1969, 1970, 1971, 1972, 1973, 1974, 1975, 1976, 1977, 1978, 1979, 1980, 1981, 1982, 1983, 1984, 1985, 1986, 1987, 1988, 1989, 1990, 1991, 1992, 1993, 1994, 1995, 1996, 1997, 1998, 1999, 2000, 2001, 2002, 2003, 2004, 2005, 2006, 2007, 2008, 2009, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2018, 2019, 2020, 2021, and 2022. Such plasmid adapted sequences may further comprise a hairpin-forming motif between the PBS and the 3′ terminal U series.

Any of the PEgRNAs of Table 11 can be used in a Prime Editing system further comprising a nick guide RNA (ngRNA). Such ngRNA can comprise a spacer comprising nucleotides 5-20 of SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 199, 200, 203, 529, 736, 737, 738, 739, or 740 and a gRNA core capable of binding to a Cas9 protein. The spacer of the ngRNA can comprise nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 199, 200, 203, 529, 736, 737, 738, 739, or 740. The spacer of the ngRNA can comprise SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 199, 200, 203, 529, 736, 737, 738, 739, or 740. The gRNA core of the ngRNA can comprise SEQ ID NO. 5857-5859. Exemplary ngRNA provided in Table 11 can comprise SEQ ID NO: 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 290, 291, 292, 293, 681, 1501, 1502, 1503, or 1504. Such ngRNA sequences may further comprise a 3′ motif at the 3′ end of the gRNA core, for example, a series of 3, 4 or more U nucleotides. Without being bound by theory, such 3′ motifs are believed to increase ngRNA stability.

Table 12 provides Prime Editing guide RNAs (PEgRNAs), which can be used in Prime Editing systems disclosed herein. Such Prime Editing systems can comprise a Cas9 protein capable of recognizing an NGG or NG PAM sequence (e.g., TGG or TG), and a reverse transcriptase. The Prime Editing systems (e.g., PE3 or PE3b systems) can further comprise a nick guide RNA (ngRNA). Such PEgRNAs and Prime Editing systems can be used, for example, to correct an H1069Q mutation in ATP7B.

The PEgRNAs of Table 12 comprise: (a) a spacer comprising nucleotides 5-20 of SEQ ID NO: 2023, (b) a gRNA core capable of binding to a Cas9 protein, and (c) an extension arm comprising: (i) an editing template comprising any one of SEQ ID NOs: 2035-2044, and (ii) a primer binding site (PBS) comprising any one of SEQ ID NOs: 2024-2034. The spacer of the PEgRNA can comprise, for example, nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 2023. The spacer of the PEgRNA can comprise SEQ ID NO: 2023. The RTT and the PBS can comprise respectively SEQ ID NO: 2035 and 2024, 2035 and 2025, 2035 and 2026, 2035 and 2027, 2035 and 2028, 2035 and 2029, 2035 and 2030, 2035 and 2031, 2035 and 2032, 2035 and 2033, 2035 and 2034, 2036 and 2024, 2036 and 2025, 2036 and 2026, 2036 and 2027, 2036 and 2028, 2036 and 2029, 2036 and 2030, 2036 and 2031, 2036 and 2032, 2036 and 2033, 2036 and 2034, 2037 and 2024, 2037 and 2025, 2037 and 2026, 2037 and 2027, 2037 and 2028, 2037 and 2029, 2037 and 2030, 2037 and 2031, 2037 and 2032, 2037 and 2033, 2037 and 2034, 2038 and 2024, 2038 and 2025, 2038 and 2026, 2038 and 2027, 2038 and 2028, 2038 and 2029, 2038 and 2030, 2038 and 2031, 2038 and 2032, 2038 and 2033, 2038 and 2034, 2039 and 2024, 2039 and 2025, 2039 and 2026, 2039 and 2027, 2039 and 2028, 2039 and 2029, 2039 and 2030, 2039 and 2031, 2039 and 2032, 2039 and 2033, 2039 and 2034, 2040 and 2024, 2040 and 2025, 2040 and 2026, 2040 and 2027, 2040 and 2028, 2040 and 2029, 2040 and 2030, 2040 and 2031, 2040 and 2032, 2040 and 2033, 2040 and 2034, 2041 and 2024, 2041 and 2025, 2041 and 2026, 2041 and 2027, 2041 and 2028, 2041 and 2029, 2041 and 2030, 2041 and 2031, 2041 and 2032, 2041 and 2033, 2041 and 2034, 2042 and 2024, 2042 and 2025, 2042 and 2026, 2042 and 2027, 2042 and 2028, 2042 and 2029, 2042 and 2030, 2042 and 2031, 2042 and 2032, 2042 and 2033, 2042 and 2034, 2043 and 2024, 2043 and 2025, 2043 and 2026, 2043 and 2027, 2043 and 2028, 2043 and 2029, 2043 and 2030, 2043 and 2031, 2043 and 2032, 2043 and 2033, 2043 and 2034, 2044 and 2024, 2044 and 2025, 2044 and 2026, 2044 and 2027, 2044 and 2028, 2044 and 2029, 2044 and 2030, 2044 and 2031, 2044 and 2032, 2044 and 2033, or 2044 and 2034. The gRNA core of the PEgRNA can comprise SEQ ID NO. 5857-5859. Exemplary PEgRNAs provided in Table 12 can comprise SEQ ID NO. 2097, 2098, 2099, 2100, 2101, 2102, 2103, 2104, 2105, 2106, 2107, 2108, 2109, 2110, 2111, 2112, 2113, 2114, 2115, 2116, 2117, 2118, 2119, 2120, 2121, 2122, 2123, 2124, 2125, 2126, 2127, 2128, 2129, 2131, 2132, 2133, 2134, 2135, 2136, 2137, 2138, 2140, 2142, 2144, 2145, 2146, 2147, 2148, 2149, 2150, 2151, 2154, 2155, 2156, 2158, 2160, 2161, 2165, 2166, 2167, 2168, 2169, 2170, 2171, 2174, 2178, 2179, 2183, 2187, 2194, 2195, 2197, 2199, 2200, 2202, 2207, 2208, 2220, 2226, or 2232. Such PEgRNA sequences may further comprise a 3Y motif at the 3′ end of the extension arm, for example, a hairpin-forming motif or a series of 3, 4 or more U nucleotides. Without being bound by theory, such 3′ motifs are believed to increase PEgRNA stability. Such PEgRNA sequences may alternatively or additionally be adapted for transcription from a DNA template, for example, by including a 5′ terminal G if the spacer of the PEgRNA begins with another nucleotide, by including 6 or 7 U nucleotides at the 3′ end of the extension arm, or both. Exemplary transcription-adapted sequences include SEQ ID NOs: 2130, 2139, 2141, 2143, 2152, 2153, 2157, 2159, 2162, 2163, 2164, 2172, 2173, 2175, 2176, 2177, 2180, 2181, 2182, 2184, 2185, 2186, 2188, 2189, 2190, 2191, 2192, 2193, 2196, 2198, 2201, 2203, 2204, 2205, 2206, 2209, 2210, 2211, 2212, 2213, 2214, 2215, 2216, 2217, 2218, 2219, 2221, 2222, 2223, 2224, 2225, 2227, 2228, 2229, 2230, 2231, 2233, 2234, 2235, 2236, 2237, 2238, 2239, 2240, 2241, 2242, 2243, 2244, 2245, 2246, 2247, 2248, 2249, 2250, 2251, 2252, 2253, 2254, 2255, and 2256. Such plasmid adapted sequences may further comprise a hairpin-forming motif between the PBS and the 3′ terminal U series.

Any of the PEgRNAs of Table 12 can be used in a Prime Editing system further comprising a nick guide RNA (ngRNA). Such ngRNA can comprise a spacer comprising nucleotides 5-20 of SEQ ID NO: 41, 60, 61, 62, 63, 64, 65, 66, 69, 71, 2045, 2046, 2047, 2048, 2049, 2050, 2051, 2052, 2053, 2054, 2055, 2056, 2057, 2058, 2059, 2060, 2061, 2062, 2063, 2064, 2065, 2066, 2067, 2068, 2069, 2070, 2071, 2072, 2073, 2074, 2075, 2076, 2077, 2078, 2079, 2080, 2081, 2082, 2083, 2084, 2085, 2086, 2087, 2088, 2089, 2090, 2091, 2092, 2093, 2094, 2095, or 2096 and a gRNA core capable of binding to a Cas9 protein. The spacer of the ngRNA can comprise nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 41, 60, 61, 62, 63, 64, 65, 66, 69, 71, 2045, 2046, 2047, 2048, 2049, 2050, 2051, 2052, 2053, 2054, 2055, 2056, 2057, 2058, 2059, 2060, 2061, 2062, 2063, 2064, 2065, 2066, 2067, 2068, 2069, 2070, 2071, 2072, 2073, 2074, 2075, 2076, 2077, 2078, 2079, 2080, 2081, 2082, 2083, 2084, 2085, 2086, 2087, 2088, 2089, 2090, 2091, 2092, 2093, 2094, 2095, or 2096. The spacer of the ngRNA can comprise SEQ ID NO: 41, 60, 61, 62, 63, 64, 65, 66, 69, 71, 2045, 2046, 2047, 2048, 2049, 2050, 2051, 2052, 2053, 2054, 2055, 2056, 2057, 2058, 2059, 2060, 2061, 2062, 2063, 2064, 2065, 2066, 2067, 2068, 2069, 2070, 2071, 2072, 2073, 2074, 2075, 2076, 2077, 2078, 2079, 2080, 2081, 2082, 2083, 2084, 2085, 2086, 2087, 2088, 2089, 2090, 2091, 2092, 2093, 2094, 2095, or 2096. The gRNA core of the ngRNA can comprise SEQ ID NO. 5857-5859. Exemplary ngRNA provided in Table 12 can comprise any one of SEQ ID NOs: 2257-2289. Such ngRNA sequences may further comprise a 3′ motif at the 3′ end of the gRNA core, for example, a series of 3, 4 or more U nucleotides. Exemplary ngRNA sequences with such 3′ adaptations include SEQ ID NOs: 2290-2292. Without being bound by theory, such 3′ motifs are believed to increase ngRNA stability.

Table 15 provides Prime Editing guide RNAs (PEgRNAs), which can be used in Prime Editing systems disclosed herein. Such Prime Editing systems can comprise a Cas9 protein capable of recognizing an NGG, NG, or NNGG PAM sequence (e.g., AGG, AG, or AGGG), and a reverse transcriptase. The Prime Editing systems (e.g., PE3 or PE3b systems) can further comprise a nick guide RNA (ngRNA). Such PEgRNAs and Prime Editing systems can be used, for example, to correct an H1069Q mutation in ATP7B.

The PEgRNAs of Table 15 comprise: (a) a spacer comprising nucleotides 5-20 of SEQ ID NO: 2293, (b) a gRNA core capable of binding to a Cas9 protein, and (c) an extension arm comprising: (i) an editing template comprising any one of SEQ ID NOs: 2305-2422, and (ii) a primer binding site (PBS) comprising any one of SEQ ID NOs: 2294-2304. The spacer of the PEgRNA can comprise, for example, nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 2293. The spacer of the PEgRNA can comprise SEQ ID NO: 2293. The RTT and the PBS can comprise respectively SEQ ID NOs: 2305 and 2294, 2305 and 2295, 2305 and 2296, 2305 and 2297, 2305 and 2298, 2305 and 2299, 2305 and 2300, 2305 and 2301, 2305 and 2302, 2305 and 2303, 2305 and 2304, 2306 and 2294, 2306 and 2295, 2306 and 2296, 2306 and 2297, 2306 and 2298, 2306 and 2299, 2306 and 2300, 2306 and 2301, 2306 and 2302, 2306 and 2303, 2306 and 2304, 2307 and 2294, 2307 and 2295, 2307 and 2296, 2307 and 2297, 2307 and 2298, 2307 and 2299, 2307 and 2300, 2307 and 2301, 2307 and 2302, 2307 and 2303, 2307 and 2304, 2308 and 2294, 2308 and 2295, 2308 and 2296, 2308 and 2297, 2308 and 2298, 2308 and 2299, 2308 and 2300, 2308 and 2301, 2308 and 2302, 2308 and 2303, 2308 and 2304, 2309 and 2294, 2309 and 2295, 2309 and 2296, 2309 and 2297, 2309 and 2298, 2309 and 2299, 2309 and 2300, 2309 and 2301, 2309 and 2302, 2309 and 2303, 2309 and 2304, 2310 and 2294, 2310 and 2295, 2310 and 2296, 2310 and 2297, 2310 and 2298, 2310 and 2299, 2310 and 2300, 2310 and 2301, 2310 and 2302, 2310 and 2303, 2310 and 2304, 2311 and 2294, 2311 and 2295, 2311 and 2296, 2311 and 2297, 2311 and 2298, 2311 and 2299, 2311 and 2300, 2311 and 2301, 2311 and 2302, 2311 and 2303, 2311 and 2304, 2312 and 2294, 2312 and 2295, 2312 and 2296, 2312 and 2297, 2312 and 2298, 2312 and 2299, 2312 and 2300, 2312 and 2301, 2312 and 2302, 2312 and 2303, 2312 and 2304, 2313 and 2294, 2313 and 2295, 2313 and 2296, 2313 and 2297, 2313 and 2298, 2313 and 2299, 2313 and 2305, 2313 and 2301, 2313 and 2302, 2313 and 2303, 2313 and 2304, 2314 and 2294, 2314 and 2295, 2314 and 2296, 2314 and 2297, 2314 and 2298, 2314 and 2299, 2314 and 2300, 2314 and 2301, 2314 and 2302, 2314 and 2303, 2314 and 2304, 2315 and 2294, 2315 and 2295, 2315 and 2296, 2315 and 2297, 2315 and 2298, 2315 and 2299, 2315 and 2300, 2315 and 2301, 2315 and 2302, 2315 and 2303, 2315 and 2304, 2316 and 2294, 2316 and 2295, 2316 and 2296, 2316 and 2297, 2316 and 2298, 2316 and 2299, 2316 and 2300, 2316 and 2301, 2316 and 2302, 2316 and 2303, 2316 and 2304, 2317 and 2294, 2317 and 2295, 2317 and 2296, 2317 and 2297, 2317 and 2298, 2317 and 2299, 2317 and 2300, 2317 and 2301, 2317 and 2302, 2317 and 2303, 2317 and 2304, 2318 and 2294, 2318 and 2295, 2318 and 2296, 2318 and 2297, 2318 and 2298, 2318 and 2299, 2318 and 2300, 2318 and 2301, 2318 and 2302, 2318 and 2303, 2318 and 2304, 2319 and 2294, 2319 and 2295, 2319 and 2296, 2319 and 2297, 2319 and 2298, 2319 and 2299, 2319 and 2300, 2319 and 2301, 2319 and 2302, 2319 and 2303, 2319 and 2304, 2320 and 2294, 2320 and 2295, 2320 and 2296, 2320 and 2297, 2320 and 2298, 2320 and 2299, 2320 and 2300, 2320 and 2301, 2320 and 2302, 2320 and 2303, 2320 and 2304, 2321 and 2294, 2321 and 2295, 2321 and 2296, 2321 and 2297, 2321 and 2298, 2321 and 2299, 2321 and 2300, 2321 and 2301, 2321 and 2302, 2321 and 2303, 2321 and 2304, 2322 and 2294, 2322 and 2295, 2322 and 2296, 2322 and 2297, 2322 and 2298, 2322 and 2299, 2322 and 2300, 2322 and 2301, 2322 and 2302, 2322 and 2303, 2322 and 2304, 2323 and 2294, 2323 and 2295, 2323 and 2296, 2323 and 2297, 2323 and 2298, 2323 and 2299, 2323 and 23004, 2323 and 2301, 2323 and 2302, 2323 and 2303, 2323 and 2304, 2324 and 2294, 2324 and 2295, 2324 and 2296, 2324 and 2297, 2324 and 2298, 2324 and 2299, 2324 and 2300, 2324 and 2301, 2324 and 2302, 2324 and 2303, 2324 and 2304, 2325 and 2294, 2325 and 2295, 2325 and 2296, 2325 and 2297, 2325 and 2298, 2325 and 2299, 2325 and 2300, 2325 and 2301, 2325 and 2302, 2325 and 2303, 2325 and 2304, 2326 and 2294, 2326 and 2295, 2326 and 2296, 2326 and 2297, 2326 and 2298, 2326 and 2299, 2326 and 2300, 2326 and 2301, 2326 and 2302, 2326 and 2303, 2326 and 2304, 2327 and 2294, 2327 and 2295, 2327 and 2296, 2327 and 2297, 2327 and 2298, 2327 and 2299, 2327 and 2300, 2327 and 2301, 2327 and 2302, 2327 and 2303, 2327 and 2304, 2328 and 2294, 2328 and 2295, 2328 and 2296, 2328 and 2297, 2328 and 2298, 2328 and 2299, 2328 and 2300, 2328 and 2301, 2328 and 2302, 2328 and 2303, 2328 and 2304, 2329 and 2294, 2329 and 2295, 2329 and 2296, 2329 and 2297, 2329 and 2298, 2329 and 2299, 2329 and 2300, 2329 and 2301, 2329 and 2302, 2329 and 2303, 2329 and 2304, 2330 and 2294, 2330 and 2295, 2330 and 2296, 2330 and 2297, 2330 and 2298, 2330 and 2299, 2330 and 2300, 2330 and 2301, 2330 and 2302, 2330 and 2303, 2330 and 2304, 2331 and 2294, 2331 and 2295, 2331 and 2296, 2331 and 2297, 2331 and 2298, 2331 and 2299, 2331 and 2300, 2331 and 2301, 2331 and 2302, 2331 and 2303, 2331 and 2304, 2332 and 2294, 2332 and 2295, 2332 and 2296, 2332 and 2297, 2332 and 2298, 2332 and 2299, 2332 and 2300, 2332 and 2301, 2332 and 2302, 2332 and 2303, 2332 and 2304, 2333 and 2294, 2333 and 2295, 2333 and 2296, 2333 and 2297, 2333 and 2298, 2333 and 2299, 2333 and 2300, 2333 and 2301, 2333 and 2302, 2333 and 2303, 2333 and 2304, 2334 and 2294, 2334 and 2295, 2334 and 2296, 2334 and 2297, 2334 and 2298, 2334 and 2299, 2334 and 2300, 2334 and 2301, 2334 and 2302, 2334 and 2303, 2334 and 2304, 2335 and 2294, 2335 and 2295, 2335 and 2296, 2335 and 2297, 2335 and 2298, 2335 and 2299, 2335 and 2300, 2335 and 2301, 2335 and 2302, 2335 and 2303, 2335 and 2304, 2336 and 2294, 2336 and 2295, 2336 and 2296, 2336 and 2297, 2336 and 2298, 2336 and 2299, 2336 and 2300, 2336 and 2301, 2336 and 2302, 2336 and 2303, 2336 and 2304, 2337 and 2294, 2337 and 2295, 2337 and 2296, 2337 and 2297, 2337 and 2298, 2337 and 2299, 2337 and 2300, 2337 and 2301, 2337 and 2302, 2337 and 2303, 2337 and 2304, 2338 and 2294, 2338 and 2295, 2338 and 2296, 2338 and 2297, 2338 and 2298, 2338 and 2299, 2338 and 2300, 2338 and 2301, 2338 and 2302, 2338 and 2303, 2338 and 2304, 2339 and 2294, 2339 and 2295, 2339 and 2296, 2339 and 2297, 2339 and 2298, 2339 and 2299, 2339 and 2300, 2339 and 2301, 2339 and 2302, 2339 and 2303, 2339 and 2304, 2340 and 2294, 2340 and 2295, 2340 and 2296, 2340 and 2297, 2340 and 2298, 2340 and 2299, 2340 and 2300, 2340 and 2301, 2340 and 2302, 2340 and 2303, 2340 and 2304, 2341 and 2294, 2341 and 2295, 2341 and 2296, 2341 and 2297, 2341 and 2298, 2341 and 2299, 2341 and 2300, 2341 and 2301, 2341 and 2302, 2341 and 2303, 2341 and 2304, 2342 and 2294, 2342 and 2295, 2342 and 2296, 2342 and 2297, 2342 and 2298, 2342 and 2299, 2342 and 2300, 2342 and 2301, 2342 and 2302, 2342 and 2303, 2342 and 2304, 2343 and 2294, 2343 and 2295, 2343 and 2296, 2343 and 2297, 2343 and 2298, 2343 and 2299, 2343 and 2300, 2343 and 2301, 2343 and 2302, 2343 and 2303, 2343 and 2304, 2344 and 2294, 2344 and 2295, 2344 and 2296, 2344 and 2297, 2344 and 2298, 2344 and 2299, 2344 and 2300, 2344 and 2301, 2344 and 2302, 2344 and 2303, 2344 and 2304, 2345 and 2294, 2345 and 2295, 2345 and 2296, 2345 and 2297, 2345 and 2298, 2345 and 2299, 2345 and 2300, 2345 and 2301, 2345 and 2302, 2345 and 2303, 2345 and 2304, 2346 and 2294, 2346 and 2295, 2346 and 2296, 2346 and 2297, 2346 and 2298, 2346 and 2299, 2346 and 2300, 2346 and 2301, 2346 and 2302, 2346 and 2303, 2346 and 2304, 2347 and 2294, 2347 and 2295, 2347 and 2296, 2347 and 2297, 2347 and 2298, 2347 and 2299, 2347 and 2300, 2347 and 2301, 2347 and 2302, 2347 and 2303, 2347 and 2304, 2348 and 2294, 2348 and 2295, 2348 and 2296, 2348 and 2297, 2348 and 2298, 2348 and 2299, 2348 and 2300, 2348 and 2301, 2348 and 2302, 2348 and 2303, 2348 and 2304, 2349 and 2294, 2349 and 2295, 2349 and 2296, 2349 and 2297, 2349 and 2298, 2349 and 2299, 2349 and 230, 2349 and 2301, 2349 and 2302, 2349 and 2303, 2349 and 2304, 2350 and 2294, 2350 and 2295, 2350 and 2296, 2350 and 2297, 2350 and 2298, 2350 and 2299, 2350 and 2300, 2350 and 2301, 2350 and 2302, 2350 and 2303, 2350 and 2304, 2351 and 2294, 2351 and 2295, 2351 and 2296, 2351 and 2297, 2351 and 2298, 2351 and 2299, 2351 and 2300, 2351 and 2301, 2351 and 2302, 2351 and 2303, 2351 and 2304, 2352 and 2294, 2352 and 2295, 2352 and 2296, 2352 and 2297, 2352 and 2298, 2352 and 2299, 2352 and 2300, 2352 and 2301, 2352 and 2302, 2352 and 2303, 2352 and 2304, 2353 and 2294, 2353 and 2295, 2353 and 2296, 2353 and 2297, 2353 and 2298, 2353 and 2299, 2353 and 2300, 2353 and 2301, 2353 and 2302, 2353 and 2303, 2353 and 2304, 2354 and 2294, 2354 and 2295, 2354 and 2296, 2354 and 2297, 2354 and 2298, 2354 and 2299, 2354 and 2300, 2354 and 2301, 2354 and 2302, 2354 and 2303, 2354 and 2304, 2355 and 2294, 2355 and 2295, 2355 and 2296, 2355 and 2297, 2355 and 2298, 2355 and 2299, 2355 and 2300, 2355 and 2301, 2355 and 2302, 2355 and 2303, 2355 and 2304, 2356 and 2294, 2356 and 2295, 2356 and 2296, 2356 and 2297, 2356 and 2298, 2356 and 2299, 2356 and 2300, 2356 and 2301, 2356 and 2302, 2356 and 2303, 2356 and 2304, 2357 and 2294, 2357 and 2295, 2357 and 2296, 2357 and 2297, 2357 and 2298, 2357 and 2299, 2357 and 2300, 2357 and 2301, 2357 and 2302, 2357 and 2303, 2357 and 2304, 2358 and 2294, 2358 and 2295, 2358 and 2296, 2358 and 2297, 2358 and 2298, 2358 and 2299, 2358 and 2300, 2358 and 2301, 2358 and 2302, 2358 and 2303, 2358 and 2304, 2359 and 2294, 2359 and 2295, 2359 and 2296, 2359 and 2297, 2359 and 2298, 2359 and 2299, 2359 and 2300, 2359 and 2301, 2359 and 2302, 2359 and 2303, 2359 and 2304, 2360 and 2294, 2360 and 2295, 2360 and 2296, 2360 and 2297, 2360 and 2298, 2360 and 2299, 2360 and 2300, 2360 and 2301, 2360 and 2302, 2360 and 2303, 2360 and 2304, 2361 and 2294, 2361 and 2295, 2361 and 2296, 2361 and 2297, 2361 and 2298, 2361 and 2299, 2361 and 2300, 2361 and 2301, 2361 and 2302, 2361 and 2303, 2361 and 2304, 2362 and 2294, 2362 and 2295, 2362 and 2296, 2362 and 2297, 2362 and 2298, 2362 and 2299, 2362 and 2300, 2362 and 2301, 2362 and 2302, 2362 and 2303, 2362 and 2304, 2363 and 2294, 2363 and 2295, 2363 and 2296, 2363 and 2297, 2363 and 2298, 2363 and 2299, 2363 and 2300, 2363 and 2301, 2363 and 2302, 2363 and 2303, 2363 and 2304, 2364 and 2294, 2364 and 2295, 2364 and 2296, 2364 and 2297, 2364 and 2298, 2364 and 2299, 2364 and 2300, 2364 and 2301, 2364 and 2302, 2364 and 2303, 2364 and 2304, 2365 and 2294, 2365 and 2295, 2365 and 2296, 2365 and 2297, 2365 and 2298, 2365 and 2299, 2365 and 2300, 2365 and 2301, 2365 and 2302, 2365 and 2303, 2365 and 2304, 2366 and 2294, 2366 and 2295, 2366 and 2296, 2366 and 2297, 2366 and 2298, 2366 and 2299, 2366 and 2300, 2366 and 2301, 2366 and 2302, 2366 and 2303, 2366 and 2304, 2367 and 2294, 2367 and 2295, 2367 and 2296, 2367 and 2297, 2367 and 2298, 2367 and 2299, 2367 and 2300, 2367 and 2301, 2367 and 2302, 2367 and 2303, 2367 and 2304, 2368 and 2294, 2368 and 2295, 2368 and 2296, 2368 and 2297, 2368 and 2298, 2368 and 2299, 2368 and 2300, 2368 and 2301, 2368 and 2302, 2368 and 2303, 2368 and 2304, 2369 and 2294, 2369 and 2295, 2369 and 2296, 2369 and 2297, 2369 and 2298, 2369 and 2299, 2369 and 2300, 2369 and 2301, 2369 and 2302, 2369 and 2303, 2369 and 2304, 2370 and 2294, 2370 and 2295, 2370 and 2296, 2370 and 2297, 2370 and 2298, 2370 and 2299, 2370 and 2300, 2370 and 2301, 2370 and 2302, 2370 and 2303, 2370 and 2304, 2371 and 2294, 2371 and 2295, 2371 and 2296, 2371 and 2297, 2371 and 2298, 2371 and 2299, 2371 and 2300, 2371 and 2301, 2371 and 2302, 2371 and 2303, 2371 and 2304, 2372 and 2294, 2372 and 2295, 2372 and 2296, 2372 and 2297, 2372 and 2298, 2372 and 2299, 2372 and 2300, 2372 and 2301, 2372 and 2302, 2372 and 2303, 2372 and 2304, 2373 and 2294, 2373 and 2295, 2373 and 2296, 2373 and 2297, 2373 and 2298, 2373 and 2299, 2373 and 2300, 2373 and 2301, 2373 and 2302, 2373 and 2303, 2373 and 2304, 2374 and 2294, 2374 and 2295, 2374 and 2296, 2374 and 2297, 2374 and 2298, 2374 and 2299, 2374 and 2300, 2374 and 2301, 2374 and 2302, 2374 and 2303, 2374 and 2304, 2375 and 2294, 2375 and 2295, 2375 and 2296, 2375 and 2297, 2375 and 2298, 2375 and 2299, 2375 and 2300, 2375 and 2301, 2375 and 2302, 2375 and 2303, 2375 and 2304, 2376 and 2294, 2376 and 2295, 2376 and 2296, 2376 and 2297, 2376 and 2298, 2376 and 2299, 2376 and 2300, 2376 and 2301, 2376 and 2302, 2376 and 2303, 2376 and 2304, 2377 and 2294, 2377 and 2295, 2377 and 2296, 2377 and 2297, 2377 and 2298, 2377 and 2299, 2377 and 2300, 2377 and 2301, 2377 and 2302, 2377 and 2303, 2377 and 2304, 2378 and 2294, 2378 and 2295, 2378 and 2296, 2378 and 2297, 2378 and 2298, 2378 and 2299, 2378 and 2300, 2378 and 2301, 2378 and 2302, 2378 and 2303, 2378 and 2304, 2379 and 2294, 2379 and 2295, 2379 and 2296, 2379 and 2297, 2379 and 2298, 2379 and 2299, 2379 and 2300, 2379 and 2301, 2379 and 2302, 2379 and 2303, 2379 and 2304, 2380 and 2294, 2380 and 2295, 2380 and 2296, 2380 and 2297, 2380 and 2298, 2380 and 2299, 2380 and 2300, 2380 and 2301, 2380 and 2302, 2380 and 2303, 2380 and 2304, 2381 and 2294, 2381 and 2295, 2381 and 2296, 2381 and 2297, 2381 and 2298, 2381 and 2299, 2381 and 2300, 2381 and 2301, 2381 and 2302, 2381 and 2303, 2381 and 2304, 2382 and 2294, 2382 and 2295, 2382 and 2296, 2382 and 2297, 2382 and 2298, 2382 and 2299, 2382 and 2300, 2382 and 2301, 2382 and 2302, 2382 and 2303, 2382 and 2304, 2383 and 2294, 2383 and 2295, 2383 and 2296, 2383 and 2297, 2383 and 2298, 2383 and 2299, 2383 and 2300, 2383 and 2301, 2383 and 2302, 2383 and 2303, 2383 and 2304, 2384 and 2294, 2384 and 2295, 2384 and 2296, 2384 and 2297, 2384 and 2298, 2384 and 2299, 2384 and 2300, 2384 and 2301, 2384 and 2302, 2384 and 2303, 2384 and 2304, 2385 and 2294, 2385 and 2295, 2385 and 2296, 2385 and 2297, 2385 and 2298, 2385 and 2299, 2385 and 2300, 2385 and 2301, 2385 and 2302, 2385 and 2303, 2385 and 2304, 2386 and 2294, 2386 and 2295, 2386 and 2296, 2386 and 2297, 2386 and 2298, 2386 and 2299, 2386 and 2300, 2386 and 2301, 2386 and 2302, 2386 and 2303, 2386 and 2304, 2387 and 2294, 2387 and 2295, 2387 and 2296, 2387 and 2297, 2387 and 2298, 2387 and 2299, 2387 and 2300, 2387 and 2301, 2387 and 2302, 2387 and 2303, 2387 and 2304, 2388 and 2294, 2388 and 2295, 2388 and 2296, 2388 and 2297, 2388 and 2298, 2388 and 2299, 2388 and 2300, 2388 and 2301, 2388 and 2302, 2388 and 2303, 2388 and 2304, 2389 and 2294, 2389 and 2295, 2389 and 2296, 2389 and 2297, 2389 and 2298, 2389 and 2299, 2389 and 2300, 2389 and 2301, 2389 and 2302, 2389 and 2303, 2389 and 2304, 2390 and 2294, 2390 and 2295, 2390 and 2296, 2390 and 2297, 2390 and 2298, 2390 and 2299, 2390 and 2300, 2390 and 2301, 2390 and 2302, 2390 and 2303, 2390 and 2304, 2391 and 2294, 2391 and 2295, 2391 and 2296, 2391 and 2297, 2391 and 2298, 2391 and 2299, 2391 and 2300, 2391 and 2301, 2391 and 2302, 2391 and 2303, 2391 and 2304, 2392 and 2294, 2392 and 2295, 2392 and 2296, 2392 and 2297, 2392 and 2298, 2392 and 2299, 2392 and 2300, 2392 and 2301, 2392 and 2302, 2392 and 2303, 2392 and 2304, 2393 and 2294, 2393 and 2295, 2393 and 2296, 2393 and 2297, 2393 and 2298, 2393 and 2299, 2393 and 2300, 2393 and 2301, 2393 and 2302, 2393 and 2303, 2393 and 2304, 2394 and 2294, 2394 and 2295, 2394 and 2296, 2394 and 2297, 2394 and 2298, 2394 and 2299, 2394 and 2300, 2394 and 2301, 2394 and 2302, 2394 and 2303, 2394 and 2304, 2395 and 2294, 2395 and 2295, 2395 and 2296, 2395 and 2297, 2395 and 2298, 2395 and 2299, 2395 and 2300, 2395 and 2301, 2395 and 2302, 2395 and 2303, 2395 and 2304, 2396 and 2294, 2396 and 2295, 2396 and 2296, 2396 and 2297, 2396 and 2298, 2396 and 2299, 2396 and 2300, 2396 and 2301, 2396 and 2302, 2396 and 2303, 2396 and 2304, 2397 and 2294, 2397 and 2295, 2397 and 2296, 2397 and 2297, 2397 and 2298, 2397 and 2299, 2397 and 2300, 2397 and 2301, 2397 and 2302, 2397 and 2303, 2397 and 2304, 2398 and 2294, 2398 and 2295, 2398 and 2296, 2398 and 2297, 2398 and 2298, 2398 and 2299, 2398 and 2300, 2398 and 2301, 2398 and 2302, 2398 and 2303, 2398 and 2304, 2399 and 2294, 2399 and 2295, 2399 and 2296, 2399 and 2297, 2399 and 2298, 2399 and 2299, 2399 and 2300, 2399 and 2301, 2399 and 2302, 2399 and 2303, 2399 and 2304, 2400 and 2294, 2400 and 2295, 2400 and 2296, 2400 and 2297, 2400 and 2298, 2400 and 2299, 2400 and 2300, 2400 and 2301, 2400 and 2302, 2400 and 2303, 2400 and 2304, 2401 and 2294, 2401 and 2295, 2401 and 2296, 2401 and 2297, 2401 and 2298, 2401 and 2299, 2401 and 2300, 2401 and 2301, 2401 and 2302, 2401 and 2303, 2401 and 2304, 2402 and 2294, 2402 and 2295, 2402 and 2296, 2402 and 2297, 2402 and 2298, 2402 and 2299, 2402 and 2300, 2402 and 2301, 2402 and 2302, 2402 and 2303, 2402 and 2304, 2403 and 2294, 2403 and 2295, 2403 and 2296, 2403 and 2297, 2403 and 2298, 2403 and 2299, 2403 and 2300, 2403 and 2301, 2403 and 2302, 2403 and 2303, 2403 and 2304, 2404 and 2294, 2404 and 2295, 2404 and 2296, 2404 and 2297, 2404 and 2298, 2404 and 2299, 2404 and 2300, 2404 and 2301, 2404 and 2302, 2404 and 2303, 2404 and 2304, 2405 and 2294, 2405 and 2295, 2405 and 2296, 2405 and 2297, 2405 and 2298, 2405 and 2299, 2405 and 2300, 2405 and 2301, 2405 and 2302, 2405 and 2303, 2405 and 2304, 2406 and 2294, 2406 and 2295, 2406 and 2296, 2406 and 2297, 2406 and 2298, 2406 and 2299, 2406 and 2300, 2406 and 2301, 2406 and 2302, 2406 and 2303, 2406 and 2304, 2407 and 2294, 2407 and 2295, 2407 and 2296, 2407 and 2297, 2407 and 2298, 2407 and 2299, 2407 and 2300, 2407 and 2301, 2407 and 2302, 2407 and 2303, 2407 and 2304, 2408 and 2294, 2408 and 2295, 2408 and 2296, 2408 and 2297, 2408 and 2298, 2408 and 2299, 2408 and 2300, 2408 and 2301, 2408 and 2302, 2408 and 2303, 2408 and 2304, 2409 and 2294, 2409 and 2295, 2409 and 2296, 2409 and 2297, 2409 and 2298, 2409 and 2299, 2409 and 2300, 2409 and 2301, 2409 and 2302, 2409 and 2303, 2409 and 2304, 2410 and 2294, 2410 and 2295, 2410 and 2296, 2410 and 2297, 2410 and 2298, 2410 and 2299, 2410 and 2300, 2410 and 2301, 2410 and 2302, 2410 and 2303, 2410 and 2304, 2411 and 2294, 2411 and 2295, 2411 and 2296, 2411 and 2297, 2411 and 2298, 2411 and 2299, 2411 and 2300, 2411 and 2301, 2411 and 2302, 2411 and 2303, 2411 and 2304, 2412 and 2294, 2412 and 2295, 2412 and 2296, 2412 and 2297, 2412 and 2298, 2412 and 2299, 2412 and 2300, 2412 and 2301, 2412 and 2302, 2412 and 2303, 2412 and 2304, 2413 and 2294, 2413 and 2295, 2413 and 2296, 2413 and 2297, 2413 and 2298, 2413 and 2299, 2413 and 2300, 2413 and 2301, 2413 and 2302, 2413 and 2303, 2413 and 2304, 2414 and 2294, 2414 and 2295, 2414 and 2296, 2414 and 2297, 2414 and 2298, 2414 and 2299, 2414 and 2300, 2414 and 2301, 2414 and 2302, 2414 and 2303, 2414 and 2304, 2415 and 2294, 2415 and 2295, 2415 and 2296, 2415 and 2297, 2415 and 2298, 2415 and 2299, 2415 and 2300, 2415 and 2301, 2415 and 2302, 2415 and 2303, 2415 and 2304, 2416 and 2294, 2416 and 2295, 2416 and 2296, 2416 and 2297, 2416 and 2298, 2416 and 2299, 2416 and 2300, 2416 and 2301, 2416 and 2302, 2416 and 2303, 2416 and 2304, 2417 and 2294, 2417 and 2295, 2417 and 2296, 2417 and 2297, 2417 and 2298, 2417 and 2299, 2417 and 2300, 2417 and 2301, 2417 and 2302, 2417 and 2303, 2417 and 2304, 2418 and 2294, 2418 and 2295, 2418 and 2296, 2418 and 2297, 2418 and 2298, 2418 and 2299, 2418 and 2300, 2418 and 2301, 2418 and 2302, 2418 and 2303, 2418 and 2304, 2419 and 2294, 2419 and 2295, 2419 and 2296, 2419 and 2297, 2419 and 2298, 2419 and 2299, 2419 and 2300, 2419 and 2301, 2419 and 2302, 2419 and 2303, 2419 and 2304, 2420 and 2294, 2420 and 2295, 2420 and 2296, 2420 and 2297, 2420 and 2298, 2420 and 2299, 2420 and 2300, 2420 and 2301, 2420 and 2302, 2420 and 2303, 2420 and 2304, 2421 and 2294, 2421 and 2295, 2421 and 2296, 2421 and 2297, 2421 and 2298, 2421 and 2299, 2421 and 2300, 2421 and 2301, 2421 and 2302, 2421 and 2303, 2421 and 2304, 2422 and 2294, 2422 and 2295, 2422 and 2296, 2422 and 2297, 2422 and 2298, 2422 and 2299, 2422 and 2300, 2422 and 2301, 2422 and 2302, 2422 and 2303, or 2422 and 2304, The gRNA core of the PEgRNA can comprise SEQ ID NO. 5857-5859. Exemplary PEgRNAs provided in Table 15 can comprise SEQ ID NO. 2445, 2446, 2447, 2448, 2449, 2450, 2451, 2452, 2453, 2454, 2455, 2456, 2457, 2458, 2459, 2460, 2461, 2462, 2463, 2464, 2465, 2466, 2467, 2468, 2469, 2470, 2471, 2472, 2473, 2474, 2475, 2476, 2477, 2478, 2479, 2480, 2481, 2482, 2483, 2484, 2485, 2486, 2487, 2488, 2489, 2490, 2491, 2492, 2493, 2494, 2495, 2496, 2497, 2498, 2499, 2500, 2501, 2502, 2503, 2504, 2505, 2506, 2507, 2508, 2509, 2510, 2511, 2512, 2513, 2514, 2515, 2516, 2517, 2518, 2519, 2520, 2521, 2522, 2523, 2524, 2525, 2526, 2527, 2528, 2529, 2530, 2531, 2532, 2533, 2534, 2535, 2538, 2539, 2540, 2541, 2542, 2543, 2544, 2545, 2546, 2547, 2548, 2549, 2550, 2551, 2553, 2554, 2555, 2556, 2557, 2558, 2559, 2560, 2561, 2562, 2563, 2564, 2565, 2566, 2567, 2568, 2569, 2570, 2571, 2572, 2573, 2574, 2575, 2576, 2577, 2578, 2580, 2582, 2584, 2585, 2586, 2587, 2588, 2589, 2590, 2591, 2592, 2593, 2594, 2595, 2596, 2597, 2598, 2600, 2601, 2602, 2603, 2605, 2606, 2607, 2608, 2609, 2610, 2611, 2612, 2613, 2614, 2615, 2616, 2617, 2618, 2619, 2620, 2621, 2623, 2624, 2628, 2629, 2630, 2631, 2632, 2633, 2634, 2635, 2636, 2637, 2638, 2643, 2645, 2646, 2647, 2648, 2649, 2650, 2651, 2652, 2653, 2654, 2655, 2656, 2657, 2658, 2659, 2660, 2663, 2664, 2665, 2667, 2668, 2669, 2670, 2671, 2672, 2674, 2675, 2676, 2677, 2678, 2680, 2681, 2683, 2685, 2687, 2688, 2689, 2690, 2692, 2694, 2695, 2696, 2697, 2699, 2701, 2702, 2704, 2706, 2708, 2711, 2713, 2715, 2716, 2717, 2720, 2721, 2722, 2723, 2725, 2726, 2727, 2728, 2729, 2730, 2733, 2734, 2735, 2744, 2747, 2748, 2749, 2752, 2753, 2757, 2758, 2759, 2760, 2761, 2762, 2764, 2765, 2768, 2769, 2770, 2772, 2773, 2774, 2777, 2786, 2788, 2789, 2790, 2791, 2792, 2793, 2794, 2795, 2796, 2797, 2798, 2799, 2800, 2801, 2802, 2807, 2810, 2811, 2812, 2814, 2816, 2824, 2825, 2826, 2828, 2829, 2830, 2832, 2833, 2834, 2841, 2842, 2843, 2844, 2846, 2847, 2854, 2855, 2856, 2857, 2862, 2864, 2866, 2867, 2868, 2869, 2870, 2871, 2885, 2886, 2887, 2888, 2889, 2890, 2891, 2893, 2894, 2896, 2898, 2899, 2901, 2902, 2909, 2910, 2914, 2916, 2918, 2919, 2920, 2926, 2927, 2932, 2933, 2937, 2938, 2939, 2941, 2942, 2945, 2953, 2954, 2956, 2957, 2960, 2962, 2963, 2964, 2965, 2967, 2972, 2973, 2977, 2979, 2980, 2982, 2983, 2988, 2991, 2993, 2994, 2995, 2997, 3006, 3008, 3012, 3013, 3015, 3023, 3024, 3027, 3028, 3029, 3030, 3031, 3032, 3033, 3034, 3035, 3036, 3037, 3038, 3039, 3043, 3044, 3045, 3046, 3048, 3049, 3050, 3051, 3052, 3053, 3054, 3055, 3059, 3064, 3065, 3071, 3072, 3075, 3076, 3080, 3082, 3084, 3093, 3096, 3098, 3099, 3101, 3119, 3121, 3122, 3123, 3124, 3126, 3128, 3130, 3133, 3142, 3144, 3148, 3159, 3161, 3162, 3163, 3164, 3165, 3166, 3168, 3169, 3170, 3176, 3182, 3188, 3190, 3191, 3195, 3200, 3202, 3203, 3210, 3212, 3216, 3218, 3226, 3227, 3228, 3229, 3230, 3231, 3232, 3234, 3235, 3238, 3239, 3241, 3243, 3244, 3245, 3246, 3247, 3248, 3249, 3250, 3260, 3262, 3263, 3271, 3273, 3275, 3281, 3282, 3283, 3287, 3288, 3289, 3300, 3301, 3302, 3303, 3304, 3305, 3307, 3310, 3311, 3312, 3313, 3314, 3315, 3316, 3317, 3318, 3322, 3324, 3325, 3328, 3330, 3346, 3347, 3348, 3349, 3350, 3358, 3359, 3362, 3364, 3365, 3366, 3367, 3368, 3372, 3373, 3382, 3385, 3387, 3388, 3389, 3390, 3391, 3392, 3393, 3400, 3403, 3404, 3405, 3407, 3408, 3409, 3412, 3414, 3420, 3423, 3425, 3426, 3427, 3428, 3429, 3430, 3431, 3434, 3438, 3441, 3442, 3446, 3449, 3450, 3451, 3452, 3453, 3454, 3455, 3463, 3466, 3469, 3470, 3471, 3472, 3473, 3474, 3477, 3478, 3480, 3481, 3482, 3487, 3490, 3494, 3498, 3499, 3502, 3503, 3505, 3506, 3508, 3509, 3510, 3511, 3513, 3520, 3522, 3523, 3526, 3529, 3533, 3535, 3536, 3542, 3543, 3546, 3547, 3549, 3550, 3553, 3554, 3555, 3557, 3560, 3561, 3563, 3564, 3567, 3568, 3569, 3571, 3574, 3575, 576, 3578, 3579, 3580, 3581, 3583, 3584, 3585, 3592, 3594, 3595, 3596, 3597, 3603, 3612, 3613, 3617, 3622, 3625, 3626, 3627, 3628, 3630, 3631, 3632, 3633, 3635, 3636, 3638, 3639, 3640, 3641, 3642, 3646, 3647, 3648, 3654, 3657, 3659, 3660, 3661, 3664, 3668, 3669, 3673, 3674, 3678, 3679, 3680, 3681, 3684, 3685, 3687, 3688, 3697, 3699, 3702, 3703, 3704, 3705, 3706, 3708, 3710, 3711, 3712, 3714, 3715, 3721, 3722, 3724, 3725, 3728, 3729, 3730, 3731, 3732, 3733, 3734, 3735, 3736, 3737, 3739, 3740, 3741, 3743, 3744, 3746, 3748, 3755, 3761, 3770, 3771, 3773, 3774, 3776, 3778, 3779, 3781, 3782, 3784, 3785, 3792, 3793, 3794, 3795, 3796, 3797, 3798, 3799, 3800, 3801, 3802, 3803, 3804, 3805, 3806, 3807, 3808, 3809, 3810, 3811, 3812, 3814, 3815, 3816, 3820, 3829, 3839, 3841, 3842, 3843, 3844, 3845, 3851, 3852, 3853, 3854, 3855, 3856, 3857, 3858, 3859, 3860, 3861, 3862, 3863, 3864, 3865, 3868, 3869, 3871, 3874, 3875, 3876, 3877, 3878, 3879, 3880, 3882, 3883, 3884, 3885, 3887, 3895, 3899, 3904, 3907, 3908, 3909, 3910, 3911, 3912, 3913, 3914, 3915, 3916, 3917, 3921, 3924, 3927, 3928, 3929, 3931, 3932, 3935, 3936, 3937, 3938, 3939, 3940, 3941, 3942, 3943, 3945, 3946, 3956, 3957, 3961, 3962, 3965, 3971, 3977, 3978, 3979, 3980, 3981, 3982, 3983, 3985, 3988, 3989, 3990, 3991, 3992, 3993, 3994, 3995, 3997, 3998, 3999, 4001, 4002, 4003, 4004, 4009, 4011, 4012, 4013, 4015, 4016, 4017, 4020, 4021, 4023, 4025, 4026, 4028, 4029, 4031, 4032, 4034, 4035, 4036, 4037, 4038, 4040, 4052, 4055, 4056, 4060, 4061, 4066, 4067, 4070, 4077, 4078, 4080, 4081, 4082, 4083, 4084, 4085, 4086, 4087, 4088, 4089, 4090, 4102, 4105, 4106, 4108, 4109, 4110, 4114, 4115, 4117, 4118, 4119, 4128, 4129, 4132, 4136, 4137, 4142, 4147, 4159, 4163, 4168, 4170, 4171, 4172, 4173, 4175, 4182, 4183, 4186, 4188, 4192, 4194, 4199, 4208, 4225, 4226, 4227, 4228, 4232, 4239, 4240, or 4258. Such PEgRNA sequences may further comprise a 3′ motif at the 3′ end of the extension arm, for example, a hairpin-forming motif or a series of 3, 4 or more U nucleotides. Exemplary PEgRNA sequences with such 3′ adaptations include SEQ ID NOs: 2536, 2537, 2552, 2579, 2581, 2583, 2599, 2604, 2622, 2644, 2661, 2662, 2682, 2686, 2691, 2693, 2698, 2710, 2738, 2739, 2745, 2750, 2751, 2771, 2775, 2776, 2782, 2785, 2787, 2818, 2823, 2831, 2835, 2839, 2873, 2874, 2876, 2892, 2895, 2897, 2904, 2906, 2912, 2915, 2917, 2923, 2948, 2952, 2970, 2974, 2987, 2989, 3003, 3022, 3025, 3040, 3079, 3083, 3089, 3100, 3105, 3108, 3120, 3127, 3137, 3149, 3167, 3174, 3184, 3194, 3199, 3225, 3259, 3264, 3267, 3268, 3272, 3276, 3277, 3279, 3286, 3297, 3345, 3355, 3411, 3415, 3424, 3443, 3476, 3501, 3540, 3552, 3650, and 3653. Without being bound by theory, such 3′ motifs are believed to increase PEgRNA stability. Such PEgRNA sequences may alternatively or additionally be adapted for transcription from a DNA template, for example, by including a 5′ terminal G if the spacer of the PEgRNA begins with another nucleotide, by including 6 or 7 U nucleotides at the 3′ end of the extension arm, or both. Exemplary transcription-adapted sequences include SEQ ID NOs: 2625, 2626, 2627, 2639, 2640, 2641, 2642, 2666, 2673, 2679, 2684, 2700, 2703, 2705, 2707, 2709, 2712, 2714, 2718, 2719, 2724, 2731, 2732, 2736, 2737, 2740, 2741, 2742, 2743, 2746, 2754, 2755, 2756, 2763, 2766, 2767, 2778, 2779, 2780, 2781, 2783, 2784, 2803, 2804, 2805, 2806, 2808, 2809, 2813, 2815, 2817, 2819, 2820, 2821, 2822, 2827, 2836, 2837, 2838, 2840, 2845, 2848, 2849, 2850, 2851, 2852, 2853, 2858, 2859, 2860, 2861, 2863, 2865, 2872, 2875, 2877, 2878, 2879, 2880, 2881, 2882, 2883, 2884, 2900, 2903, 2905, 2907, 2908, 2911, 2913, 2921, 2922, 2924, 2925, 2928, 2929, 2930, 2931, 2934, 2935, 2936, 2940, 2943, 2944, 2946, 2947, 2949, 2950, 2951, 2955, 2958, 2959, 2961, 2966, 2968, 2969, 2971, 2975, 2976, 2978, 2981, 2984, 2985, 2986, 2990, 2992, 2996, 2998, 2999, 3000, 3001, 3002, 3004, 3005, 3007, 3009, 3010, 3011, 3014, 3016, 3017, 3018, 3019, 3020, 3021, 3026, 3041, 3042, 3047, 3056, 3057, 3058, 3060, 3061, 3062, 3063, 3066, 3067, 3068, 3069, 3070, 3073, 3074, 3077, 3078, 3081, 3085, 3086, 3087, 3088, 3090, 3091, 3092, 3094, 3095, 3097, 3102, 3103, 3104, 3106, 3107, 3109, 3110, 3111, 3112, 3113, 3114, 3115, 3116, 3117, 3118, 3125, 3129, 3131, 3132, 3134, 3135, 3136, 3138, 3139, 3140, 3141, 3143, 3145, 3146, 3147, 3150, 3151, 3152, 3153, 3154, 3155, 3156, 3157, 3158, 3160, 3171, 3172, 3173, 3175, 3177, 3178, 3179, 3180, 3181, 3183, 3185, 3186, 3187, 3189, 3192, 3193, 3196, 3197, 3198, 3201, 3204, 3205, 3206, 3207, 3208, 3209, 3211, 3213, 3214, 3215, 3217, 3219, 3220, 3221, 3222, 3223, 3224, 3233, 3236, 3237, 3240, 3242, 3251, 3252, 3253, 3254, 3255, 3256, 3257, 3258, 3261, 3265, 3266, 3269, 3270, 3274, 3278, 3280, 3284, 3285, 3290, 3291, 3292, 3293, 3294, 3295, 3296, 3298, 3299, 3306, 3308, 3309, 3319, 3320, 3321, 3323, 3326, 3327, 3329, 3331, 3332, 3333, 3334, 3335, 3336, 3337, 3338, 3339, 3340, 3341, 3342, 3343, 3344, 3351, 3352, 3353, 3354, 3356, 3357, 3360, 3361, 3363, 3369, 3370, 3371, 3374, 3375, 3376, 3377, 3378, 3379, 3380, 3381, 3383, 3384, 3386, 3394, 3395, 3396, 3397, 3398, 3399, 3401, 3402, 3406, 3410, 3413, 3416, 3417, 3418, 3419, 3421, 3422, 3432, 3433, 3435, 3436, 3437, 3439, 3440, 3444, 3445, 3447, 3448, 3456, 3457, 3458, 3459, 3460, 3461, 3462, 3464, 3465, 3467, 3468, 3475, 3479, 3483, 3484, 3485, 3486, 3488, 3489, 3491, 3492, 3493, 3495, 3496, 3497, 3500, 3504, 3507, 3512, 3514, 3515, 3516, 3517, 3518, 3519, 2521, 3524, 3525, 3527, 3528, 3530, 3531, 3532, 3534, 3537, 3538, 3539, 3541, 3544, 3545, 3548, 3551, 3556, 3558, 3559, 3562, 3565, 3566, 3570, 3572, 3573, 3577, 3582, 3586, 3587, 3588, 3589, 3590, 3591, 3593, 3598, 3599, 3600, 3601, 3602, 3604, 3605, 3606, 3607, 3608, 3609, 3610, 3611, 3614, 3615, 3616, 3618, 3619, 3620, 3621, 3623, 3624, 3629, 3634, 3637, 3643, 3644, 3645, 3649, 3651, 3652, 3655, 3656, 3658, 3662, 3663, 3665, 3666, 3667, 3670, 3671, 3672, 3675, 3676, 3677, 3682, 3683, 3686, 3689, 3690, 3691, 3692, 3693, 3694, 3695, 3696, 3698, 3700, 3701, 3707, 3709, 3713, 3716, 3717, 3718, 3719, 3720, 3723, 3726, 3727, 3738, 3742, 3745, 3747, 3749, 3750, 3751, 3752, 3753, 3754, 3756, 3757, 3758, 3759, 3760, 3762, 3763, 3764, 3765, 3766, 3767, 3768, 3769, 3772, 3775, 3777, 3780, 3783, 3786, 3787, 3788, 3789, 3790, 3791, 3813, 3817, 3818, 3819, 3821, 3822, 3823, 3824, 3825, 3826, 3827, 3828, 3830, 3831, 3832, 3833, 3834, 3835, 3836, 3837, 3838, 3840, 3846, 3847, 3848, 3849, 3850, 3866, 3867, 3870, 3872, 3873, 3881, 3886, 3888, 3889, 3890, 3891, 3892, 3893, 3894, 3896, 3897, 3898, 3900, 3901, 3902, 3903, 3905, 3906, 3918, 3919, 3920, 3922, 3923, 3925, 3926, 3930, 3933, 3934, 3944, 3947, 3948, 3949, 3950, 3951, 3952, 3953, 3954, 3955, 3958, 3959, 3960, 3963, 3964, 3966, 3967, 3968, 3969, 3970, 3972, 3973, 3974, 3975, 3976, 3984, 3986, 3987, 3996, 4000, 4005, 4006, 4007, 4008, 4010, 4014, 4018, 4019, 4022, 4024, 4027, 4030, 4033, 4039, 4041, 4042, 4043, 4044, 4045, 4046, 4047, 4048, 4049, 4050, 4051, 4053, 4054, 4057, 4058, 4059, 4062, 4063, 4064, 4065, 4068, 4069, 4071, 4072, 4073, 4074, 4075, 4076, 4079, 4091, 4092, 4093, 4094, 4095, 4096, 4097, 4098, 4099, 4100, 4101, 4103, 4104, 4107, 4111, 4112, 4113, 4116, 4120, 4121, 4122, 4123, 4124, 4125, 4126, 4127, 4130, 4131, 4133, 4134, 4135, 4138, 4139, 4140, 4141, 4143, 4144, 4145, 4146, 4148, 4149, 4150, 4151, 4152, 4153, 4154, 4155, 4156, 4157, 4158, 4160, 4161, 4162, 4164, 4165, 4166, 4167, 4169, 4174, 4176, 4177, 4178, 4179, 4180, 4181, 4184, 4185, 4187, 4189, 4190, 4191, 4193, 4195, 4196, 4197, 4198, 4200, 4201, 4202, 4203, 4204, 4205, 4206, 4207, 4209, 4210, 4211, 4212, 4213, 4214, 4215, 4216, 4217, 4218, 4219, 4220, 4221, 4222, 4223, 4224, 4229, 4230, 4231, 4233, 4234, 4235, 4236, 4237, 4238, 4241, 4242, 4243, 4244, 4245, 4246, 4247, 4248, 4249, 4250, 4251, 4252, 4253, 4254, 4255, 4256, 4257, 4259, 4260, 4261, 4262, 4263, 4264, 4265, 4266, 4267, 4268, 4269, 4270, 4271, 4272, 4273, 4274, 4275, 4276, 4277, 4278, 4279, 4280, 4281, 4282, 4283, 4284, 4285, 4286, 4287, 4288, 4289, 4290, 4291, 4292, 4293, 4294, 4295, 4296, 4297, 4298, 4299, 4300, 4301, 4302, 4303, 4304, 4305, 4306, 4307, 4308, 4309, 4310, 4311, 4312, 4313, 4314, 4315, 4316, 4317, 4318, 4319, 4320, 4321, 4322, 4323, 4324, 4325, 4326, 4327, 4328, 4329, 4330, 4331, 4332, 4333, 4334, 4335, 4336, 4337, 4338, 4339, 4340, 4341, 4342, 4343, 4344, 4345, 4346, 4347, 4348, 4349, 4350, 4351, 4352, 4353, 4354, 4355, 4356, 4357, 4358, 4359, 4360, 4361, 4362, 4363, 4364, 4365, 4366, 4367, 4368, 4369, 4370, 4371, 4372, 4373, 4374, 4375, 4376, 4377, 4378, 4379, 4380, 4381, 4382, 4383, 4384, 4385, 4386, 4387, 4388, 4389, 4390, 4391, 4392, 4393, 4394, 4395, 4396, 4397, 4398, 4399, 4400, 4401, 4402, 4403, 4404, 4405, 4406, 4407, 4408, and 4409. Such plasmid adapted sequences may further comprise a hairpin-forming motif between the PBS and the 3′ terminal U series.

Any of the PEgRNAs of Table 15 can be used in a Prime Editing system further comprising a nick guide RNA (ngRNA). Such ngRNA can comprise a spacer comprising nucleotides 5-20 of SEQ ID NO: 41, 60, 61, 62, 63, 64, 65, 66, 69, 71, 2045, 2046, 2047, 2048, 2049, 2050, 2051, 2052, 2053, 2054, 2055, 2056, 2057, 2058, 2059, 2060, 2061, 2062, 2063, 2064, 2065, 2066, 2067, 2068, 2069, 2070, 2071, 2072, 2073, 2074, 2075, 2076, 2077, 2078, 2079, 2080, 2081, 2082, 2083, 2084, 2085, 2086, 2087, 2088, 2089, 2090, 2091, 2092, 2093, 2094, 2095, 2096, 2423, 2424, 2425, 2426, 2427, 2428, 2429, 2430, 2431, 2432, 2433, 2434, 2435, 2436, 2437, 2438, 2439, 2440, 2441, 2442, 2443, or 2444 and a gRNA core capable of binding to a Cas9 protein. The spacer of the ngRNA can comprise nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 41, 60, 61, 62, 63, 64, 65, 66, 69, 71, 2045, 2046, 2047, 2048, 2049, 2050, 2051, 2052, 2053, 2054, 2055, 2056, 2057, 2058, 2059, 2060, 2061, 2062, 2063, 2064, 2065, 2066, 2067, 2068, 2069, 2070, 2071, 2072, 2073, 2074, 2075, 2076, 2077, 2078, 2079, 2080, 2081, 2082, 2083, 2084, 2085, 2086, 2087, 2088, 2089, 2090, 2091, 2092, 2093, 2094, 2095, 2096, 2423, 2424, 2425, 2426, 2427, 2428, 2429, 2430, 2431, 2432, 2433, 2434, 2435, 2436, 2437, 2438, 2439, 2440, 2441, 2442, 2443, or 2444. The spacer of the ngRNA can comprise SEQ ID NO: 41, 60, 61, 62, 63, 64, 65, 66, 69, 71, 2045, 2046, 2047, 2048, 2049, 2050, 2051, 2052, 2053, 2054, 2055, 2056, 2057, 2058, 2059, 2060, 2061, 2062, 2063, 2064, 2065, 2066, 2067, 2068, 2069, 2070, 2071, 2072, 2073, 2074, 2075, 2076, 2077, 2078, 2079, 2080, 2081, 2082, 2083, 2084, 2085, 2086, 2087, 2088, 2089, 2090, 2091, 2092, 2093, 2094, 2095, 2096, 2423, 2424, 2425, 2426, 2427, 2428, 2429, 2430, 2431, 2432, 2433, 2434, 2435, 2436, 2437, 2438, 2439, 2440, 2441, 2442, 2443, or 2444. The gRNA core of the ngRNA can comprise SEQ ID NO. 5857-5859. Exemplary ngRNA provided in Table 15 can comprise SEQ ID NO: 2257, 2258, 2259, 2260, 2261, 2262, 2263, 2264, 2265, 2266, 2267, 2268, 2269, 2270, 2271, 2272, 2273, 2274, 2275, 2276, 2277, 2278, 2279, 2280, 2281, 2282, 2283, 2284, 2285, 2286, 2287, 2288, 2289, 4410, 4411, 4412, 4413, 4414, 4415, 4416, 4417, 4418, 4419, 4420, 4421, or 4422. Such ngRNA sequences may further comprise a 3′ motif at the 3′ end of the gRNA core, for example, a series of 3, 4 or more U nucleotides. Exemplary ngRNA sequences with such 3′ adaptations include SEQ ID NOs: 2290, 2291, 2292, 4423, or 4424. Without being bound by theory, such 3′ motifs are believed to increase ngRNA stability.

Table 16 provides Prime Editing guide RNAs (PEgRNAs), which can be used in Prime Editing systems disclosed herein. Such Prime Editing systems can comprise a Cas9 protein capable of recognizing an NGG or NG PAM sequence (e.g., GGG or GG), and a reverse transcriptase. The Prime Editing systems (e.g., PE3 or PE3b systems) can further comprise a nick guide RNA (ngRNA). Such PEgRNAs and Prime Editing systems can be used, for example, to correct an H1069Q mutation in ATP7B.

The PEgRNAs of Table 16 comprise: (a) a spacer comprising nucleotides 5-20 of SEQ ID NO: 4425, (b) a gRNA core capable of binding to a Cas9 protein, and (c) an extension arm comprising: (i) an editing template comprising any one of SEQ ID NOs: 4437-4492, and (ii) a primer binding site (PBS) comprising any one of SEQ ID NOs: 2297, 4426, 4427, 4428, 4429, 4430, 4431, 4432, 4433, 4434, 4435, and 4436. The spacer of the PEgRNA can comprise, for example, nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 4425. The spacer of the PEgRNA can comprise SEQ ID NO: 4425. The RTT and the PBS can comprise respectively SEQ ID NOs: 4437 and 2297, 4437 and 4426, 4437 and 4427, 4437 and 4428, 4437 and 4429, 4437 and 4430, 4437 and 4431, 4437 and 4432, 4437 and 4433, 4437 and 4434, 4437 and 4435, 4437 and 4436, 4438 and 2297, 4438 and 4426, 4438 and 4427, 4438 and 4428, 4438 and 4429, 4438 and 4430, 4438 and 4431, 4438 and 4432, 4438 and 4433, 4438 and 4434, 4438 and 4435, 4438 and 4436, 4439 and 2297, 4439 and 4426, 4439 and 4427, 4439 and 4428, 4439 and 4429, 4439 and 4430, 4439 and 4431, 4439 and 4432, 4439 and 4433, 4439 and 4434, 4439 and 4435, 4439 and 4436, 4440 and 2297, 4440 and 4426, 4440 and 4427, 4440 and 4428, 4440 and 4429, 4440 and 4430, 4440 and 4431, 4440 and 4432, 4440 and 4433, 4440 and 4434, 4440 and 4435, 4440 and 4436, 4441 and 2297, 4441 and 4426, 4441 and 4427, 4441 and 4428, 4441 and 4429, 4441 and 4430, 4441 and 4431, 4441 and 4432, 4441 and 4433, 4441 and 4434, 4441 and 4435, 4441 and 4436, 4442 and 2297, 4442 and 4426, 4442 and 4427, 4442 and 4428, 4442 and 4429, 4442 and 4430, 4442 and 4431, 4442 and 4432, 4442 and 4433, 4442 and 4434, 4442 and 4435, 4442 and 4436, 4443 and 2297, 4443 and 4426, 4443 and 4427, 4443 and 4428, 4443 and 4429, 4443 and 4430, 4443 and 4431, 4443 and 4432, 4443 and 4433, 4443 and 4434, 4443 and 4435, 4443 and 4436, 4444 and 2297, 4444 and 4426, 4444 and 4427, 4444 and 4428, 4444 and 4429, 4444 and 4430, 4444 and 4431, 4444 and 4432, 4444 and 4433, 4444 and 4434, 4444 and 4435, 4444 and 4436, 4445 and 2297, 4445 and 4426, 4445 and 4427, 4445 and 4428, 4445 and 4429, 4445 and 4430, 4445 and 4431, 4445 and 4432, 4445 and 4433, 4445 and 4434, 4445 and 4435, 4445 and 4436, 4446 and 2297, 4446 and 4426, 4446 and 4427, 4446 and 4428, 4446 and 4429, 44, 46 and 4430, 4446 and 4431, 4446 and 4432, 4446 and 4433, 4446 and 4434, 4446 and 4435, 4446 and 4436, 4447 and 2297, 4447 and 4426, 4447 and 4427, 4447 and 4428, 4447 and 4429, 4447 and 4430, 4447 and 4431, 4447 and 4432, 4447 and 4433, 4447 and 4434, 4447 and 4435, 4447 and 4436, 4448 and 2297, 4448 and 4426, 4448 and 4427, 4448 and 4428, 4448 and 4429, 4448 and 4430, 4448 and 4431, 4448 and 4432, 4448 and 4433, 4448 and 4434, 4448 and 4435, 4448 and 4436, 4449 and 2297, 4449 and 4426, 4449 and 4427, 4449 and 4428, 4449 and 4429, 4449 and 4430, 4449 and 4431, 4449 and 4432, 4449 and 4433, 4449 and 4434, 4449 and 4435, 4449 and 4436, 4450 and 2297, 4450 and 4426, 4450 and 4427, 4450 and 4428, 4450 and 4429, 4450 and 4430, 4450 and 4431, 4450 and 4432, 4450 and 4433, 4450 and 4434, 4450 and 4435, 4450 and 4436, 4451 and 2297, 4451 and 4426, 4451 and 4427, 4451 and 4428, 4451 and 4429, 4451 and 4430, 4451 and 4431, 4451 and 4432, 4451 and 4433, 4451 and 4434, 4451 and 4435, 4451 and 4436, 4452 and 2297, 4452 and 4426, 4452 and 4427, 4452 and 4428, 4452 and 4429, 4452 and 4430, 4452 and 4431, 4452 and 4432, 4452 and 4433, 4452 and 4434, 4452 and 4435, 4452 and 4436, 4453 and 2297, 4453 and 4426, 4453 and 4427, 4453 and 4428, 4453 and 4429, 4453 and 4430, 4453 and 4431, 4453 and 4432, 4453 and 4433, 4453 and 4434, 4453 and 4435, 4453 and 4436, 4454 and 2297, 4454 and 4426, 4454 and 4427, 4454 and 4428, 4454 and 4429, 4454 and 4430, 4454 and 4431, 4454 and 4432, 4454 and 4433, 4454 and 4434, 4454 and 4435, 4454 and 4436, 4455 and 2297, 4455 and 4426, 4455 and 4427, 4455 and 4428, 4455 and 4429, 4455 and 4430, 4455 and 4431, 4455 and 4432, 4455 and 4433, 4455 and 4434, 4455 and 4435, 4455 and 4436, 4456 and 2297, 4456 and 4426, 4456 and 4427, 4456 and 4428, 4456 and 4429, 4456 and 4430, 4456 and 4431, 4456 and 4432, 4456 and 4433, 4456 and 4434, 4456 and 4435, 4456 and 4436, 4457 and 2297, 4457 and 4426, 4457 and 4427, 4457 and 4428, 4457 and 4429, 4457 and 4430, 4457 and 4431, 4457 and 4432, 4457 and 4433, 4457 and 4434, 4457 and 4435, 4457 and 4436, 4458 and 2297, 4458 and 4426, 4458 and 4427, 4458 and 4428, 4458 and 4429, 4458 and 4430, 4458 and 4431, 4458 and 4432, 4458 and 4433, 4458 and 4434, 4458 and 4435, 4458 and 4436, 4459 and 2297, 4459 and 4426, 4459 and 4427, 4459 and 4428, 4459 and 4429, 4459 and 4430, 4459 and 4431, 4459 and 4432, 4459 and 4433, 4459 and 4434, 4459 and 4435, 4459 and 4436, 4460 and 2297, 4460 and 4426, 4460 and 4427, 4460 and 4428, 4460 and 4429, 4460 and 4430, 4460 and 4431, 4460 and 4432, 4460 and 4433, 4460 and 4434, 4460 and 4435, 4460 and 4436, 4461 and 2297, 4461 and 4426, 4461 and 4427, 4461 and 4428, 4461 and 4429, 4461 and 4430, 4461 and 4431, 4461 and 4432, 4461 and 4433, 4461 and 4434, 4461 and 4435, 4461 and 4436, 4462 and 2297, 4462 and 4426, 4462 and 4427, 4462 and 4428, 4462 and 4429, 4462 and 4430, 4462 and 4431, 4462 and 4432, 4462 and 4433, 4462 and 4434, 4462 and 4435, 4462 and 4436, 4463 and 2297, 4463 and 4426, 4463 and 4427, 4463 and 4428, 4463 and 4429, 4463 and 4430, 4463 and 4431, 4463 and 4432, 4463 and 4433, 4463 and 4434, 4463 and 4435, 4463 and 4436, 4464 and 2297, 4464 and 4426, 4464 and 4427, 4464 and 4428, 4464 and 4429, 4464 and 4430, 4464 and 4431, 4464 and 4432, 4464 and 4433, 4464 and 4434, 4464 and 4435, 4464 and 4436, 4465 and 2297, 4465 and 4426, 4465 and 4427, 4465 and 4428, 4465 and 4429, 4465 and 4430, 4465 and 4431, 4465 and 4432, 4465 and 4433, 4465 and 4434, 4465 and 4435, 4465 and 4436, 4466 and 2297, 4466 and 4426, 4466 and 4427, 4466 and 4428, 4466 and 4429, 4466 and 4430, 4466 and 4431, 4466 and 4432, 4466 and 4433, 4466 and 4434, 4466 and 4435, 4466 and 4436, 4467 and 2297, 4467 and 4426, 4467 and 4427, 4467 and 4428, 4467 and 4429, 4467 and 4430, 4467 and 4431, 4467 and 4432, 4467 and 4433, 4467 and 4434, 4467 and 4435, 4467 and 4436, 4468 and 2297, 4468 and 4426, 4468 and 4427, 4468 and 4428, 4468 and 4429, 4468 and 4430, 4468 and 4431, 4468 and 4432, 4468 and 4433, 4468 and 4434, 4468 and 4435, 4468 and 4436, 4469 and 2297, 4469 and 4426, 4469 and 4427, 4469 and 4428, 4469 and 4429, 4469 and 4430, 4469 and 4431, 4469 and 4432, 4469 and 4433, 4469 and 4434, 4469 and 4435, 4469 and 4436, 4470 and 2297, 4470 and 4426, 4470 and 4427, 4470 and 4428, 4470 and 4429, 4470 and 4430, 4470 and 4431, 4470 and 4432, 4470 and 4433, 4470 and 4434, 4470 and 4435, 4470 and 4436, 4471 and 2297, 4471 and 4426, 4471 and 4427, 4471 and 4428, 4471 and 4429, 4471 and 4430, 4471 and 4431, 4471 and 4432, 4471 and 4433, 4471 and 4434, 4471 and 4435, 4471 and 4436, 4472 and 2297, 4472 and 4426, 4472 and 4427, 4472 and 4428, 4472 and 4429, 4472 and 4430, 4472 and 4431, 4472 and 4432, 4472 and 4433, 4472 and 4434, 4472 and 4435, 4472 and 4436, 4473 and 2297, 4473 and 4426, 4473 and 4427, 4473 and 4428, 4473 and 4429, 4473 and 4430, 4473 and 4431, 4473 and 4432, 4473 and 4433, 4473 and 4434, 4473 and 4435, 4473 and 4436, 4474 and 2297, 4474 and 4426, 4474 and 4427, 4474 and 4428, 4474 and 4429, 4474 and 4430, 4474 and 4431, 4474 and 4432, 4474 and 4433, 4474 and 4434, 4474 and 4435, 4474 and 4436, 4475 and 2297, 4475 and 4426, 4475 and 4427, 4475 and 4428, 4475 and 4429, 4475 and 4430, 4475 and 4431, 4475 and 4432, 4475 and 4433, 4475 and 4434, 4475 and 4435, 4475 and 4436, 4476 and 2297, 4476 and 4426, 4476 and 4427, 4476 and 4428, 4476 and 4429, 4476 and 4430, 4476 and 4431, 4476 and 4432, 4476 and 4433, 4476 and 4434, 4476 and 4435, 4476 and 4436, 4477 and 2297, 4477 and 4426, 4477 and 4427, 4477 and 4428, 4477 and 4429, 4477 and 4430, 4477 and 4431, 4477 and 4432, 4477 and 4433, 4477 and 4434, 4477 and 4435, 4477 and 4436, 4478 and 2297, 4478 and 4426, 4478 and 4427, 4478 and 4428, 4478 and 4429, 4478 and 4430, 4478 and 4431, 4478 and 4432, 4478 and 4433, 4478 and 4434, 4478 and 4435, 4478 and 4436, 4479 and 2297, 4479 and 4426, 4479 and 4427, 4479 and 4428, 4479 and 4429, 4479 and 4430, 4479 and 4431, 4479 and 4432, 4479 and 4433, 4479 and 4434, 4479 and 4435, 4479 and 4436, 4480 and 2297, 4480 and 4426, 4480 and 4427, 4480 and 4428, 4480 and 4429, 4480 and 4430, 4480 and 4431, 4480 and 4432, 4480 and 4433, 4480 and 4434, 4480 and 4435, 4480 and 4436, 4481 and 2297, 4481 and 4426, 4481 and 4427, 4481 and 4428, 4481 and 4429, 4481 and 4430, 4481 and 4431, 4481 and 4432, 4481 and 4433, 4481 and 4434, 4481 and 4435, 4481 and 4436, 4482 and 2297, 4482 and 4426, 4482 and 4427, 4482 and 4428, 4482 and 4429, 4482 and 4430, 4482 and 4431, 4482 and 4432, 4482 and 4433, 4482 and 4434, 4482 and 4435, 4482 and 4436, 4483 and 2297, 4483 and 4426, 4483 and 4427, 4483 and 4428, 4483 and 4429, 4483 and 4430, 4483 and 4431, 4483 and 4432, 4483 and 4433, 4483 and 4434, 4483 and 4435, 4483 and 4436, 4484 and 2297, 4484 and 4426, 4484 and 4427, 4484 and 4428, 4484 and 4429, 4484 and 4430, 4484 and 4431, 4484 and 4432, 4484 and 4433, 4484 and 4434, 4484 and 4435, 4484 and 4436, 44185 and 2297, 4485 and 4426, 4485 and 4427, 4485 and 4428, 4485 and 4429, 4485 and 4430, 4485 and 4431, 4485 and 4432, 4485 and 4433, 4485 and 4434, 4485 and 4435, 4485 and 4436, 4486 and 2297, 4486 and 4426, 4486 and 4427, 4486 and 4428, 4486 and 4429, 4486 and 4430, 4486 and 4431, 4486 and 4432, 4486 and 4433, 4486 and 4434, 4486 and 4435, 4486 and 4436, 4487 and 2297, 4487 and 4426, 4487 and 4427, 4487 and 4428, 4487 and 4429, 4487 and 4430, 4487 and 4431, 4487 and 4432, 4487 and 4433, 4487 and 4434, 4487 and 4435, 4487 and 4436, 4488 and 2297, 4488 and 4426, 4488 and 4427, 4488 and 4428, 4488 and 4429, 4488 and 4430, 4488 and 4431, 4488 and 4432, 4488 and 4433, 4488 and 4434, 4488 and 4435, 4488 and 4436, 4489 and 2297, 4489 and 4426, 4489 and 4427, 4489 and 4428, 4489 and 4429, 4489 and 4430, 4489 and 4431, 4489 and 4432, 4489 and 4433, 4489 and 4434, 4489 and 4435, 4489 and 4436, 4490 and 2297, 4490 and 4426, 4490 and 4427, 4490 and 4428, 4490 and 4429, 4490 and 4430, 4490 and 4431, 4490 and 4432, 4490 and 4433, 4490 and 4434, 4490 and 4435, 4490 and 4436, 4491 and 2297, 4491 and 4426, 4491 and 4427, 4491 and 4428, 4491 and 4429, 4491 and 4430, 4491 and 4431, 4491 and 4432, 4491 and 4433, 4491 and 4434, 4491 and 4435, 4491 and 4436, 4492 and 2297, 4492 and 4426, 4492 and 4427, 4492 and 4428, 4492 and 4429, 4492 and 4430, 4492 and 4431, 4492 and 4432, 4492 and 4433, 4492 and 4434, 4492 and 4435, or 4492 and 4436. The gRNA core of the PEgRNA can comprise SEQ ID NO. 5857-5859. Exemplary PEgRNAs provided in Table 16 can comprise SEQ ID NO. 4493, 4494, 4495, 4496, 4497, 4498, 4499, 4500, 4501, 4502, 4503, 4504, 4505, 4506, 4507, 4518, 4509, 4510, 4511, 4512, 4513, 4514, 4515, 4516, 4517, 4518, 4519, 4521, 4522, 4523, 4524, 4525, 4526, 4527, 4528, 4529, 4530, 4531, 4532, 4533, 4535, 4537, 4538, 4539, 4540, 4542, 4543, 4544, 4545, 4546, 4547, 4549, 4550, 4552, 4553, 4555, 4556, 4557, 4558, 4559, 4560, 4561, 4562, 4564, 4566, 4567, 4568, 4570, 4573, 4574, 4575, 4576, 4577, 4578, 4579, 4580, 4581, 4582, 4583, 4584, 4585, 4586, 4587, 4588, 4589, 4591, 4592, 4595, 4596, 4597, 4603, 4604, 4605, 4606, 4612, 4614, 4617, 4618, 4619, 4620, 4621, 4623, 4624, 4625, 4626, 4627, 4629, 4630, 4632, 4634, 4635, 4637, 4638, 4639, 4640, 4646, 4649, 4651, 4653, 4654, 4655, 4656, 4657, 4659, 4660, 4661, 4662, 4666, 4667, 4668, 4670, 4673, 4674, 4675, 4676, 4677, 4679, 4682, 4684, 4689, 4691, 4697, 4698, 4700, 4702, 4705, 4710, 4711, 4714, 4717, 4718, 4719, 4720, 4722, 4724, 4725, 4726, 4737, 4738, 4739, 4740, 4742, 4746, 4748, 4749, 4751, 4755, 4756, 4758, 4759, 4760, 4761, 4763, 4765, 4767, 4768, 4776, 4780, 4784, 4785, 4789, 4792, 4795, 4799, 4801, 4802, 4804, 4809, 4811, 4812, 4816, 4817, 4820, 4822, 4826, 4827, 4828, 4832, 4835, 4836, 4839, 4840, 4841, 4842, 4848, 4852, 4856, 4857, 4859, 4860, 4861, 4862, 4864, 4865, 4868, 4871, 4873, 4874, 4875, 4876, 4882, 4887, 4889, 4893, 4895, 4896, 4898, 4902, 4904, 4905, 4907, 4908, 4909, 4912, 4913, 4915, 4922, 4923, 4924, 4932, 4934, 4935, 4936, 4937, 4939, 4947, 4949, 4950, 4951, 4954, 4955, 4956, 4957, 4958, 4959, 4961, 4963, 4969, 4970, 4971, 4972, 4976, 4978, 4979, 4980, 4985, 4986, 4987, 4990, 4993, 4995, 4998, 5000, 5001, 5002, 5003, 5004, 5005, 5010, 5011, 5018, 5019, 5020, 5021, 5022, 5023, 5024, 5026, 5029, 5030, 5033, 5034, 5035, 5036, 5037, 5039, 5042, 5043, 5044, 5045, 5046, 5047, 5053, 5054, 5056, 5057, 5058, 5060, 5062, 5063, 5066, 5070, 5071, 5072, 5073, 5076, 5077, 5084, 5088, 5091, 5092, 5093, 5094, 5096, 5097, 5098, 5103, 5110, 5111, 5112, 5115, 5119, 5123, 5132, 5135, 5138, 5147, 5151, 5164, 5167, 5175, 5176, 5180, 5181, 5183, 5184, 5185, 5186, 5187, 5188, 5189, 5190, 5191, 5192, 5193, 5194, 5195, 5196, 5197, 5198, 5199, 5200, 5201, 5202, 5203, 5204, or 5205. Such PEgRNA sequences may further comprise a 3′ motif at the 3′ end of the extension arm, for example, a hairpin-forming motif or a series of 3, 4 or more U nucleotides. Exemplary PEgRNA sequences with such 3′ adaptations include SEQ ID NOs: 4520, 4534, 4536, 4541, 4548, 4551, 4554, 4563, 4565, 4571, 4572, 4590, 4593, 4594, 4601, 4602, 4608, 4609, 4610, 4611, 4613, 4622, 4631, 4636, 4641, 4643, 4644, 4647, 4648, 4672, 4680, 4681, 4685, 4686, 4687, 4688, 4693, 4695, 4696, 4703, 4706, 4708, 4709, 4712, 4721, 4723, 4728, 4730, 4733, 4735, 4736, 4741, 4743, 4744, 4747, 4753, 4762, 4769, 4770, 4772, 4778, 4779, 4781, 4782, 4788, 4791, 4793, 4797, 4803, 4805, 4807, 4808, 4818, 4819, 4821, 4823, 4824, 4825, 4829, 4847, 4851, 4854, 4866, 4867, 4872, 4879, 4880, 4885, 4891, 4892, 4900, 4910, 4911, 4919, 4926, 4928, 4946, 4967, and 4983. Without being bound by theory, such 3′ motifs are believed to increase PEgRNA stability. Such PEgRNA sequences may alternatively or additionally be adapted for transcription from a DNA template, for example, by including a 5′ terminal G if the spacer of the PEgRNA begins with another nucleotide, by including 6 or 7 U nucleotides at the 3′ end of the extension arm, or both. Exemplary transcription-adapted sequences include SEQ ID NOs: 4569, 4598, 4599, 4600, 4607, 4615, 4616, 4628, 4633, 4642, 4645, 4650, 4652, 4658, 4663, 4664, 4665, 4669, 4671, 4678, 4683, 4690, 4692, 4694, 4699, 4701, 4704, 4707, 4713, 4715, 4716, 4727, 4729, 4731, 4732, 4734, 4745, 4750, 4752, 4754, 4757, 4764, 4766, 4771, 4773, 4774, 4775, 4777, 4783, 4786, 4787, 4790, 4794, 4796, 4798, 4800, 4806, 4810, 4813, 4814, 4815, 4830, 4831, 4833, 4834, 4837, 4838, 4843, 4844, 4845, 4846, 4849, 4850, 4853, 4855, 4858, 4863, 4869, 4870, 4877, 4878, 4881, 4883, 4884, 4886, 4888, 4890, 4894, 4897, 4899, 4901, 49031, 4906, 4914, 4916, 4917, 4918, 4920, 4921, 4925, 4927, 4929, 4930, 4931, 4933, 4938, 4940, 4941, 4942, 4943, 4944, 4945, 4948, 4952, 4953, 4960, 4962, 4964, 4965, 4966, 4968, 4973, 4974, 4975, 4977, 4981, 4982, 4984, 4988, 4989, 4991, 4992, 4994, 4996, 4997, 4999, 5006, 5007, 5008, 5009, 5012, 5013, 5014, 5015, 5016, 5017, 5025, 5027, 5028, 5031, 5032, 5038, 5040, 5041, 5048, 5049, 5050, 5051, 5052, 5055, 5059, 5061, 5064, 5065, 5067, 5068, 5069, 5074, 5075, 5078, 5079, 5080, 5081, 5082, 5083, 5085, 5086, 5087, 5089, 5090, 5095, 5099, 5100, 5101, 5102, 5104, 5105, 5106, 5107, 5108, 5109, 5113, 5114, 5116, 5117, 5118, 5120, 5121, 5122, 5124, 5125, 5126, 5127, 5128, 5129, 5130, 5131, 5133, 5134, 5136, 5137, 5139, 5140, 5141, 5142, 5143, 5144, 5145, 5146, 5148, 5149, 5150, 5152, 5153, 5154, 5155, 5156, 5157, 5158, 5159, 5160, 5161, 5162, 5163, 5165, 5166, 5168, 5169, 5170, 5171, 5172, 5173, 5174, 5177, 5178, 5179, and 5182. Such plasmid adapted sequences may further comprise a hairpin-forming motif between the PBS and the 3′ terminal U series.

Any of the PEgRNAs of Table 16 can be used in a Prime Editing system further comprising a nick guide RNA (ngRNA). Such ngRNA can comprise a spacer comprising nucleotides 5-20 of SEQ ID NO: 41, 60, 61, 62, 63, 64, 65, 66, 69, 71, 2045, 2046, 2047, 2048, 2049, 2050, 2051, 2052, 2053, 2054, 2055, 2056, 2057, 2058, 2059, 2060, 2061, 2062, 2063, 2064, 2065, 2066, 2067, 2068, 2069, 2070, 2071, 2072, 2073, 2074, 2075, 2076, 2077, 2078, 2079, 2080, 2081, 2082, 2083, 2084, 2085, 2086, 2087, 2088, 2089, 2090, 2091, 2092, 2093, 2094, 2095, 2096, 2437, 2439, 2442, 2443, or 2444 and a gRNA core capable of binding to a Cas9 protein. The spacer of the ngRNA can comprise nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 41, 60, 61, 62, 63, 64, 65, 66, 69, 71, 2045, 2046, 2047, 2048, 2049, 2050, 2051, 2052, 2053, 2054, 2055, 2056, 2057, 2058, 2059, 2060, 2061, 2062, 2063, 2064, 2065, 2066, 2067, 2068, 2069, 2070, 2071, 2072, 2073, 2074, 2075, 2076, 2077, 2078, 2079, 2080, 2081, 2082, 2083, 2084, 2085, 2086, 2087, 2088, 2089, 2090, 2091, 2092, 2093, 2094, 2095, 2096, 2437, 2439, 2442, 2443, or 2444. The spacer of the ngRNA can comprise SEQ ID NO: 41, 60, 61, 62, 63, 64, 65, 66, 69, 71, 2045, 2046, 2047, 2048, 2049, 2050, 2051, 2052, 2053, 2054, 2055, 2056, 2057, 2058, 2059, 2060, 2061, 2062, 2063, 2064, 2065, 2066, 2067, 2068, 2069, 2070, 2071, 2072, 2073, 2074, 2075, 2076, 2077, 2078, 2079, 2080, 2081, 2082, 2083, 2084, 2085, 2086, 2087, 2088, 2089, 2090, 2091, 2092, 2093, 2094, 2095, 2096, 2437, 2439, 2442, 2443, or 2444. The gRNA core of the ngRNA can comprise SEQ ID NO. 5857-5859. Exemplary ngRNA provided in Table 16 can comprise SEQ ID NO: 2257, 2258, 2259, 2260, 2261, 2262, 2263, 2264, 2265, 2266, 2267, 2268, 2269, 2270, 2271, 2272, 2273, 2274, 2275, 2276, 2277, 2278, 2279, 2280, 2281, 2282, 2283, 2284, 2285, 2286, 2287, 2288, 2289, 4411, 4416, 4418, 4420, or 4422. Such ngRNA sequences may further comprise a 3′ motif at the 3′ end of the gRNA core, for example, a series of 3, 4 or more U nucleotides. Exemplary ngRNA sequences with such 3′ adaptations include SEQ ID NOs: 2290-2292. Without being bound by theory, such 3′ motifs are believed to increase ngRNA stability.

Table 17 provides Prime Editing guide RNAs (PEgRNAs), which can be used in Prime Editing systems disclosed herein. Such Prime Editing systems can comprise a Cas9 protein capable of recognizing an NGA, NGN, NRN, or NG PAM sequence (e.g., GGA or GG), and a reverse transcriptase. The Prime Editing systems (e.g., PE3 or PE3b systems) can further comprise a nick guide RNA (ngRNA). Such PEgRNAs and Prime Editing systems can be used, for example, to correct an H1069Q mutation in ATP7B.

The PEgRNAs of Table 17 comprise: (a) a spacer comprising nucleotides 5-20 of SEQ ID NO: 5206, (b) a gRNA core capable of binding to a Cas9 protein, and (c) an extension arm comprising: (i) an editing template comprising SEQ ID NO: 5218, and (ii) a primer binding site (PBS) comprising any one of SEQ ID NOs: 5207-5217. The spacer of the PEgRNA can comprise, for example, nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 5206. The spacer of the PEgRNA can comprise SEQ ID NO: 5206. The RTT and the PBS can comprise respectively SEQ ID NOs: 5218 and 5207, 5218 and 5208, 5218 and 5209, 5218 and 5210, 5218 and 5211, 5218 and 5212, 5218 and 5213, 5218 and 5214, 5218 and 5215, 5218 and 5216, or 5218 and 5217. The gRNA core of the PEgRNA can comprise SEQ ID NO. 5857-5859. Exemplary PEgRNAs provided in Table 17 may further comprise a 3′ motif at the 3′ end of the extension arm, for example, a hairpin-forming motif or a series of 3, 4 or more U nucleotides. Without being bound by theory, such 3′ motifs are believed to increase PEgRNA stability. Such PEgRNA sequences may alternatively or additionally be adapted for transcription from a DNA template, for example, by including a 5′ terminal G if the spacer of the PEgRNA begins with another nucleotide, by including 6 or 7 U nucleotides at the 3′ end of the extension arm, or both. Such plasmid adapted sequences may further comprise a hairpin-forming motif between the PBS and the 3′ terminal U series.

Any of the PEgRNAs of Table 17 can be used in a Prime Editing system further comprising a nick guide RNA (ngRNA). Such ngRNA can comprise a spacer comprising nucleotides 5-20 of SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 67, 68, 70, 72, 199, 200, 203, 529, 737, 5219, 5220, 5221, 5222, 5223, 5224, 5225, 5226, or 5227 and a gRNA core capable of binding to a Cas9 protein. The spacer of the ngRNA can comprise nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 67, 68, 70, 72, 199, 200, 203, 529, 737, 5219, 5220, 5221, 5222, 5223, 5224, 5225, 5226, or 5227. The spacer of the ngRNA can comprise SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 67, 68, 70, 72, 199, 200, 203, 529, 737, 5219, 5220, 5221, 5222, 5223, 5224, 5225, 5226, or 5227. The gRNA core of the ngRNA can comprise SEQ ID NO. 5857-5859. Exemplary ngRNA provided in Table 17 can comprise SEQ ID NOs: 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 290, 291, 292, 293, 681, 1501, or 1504. Such ngRNA sequences may further comprise a 3′ motif at the 3′ end of the gRNA core, for example, a series of 3, 4 or more U nucleotides. Without being bound by theory, such 3′ motifs are believed to increase ngRNA stability.

Table 18 provides Prime Editing guide RNAs (PEgRNAs), which can be used in Prime Editing systems disclosed herein. Such Prime Editing systems can comprise a Cas9 protein capable of recognizing an NGA or NG PAM sequence (e.g., GGA or GG), and a reverse transcriptase. The Prime Editing systems (e.g., PE3 or PE3b systems) can further comprise a nick guide RNA (ngRNA). Such PEgRNAs and Prime Editing systems can be used, for example, to correct an H1069Q mutation in ATP7B.

The PEgRNAs of Table 18 comprise: (a) a spacer comprising nucleotides 5-20 of SEQ ID NO: 5228, (b) a gRNA core capable of binding to a Cas9 protein, and (c) an extension arm comprising: (i) an editing template comprising any one of SEQ ID NOs: 5240-5247, and (ii) a primer binding site (PBS) comprising any one of SEQ ID NOs: 5229-5239. The spacer of the PEgRNA can comprise, for example, nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 5228. The spacer of the PEgRNA can comprise SEQ ID NO: 5228. The RTT and the PBS can comprise respectively SEQ ID NOs: 5240 and 5229, 5240 and 5230, 5240 and 5231, 5240 and 5232, 5240 and 5233, 5240 and 5234, 5240 and 5235, 5240 and 5236, 5240 and 5237, 5240 and 5238, 5240 and 5239, 5241 and 5229, 5241 and 5230, 5241 and 5231, 5241 and 5232, 5241 and 5233, 5241 and 5234, 5241 and 5235, 5241 and 5236, 5241 and 5237, 5241 and 5238, 5241 and 5239, 5242 and 5229, 5242 and 5230, 5242 and 5231, 5242 and 5232, 5242 and 5233, 5242 and 5234, 5242 and 5235, 5242 and 5236, 5242 and 5237, 5242 and 5238, 5242 and 5239, 5243 and 5229, 5243 and 5230, 5243 and 5231, 5243 and 5232, 5243 and 5233, 5243 and 5234, 5243 and 5235, 5243 and 5236, 5243 and 5237, 5243 and 5238, 5243 and 5239, 5244 and 5229, 5244 and 5230, 5244 and 5231, 5244 and 5232, 5244 and 5233, 5244 and 5234, 5244 and 5235, 5244 and 5236, 5244 and 5237, 5244 and 5238, 5244 and 5239, 5245 and 5229, 5245 and 5230, 5245 and 5231, 5245 and 5232, 5245 and 5233, 5245 and 5234, 5245 and 5235, 5245 and 5236, 5245 and 5237, 5245 and 5238, 5245 and 5239, 5246 and 5229, 5246 and 5230, 5246 and 5231, 5246 and 5232, 5246 and 5233, 5246 and 5234, 5246 and 5235, 5246 and 5236, 5246 and 5237, 5246 and 5238, 5246 and 5239, 5247 and 5229, 5247 and 5230, 5247 and 5231, 5247 and 5232, 5247 and 5233, 5247 and 5234, 5247 and 5235, 5247 and 5236, 5247 and 5237, 5247 and 5238, or 5247 and 5239. The gRNA core of the PEgRNA can comprise SEQ ID NO. 5857-5859. Such PEgRNA sequences may further comprise a 3′ motif at the 3′ end of the extension arm, for example, a hairpin-forming motif or a series of 3, 4 or more U nucleotides. Without being bound by theory, such 3′ motifs are believed to increase PEgRNA stability. Such PEgRNA sequences may alternatively or additionally be adapted for transcription from a DNA template, for example, by including a 5′ terminal G if the spacer of the PEgRNA begins with another nucleotide, by including 6 or 7 U nucleotides at the 3′ end of the extension arm, or both. Such plasmid adapted sequences may further comprise a hairpin-forming motif between the PBS and the 3′ terminal U series.

Any of the PEgRNAs of Table 18 can be used in a Prime Editing system further comprising a nick guide RNA (ngRNA). Such ngRNA can comprise a spacer comprising nucleotides 5-20 of SEQ ID NOs: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 67, 68, 70, 72, 199, 200, 203, 529, 736, 737, 738, 739, 740, 5219, 5220, 5221, 5222, 5223, 5224, 5225, 5226, or 5227 and a gRNA core capable of binding to a Cas9 protein. The spacer of the ngRNA can comprise nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 67, 68, 70, 72, 199, 200, 203, 529, 736, 737, 738, 739, 740, 5219, 5220, 5221, 5222, 5223, 5224, 5225, 5226, or 5227. The spacer of the ngRNA can comprise SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 67, 68, 70, 72, 199, 200, 203, 529, 736, 737, 738, 739, 740, 5219, 5220, 5221, 5222, 5223, 5224, 5225, 5226, or 5227. The gRNA core of the ngRNA can comprise SEQ ID NO. 5857-5859. Exemplary ngRNA provided in Table 18 can comprise SEQ ID NO: 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 290, 291, 292, 293, 681, 1501, 1502, 1503, or 1504. Such ngRNA sequences may further comprise a 3′ motif at the 3′ end of the gRNA core, for example, a series of 3, 4 or more U nucleotides. Without being bound by theory, such 3′ motifs are believed to increase ngRNA stability.

Table 19 provides Prime Editing guide RNAs (PEgRNAs), which can be used in Prime Editing systems disclosed herein. Such Prime Editing systems can comprise a Cas9 protein capable of recognizing an NGA or NG PAM sequence (e.g., TGA or TG), and a reverse transcriptase. The Prime Editing systems (e.g., PE3 or PE3b systems) can further comprise a nick guide RNA (ngRNA). Such PEgRNAs and Prime Editing systems can be used, for example, to correct an H1069Q mutation in ATP7B.

The PEgRNAs of Table 19 comprise: (a) a spacer comprising nucleotides 5-20 of SEQ ID NO: 5248, (b) a gRNA core capable of binding to a Cas9 protein, and (c) an extension arm comprising: (i) an editing template comprising any one of SEQ ID NOs: 5260-5279, and (ii) a primer binding site (PBS) comprising any one of SEQ ID NOs: 5249-5259. The spacer of the PEgRNA can comprise, for example, nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 5248. The spacer of the PEgRNA can comprise SEQ ID NO: 5248. The RTT and the PBS can comprise respectively SEQ ID NOs: 5260 and 5249, 5260 and 5250, 5260 and 5251, 5260 and 5252, 5260 and 5253, 5260 and 5254, 5260 and 5255, 5260 and 5256, 5260 and 5257, 5260 and 5258, 5260 and 5259, 5261 and 5249, 5261 and 5250, 5261 and 5251, 5261 and 5252, 5261 and 5253, 5261 and 5254, 5261 and 5255, 5261 and 5256, 5261 and 5257, 5261 and 5258, 5261 and 5259, 5262 and 5249, 5262 and 5250, 5262 and 5251, 5262 and 5252, 5262 and 5253, 5262 and 5254, 5262 and 5255, 5262 and 5256, 5262 and 5257, 5262 and 5258, 5262 and 5259, 5263 and 5249, 5263 and 5250, 5263 and 5251, 5263 and 5252, 5263 and 5253, 5263 and 5254, 5263 and 5255, 5263 and 5256, 5263 and 5257, 5263 and 5258, 5263 and 5259, 5264 and 5249, 5264 and 5250, 5264 and 5251, 5264 and 5252, 5264 and 5253, 5264 and 5254, 5264 and 5255, 5264 and 5256, 5264 and 5257, 5264 and 5258, 5264 and 5259, 5265 and 5249, 5265 and 5250, 5265 and 5251, 5265 and 5252, 5265 and 5253, 5265 and 5254, 5265 and 5255, 5265 and 5256, 5265 and 5257, 5265 and 5258, 5265 and 5259, 5266 and 5249, 5266 and 5250, 5266 and 5251, 5266 and 5252, 5266 and 5253, 5266 and 5254, 5266 and 5255, 5266 and 5256, 5266 and 5257, 5266 and 5258, 5266 and 5259, 5267 and 5249, 5267 and 5250, 5267 and 5251, 5267 and 5252, 5267 and 5253, 5267 and 5254, 5267 and 5255, 5267 and 5256, 5267 and 5257, 5267 and 5258, 5267 and 5259, 5268 and 5249, 5268 and 5250, 5268 and 5251, 5268 and 5252, 5268 and 5253, 5268 and 5254, 5268 and 5255, 5268 and 5256, 5268 and 5257, 5268 and 5258, 5268 and 5259, 5269 and 5249, 5269 and 5250, 5269 and 5251, 5269 and 5252, 5269 and 5253, 5269 and 5254, 5269 and 5255, 5269 and 5256, 5269 and 5257, 5269 and 5258, 5269 and 5259, 5270 and 5249, 5270 and 5250, 5270 and 5251, 5270 and 5252, 5270 and 5253, 5270 and 5254, 5270 and 5255, 5270 and 5256, 5270 and 5257, 5270 and 5258, 5270 and 5259, 5271 and 5249, 5271 and 5250, 5271 and 5251, 5271 and 5252, 5271 and 5253, 5271 and 5254, 5271 and 5255, 5271 and 5256, 5271 and 5257, 5271 and 5258, 5271 and 5259, 5272 and 5249, 5272 and 5250, 5272 and 5251, 5272 and 5252, 5272 and 5253, 5272 and 5254, 5272 and 5255, 5272 and 5256, 5272 and 5257, 5272 and 5258, 5272 and 5259, 5273 and 5249, 5273 and 5250, 5273 and 5251, 5273 and 5252, 5273 and 5253, 5273 and 5254, 5273 and 5255, 5273 and 5256, 5273 and 5257, 5273 and 5258, 5273 and 5259, 5274 and 5249, 5274 and 5250, 5274 and 5251, 5274 and 5252, 5274 and 5253, 5274 and 5254, 5274 and 5255, 5274 and 5256, 5274 and 5257, 5274 and 5258, 5274 and 5259, 5275 and 5249, 5275 and 5250, 5275 and 5251, 5275 and 5252, 5275 and 5253, 5275 and 5254, 5275 and 5255, 5275 and 5256, 5275 and 5257, 5275 and 5258, 5275 and 5259, 5276 and 5249, 5276 and 5250, 5276 and 5251, 5276 and 5252, 5276 and 5253, 5276 and 5254, 5276 and 5255, 5276 and 5256, 5276 and 5257, 5276 and 5258, 5276 and 5259, 5277 and 5249, 5277 and 5250, 5277 and 5251, 5277 and 5252, 5277 and 5253, 5277 and 5254, 5277 and 5255, 5277 and 5256, 5277 and 5257, 5277 and 5258, 5277 and 5259, 5278 and 5249, 5278 and 5250, 5278 and 5251, 5278 and 5252, 5278 and 5253, 5278 and 5254, 5278 and 5255, 5278 and 5256, 5278 and 5257, 5278 and 5258, 5278 and 5259, 5279 and 5249, 5279 and 5250, 5279 and 5251, 5279 and 5252, 5279 and 5253, 5279 and 5254, 5279 and 5255, 5279 and 5256, 5279 and 5257, 5279 and 5258, or 5279 and 5259. The gRNA core of the PEgRNA can comprise SEQ ID NO. 5857-5859. Such PEgRNA sequences may further comprise a 3′ motif at the 3′ end of the extension arm, for example, a hairpin-forming motif or a series of 3, 4 or more U nucleotides. Without being bound by theory, such 3′ motifs are believed to increase PEgRNA stability. Such PEgRNA sequences may alternatively or additionally be adapted for transcription from a DNA template, for example, by including a 5′ terminal G if the spacer of the PEgRNA begins with another nucleotide, by including 6 or 7 U nucleotides at the 3′ end of the extension arm, or both. Such plasmid adapted sequences may further comprise a hairpin-forming motif between the PBS and the 3′ terminal U series.

Any of the PEgRNAs of Table 19 can be used in a Prime Editing system further comprising a nick guide RNA (ngRNA). Such ngRNA can comprise a spacer comprising nucleotides 5-20 of SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 67, 68, 70, 72, 199, 200, 203, 529, 736, 737, 738, 739, 740, 5219, 5220, 5221, 5222, 5223, 5224, 5225, 5226, 5227, 5280, or 5281 and a gRNA core capable of binding to a Cas9 protein. The spacer of the ngRNA can comprise nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 67, 68, 70, 72, 199, 200, 203, 529, 736, 737, 738, 739, 740, 5219, 5220, 5221, 5222, 5223, 5224, 5225, 5226, 5227, 5280 or 5281. The spacer of the ngRNA can comprise SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 67, 68, 70, 72, 199, 200, 203, 529, 736, 737, 738, 739, 740, 5219, 5220, 5221, 5222, 5223, 5224, 5225, 5226, 5227, 5280, or 5281. The gRNA core of the ngRNA can comprise SEQ ID NO. 5857-5859. Exemplary ngRNA provided in Table 19 can comprise SEQ ID NOs: 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 290, 291, 292, 293, 681, 1501, 1502, 1503, or 1504. Such ngRNA sequences may further comprise a 3′ motif at the 3′ end of the gRNA core, for example, a series of 3, 4 or more U nucleotides. Without being bound by theory, such 3′ motifs are believed to increase ngRNA stability.

Table 20 provides Prime Editing guide RNAs (PEgRNAs), which can be used in Prime Editing systems disclosed herein. Such Prime Editing systems can comprise a Cas9 protein capable of recogiizing an NRN or NNGG PAM sequence (e.g., GAG or GAGG), and a reverse transcriptase. The Prime Editing systems (e.g., PE3 or PE3b systems) can further comprise a nick guide RNA (ngRNA). Such PEgRNAs and Prime Editing systems can be used, for example, to correct an H1069Q mutation in ATP7B.

The PEgRNAs of Table 20 comprise: (a) a spacer comprising nucleotides 5-20 of SEQ ID NO: 5282, (b) a gRNA core capable of binding to a Cas9 protein, and (c) an extension arm comprising: (i) an editing template comprising any one of SEQ ID NOs: 5294-5302, and (ii) a primer binding site (PBS) comprising any one of SEQ ID NOs: 5283-5293. The spacer of the PEgRNA can comprise, for example, nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 5282. The spacer of the PEgRNA can comprise SEQ ID NO: 5282. The RTT and the PBS can comprise respectively SEQ ID NOs: 5294 and 5283, 5294 and 5284, 5294 and 5285, 5294 and 5286, 5294 and 5287, 5294 and 5288, 5294 and 5289, 5294 and 5290, 5294 and 5291, 5294 and 5292, 5294 and 5293, 5295 and 5283, 5295 and 5284, 5295 and 5285, 5295 and 5286, 5295 and 5287, 5295 and 5288, 5295 and 5289, 5295 and 5290, 5295 and 5291, 5295 and 5292, 5295 and 5293, 5296 and 5283, 5296 and 5284, 5296 and 5285, 5296 and 5286, 5296 and 5287, 5296 and 5288, 5296 and 5289, 5296 and 5290, 5296 and 5291, 5296 and 5292, 5296 and 5293, 5297 and 5283, 5297 and 5284, 5297 and 5285, 5297 and 5286, 5297 and 5287, 5297 and 5288, 5297 and 5289, 5297 and 5290, 5297 and 5291, 5297 and 5292, 5297 and 5293, 5298 and 5283, 5298 and 5284, 5298 and 5285, 5298 and 5286, 5298 and 5287, 5298 and 5288, 5298 and 5289, 5298 and 5290, 5298 and 5291, 5298 and 5292, 5298 and 5293, 5299 and 5283, 5299 and 5284, 5299 and 5285, 5299 and 5286, 5299 and 5287, 5299 and 5288, 5299 and 5289, 5299 and 5290, 5299 and 5291, 5299 and 5292, 5299 and 5293, 5300 and 5283, 5300 and 5284, 5300 and 5285, 5300 and 5286, 5300 and 5287, 5300 and 5288, 5300 and 5289, 5300 and 5290, 5300 and 5291, 5300 and 5292, 5300 and 5293, 5301 and 5283, 5301 and 5284, 5301 and 5285, 5301 and 5286, 5301 and 5287, 5301 and 5288, 5301 and 5289, 5301 and 5290, 5301 and 5291, 5301 and 5292, 5301 and 5293, 5302 and 5283, 5302 and 5284, 5302 and 5285, 5302 and 5286, 5302 and 5287, 5302 and 5288, 5302 and 5289, 5302 and 5290, 5302 and 5291, 5302 and 5292, or 5302 and 5293. The gRNA core of the PEgRNA can comprise SEQ ID NO. 5857-5859. Such PEgRNA sequences may further comprise a 3′ motif at the 3′ end of the extension arm, for example, a hairpin-forming motif or a series of 3, 4 or more U nucleotides. Without being bound by theory, such 3′ motifs are believed to increase PEgRNA stability. Such PEgRNA sequences may alternatively or additionally be adapted for transcription from a DNA template, for example, by including a 5′ terminal G if the spacer of the PEgRNA begins with another nucleotide, by including 6 or 7 U nucleotides at the 3′ end of the extension arm, or both. Such plasmid adapted sequences may further comprise a hairpin-forming motif between the PBS and the 3′ terminal U series.

Any of the PEgRNAs of Table 20 can be used in a Prime Editing system further comprising a nick guide RNA (ngRNA). Such ngRNA can comprise a spacer comprising nucleotides 5-20 of SEQ ID NO: 27, 30, 33, 35, 50, 68, 70, 200, 201, 202, 204, 205, 206, 207, 208, 209, 337, 736, 740, 5303, 5304, 5305, 5306, 5307, 5308, 5309, 5310, 5311, or 5312 and a gRNA core capable of binding to a Cas9 protein. The spacer of the ngRNA can comprise nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 27, 30, 33, 35, 50, 68, 70, 200, 201, 202, 204, 205, 206, 207, 208, 209, 337, 736, 740, 5303, 5304, 5305, 5306, 5307, 5308, 5309, 5310, 5311, or 5312. The spacer of the ngRNA can comprise SEQ ID NO: 27, 30, 33, 35, 50, 68, 70, 200, 201, 202, 204, 205, 206, 207, 208, 209, 337, 736, 740, 5303, 5304, 5305, 5306, 5307, 5308, 5309, 5310, 5311, or 5312. The gRNA core of the ngRNA can comprise SEQ ID NO. 5857-5859. Exemplary ngRNA provided in Table 20 can comprise SEQ ID NOs: 153, 154, 155, 157, 160, 162, 163, 165, 166, 167, 170, 171, 172, 173, 177, 178, 180, 181, 291, 292, 1501, 1503, or 1504. Such ngRNA sequences may further comprise a 3′ motif at the 3′ end of the gRNA core, for example, a series of 3, 4 or more U nucleotides. Without being bound by theory, such 3′ motifs are believed to increase ngRNA stability.

Table 21 provides Prime Editing guide RNAs (PEgRNAs), which can be used in Prime Editing systems disclosed herein. Such Prime Editing systems can comprise a Cas9 protein capable of recognizing an NRN PAM sequence (e.g., CAG), and a reverse transcriptase. The Prime Editing systems (e.g., PE3 or PE3b systems) can further comprise a nick guide RNA (ngRNA). Such PEgRNAs and Prime Editing systems can be used, for example, to correct an H1069Q mutation in ATP7B.

The PEgRNAs of Table 21 comprise: (a) a spacer comprising nucleotides 5-20 of SEQ ID NO: 5313, (b) a gRNA core capable of binding to a Cas9 protein, and (c) an extension arm comprising: (i) an editing template comprising any one of SEQ ID NOs: 5325-5338, and (ii) a primer binding site (PBS) comprising any one of SEQ ID NOs: 5314-5324. The spacer of the PEgRNA can comprise, for example, nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 5313. The spacer of the PEgRNA can comprise SEQ ID NO: 5313. The RTT and the PBS can comprise respectively SEQ ID NOs: 5325 and 5314, 5325 and 5315, 5325 and 5316, 5325 and 5317, 5325 and 5318, 5325 and 5319, 5325 and 5320, 5325 and 5321, 5325 and 5322, 5325 and 5323, 5325 and 5324, 5326 and 5314, 5326 and 5315, 5326 and 5316, 5326 and 5317, 5326 and 5318, 5326 and 5319, 5326 and 5320, 5326 and 5321, 5326 and 5322, 5326 and 5323, 5326 and 5324, 5327 and 5314, 5327 and 5315, 5327 and 5316, 5327 and 5317, 5327 and 5318, 5327 and 5319, 5327 and 5320, 5327 and 5321, 5327 and 5322, 5327 and 5323, 5327 and 5324, 5328 and 5314, 5328 and 5315, 5328 and 5316, 5328 and 5317, 5328 and 5318, 5328 and 5319, 5328 and 5320, 5328 and 5321, 5328 and 5322, 5328 and 5323, 5328 and 5324, 5329 and 5314, 5329 and 5315, 5329 and 5316, 5329 and 5317, 5329 and 5318, 5329 and 5319, 5329 and 5320, 5329 and 5321, 5329 and 5322, 5329 and 5323, 5329 and 5324, 5330 and 5314, 5330 and 5315, 5330 and 5316, 5330 and 5317, 5330 and 5318, 5330 and 5319, 5330 and 5320, 5330 and 5321, 5330 and 5322, 5330 and 5323, 5330 and 5324, 5331 and 5314, 5331 and 5315, 5331 and 5316, 5331 and 5317, 5331 and 5318, 5331 and 5319, 5331 and 5320, 5331 and 5321, 5331 and 5322, 5331 and 5323, 5331 and 5324, 5332 and 5314, 5332 and 5315, 5332 and 5316, 5332 and 5317, 5332 and 5318, 5332 and 5319, 5332 and 5320, 5332 and 5321, 5332 and 5322, 5332 and 5323, 5332 and 5324, 5333 and 5314, 5333 and 5315, 5333 and 5316, 5333 and 5317, 5333 and 5318, 5333 and 5319, 5333 and 5320, 5333 and 5321, 5333 and 5322, 5333 and 5323, 5333 and 5324, 5334 and 5314, 5334 and 5315, 5334 and 5316, 5334 and 5317, 5334 and 5318, 5334 and 5319, 5334 and 5320, 5334 and 5321, 5334 and 5322, 5334 and 5323, 5334 and 5324, 5335 and 5314, 5335 and 5315, 5335 and 5316, 5335 and 5317, 5335 and 5318, 5335 and 5319, 5335 and 5320, 5335 and 5321, 5335 and 5322, 5335 and 5323, 5335 and 5324, 5336 and 5314, 5336 and 5315, 5336 and 5316, 5336 and 5317, 5336 and 5318, 5336 and 5319, 5336 and 5320, 5336 and 5321, 5336 and 5322, 5336 and 5323, 5336 and 5324, 5337 and 5314, 5337 and 5315, 5337 and 5316, 5337 and 5317, 5337 and 5318, 5337 and 5319, 5337 and 5320, 5337 and 5321, 5337 and 5322, 5337 and 5323, 5337 and 5324, 5338 and 5314, 5338 and 5315, 5338 and 5316, 5338 and 5317, 5338 and 5318, 5338 and 5319, 5338 and 5320, 5338 and 5321, 5338 and 5322, 5338 and 5323, or 5338 and 5324. The gRNA core of the PEgRNA can comprise SEQ ID NO. 5857-5859. Such PEgRNA sequences may further comprise a 3′ motif at the 3′ end of the extension arm, for example, a hairpin-forming motif or a series of 3, 4 or more U nucleotides. Without being bound by theory, such 3′ motifs are believed to increase PEgRNA stability. Such PEgRNA sequences may alternatively or additionally be adapted for transcription from a DNA template, for example, by including a 5′ terminal G if the spacer of the PEgRNA begins with another nucleotide, by including 6 or 7 U nucleotides at the 3′ end of the extension arm, or both. Such plasmid adapted sequences may further comprise a hairpin-forming motif between the PBS and the 3′ terminal U series.

Any of the PEgRNAs of Table 21 can be used in a Prime Editing system further comprising a nick guide RNA (ngRNA). Such ngRNA can comprise a spacer comprising nucleotides 5-20 of SEQ ID NO: 201, 206, 207, 208, 337, 5303, 5305, 5307, 5308, 5309, 5310, 5311, 5312, or 5339 and a gRNA core capable of binding to a Cas9 protein. The spacer of the ngRNA can comprise nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 201, 206, 207, 208, 337, 5303, 5305, 5307, 5308, 5309, 5310, 5311, 5312, or 5339. The spacer of the ngRNA can comprise SEQ ID NO: 201, 206, 207, 208, 337, 5303, 5305, 5307, 5308, 5309, 5310, 5311, 5312, or 5339. The gRNA core of the ngRNA can comprise SEQ ID NO. 5857-5859. Exemplary ngRNA provided in Table 21 can comprise SEQ ID NO: 155, 160, 165, 166, 171, 172, 177, 291, or 292. Such ngRNA sequences may further comprise a 3′ motif at the 3′ end of the gRNA core, for example, a series of 3, 4 or more U nucleotides. Without being bound by theory, such 3′ motifs are believed to increase ngRNA stability.

Table 22 provides Prime Editing guide RNAs (PEgRNAs), which can be used in Prime Editing systems disclosed herein. Such Prime Editing systems can comprise a Cas9 protein capable of recognizing an NRN PAM sequence (e.g., CAG), and a reverse transcriptase. The Prime Editing systems (e.g., PE3 or PE3b systems) can further comprise a nick guide RNA (ngRNA). Such PEgRNAs and Prime Editing systems can be used, for example, to correct an H1069Q mutation in ATP7B.

The PEgRNAs of Table 22 comprise: (a) a spacer comprising nucleotides 5-20 of SEQ ID NO: 5340, (b) a gRNA core capable of binding to a Cas9 protein, and (c) an extension arm comprising: (i) an editing template comprising any one of SEQ ID NOs: 5352-5368, and (ii) a primer binding site (PBS) comprising any one of SEQ ID NOs: 5341-5351. The spacer of the PEgRNA can comprise, for example, nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 5340. The spacer of the PEgRNA can comprise SEQ ID NO: 5340. The RTT and the PBS can comprise respectively SEQ ID NOs: 5352 and 5341, 5352 and 5342, 5352 and 5343, 5352 and 5344, 5352 and 5345, 5352 and 5346, 5352 and 5347, 5352 and 5348, 5352 and 5349, 5352 and 5350, 5352 and 5351, 5353 and 5341, 5353 and 5342, 5353 and 5343, 5353 and 5344, 5353 and 5345, 5353 and 5346, 5353 and 5347, 5353 and 5348, 5353 and 5349, 5353 and 5350, 5353 and 5351, 5354 and 5341, 5354 and 5342, 5354 and 5343, 5354 and 5344, 5354 and 5345, 5354 and 5346, 5354 and 5347, 5354 and 5348, 5354 and 5349, 5354 and 5350, 5354 and 5351, 5355 and 5341, 5355 and 5342, 5355 and 5343, 5355 and 5344, 5355 and 5345, 5355 and 5346, 5355 and 5347, 5355 and 5348, 5355 and 5349, 5355 and 5350, 5355 and 5351, 5356 and 5341, 5356 and 5342, 5356 and 5343, 5356 and 5344, 5356 and 5345, 5356 and 5346, 5356 and 5347, 5356 and 5348, 5356 and 5349, 5356 and 5350, 5356 and 5351, 5357 and 5341, 5357 and 5342, 5357 and 5343, 5357 and 5344, 5357 and 5345, 5357 and 5346, 5357 and 5347, 5357 and 5348, 5357 and 5349, 5357 and 5350, 5357 and 5351, 5358 and 5341, 5358 and 5342, 5358 and 5343, 5358 and 5344, 5358 and 5345, 5358 and 5346, 5358 and 5347, 5358 and 5348, 5358 and 5349, 5358 and 5350, 5358 and 5351, 5359 and 5341, 5359 and 5342, 5359 and 5343, 5359 and 5344, 5359 and 5345, 5359 and 5346, 5359 and 5347, 5359 and 5348, 5359 and 5349, 5359 and 5350, 5359 and 5351, 5360 and 5341, 5360 and 5342, 5360 and 5343, 5360 and 5344, 5360 and 5345, 5360 and 5346, 5360 and 5347, 5360 and 5348, 5360 and 5349, 5360 and 5350, 5360 and 5351, 5361 and 5341, 5361 and 5342, 5361 and 5343, 5361 and 5344, 5361 and 5345, 5361 and 5346, 5361 and 5347, 5361 and 5348, 5361 and 5349, 5361 and 5350, 5361 and 5351, 5362 and 5341, 5362 and 5342, 5362 and 5343, 5362 and 5344, 5362 and 5345, 5362 and 5346, 5362 and 5347, 5362 and 5348, 5362 and 5349, 5362 and 5350, 5362 and 5351, 5363 and 5341, 5363 and 5342, 5363 and 5343, 5363 and 5344, 5363 and 5345, 5363 and 5346, 5363 and 5347, 5363 and 5348, 5363 and 5349, 5363 and 5350, 5363 and 5351, 5364 and 5341, 5364 and 5342, 5364 and 5343, 5364 and 5344, 5364 and 5345, 5364 and 5346, 5364 and 5347, 5364 and 5348, 5364 and 5349, 5364 and 5350, 5364 and 5351, 5365 and 5341, 5365 and 5342, 5365 and 5343, 5365 and 5344, 5365 and 5345, 5365 and 5346, 5365 and 5347, 5365 and 5348, 5365 and 5349, 5365 and 5350, 5365 and 5351, 5366 and 5341, 5366 and 5342, 5366 and 5343, 5366 and 5344, 5366 and 5345, 5366 and 5346, 5366 and 5347, 5366 and 5348, 5366 and 5349, 5366 and 5350, 5366 and 5351, 5367 and 5341, 5367 and 5342, 5367 and 5343, 5367 and 5344, 5367 and 5345, 5367 and 5346, 5367 and 5347, 5367 and 5348, 5367 and 5349, 5367 and 5350, 5367 and 5351, 5368 and 5341, 5368 and 5342, 5368 and 5343, 5368 and 5344, 5368 and 5345, 5368 and 5346, 5368 and 5347, 5368 and 5348, 5368 and 5349, 5368 and 5350, or 5368 and 5351. The gRNA core of the PEgRNA can comprise SEQ ID NO. 5857-5859. Such PEgRNA sequences may further comprise a 3′ motif at the 3′ end of the extension arm, for example, a hairpin-forming motif or a series of 3, 4 or more U nucleotides. Without being bound by theory, such 3′ motifs are believed to increase PEgRNA stability. Such PEgRNA sequences may alternatively or additionally be adapted for transcription from a DNA template, for example, by including a 5′ terminal G if the spacer of the PEgRNA begins with another nucleotide, by including 6 or 7 U nucleotides at the 3′ end of the extension arm, or both. Such plasmid adapted sequences may further comprise a hairpin-forming motif between the PBS and the 3′ terminal U series.

Any of the PEgRNAs of Table 22 can be used in a Prime Editing system further comprising a nick guide RNA (ngRNA). Such ngRNA can comprise a spacer comprising nucleotides 5-20 of SEQ ID NO: 201, 206, 207, 208, 337, 5303, 5305, 5307, 5308, 5309, 5310, 5311, 5312, or 5339 and a gRNA core capable of binding to a Cas9 protein. The spacer of the ngRNA can comprise nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 201, 206, 207, 208, 337, 5303, 5305, 5307, 5308, 5309, 5310, 5311, 5312, or 5339. The spacer of the ngRNA can comprise SEQ ID NO: 201, 206, 207, 208, 337, 5303, 5305, 5307, 5308, 5309, 5310, 5311, 5312, or 5339. The gRNA core of the ngRNA can comprise SEQ ID NO. 5857-5859. Exemplary ngRNA provided in Table 22 can comprise SEQ ID NO: 155, 160, 165, 166, 171, 172, 177, 291, or 292. Such ngRNA sequences may further comprise a 3′ motif at the 3′ end of the gRNA core, for example, a series of 3, 4 or more U nucleotides. Without being bound by theory, such 3′ motifs are believed to increase ngRNA stability.

Table 23 provides Prime Editing guide RNAs (PEgRNAs), which can be used in Prime Editing systems disclosed herein. Such Prime Editing systems can comprise a Cas9 protein capable of recognizing an NRN or NNGG PAM sequence (e.g., AAG or AAGG), and a reverse transcriptase. The Prime Editing systems (e.g., PE3 or PE3b systems) can further comprise a nick guide RNA (ngRNA). Such PEgRNAs and Prime Editing systems can be used, for example, to correct an H1069Q mutation in ATP7B.

The PEgRNAs of Table 23 comprise: (a) a spacer comprising nucleotides 5-20 of SEQ ID NO: 5369, (b) a gRNA core capable of binding to a Cas9 protein, and (c) an extension arm comprising: (i) an editing template comprising any one of SEQ ID NOs: 5381-5401, and (ii) a primer binding site (PBS) comprising any one of SEQ ID NOs: 5370-5380. The spacer of the PEgRNA can comprise, for example, nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 5369. The spacer of the PEgRNA can comprise SEQ ID NO: 5369. The RTT and the PBS can comprise respectively SEQ ID NOs: 5381 and 5370, 5381 and 5371, 5381 and 5372, 5381 and 5373, 5381 and 5374, 5381 and 5375, 5381 and 5376, 5381 and 5377, 5381 and 5378, 5381 and 5379, 5381 and 5380, 5382 and 5370, 5382 and 5371, 5382 and 5372, 5382 and 5373, 5382 and 5374, 5382 and 5375, 5382 and 5376, 5382 and 5377, 5382 and 5378, 5382 and 5379, 5382 and 5380, 5383 and 5370, 5383 and 5371, 5383 and 5372, 5383 and 5373, 5383 and 5374, 5383 and 5375, 5383 and 5376, 5383 and 5377, 5383 and 5378, 5383 and 5379, 5383 and 5380, 5384 and 5370, 5384 and 5371, 5384 and 5372, 5384 and 5373, 5384 and 5374, 5384 and 5375, 5384 and 5376, 5384 and 5377, 5384 and 5378, 5384 and 5379, 5384 and 5380, 5385 and 5370, 5385 and 5371, 5385 and 5372, 5385 and 5373, 5385 and 5374, 5385 and 5375, 5385 and 5376, 5385 and 5377, 5385 and 5378, 5385 and 5379, 5385 and 5380, 5386 and 5370, 5386 and 5371, 5386 and 5372, 5386 and 5373, 5386 and 5374, 5386 and 5375, 5386 and 5376, 5386 and 5377, 5386 and 5378, 5386 and 5379, 5386 and 5380, 5387 and 5370, 5387 and 5371, 5387 and 5372, 5387 and 5373, 5387 and 5374, 5387 and 5375, 5387 and 5376, 5387 and 5377, 5387 and 5378, 5387 and 5379, 5387 and 5380, 5388 and 5370, 5388 and 5371, 5388 and 5372, 5388 and 5373, 5388 and 5374, 5388 and 5375, 5388 and 5376, 5388 and 5377, 5388 and 5378, 5388 and 5379, 5388 and 5380, 5389 and 5370, 5389 and 5371, 5389 and 5372, 5389 and 5373, 5389 and 5374, 5389 and 5375, 5389 and 5376, 5389 and 5377, 5389 and 5378, 5389 and 5379, 5389 and 5380, 5390 and 5370, 5390 and 5371, 5390 and 5372, 5390 and 5373, 5390 and 5374, 5390 and 5375, 5390 and 5376, 5390 and 5377, 5390 and 5378, 5390 and 5379, 5390 and 5380, 5391 and 5370, 5391 and 5371, 5391 and 5372, 5391 and 5373, 5391 and 5374, 5391 and 5375, 5391 and 5376, 5391 and 5377, 5391 and 5378, 5391 and 5379, 5391 and 5380, 5392 and 5370, 5392 and 5371, 5392 and 5372, 5392 and 5373, 5392 and 5374, 5392 and 5375, 5392 and 5376, 5392 and 5377, 5392 and 5378, 5392 and 5379, 5392 and 5380, 5393 and 5370, 5393 and 5371, 5393 and 5372, 5393 and 5373, 5393 and 5374, 5393 and 5375, 5393 and 5376, 5393 and 5377, 5393 and 5378, 5393 and 5379, 5393 and 5380, 5394 and 5370, 5394 and 5371, 5394 and 5372, 5394 and 5373, 5394 and 5374, 5394 and 5375, 5394 and 5376, 5394 and 5377, 5394 and 5378, 5394 and 5379, 5394 and 5380, 5395 and 5370, 5395 and 5371, 5395 and 5372, 5395 and 5373, 5395 and 5374, 5395 and 5375, 5395 and 5376, 5395 and 5377, 5395 and 5378, 5395 and 5379, 5395 and 5380, 5396 and 5370, 5396 and 5371, 5396 and 5372, 5396 and 5373, 5396 and 5374, 5396 and 5375, 5396 and 5376, 5396 and 5377, 5396 and 5378, 5396 and 5379, 5396 and 5380, 5397 and 5370, 5397 and 5371, 5397 and 5372, 5397 and 5373, 5397 and 5374, 5397 and 5375, 5397 and 5376, 5397 and 5377, 5397 and 5378, 5397 and 5379, 5397 and 5380, 5398 and 5370, 5398 and 5371, 5398 and 5372, 5398 and 5373, 5398 and 5374, 5398 and 5375, 5398 and 5376, 5398 and 5377, 5398 and 5378, 5398 and 5379, 5398 and 5380, 5399 and 5370, 5399 and 5371, 5399 and 5372, 5399 and 5373, 5399 and 5374, 5399 and 5375, 5399 and 5376, 5399 and 5377, 5399 and 5378, 5399 and 5379, 5399 and 5380, 5400 and 5370, 5400 and 5371, 5400 and 5372, 5400 and 5373, 5400 and 5374, 5400 and 5375, 5400 and 5376, 5400 and 5377, 5400 and 5378, 5400 and 5379, 5400 and 5380, 5401 and 5370, 5401 and 5371, 5401 and 5372, 5401 and 5373, 5401 and 5374, 5401 and 5375, 5401 and 5376, 5401 and 5377, 5401 and 5378, 5401 and 5379, or 5401 and 5380. The gRNA core of the PEgRNA can comprise SEQ ID NO. 5857-5859. Such PEgRNA sequences may further comprise a 3′ motif at the 3′ end of the extension arm, for example, a hairpin-forming motif or a series of 3, 4 or more U nucleotides. Without being bound by theory, such 3′ motifs are believed to increase PEgRNA stability. Such PEgRNA sequences may alternatively or additionally be adapted for transcription from a DNA template, for example, by including a 5′ terminal G if the spacer of the PEgRNA begins with another nucleotide, by including 6 or 7 U nucleotides at the 3′ end of the extension arm, or both. Such plasmid adapted sequences may further comprise a hairpin-forming motif between the PBS and the 3′ terminal U series.

Any of the PEgRNAs of Table 23 can be used in a Prime Editing system further comprising a nick guide RNA (ngRNA). Such ngRNA can comprise a spacer comprising nucleotides 5-20 of SEQ ID NO: 2048, 2052, 2070, 2080, 2085, 2086, 2088, 2089, 2091, 2423, 2424, 2425, 2426, 2427, 2428, 2429, 2430, 2431, 2432, 2433, 2434, 2435, 2436, 2438, 2439, 2440, 2443, 2444, 5402, 5403, 5404, or 5405 and a gRNA core capable of binding to a Cas9 protein. The spacer of the ngRNA can comprise nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 2048, 2052, 2070, 2080, 2085, 2086, 2088, 2089, 2091, 2423, 2424, 2425, 2426, 2427, 2428, 2429, 2430, 2431, 2432, 2433, 2434, 2435, 2436, 2438, 2439, 2440, 2443, 2444, 5402, 5403, 5404, or 5405. The spacer of the ngRNA can comprise SEQ ID NO: 2048, 2052, 2070, 2080, 2085, 2086, 2088, 2089, 2091, 2423, 2424, 2425, 2426, 2427, 2428, 2429, 2430, 2431, 2432, 2433, 2434, 2435, 2436, 2438, 2439, 2440, 2443, 2444, 5402, 5403, 5404, or 5405. The gRNA core of the ngRNA can comprise SEQ ID NO. 5857-5859. Exemplary ngRNA provided in Table 23 can comprise SEQ ID NO: 2257, 2259, 2260, 2261, 2262, 2264, 2270, 2272, 2274, 2275, 2276, 2278, 2280, 2282, 2283, 2284, 2285, 2288, 4411, 4412, 4413, 4415, 4416, 4417, 4419, 4420, or 4421. Such ngRNA sequences may further comprise a 3′ motif at the 3′ end of the gRNA core, for example, a series of 3, 4 or more U nucleotides. Exemplary ngRNA sequences with such 3′ adaptations include SEQ ID NOs: 2292 and 4424. Without being bound by theory, such 3′ motifs are believed to increase ngRNA stability.

Table 24 provides Prime Editing guide RNAs (PEgRNAs), which can be used in Prime Editing systems disclosed herein. Such Prime Editing systems can comprise a Cas9 protein capable of recognizing an NG PAM sequence (e.g., TG), and a reverse transcriptase. The Prime Editing systems (e.g., PE3 or PE3b systems) can further comprise a nick guide RNA (ngRNA). Such PEgRNAs and Prime Editing systems can be used, for example, to correct an H1069Q mutation in ATP7B.

The PEgRNAs of Table 24 comprise: (a) a spacer comprising nucleotides 5-20 of SEQ ID NO: 5406, (b) a gRNA core capable of binding to a Cas9 protein, and (c) an extension arm comprising: (i) an editing template comprising any one of SEQ ID NOs: 5418-5422, and (ii) a primer binding site (PBS) comprising any one of SEQ ID NOs: 5407-5417. The spacer of the PEgRNA can comprise, for example, nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 5406. The spacer of the PEgRNA can comprise SEQ ID NO: 5406. The RTT and the PBS can comprise respectively SEQ ID NOs: 5418 and 5407, 5418 and 5408, 5418 and 5409, 5418 and 5410, 5418 and 5411, 5418 and 5412, 5418 and 5413, 5418 and 5414, 5418 and 5415, 5418 and 5416, 5418 and 5417, 5419 and 5407, 5419 and 5408, 5419 and 5409, 5419 and 5410, 5419 and 5411, 5419 and 5412, 5419 and 5413, 5419 and 5414, 5419 and 5415, 5419 and 5416, 5419 and 5417, 5420 and 5407, 5420 and 5408, 5420 and 5409, 5420 and 5410, 5420 and 5411, 5420 and 5412, 5420 and 5413, 5420 and 5414, 5420 and 5415, 5420 and 5416, 5420 and 5417, 5421 and 5407, 5421 and 5408, 5421 and 5409, 5421 and 5410, 5421 and 5411, 5421 and 5412, 5421 and 5413, 5421 and 5414, 5421 and 5415, 5421 and 5416, 5421 and 5417, 5422 and 5407, 5422 and 5408, 5422 and 5409, 5422 and 5410, 5422 and 5411, 5422 and 5412, 5422 and 5413, 5422 and 5414, 5422 and 5415, 5422 and 5416, or 5422 and 5417. The gRNA core of the PEgRNA can comprise SEQ ID NO. 5857-5859. Such PEgRNA sequences may further comprise a 3′ motif at the 3′ end of the extension arm, for example, a hairpin-forming motif or a series of 3, 4 or more U nucleotides. Without being bound by theory, such 3′ motifs are believed to increase PEgRNA stability. Such PEgRNA sequences may alternatively or additionally be adapted for transcription from a DNA template, for example, by including a 5′ terminal G if the spacer of the PEgRNA begins with another nucleotide, by including 6 or 7 U nucleotides at the 3′ end of the extension arm, or both. Such plasmid adapted sequences may further comprise a hairpin-forming motif between the PBS and the 3′ terminal U series.

Any of the PEgRNAs of Table 24 can be used in a Prime Editing system further comprising a nick guide RNA (ngRNA). Such ngRNA can comprise a spacer comprising nucleotides 5-20 of SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 67, 68, 70, 72, 199, 200, 203, 529, 736, 737, 739, or 740 and a gRNA core capable of binding to a Cas9 protein. The spacer of the ngRNA can comprise nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 67, 68, 70, 72, 199, 200, 203, 529, 736, 737, 739, or 740. The spacer of the ngRNA can comprise SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 67, 68, 70, 72, 199, 200, 203, 529, 736, 737, 739, or 740. The gRNA core of the ngRNA can comprise SEQ ID NO. 5857-5859. Exemplary ngRNA provided in Table 24 can comprise SEQ ID NO: 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 290, 291, 292, 293, 681, 1501, 1503, or 1504. Such ngRNA sequences may further comprise a 3′ motif at the 3′ end of the gRNA core, for example, a series of 3, 4 or more U nucleotides. Without being bound by theory, such 3′ motifs are believed to increase ngRNA stability.

Table 25 provides Prime Editing guide RNAs (PEgRNAs), which can be used in Prime Editing systems disclosed herein. Such Prime Editing systems can comprise a Cas9 protein capable of recognizing an NG PAM sequence (e.g., GG), and a reverse transcriptase. The Prime Editing systems (e.g., PE3 or PE3b systems) can further comprise a nick guide RNA (ngRNA). Such PEgRNAs and Prime Editing systems can be used, for example, to correct an H1069Q mutation in ATP7B.

The PEgRNAs of Table 25 comprise: (a) a spacer comprising nucleotides 5-20 of SEQ ID NO: 5423, (b) a gRNA core capable of binding to a Cas9 protein, and (c) an extension arm comprising: (i) an editing template comprising any one of SEQ ID NOs: 5435-5445, and (ii) a primer binding site (PBS) comprising any one of SEQ ID NOs: 5424-5434. The spacer of the PEgRNA can comprise, for example, nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 5423. The spacer of the PEgRNA can comprise SEQ ID NO: 5423. The RTT and the PBS can comprise respectively SEQ ID NOs: 5435 and 5424, 5435 and 5425, 5435 and 5426, 5435 and 5427, 5435 and 5428, 5435 and 5429, 5435 and 5430, 5435 and 5431, 5435 and 5432, 5435 and 5433, 5435 and 5434, 5436 and 5424, 5436 and 5425, 5436 and 5426, 5436 and 5427, 5436 and 5428, 5436 and 5429, 5436 and 5430, 5436 and 5431, 5436 and 5432, 5436 and 5433, 5436 and 5434, 5437 and 5424, 5437 and 5425, 5437 and 5426, 5437 and 5427, 5437 and 5428, 5437 and 5429, 5437 and 5430, 5437 and 5431, 5437 and 5432, 5437 and 5433, 5437 and 5434, 5438 and 5424, 5438 and 5425, 5438 and 5426, 5438 and 5427, 5438 and 5428, 5438 and 5429, 5438 and 5430, 5438 and 5431, 5438 and 5432, 5438 and 5433, 5438 and 5434, 5439 and 5424, 5439 and 5425, 5439 and 5426, 5439 and 5427, 5439 and 5428, 5439 and 5429, 5439 and 5430, 5439 and 5431, 5439 and 5432, 5439 and 5433, 5439 and 5434, 5440 and 5424, 5440 and 5425, 5440 and 5426, 5440 and 5427, 5440 and 5428, 5440 and 5429, 5440 and 5430, 5440 and 5431, 5440 and 5432, 5440 and 5433, 5440 and 5434, 5441 and 5424, 5441 and 5425, 5441 and 5426, 5441 and 5427, 5441 and 5428, 5441 and 5429, 5441 and 5430, 5441 and 5431, 5441 and 5432, 5441 and 5433, 5441 and 5434, 5442 and 5424, 5442 and 5425, 5442 and 5426, 5442 and 5427, 5442 and 5428, 5442 and 5429, 5442 and 5430, 5442 and 5431, 5442 and 5432, 5442 and 5433, 5442 and 5434, 5443 and 5424, 5443 and 5425, 5443 and 5426, 5443 and 5427, 5443 and 5428, 5443 and 5429, 5443 and 5430, 5443 and 5431, 5443 and 5432, 5443 and 5433, 5443 and 5434, 5444 and 5424, 5444 and 5425, 5444 and 5426, 5444 and 5427, 5444 and 5428, 5444 and 5429, 5444 and 5430, 5444 and 5431, 5444 and 5432, 5444 and 5433, 5444 and 5434, 5445 and 5424, 5445 and 5425, 5445 and 5426, 5445 and 5427, 5445 and 5428, 5445 and 5429, 5445 and 5430, 5445 and 5431, 5445 and 5432, 5445 and 5433, or 5445 and 5434. The gRNA core of the PEgRNA can comprise SEQ ID NO. 5857-5859. Such PEgRNA sequences may further comprise a 3′ motif at the 3′ end of the extension arm, for example, a hairpin-forming motif or a series of 3, 4 or more U nucleotides. Without being bound by theory, such 3′ motifs are believed to increase PEgRNA stability. Such PEgRNA sequences may alternatively or additionally be adapted for transcription from a DNA template, for example, by including a 5′ terminal G if the spacer of the PEgRNA begins with another nucleotide, by including 6 or 7 U nucleotides at the 3′ end of the extension arm, or both. Such plasmid adapted sequences may further comprise a hairpin-forming motif between the PBS and the 3′ terminal U series.

Any of the PEgRNAs of Table 25 can be used in a Prime Editing system further comprising a nick guide RNA (ngRNA). Such ngRNA can comprise a spacer comprising nucleotides 5-20 of SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 67, 68, 70, 72, 199, 200, 203, 529, 736, 737, 738, 739, 740, or 5280 and a gRNA core capable of binding to a Cas9 protein. The spacer of the ngRNA can comprise nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 67, 68, 70, 72, 199, 200, 203, 529, 736, 737, 738, 739, 740, or 5280. The spacer of the ngRNA can comprise SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 67, 68, 70, 72, 199, 200, 203, 529, 736, 737, 738, 739, 740, or 5280. The gRNA core of the ngRNA can comprise SEQ ID NO. 5857-5859. Exemplary ngRNA provided in Table 25 can comprise SEQ ID NO: 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 290, 291, 292, 293, 681, 1501, 1502, 1503, or 1504. Such ngRNA sequences may further comprise a 3′ motif at the 3′ end of the gRNA core, for example, a series of 3, 4 or more U nucleotides. Without being bound by theory, such 3′ motifs are believed to increase ngRNA stability.

Table 26 provides Prime Editing guide RNAs (PEgRNAs), which can be used in Prime Editing systems disclosed herein. Such Prime Editing systems can comprise a Cas9 protein capable of recognizing an NG or NNNRRT PAM sequence (e.g., AG or AGCAGT), and a reverse transcriptase. The Prime Editing systems (e.g., PE3 or PE3b systems) can further comprise a nick guide RNA (ngRNA). Such PEgRNAs and Prime Editing systems can be used, for example, to correct an H1069Q mutation in ATP7B.

The PEgRNAs of Table 26 comprise: (a) a spacer comprising nucleotides 5-20 of SEQ ID NO: 5446, (b) a gRNA core capable of binding to a Cas9 protein, and (c) an extension arm comprising: (i) an editing template comprising any one of SEQ ID NOs: 5458-5472, and (ii) a primer binding site (PBS) comprising any one of SEQ ID NOs: 5447-5457. The spacer of the PEgRNA can comprise, for example, nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 5446. The spacer of the PEgRNA can comprise SEQ ID NO: 5446. The RTT and the PBS can comprise respectively SEQ ID NOs: 5458 and 5447, 5458 and 5448, 5458 and 5449, 5458 and 5450, 5458 and 5451, 5458 and 5452, 5458 and 5453, 5458 and 5454, 5458 and 5455, 5458 and 5456, 5458 and 5457, 5459 and 5447, 5459 and 5448, 5459 and 5449, 5459 and 5450, 5459 and 5451, 5459 and 5452, 5459 and 5453, 5459 and 5454, 5459 and 5455, 5459 and 5456, 5459 and 5457, 5460 and 5447, 5460 and 5448, 5460 and 5449, 5460 and 5450, 5460 and 5451, 5460 and 5452, 5460 and 5453, 5460 and 5454, 5460 and 5455, 5460 and 5456, 5460 and 5457, 5461 and 5447, 5461 and 5448, 5461 and 5449, 5461 and 5450, 5461 and 5451, 5461 and 5452, 5461 and 5453, 5461 and 5454, 5461 and 5455, 5461 and 5456, 5461 and 5457, 5462 and 5447, 5462 and 5448, 5462 and 5449, 5462 and 5450, 5462 and 5451, 5462 and 5452, 5462 and 5453, 5462 and 5454, 5462 and 5455, 5462 and 5456, 5462 and 5457, 5463 and 5447, 5463 and 5448, 5463 and 5449, 5463 and 5450, 5463 and 5451, 5463 and 5452, 5463 and 5453, 5463 and 5454, 5463 and 5455, 5463 and 5456, 5463 and 5457, 5464 and 5447, 5464 and 5448, 5464 and 5449, 5464 and 5450, 5464 and 5451, 5464 and 5452, 5464 and 5453, 5464 and 5454, 5464 and 5455, 5464 and 5456, 5464 and 5457, 5465 and 5447, 5465 and 5448, 5465 and 5449, 5465 and 5450, 5465 and 5451, 5465 and 5452, 5465 and 5453, 5465 and 5454, 5465 and 5455, 5465 and 5456, 5465 and 5457, 5466 and 5447, 5466 and 5448, 5466 and 5449, 5466 and 5450, 5466 and 5451, 5466 and 5452, 5466 and 5453, 5466 and 5454, 5466 and 5455, 5466 and 5456, 5466 and 5457, 5467 and 5447, 5467 and 5448, 5467 and 5449, 5467 and 5450, 5467 and 5451, 5467 and 5452, 5467 and 5453, 5467 and 5454, 5467 and 5455, 5467 and 5456, 5467 and 5457, 5468 and 5447, 5468 and 5448, 5468 and 5449, 5468 and 5450, 5468 and 5451, 5468 and 5452, 5468 and 5453, 5468 and 5454, 5468 and 5455, 5468 and 5456, 5468 and 5457, 5469 and 5447, 5469 and 5448, 5469 and 5449, 5469 and 5450, 5469 and 5451, 5469 and 5452, 5469 and 5453, 5469 and 5454, 5469 and 5455, 5469 and 5456, 5469 and 5457, 5470 and 5447, 5470 and 5448, 5470 and 5449, 5470 and 5450, 5470 and 5451, 5470 and 5452, 5470 and 5453, 5470 and 5454, 5470 and 5455, 5470 and 5456, 5470 and 5457, 5471 and 5447, 5471 and 5448, 5471 and 5449, 5471 and 5450, 5471 and 5451, 5471 and 5452, 5471 and 5453, 5471 and 5454, 5471 and 5455, 5471 and 5456, 5471 and 5457, 5472 and 5447, 5472 and 5448, 5472 and 5449, 5472 and 5450, 5472 and 5451, 5472 and 5452, 5472 and 5453, 5472 and 5454, 5472 and 5455, 5472 and 5456, or 5472 and 5457. The gRNA core of the PEgRNA can comprise SEQ ID NO, 5857-5859. Such PEgRNA sequences may further comprise a 3′ motif at the 3′ end of the extension arm, for example, a hairpin-forming motif or a series of 3, 4 or more U nucleotides. Without being bound by theory, such 3′ motifs are believed to increase PEgRNA stability. Such PEgRNA sequences may alternatively or additionally be adapted for transcription from a DNA template, for example, by including a 5′ terminal G if the spacer of the PEgRNA begins with another nucleotide, by including 6 or 7 U nucleotides at the 3′ end of the extension arm, or both. Such plasmid adapted sequences may further comprise a hairpin-forming motif between the PBS and the 3′ terminal U series.

Any of the PEgRNAs of Table 26 can be used in a Prime Editing system further comprising a nick guide RNA (ngRNA). Such ngRNA can comprise a spacer comprising nucleotides 5-20 of SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 67, 68, 70, 72, 199, 200, 201, 203, 529, 736, 737, 738, 739, 740, 5280, or 5281 and a gRNA core capable of binding to a Cas9 protein. The spacer of the ngRNA can comprise nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 67, 68, 70, 72, 199, 200, 201, 203, 529, 736, 737, 738, 739, 740, 5280, or 5281. The spacer of the ngRNA can comprise SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 67, 68, 70, 72, 199, 200, 201, 203, 529, 736, 737, 738, 739, 740, 5280, or 5281. The gRNA core of the ngRNA can comprise SEQ ID NO. 5857-5859. Exemplary ngRNA provided in Table 26 can comprise SEQ ID NO: 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 290, 291, 292, 293, 681, 1501, 1502, 1503, or 1504. Such ngRNA sequences may further comprise a 3′ motif at the 3′ end of the gRNA core, for example, a series of 3, 4 or more U nucleotides. Without being bound by theory, such 3′ motifs are believed to increase ngRNA stability.

Table 27 provides Prime Editing guide RNAs (PEgRNAs), which can be used in Prime Editing systems disclosed herein. Such Prime Editing systems can comprise a Cas9 protein capable of recognizing an NG PAM sequence (e.g., AG), and a reverse transcriptase. The Prime Editing systems (e.g., PE3 or PE3b systems) can further comprise a nick guide RNA (ngRNA). Such PEgRNAs and Prime Editing systems can be used, for example, to correct an H1069Q mutation in ATP7B.

The PEgRNAs of Table 27 comprise: (a) a spacer comprising nucleotides 5-20 of SEQ ID NO: 5473, (b) a gRNA core capable of binding to a Cas9 protein, and (c) an extension arm comprising: (i) an editing template comprising any one of SEQ ID NOs: 5485-5502, and (ii) a primer binding site (PBS) comprising any one of SEQ ID NOs: 5474-5484. The spacer of the PEgRNA can comprise, for example, nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 5473. The spacer of the PEgRNA can comprise SEQ ID NO: 5473. The RTT and the PBS can comprise respectively SEQ ID NOs: 5485 and 5474, 5485 and 5475, 5485 and 5476, 5485 and 5477, 5485 and 5478, 5485 and 5479, 5485 and 5480, 5485 and 5481, 5485 and 5482, 5485 and 5483, 5485 and 5484, 5486 and 5474, 5486 and 5475, 5486 and 5476, 5486 and 5477, 5486 and 5478, 5486 and 5479, 5486 and 5480, 5486 and 5481, 5486 and 5482, 5486 and 5483, 5486 and 5484, 5487 and 5474, 5487 and 5475, 5487 and 5476, 5487 and 5477, 5487 and 5478, 5487 and 5479, 5487 and 5480, 5487 and 5481, 5487 and 5482, 5487 and 5483, 5487 and 5484, 5488 and 5474, 5488 and 5475, 5488 and 5476, 5488 and 5477, 5488 and 5478, 5488 and 5479, 5488 and 5480, 5488 and 5481, 5488 and 5482, 5488 and 5483, 5488 and 5484, 5489 and 5474, 5489 and 5475, 5489 and 5476, 5489 and 5477, 5489 and 5478, 5489 and 5479, 5489 and 5480, 5489 and 5481, 5489 and 5482, 5489 and 5483, 5489 and 5484, 5490 and 5474, 5490 and 5475, 5490 and 5476, 5490 and 5477, 5490 and 5478, 5490 and 5479, 5490 and 5480, 5490 and 5481, 5490 and 5482, 5490 and 5483, 5490 and 5484, 5491 and 5474, 5491 and 5475, 5491 and 5476, 5491 and 5477, 5491 and 5478, 5491 and 5479, 5491 and 5480, 5491 and 5481, 5491 and 5482, 5491 and 5483, 5491 and 5484, 5492 and 5474, 5492 and 5475, 5492 and 5476, 5492 and 5477, 5492 and 5478, 5492 and 5479, 5492 and 5480, 5492 and 5481, 5492 and 5482, 5492 and 5483, 5492 and 5484, 5493 and 5474, 5493 and 5475, 5493 and 5476, 5493 and 5477, 5493 and 5478, 5493 and 5479, 5493 and 5480, 5493 and 5481, 5493 and 5482, 5493 and 5483, 5493 and 5484, 5494 and 5474, 5494 and 5475, 5494 and 5476, 5494 and 5477, 5494 and 5478, 5494 and 5479, 5494 and 5480, 5494 and 5481, 5494 and 5482, 5494 and 5483, 5494 and 5484, 5495 and 5474, 5495 and 5475, 5495 and 5476, 5495 and 5477, 5495 and 5478, 5495 and 5479, 5495 and 5480, 5495 and 5481, 5495 and 5482, 5495 and 5483, 5495 and 5484, 5496 and 5474, 5496 and 5475, 5496 and 5476, 5496 and 5477, 5496 and 5478, 5496 and 5479, 5496 and 5480, 5496 and 5481, 5496 and 5482, 5496 and 5483, 5496 and 5484, 5497 and 5474, 5497 and 5475, 5497 and 5476, 5497 and 5477, 5497 and 5478, 5497 and 5479, 5497 and 5480, 5497 and 5481, 5497 and 5482, 5497 and 5483, 5497 and 5484, 5498 and 5474, 5498 and 5475, 5498 and 5476, 5498 and 5477, 5498 and 5478, 5498 and 5479, 5498 and 5480, 5498 and 5481, 5498 and 5482, 5498 and 5483, 5498 and 5484, 5499 and 5474, 5499 and 5475, 5499 and 5476, 5499 and 5477, 5499 and 5478, 5499 and 5479, 5499 and 5480, 5499 and 5481, 5499 and 5482, 5499 and 5483, 5499 and 5484, 5500 and 5474, 5500 and 5475, 5500 and 5476, 5500 and 5477, 5500 and 5478, 5500 and 5479, 5500 and 5480, 5500 and 5481, 5500 and 5482, 5500 and 5483, 5500 and 5484, 5501 and 5474, 5501 and 5475, 5501 and 5476, 5501 and 5477, 5501 and 5478, 5501 and 5479, 5501 and 5480, 5501 and 5481, 5501 and 5482, 5501 and 5483, 5501 and 5484, 5502 and 5474, 5502 and 5475, 5502 and 5476, 5502 and 5477, 5502 and 5478, 5502 and 5479, 5502 and 5480, 5502 and 5481, 5502 and 5482, 5502 and 5483, or 5502 and 5484. The gRNA core of the PEgRNA can comprise SEQ ID NO. 5857-5859. Such PEgRNA sequences may further comprise a 3′ motif at the 3′ end of the extension arm, for example, a hairpin-forming motif or a series of 3, 4 or more U nucleotides. Without being bound by theory, such 3′ motifs are believed to increase PEgRNA stability. Such PEgRNA sequences may alternatively or additionally be adapted for transcription from a DNA template, for example, by including a 5′ terminal G if the spacer of the PEgRNA begins with another nucleotide, by including 6 or 7 U nucleotides at the 3′ end of the extension arm, or both. Such plasmid adapted sequences may further comprise a hairpin-forming motif between the PBS and the 3′ terminal U series.

Any of the PEgRNAs of Table 27 can be used in a Prime Editing system further comprising a nick guide RNA (ngRNA). Such ngRNA can comprise a spacer comprising nucleotides 5-20 of SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 67, 68, 70, 72, 199, 200, 203, 529, 736, 737, 738, 739, 740, 5280, or 5281 and a gRNA core capable of binding to a Cas9 protein. The spacer of the ngRNA can comprise nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 67, 68, 70, 72, 199, 200, 203, 529, 736, 737, 738, 739, 740, 5280, or 5281. The spacer of the ngRNA can comprise SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 67, 68, 70, 72, 199, 200, 203, 529, 736, 737, 738, 739, 740, 5280, or 5281. The gRNA core of the ngRNA can comprise SEQ ID NO. 5857-5859. Exemplary ngRNA provided in Table 27 can comprise SEQ ID NO: 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 290, 291, 292, 293, 681, 1501, 1502, 1503, or 1504. Such ngRNA sequences may further comprise a 3′ motif at the 3′ end of the gRNA core, for example, a series of 3, 4 or more U nucleotides. Without being bound by theory, such 3′ motifs are believed to increase ngRNA stability.

Table 28 provides Prime Editing guide RNAs (PEgRNAs), which can be used in Prime Editing systems disclosed herein. Such Prime Editing systems can comprise a Cas9 protein capable of recognizing an NG PAM sequence (e.g., TG), and a reverse transcriptase. The Prime Editing systems (e.g., PE3 or PE3b systems) can further comprise a nick guide RNA (ngRNA). Such PEgRNAs and Prime Editing systems can be used, for example, to correct an H1069Q mutation in ATP7B.

The PEgRNAs of Table 28 comprise: (a) a spacer comprising nucleotides 5-20 of SEQ ID NO: 5503, (b) a gRNA core capable of binding to a Cas9 protein, and (c) an extension arm comprising: (i) an editing template comprising any one of SEQ ID NOs: 5515-5535, and (ii) a primer binding site (PBS) comprising any one of SEQ ID NOs: 5504-5514. The spacer of the PEgRNA can comprise, for example, nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 5503. The spacer of the PEgRNA can comprise SEQ ID NO: 5503. The RTT and the PBS can comprise respectively SEQ ID NOs: 5515 and 5504, 5515 and 5505, 5515 and 5506, 5515 and 5507, 5515 and 5508, 5515 and 5509, 5515 and 5510, 5515 and 5511, 5515 and 5512, 5515 and 5513, 5515 and 5514, 5516 and 5504, 5516 and 5505, 5516 and 5506, 5516 and 5507, 5516 and 5508, 5516 and 5509, 5516 and 5510, 5516 and 5511, 5516 and 5512, 5516 and 5513, 5516 and 5514, 5517 and 5504, 5517 and 5505, 5517 and 5506, 5517 and 5507, 5517 and 5508, 5517 and 5509, 5517 and 5510, 5517 and 5511, 5517 and 5512, 5517 and 5513, 5517 and 5514, 5518 and 5504, 5518 and 5505, 5518 and 5506, 5518 and 5507, 5518 and 5508, 5518 and 5509, 5518 and 5510, 5518 and 5511, 5518 and 5512, 5518 and 5513, 5518 and 5514, 5519 and 5504, 5519 and 5505, 5519 and 5506, 5519 and 5507, 5519 and 5508, 5519 and 5509, 5519 and 5510, 5519 and 5511, 5519 and 5512, 5519 and 5513, 5519 and 5514, 5520 and 5504, 5520 and 5505, 5520 and 5506, 5520 and 5507, 5520 and 5508, 5520 and 5509, 5520 and 5510, 5520 and 5511, 5520 and 5512, 5520 and 5513, 5520 and 5514, 5521 and 5504, 5521 and 5505, 5521 and 5506, 5521 and 5507, 5521 and 5508, 5521 and 5509, 5521 and 5510, 5521 and 5511, 5521 and 5512, 5521 and 5513, 5521 and 5514, 5522 and 5504, 5522 and 5505, 5522 and 5506, 5522 and 5507, 5522 and 5508, 5522 and 5509, 5522 and 5510, 5522 and 5511, 5522 and 5512, 5522 and 5513, 5522 and 5514, 5523 and 5504, 5523 and 5505, 5523 and 5506, 5523 and 5507, 5523 and 5508, 5523 and 5509, 5523 and 5510, 5523 and 5511, 5523 and 5512, 5523 and 5513, 5523 and 5514, 5524 and 5504, 5524 and 5505, 5524 and 5506, 5524 and 5507, 5524 and 5508, 5524 and 5509, 5524 and 5510, 5524 and 5511, 5524 and 5512, 5524 and 5513, 5524 and 5514, 5525 and 5504, 5525 and 5505, 5525 and 5506, 5525 and 5507, 5525 and 5508, 5525 and 5509, 5525 and 5510, 5525 and 5511, 5525 and 5512, 5525 and 5513, 5525 and 5514, 5526 and 5504, 5526 and 5505, 5526 and 5506, 5526 and 5507, 5526 and 5508, 5526 and 5509, 5526 and 5510, 5526 and 5511, 5526 and 5512, 5526 and 5513, 5526 and 5514, 5527 and 5504, 5527 and 5505, 5527 and 5506, 5527 and 5507, 5527 and 5508, 5527 and 5509, 5527 and 5510, 5527 and 5511, 5527 and 5512, 5527 and 5513, 5527 and 5514, 5528 and 5504, 5528 and 5505, 5528 and 5506, 5528 and 5507, 5528 and 5508, 5528 and 5509, 5528 and 5510, 5528 and 5511, 5528 and 5512, 5528 and 5513, 5528 and 5514, 5529 and 5504, 5529 and 5505, 5529 and 5506, 5529 and 5507, 5529 and 5508, 5529 and 5509, 5529 and 5510, 5529 and 5511, 5529 and 5512, 5529 and 5513, 5529 and 5514, 5530 and 5504, 5530 and 5505, 5530 and 5506, 5530 and 5507, 5530 and 5508, 5530 and 5509, 5530 and 5510, 5530 and 5511, 5530 and 5512, 5530 and 55131, 5530 and 5514, 5531 and 5504, 5531 and 5505, 5531 and 5506, 5531 and 5507, 5531 and 5508, 5531 and 5509, 5531 and 5510, 5531 and 5511, 5531 and 5512, 5531 and 5513, 5531 and 5514, 5532 and 5504, 5532 and 5505, 5532 and 5506, 5532 and 5507, 5532 and 5508, 5532 and 5509, 5532 and 5510, 5532 and 5511, 5532 and 5512, 5532 and 5513, 5532 and 5514, 5533 and 5504, 5533 and 5505, 5533 and 5506, 5533 and 5507, 5533 and 5508, 5533 and 5509, 5533 and 5510, 5533 and 5511, 5533 and 5512, 5533 and 5513, 5533 and 5514, 5534 and 5504, 5534 and 5505, 5534 and 5506, 5534 and 5507, 5534 and 5508, 5534 and 5509, 5534 and 5510, 5534 and 5511, 5534 and 5512, 5534 and 5513, 5534 and 5514, 5535 and 5504, 5535 and 5505, 5535 and 5506, 5535 and 5507, 5535 and 5508, 5535 and 5509, 5535 and 5510, 5535 and 5511, 5535 and 5512, 5535 and 5513, or 5535 and 5514. The gRNA core of the PEgRNA can comprise SEQ ID NO. 5857-5859. Such PEgRNA sequences may further comprise a 3′ motif at the 3′ end of the extension arm, for example, a hairpin-forming motif or a series of 3, 4 or more U nucleotides. Without being bound by theory, such 3′ motifs are believed to increase PEgRNA stability. Such PEgRNA sequences may alternatively or additionally be adapted for transcription from a DNA template, for example, by including a 5′ terminal G if the spacer of the PEgRNA begins with another nucleotide, by including 6 or 7 U nucleotides at the 3′ end of the extension arm, or both. Such plasmid adapted sequences may further comprise a hairpin-forming motif between the PBS and the 3′ terminal U series.

Any of the PEgRNAs of Table 28 can be used in a Prime Editing system further comprising a nick guide RNA (ngRNA). Such ngRNA can comprise a spacer comprising nucleotides 5-20 of SEQ ID NO: 2045, 2046, 2047, 2048, 2049, 2050, 2051, 2052, 2053, 2054, 2055, 2056, 2057, 2058, 2059, 2060, 2061, 2062, 2063, 2064, 2065, 2066, 2067, 2068, 2069, 2070, 2071, 2072, 2073, 2074, 2075, 2076, 2077, 2078, 2079, 2080, 2081, 2082, 2083, 2084, 2085, 2086, 2087, 2088, 2089, 2090, 2091, 2092, 2093, 2094, 2095, 2096, 2437, 2439, 2442, 2443, 2444, or 5536 and a gRNA core capable of binding to a Cas9 protein. The spacer of the ngRNA can comprise nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 2045, 2046, 2047, 2048, 2049, 2050, 2051, 2052, 2053, 2054, 2055, 2056, 2057, 2058, 2059, 2060, 2061, 2062, 2063, 2064, 2065, 2066, 2067, 2068, 2069, 2070, 2071, 2072, 2073, 2074, 2075, 2076, 2077, 2078, 2079, 2080, 2081, 2082, 2083, 2084, 2085, 2086, 2087, 2088, 2089, 2090, 2091, 2092, 2093, 2094, 2095, 2096, 2437, 2439, 2442, 2443, 2444, or 5536. The spacer of the ngRNA can comprise SEQ ID NO: 2045, 2046, 2047, 2048, 2049, 2050, 2051, 2052, 2053, 2054, 2055, 2056, 2057, 2058, 2059, 2060, 2061, 2062, 2063, 2064, 2065, 2066, 2067, 2068, 2069, 2070, 2071, 2072, 2073, 2074, 2075, 2076, 2077, 2078, 2079, 2080, 2081, 2082, 2083, 2084, 2085, 2086, 2087, 2088, 2089, 2090, 2091, 2092, 2093, 2094, 2095, 2096, 2437, 2439, 2442, 2443, 2444, or 5536. The gRNA core of the ngRNA can comprise SEQ ID NO. 5857-5859. Exemplary ngRNA provided in Table 28 can comprise SEQ ID NO: 2257, 2258, 2259, 2260, 2261, 2262, 2263, 2264, 2265, 2266, 2267, 2268, 2269, 2270, 2271, 2272, 2273, 2274, 2275, 2276, 2277, 2278, 2279, 2280, 2281, 2282, 2283, 2284, 2285, 2286, 2287, 2288, 2289, 4411, 4416, 4418, 4420, or 4422. Such ngRNA sequences may further comprise a 3′ motif at the 3′ end of the gRNA core, for example, a series of 3, 4 or more U nucleotides. Exemplary ngRNA sequences with such 3′ adaptations include SEQ ID NOs: 2290-2292. Without being bound by theory, such 3′ motifs are believed to increase ngRNA stability.

Table 29 provides Prime Editing guide RNAs (PEgRNAs), which can be used in Prime Editing systems disclosed herein. Such Prime Editing systems can comprise a Cas9 protein capable of recognizing an NNGG PAM sequence (e.g., GCGG), and a reverse transcriptase. The Prime Editing systems (e.g., PE3 or PE3b systems) can further comprise a nick guide RNA (ngRNA). Such PEgRNAs and Prime Editing systems can be used, for example, to correct an H1069Q mutation in ATP7B.

The PEgRNAs of Table 29 comprise: (a) a spacer comprising nucleotides 5-20 of SEQ ID NO: 5537, (b) a gRNA core capable of binding to a Cas9 protein, and (c) an extension arm comprising: (i) an editing template comprising any one of SEQ ID NOs: 5549-5554, and (ii) a primer binding site (PBS) comprising any one of SEQ ID NOs: 5538-5548. The spacer of the PEgRNA can comprise, for example, nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 5537. The spacer of the PEgRNA can comprise SEQ ID NO: 5537. The RTT and the PBS can comprise respectively SEQ ID NOs: 5549 and 5538, 5549 and 5539, 5549 and 5540, 5549 and 5541, 5549 and 5542, 5549 and 5543, 5549 and 5544, 5549 and 5545, 5549 and 5546, 5549 and 5547, 5549 and 5548, 5550 and 5538, 5550 and 5539, 5550 and 5540, 5550 and 5541, 5550 and 5542, 5550 and 5543, 5550 and 5544, 5550 and 5545, 5550 and 5546, 5550 and 5547, 5550 and 5548, 5551 and 5538, 5551 and 5539, 5551 and 5540, 5551 and 5541, 5551 and 5542, 5551 and 5543, 5551 and 5544, 5551 and 5545, 5551 and 5546, 5551 and 5547, 5551 and 5548, 5552 and 5538, 5552 and 5539, 5552 and 5540, 5552 and 5541, 5552 and 5542, 5552 and 5543, 5552 and 5544, 5552 and 5545, 5552 and 5546, 5552 and 5547, 5552 and 5548, 5553 and 5538, 5553 and 5539, 5553 and 5540, 5553 and 5541, 5553 and 5542, 5553 and 5543, 5553 and 5544, 5553 and 5545, 5553 and 5546, 5553 and 5547, 5553 and 5548, 5554 and 5538, 5554 and 5539, 5554 and 5540, 5554 and 5541, 5554 and 5542, 5554 and 5543, 5554 and 5544, 5554 and 5545, 5554 and 5546, 5554 and 5547, or 5554 and 5548. The gRNA core of the PEgRNA can comprise SEQ ID NO. 5857-5859. Such PEgRNA sequences may further comprise a 3′ motif at the 3′ end of the extension arm, for example, a hairpin-forming motif or a series of 3, 4 or more U nucleotides. Without being bound by theory, such 3′ motifs are believed to increase PEgRNA stability. Such PEgRNA sequences may alternatively or additionally be adapted for transcription from a DNA template, for example, by including a 5′ terminal G if the spacer of the PEgRNA begins with another nucleotide, by including 6 or 7 U nucleotides at the 3′ end of the extension arm, or both. Such plasmid adapted sequences may further comprise a hairpin-forming motif between the PBS and the 3′ terminal U series.

Any of the PEgRNAs of Table 29 can be used in a Prime Editing system further comprising a nick guide RNA (ngRNA). Such ngRNA can comprise a spacer comprising nucleotides 5-20 of SEQ ID NO: 27, 30, 33, 35, 50, 68, 70, 200, 201, 202, 204, 205, 206, 207, 208, 209, 337, 736, 740, or 5306 and a gRNA core capable of binding to a Cas9 protein. The spacer of the ngRNA can comprise nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 27, 30, 33, 35, 50, 68, 70, 200, 201, 202, 204, 205, 206, 207, 208, 209, 337, 736, 740, or 5306. The spacer of the ngRNA can comprise SEQ ID NO: 27, 30, 33, 35, 50, 68, 70, 200, 201, 202, 204, 205, 206, 207, 208, 209, 337, 736, 740, or 5306. The gRNA core of the ngRNA can comprise SEQ ID NO. 5857-5859. Exemplary ngRNA provided in Table 29 can comprise SEQ ID NO: 153, 154, 157, 160, 162, 163, 165, 166, 167, 170, 171, 172, 173, 177, 178, 180, 181, 291, 292, 1501, 1503, or 1504. Such ngRNA sequences may further comprise a 3′ motif at the 3′ end of the gRNA core, for example, a series of 3, 4 or more U nucleotides. Without being bound by theory, such 3′ motifs are believed to increase ngRNA stability.

Table 30 provides Prime Editing guide RNAs (PEgRNAs), which can be used in Prime Editing systems disclosed herein. Such Prime Editing systems can comprise a Cas9 protein capable of recognizing an NGG or NG PAM sequence (e.g., TGG or TG), and a reverse transcriptase. The Prime Editing systems (e.g., PE3 or PE3b systems) can further comprise a nick guide RNA (ngRNA). Such PEgRNAs and Prime Editing systems can be used, for example, to correct an H1069Q mutation in ATP7B.

The PEgRNAs of Table 30 comprise: (a) a spacer comprising nucleotides 5-20 of SEQ ID NO: 5555, (b) a gRNA core capable of binding to a Cas9 protein, and (c) an extension arm comprising: (i) an editing template comprising any one of SEQ ID NOs: 5567-5590, and (ii) a primer binding site (PBS) comprising any one of SEQ ID NOs: 5556-5566. The spacer of the PEgRNA can comprise, for example, nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 5555. The spacer of the PEgRNA can comprise SEQ ID NO: 5555. The RTT and the PBS can comprise respectively SEQ ID NOs: 5567 and 5556, 5567 and 5557, 5567 and 5558, 5567 and 5559, 5567 and 5560, 5567 and 5561, 5567 and 5562, 5567 and 5563, 5567 and 5564, 5567 and 5565, 5567 and 5566, 5568 and 5556, 5568 and 5557, 5568 and 5558, 5568 and 5559, 5568 and 5560, 5568 and 5561, 5568 and 5562, 5568 and 5563, 5568 and 5564, 5568 and 5565, 5568 and 5566, 5569 and 5556, 5569 and 5557, 5569 and 5558, 5569 and 5559, 5569 and 5560, 5569 and 5561, 5569 and 5562, 5569 and 5563, 5569 and 5564, 5569 and 5565, 5569 and 5566, 5570 and 5556, 5570 and 5557, 5570 and 5558, 5570 and 5559, 5570 and 5560, 5570 and 5561, 5570 and 5562, 5570 and 5563, 5570 and 5564, 5570 and 5565, 5570 and 5566, 5571 and 5556, 5571 and 5557, 5571 and 5558, 5571 and 5559, 5571 and 5560, 5571 and 5561, 5571 and 5562, 5571 and 5563, 5571 and 5564, 5571 and 5565, 5571 and 5566, 5572 and 5556, 5572 and 5557, 5572 and 5558, 5572 and 5559, 5572 and 5560, 5572 and 5561, 5572 and 5562, 5572 and 5563, 5572 and 5564, 5572 and 5565, 5572 and 5566, 5573 and 5556, 5573 and 5557, 5573 and 5558, 5573 and 5559, 5573 and 5560, 5573 and 5561, 5573 and 5562, 5573 and 5563, 5573 and 5564, 5573 and 5565, 5573 and 5566, 5574 and 5556, 5574 and 5557, 5574 and 5558, 5574 and 5559, 5574 and 5560, 5574 and 5561, 5574 and 5562, 5574 and 5563, 5574 and 5564, 5574 and 5565, 5574 and 5566, 5575 and 5556, 5575 and 5557, 5575 and 5558, 5575 and 5559, 5575 and 5560, 5575 and 5561, 5575 and 5562, 5575 and 5563, 5575 and 5564, 5575 and 5565, 5575 and 5566, 5576 and 5556, 5576 and 5557, 5576 and 5558, 5576 and 5559, 5576 and 5560, 5576 and 5561, 5576 and 5562, 5576 and 5563, 5576 and 5564, 5576 and 5565, 5576 and 5566, 5577 and 5556, 5577 and 5557, 5577 and 5558, 5577 and 5559, 5577 and 5560, 5577 and 5561, 5577 and 5562, 5577 and 5563, 5577 and 5564, 5577 and 5565, 5577 and 5566, 5578 and 5556, 5578 and 5557, 5578 and 5558, 5578 and 5559, 5578 and 5560, 5578 and 5561, 5578 and 5562, 5578 and 5563, 5578 and 5564, 5578 and 5565, 5578 and 5566, 5579 and 5556, 5579 and 5557, 5579 and 5558, 5579 and 5559, 5579 and 5560, 5579 and 5561, 5579 and 5562, 5579 and 5563, 5579 and 5564, 5579 and 5565, 5579 and 5566, 5580 and 5556, 5580 and 5557, 5580 and 5558, 5580 and 5559, 5580 and 5560, 5580 and 5561, 5580 and 5562, 5580 and 5563, 5580 and 5564, 5580 and 5565, 5580 and 5566, 5581 and 5556, 5581 and 5557, 5581 and 5558, 5581 and 5559, 5581 and 5560, 5581 and 5561, 5581 and 5562, 5581 and 5563, 5581 and 5564, 5581 and 5565, 5581 and 5566, 5582 and 5556, 5582 and 5557, 5582 and 5558, 5582 and 5559, 5582 and 5560, 5582 and 5561, 5582 and 5562, 5582 and 5563, 5582 and 5564, 5582 and 5565, 5582 and 5566, 5583 and 5556, 5583 and 5557, 5583 and 5558, 5583 and 5559, 5583 and 5560, 5583 and 5561, 5583 and 5562, 5583 and 5563, 5583 and 5564, 5583 and 5565, 5583 and 5566, 5584 and 5556, 5584 and 5557, 5584 and 5558, 5584 and 5559, 5584 and 5560, 5584 and 5561, 5584 and 5562, 5584 and 5563, 5584 and 5564, 5584 and 5565, 5584 and 5566, 5585 and 5556, 5585 and 5557, 5585 and 5558, 5585 and 5559, 5585 and 5560, 5585 and 5561, 5585 and 5562, 5585 and 5563, 5585 and 5564, 5585 and 5565, 5585 and 5566, 5586 and 5556, 5586 and 5557, 5586 and 5558, 5586 and 5559, 5586 and 5560, 5586 and 5561, 5586 and 5562, 5586 and 5563, 5586 and 5564, 5586 and 5565, 5586 and 5566, 5587 and 5556, 5587 and 5557, 5587 and 5558, 5587 and 5559, 5587 and 5560, 5587 and 5561, 5587 and 5562, 5587 and 5563, 5587 and 5564, 5587 and 5565, 5587 and 5566, 5588 and 5556, 5588 and 5557, 5588 and 5558, 5588 and 5559, 5588 and 5560, 5588 and 5561, 5588 and 5562, 5588 and 5563, 5588 and 5564, 5588 and 5565, 5588 and 5566, 5589 and 5556, 5589 and 5557, 5589 and 5558, 5589 and 5559, 5589 and 5560, 5589 and 5561, 5589 and 5562, 5589 and 5563, 5589 and 5564, 5589 and 5565, 5589 and 5566, 5590 and 5556, 5590 and 5557, 5590 and 5558, 5590 and 5559, 5590 and 5560, 5590 and 5561, 5590 and 5562, 5590 and 5563, 5590 and 5564, 5590 and 5565, or 5590 and 5566. The gRNA core of the PEgRNA can comprise SEQ ID NO. 5857-5859. Exemplary PEgRNAs provided in Table 30 can comprise and one of SEQ ID NOs. 5591-5637. Such PEgRNA sequences may further comprise a 3 motif at the 3 end of the extension arm, for example, a hairpin-forming motif or a series of 3, 4 or more U nucleotides. Without being bound by theory, such 3′ motifs are believed to increase PEgRNA stability. Such PEgRNA sequences may alternatively or additionally be adapted for transcription from a DNA template, for example, by including a 5′ terminal G if the spacer of the PEgRNA begins with another nucleotide, by including 6 or 7 U nucleotides at the 3′ end of the extension arm, or both. Such plasmid adapted sequences may further comprise a hairpin-forming motif between the PBS and the 3′ terminal U series.

Any of the PEgRNAs of Table 30 can be used in a Prime Editing system further comprising a nick guide RNA (ngRNA). Such ngRNA can comprise a spacer comprising nucleotides 5-20 of SEQ ID NO: 41, 60, 61, 62, 63, 64, 65, 66, 69, or 71 and a gRNA core capable of binding to a Cas9 protein. The spacer of the ngRNA can comprise nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 41, 60, 61, 62, 63, 64, 65, 66, 69, or 71. The spacer of the ngRNA can comprise SEQ ID NO: 41, 60, 61, 62, 63, 64, 65, 66, 69, or 71. The gRNA core of the ngRNA can comprise SEQ ID NO. 5857-5859. Such ngRNA sequences may further comprise a 3′ motif at the 3′ end of the gRNA core, for example, a series of 3, 4 or more U nucleotides. Without being bound by theory, such 3′ motifs are believed to increase ngRNA stability.

Table 31 provides Prime Editing guide RNAs (PEgRNAs), which can be used in Prime Editing systems disclosed herein. Such Prime Editing systems can comprise a Cas9 protein capable of recognizing an NGG or NG PAM sequence (e.g., AGG or AG), and a reverse transcriptase. The Prime Editing systems (e.g., PE3 or PE3b systems) can further comprise a nick guide RNA (ngRNA). Such PEgRNAs and Prime Editing systems can be used, for example, to correct an H1069Q mutation in ATP7B.

The PEgRNAs of Table 31 comprise: (a) a spacer comprising nucleotides 5-20 of SEQ ID NO: 5638, (b) a gRNA core capable of binding to a Cas9 protein, and (c) an extension arm comprising: (i) an editing template comprising any one of SEQ ID NOs: 5650-5668, and (ii) a primer binding site (PBS) comprising any one of SEQ ID NOs: 5639-5649. The spacer of the PEgRNA can comprise, for example, nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 5638. The spacer of the PEgRNA can comprise SEQ ID NO: 5638. The RTT and the PBS can comprise respectively SEQ ID NOs: 5650 and 5639, 5650 and 5640, 5650 and 5641, 5650 and 5642, 5650 and 5643, 5650 and 5644, 5650 and 5645, 5650 and 5646, 5650 and 5647, 5650 and 5648, 5650 and 5649, 5651 and 5639, 5651 and 5640, 5651 and 5641, 5651 and 5642, 5651 and 5643, 5651 and 5644, 5651 and 5645, 5651 and 5646, 5651 and 5647, 5651 and 5648, 5651 and 5649, 5652 and 5639, 5652 and 5640, 5652 and 5641, 5652 and 5642, 5652 and 5643, 5652 and 5644, 5652 and 5645, 5652 and 5646, 5652 and 5647, 5652 and 5648, 5652 and 5649, 5653 and 5639, 5653 and 5640, 5653 and 5641, 5653 and 5642, 5653 and 5643, 5653 and 5644, 5653 and 5645, 5653 and 5646, 5653 and 5647, 5653 and 5648, 5653 and 5649, 5654 and 5639, 5654 and 5640, 5654 and 5641, 5654 and 5642, 5654 and 5643, 5654 and 5644, 5654 and 5645, 5654 and 5646, 5654 and 5647, 5654 and 5648, 5654 and 5649, 5655 and 5639, 5655 and 5640, 5655 and 5641, 5655 and 5642, 5655 and 5643, 5655 and 5644, 5655 and 5645, 5655 and 5646, 5655 and 5647, 5655 and 5648, 5655 and 5649, 5656 and 5639, 5656 and 5640, 5656 and 5641, 5656 and 5642, 5656 and 5643, 5656 and 5644, 5656 and 5645, 5656 and 5646, 5656 and 5647, 5656 and 5648, 5656 and 5649, 5657 and 5639, 5657 and 5640, 5657 and 5641, 5657 and 5642, 5657 and 5643, 5657 and 5644, 5657 and 5645, 5657 and 5646, 5657 and 5647, 5657 and 5648, 5657 and 5649, 5658 and 5639, 5658 and 5640, 5658 and 5641, 5658 and 5642, 5658 and 5643, 5658 and 5644, 5658 and 5645, 5658 and 5646, 5658 and 5647, 5658 and 5648, 5658 and 5649, 5659 and 5639, 5659 and 5640, 5659 and 5641, 5659 and 5642, 5659 and 5643, 5659 and 5644, 5659 and 5645, 5659 and 5646, 5659 and 5647, 5659 and 5648, 5659 and 5649, 5660 and 5639, 5660 and 5640, 5660 and 5641, 5660 and 5642, 5660 and 5643, 5660 and 5644, 5660 and 5645, 5660 and 5646, 5660 and 5647, 5660 and 5648, 5660 and 5649, 5661 and 5639, 5661 and 5640, 5661 and 5641, 5661 and 5642, 5661 and 5643, 5661 and 5644, 5661 and 5645, 5661 and 5646, 5661 and 5647, 5661 and 5648, 5661 and 5649, 5662 and 5639, 5662 and 5640, 5662 and 5641, 5662 and 5642, 5662 and 5643, 5662 and 5644, 5662 and 5645, 5662 and 5646, 5662 and 5647, 5662 and 5648, 5662 and 5649, 5663 and 5639, 5663 and 5640, 5663 and 5641, 5663 and 5642, 5663 and 5643, 5663 and 5644, 5663 and 5645, 5663 and 5646, 5663 and 5647, 5663 and 5648, 5663 and 5649, 5664 and 5639, 5664 and 5640, 5664 and 5641, 5664 and 5642, 5664 and 5643, 5664 and 5644, 5664 and 5645, 5664 and 5646, 5664 and 5647, 5664 and 5648, 5664 and 5649, 5665 and 5639, 5665 and 5640, 5665 and 5641, 5665 and 5642, 5665 and 5643, 5665 and 5644, 5665 and 5645, 5665 and 5646, 5665 and 5647, 5665 and 5648, 5665 and 5649, 5666 and 5639, 5666 and 5640, 5666 and 5641, 5666 and 5642, 5666 and 5643, 5666 and 5644, 5666 and 5645, 5666 and 5646, 5666 and 5647, 5666 and 5648, 5666 and 5649, 5667 and 5639, 5667 and 5640, 5667 and 5641, 5667 and 5642, 5667 and 5643, 5667 and 5644, 5667 and 5645, 5667 and 5646, 5667 and 5647, 5667 and 5648, 5667 and 5649, 5668 and 5639, 5668 and 5640, 5668 and 5641, 5668 and 5642, 5668 and 5643, 5668 and 5644, 5668 and 5645, 5668 and 5646, 5668 and 5647, 5668 and 5648, or 5668 and 5649. The gRNA core of the PEgRNA can comprise SEQ ID NO. 5857-5859. Exemplary PEgRNAs provided in Table 31 can comprise any one of SEQ ID NOs. 5669-5705. Such PEgRNA sequences may further comprise a 3′ motif at the 3′ end of the extension arm, for example, a hairpin-forming motif or a series of 3, 4 or more U nucleotides. Without being bound by theory, such 3′ motifs are believed to increase PEgRNA stability. Such PEgRNA sequences may alternatively or additionally be adapted for transcription from a DNA template, for example, by including a 5′ terminal G if the spacer of the PEgRNA begins with another nucleotide, by including 6 or 7 U nucleotides at the 3′ end of the extension arm, or both. Exemplary transcription-adapted sequences include None. Such plasmid adapted sequences may further comprise a hairpin-forming motif between the PBS and the 3′ terminal U series.

Any of the PEgRNAs of Table 31 can be used in a Prime Editing system further comprising a nick guide RNA (ngRNA). Such ngRNA can comprise a spacer comprising nucleotides 5-20 of SEQ ID NO: 41, 60, 61, 62, 63, 64, 65, 66, 69, or 71 and a gRNA core capable of binding to a Cas9 protein. The spacer of the ngRNA can comprise nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 41, 60, 61, 62, 63, 64, 65, 66, 69, or 71. The spacer of the ngRNA can comprise SEQ ID NO: 41, 60, 61, 62, 63, 64, 65, 66, 69, or 71. The gRNA core of the ngRNA can comprise SEQ ID NO. 5857-5859. Such ngRNA sequences may further comprise a 3′ motif at the 3′ end of the gRNA core, for example, a series of 3, 4 or more U nucleotides. Without being bound by theory, such 3′ motifs are believed to increase ngRNA stability.

Table 32 provides Prime Editing guide RNAs (PEgRNAs), which can be used in Prime Editing systems disclosed herein. Such Prime Editing systems can comprise a Cas9 protein capable of recognizing an NGG or NG PAM sequence (e.g., AGG or AG), and a reverse transcriptase. The Prime Editing systems (e.g., PE3 or PE3b systems) can further comprise a nick guide RNA (ngRNA). Such PEgRNAs and Prime Editing systems can be used, for example, to correct an H1069Q mutation in ATP7B.

The PEgRNAs of Table 32 comprise: (a) a spacer comprising nucleotides 5-20 of SEQ ID NO: 5706. (b) a gRNA core capable of binding to a Cas9 protein, and (c) an extension arm comprising: (i) an editing template comprising any one of SEQ ID NOs: 5718-5738, and (ii) a primer binding site (PBS) comprising any one of SEQ ID NOs: 5707-5717. The spacer of the PEgRNA can comprise, for example, nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 5706. The spacer of the PEgRNA can comprise SEQ ID NO: 5706. The RTT and the PBS can comprise respectively SEQ ID NOs: 5718 and 5707, 5718 and 5708, 5718 and 5709, 5718 and 5710, 5718 and 5711, 5718 and 5712, 5718 and 5713, 5718 and 5714, 5718 and 5715, 5718 and 5716, 5718 and 5717, 5719 and 5707, 5719 and 5708, 5719 and 5709, 5719 and 5710, 5719 and 5711, 5719 and 5712, 5719 and 5713, 5719 and 5714, 5719 and 5715, 5719 and 5716, 5719 and 5717, 5720 and 5707, 5720 and 5708, 5720 and 5709, 5720 and 5710, 5720 and 5711, 5720 and 5712, 5720 and 5713, 5720 and 5714, 5720 and 5715, 5720 and 5716, 5720 and 5717, 5721 and 5707, 5721 and 5708, 5721 and 5709, 5721 and 5710, 5721 and 5711, 5721 and 5712, 5721 and 5713, 5721 and 5714, 5721 and 5715, 5721 and 5716, 5721 and 5717, 5722 and 5707, 5722 and 5708, 5722 and 5709, 5722 and 5710, 5722 and 5711, 5722 and 5712, 5722 and 5713, 5722 and 5714, 5722 and 5715, 5722 and 5716, 5722 and 5717, 5723 and 5707, 5723 and 5708, 5723 and 5709, 5723 and 5710, 5723 and 5711, 5723 and 5712, 5723 and 5713, 5723 and 5714, 5723 and 5715, 5723 and 5716, 5723 and 5717, 5724 and 5707, 5724 and 5708, 5724 and 5709, 5724 and 5710, 5724 and 5711, 5724 and 5712, 5724 and 5713, 5724 and 5714, 5724 and 5715, 5724 and 5716, 5724 and 5717, 5725 and 5707, 5725 and 5708, 5725 and 5709, 5725 and 5710, 5725 and 5711, 5725 and 5712, 5725 and 5713, 5725 and 5714, 5725 and 5715, 5725 and 5716, 5725 and 5717, 5726 and 5707, 5726 and 5708, 5726 and 5709, 5726 and 5710, 5726 and 5711, 5726 and 5712, 5726 and 5713, 5726 and 5714, 5726 and 57151, 5726 and 5716, 5726 and 5717, 5727 and 5707, 5727 and 5708, 5727 and 5709, 5727 and 5710, 5727 and 5711, 5727 and 5712, 5727 and 5713, 5727 and 5714, 5727 and 5715, 5727 and 5716, 5727 and 5717, 5728 and 5707, 5728 and 5708, 5728 and 5709, 5728 and 5710, 5728 and 5711, 5728 and 5712, 5728 and 5713, 5728 and 5714, 5728 and 5715, 5728 and 5716, 5728 and 5717, 5729 and 5707, 5729 and 5708, 5729 and 5709, 5729 and 5710, 5729 and 5711, 5729 and 5712, 5729 and 5713, 5729 and 57141, 5729 and 5715, 5729 and 5716, 5729 and 5717, 5730 and 5707, 5730 and 5708, 5730 and 5709, 5730 and 5710, 5730 and 5711, 5730 and 5712, 5730 and 5713, 5730 and 5714, 5730 and 5715, 5730 and 5716, 5730 and 5717, 5731 and 5707, 5731 and 5708, 5731 and 5709, 5731 and 5710, 5731 and 5711, 5731 and 5712, 5731 and 5713, 5731 and 5714, 5731 and 5715, 5731 and 5716, 5731 and 5717, 5732 and 5707, 5732 and 5708, 5732 and 5709, 5732 and 5710, 5732 and 5711, 5732 and 5712, 5732 and 5713, 5732 and 5714, 5732 and 5715, 5732 and 5716, 5732 and 5717, 5733 and 5707, 5733 and 5708, 5733 and 5709, 5733 and 5710, 5733 and 5711, 5733 and 5712, 5733 and 5713, 5733 and 5714, 5733 and 5715, 5733 and 5716, 5733 and 5717, 5734 and 5707, 5734 and 5708, 5734 and 5709, 5734 and 5710, 5734 and 5711, 5734 and 5712, 5734 and 5713, 5734 and 5714, 5734 and 5715, 5734 and 5716, 5734 and 5717, 5735 and 5707, 5735 and 5708, 5735 and 5709, 5735 and 5710, 5735 and 5711, 5735 and 5712, 5735 and 5713, 5735 and 5714, 5735 and 5715, 5735 and 5716, 5735 and 5717, 5736 and 5707, 5736 and 5708, 5736 and 5709, 5736 and 5710, 5736 and 5711, 5736 and 5712, 5736 and 5713, 5736 and 5714, 5736 and 5715, 5736 and 5716, 5736 and 5717, 5737 and 5707, 5737 and 5708, 5737 and 5709, 5737 and 5710, 5737 and 5711, 5737 and 5712, 5737 and 5713, 5737 and 5714, 5737 and 5715, 5737 and 5716, 5737 and 5717, 5738 and 5707, 5738 and 5708, 5738 and 5709, 5738 and 5710, 5738 and 5711, 5738 and 5712, 5738 and 5713, 5738 and 5714, 5738 and 5715, 5738 and 5716, or 5738 and 5717. The gRNA core of the PEgRNA can comprise SEQ ID NO. 5857-5859. Exemplary PEgRNAs provided in Table 32 can comprise any one of SEQ ID NOs. 5739-5779. Such PEgRNA sequences may further comprise a 3′ motif at the 3′ end of the extension arm, for example, a hairpin-forming motif or a series of 3, 4 or more U nucleotides. Without being bound by theory, such 3′ motifs are believed to increase PEgRNA stability. Such PEgRNA sequences may alternatively or additionally be adapted for transcription from a DNA template, for example, by including a 5′ terminal G if the spacer of the PEgRNA begins with another nucleotide, by including 6 or 7 U nucleotides at the 3′ end of the extension arm, or both. Such plasmid adapted sequences may further comprise a hairpin-forming motif between the PBS and the 3′ terminal U series.

Any of the PEgRNAs of Table 32 can be used in a Prime Editing system further comprising a nick guide RNA (ngRNA). Such ngRNA can comprise a spacer comprising nucleotides 5-20 of SEQ ID NO: 41, 60, 61, 62, 63, 64, 65, 66, 69, or 71 and a gRNA core capable of binding to a Cas9 protein. The spacer of the ngRNA can comprise nucleotides 4-20, 3-20, 2-20, or 1-20 of SEQ ID NO: 41, 60, 61, 62, 63, 64, 65, 66, 69, or 71. The spacer of the ngRNA can comprise SEQ ID NO: 41, 60, 61, 62, 63, 64, 65, 66, 69, or 71. The gRNA core of the ngRNA can comprise SEQ ID NO. 5857-5859. Such ngRNA sequences may further comprise a 3′ motif at the 3′ end of the gRNA core, for example, a series of 3, 4 or more U nucleotides. Without being bound by theory, such 3′ motifs are believed to increase ngRNA stability.

Table 33 provides Prime Editing guide RNAs (PEgRNAs) and nick guide RNAs (ngRNAs) that can be used in Prime Editing systems disclosed herein. Any of the PEgRNAs of Table 33 can be used in a Prime Editing system further comprising any ngRNA of Table 33.

TABLE 33

exemplary PEgRNA and ngRNA sequences.

SEQ

ID
DESCRIP-

NO.
TION
SEQUENCE

2739
PEgRNA
UUUGGUGACUGCCACGCCCAGUUUUAGAGCUAGAAA

UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU

GAAAAAGUGGCACCGAGUCGGUGCAGUGAACAcCCCU

UGGGCGUGGCAuuuu

3277
PEgRNA
UUUGGUGACUGCCACGCCCAGUUUUAGAGCUAGAAA

UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU

GAAAAAGUGGCACCGAGUCGGUGCAGUGAACAcCCgU

UGGGCGUGGCAGUCACCAAuuuu

3276
PEgRNA
UUUGGUGACUGCCACGCCCAGUUUUAGAGCUAGAAA

UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU

GAAAAAGUGGCACCGAGUCGGUGCAGUGAACAcCCCU

UGGGCGUGGCAGUCACCAAuuuu

2785
PERNA
UUUGGUGACUGCCACGCCCAGUUUUAGAGCUAGAAA

UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU

GAAAAAGUGGCACCGAGUCGGUGCGAACAcCCCUUGG

GCGUGGCAGUCAuuuu

4695
PEgRNA
UUGGUGACUGCCACGCCCAAGUUUUAGAGCUAGAAA

UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU

GAAAAAGUGGCACCGAGUCGGUGCGUGAACAcCCCUU

GGGCGUGGCAGUCuuuu

4762
PERNA
UUGGUGACUGCCACGCCCAAGUUUUAGAGCUAGAAA

UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU

GAAAAAGUGGCACCGAGUCGGUGCAGUGAACAcCCCU

UGGGCGUGGCAGUCAuuuu

4824
PEgRNA
UUGGUGACUGCCACGCCCAAGUUUUAGAGCUAGAAA

UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU

GAAAAAGUGGCACCGAGUCGGUGCGCAGUGAACAcCC

CUUGGGCGUGGCAGUCAuuuu

4721
PEgRNA
UUGGUGACUGCCACGCCCAAGUUUUAGAGCUAGAAA

UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU

GAAAAAGUGGCACCGAGUCGGUGCAGUGAACAcCCCU

UGGGCGUGGCAGUuuuu

4743
PEgRNA
UUGGUGACUGCCACGCCCAAGUUUUAGAGCUAGAAA

UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU

GAAAAAGUGGCACCGAGUCGGUGCAGUGAACAcCCCU

UGGGCGUGGCAGUCuuuu

4536
PEgRNA
UUGGUGACUGCCACGCCCAAGUUUUAGAGCUAGAAA

UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU

GAAAAAGUGGCACCGAGUCGGUGCAACAcCCCUUGGG

CGUGGCuuuu

4741
PEgRNA
UUGGUGACUGCCACGCCCAAGUUUUAGAGCUAGAAA

UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU

GAAAAAGUGGCACCGAGUCGGUGCUGAACAcCCCUUG

GGCGUGGCAGUCACuuuu

4788
PEgRNA
UUGGUGACUGCCACGCCCAAGUUUUAGAGCUAGAAA

UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU

GAAAAAGUGGCACCGAGUCGGUGCAGCAGUGAACAcC

CCUUGGGCGUGGCAGuuuu

4613
PEgRNA
UUGGUGACUGCCACGCCCAAGUUUUAGAGCUAGAAA

UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU

GAAAAAGUGGCACCGAGUCGGUGCUGAACAcCCCUUG

GGCGUGGCAGuuuu

2291
ngRNA
GCCAGCAGUGAACAcCCCUUGUUUUAGAGCUAGAAAU

AGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG

AAAAAGUGGCACCGAGUCGGUGCuuuu

2292
ngRNA
GGCCAGCAGUGAACAcCCCUGUUUUAGAGCUAGAAAU

AGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG

AAAAAGUGGCACCGAGUCGGUGCuuuu

4423
ngRNA
GCCAGCAGUGAACAcCCGUUGUUUUAGAGCUAGAAA

UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU

GAAAAAGUGGCACCGAGUCGGUGCuuuu

4424
ngRNA
GGCCAGCAGUGAACAcCCGUGUUUUAGAGCUAGAAA

UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU

GAAAAAGUGGCACCGAGUCGGUGCuuuu

2290
ngRNA
CAUGCGGGUGCUCCUGCUGGGUUUUAGAGCUAGAAA

UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUU

GAAAAAGUGGCACCGAGUCGGUGCuuuu

A PEgRNA and/or an ngRNA of this disclosure, in some embodiments, may include modified nucleotides, e.g., chemically modified DNA or RNA nucleobases, and may include one or more nucleobase analogs (e.g., modifications which might add functionality, such as temperature resilience). In some embodiments, PEgRNAs and/or ngRNAs as described herein may be chemically modified. The phrase “chemical modifications,” as used herein, can include modifications which introduce chemistries which differ from those seen in naturally occurring DNA or RNAs, for example, covalent modifications such as the introduction of modified nucleotides, (e.g., nucleotide analogs, or the inclusion of pendant groups which are not naturally found in DNA or RNA molecules).

In some embodiments, the PEgRNAs and/or ngRNAs provided in this disclosure may have undergone a chemical or biological modifications. Modifications may be made at any position within a PEgRNA or ngRNA, and may include modification to a nucleobase or to a phosphate backbone of the PEgRNA or ngRNA. In some embodiments, chemical modifications can be a structure guided modifications. In some embodiments, a chemical modification is at the 5′ end and/or the 3′ end of a PEgRNA. In some embodiments, a chemical modification is at the 5′ end and/or the 3′ end of a ngRNA. In some embodiments, a chemical modification may be within the spacer sequence, the extension arm, the editing template sequence, or the primer binding site of a PEgRNA. In some embodiments, a chemical modification may be within the spacer sequence or the gRNA core of a PEgRNA or a ngRNA. In some embodiments, a chemical modification may be within the 3′ most nucleotides of a PEgRNA or ngRNA. In some embodiments, a chemical modification may be within the 3′ most end of a PEgRNA or ngRNA. In some embodiments, a chemical modification may be within the 5′ most end of a PEgRNA or ngRNA. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more chemically modified nucleotides at the 3′ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more chemically modified nucleotides at the 5′ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, 3, 4, or 5 or more chemically modified nucleotides at the 3′ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, 3, 4, or 5 more chemically modified nucleotides at the 5′ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, or 3 or more chemically modified nucleotides at the 3′ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, or 3 more chemically modified nucleotides at the 5′ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more contiguous chemically modified nucleotides at the 3′ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more contiguous chemically modified nucleotides at the 5′ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, 3, 4, or 5 contiguous chemically modified nucleotides at the 3′ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, 3, 4, or 5 contiguous chemically modified nucleotides at the 5′ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, or 3 contiguous chemically modified nucleotides at the 3′ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, or 3 contiguous chemically modified nucleotides at the 5′ end. In some embodiments, a PEgRNA or ngRNA comprises 3 contiguous chemically modified nucleotides at the 3′ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, 3, 4, 5, or more chemically modified nucleotides near the 3′ end. In some embodiments, a PEgRNA or ngRNA comprises 3 contiguous chemically modified nucleotides at the 3′ end. In some embodiments, a PEgRNA or ngRNA comprises 3 contiguous chemically modified nucleotides at the 5′ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, 3, 4, 5, or more chemically modified nucleotides near the 3′ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, 3, 4, 5, or more contiguous chemically modified nucleotides near the 3′ end. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, 3, 4, 5, or more chemically modified nucleotides near the 3′ end, where the 3′ most nucleotide is not modified, and the 1, 2, 3, 4, 5, or more chemically modified nucleotides precede the 3′ most nucleotide in a 5′-to-3′ order. In some embodiments, a PEgRNA or ngRNA comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more chemically modified nucleotides near the 3′ end, where the 3′ most nucleotide is not modified, and the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more chemically modified nucleotides precede the 3′ most nucleotide in a 5′-to-3′ order.

In some embodiments, a PEgRNA or ngRNA comprises one or more chemical modified nucleotides in the gRNA core. As exemplified in FIG. 5, the gRNA core of a PEgRNA may comprise one or more regions of a base paired lower stein, a base paired upper stem, where the lower stein and tipper stem may be connected by a bulge comprising unpaired RNAs. The gRNA core may further comprise a nexus distal from the spacer sequence. In some embodiments, the gRNA core comprises one or more chemically modified nucleotides in the lower stem, upper stein, and/or the hairpin regions. In some embodiments, all of the nucleotides in the lower stem, upper stem, and/or the hairpin regions are chemically modified.

A chemical modification to a PEgRNA or ngRNA can comprise a 2′-O-thionocarbamate-protected nucleoside phosphoramidite, a 2′-O-methyl (M), a 2′-O-methyl 3′ phosphorothioate (MS), or a 2′-O-methyl 3′ thioPACE (MSP), or any combination thereof. In some embodiments, a chemically modified PEgRNA and/or ngRNA can comprise a ′—O-methyl (M) RNA, a 2′-O-methyl 3′ phosphorothioate (MS) RNA, a 2′-O-methyl 3′ thioPACE (MSP) RNA, a 2′-F RNA, a phosphorothioate bond modification, any other chemical modifications known in the art, or any combination thereof. A chemical modification may also include, for example, the incorporation of non-nucleotide linkages or modified nucleotides into the PEgRNA and/or ngRNA (e.g., modifications to one or both of the 3′ and 5′ ends of a guide RNA molecule). Such modifications can include the addition of bases to an RNA sequence, complexing the RNA with an agent (e.g., a protein or a complementary nucleic acid molecule), and inclusion of elements which change the structure of an RNA molecule (e.g., which form secondary structures).

Prime Editing Compositions

Disclosed herein, in some embodiments, are compositions, systems, and methods using a prime editing composition. The term “prime editing composition” or “prime editing system” refers to compositions involved in the method of prime editing as described herein. A prime editing composition may include a prime editor, e.g., a prime editor fusion protein, and a PEgRNA. A prime editing composition may further comprise additional elements, such as second strand nicking ngRNAs. Components of a prime editing composition may be combined to form a complex for prime editing, or may be kept separately, e.g., for administration purposes. In some embodiments, a prime editing composition comprises a prime editor fusion protein complexed with a PEgRNA and optionally complexed with a ngRNA. In some embodiments, the prime editing composition comprises a prime editor comprising a DNA binding domain and a DNA polymerase domain associated with each other through a PEgRNA. For example, the prime editing composition may comprise a prime editor comprising a DNA binding domain and a DNA polymerase domain linked to each other by an RNA-protein recruitment aptamer RNA sequence, which is linked to a PEgRNA. In some embodiments, a prime editing composition comprises a PEgRNA and a polynucleotide, a polynucleotide construct, or a vector that encodes a prime editor fusion protein. In some embodiments, a prime editing composition comprises a PEgRNA, a ngRNA, and a polynucleotide, a polynucleotide construct, or a vector that encodes a prime editor fusion protein. In some embodiments, a prime editing composition comprises multiple polynucleotides, polynucleotide constructs, or vectors, each of which encodes one or more prime editing composition components. In some embodiments, the PEgRNA of a prime editing composition is associated with the DNA binding domain, e.g., a Cas9 nickase, of the prime editor. In some embodiments, the PEgRNA of a prime editing composition complexes with the DNA binding domain of a prime editor and directs the prime editor to the target DNA.

In some embodiments, a prime editing composition comprises one or more polynucleotides that encode prime editor components and/or PEgRNA or ngRNAs. In some embodiments, a prime editing composition comprises a polynucleotide encoding a fusion protein comprising a DNA binding domain and a DNA polymerase domain. In some embodiments, a prime editing composition comprises (i) a polynucleotide encoding a fusion protein comprising a DNA binding domain and a DNA polymerase domain, and (ii) a PEgRNA or a polynucleotide encoding the PEgRNA. In some embodiments, a prime editing composition comprises (i) a polynucleotide encoding a fusion protein comprising a DNA binding domain and a DNA polymerase domain, (ii) a PEgRNA or a polynucleotide encoding the PEgRNA, and (iii) an ngRNA or a polynucleotide encoding the ngRNA. In some embodiments, a prime editing composition comprises (i) a polynucleotide encoding a DNA binding domain of a prime editor, e.g., a Cas9 nickase, (ii) a polynucleotide encoding a DNA polymerase domain of a prime editor, e.g., a reverse transcriptase, and (iii) a PEgRNA or a polynucleotide encoding the PEgRNA. In some embodiments, a prime editing composition comprises (i) a polynucleotide encoding a DNA binding domain of a prime editor, e.g., a Cas9 nickase, (ii) a polynucleotide encoding a DNA polymerase domain of a prime editor, e.g., a reverse transcriptase, (iii) a PEgRNA or a polynucleotide encoding the PEgRNA, and (iv) an ngRNA or a polynucleotide encoding the ngRNA. In some embodiments, the polynucleotide encoding the DNA biding domain or the polynucleotide encoding the DNA polymerase domain further encodes an additional polypeptide domain, e.g., an RNA-protein recruitment domain, such as a MS2 coat protein domain. In some embodiments, a prime editing composition comprises (i) a polynucleotide encoding a N-terminal half of a prime editor fusion protein and an intein-N and (ii) a polynucleotide encoding a C-terminal half of a prime editor fusion protein and an intein-C. In some embodiments, a prime editing composition comprises (i) a polynucleotide encoding a N-terminal half of a prime editor fusion protein and an intein-N (ii) a polynucleotide encoding a C-terminal half of a prime editor fusion protein and an intein-C, (iii) a PEgRNA or a polynucleotide encoding the PEgRNA, and/or (iv) an ngRNA or a polynucleotide encoding the ngRNA. In some embodiments, a prime editing composition comprises (i) a polynucleotide encoding a N-terminal portion of a DNA binding domain and an intein-N, (ii) a polynucleotide encoding a C-terminal portion of the DNA binding domain, an intein-C, and a DNA polymerase domain. In some embodiments, the DNA binding domain is a Cas protein domain, e.g., a Cas9 nickase. In some embodiments, the prime editing composition comprises (i) a polynucleotide encoding a N-terminal portion of a DNA binding domain and an intein-N, (ii) a polynucleotide encoding a C-terminal portion of the DNA binding domain, an intein-C, and a DNA polymerase domain, (iii) a PEgRNA or a polynucleotide encoding the PEgRNA, and/or (iv) a ngRNA or a polynucleotide encoding the ngRNA.

In some embodiments, a prime editing system comprises one or more polynucleotides encoding one or more prime editor polypeptides, wherein activity of the prime editing system can be temporally regulated by controlling the timing in which the vectors are delivered. For example, in some embodiments, a polynucleotide encoding the prime editor and a polynucleotide encoding a PEgRNA can be delivered simultaneously. For example, in some embodiments, a polynucleotide encoding the prime editor and a polynucleotide encoding a PEgRNA can be delivered sequentially.

In some embodiments, a polynucleotide encoding a component of a prime editing system can further comprise an element that is capable of modifying the intracellular half-life of the polynucleotide and/or modulating translational control. In some embodiments, the polynucleotide is a RNA, for example, an mRNA. In some embodiments, the half-life of the polynucleotide, e.g., the RNA may be increased. In some embodiments, the half-life of the polynucleotide, e.g., the RNA may be decreased. In some embodiments, the element may be capable of increasing the stability of the polynucleotide, e.g., the RNA. In some embodiments, the element may be capable of decreasing the stability of the polynucleotide, e.g., the RNA. In some embodiments, the element may be within the 3′ UTR of the RNA. In some embodiments, the element may include a polyadenylation signal (PA). In some embodiments, the element may include a cap, e.g., an upstream mRNA or PEgRNA end. In some embodiments, the RNA may comprise no PA such that it is subject to quicker degradation in the cell after transcription.

In some embodiments, the element may include at least one AU-rich element (ARE). The AREs may be bound by ARE binding proteins (ARE-BPs) in a manner that is dependent upon tissue type, cell type, timing, cellular localization, and environment. In some embodiments the destabilizing element may promote RNA decay, affect RNA stability, or activate translation. In some embodiments, the ARE may comprise 50 to 150 nucleotides in length. In some embodiments, the ARE may comprise at least one copy of the sequence AUUUA. In some embodiments, at least one ARE may be added to the 3′ UTR of the RNA. In some embodiments, the element may be a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE). In further embodiments, the element is a modified and/or truncated WPRE sequence that is capable of enhancing expression from the transcript. In some embodiments, the WPRE or equivalent may be added to the 3′ UTR of the RNA. In some embodiments, the element may be selected from other RNA sequence motifs that are enriched in either fast- or slow-decaying transcripts. In some embodiments, the polynucleotide, e.g., a vector, encoding the PE or the PEgRNA may be self-destroyed via cleavage of a target sequence present on the polynucleotide, e.g., a vector. The cleavage may prevent continued transcription of a PE or a PEgRNA.

Polynucleotides encoding prime editing composition components can be DNA, RNA, or any combination thereof. In some embodiments, a polynucleotide encoding a prime editing composition component is an expression construct. In some embodiments, a polynucleotide encoding a prime editing composition component is a vector. In some embodiments, the vector is a DNA vector. In some embodiments, the vector is a plasmid. In some embodiments, the vector is a virus vector, e.g., a retroviral vector, adenoviral vector, lentiviral vector, herpesvirus vector, or an adeno-associated virus vector (AAV).

In some embodiments, polynucleotides encoding polypeptide components of a prime editing composition are codon optimized by replacing at least one codon (e.g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. In some embodiments, a polynucleotide encoding a polypeptide component of a prime editing composition are operably linked to one or more expression regulatory elements, for example, a promoter, a 3′ UTR, a 5′ UTR, or any combination thereof. In some embodiments, a polynucleotide encoding a prime editing composition component is a messenger RNA (mRNA). In some embodiments, the mRNA comprises a Cap at the 5′ end and/or a poly A tail at the 3′ end.

In some embodiments, the PEgRNA as described herein comprises a spacer that comprises a sequence selected from the group consisting of SEQ ID Nos. 1, 182, 294, 483, 682, 1505, 2023, 2293, 4425, 5206, 5228, 5248, 5282, 5313, 5340, 5369, 5406, 5423, 5446, 5473, 5503, 5537, 5555, 5638, and 5706.

In some embodiments, the PEgRNA as described herein comprises a editing template that comprises a sequence selected from the group consisting of SEQ ID Nos.: 13-17, 194-198, 306-336, 495-528, 694-735, 1517-1546, 2035-2044, 2305-2422, 4437-4492, 5218, 5240-5247, 5260-5279, 5294-5302, 5325-5338, 5352-5368, 5381-5401, 5418-5422, 5435-5445, 5458-5472, 5485-5502, 5515-5535, 5549-5554, 5567-5590, 5650-5668, and 5718-5738.

In some embodiments, the PEgRNA as described herein comprises a PBS that comprises a sequence selected from the group consisting of SEQ ID Nos. 2-12, 183-193, 295-305, 484-494, 683-693, 1506-1516, 2024-2034, 2294-2304, 4426-4436, 5207-5217, 5229-5239, 5249-5259, 5283-5293, 5314-5324, 5341-5351, 5370-5380, 5407-5417, 5424-5434, 5447-5457, 5474-5484, 5504-5514, 5538-5548, 5556-5566, 5639-5649, and 5707-5717.

In some embodiments, the PEgRNA as described herein comprises a sequence selected from the group consisting of SEQ ID Nos. 73-152, 210-289, 338-482, 530-680, 741-1500, 1547-2022, 2097-2256, 2445-4409, 4493-5205, 5591-5637, 5669-5705, and 5739-5779.

In some embodiments, the ngRNA disclosed herein comprises a ng spacer that comprises a sequence selected from the group consisting of SEQ ID Nos. 18-72, 199-209, 337, 529, 736-740, 2045-2096, 2423-2444, 5219-5227, 5280-5281, 5303-5312, 5339, 5402-5405, and 5536.

In some embodiments, the ngRNA disclosed herein comprises a sequence selected from the group consisting of SEQ ID NOs: 153-181.

Pharmaceutical Compositions

Disclosed herein are pharmaceutical compositions comprising any of the prime editing composition components, for example, prime editors, fusion proteins, polynucleotides encoding prime editor polypeptides, PEgRNAs, ngRNAs, and/or prime editing complexes described herein.

The term “pharmaceutical composition”, as used herein, refers to a composition formulated for pharmaceutical use. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises additional agents, e.g., for specific delivery, increasing half-life, or other therapeutic compounds.

In some embodiments, a pharmaceutically-acceptable carrier comprises any vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, tale magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the compound from one site (e.g., the delivery site) of the body, to another site (e.g., organ, tissue or portion of the body). A pharmaceutically acceptable carrier is “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the tissue of the subject (e.g., physiologically compatible, sterile, physiologic pH, etc.).

Formulations of the pharmaceutical compositions described herein can be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient(s) into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping and/or packaging the product into a desired single- or multi-dose unit. Pharmaceutical formulations can additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.

Methods of Editing

The methods and compositions disclosed herein can be used to edit a target gene of interest by prime editing.

In some embodiments, the prime editing method comprises contacting a target gene, e.g., an ATP7B gene, with a PEgRNA and a prime editor (PE) polypeptide described herein. In some embodiments, the target gene is double stranded, and comprises two strands of DNA complementary to each other. In some embodiments, the contacting with a PEgRNA and the contacting with a prime editor are performed sequentially. In some embodiments, the contacting with a prime editor is performed after the contacting with a PEgRNA. In some embodiments, the contacting with a PEgRNA is performed after the contacting with a prime editor. In some embodiments, the contacting with a PEgRNA, and the contacting with a prime editor are performed simultaneously. In some embodiments, the PEgRNA and the prime editor are associated in a complex prior to contacting a target gene.

In some embodiments, contacting the target gene with the prime editing composition results in binding of the PEgRNA to a target strand of the target gene, e.g., an ATP7B gene. In some embodiments, contacting the target gene with the prime editing composition results in binding of the PEgRNA to a search target sequence on the target strand of the target gene upon contacting with the PEgRNA. In some embodiments, contacting the target gene with the prime editing composition results in binding of a spacer sequence of the PEgRNA to a search target sequence with the search target sequence on the target strand of the target gene upon said contacting of the PEgRNA.

In some embodiments, contacting the target gene with the prime editing composition results in binding of the prime editor to the target gene, e.g. the target ATP7B gene, upon the contacting of the PE composition with the target gene. In some embodiments, the DNA binding domain of the PE associates with the PEgRNA. In some embodiments, the PE binds the target gene, e.g. an ATP7B gene, directed by the PEgRNA. Accordingly, in some embodiments, the contacting of the target gene result in binding of a DNA binding domain of a prime editor of the target ATP7B gene directed by the PEgRNA.

In some embodiments, contacting the target gene with the prime editing composition results in a nick in an edit strand of the target gene, e.g. an ATP7B gene by the prime editor upon contacting with the target gene, thereby generating a nicked on the edit strand of the target gene. In some embodiments, contacting the target gene with the prime editing composition results in a single-stranded DNA comprising a free 3′ end at the nick site of the edit strand of the target gene. In some embodiments, contacting the target gene with the prime editing composition results in a nick in the edit strand of the target gene by a DNA binding domain of the prime editor, thereby generating a single-stranded DNA comprising a free 3′ end at the nick site. In some embodiments, the DNA binding domain of the prime editor is a Cas domain. In some embodiments, the DNA binding domain of the prime editor is a Cas9. In some embodiments, the DNA binding domain of the prime editor is a Cas9 nickase.

In some embodiments, contacting the target gene with the prime editing composition results in hybridization of the PEgRNA with the 3′ end of the nicked single-stranded DNA, thereby priming DNA polymerization by a DNA polymerase domain of the prime editor. In some embodiments, the free 3′ end of the single-stranded DNA generated at the nick site hybridizes to a primer binding site sequence (PBS) of the contacted PEgRNA, thereby priming DNA polymerization. In some embodiments, the DNA polymerization is reverse transcription catalyzed by a reverse transcriptase domain of the prime editor. In some embodiments, the method comprises contacting the target gene with a DNA polymerase, e.g., a reverse transcriptase, as a pail of a prime editor fusion protein or prime editing complex (in cis), or as a separate protein (in trans).

In some embodiments, contacting the target gene with the prime editing composition generates an edited single stranded DNA that is coded by the editing template of the PEgRNA by DNA polymerase mediated polymerization from the 3′ free end of the single-stranded DNA at the nick site. In some embodiments, the editing template of the PEgRNA comprises one or more intended nucleotide edits compared to endogenous sequence of the target gene, e.g., an ATP7B gene. In some embodiments, the intended nucleotide edits are incorporated in the target gene, by excision of the 5′ single stranded DNA of the edit strand of the target gene generated at the nick site and DNA repair. In some embodiments, the intended nucleotide edits are incorporated in the target gene by excision of the editing target sequence and DNA repair. In some embodiments, excision of the 5′ single stranded DNA of the edit strand generated at the nick site is by a flap endonuclease. In some embodiments, the flap nuclease is FEN1. In some embodiments, the method further comprises contacting the target gene with a flap endonuclease. In some embodiments, the flap endonuclease is provided as a part of a prime editor fusion protein. In some embodiments, the flap endonuclease is provided in trans.

In some embodiments, contacting the target gene with the prime editing composition generates a mismatched heteroduplex comprising the edit strand of the target gene that comprises the edited single stranded DNA, and the unedited target strand of the target gene. Without being bound by theory, the endogenous DNA repair and replication may resolve the mismatched edited DNA to incorporate the nucleotide change(s) to form the desired edited target gene.

In some embodiments, the method further comprises contacting the target gene, e.g. an ATP7B gene, with a nick guide (ngRNA) disclosed herein. In some embodiments, the ngRNA comprises a spacer that binds a second search target sequence on the edit strand of the target gene. In some embodiments, the contacted ngRNA directs the PE to introduce a nick in the target strand of the target gene. In some embodiments, the nick on the target strand (non-edit strand) results in endogenous DNA repair machinery to use the edit strand to repair the non-edit strand, thereby incorporating the intended nucleotide edit in both strand of the target gene and modifying the target gene. In some embodiments, the ngRNA comprises a spacer sequence that is complementary to, and may hybridize with, the second search target sequence on the edit strand only after the intended nucleotide edit(s) are incorporated in the edit strand of the target gene.

In some embodiments, the target gene is contacted by the ngRNA, the PEgRNA, and the IE simultaneously. In some embodiments, the ngRNA, the PEgRNA, and the PE form a complex when they contact the target gene. In some embodiments, the target gene is contacted with the ngRNA, the PEgRNA, and the prime editor sequentially. In some embodiments, the target gene is contacted with the ngRNA and/or the PEgRNA after contacting the target gene with the PE. In some embodiments, the target gene is contacted with the ngRNA and/or the PEgRNA before contacting the target gene with the prime editor.

In some embodiments, the target gene. e.g., an ATP7B gene, is in a cell. Accordingly, also provided herein are methods of modifying a cell.

In some embodiments, the prime editing method comprises introducing a PEgRNA, a prime editor, and/or a ngRNA into the cell that has the target gene. In some embodiments, the prime editing method comprises introducing into the cell that has the target gene with a prime editing composition comprising a PEgRNA, a prime editor polypeptide, and/or a ngRNA. In some embodiments, the PEgRNA, the prime editor polypeptide, and/or the ngRNA form a complex prior to the introduction into the cell. In some embodiments, the PEgRNA, the prime editor polypeptide, and/or the ngRNA form a complex after the introduction into the cell. The prime editors, PEgRNA and/or ngRNAs, and prime editing complexes may be introduced into the cell by any delivery approaches described herein or any delivery approach known in the art, including ribonucleoprotein (RNPs), lipid nanoparticles (LNPs), viral vectors, non-viral vectors, mRNA delivery, and physical techniques such as cell membrane disruption by a microfluidics device. The prime editors, PEgRNA and/or ngRNAs. and prime editing complexes may be introduced into the cell simultaneously or sequentially.

In some embodiments, the prime editing method comprises introducing into the cell a PEgRNA or a polynucleotide encoding the PEgRNA, a prime editor polynucleotide encoding a prime editor polypeptide, and optionally an ngRNA or a polynucleotide encoding the ngRNA. In some embodiments, the method comprises introducing the PEgRNA or the polynucleotide encoding the PEgRNA, the polynucleotide encoding the prime editor polypeptide, and/or the ngRNA or the polynucleotide encoding the ngRNA into the cell simultaneously. In some embodiments, the method comprises introducing the PEgRNA or the polynucleotide encoding the PEgRNA, the polynucleotide encoding the prime editor polypeptide, and/or the ngRNA or the polynucleotide encoding the ngRNA into the cell sequentially. In some embodiments, the method comprises introducing the polynucleotide encoding the prime editor polypeptide into the cell before introduction of the PEgRNA or the polynucleotide encoding the PEgRNA and/or the ngRNA or the polynucleotide encoding the ngRNA. In some embodiments, the polynucleotide encoding the prime editor polypeptide is introduced into and expressed in the cell before introduction of the PEgRNA or the polynucleotide encoding the PEgRNA and/or the ngRNA or the polynucleotide encoding the ngRNA into the cell. In some embodiments, the polynucleotide encoding the prime editor polypeptide is introduced into the cell after the PEgRNA or the polynucleotide encoding the PEgRNA and/or the ngRNA or the polynucleotide encoding the ngRNA are introduced into the cell. The polynucleotide encoding the prime editor polypeptide, the PEgRNA or the polynucleotide encoding the PEgRNA, and/or the ngRNA or the polynucleotide encoding the ngRNA, may be introduced into the cell by any delivery approaches described herein or any delivery approach known in the art, for example, by RNPs, LNPs, viral vectors, non-viral vectors, mRNA delivery, and physical.

In some embodiments, the polynucleotide encoding the prime editor polypeptide, the poly nucleotide encoding the PEgRNA, and/or the polynucleotide encoding the ngRNA integrate into the genome of the cell after being introduced into the cell. In some embodiments, the polynucleotide encoding the prime editor polypeptide, the polynucleotide encoding the PEgRNA, and/or the polynucleotide encoding the ngRNA are introduced into the cell for transient expression. Accordingly, also provided herein are cells modified by prime editing.

In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a non-human primate cell, bovine cell, porcine cell, rodent or mouse cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a primary cell. In some embodiments, the cell is a human primary cell. In some embodiments, the cell is a progenitor cell. In some embodiments, the cell is a human progenitor cell. In some embodiments, the cell is a hepatocyte. In some embodiments, the cell is a human hepatocyte. In some embodiments, the cell is a primary human hepatocyte derived from an induced human pluripotent stem cell (iPSC). In some embodiments, the cell is a neuron. In some embodiments, the cell is a neuron from basal ganglia. In some embodiments, the cell is a neuron from basal ganglia of a subject.

In some embodiments, the target gene edited by prime editing is in a chromosome of the cell. In some embodiments, the intended nucleotide edits incorporate in the chromosome of the cell and are inheritable by progeny cells. In some embodiments, the intended nucleotide edits introduced to the cell by the prime editing compositions and methods are such that the cell and progeny of the cell also include the intended nucleotide edits. In some embodiments, the cell is autologous, allogeneic, or xenogeneic to a subject. In some embodiments, the cell is from or derived from a subject. In some embodiments, the cell is from or derived from a human subject. In some embodiments, the cell is introduced back into the subject, e.g., a human subject, after incorporation of the intended nucleotide edits by prime editing.

In some embodiments, the method provided herein comprises introducing the prime editor polypeptide or the polynucleotide encoding the prime editor polypeptide, the PEgRNA or the polynucleotide encoding the PEgRNA, and/or the ngRNA or the polynucleotide encoding the ngRNA into a plurality or a population of cells that comprise the target gene. In some embodiments, the population of cells is of the same cell type. In some embodiments, the population of cells is of the same tissue or organ. In some embodiments, the population of cells is heterogeneous. In some embodiments, the population of cells is homogeneous. In some embodiments, the population of cells is from a single tissue or organ, and the cells are heterogeneous. In some embodiments, the introduction into the population of cells is ex vivo. In some embodiments, the introduction into the population of cells is in vivo, e.g., into a human subject.

In some embodiments, the target gene is in a genome of each cell of the population. In some embodiments, introduction of the prime editor polypeptide or the polynucleotide encoding the prime editor polypeptide, the PEgRNA or the polynucleotide encoding the PEgRNA, and/or the ngRNA or the polynucleotide encoding the ngRNA results in incorporation of one or more intended nucleotide edits in the target gene in at least one of the cells in the population of cells. In some embodiments, introduction of the prime editor polypeptide or the polynucleotide encoding the prime editor polypeptide, the PEgRNA or the polynucleotide encoding the PEgRNA, and/or the ngRNA or the polynucleotide encoding the ngRNA results in incorporation of the one or more intended nucleotide edits in the target gene in a plurality of the population of cells. In some embodiments, introduction of the prime editor polypeptide or the poly nucleotide encoding the prime editor polypeptide, the PEgRNA or the polynucleotide encoding the PEgRNA, and/or the ngRNA or the polynucleotide encoding the ngRNA results in incorporation of the one or more intended nucleotide edits in the target gene in each cell of the population of cells. In some embodiments, introduction of the prime editor polypeptide or the polynucleotide encoding the prime editor polypeptide, the PEgRNA or the polynucleotide encoding the PEgRNA, and/or the ngRNA or the polynucleotide encoding the ngRNA results in incorporation of the one or more intended nucleotide edits in the target gene in sufficient number of cells such that the disease or disorder is treated, prevented or ameliorated.

In some embodiments, editing efficiency of the prime editing compositions and method described herein can be measured by calculating the percentage of edited target genes in a population of cells introduced with the prime editing composition. In some embodiments, the editing efficiency is determined after 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 4 days, 5 days, 7 days. 10 days, or 14 days of exposing a target gene (e.g., a ATP7B gene within the genome of a cell) to a prime editing composition. In some embodiments, the population of cells introduced with the prime editing composition is ex vivo. In some embodiments, the population of cells introduced with the prime editing composition is in vitro. In some embodiments, the population of cells introduced with the prime editing composition is in vivo. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or at least about 99% relative to a suitable control. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least 25% relative to a suitable control. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least 35% relative to a suitable control, prime editing method disclosed herein has an editing efficiency of at least 30% relative to a suitable control. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least 45% relative to a suitable control. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least 50% relative to a suitable control.

In some embodiments, the methods disclosed herein have an editing efficiency of at least about 1%, at least about 5%, at least about 7.5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% of editing a primary cell relative to a suitable control.

In some embodiments, the methods disclosed herein have an editing efficiency of at least about 5%, at least about 7.5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% of editing a hepatocyte relative to a corresponding control hepatocyte. In some embodiments, the hepatocyte is a human hepatocyte.

In some embodiments, the prime editing compositions provided herein are capable of incorporated one or more intended nucleotide edits without generating a significant proportion of indels. The term “indel(s)”, as used herein, refers to the insertion or deletion of a nucleotide base within a polynucleotide, for example, a target gene. Such insertions or deletions can lead to frame shift mutations within a coding region of a gene. Indel frequency of editing can be calculated by methods known in the art. In some embodiments, indel frequency can be calculated based on sequence alignment such as the CRISPResso 2 algorithm as described in Clement et al., Nat. Biotechnol. 37(3): 224-226 (2019), which is incorporated herein in its entirety. In some embodiments, the methods disclosed herein can have an indel frequency of less than 20%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1.5%, or less than 1%. In some embodiments, any number of indels is determined after at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 3 days, at least 4 days, at least 5 days, at least 7 days, at least 10 days, or at least 14 days of exposing a target gene (e.g., a ATP7B gene within the genome of a cell) to a prime editing composition.

In some embodiments, the prime editing compositions provided herein are capable of incorporated one or more intended nucleotide edits efficiently without generating a significant proportion of indels. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 1% and an indel frequency of less than 1% in a target cell, e.g., a human primary cell or hepatocyte. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 1% and an indel frequency of less than 0.5% in a target cell, e.g., a human primary cell or hepatocyte. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 1% and an indel frequency of less than 0.1% in a target cell, e.g., a human primary cell or hepatocyte. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 5% and an indel frequency of less than 1% in a target cell, e.g., a human primary cell or hepatocyte. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 5% and an indel frequency of less than 0.5% in a target cell, e.g., a human primary cell or hepatocyte. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 5% and an indel frequency of less than 0.1% in a target cell, e.g., a human primary cell or hepatocyte.

In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 7.5% and an indel frequency of less than 1% in a target cell, e.g., a human primary cell or hepatocyte. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 7.5% and an indel frequency of less than 0.5% in a target cell, e.g., a human primary cell or hepatocyte. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 7.5% and an indel frequency of less than 0.1% in a target cell, e.g., a human primary cell or hepatocyte.

In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 10% and an indel frequency of less than 1% in a target cell, e.g., a human primary cell or hepatocyte. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 10% and an indel frequency of less than 0.5% in a target cell, e.g., a human primary cell or hepatocyte. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 10% and an indel frequency of less than 0.1% in a target cell, e.g., a human primary cell or hepatocyte.

In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 15% and an indel frequency of less than 1% in a target cell, e.g., a human primary cell or hepatocyte. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 15% and an indel frequency of less than 0.5% in a target cell, e.g., a human primary cell or hepatocyte. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 15% and an indel frequency of less than 0.1% in a target cell, e.g., a human primary cell or hepatocyte.

In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 20% and an indel frequency of less than 1% in a target cell, e.g., a human primary cell or hepatocyte. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 20% and an indel frequency of less than 0.5% in a target cell, e.g., a human primary cell or hepatocyte. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 20% and an indel frequency of less than 0.1% in a target cell, e.g., a human primary cell or hepatocyte.

In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 30% and an indel frequency of less than 1% in a target cell, e.g., a human primary cell or hepatocyte. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 30% and an indel frequency of less than 0.5% in a target cell, e.g., a human primary cell or hepatocyte. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 30% and an indel frequency of less than 0.1% in a target cell, e.g., a human primary cell or hepatocyte.

In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 40% and an indel frequency of less than 1% in a target cell, e.g., a human primary cell or hepatocyte. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 40% and an indel frequency of less than 0.5% in a target cell, e.g., a human primary cell or hepatocyte. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 40% and an indel frequency of less than 0.1% in a target cell, e.g., a human primary cell or hepatocyte.

In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 50% and an indel frequency of less than 1% in a target cell, e.g., a human primary cell or hepatocyte. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 50% and an indel frequency of less than 0.5% in a target cell, e.g., a human primary cell or hepatocyte. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 50% and an indel frequency of less than 0.1% in a target cell, e.g., a human primary cell or hepatocyte.

In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 60% and an indel frequency of less than 1% in a target cell, e.g., a human primary cell or hepatocyte. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 60% and an indel frequency of less than 0.5% in a target cell, e.g., a human primary cell or hepatocyte. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 60% and an indel frequency of less than 0.1% in a target cell, e.g., a human primary cell or hepatocyte.

In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 70% and an indel frequency of less than 1% in a target cell, e.g., a human primary cell or hepatocyte. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 70% and an indel frequency of less than 0.5% in a target cell, e.g., a human primary cell or hepatocyte. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 70% and an indel frequency of less than 0.1% in a target cell, e.g., a human primary cell or hepatocyte.

In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 80% and an indel frequency of less than 1% in a target cell, e.g., a human primary cell or hepatocyte. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 80% and an indel frequency of less than 0.5% in a target cell, e.g., a human primary cell or hepatocyte. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 80% and an indel frequency of less than 0.1% in a target cell, e.g., a human primary cell or hepatocyte.

In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 1% in a target cell, e.g., a human primary cell or hepatocyte. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 0.5% in a target cell, e.g., a human primary cell or hepatocyte. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 90% and an indel frequency of less than 0.1% in a target cell, e.g., a human primary cell or hepatocyte.

In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 95% and an indel frequency of less than 1% in a target cell, e.g., a human primary cell or hepatocyte. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 95% and an indel frequency of less than 0.5% in a target cell, e.g., a human primary cell or hepatocyte. In some embodiments, the prime editing methods disclosed herein have an editing efficiency of at least about 95% and an indel frequency of less than 0.1% in a target cell, e.g., a human primary cell or hepatocyte. In some embodiments, any number of indels is determined after at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 3 days, at least 4 days, at least 5 days, at least 7 days, at least 10 days, or at least 14 days of exposing a target gene (e.g., a ATP7B gene within the genome of a cell) to a prime editing composition. In some embodiments, the editing efficiency is determined after 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 4 days, 5 days, 7 days, 10 days, or 14 days of exposing a target gene (e.g., a ATP7B gene within the genome of a cell) to a prime editing composition.

In some embodiments, the prime editing composition described herein result in less than 50%, less than 40%, less than 30%, less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.09%, less than 0.08%, less than 0.07%, less than 0.06%, less than 0.05%, less than 0.04%, less than 0.03%, less than 0.02%, or less than 0.01% off-target editing in a chromosome that includes the target gene. In some embodiments, off-target editing is determined after at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 3 days, at least 4 days, at least 5 days, at least 7 days, at least 10 days, or at least 14 days of exposing a target gene (e.g., a nucleic acid within the genome of a cell) to a prime editing composition.

In some embodiments, the prime editing compositions (e.g., PEgRNAs and prime editors as described herein) and prime editing; methods disclosed herein can be used to edit a target ATP7B gene. In some embodiments, the target ATP7B gene comprises a mutation compared to a wild type ATP7B gene. In some embodiments, the mutation is associated with Wilson's disease. In some embodiments, the target ATP7B gene comprises an editing target sequence that contains the mutation associated with Wilson's disease. In some embodiments, the mutation is in a coding region of the target ATP7B gene. In some embodiments, the mutation is in an exon of the target ATP7B gene. In some embodiments, the mutation is in exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, or exon 21 of the ATP7B gene as compared to a wild type ATP7B gene. In some embodiments, the mutation is exon 8, exon 13, exon 14, exon 15, or exon 17 of the ATP71B gene as compared to a wild type ATP7B gene. In some embodiments, the mutation is in exon 3 of the ATP7B gene as compared to a wild type ATP7B gene. In some embodiments, the mutation is located in exon 8 of the ATP7B gene as compared to a wild type ATP7B gene. In some embodiments, the mutation is not a c.1288dup duplication. In some embodiments, the mutation is in exon 14 of the target ATP7B gene. In some embodiments, the mutation is located between positions 51944045 and 51944245 of human chromosome 13 as set forth in human genome assembly consortium Human build 38 (GRCh38), GenBank accession GCA_000001405.15. In some embodiments, the mutation encodes an amino acid substitution H1069Q relative to a wild type ATP7B polypeptide set forth in SEQ ID NO: 5861. In some embodiments, the editing target sequence comprises a C>A mutation at position 51944145 in human chromosome 13 as set forth in human genome assembly consortium Human build 38 (GRCh38), GenBank accession GCA_000001405.15. In some embodiments, the prime editing method comprises contacting a target ATP7B gene with a prime editing composition comprising a prime editor, a PEgRNA, and/or a ngRNA. In some embodiments, contacting the target ATP7B gene with the prime editing composition results in incorporation of one or more intended nucleotide edits in the target ATP7B gene. In some embodiments, the incorporation is in a region of the target ATP7B gene that corresponds to an editing target sequence inn the ATP7B gene. In some embodiments, the one or more intended nucleotide edits comprises a single nucleotide substitution, an insertion, a deletion, or any combination thereof, compared to the endogenous sequence of the target ATP7B gene. In some embodiments, incorporation of the one or more intended nucleotide edits results in replacement of one or more mutations with the corresponding sequence that encodes a wild type ATP7B polypeptide set forth in SEQ ID NO: 5861. In some embodiments, incorporation of the one or more intended nucleotide edits results in replacement of the one or more mutations with the corresponding sequence in a wild type ATP7B gene. In some embodiments, incorporation of the one more intended nucleotide edits results in correction of a mutation in the target ATP7B gene. In some embodiments, the target A P7B1 gene comprises an editing template sequence that contains the mutation. In some embodiments, contacting the target ATP7B gene with the prime editing composition results in incorporation of one or more intended nucleotide edits in the target ATP7B gene, which corrects the mutation in the editing target sequence (or a double stranded region comprising the editing target sequence and the complementary sequence to the editing target sequence on a target strand) in the target ATP73 gene.

In some embodiments, incorporation of the one more intended nucleotide edits results in correction of a mutation in exon 14 of the target ATP7B gene as compared to a wild type ATP7B gene. In some embodiments, incorporation of the one more intended nucleotide edits results in correction of a mutation located between positions 51944045 and 51944245 of human chromosome 13 in the target ATP7B gene as set forth in human genome assembly consortium Human build 38 (GRCh38), GenBank accession GCA_000001405.15. In some embodiments, incorporation of the one more intended nucleotide edits results in an A>C nucleotide substitution at position 51944145 in human chromosome 13 in the target ATP7B gene as compared to the endogenous sequence of the target ATP7B gene, thereby correcting a C>A mutation at position 51944145 in human chromosome 13 in the target ATP7B gene as set forth in human genome assembly consortium Human build 38 (GRCh38), GenBank accession GCA_000001405.15. In some embodiments, incorporation of the one more intended nucleotide edits results in correction of an ATP7B gene sequence that encodes a H1069Q amino acid substitution, and restores wild type expression and function of the ATP7B protein.

In some embodiments, the target ATP7B gene is in a target cell. Accordingly, in one aspect provided herein is a method of editing a target cell comprising a target ATP7B gene that encodes a polypeptide that comprises one or more mutations relative to a wild type ATP7B gene. In some embodiments, the methods of the present disclosure comprise introducing a prime editing composition comprising a PEgRNA, a prime editor polypeptide, and/or a ngRNA into the target cell that has the target ATP7B gene to edit the target ATP7B gene, thereby generating an edited cell. In some embodiments, the target cell is a mammalian cell. In some embodiments, the target cell is a human cell. In some embodiments, the target cell is a primary cell. In some embodiments, the target cell is a human primary cell. In some embodiments, the target cell is a progenitor cell. In some embodiments, the target cell is a human progenitor cell. In some embodiments, the target cell is a stein cell. In some embodiments, the target cell is a human stem cell. In some embodiments, the target cell is a hepatocyte. In some embodiments, the target cell is a human hepatocyte. In some embodiments, the target cell is a primary human hepatocyte derived from an induced human pluripotent stem cell (iPSC). In some embodiments, the cell is a neuron. In some embodiments, the cell is a neuron from basal ganglia. In some embodiments, the cell is a neuron from basal ganglia of a subject. In some embodiments, the cell is a neuron in the basal ganglia of a subject.

In some embodiments, components of a prime editing composition described herein are provided to a target cell in vitro. In some embodiments, components of a prime editing composition described herein are provided to a target cell ex vivo. In some embodiments, components of a prime editing composition described herein are provided to a target cell in vivo.

In some embodiments, incorporation of the one or more intended nucleotide edits in the target ATP7B gene that comprises one or more mutations restores wild type expression and function of the ATP7B protein encoded by the ATP7B gene. In some embodiments, the target ATP7 gene encodes a H1069Q amino acid substitution as compared to the wild type ATP7B protein prior to incorporation of the one or more intended nucleotide edits. In some embodiments, expression and/or function of the ATP7B protein may be measured when expressed in a target cell. In some embodiments, incorporation of the one or more intended nucleotide edits in the target ATP7B gene comprising one or more mutations lead to a fold change in a level of ATP7B gene expression, ATP7B protein expression, or a combination thereof. In some embodiments, a change in the level of ATP7B expression level can comprise a fold change of, e.g., 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold. 10-fold, 15-fold, 20-fold, 25-fold, 30-fold. 40-fold, 50-fold. 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or greater as compared to expression in a suitable control cell not introduced with a prime editing composition described herein. In some embodiments, incorporation of the one or more intended nucleotide edits in the target ATP7B gene that comprises one or more mutations restores wild type expression of the ATP7B protein by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%. 90% 95%, o99% or more as compared to wild type expression of the ATP7B protein in a suitable control cell that comprises a wild type ATP7B gene.

In some embodiments, an ATP7B expression increase can be measured by a functional assay. In some embodiments, the functional assay can comprise a copper sensitivity assay, a cell viability assay, or a combination thereof. In some embodiments, protein expression can be measured using a protein assay. In some embodiments, protein expression can be measured using antibody testing. In some embodiments, an antibody can comprise anti-ATP7B. In some embodiments, protein expression can be measured using ELISA, mass spectrometry, Western blot, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), high performance liquid chromatography (HPLC), electrophoresis, or any combination thereof. In some embodiments, a protein assay can comprise SDS-PAGE and densitometric analysis of a Coomassie Blue-stained gel. In some embodiments, ATP7B activity can be measured by measuring ATPase activity. In some embodiments, ATPase activity can be measured using an ATPase assay.

Methods of Treating Wilson's Disease

In some embodiments, provided herein are methods for treatment of a subject diagnosed with a disease associated with or caused by one or more pathogenic mutations that can be corrected by prime editing. In some embodiments, provided herein are methods for treating Wilson's disease that comprise administering to a subject a therapeutically effective amount of a prime editing composition, or a pharmaceutical composition comprising a prime editing composition as described herein. In some embodiments, administration of the prime editing composition results in incorporation of one or more intended nucleotide edits in the target gene in the subject. In some embodiments, administration of the prime editing composition results in correction of one or more pathogenic mutations, e.g. point mutations, insertions, or deletions, associated with Wilson's disease in the subject. In some embodiments, the target gene comprise an editing target sequence that contains the pathogenic mutation. In some embodiments, administration of the prime editing composition results in incorporation of one or more intended nucleotide edits in the target gene that corrects the pathogenic mutation in the editing target sequence (or a double stranded region comprising the editing target sequence and the complementary sequence to the editing target sequence on a target strand) of the target gene in the subject.

In some embodiments, the method provided herein comprises administering to a subject an effective amount of a prime editing composition, for example, a PEgRNA, a prime editor, and/or a ngRNA. In some embodiments, the method comprises administering to the subject an effective amount of a prime editing composition described herein, for example, polynucleotides, vectors, or constructs that encode prime editing composition components, or RNPs, LNPs, and/or polypeptides comprising prime editing composition components. Prime editing compositions can be administered to target the ATP7B gene in a subject, e.g., a human subject, suffering from, having, susceptible to, or at risk for Wilsons' disease. Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method). In some embodiments, the subject has Wilson's disease.

In some embodiments, the subject has been diagnosed with Wilson's disease by sequencing of a ATP7B gene in the subject. In some embodiments, the subject comprises at least a copy of ATP7B gene that comprises one or more mutations compared to a wild type ATP7B gene. In some embodiments, the subject comprises at least a copy of ATP7B gene that comprises a mutation in a coding region of the ATP7B gene. In some embodiments, the subject comprises at least a copy of ATP7B gene that comprises a mutation in exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, or exon 21, as compared to a wild type ATP7B gene. In some embodiments, the subject comprises at least a copy of ATP7B gene that comprises a mutation in exon 8, exon 13, exon 14, exon 15, or exon 17 as compared to a wild type ATP7B gene. In some embodiments, the subject comprises at least a copy of ATP7B gene that comprises a mutation in exon 14 of the ATP7B gene as compared to a wild type ATP7B gene. In some embodiments, the subject comprises at least a copy of ATP7B gene that comprises a mutation in exon 3 as compared to a wild type ATP7B gene. In some embodiments, the mutation is not a c.1288dup duplication. In some embodiments, the subject comprises at least a copy of ATP7B gene that encodes a polypeptide that comprises an amino acid substitution H1069Q relative to a wild type ATP7B polypeptide set forth in SEQ ID NO: 5861.

In some embodiments, the method comprises directly administering prime editing compositions provided herein to a subject. The prime editing compositions described herein can be delivered with in any form as described herein, e.g., as LNPs, RNPs, polynucleotide vectors such as viral vectors, or mRNAs. The prime editing compositions can be formulated with any pharmaceutically acceptable carrier described herein or known in the art for administering directly to a subject. Components of a prime editing composition or a pharmaceutical composition thereof may be administered to the subject simultaneously or sequentially. For example, in some embodiments, the method comprises administering a prime editing composition, or pharmaceutical composition thereof, comprising a complex that comprises a prime editor fusion protein and a PEgRNA and/or a ngRNA, to a subject. In some embodiments, the method comprises administering a polynucleotide or vector encoding a prime editor to a subject simultaneously with a PEgRNA and/or a ngRNA. In some embodiments, the method comprises administering a polynucleotide or vector encoding a prime editor to a subject before administration with a PEgRNA and/or a ngRNA.

Suitable routes of administrating the prime editing compositions to a subject include, without limitation: topical, subcutaneous, transdermal, intradermal, intralesional, intraarticular, intraperitoneal, intravesical, transmucosal, gingival, intradental, intracochlear, transtympanic, intraorgan, epidural, intrathecal, intramuscular, intravenous, intravascular, intraosseus, periocular, intratumoral, intracerebral, and intracerebroventricular administration. In some embodiments, the compositions described are administered intraperitoneally, intravenously, or by direct injection or direct infusion. In some embodiments, the compositions described are administered by direct injection or infusion into the liver of a subject. In some embodiments, the compositions described herein are administered to a subject by injection, by means of a catheter, by means of a suppository, or by means of an implant.

In some embodiments, the method comprises administering cells edited with a prime editing composition described herein to a subject. In some embodiments, the cells are allogeneic. In some embodiments, allogeneic cells are or have been contacted ex vivo with a prime editing composition or pharmaceutical composition thereof and are introduced into a human subject in need thereof. In some embodiments, the cells are autologous to the subject. In some embodiments, cells are removed from a subject and contacted ex vivo with a prime editing composition or pharmaceutical composition thereof and are re-introduced into the subject.

In some embodiments, cells are contacted ex vivo with one or more components of a prime editing composition. In some embodiments, the ex vivo-contacted cells are introduced into the subject, and the subject is administered in vivo with one or more components of a prime editing composition. For example, in some embodiments, cells are contacted ex vivo with a prime editor and introduced into a subject. In some embodiments, the subject is then administered with a PEgRNA and/or a ngRNA, or a polynucleotide encoding the PEgRNA and/or the ngRNA.

In some embodiments, cells contacted with the prime editing composition are determined for incorporation of the one or more intended nucleotide edits in the genome before re-introduction into the subject. In some embodiments, the cells are enriched for incorporation of the one or more intended nucleotide edits in the genome before re-introduction into the subject. In some embodiments, the edited cells are primary cells. In some embodiments, the edited cells are progenitor cells. In some embodiments, the edited cells are stem cells. In some embodiments, the edited cells are hepatocytes. In some embodiments, the edited cells are primary human cells. In some embodiments, the edited cells are human progenitor cells. In some embodiments, the edited cells are human stem cells. In some embodiments, the edited cells are human hepatocytes. In some embodiments, the cell is a neuron. In some embodiments, the cell is a neuron from basal ganglia. In some embodiments, the cell is a neuron from basal ganglia of a subject. In some embodiments, the cell is a neuron in the basal ganglia of a subject. The prime editing composition or components thereof may be introduced into a cell by any delivery approaches as described herein, including LNP administration, RNP administration, electroporation, nucleofection, transfection, viral transduction, microinjection, cell membrane disruption and diffusion, or any other approach known in the art.

The cells edited with prime editing can be introduced into the subject by any route known in the art. In some embodiments, the edited cells are administered to a subject by direct infusion. In some embodiments, the edited cells are administered to a subject by intravenous infusion. In some embodiments, the edited cells are administered to a subject as implants.

The pharmaceutical compositions, prime editing compositions, and cells, as described herein, can be administered in effective amounts. In some embodiments, the effective amount depends upon the mode of administration. In some embodiments, the effective amount depends upon the stage of the condition, the age and physical condition of the subject, the nature of concurrent therapy, if any, and like factors well-known to the medical practitioner.

The specific dose administered can be a uniform dose for each subject. Alternatively, a subject's dose can be tailored to the approximate body weight of the subject. Other factors in determining the appropriate dosage can include the disease or condition to be treated or prevented, the severity of the disease, the route of administration, and the age, sex and medical condition of the patient.

In embodiments wherein components of a prime editing composition are administered sequentially, the time between sequential administration can be at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 3 days, at least 4 days, at least 5 days, at least 7 days, at least 10 days, or at least 14 days.

In some embodiments, a method of monitoring treatment progress is provided. In some embodiments, the method includes the step of determining a level of diagnostic marker, for example, correction of a mutation in ATP7B gene, or diagnostic measurement associated with Wilson's disease, (e.g., copper sensitivity screen or assay) in a subject suffering from Wilson's disease symptoms and has been administered an effective amount of a prime editing composition described herein. The level of the diagnostic marker determined in the method can be compared to known levels of the marker in either healthy normal controls or in other afflicted subjects to establish the subject's disease status.

Delivery

Prime editing compositions described herein can be delivered to a cellular environment with any approach known in the art. Components of a prime editing composition can be delivered to a cell by the same mode or different modes. For example, in some embodiments, a prime editor can be delivered as a polypeptide or a polynucleotide (DNA or RNA) encoding the polypeptide. In some embodiments, a PEgRNA can be delivered directly as an RNA or as a DNA encoding the PEgRNA.

In some embodiments, a prime editing composition component is encoded by a polynucleotide, a vector, or a construct. In some embodiments, a prime editor polypeptide, a PEgRNA and/or a ngRNA is encoded by a polynucleotide. In some embodiments, the polynucleotide encodes a prime editor fusion protein comprising a DNA binding domain and a DNA polymerase domain. In some embodiments, the polynucleotide encodes a DNA polymerase domain of a prime editor. In some embodiments, the polynucleotide encodes a DNA polymerase domain of a prime editor. In some embodiments, the polynucleotide encodes a portion of a prime editor protein, for example, a N-terminal portion of a prime editor fusion protein connected to an intein-N. In some embodiments, the polynucleotide encodes a portion of a prime editor protein, for example, a C-terminal portion of a prime editor fusion protein connected to an intein-C. In some embodiments, the polynucleotide encodes a PEgRNA and/or a ngRNA. In some embodiments, the polypeptide encodes two or more components of a prime editing composition, for example, a prime editor fusion protein and a PEgRNA.

In some embodiments, the polynucleotide encoding one or more prime editing composition components is delivered to a target cell is integrated into the genome of the cell for long-term expression, for example, by a retroviral vector. In some embodiments, the polynucleotide delivered to a target cell is expressed transiently. For example, the polynucleotide may be delivered in the form of a mRNA, or a non-integrating vector (non-integrating virus, plasmids, minicircle DNAs) for episomal expression.

In some embodiments, a polynucleotide encoding one or more prime editing system components can be operably linked to a regulatory element, e.g., a transcriptional control element, such as a promoter. In some embodiments, the polynucleotide is operably linked to multiple control elements. Depending on the expression system utilized, any of a number of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector (e.g., U6 promoter, H1 promoter).

In some embodiments, the polynucleotide encoding one or more prime editing composition components is a part of, or is encoded by, a vector. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a non-viral vector.

Non-viral vector delivery systems can include DNA plasmids. RNA (e.g., a transcript of a vector described herein), naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome. In some embodiments, the polynucleotide is provided as an RNA, e.g., a mRNA or a transcript. Any RNA of the prime editing systems, for example a guide RNA or a base editor-encoding mRNA, can be delivered in the form of RNA. In some embodiments, one or more components of the prime editing system that are RNAs is produced by direct chemical synthesis or may be transcribed in vitro from a DNA. In some embodiments, a mRNA that encodes a prime editor polypeptide is generated using ii vitro transcription. Guide polynucleotides (e.g., PEgRNA or ngRNA) can also be transcribed using in vitro transcription from a cassette containing a T7 promoter, followed by the sequence “GG”, and guide polynucleotide sequence. In some embodiments, the prime editor encoding mRNA. PEgRNA, and/or ngRNA are synthesized in vitro using an RNA polymerase enzyme (e.g., T7 polymerase, T3 polymerase, SP6 polymerase, etc.). Once synthesized, the RNA can directly contact a target ATP7B gene or can be introduced into a cell using any suitable technique for introducing nucleic acids into cells (e.g., microinjection, electroporation, transfection). In some embodiments, the prime editor-coding sequences, the PEgRNAs, and/or the ngRNAs are modified to include one or more modified nucleoside e.g. using pseudo-U or 5-Methyl-C.

Methods of non-viral delivery of nucleic acids can include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, cell membrane disruption by a microfluidics device, and agent-enhanced uptake of DNA. Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides can be used. Delivery can be to cells (e.g., in vitro or ex vivo administration) or target tissues (e.g., in vivo administration). The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, can be used.

Viral vector delivery systems can include DNA and RNA viruses, which can have either episomal or integrated genomes after delivery to the cell. RNA or DNA viral based systems can be used to target specific cells and trafficking the viral payload to an organelle of the cell. Viral vectors can be administered directly (in vivo) or they can be used to treat cells in vitro, and the modified cells can optionally be administered (ex vivo).

In some embodiments, the viral vector is a retroviral, lentiviral, adenoviral, adeno-associated viral or herpes simplex viral vector. Retroviral vectors can include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus (SIV), human immuno deficiency virus (HIV), and combinations thereof. In some embodiments, the retroviral vector is a lentiviral vector. In some embodiments, the retroviral vector is a gamma retroviral vector. In some embodiments, the viral vector is an adenoviral vector. In some embodiments, the viral vector is an adeno-associated virus (“AAV”) vector.

In some embodiments, polynucleotides encoding one or more prime editing composition components are packaged in a virus particle. Packaging cells can be used to form virus particles that can infect a target cell. Such cells can include 293 cells, (e.g., for packaging adenovirus), and .psi.2 cells or PA317 cells (e.g., for packaging retrovirus). Viral vectors can be generated by producing a cell line that packages a nucleic acid vector into a viral particle. The vectors can contain the minimal viral sequences required for packaging and subsequent integration into a host. The vectors can contain other viral sequences being replaced by an expression cassette for the polynucleotide(s) to be expressed. The missing viral functions can be supplied in trans by the packaging cell line. For example, AAV vectors can comprise ITR sequences from the AAV genome which are required for packaging and integration into the host genome.

In some embodiments, dual AAV vectors are generated by splitting a large transgene expression cassette in two separate halves (5′ and 3′ ends that encode N-terminal portion and C-terminal portion of, e.g., a prime editor polypeptide), where each half of the cassette is no more than 5 kb in length, optionally no more than 4.7 kb in length, and is packaged in a single AAV vector. In some embodiments, the full-length transgene expression cassette is reassembled upon co-infection of the same cell by both dual AAV vectors. In some embodiments, a portion or fragment of a prime editor polypeptide, e.g. a Cas9 nickase, is fused to an intein. The portion or fragment of the polypeptide can be fused to the N-terminus or the C-terminus of the intein. In some embodiments, a N-terminal portion of the polypeptide is fused to an intein-N, and a C-terminal portion of the polypeptide is separately fused to an intein-C. In some embodiments, a portion or fragment of a prime editor fusion protein is fused to an intein and fused to an AAV capsid protein. The intein, nuclease and capsid protein can be fused together in any arrangement (e.g., nuclease-intein-capsid, intein-nuclease-capsid, capsid-intein-nuclease, etc.). In some embodiments, a polynucleotide encoding a prime editor fusion protein is split in two separate halves, each encoding a portion of the prime editor fusion protein and separately fused to an intein. In some embodiments, each of the two halves of the polynucleotide is packaged in an individual AAV vector of a dual AAV vector system. In some embodiments, each of the two halves of the polynucleotide is no more than 5 kb in length, optionally no more than 4.7 kb in length. In some embodiments, the full-length prime editor fusion protein is reassembled upon co-infection of the same cell by both dual AAV vectors, expression of both halves of the prime editor fusion protein, and self-excision of the inteins.

A target cell can be transiently or non-transiently transfected with one or more vectors described herein. A cell can be transfected as it naturally occurs in a subject. A cell can be taken or derived from a subject and transfected. A cell can be derived from cells taken from a subject, such as a cell line. In some embodiments, a cell transfected with one or more vectors described herein can be used to establish a new cell line comprising one or more vector-derived sequences. In some embodiments, a cell transiently transfected with the compositions of the disclosure (such as by transient transfection of one or more vectors, or transfection with RNA), and modified through the activity of a prime editor, can be used to establish a new cell line comprising cells containing the modification but lacking any other exogenous sequence. Any suitable vector compatible with the host cell can be used with the methods of the disclosure. Non-limiting examples of vectors include pXT1, pSG5, pSVK3, pBPV, pMSG, and pSVLSV40.

In some embodiments, a prime editor protein can be provided to cells as a polypeptide. In some embodiments, the prime editor protein is fused to a polypeptide domain that increases solubility of the protein. In some embodiments, the prime editor protein is formulated to improve solubility of the protein.

In some embodiment, a prime editor polypeptide is fused to a polypeptide permeant domain to promote uptake by the cell. In some embodiments, the permeant domain is a including peptide, a peptidomimetic, or a non-peptide carrier. For example, a permeant peptide may be derived from the third alpha helix of Drosophila melanogaster transcription factor Antennapaedia, referred to as penetratin, which comprises the amino acid sequence RQIKIWFQNRRMKWKK (SEQ ID NO: 5897). As another example, the permeant peptide can comprise the HIV-1 tat basic region amino acid sequence, which may include, for example, amino acids 49-57 of naturally-occurring tat protein. Other permeant domains can include poly-arginine motifs, for example, the region of amino acids 34-56 of HIV-1 rev protein, nona-arginine (SEQ ID NO: 5898), and octa-arginine (SEQ ID NO: 5899). The nona-arginine (R9) sequence (SEQ ID NO: 5898) can be used. The site at which the fusion can be made may be selected in order to optimize the biological activity, secretion or binding characteristics of the polypeptide.

In some embodiments, a prime editor polypeptide is produced in vitro or by host cells, and it may be further processed by unfolding, e.g., heat denaturation, DTT reduction, etc. and may be further refolded. In some embodiments, a prime editor polypeptide is prepared by in vitro synthesis. Various commercial synthetic apparatuses can be used. By using synthesizers, naturally occurring amino acids can be substituted with unnatural amino acids. In some embodiments, a prime editor polypeptide is isolated and purified in accordance with recombinant synthesis methods, for example, by expression in a host cell and the lysate purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique.

In some embodiments, a prime editing composition, for example, prime editor polypeptide components and PEgRNA/ngRNA are introduced to a target cell by nanoparticles. In some embodiments, the prime editor polypeptide components and the PEgRNA and/or ngRNA form a complex in the nanoparticle. Any suitable nanoparticle design can be used to deliver genome editing system components or nucleic acids encoding such components. In some embodiments, the nanoparticle is inorganic. In some embodiments, the nanoparticle is organic. In some embodiments, a prime editing composition is delivered to a target cell, e.g., a hepatocyte, in an organic nanoparticle, e.g. a lipid nanoparticle (LNP) or polymer nanoparticle.

In some embodiments, LNPs are formulated from cationic, anionic, neutral lipids, or combinations thereof. In some embodiments, neutral lipids, such as the fusogenic phospholipid DOPE or the membrane component cholesterol, are included to enhance transfection activity and nanoparticle stability. In some embodiments, LNPs are formulated with hydrophobic lipids, hydrophilic lipids, or combinations thereof. Lipids may be formulated in a wide range of molar ratios to produce an LNP. Any lipid or combination of lipids that are known in the art can be used to produce an LNP. Exemplary lipids used to produce LNPs are provided in Table 3 below.

In some embodiments, components of a prime editing composition form a complex prior to delivery to a target cell. For example, a prime editor fusion protein, a PEgRNA, and/or a ngRNA can form a complex prior to delivery to the target cell. In some embodiments, a prime editing polypeptide (e.g. a prime editor fusion protein) and a guide polynucleotide (e.g. a PEgRNA or ngRNA) form a ribonucleoprotein (RNP) for delivery to a target cell. In some embodiments, the RNP comprises a prime editor fusion protein in complex with a PEgRNA. RNPs may be delivered to cells using known methods, such as electroporation, nucleofection, or cationic lipid-mediated methods, or any other approaches known in the art. In some embodiments, delivery of a prime editing composition or complex to the target cell does not require the delivery of foreign DNA into the cell. In some embodiments, the RNP comprising the prime editing complex is degraded over time in the target cell. Exemplary lipids for use in nanoparticle formulations and/or gene transfer are shown in Table 3 below.

TABLE 3

Exemplary lipids for nanoparticle formulation or gene transfer

Lipid
Abbreviation
Feature

1,2-Dioleoyl-sn-glycero-3-phosphatidylcholine
DOPC
Helper

1,2-Dioleoyl-sn-glycero-3-phosphatidylethanolamine
DOPE
Helper

Cholesterol

Helper

N41-(2,3-Dioleyloxy)prophyliN,N,N-trimethylammonium
DOTMA
Cationic

chloride

1,2-Dioleoyloxy-3-trimethylammonium-propane
DOGS
Cationic

Dioctadecylamidoglycylspermine

N-(3-Aminopropy1)-N,N-dimethy 1-2,3-bis(dodecyloxy)-1-
GAP-DLRIE
Cationic

propanaminium bromide

Cetyltrimethylammonium bromide
CTAB
Cationic

6-Lauroxyhexyl omithinate
LHON
Cationic

1-(2,3-Dioleoyloxypropy1)-2,4,6-trimethylpyridinium
2Oc
Cationic

2,3-Dioleyloxy-N-P(spenninecarboxamido-ethy1J-N,Ndimethyl-
DOSPA
Cationic

1-propanatninium trifluoroacetate

1,2-Dioley 1-3-trimethylamtnonium-propane
DOPA
Cationic

N-(2-Hydroxyethyl)-N,N-dimethy 1-2,3-bis(tetradecyloxy)-1-
MDRIE
Cationic

propanaminium bromide

Dimyristooxypropyl dimethyl hydroxyethyl ammonium bromide
DMRI
Cationic

3β-[N-(N′,N′-Dimethylaminoethane)-carbamoyl]cholesterol
DC-Chol
Cationic

Bis-guanidium-tren-cholesterol
BGTC
Cationic

1,3-Diodeoxy-2-(6-carboxy-spermy1)-propylamide
DOSPER
Cationic

Dimethyloctadecylammonium bromide
DDAB
Cationic

Dioctadecylamidoglicylspermidin
DSL
Cationic

rac-[(2,3-Dioctadecyloxypropyl)(2-hydroxyethyl)]-
CLIP-1
Cationic

dimethylammonium chloride

rac-[2(2,3-Dihexadecyloxypropyloxymethyloxy)
CLIP-6
Cationic

ethyl|trimethylammoniun bromide

Ethyldimyristoylphosphatidylcholine
EDMPC
Cationic

1,2-Distearyloxy-N,N-dimethyl-3-aminopropane
DSDMA
Cationic

1,2-Dimyristoyl-trimethylammonium propane
DMTAP
Cationic

O,O′-Dimyristyl-N-lysyl aspartate
DMKE
Cationic

1,2-Distearoyl-sn-glycero-3-ethylpho sphocholine
DSEPC
Cationic

N-Palmitoyl D-erythro-sphingosyl carbamoyl-spenmine
CCS
Cationic

N-t-Butyl-N0-tetradecy1-3-tetradecylaminopropionamidine
diC14-amidine
Cationic

Octadecenolyoxy[ethyl-2-heptadeceny1-3 hydroxyethyl]
DOTIM
Cationic

imidazolinium chloride

N1-Cholesteryloxycarbonyl-3,7-diazanonane-1,9-diamine
CDAN
Cationic

2-(3-Bis(3-amino-propy1)-amino]propylamino)-
RPR209120
Cationic

Nditetradecylcarbamoylme-ethyl-acetamide

1,2-dilinoleyloxy-3-dimethylaminopropane
DLinDMA
Cationic

2,2-dilinoley1-4-dimethylaminoethyl-[1,3]-dioxolane
DLin-KC2-
Cationic

DMA

dilinoleyl-methyl-4-dimethylaminobutyrate
DLin-MC3-
Cationic

DMA

Exemplary polymers for use in nanoparticle formulations and/or gene transfer are shown in Table 4 below.

TABLE 4

Exemplary lipids for nanoparticle formulation or gene transfer

Polymer
Abbreviation

Poly(ethylene)glycol
PEG

Polyethylenimine
PEI

Dithiobis (succinimidylpropionate)
DSP

Dimethyl-3,3′-dithiobispropionimidate
DTBP

Poly(ethyleneimine)biscarbamate
PEIC

Poly(L-lysine)
PLL

Histidine modified PLL

Poly(N-vinylpyrrolidone)
PVP

Poly(propylenimine)
PPI

Poly(amidoamine)
PAMAM

Poly(amidoethylenimine)
SS_PAEI

Triethylenetetramine
TETA

Poly(β-aminoester)

Poly(4-hydroxy-L-proline ester)
PHP

Poly(allylamine)

Poly(α-[4-aminobutyl]-L-glycolic acid)
PAGA

Poly(D,L-lactic-co-glycolic acid)
PLGA

Poly(N-ethyl-4-vinylpyridinium bromide)

Poly(phosphazene)s
PPZ

Poly(phosphoester)s
PPE

Poly(phosphoramidate)s
PPA

Poly(N-2-hydroxypropylmethacrylamide)
pHPMA

Poly (2-(dimethylamino)ethyl methacrylate)
pDMAEMA

Poly(2-aminoethyl propylene phosphate)
PPE-EA

Chitosan

Galactosylated chitosan

N-dodacylated chitosam

Histone

Collagen

Dextran-spermine
D-SPM

Exemplary delivery methods for polynucleotides encoding prime editing composition components are shown in Table 5 below.

TABLE 5

Exemplary polynucleotide delivery methods

Delivery into

Type of

Non-Dividing
Duration of
Genome
Molecule

Delivery
Vector/Mode
Cells
Expression
Integration
Delivered

Physical
(e.g.,
YES
Transient
NO
Nucleic Acids

electroporation,

and Proteins

particle gun,

Calcium phosphate

transfection)

Viral
Retrovirus
NO
Stable
YES
RNA

Lentivirus
YES
Stable
YES/NO with
RNA

modification

Adenovirus
YES
Transient
NO
DNA

Adeno-Associated
YES
Stable
NO
DNA

Virus (AAV)

Vaccinia Virus
YES
Very Transient
NO
DNA

Herpes Simplex
YES
Stable
NO
DNA

Virus

Non-Viral
Cationic
YES
Transient
Depends on
Nucleic acids

what is
and Proteins

delivered

Polymeric
YES
Transient
NO
Nucleic Acids

Nanoparticles

Biological
Attenuated Bacteria
YES
Transient
NO
Nucleic Acids

Non-Viral
Engineered
YES
Transient
NO
Nucleic Acids

Delivery
Bacteriophages

Vehicles
Mammalian Virus-
YES
Transient
NO
Nucleic Acids

like Particles

Biological
YES
Transient
NO
Nucleic Acids

liposomes:

Erythrocyte Ghosts

and Exosomes

The prime editing compositions of the disclosure, whether introduced as polynucleotides or polypeptides, can be provided to the cells for about 30 minutes to about 24 hours, e.g., 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 16 hours, 18 hours, 20 hours, or any other period from about 30 minutes to about 24 hours, which can be repeated with a frequency of about every day to about every 4 days, e.g., every 1.5 days, every 2 days, every 3 days, or any other Frequency from about every day to about every four days. The compositions may be provided to the subject cells one or more times, e.g., one time, twice, three times, or more than three times, and the cells allowed to incubate with the agent(s) for some amount of time following each contacting event e.g., 16-24 hours. In cases in which two or more different prime editing system components, e.g., two different polynucleotide constructs are provided to the cell (e.g., different components of the same prim editing system, or two different guide nucleic acids that are complementary to different sequences within the same or different target genes), the compositions may be delivered simultaneously (e.g., as two polypeptides and/or nucleic acids). Alternatively, they may be provided sequentially, e.g., one composition being provided first, followed by a second composition.

The prime editing compositions and pharmaceutical compositions of the disclosure, whether introduced as polynucleotides or polypeptides, can be administered to subjects in need thereof for about 30 minutes to about 24 hours, e.g., 1 hour, 1.5 hours, 2 hours, 2.5 hours. 3 hours, 3.5 hours 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 16 hours, 18 hours, 20 hours, or any other period from about 30 minutes to about 24 hours, which can be repeated with a frequency of about every day to about every 4 days, e.g., every 1.5 days, every 2 days, every 3 days, or any other frequency from about every day to about every four days. The compositions may be provided to the subject one or more times, e.g., one time, twice, three times, or more than three times. In cases in which two or more different prime editing system components, e.g. two different polynucleotide constructs are administered to the subject (e.g., different components of the same prim editing system, or two different guide nucleic acids that are complementary to different sequences within the same or different target genes), the compositions may be administered simultaneously (e.g., as two polypeptides and/or nucleic acids). Alternatively, they may be provided sequentially, e.g., one composition being provided first, followed by a second composition.

EXAMPLES

The following examples are provided for illustrative purposes only and are not intended to limit the scope of the claims provided herein.

Example 1—General Methods

PEgRNA assembly: PEgRNA libraries are assembled by one of three methods: in the first method, pooled synthesized DNA oligos encoding the PEgRNA and flanking U6 expression plasmid homology regions are cloned into U6 expression plasmids via Gibson cloning and sequencing of bacterial colonies via Sanger or Next-generation sequencing. In the second method, double-stranded linear DNA fragments encoding PEgRNA and homology sequences as above are individually Gibson-cloned into U6 expression plasmids. In the third method, for each PEgRNA, separate oligos encoding a protospacer, a gRNA scaffold, and PEgRNA extension (PBS and RTT) are ligated, and then cloned into a U6 expression plasmid as described in Anzalone et al., Nature. 2019 December; 576(7785):149-157. Bacterial colonies carrying sequence-verified plasmids are propagated in LB or TB. Plasmid DNA is purified by minipreps for mammalian transfection.

Mammalian cell culture and transfection: HEK293T and Huh-7 cells are propagated in DMEM with 10% FBS. HepG2 cells are propagated in EMEM with 10% FBS. Cells are seeded in 96-well plates and then transfected with Lipofectamine 2000 according to the manufacturer's directions with DNA encoding a prime editor, PEgRNA, and (if applicable) ngRNA. Alternatively, cells are transfected with MessengerMax according to the manufacturer's directions with mRNA encoding a prime editor, synthetic PEgRNA, and (if applicable) ngRNA. Three days after transfection, gDNA is harvested in lysis buffer for high throughput sequencing and sequenced using miseq.

Lentiviral production and cell line generation: Lentiviral transfer plasmids containing the H1069Q mutation with flanking sequences from the ATP7B gene on each side, and an IRES-Puromycin selection cassette, are cloned behind an EF1α short promoter. HEK 293T cells are transiently transfected with the transfer plasmids and packaging plasmids containing VSV glycoprotein and lentiviral gag/pol coding sequences. After transfection, lentiviral particles are harvested from the cell media and concentrated. HEK 293T cells are transduced using serial dilutions of the lentiviral particles described above. Cells generated at a dilution of MOI<1, as determined by survival following puromycin, are selected for expansion. A resulting HEK293T cell line carrying the H1069Q mutation is used to screen PEgRNAs.

ATP7B H1069Q mutation installation: An ATP7B H1069Q mutation is installed at the endogenous ATP7B locus in HEK 293T, Huh-7, and HepG2 cells by prime editing and single-cell clones are obtained via limiting dilution and clonal expansion.

Prime Editing in primary human hepatocytes: Primary human hepatocytes are transduced with lentivirus encoding the H1069Q cassette 2 days after cryorecovery, followed 6 days later by transfection with RNA encoding a prime editor, PEgRNA, and (if applicable) ngRNA. Genomic DNA is harvested after a 1-week incubation.

Example 2—Screen of Cas9 Cutting Activity at Spacers within 200 nt of the ATP7B H1069Q Mutation Site

A spacer screen was performed to investigate Cas9 cutting activity at sites within 200 nucleotides (nts) of the H1069Q mutation site in the ATP7B gene. HEK293T cells were cultured and transfected with mRNA encoding a Cas9 and synthetic sgRNA as described above. The results are shown in Table XA.

TABLE XA

Spacer screen for Cas9 cutting activity within 200

nt of the H1069Q mutation site in the ATP7B gene

PAM Location

Nick-to-edit

Relative to

Spacer¹
distance to H1069

Percent
H1069Q Mutation

SEQ ID NO:
(nt)²
PAM
Indel³
site⁴

5281
−85
NGA
+++
5′

738
−78
NGG
+++
5′

740
−75
NGG
++
5′

736
−74
NGG
++
5′

737
−73
NGG
++
5′

529
−72
NGA
++
5′

200
−70
NGG
+
5′

199
−69
NGG
++
5′

35
−65
NGG
+
5′

55
−64
NGG
+
5′

2090
−51
NGG
+
3′

2063
−46
NGG
++
3′

2048
−45
NGG
+
3′

2066
−40
NGA
+
3′

2051
−39
NGG
+++
3′

2045
−32
NGG
++++
3′

2091
−30
NGA
+++
3′

45; 2023
−27
NGG
++
5′

43
−24
NGA
+
5′

33; 2293
−9
NGG
++
5′

58; 4425
−8
NGG
+++
5′

53
−7
NGG
++
5′

2076; 5248
9
NGA
++
5′

1505; 2055
19
NGG
+++
5′

2081; 5228
21
NGA
+
5′

682; 2087
22
NGG
+
5′

2083; 5206
28
NGA
+
5′

483; 2061
29
NGG
++++
5′

294; 2070
30
NGG
++++
5′

182; 2080
31
NGG
+++
5′

42
32
NGA
+
3′

1; 2058
34
NGG
+++
5′

2069; 5555
40
NGG
++++
5′

39
42
NGA
++++
3′

70
44
NGG
++++
3′

68
45
NGG
+++
3′

47
46
NGG
++++
3′

2057; 5706
46
NGG
++++
5′

2064
49
NGA
+
5′

2056; 5638
50
NGG
+++
5′

32
54
NGG
+++
3′

2068
67
NGG
+
5′

31
71
NGG
+++
3′

40
72
NGA
++++
3′

2046
72
NGA
+
5′

2059
73
NGG
++
5′

2089
74
NGG
+++
5′

2443
75
NGG
++
5′

2444
76
NGG
+++
5′

20
84
NGA
+
3′

2437
88
NGG
++++
5′

2439
89
NGG
++++
5′

30
90
NGG
+++
3′

27
91
NGG
+++
3′

36
92
NGG
+++
3′

21
93
NGA
+
3′

5536
97
NGG
++++
5′

69
98
NGG
++++
5′

50
101
NGG
+++
3′

34
102
NGG
++
3′

62
108
NGG
+++
5′

72
108
NGG
++
5′

65
123
NGG
++++
5′

5219
127
NGA
+
5′

5220
132
NGA
+
5′

5226
139
NGA
++
5′

5221
145
NGA
+
5′

5222
151
NGA
+
5′

41
155
NGG
++
5′

63
165
NGG
++++
5′

5223
166
NGA
++
3′

5227
172
NGA
++++
5′

5224
178
NGA
++
3′

64
179
NGG
++++
5′

66
181
NGG
++
3′

5225
182
NGA
+++
3′

61
184
NGG
++++
3′

71
185
NGG
++++
3′

60
186
NGG
++++
3′

¹The indicated sequence sequences recite only the spacer; the sgRNA used experimentally contained the gRNA core of SEQ ID NO: 5957, a 3′ mU*mU*mU*U modification, and a 5′mN*mN*mN* modification, where m indicates that the indicated nucleotide contains a 2′-O— Me modification and a * indicates a phosphorothioate bond. Some spacers are identified by two SEQ ID NOs because the same spacer sequence was assigned a different SEQ ID NO in the cluster tables depending upon whether it was included as a ngRNA spacer or a PEgRNA spacer.

²A (+) nick-to-edit distance indicates the PAM is on the sense strand whereas a (−) nick-to-edit distance indicates the PAM is on the antisense strand.

³+ = 0.0%-3.3%; ++ = 3.3%-7.2%; +++ 7.2%-20%; ++++ = 20%-52.9%

⁴The indication of 5′ or 3′ refers to the position of the PAM relative to the H1069Q mutation site on the PAM strand in the ATP7B gene. The H1069Q mutation site may therefore refer to the sense or antisense strand, depending upon which strand contains the PAM sequence.

Example 3—Prime Editing at a Lentivirus-Introduced ATP7B H1069Q Mutation Site in HEK293T Cells Using a PE2 System

Four exemplary PEgRNA spacers close to the H1069Q mutation are shown in FIG. 3A. In FIG. 3A, pegRNA spacer #1 corresponds to SEQ ID NO: 2293, pegRNA spacer #2 corresponds to SEQ ID NO: 4452, pegRNA spacer #3 corresponds to SEQ ID NO: 1505, and pegRNA spacer #4 corresponds to SEQ ID NO: 682. PEgRNA incorporating these spacers were designed and screened for Prime Editing efficiency in a HEK293T cell line containing a lentivirus-introduced H1069Q mutation. The cell line was generated as described in Example 1. These spacers were selected because they are close to the H1069Q mutation site and would produce a nick that is 5′ of the H1069Q mutation site when used in conjunction with a prime editor having a Cas9 protein containing an inactivating mutation in the HNH nuclease domain. Each of these spacers also showed at least some activity in the spacer screen of Example 2.

375 PEgRNA were designed and screened in a PE2 system (i.e., without a ngRNA). In this initial screen, multiple primer binding site (PBS) and reverse transcription template (RTT) lengths were tested for each of the four exemplary spacers. All the PEgRNA were designed to restore the wild-type nucleic acid sequence at the H1069Q site. Where possible, additional PEgRNAs were designed that also introduce a silent mutation that destroys the PAM sequence (i.e., a PAM silencing mutation).

The results for individual PEgRNA are shown in Table XB. Successful Prime Editing was observed across PBS and RTT lengths, with and without PAM silencing. The percent editing observed for all PEgRNA having the same spacer were averaged, and the results reported in Table XC. The relative levels of Prime Editing observed between spacers is similar to the relative levels of Cas9 cutting for these spacers in the spacer screen of Example 2.

TABLE XB

PE2 Screen at H1069Q mutation site in HEK293T cells

PEgRNA¹
Spacer
RTT²

PBS

SEQ ID
SEQ
SEQ
RTT
SEQ
PBS
Percent

NO:
ID NO:
ID NO:
Length
ID NO:
Length
Edit³

1565
1505; 2055
1518
21
1509
10
+++

1564
1505; 2055
1517*
21
1509
10
+

1569
1505; 2055
1519
22
1509
10
++++

1575
1505; 2055
1520*
22
1509
10
+

1580
1505; 2055
1522
23
1509
10
+

1577
1505; 2055
1521*
23
1509
10
+++

1593
1505; 2055
1524
24
1509
10
++

1590
1505; 2055
1523*
24
1509
10
+++

1600
1505; 2055
1526
25
1509
10
+++

1598
1505; 2055
1525*
25
1509
10
++++

1578
1505; 2055
1518
21
1511
12
+++

1586
1505; 2055
1517*
21
1511
12
+

1591
1505; 2055
1519
22
1511
12
+++

1587
1505; 2055
1520*
22
1511
12
+++

1609
1505; 2055
1522
23
1511
12
++

1607
1505; 2055
1521*
23
1511
12
+++

1616
1505; 2055
1524
24
1511
12
++++

1622
1505; 2055
1523*
24
1511
12
++++

1643
1505; 2055
1526
25
1511
12
+++

1637
1505; 2055
1525*
25
1511
12
++++

1602
1505; 2055
1518
21
1513
14
++

1606
1505; 2055
1517*
21
1513
14
+

1626
1505; 2055
1519
22
1513
14
+++

1615
1505; 2055
1520*
22
1513
14
++++

1628
1505; 2055
1522
23
1513
14
+++

1645
1505; 2055
1521*
23
1513
14
++

1664
1505; 2055
1524
24
1513
14
++

1668
1505; 2055
1523*
24
1513
14
+++

1671
1505; 2055
1526
25
1513
14
++

1679
1505; 2055
1525*
25
1513
14
++++

1640
1505; 2055
1518
21
1515
16
++

1646
1505; 2055
1517*
21
1515
16
+

1654
1505; 2055
1519
22
1515
16
+

1663
1505; 2055
1520*
22
1515
16
++++

1691
1505; 2055
1522
23
1515
16
+

1678
1505; 2055
1521*
23
1515
16
++

1708
1505; 2055
1524
24
1515
16
++

1694
1505; 2055
1523*
24
1515
16
+++

1725
1505; 2055
1526
25
1515
16
++++

1721
1505; 2055
1525*
25
1515
16
++++

1550
1505; 2055
1518
21
1507
8
+++

1551
1505; 2055
1517*
21
1507
8
+++

1554
1505; 2055
1519
22
1507
8
+++

1553
1505; 2055
1520*
22
1507
8
++

1562
1505; 2055
1522
23
1507
8
+

1566
1505; 2055
1521*
23
1507
8
+++

1570
1505; 2055
1524
24
1507
8
++

1567
1505; 2055
1523*
24
1507
8
++

1583
1505; 2055
1526
25
1507
8
++

1584
1505; 2055
1525*
25
1507
8
++

777
682; 2087
697
24
686
10
+

778
682; 2087
694*
24
686
10
+

775
682; 2087
695*
24
686
10
+

772
682; 2087
696*
24
686
10
+

781
682; 2087
699
25
686
10
+

787
682; 2087
701*
25
686
10
+

784
682; 2087
698*
25
686
10
+

791
682; 2087
700*
25
686
10
+

807
682; 2087
702
26
686
10
+

820
682; 2087
704*
26
686
10
+

801
682; 2087
703*
26
686
10
+

813
682; 2087
705*
26
686
10
+

838
682; 2087
707
27
686
10
++

828
682; 2087
708*
27
686
10
+++

829
682; 2087
709*
27
686
10
++

824
682; 2087
706*
27
686
10
++

852
682; 2087
711
28
686
10
+++

854
682; 2087
712*
28
686
10
++

860
682; 2087
710*
28
686
10
+++

856
682; 2087
713*
28
686
10
++

819
682; 2087
697
24
688
12
+

811
682; 2087
694*
24
688
12
+

815
682; 2087
695*
24
688
12
+

805
682; 2087
696*
24
688
12
+

825
682; 2087
699
25
688
12
+

822
682; 2087
701*
25
688
12
+

832
682; 2087
698*
25
688
12
+

835
682; 2087
700*
25
688
12
+

843
682; 2087
702
26
688
12
+

849
682; 2087
704*
26
688
12
+

853
682; 2087
703*
26
688
12
+

858
682; 2087
705*
26
688
12
+++

882
682; 2087
707
27
688
12
++

880
682; 2087
708*
27
688
12
++

870
682; 2087
709*
27
688
12
++

879
682; 2087
706*
27
688
12
++

896
682; 2087
711
28
688
12
+

920
682; 2087
712*
28
688
12
+++

900
682; 2087
710*
28
688
12
++

908
682; 2087
713*
28
688
12
++

851
682; 2087
697
24
690
14
+

857
682; 2087
694*
24
690
14
+

841
682; 2087
695*
24
690
14
+

846
682; 2087
696*
24
690
14
+

861
682; 2087
699
25
690
14
++

873
682; 2087
701*
25
690
14
+

878
682; 2087
698*
25
690
14
+

887
682; 2087
700*
25
690
14
+

906
682; 2087
702
26
690
14
+

912
682; 2087
704*
26
690
14
+

893
682; 2087
703*
26
690
14
+

916
682; 2087
705*
26
690
14
+

929
682; 2087
707
27
690
14
+

960
682; 2087
708*
27
690
14
++

923
682; 2087
709*
27
690
14
++

934
682; 2087
706*
27
690
14
+

989
682; 2087
711
28
690
14
+++

961
682; 2087
712*
28
690
14
+++

997
682; 2087
710*
28
690
14
++

971
682; 2087
713*
28
690
14
++

914
682; 2087
697
24
692
16
+

905
682; 2087
694*
24
692
16
+

902
682; 2087
695*
24
692
16
+

898
682; 2087
696*
24
692
16
+

926
682; 2087
699
25
692
16
+

927
682; 2087
701*
25
692
16
1

938
682; 2087
698*
25
692
16
+

935
682; 2087
700*
25
692
16
+

977
682; 2087
702
26
692
16
++

983
682; 2087
704*
26
692
16
+

963
682; 2087
703*
26
692
16
+

970
682; 2087
705*
26
692
16
+

1041
682; 2087
707
27
692
16
+

1020
682; 2087
708*
27
692
16
++

1043
682; 2087
709*
27
692
16
+

1024
682; 2087
706*
27
692
16
+

1074
682; 2087
711
28
692
16
++

1057
682; 2087
712*
28
692
16
++

1068
682; 2087
710*
28
692
16
++

1085
682; 2087
713*
28
692
16
+++

752
682; 2087
697
24
684
8
+

745
682; 2087
694*
24
684
8
+

751
682; 2087
695*
24
684
8
+

746
682; 2087
696
24
684
8
+

760
682; 2087
699
25
684
8
+

758
682; 2087
701*
25
684
8
+

756
682; 2087
698*
25
684
8
+

761
682; 2087
700*
25
684
8
+

765
682; 2087
702
26
684
8
+

767
682; 2087
704*
26
684
8
+

770
682; 2087
703*
26
684
8
+

780
682; 2087
705*
26
684
8
+

783
682; 2087
707
27
684
8
+

796
682; 2087
708*
27
684
8
++

790
682; 2087
709*
27
684
8
+

789
682; 2087
706*
27
684
8
+

804
682; 2087
711
28
684
8
++

812
682; 2087
712*
28
684
8
+++

809
682; 2087
710*
28
684
8
+

816
682; 2087
713*
28
684
8
++

4507
58; 4425
4438
10
4429
10
+++

4527
58; 4425
4441
12
4429
10
++++

4533
58; 4425
4444
13
4429
10
++++

4555
58; 4425
4446
14
4429
10
++

4564
58; 4425
4448
15
4429
10
+

4649
58; 4425
4452
18
4429
10
++++

4756
58; 4425
4456
21
4429
10
++++

4519
58; 4425
4438
10
4431
12
++++

4542
58; 4425
4440
11
4431
12
++++

4559
58; 4425
4441
12
4431
12
++

4576
58; 4425
4444
13
4431
12
++++

4588
58; 4425
4446
14
4431
12
+

4624
58; 4425
4448
15
4431
12
+

4679
58; 4425
4450
17
4431
12
++++

4697
58; 4425
4452
18
4431
12
++

4817
58; 4425
4456
21
4431
12
++++

4553
58; 4425
4438
10
4433
14
+++

4562
58; 4425
4440
11
4433
14
++

4592
58; 4425
4441
12
4433
14
++++

4640
58; 4425
4446
14
4433
14
++++

4691
58; 4425
4448
15
4433
14
++++

4726
58; 4425
4450
17
4433
14
++++

4761
58; 4425
4452
18
4433
14
++++

4852
58; 4425
4456
21
4433
14
++++

4629
58; 4425
4440
11
4435
16
++

4653
58; 4425
4441
12
4435
16
++++

4668
58; 4425
4444
13
4435
16
++++

4724
58; 4425
4446
14
4435
16
++

4738
58; 4425
4448
15
4435
16
+

4809
58; 4425
4450
17
4435
16
+++

4840
58; 4425
4452
18
4435
16
+++

4893
58; 4425
4456
21
4435
16
++++

4500
58; 4425
4440
11
4427
8
++++

4504
58; 4425
4441
12
4427
8
+

4517
58; 4425
4444
13
4427
8
+++

4522
58; 4425
4446
14
4427
8
++++

4540
58; 4425
4448
15
4427
8
++++

4580
58; 4425
4450
17
4427
8
++++

4591
58; 4425
4452
18
4427
8
++++

2480
33; 2293
2307
11
2297
10
+++

2478
33; 2293
2308*
11
2297
10
+

2487
33; 2293
2310*
11
2297
10
++

2488
33; 2293
2306*
11
2297
10
++

2510
33; 2293
2313
12
2297
10
++++

2514
33; 2293
2312*
12
2297
10
+

2513
33; 2293
2311*
12
2297
10
++

2493
33; 2293
2316*
12
2297
10
++

2533
33; 2293
2320
13
2297
10
++++

2520
33; 2293
2318*
13
2297
10
++

2531
33; 2293
2321*
13
2297
10
+

2527
33; 2293
2319*
13
2297
10
+++

2559
33; 2293
2326
14
2297
10
+++

2553
33; 2293
2324*
14
2297
10
++

2561
33; 2293
2328*
14
2297
10
+++

2571
33; 2293
2325*
14
2297
10
++++

2605
33; 2293
2329
15
2297
10
+++

2618
33; 2293
2333*
15
2297
10

2615
33; 2293
2334*
15
2297
10
++++

2598
33; 2293
2332*
15
2297
10
+++

2633
33; 2293
2335
16
2297
10
++++

2632
33; 2293
2338*
16
2297
10
+++

2645
33; 2293
2339*
16
2297
10
++++

2634
33; 2293
2337*
16
2297
10
+++

2769
33; 2293
2342
18
2297
10
++++

2773
33; 2293
2346*
18
2297
10
++

2772
33; 2293
2343*
18
2297
10
+++

2765
33; 2293
2344*
18
2297
10
+++

2801
33; 2293
2351
19
2297
10
++++

2829
33; 2293
2347*
19
2297
10
++

2826
33; 2293
2348*
19
2297
10
+++

2807
33; 2293
2352*
19
2297
10
+++

2983
33; 2293
2353
22
2297
10
+++

3023
33; 2293
2356*
22
2297
10
++

3012
33; 2293
2355*
22
2297
10
+++

2977
33; 2293
2358*
22
2297
10
+++

2522
33; 2293
2307
11
2299
12
+++

2528
33; 2293
2308*
11
2299
12
++

2526
33; 2293
2310*
11
2299
12
++

2544
33; 2293
2306*
11
2299
12
++++

2575
33; 2293
2313
12
2299
12
++++

2562
33; 2293
2312*
12
2299
12
++

2554
33; 2293
2311*
12
2299
12
++

2563
33; 2293
2316*
12
2299
12
++

2585
33; 2293
2320
13
2299
12
++++

2596
33; 2293
2318*
13
2299
12
+

2610
33; 2293
2321*
13
2299
12
++

2590
33; 2293
2319*
13
2299
12
+++

2628
33; 2293
2326
14
2299
12
++++

2646
33; 2293
2324*
14
2299
12
+

2643
33; 2293
2328*
14
2299
12
+++

2665
33; 2293
2325*
14
2299
12
+++

2667
33; 2293
2329
15
2299
12
++++

2699
33; 2293
2333*
15
2299
12
++

2701
33; 2293
2334*
15
2299
12
+++

2674
33; 2293
2332*
15
2299
12
+++

2744
33; 2293
2335
16
2299
12
++++

2734
33; 2293
2338*
16
2299
12
++

2735
33; 2293
2339*
16
2299
12
+++

2733
33; 2293
2337*
16
2299
12
++++

2886
33; 2293
2342
18
2299
12
++++

2899
33; 2293
2346*
18
2299
12
+++

2885
33; 2293
2343*
18
2299
12
+++

2890
33; 2293
2344*
18
2299
12
++++

2916
33; 2293
2351
19
2299
12
++++

2941
33; 2293
2347*
19
2299
12
++

2960
33; 2293
2348*
19
2299
12
+++

2962
33; 2293
2352*
19
2299
12
+++

3163
33; 2293
2353
22
2299
12
+++

3122
33; 2293
2356*
22
2299
12
+++

3188
33; 2293
2355*
22
2299
12
+++

3166
33; 2293
2358*
22
2299
12
+++

2606
33; 2293
2307
11
2301
14
++

2602
33; 2293
2308*
11
2301
14
+

2609
33; 2293
2310*
11
2301
14
++

2584
33; 2293
2306*
11
2301
14
++

2623
33; 2293
2313
12
2301
14
++++

2647
33; 2293
2312*
12
2301
14
++

2660
33; 2293
2311*
12
2301
14
++

2619
33; 2293
2316*
12
2301
14
+++

2675
33; 2293
2320
13
2301
14
++++

2677
33; 2293
2318*
13
2301
14
++

2685
33; 2293
2321*
13
2301
14
++

2670
33; 2293
2319*
13
2301
14
+++

2729
33; 2293
2326
14
2301
14
++++

2749
33; 2293
2324*
14
2301
14
++

2764
33; 2293
2328*
14
2301
14
+++

2730
33; 2293
2325*
14
2301
14
+++

2802
33; 2293
2329
15
2301
14
++++

2816
33; 2293
2333*
15
2301
14
+++

2777
33; 2293
2334*
15
2301
14
++++

2832
33; 2293
2332*
15
2301
14
++++

2898
33; 2293
2335
16
2301
14
++++

2834
33; 2293
2338*
16
2301
14
+++

2893
33; 2293
2339*
16
2301
14
++++

2889
33; 2293
2337*
16
2301
14
++++

3043
33; 2293
2342
18
2301
14
++++

3013
33; 2293
2346*
18
2301
14
++

3015
33; 2293
2343*
18
2301
14
++++

2982
33; 2293
2344*
18
2301
14
++++

3076
33; 2293
2351
19
2301
14
++++

3071
33; 2293
2347*
19
2301
14
+++

3080
33; 2293
2348*
19
2301
14
++++

3072
33; 2293
2352*
19
2301
14
++++

3287
33; 2293
2353
22
2301
14
+++

3275
33; 2293
2356*
22
2301
14
++

3301
33; 2293
2355*
22
2301
14
+++

3300
33; 2293
2358*
22
2301
14
+++

2683
33; 2293
2307
11
2303
16
++++

2711
33; 2293
2308*
11
2303
16
+

2680
33; 2293
2310*
11
2303
16
++

2702
33; 2293
2306*
11
2303
16
++++

2758
33; 2293
2313
12
2303
16
++++

2760
33; 2293
2312*
12
2303
16
++

2759
33; 2293
2311*
12
2303
16
++

2757
33; 2293
2316*
12
2303
16
++

2825
33; 2293
2320
13
2303
16
+++

2830
33; 2293
2318*
13
2303
16
++

2814
33; 2293
2321*
13
2303
16
++

2800
33; 2293
2319*
13
2303
16
+

2844
33; 2293
2326
14
2303
16
++++

2842
33; 2293
2324*
14
2303
16
++++

2843
33; 2293
2328*
14
2303
16
+++

2841
33; 2293
2325*
14
2303
16
+++

2932
33; 2293
2329
15
2303
16
++++

2926
33; 2293
2333*
15
2303
16
++++

2964
33; 2293
2334*
15
2303
16
+++

2933
33; 2293
2332*
15
2303
16
+++

2993
33; 2293
2335
16
2303
16
++++

2972
33; 2293
2338*
16
2303
16
++

2988
33; 2293
2339*
16
2303
16
++++

2979
33; 2293
2337*
16
2303
16
+++

3144
33; 2293
2342
18
2303
16
+++

3148
33; 2293
2346*
18
2303
16
++

3123
33; 2293
2343*
18
2303
16
++++

3165
33; 2293
2344*
18
2303
16
++++

3263
33; 2293
2351
19
2303
16
++++

3200
33; 2293
2347*
19
2303
16
++

3248
33; 2293
2348*
19
2303
16
+

3210
33; 2293
2352*
19
2303
16
+++

3441
33; 2293
2353
22
2303
16
++++

3420
33; 2293
2356*
22
2303
16
++

3430
33; 2293
2355*
22
2303
16
+++

3442
33; 2293
2358*
22
2303
16
+++

2453
33; 2293
2307
11
2295
8
++

2451
33; 2293
2308*
11
2295
8
+

2458
33; 2293
2310*
11
2295
8
+++

2457
33; 2293
2306*
11
2295
8
++++

2459
33; 2293
2313
12
2295
8
++++

2460
33; 2293
2312*
12
2295
8
++++

2466
33; 2293
2311*
12
2295
8
++

2461
33; 2293
2316*
12
2295
8
++

2489
33; 2293
2320
13
2295
8
++++

2473
33; 2293
2318*
13
2295
8
++

2490
33; 2293
2321*
13
2295
8
++++

2484
33; 2293
2319*
13
2295
8
+++

2515
33; 2293
2326
14
2295
8
++++

2501
33; 2293
2324*
14
2295
8
++

2512
33; 2293
2328*
14
2295
8
+++

2509
33; 2293
2325*
14
2295
8
+++

2548
33; 2293
2329
15
2295
8
++++

2535
33; 2293
2333*
15
2295
8
++

2546
33; 2293
2334*
15
2295
8
+++

2541
33; 2293
2332*
15
2295
8
+++

2557
33; 2293
2335
16
2295
8
++++

2564
33; 2293
2338*
16
2295
8
++

2568
33; 2293
2339*
16
2295
8
++++

2551
33; 2293
2337*
16
2295
8
+++

2637
33; 2293
2342
18
2295
8
++++

2635
33; 2293
2346*
18
2295
8
++

2638
33; 2293
2343*
18
2295
8
+++

2636
33; 2293
2344*
18
2295
8
+++

2689
33; 2293
2351
19
2295
8
++++

2669
33; 2293
2347*
19
2295
8
++

2690
33; 2293
2348*
19
2295
8
++++

2706
33; 2293
2352*
19
2295
8
++++

2871
33; 2293
2353
22
2295
8
++++

2856
33; 2293
2356*
22
2295
8
++

2891
33; 2293
2355*
22
2295
8
+++

2894
33; 2293
2358*
22
2295
8
+++

¹Indicated PEgRNA sequence does not contain the adaptations for transcription from a DNA template used experimentally (i.e., addition of a 5'G if the spacer did not already start with a G and addition of 1-6 3'U from the U6 transcription termination sequence).

²* = RTT contains a PAM silencing mutation

³+ = 0.4%-3.3%; ++ = 3.3%-7.2%; +++ 7.2%-20%; ++++ = 20%-52.9%

TABLE XC

Average Percent Edit by Spacer in PE2 Screen

at H1069Q mutation site in HEK293T cells

Spacer

SEQ ID NO:
Avg. % Edit¹

1505; 2055
+++

682; 2087
+

58; 4425
++++

33; 2293
+++

¹+ = 0.4%-3.3%; ++ = 3.3%-7.2%; +++ 7.2%-20%; ++++ = 20%-52.9%

A subset of the PEgRNAs from Table XB were further examined for indels, the results of which are shown in Table XC. Indel frequency was quantified using standard quantification techniques via CRISPResso2 algorithm as described in Clement, K. et al., Nat. Biotechnol. 37, 224-226 (2019), with the quantification window defined as at least 20 bases 5′ and 3′ of pegRNA and ngRNA nick site.

TABLE XD

PE2 screen at the H1069 mutation site in HEK293T cells

PEgRNA¹

RTT²

PBS

SEQ ID
Spacer
SEQ ID
RTT
SEQ ID
PBS

NO:
SEQ ID NO:
NO:
Length
NO:
Length
% Edit³
% Indel³

2557
33; 2293
2335
16
2295
8
++++
+

2568
33; 2293
2339*
16
2295
8
++++
++

2871
33; 2293
2353
22
2295
8
+++
++

2533
33; 2293
2320
13
2297
10
++++
++

2615
33; 2293
2334*
15
2297
10
++++
++

2633
33; 2293
2335
16
2297
10
+++
+

2585
33; 2293
2320
13
2299
12
++++
++

2667
33; 2293
2329
15
2299
12
++
++

2744
33; 2293
2335
16
2299
12
+++
+

2890
33; 2293
2344*
18
2299
12
+++
+

2675
33; 2293
2320
13
2301
14
++++
++

2729
33; 2293
2326
14
2301
14
++++
++

2898
33; 2293
2335
16
2301
14
+++
++

3165
33; 2293
2344*
18
2303
16
++++
++

2802
33; 2293
2329
15
2301
14
++++
+

4540
58; 4425
4448
15
4427
8
+++
+

4533
58; 4425
4444
13
4429
10
++++
+

4756
58; 4425
4456
21
4429
10
+++
+

4624
58; 4425
4448
15
4431
12
++++
+

4592
58; 4425
4441
12
4433
14
++++
+

4640
58; 4425
4446
14
4433
14
++++
+

4691
58; 4425
4448
15
4433
14
++++
+

4852
58; 4425
4456
21
4433
14
+++
+

4761
58; 4425
4452
18
4433
14
++++
+

1622
1505; 2055
1523
24
1511
12
+++
++

1615
1505; 2055
1520
22
1513
14
++++
++

1663
1505; 2055
1520
22
1515
16
+++
+++

1721
1505; 2055
1525
25
1515
16
+++
++

¹Indicated PEgRNA sequence does not contain the adaptations for transcription from a DNA template used experimentally (i.e., addition of a 5′G if the spacer did not already start with a G and addition of 1-6 3′U from the U6 transcription termination sequence).

²* = RTT contains a PAM silencing mutation

³+ = 0.3%-3.4%; ++ = 3.4%-13.5%; +++ = 13.5%-23.0%; ++++ = 23.0%-36.0%

Example 4—Prime Editing at the Endogenous ATP7B H1069Q Mutation Site in HEK293T Cells Using a PE3 System

An ATP7B H1069Q mutation was installed at the endogenous ATP7B locus in HEK 293T cells by prime editing and single-cell clones were obtained via limiting dilution and clonal expansion. A PE3 screen measuring correction and indel formation was performed at the endogenous ATP7B H1069Q locus. The HEK293T cells were transfected with DNA encoding a prime editor, PEgRNA, and ngRNA, as described in Example 1.

The results of the PE3 screen are provided in Tables XEa-XEd. Below each of Tables XEa-XEd is a table summarizing the PEgRNAs used experimentally. Each of the PEgRNA were tested in combination with multiple ngRNA. Some of the ngRNA were designed for a PE3B strategy and contain spacers complementary to the portion of the edit strand containing the edit. These results demonstrate the successful correction of the H1069Q mutation at the endogenous ATP7B locus in mammalian cells using both PE3 and PE3B Prime Editing systems.

TABLE XEa

PE3 screen at the H1069 mutation site in HEK293T cells

PEgRNA²SEQ ID NO:

2557
2568
2871
2533
2615

%
%
%
%
%
%
%
%
%
%

Edit¹
indel¹
Edit¹
indel¹
Edit¹
indel¹
Edit¹
indel¹
Edit¹
indel¹

ngRNA²
PE2
++++
+
++++
+
+++
+
++++
++
++++
+

SEQ
2263
++
++
+
+
+
++
++
+
++
++

ID
2266
++++
++
+
+
++
++
+++
+
+++
+

NO:
2276
+++
++++
++
++
+
+
+
+
++
+++

2277
++++
+++
++
++
+
++
+++
++
++++
++

2259
++++
+++
+++
++
+
++
++++
++
++++
++

2257
+++
++
X
X
+
+
++++
++
X
X

2268
+++
+++
X
X
++
++
+++
++
X
X

2264
++++
+
X
X
++
++
++++
+
X
X

2275
++++
++
+++
++
++
++
++++
++
+++
+

2273
++++
++++
++
++
+
+
++++
+++
++++
++

2271
++++
++++
++
++
++
+
+++
+++
+++
+++

2261
++++
+++
++
++
+
++
++++
+++
++++
++

2274
++++
+
+++
+
++
+
++++
+
++++
+

2269
++++
+
+++
++
++
+
++++
+
++++
+

2258
+++
++++
++
++
+
++
+++
+++
+++
+++

2270
++++
++++
++
++
+
++
+++
+++
+++
++

2262
++++
+++
++
+++
+
+
++++
++
++++
++

2267
++++
+
+++
++
+
+
++++
+
++++
+

2265
++++
++
++
+
++
++
++++
+
+++
+

2260
++++
+
+++
++
+
++
++++
+
++++
+

2272
++++
++
+++
+
++
+
+++
+
++++
+

4411
++++
++
++
++
+
+
++++
+
++++
+

4418
++++
++++
++
++
+
+
++++
+++
++++
++

4416
++++
+++
+++
++
++
++
++++
++
++++
+

¹+ = 0.3%-6.3%; ++ = 6.3%-12.0%; +++ = 12%-20.3%; +++ = 20.3%-55.9%; X indicates successful PE3B editing was observed with the PEgRNA (data not shown)

Summary of PEgRNA Used:

PBS

PEgRNA²
Spacer
RTT³
RTT
SEQ
PBS

SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
Length
ID NO:
Length

2557
33; 2293
2335
16
2295
8

2568
33; 2293
2339*
16
2295
8

2871
33; 2293
2353
22
2295
8

2533
33; 2293
2320
13
2297
10

2615
33; 2293
2334*
15
2297
10

²Indicated sequence does not contain the transcription adaptations used experimentally (i.e., addition of a 5′G if the spacer did not already start with a G and addition of 1-6 3′U from the U6 transcription termination sequence). The first 20 nt of the ngRNA sequence are the spacer; italics indicate that the spacer is a PE3B spacer.

³* = RTT contains a PAM silencing mutation.

TABLE XEb

PE3 screen at the H1069 mutation site in HEK293T cells

PEgRNA²SEQ ID NO:

2633
2585
2667
2744
2890

%
%
%
%
%
%
%
%
%
%

Edit¹
indel¹
Edit¹
indel¹
Edit¹
indel¹
Edit¹
indel¹
Edit¹
indel¹

ngRNA²
PE2
++++
+
++++
++
++
+++
+++
+
+++
+

SEQ
2263
++
++
++
++
++
++
++
++
++
++

ID
2266
+++
+
+++
+
+++
+
+++
+
+++
+

NO:
2276
++
+++
++
+++
++
+++
++
++
++
+++

2277
++++
+++
+++
++
+++
+
+++
++
++++
++

2259
++++
++
++++
++
++++
++
++++
++
++++
++

2257
++++
+++
++++
++
++++
++
++++
++
X
X

2268
+++
++
+++
++
+++
++
+++
++
X
X

2264
++++
+
++++
+
++++
+
+++
+
X
X

2275
++++
++
++++
++
++++
++
++++
++
++++
++

2273
++++
+++
++++
+++
++++
+++
++++
+++
++++
++

2271
+++
+++
+++
+++
+++
+++
+++
+++
++++
+++

2261
++++
+++
++++
++
++++
+++
++++
++
++++
++

2274
++++
+
++++
+
++++
+
++++
+
++++
+

2269
++++
+
++++
+
++++
+
++++
+
++++
+

2258
+++
++++
+++
+++
+++
+++
+++
+++
+++
+++

2270
+++
+++
+++
+++
++++
+++
+++
+++
++++
+++

2262
++++
+++
++++
+++
++++
+++
++++
+++
++++
++

2267
++++
+
++++
+
++++
+
++++
+
++++
+

2265
++++
+
++++
+
++++
+
+++
+
++++
+

2260
++++
+
++++
+
++++
+
++++
+
++++
+

2272
++++
+
++++
+
++++
+
+++
+
++++
+

4411
++++
+
++++
+
++++
+
++++
+
++++
+

4418
++++
+++
++++
+++
++++
+++
++++
+++
++++
++

4416
++++
++
++++
+
++++
++
++++
++
+
+

¹+ = 0.3%-6.3%; ++ = 6.3%-12.0%; +++ = 12%-20.3%; +++ = 20.3%-55.9%; X indicates successful PE3B editing was observed with the PEgRNA (data not shown)

Summary of PEgRNA Used:

PBS

PEgRNA²
Spacer
RTT³
RTT
SEQ
PBS

SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
Length
ID NO:
Length

2633
33; 2293
2335
16
2297
10

2585
33; 2293
2320
13
2299
12

2667
33; 2293
2329
15
2299
12

2744
33; 2293
2335
16
2299
12

2890
33; 2293
2344*
18
2299
12

²Indicated sequence does not contain the transcription adaptations used experimentally (i.e., addition of a 5′G if the spacer did not already start with a G and addition of 1-6 3′U from the U6 transcription termination sequence). The first 20 nt of the ngRNA sequence are the spacer; italics indicate that the spacer is a PE3B spacer.

³* = RTT contains a PAM silencing mutation

TABLE XEc

PE3 screen at the H1069 mutation site in HEK293T cells

PEgRNA²SEQ ID NO:

2675
2729
2898
3165

%
%
%
%
%
%
%
%

Edit¹
indel¹
Edit¹
indel¹
Edit¹
indel¹
Edit¹
indel¹

ngRNA²
PE2
++++
+
++++
++
+++
++
++++
++

SEQ
2263
+++
+
++
++
++
++
+
+

ID
2266
+++
+
+++
++
++++
+
++
+

NO:
2276
+++
+
++
+++
+++
+++
+++
++

2277
++++
+
+++
+++
++++
+++
+++
++

2259
+++
+
++++
++
++++
++
+++
++

2257
+++
+
++++
++
++++
++
X
X

2268
+++
+
+++
++
+++
++
X
X

2264
+++
+
++++
+
++++
+
X
X

2275
++++
+
++++
++
++++
++
++++
++

2273
+++
+
++++
+++
++++
+++
+++
+++

2271
++++
+
+++
+++
++++
++++
+++
+++

2261
+++
+
++++
++
++++
+++
+++
+++

2274
++++
+
++++
+
++++
+
++++
+

2269
++++
++
++++
+
++++
+
++++
++

2258
++++
+
++
+++
+++
++++
++
+++

2270
++++
+
+++
+++
++++
+++
+++
++

2262
++++
+
++++
++
++++
+++
++
+++

2267
+++
+
++++
+
++++
+
+++
++

2265
++++
+
++++
+
++++
+
++
+

2260
++++
+
++++
+
++++
+
++++
+

2272
+
+
++++
+
++++
+
+++
+

4411
+++
+
++++
+
++++
+
+++
++

4418
+++
+
++++
+++
++++
+++
++
+++

4416
++++
+
++++
+
++++
++
+++
++

¹+ = 0.3%-6.3%; ++ = 6.3%-12.0%; +++ = 12%-20.3%; +++ = 20.3%-55.9%; X indicates successful PE3B editing was observed with the PEgRNA (data not shown)

Summary of PEgRNA Used:

PBS

PEgRNA²
Spacer
RTT³
RTT
SEQ
PBS

SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
Length
ID NO:
Length

2675
33; 2293
2320
13
2301
14

2729
33; 2293
2326
14
2301
14

2898
33; 2293
2335
16
2301
14

3165
33; 2293
2344*
18
2303
16

²Indicated sequence does not contain the transcription adaptations used experimentally (i.e., addition of a 5′G if the spacer did not already start with a G and addition of 1-6 3′U from the U6 transcription termination sequence). The first 20 nt of the ngRNA sequence are the spacer; italics indicate that the spacer is a PE3B spacer.

³* = RTT contains a PAM silencing mutation

TABLE XEd

PE3 screen at the H1069 mutation site in HEK293T cells

PEgRNA²SEQ ID NO:

4540
4756
4624
4592
4691

%
%
%
%
%
%
%
%
%
%

Edit¹
indel¹
Edit¹
indel¹
Edit¹
indel¹
Edit¹
indel¹
Edit¹
indel¹

ngRNA²
PE2
++++
+++
+++
++
++++
+
+++
+
++++
+

SEQ
2263
++
+
+
+
++
+
++
+
++
+

ID
2266
+++
+
++
+
+++
+
+++
+
+++
+

NO:
2276
++
+++
++
+++
++
+++
++
+++
++
+++

2277
+++
+++
+++
++
+++
++
+++
++
++++
+++

2259
++++
+++
++++
++
++++
++
++++
++
++++
++

2257
++++
+++
++++
++
++++
++
++++
++
++++
+++

2268
+++
++
+++
+
+++
+
++++
+
++++
++

2264
++++
+
+++
+
+++
+
+++
+
++++
+

2275
++++
++
++++
+
++++
+
++++
+
++++
++

2273
++++
+++
++++
++
++++
++
++++
+++
++++
+++

2271
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++

2261
++++
+++
++++
++
++++
++
++++
++
++++
+++

2274
++++
+
++++
+
++++
+
++++
+
++++
+

2269
++++
++
++++
+
++++
+
++++
+
++++
++

2258
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++

2270
++++
+++
+++
++
+++
+++
++++
+++
+++
+++

2262
+++
+++
+++
+++
++++
++
++++
+++
++++
+++

2267
++++
++
++++
+
++++
+
++++
+
++++
++

2265
++++
+
+++
+
+++
+
+++
+
++++
+

2260
++++
++
++++
+
++++
+
++++
+
++++
++

2272
++++
++
+++
+
++++
+
++++
+
+++
+

4411
++++
++
++++
++
++++
+
++++
++
++++
++

4418
++++
+++
++++
++
++++
+++
++++
+++
++++
+++

4416
++++
+++
++++
++
++++
++
++++
++
++
+

¹+ = 0.3%-6.3%; ++ = 6.3%-12.0%; +++ = 12%-20.3%; +++ = 20.3%-55.9%

Summary of PEgRNA Used:

PBS

PEgRNA²
Spacer
RTT
RTT
SEQ
PBS

SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
Length
ID NO:
Length

4540
58; 4425
4448
15
4427
8

4756
58; 4425
4456
21
4429
10

4624
58; 4425
4448
15
4431
12

4592
58; 4425
4441
12
4433
14

4691
58; 4425
4448
15
4433
14

²Indicated sequence does not contain the transcription adaptations used experimentally (i.e., addition of a 5′G if the spacer did not already start with a G and addition of 1-6 3′U from the U6 transcription termination sequence). The first 20 nt of the ngRNA sequence are the spacer; italics indicate that the spacer is a PE3B spacer.

³* = RTT contains a PAM silencing mutation

Example 5—Prime Editing at a Lentivirus-Introduced ATP7B H1069Q Mutation Site in Primary Human Hepatocytes Using a PE3 System

PEgRNAs from the PE3 screen above were tested in hepatocytes. Primary human hepatocytes were transduced with lentivirus encoding the H1069Q cassette 2 days after cryorecovery, followed 6 days later by transfection with RNA encoding a prime editor, PEgRNA, and (where applicable) ngRNA. Genomic DNA was harvested after a 1-week incubation. Correction of the H1069Q mutation was examined and the results are provided in Table XD. “PET” in the ngRNA SEQ ID NO: column indicates a PE2 editing strategy was used instead of a PE3 editing strategy. These results demonstrate successful Prime Editing of the H1069 mutation site in clinically relevant cells types using both PE2 and PE3 editing strategies.

TABLE XF

PE3 screen at H1069Q mutation site in primary human hepatocytes

PEgRNA¹

RTT

PBS

ngRNA¹

SEQ ID
Spacer
SEQ ID
RTT
SEQ ID
PBS
SEQ ID
%
%

NO:
SEQ ID NO:
NO:
Len.
NO:
Len.
NO:
Edit
Indel

2557
33; 2293
2335
16
2295
8
PE2
++
++

2557
33; 2293
2335
16
2295
8
4414
++
+

2557
33; 2293
2335
16
2295
8
4412
+++
++

2557
33; 2293
2335
16
2295
8
2269
+++
+++

2557
33; 2293
2335
16
2295
8
2260
+++
++

2557
33; 2293
2335
16
2295
8
PE2
+++
++

2557
33; 2293
2335
16
2295
8
2268
++++
++

2557
33; 2293
2335
16
2295
8
2264
++++
++

2557
33; 2293
2335
16
2295
8
2269
+++
+++

2557
33; 2293
2335
16
2295
8
2260
+++
++

4624
58; 4425
4448
15
4431
12
PE2
++++
+

4624
58; 4425
4448
15
4431
12
2268
+
+

4624
58; 4425
4448
15
4431
12
2264
++++
+

4624
58; 4425
4448
15
4431
12
2269
++++
+++

4624
58; 4425
4448
15
4431
12
2260
++++
+

2898
33; 2293
2335
16
2301
14
PE2
++++
+

2898
33; 2293
2335
16
2301
14
2268
++++
++

2898
33; 2293
2335
16
2301
14
2264
++++
+

2898
33; 2293
2335
16
2301
14
2269
++++
++

2898
33; 2293
2335
16
2301
14
2260
+++
+

¹Indicated PEgRNA or ngRNA sequence does not contain the adaptations for transcription from a DNA template used experimentally (i.e., addition of a 5′G if the spacer did not already start with a G and addition of 1-6 3′U from the U6 transcription termination sequence). The first 20 nts of the ngRNA sequence are the spacer; italics indicates the spacer is a PE3B spacer.

²+ = 0.03%-0.15%; ++ = 0.15%-0.55%; +++ = 0.55%-2.67%; ++++ = 2.67%-9.54%

Example 6—Prime Editing at the Endogenous ATP7B H1069 Mutation Site in Mammalian Cells Using Synthetic PEgRNA in a PE2 System

An ATP7B H1069Q mutation was installed at the endogenous ATP7B locus in HEK293T and HepG2 cells by prime editing and single-cell clones were obtained via limiting dilution and clonal expansion, as described in Example 1. A PE2 screen measuring percent correction was performed at the endogenous ATP7B H1069Q locus. The cells were transfected with mRNA encoding a prime editor, and synthetic PEgRNA, as described in Example 1.

The results of the PE2 screen for the HEK and HepG2 are provided in Table XG. These data demonstrate successful Prime Editing at the endogenous ATP7B H1069Q mutation site in multiple mammalian cell models. Successful editing was observed with PEgRNAs containing multiple PBS lengths, multiple RTT lengths, and both with and without PAM silencing mutations.

These experiments were also performed in Huh cells (data not shown).

TABLE XG

PE2 screen at H1069Q mutation site in mammalian

cell culture using synthetic PEgRNAs

PERNA¹

RTT²

% Edit³
% Edit⁴

SEQ ID
Spacer
SEQ ID
RTT
PBS SEQ
PBS
in HEK
in HepG2

NO:
SEQ ID NO:
NO:
Length
ID NO:
Length
cells
cells

4495
58; 4425
4438
10
4427
8
+++
+++

4500
58; 4425
4440
11
4427
8
++++
++++

4504
58; 4425
4441
12
4427
8
++++
++

4517
58; 4425
4444
13
4427
8
++++
+++

4522
58; 4425
4446
14
4427
8
++++
+

4540
58; 4425
4448
15
4427
8
++++
++++

4580
58; 4425
4450
17
4427
8
++++
++++

4591
58; 4425
4452
18
4427
8
++++
+++

4667
58; 4425
4456
21
4427
8
++++
+++

4499
58; 4425
4438
10
4428
9
++++
++++

4508
58; 4425
4440
11
4428
9
+++

4509
58; 4425
4441
12
4428
9
++++
+++

4524
58; 4425
4444
13
4428
9
++++
++++

4531
58; 4425
4446
14
4428
9
++++
++++

4545
58; 4425
4448
15
4428
9
++++
++

4603
58; 4425
4450
17
4428
9
++++
++

4627
58; 4425
4452
18
4428
9
++++
++++

4710
58; 4425
4456
21
4428
9
++++
+++

4507
58; 4425
4438
10
4429
10
+++
+++

4514
58; 4425
4440
11
4429
10
++++
+++

4527
58; 4425
4441
12
4429
10
+++
++++

4533
58; 4425
4444
13
4429
10
++++
++++

4555
58; 4425
4446
14
4429
10
++++
+++

4564
58; 4425
4448
15
4429
10
++++
++++

4612
58; 4425
4450
17
4429
10
++++
+++

4649
58; 4425
4452
18
4429
10
++++
+++

4756
58; 4425
4456
21
4429
10
++++
++++

4510
58; 4425
4438
10
4430
11
+++
++++

4529
58; 4425
4440
11
4430
11
++++
++++

4544
58; 4425
4441
12
4430
11
++++
+++

4556
58; 4425
4444
13
4430
11
++++
++++

4579
58; 4425
4446
14
4430
11
++++
++++

4589
58; 4425
4448
15
4430
11
++++
++++

4634
58; 4425
4450
17
4430
11
++++
++++

4675
58; 4425
4452
18
4430
11
++++
+++

4759
58; 4425
4456
21
4430
11
++++
++++

4519
58; 4425
4438
10
4431
12
++
++++

4542
58; 4425
4440
11
4431
12
++++
+++

4559
58; 4425
4441
12
4431
12
++++
+++

4576
58; 4425
4444
13
4431
12
++++
+++

4588
58; 4425
4446
14
4431
12
++++
++++

4624
58; 4425
4448
15
4431
12
++++
++++

4679
58; 4425
4450
17
4431
12
++++
++++

4697
58; 4425
4452
18
4431
12
+++
++++

4817
58; 4425
4456
21
4431
12
+++
+++

4532
58; 4425
4438
10
4432
13
++++
++++

4558
58; 4425
4440
11
4432
13
+++
++++

4577
58; 4425
4441
12
4432
13
++++
++++

4605
58; 4425
4444
13
4432
13
++++
++++

4623
58; 4425
4446
14
4432
13
++++
++

4657
58; 4425
4448
15
4432
13
++++
++++

4719
58; 4425
4450
17
4432
13
++++

4740
58; 4425
4452
18
4432
13
++++
++++

4827
58; 4425
4456
21
4432
13
++++
++++

4553
58; 4425
4438
10
4433
14
+++
++

4562
58; 4425
4440
11
4433
14
++++
++++

4592
58; 4425
4441
12
4433
14
++++
++++

4618
58; 4425
4444
13
4433
14
++++
+++

4640
58; 4425
4446
14
4433
14
++++
+++

4691
58; 4425
4448
15
4433
14
++++
+++

4726
58; 4425
4450
17
4433
14
++
++++

4761
58; 4425
4452
18
4433
14
+++
+++

4852
58; 4425
4456
21
4433
14
++
++++

4575
58; 4425
4438
10
4434
15
++
++++

4595
58; 4425
4440
11
4434
15
+++
+++

4619
58; 4425
4441
12
4434
15
+++
++++

4638
58; 4425
4444
13
4434
15
++
+++

4684
58; 4425
4446
14
4434
15
++
+++

4720
58; 4425
4448
15
4434
15
++
++++

4758
58; 4425
4450
17
4434
15
++
+++

4812
58; 4425
4452
18
4434
15
++
++++

4875
58; 4425
4456
21
4434
15
+
+++

4586
58; 4425
4438
10
4435
16
+++
++++

4629
58; 4425
4440
11
4435
16
++++
++++

4653
58; 4425
4441
12
4435
16
++++
+++

4668
58; 4425
4444
13
4435
16
++++
++++

4724
58; 4425
4446
14
4435
16
++++
++++

4738
58; 4425
4448
15
4435
16
++++
+++

4809
58; 4425
4450
17
4435
16
++++
++

4840
58; 4425
4452
18
4435
16
+
++++

4893
58; 4425
4456
21
4435
16
++++
++++

4614
58; 4425
4438
10
4436
17
++++
+++

4655
58; 4425
4440
11
4436
17
+++
++++

4674
58; 4425
4441
12
4436
17
++++
+++

4722
58; 4425
4444
13
4436
17
++
++

4748
58; 4425
4446
14
4436
17
++
+++

4785
58; 4425
4448
15
4436
17
+++
+++

4835
58; 4425
4450
17
4436
17
+++
+++

4856
58; 4425
4452
18
4436
17
+++
++++

4912
58; 4425
4456
21
4436
17
+++
+++

2453
33; 2293
2307
11
2295
8
++
++

2480
33; 2293
2307
11
2297
10
+++
+++

2522
33; 2293
2307
11
2299
12
+++
+++

2606
33; 2293
2307
11
2301
14
++
+++

2683
33; 2293
2307
11
2303
16
+++
+++

2459
33; 2293
2313
12
2295
8
++++
++++

2510
33; 2293
2313
12
2297
10
++++
++++

2575
33; 2293
2313
12
2299
12
+++
++++

2623
33; 2293
2313
12
2301
14
++++
++++

2758
33; 2293
2313
12
2303
16
++++
++++

2489
33; 2293
2320
13
2295
8

++++

2533
33; 2293
2320
13
2297
10

++++

2585
33; 2293
2320
13
2299
12

++++

2675
33; 2293
2320
13
2301
14
+++
+++

2825
33; 2293
2320
13
2303
16
+++
+++

2515
33; 2293
2326
14
2295
8
+++
+++

2559
33; 2293
2326
14
2297
10
++++
++++

2628
33; 2293
2326
14
2299
12
+++
++++

2729
33; 2293
2326
14
2301
14
++++
++++

2844
33; 2293
2326
14
2303
16
+++
+++

2548
33; 2293
2329
15
2295
8
++++
++++

2605
33; 2293
2329
15
2297
10
++++
++++

2667
33; 2293
2329
15
2299
12
++++
+++

2802
33; 2293
2329
15
2301
14
++++
++++

2932
33; 2293
2329
15
2303
16
+++
++++

2557
33; 2293
2335
16
2295
8
++++
+++

2633
33; 2293
2335
16
2297
10
++++
++++

2744
33; 2293
2335
16
2299
12
++++
++++

2898
33; 2293
2335
16
2301
14
++++
+++

2993
33; 2293
2335
16
2303
16
++++
++++

2637
33; 2293
2342
18
2295
8
++++
+++

2769
33; 2293
2342
18
2297
10
++++
++++

2886
33; 2293
2342
18
2299
12
++++
+++

3043
33; 2293
2342
18
2301
14
++++
++++

3144
33; 2293
2342
18
2303
16
++++
++++

2689
33; 2293
2351
19
2295
8
++++
+++

2801
33; 2293
2351
19
2297
10
+++
+++

2916
33; 2293
2351
19
2299
12
+++
+++

3076
33; 2293
2351
19
2301
14
+++
+++

3263
33; 2293
2351
19
2303
16
+++
+++

2871
33; 2293
2353
22
2295
8
+++
++

2983
33; 2293
2353
22
2297
10
+++
+++

3163
33; 2293
2353
22
2299
12
+++
+++

3287
33; 2293
2353
22
2301
14
+++
+++

3441
33; 2293
2353
22
2303
16
+++
++

2458
33; 2293
2310*
11
2295
8
+
++

2487
33; 2293
2310*
11
2297
10
+
++

2526
33; 2293
2310*
11
2299
12
+
++

2609
33; 2293
2310*
11
2301
14
+
++

2680
33; 2293
2310*
11
2303
16
+
+

2466
33; 2293
2311*
12
2295
8
+
+

2513
33; 2293
2311*
12
2297
10
+
++

2554
33; 2293
2311*
12
2299
12
+
++

2660
33; 2293
2311*
12
2301
14
+
++

2759
33; 2293
2311*
12
2303
16
+
++

2490
33; 2293
2321*
13
2295
8
+
++

2531
33; 2293
2321*
13
2297
10
+
+

2610
33; 2293
2321*
13
2299
12
++
++

2685
33; 2293
2321*
13
2301
14
+
+

2814
33; 2293
2321*
13
2303
16
+
++

2512
33; 2293
2328*
14
2295
8
++
+

2561
33; 2293
2328*
14
2297
10
+
++

2643
33; 2293
2328*
14
2299
12
+
+

2764
33; 2293
2328*
14
2301
14
+
+

2843
33; 2293
2328*
14
2303
16
+
++

2546
33; 2293
2334*
15
2295
8
++
++

2615
33; 2293
2334*
15
2297
10
++
++

2701
33; 2293
2334*
15
2299
12
++
+++

2777
33; 2293
2334*
15
2301
14
++
++

2964
33; 2293
2334*
15
2303
16
++
++

2568
33; 2293
2339*
16
2295
8
++
++

2645
33; 2293
2339*
16
2297
10
++
++

2735
33; 2293
2339*
16
2299
12
++
+++

2893
33; 2293
2339*
16
2301
14
+++
+++

2988
33; 2293
2339*
16
2303
16
+++
+++

2638
33; 2293
2343*
18
2295
8
+++
++

2772
33; 2293
2343*
18
2297
10
+++
++

2885
33; 2293
2343*
18
2299
12
+++
+++

3015
33; 2293
2343*
18
2301
14
+++
++

3123
33; 2293
2343*
18
2303
16
+++
++

2690
33; 2293
2348*
19
2295
8
+++
+

2826
33; 2293
2348*
19
2297
10
+++
+++

2960
33; 2293
2348*
19
2299
12
+++
++

3080
33; 2293
2348*
19
2301
14
+++
++

3248
33; 2293
2348*
19
2303
16
+++
++

2891
33; 2293
2355*
22
2295
8
++
++

3012
33; 2293
2355*
22
2297
10
++
++

3188
33; 2293
2355*
22
2299
12
++
+++

3301
33; 2293
2355*
22
2301
14
+
++

3430
33; 2293
2355*
22
2303
16
++
++

2457
33; 2293
2306*
11
2295
8
+
++

2488
33; 2293
2306*
11
2297
10
+
++

2544
33; 2293
2306*
11
2299
12
+
+

2584
33; 2293
2306*
11
2301
14
+
++

2702
33; 2293
2306*
11
2303
16
++
+

2461
33; 2293
2316*
12
2295
8
+
++

2493
33; 2293
2316*
12
2297
10
+
++

2563
33; 2293
2316*
12
2299
12
++

2619
33; 2293
2316*
12
2301
14
+

2757
33; 2293
2316*
12
2303
16
+

2484
33; 2293
2319*
13
2295
8
++

2527
33; 2293
2319*
13
2297
10
++

2590
33; 2293
2319*
13
2299
12
+++

2670
33; 2293
2319*
13
2301
14
++

2800
33; 2293
2319*
13
2303
16
+++

2509
33; 2293
2325*
14
2295
8
++

2571
33; 2293
2325*
14
2297
10
++

2665
33; 2293
2325*
14
2299
12
+++

2730
33; 2293
2325*
14
2301
14
++

2841
33; 2293
2325*
14
2303
16
++
++

2541
33; 2293
2332*
15
2295
8
++
+

2598
33; 2293
2332*
15
2297
10
++++
+++

2674
33; 2293
2332*
15
2299
12
+++
++

2832
33; 2293
2332*
15
2301
14
+++
++

2933
33; 2293
2332*
15
2303
16
+++
++

2551
33; 2293
2337*
16
2295
8
+++
+++

2634
33; 2293
2337*
16
2297
10
+++
++

2733
33; 2293
2337*
16
2299
12
+++
++

2889
33; 2293
2337*
16
2301
14
+++
+++

2979
33; 2293
2337*
16
2303
16
+++
++

2636
33; 2293
2344*
18
2295
8
+++
+

2765
33; 2293
2344*
18
2297
10
++

2890
33; 2293
2344*
18
2299
12
+

2982
33; 2293
2344*
18
2301
14
+

3165
33; 2293
2344*
18
2303
16
++

2706
33; 2293
2352*
19
2295
8
++

2807
33; 2293
2352*
19
2297
10
+

2962
33; 2293
2352*
19
2299
12
+++

3072
33; 2293
2352*
19
2301
14
++

3210
33; 2293
2352*
19
2303
16
+

2894
33; 2293
2358*
22
2295
8
++

2977
33; 2293
2358*
22
2297
10
++

3166
33; 2293
2358*
22
2299
12
++

3300
33; 2293
2358*
22
2301
14
+++
++

3442
33; 2293
2358*
22
2303
16
+++
+

2451
33; 2293
2308*
11
2295
8
+
++

2478
33; 2293
2308*
11
2297
10
+
++

2528
33; 2293
2308*
11
2299
12
+
++

2602
33; 2293
2308*
11
2301
14
+
++

2711
33; 2293
2308*
11
2303
16
+
+

2460
33; 2293
2312*
12
2295
8
++
+

2514
33; 2293
2312*
12
2297
10
+
++

2562
33; 2293
2312*
12
2299
12
+
++

2647
33; 2293
2312*
12
2301
14
++
++

2760
33; 2293
2312*
12
2303
16
+
++

2473
33; 2293
2318*
13
2295
8
++

2520
33; 2293
2318*
13
2297
10
+

2596
33; 2293
2318*
13
2299
12
+

2677
33; 2293
2318*
13
2301
14
+

2830
33; 2293
2318*
13
2303
16
+

2501
33; 2293
2324*
14
2295
8
++

2553
33; 2293
2324*
14
2297
10
++

2646
33; 2293
2324*
14
2299
12
++

2749
33; 2293
2324*
14
2301
14
+

2842
33; 2293
2324*
14
2303
16
++

2535
33; 2293
2333*
15
2295
8
+

2618
33; 2293
2333*
15
2297
10
++

2699
33; 2293
2333*
15
2299
12
+++
+

2816
33; 2293
2333*
15
2301
14
+++
++

2926
33; 2293
2333*
15
2303
16
+++
++

2564
33; 2293
2338*
16
2295
8
+++
++

2632
33; 2293
2338*
16
2297
10
+++
+

2734
33; 2293
2338*
16
2299
12
+++
+++

2834
33; 2293
2338*
16
2301
14
++
+++

2972
33; 2293
2338*
16
2303
16
++
+

2635
33; 2293
2346*
18
2295
8
+++
+

2773
33; 2293
2346*
18
2297
10
+++
++

2899
33; 2293
2346*
18
2299
12
+++
++

3013
33; 2293
2346*
18
2301
14
+++
+

3148
33; 2293
2346*
18
2303
16
++

2669
33; 2293
2347*
19
2295
8
++

2829
33; 2293
2347*
19
2297
10
++
+

2941
33; 2293
2347*
19
2299
12
++
+

3071
33; 2293
2347*
19
2301
14
++
+

3200
33; 2293
2347*
19
2303
16
++
+

2856
33; 2293
2356*
22
2295
8
++
+

3023
33; 2293
2356*
22
2297
10
++
+

3122
33; 2293
2356*
22
2299
12
++
+

3275
33; 2293
2356*
22
2301
14
++
+

3420
33; 2293
2356*
22
2303
16
+
+

1550
1505; 2055
1518
21
1507
8
+

1565
1505; 2055
1518
21
1509
10
+
+

1578
1505; 2055
1518
21
1511
12
+
+

1602
1505; 2055
1518
21
1513
14
+
+

1640
1505; 2055
1518
21
1515
16
+
+

1554
1505; 2055
1519
22
1507
8
+
+

1569
1505; 2055
1519
22
1509
10
+
+

1591
1505; 2055
1519
22
1511
12
+
+

1626
1505; 2055
1519
22
1513
14
+
+

1654
1505; 2055
1519
22
1515
16
+
+

1562
1505; 2055
1522
23
1507
8
+
+

1580
1505; 2055
1522
23
1509
10
+
+

1609
1505; 2055
1522
23
1511
12
+
+

1628
1505; 2055
1522
23
1513
14

1691
1505; 2055
1522
23
1515
16

1570
1505; 2055
1524
24
1507
8

1593
1505; 2055
1524
24
1509
10

1616
1505; 2055
1524
24
1511
12

1664
1505; 2055
1524
24
1513
14

1708
1505; 2055
1524
24
1515
16

1583
1505; 2055
1526
25
1507
8

1600
1505; 2055
1526
25
1509
10

1643
1505; 2055
1526
25
1511
12

1671
1505; 2055
1526
25
1513
14

1725
1505; 2055
1526
25
1515
16

1551
1505; 2055
1517*
21
1507
8
+
+

1564
1505; 2055
1517*
21
1509
10
+
+

1586
1505; 2055
1517*
21
1511
12
+
+

1606
1505; 2055
1517*
21
1513
14
+
+

1646
1505; 2055
1517*
21
1515
16
+
+

1553
1505; 2055
1520*
22
1507
8
+
+

1575
1505; 2055
1520*
22
1509
10
+
+

1587
1505; 2055
1520*
22
1511
12
+
+

1615
1505; 2055
1520*
22
1513
14
+
+

1663
1505; 2055
1520*
22
1515
16
+
+

1566
1505; 2055
1521*
23
1507
8
+
+

1577
1505; 2055
1521*
23
1509
10
++
+

1607
1505; 2055
1521*
23
1511
12
+

1645
1505; 2055
1521*
23
1513
14
+

1678
1505; 2055
1521*
23
1515
16
++

1567
1505; 2055
1523*
24
1507
8
+

1590
1505; 2055
1523*
24
1509
10
++

1622
1505; 2055
1523*
24
1511
12
++

1668
1505; 2055
1523*
24
1513
14
+++

1694
1505; 2055
1523*
24
1515
16
+

1584
1505; 2055
1525*
25
1507
8
+++

1598
1505; 2055
1525*
25
1509
10
+++

1637
1505; 2055
1525*
25
1511
12
++

1679
1505; 2055
1525*
25
1513
14
+++

1721
1505; 2055
1525*
25
1515
16
+++
+

804
682; 2087
711
28
684
8
+++
+

852
682; 2087
711
28
686
10
++
+

896
682; 2087
711
28
688
12
++
+

989
682; 2087
711
28
690
14
++
+

911
682; 2087
719
32
684
8
++
+

979
682; 2087
719
32
686
10
++
+

1046
682; 2087
719
32
688
12
+
+

1147
682; 2087
719
32
690
14
+
+

992
682; 2087
720
34
684
8
++
+

1045
682; 2087
720
34
686
10
++
+

1172
682; 2087
720
34
688
12
++
+

1244
682; 2087
720
34
690
14
+

1124
682; 2087
724
37
684
8
++

1220
682; 2087
724
37
686
10
+

1270
682; 2087
724
37
688
12
++

1342
682; 2087
724
37
690
14
++

¹Indicated PEgRNA sequence does not contain the 3′ linker and hairpin motif used experimentally. The experimental PEgRNA further contained 3′ mN*mN*mN*N and 5′mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2′-O-Me modification and a * indicates a phosphorothioate bond.

²* = RTT contains a PAM silencing mutation

³+ = 0.01%-0.77%, ++ = 0.77%-5.09%, +++ = 5.09%-15.96%, ++++ = 15.96%-45.89%

⁴+ = 0.01%-1.66%, ++ = 1.66%-2.62%, +++ = 2.62%-5.25%, ++++ = 5.25%-17.71%

Example 7—Prime Editing with Silent Recoding at H1069Q Mutation Site

A PE2 screen measuring percent correction and percent indel formation was performed in HEK 293T at the endogenous ATP7B H1069Q locus. The cells were transfected with mRNA encoding a prime editor, and synthetic PEgRNA, as described in Example 1. Unlike previous examples, the PEgRNA used here were not designed to restore the wild-type nucleic acid sequence at the H1069Q site. Rather, the PEgRNA used here were designed to restore the wild-type amino acid sequence using an alternative histidine codon. The results presented in Table XH demonstrate successful recoding at the H1069Q mutation site using PEgRNA having multiple RTT and PBS length combinations.

TABLE XH

PE2 screen with silent recoding at H1069Q mutation site

in mammalian cell culture using synthetic PEgRNAs

PEgRNA¹

SEQ ID
Spacer
RTT SEQ
RTT
PBS SEQ
PBS

NO:
SEQ ID NO:
ID NO:
Length
ID NO:
Length
% Edit²
% Indel²

4494
58; 4425
4437
10
4427
8
++
+

4501
58; 4425
4439
11
4427
8
+++
+

4503
58; 4425
4442
12
4427
8
+++
+

4513
58; 4425
4443
13
4427
8
+++
+

4525
58; 4425
4445
14
4427
8
++++
+

4530
58; 4425
4447
15
4427
8
+++
+

4568
58; 4425
4451
17
4427
8
+++
+

4597
58; 4425
4453
18
4427
8
++++
++

4682
58; 4425
4455
21
4427
8
+++
+

4498
58; 4425
4437
10
4428
9
+++
+

4505
58; 4425
4439
11
4428
9
+++
+

4512
58; 4425
4442
12
4428
9
++++
+

4526
58; 4425
4443
13
4428
9
++++
+

4538
58; 4425
4445
14
4428
9
++++
++

4547
58; 4425
4447
15
4428
9
++++
++

4583
58; 4425
4451
17
4428
9
++++
++

4617
58; 4425
4453
18
4428
9
++++
++

4718
58; 4425
4455
21
4428
9
++++
++

4502
58; 4425
4437
10
4429
10
+++
+

4511
58; 4425
4439
11
4429
10
+++
+

4521
58; 4425
4442
12
4429
10
+++
+

4543
58; 4425
4443
13
4429
10
+++
+

4561
58; 4425
4445
14
4429
10
++++
++

4581
58; 4425
4447
15
4429
10
++++
++

4626
58; 4425
4451
17
4429
10
++++
++

4651
58; 4425
4453
18
4429
10
++++
++

4739
58; 4425
4455
21
4429
10
+++
++

4515
58; 4425
4437
10
4430
11
+++
+

4523
58; 4425
4439
11
4430
11
+++
+

4537
58; 4425
4442
12
4430
11
++++
+

4557
58; 4425
4443
13
4430
11
+++
+

4567
58; 4425
4445
14
4430
11
++++
++

4584
58; 4425
4447
15
4430
11
++++
++

4639
58; 4425
4451
17
4430
11
++++
++

4662
58; 4425
4453
18
4430
11
++++
++

4776
58; 4425
4455
21
4430
11
+++
++

4528
58; 4425
4437
10
4431
12
+++
+

4535
58; 4425
4439
11
4431
12
++++
+

4552
58; 4425
4442
12
4431
12
++++
+

4570
58; 4425
4443
13
4431
12
++++
+

4596
58; 4425
4445
14
4431
12
+++
+++

4621
58; 4425
4447
15
4431
12
+++
+

4676
58; 4425
4451
17
4431
12
++++
++

4702
58; 4425
4453
18
4431
12
+++
+

4795
58; 4425
4455
21
4431
12
+++
+

4539
58; 4425
4437
10
4432
13
+++
+

4546
58; 4425
4439
11
4432
13
++++
+

4573
58; 4425
4442
12
4432
13
++++
+

4585
58; 4425
4443
13
4432
13
++++
++

4630
58; 4425
4445
14
4432
13
++++
++

4660
58; 4425
4447
15
4432
13
++++
+++

4714
58; 4425
4451
17
4432
13
++++
++

4751
58; 4425
4453
18
4432
13
++++
++

4820
58; 4425
4455
21
4432
13
+++
++

4549
58; 4425
4437
10
4433
14
+++
+

4574
58; 4425
4439
11
4433
14
++++
++

4582
58; 4425
4442
12
4433
14
++++
++

4625
58; 4425
4443
13
4433
14
++++
+

4646
58; 4425
4445
14
4433
14
++++
++

4670
58; 4425
4447
15
4433
14
++++
++

4725
58; 4425
4451
17
4433
14
++++
+++

4784
58; 4425
4453
18
4433
14
++++
++

4857
58; 4425
4455
21
4433
14
+++
++

4578
58; 4425
4437
10
4434
15
+++
+

4604
58; 4425
4439
11
4434
15
+++
+

4620
58; 4425
4442
12
4434
15
++++
+

4659
58; 4425
4443
13
4434
15
+++
+

4677
58; 4425
4445
14
4434
15
++++
++

4717
58; 4425
4447
15
4434
15
++++
++

4760
58; 4425
4451
17
4434
15
+++
++

4802
58; 4425
4453
18
4434
15
+++
++

4874
58; 4425
4455
21
4434
15
+++
++

4587
58; 4425
4437
10
4435
16
++
+

4606
58; 4425
4439
11
4435
16
+++
+

4654
58; 4425
4442
12
4435
16
++++
+

4673
58; 4425
4443
13
4435
16
+++
+

4698
58; 4425
4445
14
4435
16
++++
+

4737
58; 4425
4447
15
4435
16
++++
++

4804
58; 4425
4451
17
4435
16
+++
++

4822
58; 4425
4453
18
4435
16
+++
++

4902
58; 4425
4455
21
4435
16
+++
++

4632
58; 4425
4437
10
4436
17
++
+

4656
58; 4425
4439
11
4436
17
+++
+

4689
58; 4425
4442
12
4436
17
++++
+

4711
58; 4425
4443
13
4436
17
+++
+

4746
58; 4425
4445
14
4436
17
++++
++

4780
58; 4425
4447
15
4436
17
++++
++

4841
58; 4425
4451
17
4436
17
+++
++

4860
58; 4425
4453
18
4436
17
+++
++

4915
58; 4425
4455
21
4436
17
+++
++

¹Indicated PEgRNA sequence does not contain the 3′ mU*mU*mU*U and 5′mN*mN*mN* modifications used experimentally, where m indicates that the indicated nucleotide contains a 2′-O-Me modification and a * indicates a phosphorothioate bond.

²+ = 0-0.72%, ++ = 0.72%-2.33%, +++ = 2.33%-11.14%, ++++ = 11.14%-22.15%

Example 8—Phenotypic Rescue by Prime Editing

Patient Fibroblast cells (GM05798) harboring homozygous H1069Q mutation were obtained from Coriell Institute. Fibroblast cells were propagated in EMEM with 15% FBS (not HI). 10 K cells were plated in 96-well plate and twenty-fours later cells were transfected with Messenger Max according to the manufacturer's directions with mRNA encoding a prime editor fusion protein, PEgRNA and NgRNA. Following transfection, the cells were challenged with copper (Cu) at a concentration of 500 uM. Twenty-four hours later, phenotypic rescue of the edited was measured by cell viability assay using cell titer glow from Promega according to the manufacture's protocol. The viability of the edited cells was normalized to the transfected cells with 0 Cu treatment and the phenotypic recue was measured relative to the untransfected cells challenged with the Cu at 500 uM. Editing of these cells were measured in parallel by harvesting cells in quick DNA extract for high throughput sequencing and sequenced using miseq.

The correlation between the percent correction and percent cell viability rescue data sets was analyzed assuming Gaussian distribution yielding Pearson correlation coefficients of 0.8970 with 95% confidence interval and R²value of 0.80. The P-value<0.0001 was calculated using 2-tailed test with 95% confidence interval. These data and analyses show that the level of phenotypic rescue in patient fibroblast cell populations correlates positively with percent correction observed in those populations.

TABLE XI

Phenotypic Rescue in Patient Fibroblast cells

RNA Dose Level
Percent
Percent Cell

(1-high to 20-low)
Correction¹
Viability Rescue²

1
++++
++++

2
++++
++++

3
++++
++++

4
++++
++++

5
++++
++++

6
++++
++++

7
+++
+++

8
+++
+++

9
+++
++

10
+++
+++

11
+++
+++

12
++
++

13
++
+

14
++
++

15
++
+

16
++
++

17
+
+

18
+
+++

19
+
++

20
+
+

21
+
+

¹+ = 0.06-0.48%, ++ = 0.48%-3.84%, + ++ = 3.84%-30.05%, +++ + = 30.05%-47.97%

²+ = −10.3-5.14%, ++ = 5.14%-11.4%, +++ = 11.4%-31.58%, ++++ = 31.58%-55.66%

Example 9—Prime Editing at the Endogenous ATP7B H1069 Mutation Site in HEK293T Cells Using Synthetic PEgRNA and ngRNA

A PE3 screen measuring percent correction and percent indels was performed at the endogenous ATP7B H1069Q locus. The cells were transfected with mRNA encoding a prime editor, and synthetic PEgRNA and ngRNA, as described in Example 1.

The results of the PE3 screen are provided in Tables XEa-XEe. Below each of Tables XEa-XEe is a table summarizing the PEgRNAs used experimentally. Each of the PEgRNA were tested in combination with multiple ngRNA. Some of the ngRNA were designed for a PE3B strategy and contain spacers complementary to the portion of the edit strand containing the edit. These results demonstrate the successful correction of the H1069Q mutation at the endogenous ATP7B locus in mammalian cells using both PE3 and PE3B Prime Editing systems.

TABLE Ya

PE3 screen at the H1069 mutation site in HEK293T

cells with synthetic PEgRNA and ngRNA

PEgRNA²SEQ ID NO:

4499
4533
4522
4518
4540

%
%
%
%
%
%
%
%
%
%

Edit¹
indel¹
Edit¹
indel¹
Edit¹
indel¹
Edit¹
indel¹
Edit¹
indel¹

ngRNA²
PE2
+++
+
+++
+
+++
+
+++
+
++++
+

SEQ
2257
+++
+
++++
++
++++
+
+++
++
++++
++

ID
2268
++++
++
++++
++
++++
++
++++
+
++++
+

NO:
2264
+++
+
++++
+
++++
++
++++
+
++++
+

2261
++
++
+++
++
+++
++
+++
++
+++
+++

2274
++
+++
++++
+++
+++
+++
+++
++
++++
++

2269
+++
++
++++
+++
+++
+++
+++
++
+++
++

2270
++
++
++++
++
++++
++
+++
+
++++
++

2265
+++
++
++++
++
++++
++
++++
++
++++
++

2260
+++
++
++++
++
++++
++
++++
++
++++
++

2272
+++
++
++++
++
++++
++
+++
+
++++
++

4415
++
+
++++
+
++++
+
+++
+
+++
+

4414
+++
+
++++
+
++++
+
++++
+
++++
+

4414
++++
+
++++
+
++++
+
++++
+
++++
+

4412
++++
+
++++
+
+
+
++++
+
++++
+

4412
+++
+
++++
+
++++
+
++++
+
++++
+

4. + = 0.1%-2.0%; ++ = 2.0%-11.7%; +++ = 11.7%-34.85%; ++++ = 20.3%-55.9%

Summary of PEgRNA Used:

PBS

PEgRNA²
Spacer
RTT³
RTT
SEQ
PBS

SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
Length
ID NO:
Length

4499
58
4438
10
4428
9

4533
58
4444
13
4429
10

4522
58
4446
14
4427
8

4518
58
4448
15
4426
7

4540
58
4448
15
4427
8

5. Indicated sequence does not contain the 3′ linker and hairpin motif used experimentally. The experimental PEgRNA further contained 3′ mN*mN*mN*N and 5′mN*mN*mN* modifications, where m

indicates that the nucleotide contains a 2′-O—Me modification and a * indicates a phosphorothioate

bond .. The first 20 nt of the ngRNA sequence are the spacer; italics indicate that the spacer is a PE3B

spacer.

6. * = RTT contains a PAM silencing mutation.

TABLE Yb

PE3 screen at the H1069 mutation site in HEK293T

cells with synthetic PERNA and ngRNA

PEgRNA²SEQ ID NO:

4657
4691
4560
#
4603

%
%
%
%
%
%
%
%
%
%

Edit¹
indel¹
Edit¹
indel¹
Edit¹
indel¹
Edit¹
indel¹
Edit¹
indel¹

ngRNA²
PE2
+++
+
+++
+
+++
+
+++
+
++++
+

SEQ
2257
++++
++
++++
++
+++
++
+++
+
++++
++

ID
2268
++++
++
++++
++
++++
+
++++
+
++++
++

NO:
2264
++++
+
++++
+
++++
+
++++
+
++++
+

2261
+++
++
+++
++
+++
++
+++
++
++++
++

2274
+++
++
+++
++
+++
++
+++
++
++++
++

2269
+++
+++
++++
+++
+++
++
+++
++
+++
++

2270
++++
++
++++
++
++++
++
+++
++
++++
++

2265
++++
++
++++
++
++++
++
+++
++
++++
++

2260
++++
++
++++
++
++++
++
+++
++
++++
++

2272
++++
++
+++
++
+++
++
+++
+
++++
++

4415
++++
+
++++
+
+++
+
+++
+
++++
+

4414
++++
+
++++
+
++++
+
++++
+
++++
+

4414
++++
+
++++
+
++++
+
++++
+
++++
+

4412
++++
+
++++
+
++++
+
++++
+
++++
+

4412
++++
+
++++
+
++++
+
++++
+
++++
+

¹+ = 0.1%-2.0%; ++ = 2.0%-11.7%; +++ = 11.7%-34.85%; ++++ = 20.3%-55.9%

Summary of PEgRNA Used:

PBS

PEgRNA²
Spacer
RTT³
RTT
SEQ
PBS

SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
Length
ID NO:
Length

4657
58
4448
15
4432
13

4691
58
4448
15
4433
14

4560
58
4449
16
4427
8

#
58
4449
16
4433
14

4603
58
4450
17
4428
9

²Indicated sequence does not contain the 3′ linker and hairpin motif used experimentally. The experimental PEgRNA further contained 3′ mN*mN*mN*N and 5′mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2′-O—Me modification and a * indicates a phosphorothioate bond. The first 20 nt of the ngRNA sequence are the spacer; italics indicate that the spacer is a PE3B spacer.

³* = RTT contains a PAM silencing mutation.

TABLE Yc

PE3 screen at the H1069 mutation site in HEK293T

cells with synthetic PEgRNA and ngRNA

PEgRNA²SEQ ID NO:

4719
4649
4592
4592
4624

%
%
%
%
%
%
%
%
%
%

Edit¹
indel¹
Edit¹
indel¹
Edit¹
indel¹
Edit¹
indel¹
Edit¹
indel¹

ngRNA²
PE2
+++
+
+++
+
+++
+
+++
+
+++
+

SEQ
2257
+++
++
+++
+
+++
+
+++
+
++++
++

ID
2268
++++
++
++++
+
++++
+
++++
++
++++
++

NO:
2264
++++
+
++++
+
++++
+
++++
+
++++
+

2261
+++
++
+++
++
+++
++
+++
++
++++
+++

2274
+++
++
+++
++
+++
++
+++
++
+++
+++

2269
+++
++
+++
++

+++
++
+++
+++

2270
+++
+
+++
++
+++
+
+++
++
++++
++

2265
++++
++
++++
++
++++
++
+++
++
++++
++

2260
++++
++
++++
++
+++
++
++++
++
++++
++

2272
++++
++
+++
+
+++
++
+++
++
++++
++

4415
++++
+
+++
+

++++
+

4414
++++
+
++++
+
++++
+
++++
+
++++
+

4414
++++
+
++++
++
++++
+
++++
+
++++
+

4412
++++
+
++++
+
++++
+
++++
+
++++
+

4412
++++
+
++++
+
++++
+
++++
+
++++
+

¹+ = 0.1%-2.0%; ++ = 2.0%-11.7%; +++ = 11.7%-34.85%; ++++ = 20.3%-55.9%

Summary of PEgRNA Used:

PBS

PEgRNA²
Spacer
RTT³
RTT
SEQ
PBS

SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
Length
ID NO:
Length

4719
58
4450
17
4432
13

4649
58
4452
18
4429
10

4592
58
4441
12
4433
14

4592
58
4441
12
4433
14

4624
58
4448
15
4431
12

²Indicated sequence does not contain the 3′ linker and hairpin motif used experimentally. The experimental PEgRNA further contained 3′ mN*mN*mN*N and 5′mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2′-O—Me modification and a * indicates a phosphorothioate bond. The first 20 nt of the ngRNA sequence are the spacer; italics indicate that the spacer is a PE3B spacer.

³* = RTT contains a PAM silencing mutation.

TABLE Yd

PE3 screen at the H1069 mutation site in HEK293T

cells with synthetic PEgRNA and ngRNA

PEgRNA²SEQ ID NO:

4624
4720
4720
4852
4852

%
%
%
%
%
%
%
%
%
%

Edit¹
indel¹
Edit¹
indel¹
Edit¹
indel¹
Edit¹
indel¹
Edit¹
indel¹

ngRNA²
PE2
+++
+
+++
+
+++
+
+++
+
+++
+

SEQ
2257
+++
++
+++
++
+++
++
+++
++
+++
+

ID
2268
++++
+
++++
++
++++
++
++++
+
++++
+

NO:
2264
++++
+
++++
+
++++
+
++++
+
++++
+

2261
+++
++
+++
++
+++
++
+++
++
+++
++

2274
+++
++
+++
++
+++
++
+++
++
+++
++

2269
+++
++
+++
++
+++
++

+++
++

2270
+++
+
+++
++
+++
++

2265
+++
++
+++
++
+++
++

++++
++

2260
++++
++
+++
++
+++
+
++++
++
++++
++

2272
+++
++
+++
++
+++
+
++++
++
+++
++

4415
+++
+
+++
+
+++
+

4414
++++
+
++++
+
++++
+

4414
++++
+
++++
+
++++
+
++++
+
++++
+

4412
++++
++
++++
+
++++
+

4412
++++
+
++++
+
++++
+
++++
+
++++
+

¹+ = 0.1%-2.0%; ++ = 2.0%-11.7%; +++ = 11.7%-34.85%; ++++ = 20.3%-55.9%

Summary of PEgRNA Used:

PBS

PEgRNA²
Spacer
RTT³
RTT
SEQ
PBS

SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
Length
ID NO:
Length

4624
58
4448
15
4431
12

4720
58
4448
15
4434
15

4720
58
4448
15
4434
15

4852
58
4456
21
4433
14

4852
58
4456
21
4433
14

²Indicated sequence does not contain the 3′ linker and hairpin motif used experimentally. The experimental PEgRNA further contained 3′ mN*mN*mN*N and 5′mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2′-O—Me modification and a * indicates a phosphorothioate bond. The first 20 nt of the ngRNA sequence are the spacer; italics indicate that the spacer is a PE3B spacer.

³* = RTT contains a PAM silencing mutation.

TABLE Ye

PE3 screen at the H1069 mutation site in HEK293T

cells with synthetic PEgRNA and ngRNA

PEgRNA²SEQ ID NO:

2548
2557
2582
2633
##

%
%
%
%
%
%
%
%
%
%

Edit¹
indel¹
Edit¹
indel¹
Edit¹
indel¹
Edit¹
indel¹
Edit¹
indel¹

ngRNA²
PE2
+++
+

+++
+
+++
+
+++
++

SEQ
2257
+++
++
+++
++
+++
++
+++
++
+++
+

ID
2268
++++
+
++++
+
++++
++
++++
++
++++
+

NO:
2264
++++
+
++++
+
++++
+
++++
+
++++
+

2261
+++
++
+++
++
+++
++
+++
++
+++
++

2274
+++
++
+++
++
+++
++
+++
++
+++
++

2269
+++
++
+++
++
+++
++
+++
+++
+++
++

2270
+++
++
+++
++
+++
+
+++
++
+++
+

2265
+++
++
+++
++
+++
++
+++
++
+++
+

2260
+++
++
+++
++
+++
++
+++
++
+++
++

2272
+++
++
+++
+
+++
++
+++
++
+++
++

4415
+++
+
+++
+
+++
+
+++
++
+++
+

4414
++++
+
++++
+
++++
++
++++
+
+++
+

4414
++++
+
++++
+

++++
+
++++
+

4412
++++
+
++++
+
++++
+
++++
+
+++
+

4412
+++
+
++++
+
++++
+
++++
++
+++
+

¹+ = 0.1%-2.0%; ++ = 2.0%-11.7%; +++ = 11.7%-34.85%; ++++ = 20.3%-55.9%

Summary of PEgRNA Used:

PBS

PEgRNA²
Spacer
RTT³
RTT
SEQ
PBS

SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
Length
ID NO:
Length

2548
33
2329
15
2295
8

2557
33
2335
16
2295
8

2582
33
2335
16
2296
9

2633
33
2335
16
2297
10

##
33
##
17
2295
8

²Indicated sequence does not contain the 3′ linker and hairpin motif used experimentally. The experimental PEgRNA further contained 3′ mN*mN*mN*N and 5′mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2′-O—Me modification and a * indicates a phosphorothioate bond. The first 20 nt of the ngRNA sequence are the spacer; italics indicate that the spacer is a PE3B spacer.

³* = RTT contains a PAM silencing mutation.

TABLE Yf

PE3 screen at the H1069 mutation site in HEK293T

cells with synthetic PEgRNA and ngRNA

PEgRNA²SEQ ID NO:

2957
2871
2568
2645
2551

%
%
%
%
%
%
%
%
%
%

Edit¹
indel¹
Edit¹
indel¹
Edit¹
indel¹
Edit¹
indel¹
Edit¹
indel¹

ngRNA²
PE2
+++
+

+
++
+
++
++
+++
+

SEQ
2257
+++
+
+++
+
++++
+
+++
++
++++
+

ID
2268
++++
++
+++

++++
+

++++
++

NO:
2264
++++
+
+++
+
++++
+
+++
+
++++
+

2261
+++
++
++
++
+++
++
+++
++
++++
++

2274
+++
++
++
++
+++
++
+++
++
+++
++

2269
+++
++
+++
++
+++
++
+++
++
++++
++

2270
+++
++
++
+
++++
++
+++
++
++++
++

2265
+++
++
+++
+
++++
++
+++
++
++++
++

2260
+++
++
+++
+
+++
++
+++
++
++++
++

2272
+++
++
+++
++
+++
+
+++
++
+++
++

4415
+++
+
++
+
+++
+
+++
+
+++
+

4414
++++
+
+++
+
++++
++
++++
++
++++
+

4414
++++
+
+++
+
++++
+
+++
+
++++
++

4412
++++
+
+++
+
++++
++
++++
++
++++
+

4412
++++
+
+++
+
+++
+
+++
+
++++
+

¹+ = 0.1%-2.0%; ++ = 2.0%-11.7%; +++ = 11.7%-34.85%; ++++ = 20.3%-55.9%

Summary of PEgRNA Used:

PBS

PEgRNA²
Spacer
RTT³
RTT
SEQ
PBS

SEQ ID NO:
SEQ ID NO:
SEQ ID NO:
Length
ID NO:
Length

2957
33
2342
18
2300
13

2871
33
2353
22
2295
8

2568
33
2339*
16
2295
8

2645
33
2339*
16
2297
10

2551
33
2337*
16
2295
8

²Indicated sequence does not contain the 3′ linker and hairpin motif used experimentally. The experimental PEgRNA further contained 3′ mN*mN*mN*N and 5′mN*mN*mN* modifications, where m indicates that the nucleotide contains a 2′-O—Me modification and a * indicates a phosphorothioate bond. The first 20 nt of the ngRNA sequence are the spacer; italics indicate that the spacer is a PE3B spacer.

³* = RTT contains a PAM silencing mutation.

Wild-Type ATP7B Protein Sequence (SEO ID NO: 5861)

>sp|P35670|ATP7B_HUMAN Copper-transporting

ATPase 2 OS = Homo sapiens OX = 9606

GN = ATP7B PE = 1 SV = 4

MPEQERQITAREGASRKILSKLSLPTRAWEPAMKKSFAFDNVGYEGGLDG

LGPSSQVATSTVRILGMTCQSCVKSIEDRISNLKGIISMKVSLEQGSATV

KYVPSVVCLQQVCHQIGDMGFEASIAEGKAASWPSRSLPAQEAVVKLRVE

GMTCQSCVSSIEGKVRKLQGVVRVKVSLSNQEAVITYQPYLIQPEDLRDH

VNDMGFEAAIKSKVAPLSLGPIDIERLQSTNPKRPLSSANQNFNNSETLG

HQGSHVVTLQLRIDGMHCKSCVLNIEENIGQLLGVQSIQVSLENKTAQVK

YDPSCTSPVALQRAIEALPPGNFKVSLPDGAEGSGTDHRSSSSHSPGSPP

RNQVQGTCSTTLIAIAGMTCASCVHSIEGMISQLEGVQQISVSLAEGTAT

VLYNPSVISPEELRAAIEDMGFEASVVSESCSTNPLGNHSAGNSMVQTTD

GTPTSVQEVAPHTGRLPANHAPDILAKSPQSTRAVAPQKCFLQIKGMTCA

SCVSNIERNLQKEAGVLSVLVALMAGKAEIKYDPEVIQPLEIAQFIQDLG

FEAAVMEDYAGSDGNIELTITGMTCASCVHNIESKLTRINGITYASVALA

TSKALVKFDPEIIGPRDIIKIIEEIGFHASLAQRNPNAHHLDHKMEIKQW

KKSFLCSLVFGIPVMALMIYMLIPSNEPHQSMVLDHNIIPGLSILNLIFF

ILCTFVQLLGGWYFYVQAYKSLRHRSANMDVLIVLATSIAYVYSLVILVV

AVAEKAERSPVTFFDTPPMLFVFIALGRWLEHLAKSKTSEALAKLMSLQA

TEATVVTLGEDNLIIREEQVPMELVQRGDIVKVVPGGKFPVDGKVLEGNT

MADESLITGEAMPVTKKPGSTVIAGSINAHGSVLIKATHVGNDTTLAQIV

KLVEEAQMSKAPIQQLADRFSGYFVPFIIIMSTLTLVVWIVIGFIDFGVV

QRYFPNPNKHISQTEVIIRFAFQTSITVLCIACPCSLGLATPTAVMVGTG

VAAQNGILIKGGKPLEMAHKIKTVMFDKTGTITHGVPRVMRVLLLGDVAT

LPLRKVLAVVGTAEASSEHPLGVAVTKYCKEELGTETLGYCTDFQAVPGC

GIGCKVSNVEGILAHSERPLSAPASHLNEAGSLPAEKDAVPQTFSVLIGN

REWLRRNGLTISSDVSDAMTDHEMKGQTAILVAIDGVLCGMIAIADAVKQ

EAALAVHTLQSMGVDVVLITGDNRKTARAIATQVGINKVFAEVLPSHKVA

KVQELQNKGKKVAMVGDGVNDSPALAQADMGVAIGTGTDVAIEAADVVLI

RNDLLDVVASIHLSKRTVRRIRINLVLALIYNLVGIPIAAGVFMPIGIVL

QPWMGSAAMAASSVSVVLSSLQLKCYKKPDLERYEAQAHGHMKPLTASQV

SVHIGMDDRWRDSPRATPWDQVSYVSQVSLSSLTSDKPSRHSAAADDDGD

KWSLLLNGRDEEQYI

	Number	Date	Country
Parent	PCT/US2022/032267	Jun 2022	US
Child	18526247		US

GENOME EDITING COMPOSITIONS AND METHODS FOR TREATMENT OF WILSON'S DISEASE

Information

Publication Number

Date Filed

Date Published

Inventors

Original Assignees

CPC

International Classifications

Abstract

Description

Claims

CROSS-REFERENCE

Provisional Applications (1)

Continuations (1)