Patent Application
20220186216
This application includes an electronically submitted sequence listing in .txt format. The .txt file contains a sequence listing entitled “2022-02-25 01245-0002-00PCT_ST25.txt” created on Feb. 25, 2022 and is size 11.7 MB in size. The sequence listing contained in this .txt file is part of the specification and is hereby incorporated by reference herein in its entirety.
Repetitive DNA sequences, including trinucleotide repeats and other sequences with self-complementarity, tend to show marked genetic instability and are recognized as a major cause of neurological and neuromuscular diseases. In particular, trinucleotide repeats (TNRs) in or near various genes are associated with a number of neurological and neuromuscular conditions, including degenerative conditions such as myotonic dystrophy type 1 (DM1), Huntington's disease, and various types of spinocerebellar ataxia.
CRISPR-based genome editing can provide sequence-specific cleavage of genomic DNA using an RNA-targeted endonuclease and a guide RNA. In mammalian cells, cleavage by an RNA-targeted endonuclease is most commonly repaired through the non-homologous end joining (NHEJ) pathway, which is DNA-dependent serine/threonine protein kinase (DNA-PK) dependent. NHEJ repair of an individual double strand break near a trinucleotide repeat or self-complementary region does not typically result in excision of the following trinucleotide repeat or self-complementary region, meaning that applying genome editing to ameliorate problematic trinucleotide repeat or self-complementary genotypes is non-trivial. Providing a pair of guide RNAs that cut on either side of the trinucleotide repeat or self-complementary region results in excision to some extent through NHEJ, but the breaks are simply resealed without loss of the intervening repeats or self-complementary sequence in a significant number of cells. Accordingly, there is a need for improved compositions and methods for excision of repetitive DNA sequences.
Disclosed herein are compositions and methods using an RNA-targeted endonuclease, at least one guide RNA that targets the endonuclease to a target in or near trinucleotide repeats or a self-complementary region to excise repeats or self-complementary sequence from the DNA, and optionally a DNA-PK inhibitor. Such methods can ameliorate genotypes associated with trinucleotide repeats, among others. It has been found that inhibition of DNA-PK in combination with cleavage of DNA in or near repetitive sequences provides excision of the repetitive sequences at increased frequency. Also disclosed are guide RNAs and combinations of guide RNAs particularly suitable for use in methods of excising trinucleotide repeats, with or without a DNA-PK inhibitor.
Accordingly, the following embodiments are provided.
Reference will now be made in detail to certain embodiments of the invention, examples of which are illustrated in the accompanying drawings. While the invention is described in conjunction with the illustrated embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the invention as defined by the appended claims and included embodiments.
Before describing the present teachings in detail, it is to be understood that the disclosure is not limited to specific compositions or process steps, as such may vary. It should be noted that, as used in this specification and the appended claims, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, reference to “a guide” includes a plurality of guides and reference to “a cell” includes a plurality of cells and the like.
Numeric ranges are inclusive of the numbers defining the range. Measured and measurable values are understood to be approximate, taking into account significant digits and the error associated with the measurement. Also, the use of “comprise”, “comprises”, “comprising”, “contain”, “contains”, “containing”, “include”, “includes”, and “including” are not intended to be limiting. It is to be understood that both the foregoing general description and detailed description are exemplary and explanatory only and are not restrictive of the teachings.
Unless specifically noted in the specification, embodiments in the specification that recite “comprising” various components are also contemplated as “consisting of” or “consisting essentially of” the recited components; embodiments in the specification that recite “consisting of” various components are also contemplated as “comprising” or “consisting essentially of” the recited components; and embodiments in the specification that recite “consisting essentially of” various components are also contemplated as “consisting of” or “comprising” the recited components (this interchangeability does not apply to the use of these terms in the claims). The term “or” is used in an inclusive sense, i.e., equivalent to “and/or,” unless the context clearly indicates otherwise.
The section headings used herein are for organizational purposes only and are not to be construed as limiting the desired subject matter in any way. In the event that any material incorporated by reference contradicts any term defined in this specification or any other express content of this specification, this specification controls. While the present teachings are described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art. -
Unless stated otherwise, the following terms and phrases as used herein are intended to have the following meanings:
“Polynucleotide” and “nucleic acid” are used herein to refer to a multimeric compound comprising nucleosides or nucleoside analogs which have nitrogenous heterocyclic bases or base analogs linked together along a backbone, including conventional RNA, DNA, mixed RNA-DNA, and polymers that are analogs thereof. A nucleic acid “backbone” can be made up of a variety of linkages, including one or more of sugar-phosphodiester linkages, peptide-nucleic acid bonds (“peptide nucleic acids” or PNA; PCT No. WO 95/32305), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. Sugar moieties of a nucleic acid can be ribose, deoxyribose, or similar compounds with substitutions, e.g., 2′ methoxy or 2′ halide substitutions. Nitrogenous bases can be conventional bases (A, G, C, T, U), analogs thereof (e.g., modified uridines such as 5-methoxyuridine, pseudouridine, or N1-methylpseudouridine, or others); inosine; derivatives of purines or pyrimidines (e.g., N4-methyl deoxyguanosine, deaza- or aza-purines, deaza- or aza-pyrimidines, pyrimidine bases with substituent groups at the 5 or 6 position (e.g., 5-methylcytosine), purine bases with a substituent at the 2, 6, or 8 positions, 2-amino-6-methylaminopurine, O6-methylguanine, 4-thio-pyrimidines, 4-amino-pyrimidines, 4-dimethylhydrazine-pyrimidines, and O4-alkyl-pyrimidines; U.S. Pat. No. 5,378,825 and PCT No. WO 93/13121). For general discussion see The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11th ed., 1992). Nucleic acids can include one or more “abasic” residues where the backbone includes no nitrogenous base for position(s) of the polymer (US Pat. No. 5,585,481). A nucleic acid can comprise only conventional RNA or DNA sugars, bases and linkages, or can include both conventional components and substitutions (e.g., conventional bases with 2′ methoxy linkages, or polymers containing both conventional bases and one or more base analogs). Nucleic acid includes “locked nucleic acid” (LNA), an analogue containing one or more LNA nucleotide monomers with a bicyclic furanose unit locked in an RNA mimicking sugar conformation, which enhance hybridization affinity toward complementary RNA and DNA sequences (Vester and Wengel, 2004, Biochemistry 43(42):13233-41). RNA and DNA have different sugar moieties and can differ by the presence of uracil or analogs thereof in RNA and thymine or analogs thereof in DNA.
“Guide RNA”, “gRNA”, and simply “guide” are used herein interchangeably to refer to either a crRNA (also known as CRISPR RNA), or the combination of a crRNA and a trRNA (also known as tracrRNA). The crRNA and trRNA may be associated as a single RNA molecule (single guide RNA, sgRNA) or in two separate RNA molecules (dual guide RNA, dgRNA). “Guide RNA” or “gRNA” refers to each type. The trRNA may be a naturally-occurring sequence, or a trRNA sequence with modifications or variations compared to naturally-occurring sequences.
As used herein, a “spacer sequence,” sometimes also referred to herein and in the literature as a “guide sequence,” or “targeting sequence” refers to a sequence within a guide RNA that is complementary to a target sequence and functions to direct a guide RNA to a target sequence for cleavage by an RNA-targeted endonuclease. A guide sequence can be 20 base pairs in length, e.g., in the case of Streptococcus pyogenes (i.e., Spy Cas9, SpCas9) and related Cas9 homologs/orthologs. Shorter or longer sequences can also be used as guides, e.g., 15-, 16-, 17-, 18-, 19-, 21-, 22-, 23-, 24-, or 25-nucleotides in length. For example, in some embodiments, the guide sequence comprises at least 17, 18, 19, 20, 21, 22, 23, 24, or 25 contiguous nucleotides of a sequence selected from SEQ ID NOs: 101-4988, 5001-7264, or 7301-53372. In some embodiments, the guide sequence comprises a sequence selected from SEQ ID NOs: 101-4988, 5001-7264, or 7301-53372. In some embodiments, the target sequence is in a gene or on a chromosome, for example, and is complementary to the guide sequence. In some embodiments, the degree of complementarity or identity between a guide sequence and its corresponding target sequence may be about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. For example, in some embodiments, the guide sequence comprises a sequence with about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to at least 17, 18, 19, 20, 21, 22, 23, 24, or 25 contiguous nucleotides of a sequence selected from SEQ ID NOs: 101-4988, 5001-7264, or 7301-53372. In some embodiments, the guide sequence comprises a sequence with about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a sequence selected from SEQ ID NOs: 101-4988, 5001-7264, or 7301-53372. In some embodiments, the guide sequence and the target region may be 100% complementary or identical. In other embodiments, the guide sequence and the target region may contain at least one mismatch. For example, the guide sequence and the target sequence may contain 1, 2, 3, or 4 mismatches, where the total length of the target sequence is at least 17, 18, 19, 20 or more base pairs. In some embodiments, the guide sequence and the target region may contain 1-4 mismatches where the guide sequence comprises at least 17, 18, 19, 20 or more nucleotides. In some embodiments, the guide sequence and the target region may contain 1, 2, 3, or 4 mismatches where the guide sequence comprises 20 nucleotides.
In some embodiments, the guide sequence comprises a sequence selected from SEQ ID NOs: 101-4988, 5001-7264, or 7301-53372, wherein if the 5′ terminal nucleotide is not guanine, one or more guanine (g) is added to the sequence at its 5′ end. The 5′ g or gg is required in some instances for transcription, for example, for expression by the RNA polymerase III-dependent U6 promoter or the T7 promoter. In some embodiments, a 5′ guanine is added to any one of the guide sequences or pairs of guide sequences disclosed herein.
Target sequences for RNA-targeted endonucleases include both the positive and negative strands of genomic DNA (i.e., the sequence given and the sequence's reverse compliment), as a nucleic acid substrate for an RNA-targeted endonuclease is a double stranded nucleic acid. Accordingly, where a guide sequence is said to be “complementary to a target sequence”, it is to be understood that the guide sequence may direct a guide RNA to bind to the reverse complement of a target sequence. Thus, in some embodiments, where the guide sequence binds the reverse complement of a target sequence, the guide sequence is identical to certain nucleotides of the target sequence (e.g., the target sequence not including the PAM) except for the substitution of U for T in the guide sequence.
As used herein, a “pair of guide RNAs” or “guide pair” or “gRNA pair” or “paired guide RNAs” refers to two guide RNAs that do not have identical spacer sequences. The first spacer sequence refers to the spacer sequence of one of the gRNAs of the pair, and the second spacer sequence refers to the spacer sequence of the other gRNA of the pair. In some embodiments, use of a pair of guide RNAs is also referred to as a “double cut” or “DoubleCut” strategy, in which two cuts are made. In contrast, in some embodiments, use of only one guide RNA is referred to as a “single cut” or “SingleCut” strategy, in which one cut is made.
As used herein, an “RNA-targeted endonuclease” means a polypeptide or complex of polypeptides having RNA and DNA binding activity and DNA cleavage activity, or a DNA-binding subunit of such a complex, wherein the DNA binding activity is sequence-specific and depends on the sequence of the RNA. Exemplary RNA-targeted endonucleases include Cas cleavases/nickases. “Cas nuclease”, also called “Cas protein” as used herein, encompasses Cas cleavases and Cas nickases. Cas cleavases/nickases include a Csm or Cmr complex of a type III CRISPR system, the Cas10, Csm1, or Cmr2 subunit thereof, a Cascade complex of a type I CRISPR system, the Cas3 subunit thereof, and Class 2 Cas nucleases. In some embodiments, the RNA-targeted endonuclease is Class 1 Cas nuclease. In some embodiments, the RNA-targeted endonuclease is Class 2 Cas nuclease. As used herein, a “Class 2 Cas nuclease” is a single-chain polypeptide with RNA-targeted endonuclease activity. Class 2 Cas nucleases include Class 2 Cas cleavases/nickases (e.g., H840A, D10A, or N863A variants), which further have RNA-guided DNA cleavases or nickase activity. Class 2 Cas nucleases include, for example, Cas9, Cpf1, C2c1, C2c2, C2c3, HF Cas9 (e.g., N497A, R661A, Q695A, Q926A variants), HypaCas9 (e.g., N692A, M694A, Q695A, H698A variants), eSPCas9(1.0) (e.g, K810A, K1003A, R1060A variants), and eSPCas9(1.1) (e.g., K848A, K1003A, R1060A variants) proteins and modifications thereof. Cpf1 protein, Zetsche et al., Cell, 163: 1-13 (2015), is homologous to Cas9, and contains a RuvC-like nuclease domain. Cpf1 sequences of Zetsche are incorporated by reference in their entirety. See, e.g., Zetsche, Tables S1 and S3. See, e.g., Makarova et al., Nat Rev Microbiol, 13(11): 722-36 (2015); Shmakov et al., Molecular Cell, 60:385-397 (2015). Class 1 is divided into types I, III, and IV Cas nucleases. Class 2 is divided into types II, V, and VI Cas nucleases. In some embodiments, the RNA-targeted endonuclease is a Type I, II, III, IV, V, or VI Cas nuclease.
As used herein, “ribonucleoprotein” (RNP) or “RNP complex” refers to a guide RNA together with an RNA-targeted endonuclease, such as a Cas nuclease, e.g., a Cas cleavase or Cas nickase (e.g., Cas9). In some embodiments, the guide RNA guides the RNA-targeted endonuclease such as Cas9 to a target sequence, and the guide RNA hybridizes with and the agent binds to the target sequence, which can be followed by cleaving or nicking.
As used herein, a “self-complementary region” refers to any portion of a nucleic acid that can form secondary structure (e.g., hairpins, cruciforms, etc.) through hybridization to itself, e.g., when the region has at least one free double-strand end. Various forms of repeats and GC-rich or AT-rich nucleic acids qualify as self-complementary and can form secondary structures. Self-complementarity does not require perfect self-complementarity, as secondary structures may form despite the presence of some mismatched bases and/or non-canonical base pairs. In some embodiments, a self-complementary region comprises 40 nucleotides. Self-complementary regions may be interrupted by a loop-forming sequence, which is not necessarily self-complementary and may exist in a single-stranded state between segments of the self-complementary region that form the stem in a hairpin or other secondary structure.
As used herein, a first sequence is considered to “comprise a sequence with at least X % identity to” a second sequence if an alignment of the first sequence to the second sequence shows that X % or more of the positions of the second sequence in its entirety are matched by the first sequence. For example, the sequence AAGA comprises a sequence with 100% identity to the sequence AAG because an alignment would give 100% identity in that there are matches to all three positions of the second sequence. The differences between RNA and DNA (generally the exchange of uridine for thymidine or vice versa) and the presence of nucleoside analogs such as modified uridines do not contribute to differences in identity or complementarity among polynucleotides as long as the relevant nucleotides (such as thymidine, uridine, or modified uridine) have the same complement (e.g., adenosine for all of thymidine, uridine, or modified uridine; another example is cytosine and 5-methylcytosine, both of which have guanosine or modified guanosine as a complement). Thus, for example, the sequence 5′-AXG where X is any modified uridine, such as pseudouridine, N1-methyl pseudouridine, or 5-methoxyuridine, is considered 100% identical to AUG in that both are perfectly complementary to the same sequence (5′-CAU). Exemplary alignment algorithms are the Smith-Waterman and Needleman-Wunsch algorithms, which are well-known in the art. One skilled in the art will understand what choice of algorithm and parameter settings are appropriate for a given pair of sequences to be aligned; for sequences of generally similar length and expected identity >50% for amino acids or >75% for nucleotides, the Needleman-Wunsch algorithm with default settings of the Needleman-Wunsch algorithm interface provided by the EBI at the www.ebi.ac.uk web server is generally appropriate.
“mRNA” is used herein to refer to a polynucleotide that is not DNA and comprises an open reading frame that can be translated into a polypeptide (i.e., can serve as a substrate for translation by a ribosome and amino-acylated tRNAs). mRNA can comprise a phosphate-sugar backbone including ribose residues or analogs thereof, e.g., 2′-methoxy ribose residues. In some embodiments, the sugars of an mRNA phosphate-sugar backbone consist essentially of ribose residues, 2′-methoxy ribose residues, or a combination thereof.
Guide sequences useful in the guide RNA compositions and methods described herein are shown in Table 2 and the Sequence Listing and throughout the application.
As used herein, a “target sequence” refers to a sequence of nucleic acid in a target gene that has complementarity to the guide sequence of the gRNA. The interaction of the target sequence and the guide sequence directs an RNA-targeted endonuclease to bind, and potentially nick or cleave (depending on the activity of the agent), within the target sequence.
As used herein, “treatment” refers to any administration or application of a therapeutic for disease or disorder in a subject, and includes inhibiting the disease or development of the disease (which may occur before or after the disease is formally diagnosed, e.g., in cases where a subject has a genotype that has the potential or is likely to result in development of the disease), arresting its development, relieving one or more symptoms of the disease, curing the disease, or preventing reoccurrence of one or more symptoms of the disease. For example, treatment of DM1 may comprise alleviating symptoms of DM1.
As used herein, “ameliorating” refers to any beneficial effect on a phenotype or symptom, such as reducing its severity, slowing or delaying its development, arresting its development, or partially or completely reversing or eliminating it. In the case of quantitative phenotypes such as expression levels, ameliorating encompasses changing the expression level so that it is closer to the expression level seen in healthy or unaffected cells or individuals.
As used herein, a target sequence is “near” a trinucleotide repeat or self-complementary sequence if cleavage of the target followed by MMEJ or other non-NHEJ repair results in excision of the trinucleotide repeat or self-complementary sequence to a detectable extent. In some embodiments, a target sequence is within 10, 20, 30, 40, 50 or 100 nucleotides of the trinucleotide repeat or self-complementary sequence, where the distance from the target to the trinucleotide repeat or self-complementary sequence is measured as the number of nucleotides between the closest nucleotide of the trinucleotide repeat or self-complementary sequence and the site in the target that undergoes cleavage.
As used herein, “excision” of a sequence means and process that results in removal of the sequence from nucleic acid (e.g., DNA, such as gDNA) in which it originally occurred, including but not limited to processes comprising one or two double strand cleavage events or two or more nicking events followed by any repair process that does not include the sequence in the repair product, which may comprise one or more of ligation of distal ends (e.g.,
As used herein, an “expanded amino acid repeat” refers to a segment of a given amino acid (e.g., one of glutamine, alanine, etc.) in a polypeptide that contains more instances of the amino acid than normally appears in wild-type versions of the polypeptide. For trinucleotide repeats in Table 1 that are listed as occurring in exons, the normal range indicates the range of instances of the amino acid than normally appears in wild-type versions of the corresponding polypeptide.
As used herein, “DM1 myoblasts” refer to precursors of muscle cells that have a genotype associated with DM1, and include e.g., cells derived from or isolated from a subject with DM1. DM1 myoblasts include primary cells, cultured cells, or cell lines.
A “pharmaceutically acceptable excipient” refers to an agent that is included in a pharmaceutical formulation that is not the active ingredient. Pharmaceutically acceptable excipients may e.g., aid in drug delivery or support or enhance stability or bioavailability.
The term “about” or “approximately” means an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined.
Disclosed herein are compositions and methods based on our discovery that RNA-directed endonucleases can excise trinucleotide repeats or self-complementary regions in combination with single or paired guide RNAs that target the endonuclease to sites flanking the TNR, as well as our finding that DNA-PK inhibitors provide improved excision of such sequences. As illustrated in
Additionally, we have also found that DNA-PK inhibitors can facilitate excision of trinucleotide repeats by an RNA-directed nuclease such as Cas9 or Cpf1 in combination with one gRNA, as illustrated in
Methods and compositions provided herein can be used to excise trinucleotide repeats or self-complementary sequences to ameliorate genotypes associated with various disorders. Table 1 provides information regarding exemplary genes, disorders, and trinucleotide repeats.
This disclosure provides compositions for use in, and methods, of excising trinucleotide repeats or self-complementary regions and/or treating a disease or disorder characterized by a trinucleotide repeat (TNR) in DNA. In some embodiments, one or more gRNAs described herein (e.g., a pair of gRNAs) or a vector encoding the gRNAs are delivered to a cell in combination with an RNA-targeted endonuclease or a nucleic acid encoding the RNA-targeted endonuclease. Exemplary gRNAs, vectors, and RNA-targeted endonucleases are described herein, e.g., in the Summary and Composition sections. In some embodiments, the method further comprises delivering a DNA-PK inhibitor to the cell.
Provided is a method of treating a disease or disorder characterized by a trinucleotide repeat (TNR) in DNA, the method comprising delivering to a cell that comprises a TNR i) a guide RNA or a pair of guide RNAs comprising a spacer sequence or a pair of spacer sequences that directs an RNA-targeted endonuclease to or near the TNR, or a nucleic acid encoding the guide RNA or pair of guide RNAs; ii) an RNA-targeted endonuclease or a nucleic acid encoding the RNA-targeted endonuclease; and optionally iii) a DNA-PK inhibitor. In some embodiments, the method comprises a DNA-PK inhibitor. In some embodiments, the DNA-PK inhibitor is Compound 3 or Compound 6.
In some embodiments, a method is provided of treating a disease or disorder characterized by a trinucleotide repeat (TNR) in DNA, the method comprising delivering to a cell that comprises a TNR i) a guide RNA or a pair of guide RNAs comprising a spacer or a pair of spacer sequences that directs an RNA-targeted endonuclease to or near the TNR, or a nucleic acid encoding the guide RNA or pair of guide RNAs; ii) an RNA-targeted endonuclease or a nucleic acid encoding the RNA-targeted endonuclease; and iii) a DNA-PK inhibitor which is Compound 3 or Compound 6.
Also provided is a method of excising a self-complementary region comprising delivering to a cell that comprises the self-complementary region i) a guide RNA or pair of guide RNAs comprising a spacer or a pair of spacer sequences that directs an RNA-targeted endonuclease to or near the self-complementary region, or a nucleic acid encoding the guide RNA or pair of guide RNAs; ii) an RNA-targeted endonuclease or a nucleic acid encoding the RNA-targeted endonuclease; and optionally iii) a DNA-PK inhibitor, wherein the self-complementary region is excised. In some embodiments, the method comprises a DNA-PK inhibitor. In some embodiments, the DNA-PK inhibitor is Compound 3 or Compound 6.
In some embodiments, a method is provided of excising a trinucleotide repeat (TNR) in DNA comprising delivering to a cell that comprises the TNR i) a guide RNA comprising a spacer that directs an RNA-targeted endonuclease to or near the TNR, or a nucleic acid encoding the guide RNA; ii) an RNA-targeted endonuclease or a nucleic acid encoding the RNA-targeted endonuclease; and optionally iii) a DNA-PK inhibitor, wherein at least one TNR is excised. In some embodiments, the method comprises a DNA-PK inhibitor. In some embodiments, the DNA-PK inhibitor is Compound 3 or Compound 6.
In some embodiments, the method of excising a self-complementary region and/or method of excising a TNR in DNA is for the treatment of a disease or disorder provided in Table 1.
Also provided is a method of treating a disease or disorder characterized by a trinucleotide repeat (TNR) in the 3′ UTR of the DMPK gene, the method comprising delivering to a cell that comprises a TNR in the 3′ UTR of the DMPK gene i) a guide RNA comprising a spacer comprising a sequence of any one of SEQ ID NOs 101-4988, or a nucleic acid encoding the guide RNA; ii) an RNA-targeted endonuclease or a nucleic acid encoding the RNA-targeted endonuclease; and iii) optionally a DNA-PK inhibitor. In some embodiments, the method comprises a DNA-PK inhibitor. In some embodiments, the DNA-PK inhibitor is Compound 3 or Compound 6.
Also provided is a method of treating a disease or disorder characterized by a trinucleotide repeat (TNR) in the 3′ UTR of the DMPK gene, the method comprising delivering to a cell that comprises a TNR in the 3′ UTR of the DMPK gene i) a guide RNA comprising a spacer comprising a sequence of any one of SEQ ID NOs: 4018, 4010, 4002, 4042, 4034, 4026, 3954, 3946, 3994, 3914, 3978, 3906, 3898, 3938, 3922, 3858, 3850, 3882, 3826, 3818, 3842, 3794, 3786, 3762, 3810, 3746, 3778, 3738, 3770, 3722, 3754, 3690, 3666, 3658, 3634, 3586, 3546, 3530, 3642, 3514, 3506, 3490, 3618, 3610, 3602, 3578, 3442, 3522, 3410, 3378, 3434, 3370, 3426, 3418, 3394, 3386, 3330, 3354, 3346, 3314, 3930, 3890, 3834, 3802, 3706, 3698, 3682, 3674, 3570, 3554, 3538, 3498, 3482, 3458, 3474, 3450, 2667, 2666, 2650, 2642, 2626, 2618, 2706, 2690, 2682, 2610, 2674, 2658, 2602, 2594, 2634, 2554, 2546, 2586, 2538, 2578, 2570, 2522, 2498, 2490, 2466, 2458, 2450, 2514, 2506, 2418, 2482, 2474, 2394, 2442, 2434, 2370, 2378, 2354, 2346, 2338, 2314, 2298, 2282, 2274, 2266, 2330, 2258, 2322, 2242, 2234, 2290, 2250, 2218, 2226, 2210, 2194, 2146, 2138, 2122, 2106, 2098, 2090, 2130, 2114, 2034, 2026, 2058, 2050, 2042, 1914, 1786, 1778, 1770, 1842, 1738, 1706, 1690, 1746, 1714, 1650, 1642, 1610, 1586, 1562, 1546, 1578, 1538, 1378, 1370, 1922, 1898, 1906, 1794, 1762, 1698, 1674, 1722, 1362, 1450, 2202, 2178, 2170, 2162, 2018, 2010, 1890, 1962, 1946, 1850, 1818, 1658, 1634, 1602, 1554, 1434, 1426, 1338, 1346, 1978, 1994, 1986, 1970, 1938, 1930, 1810, 1834, 1826, 1802, 1626, 1594, 1514, 1498, 1490, 1482, 1474, 1458, 1442, 1418, 1410, 1402, 1394, or 1386, or a nucleic acid encoding the guide RNA; ii) an RNA-targeted endonuclease or a nucleic acid encoding the RNA-targeted endonuclease; and iii) optionally a DNA-PK inhibitor. Also provided is a method of excising a trinucleotide repeat (TNR) in the 3′ UTR of the DMPK gene comprising delivering to a cell that comprises the TNR i) a guide RNA comprising a spacer comprising a sequence of any one of SEQ ID NOs 4018, 4010, 4002, 4042, 4034, 4026, 3954, 3946, 3994, 3914, 3978, 3906, 3898, 3938, 3922, 3858, 3850, 3882, 3826, 3818, 3842, 3794, 3786, 3762, 3810, 3746, 3778, 3738, 3770, 3722, 3754, 3690, 3666, 3658, 3634, 3586, 3546, 3530, 3642, 3514, 3506, 3490, 3618, 3610, 3602, 3578, 3442, 3522, 3410, 3378, 3434, 3370, 3426, 3418, 3394, 3386, 3330, 3354, 3346, 3314, 3930, 3890, 3834, 3802, 3706, 3698, 3682, 3674, 3570, 3554, 3538, 3498, 3482, 3458, 3474, 3450, 2667, 2666, 2650, 2642, 2626, 2618, 2706, 2690, 2682, 2610, 2674, 2658, 2602, 2594, 2634, 2554, 2546, 2586, 2538, 2578, 2570, 2522, 2498, 2490, 2466, 2458, 2450, 2514, 2506, 2418, 2482, 2474, 2394, 2442, 2434, 2370, 2378, 2354, 2346, 2338, 2314, 2298, 2282, 2274, 2266, 2330, 2258, 2322, 2242, 2234, 2290, 2250, 2218, 2226, 2210, 2194, 2146, 2138, 2122, 2106, 2098, 2090, 2130, 2114, 2034, 2026, 2058, 2050, 2042, 1914, 1786, 1778, 1770, 1842, 1738, 1706, 1690, 1746, 1714, 1650, 1642, 1610, 1586, 1562, 1546, 1578, 1538, 1378, 1370, 1922, 1898, 1906, 1794, 1762, 1698, 1674, 1722, 1362, 1450, 2202, 2178, 2170, 2162, 2018, 2010, 1890, 1962, 1946, 1850, 1818, 1658, 1634, 1602, 1554, 1434, 1426, 1338, 1346, 1978, 1994, 1986, 1970, 1938, 1930, 1810, 1834, 1826, 1802, 1626, 1594, 1514, 1498, 1490, 1482, 1474, 1458, 1442, 1418, 1410, 1402, 1394, or 1386, or a nucleic acid encoding the guide RNA; ii) an RNA-targeted endonuclease or a nucleic acid encoding the RNA-targeted endonuclease; and iii) optionally a DNA-PK inhibitor, wherein at least one TNR is excised. In some embodiments, the gRNA comprises a spacer sequence comprising a sequence of any one of SEQ ID NOs: 3330, 3914, 3418, 3746, 3778, 3394, 4026, 3690, 3794, 3386, 3938, 3682, 3818, 3658, 3722, 3802, 3858, 3514, 3770, 3370, 3354, 4010, 2202, 1706, 2210, 2170, 1778, 2258, 2114, 2178, 1642, 1738, 1746, 2322, 1770, 1538, 2514, 2458, 2194, 2594, 2162, or 2618. In some embodiments, the gRNA comprises a spacer sequence comprising a sequence of any one of SEQ ID NOs: 3746, 3778, 3394, 3386, 3938, 3818, 3722, 3858, 3370, 1706, 2210, 2114, 1538, or 2594. In some embodiments, the gRNA comprises a spacer sequence comprising a sequence of any one of SEQ ID NOs: 3330, 3746, 3778, 3394, 4026, 3386, 3938, 3818, 3722, 3802, 3858, 3514, 3770, 3370, 2202, 1706, 2210, 1778, 2114, 1738, 1746, 2322, 1538, 2514, 2458, 2194, or 2594. In some embodiments, the gRNA comprises a spacer sequence comprising a sequence of any one of SEQ ID NOs: 3330, 3914, 3418, 3746, 3778, 3394, 4026, 3690, 3794, 3386, 3938, 3682, 3818, 3658, or 3722. In some embodiments, the gRNA comprises a spacer sequence comprising a sequence of any one of SEQ ID NOs: 2202, 1706, 2210, 2170, 1778, 2258, 2114, 2178, 1642, 1738, 1746, or 2322. In some embodiments, the gRNA comprises a spacer sequence comprising a sequence of any one of SEQ ID NOs: 3778, 4026, 3794, 4010, 3906, 3746, 1778, 1746, 1770, 1586, 1914, or 2210. In some embodiments, the gRNA comprises a spacer sequence comprising a sequence of any one of SEQ ID NOs: 3378, 3354, 3346, 3330, 3314, 2658, 2690, 2546, 2554, 2498, or 2506. In some embodiments, the gRNA comprises a spacer sequence comprising a sequence of any one of SEQ ID NOs: 3330, 3314, 2658, 2690, 2554, or 2498. In some embodiments, the gRNA comprises a spacer sequence comprising a sequence of any one of SEQ ID NOs: 3314, 2690, 2554, or 2498. In some embodiments, the gRNA comprises a spacer sequence comprising a sequence of any one of SEQ ID NOs: 3914, 3514, 1778, 2458, 3858, 3418, 1706, or 2258. . In some embodiments, the gRNA comprises a spacer sequence comprising a sequence of any one of SEQ ID NOs: 3916, 3420, or 3940. In some embodiments, the gRNA comprises a spacer sequence comprising SEQ ID NO: 3914. In some embodiments, the gRNA comprises a spacer sequence comprising SEQ ID NO: 3418. In some embodiments, the gRNA comprises a spacer sequence comprising SEQ ID NO: 3938. In some embodiments, the methods further comprise administering a DNA-PK inhibitor. In some embodiments, the DNA-PK inhibitor is Compound 6. In some embodiments, the DNA-PK inhibitor is Compound 3.
Also provided is a method of treating a disease or disorder characterized by a trinucleotide repeat (TNR) in the 5′ UTR of the FMR1 gene, the method comprising delivering to a cell that comprises a TNR in the 5′ UTR of the FMR1 gene i) a guide RNA comprising a spacer comprising a sequence of any one of SEQ ID NOs 5001-7264, or a nucleic acid encoding the guide RNA; ii) an RNA-targeted endonuclease or a nucleic acid encoding the RNA-targeted endonuclease; and iii) optionally a DNA-PK inhibitor. In some embodiments, the method comprises a DNA-PK inhibitor. In some embodiments, the DNA-PK inhibitor is Compound 3 or Compound 6.
Also provided is a method of treating a disease or disorder characterized by a trinucleotide repeat (TNR) in the 5′ UTR of the FMR1 gene, the method comprising delivering to a cell that comprises a TNR i) a guide RNA comprising a spacer having a sequence of any one of SEQ ID NOs 5262, 5782, 5830, 5926, 5950, 5998, 6022, 5310, and 5334, or a nucleic acid encoding the guide RNA; ii) an RNA-targeted endonuclease or a nucleic acid encoding the RNA-targeted endonuclease; and iii) optionally a DNA-PK inhibitor. Also provided is a method of excising a trinucleotide repeat (TNR) in the 5′ UTR of the FMR1 gene comprising delivering to a cell that comprises the TNR i) a guide RNA comprising a spacer comprising a sequence of any one of SEQ ID NOs 5262, 5782, 5830, 5926, 5950, 5998, 6022, 5310, and 5334, or a nucleic acid encoding the guide RNA; ii) an RNA-targeted endonuclease or a nucleic acid encoding the RNA-targeted endonuclease; and iii) optionally a DNA-PK inhibitor, wherein at least one TNR is excised. In some embodiments, the gRNA comprises a spacer sequence comprising a sequence of any one of SEQ ID NOs: 5830, 6022, 5262, or 5310. In some embodiments, the gRNA comprises a spacer sequence comprising a sequence of any one of SEQ ID NOs: 5262, 5334, and 5830. In some embodiments, the gRNA comprises a spacer sequence comprising a sequence of any one of SEQ ID NOs: 5264, 5336, 5832, 6024, or 5312. In some embodiments, the gRNA comprises a spacer sequence comprising SEQ ID NO: 5262. In some embodiments, the gRNA comprises a spacer sequence comprising SEQ ID NO: 5264. In some embodiments, the methods further comprise administering a DNA-PK inhibitor. In some embodiments, the DNA-PK inhibitor is Compound 6. In some embodiments, the DNA-PK inhibitor is Compound 3.
Also provided is a method of treating a disease or disorder characterized by a trinucleotide repeat (TNR) in the 5′ UTR of the FXN gene, the method comprising delivering to a cell that comprises a TNR in the 5′ UTR of the FXN gene i) a guide RNA comprising a spacer comprising a sequence of any one of SEQ ID NOs 7301-53372, or a nucleic acid encoding the guide RNA; ii) an RNA-targeted endonuclease or a nucleic acid encoding the RNA-targeted endonuclease; and iii) optionally a DNA-PK inhibitor. In some embodiments, the method comprises a DNA-PK inhibitor. In some embodiments, the DNA-PK inhibitor is Compound 3 or Compound 6.
Also provided is a method of treating a disease or disorder characterized by a trinucleotide repeat (TNR) in an intron of the FXN gene, the method comprising delivering to a cell that comprises a TNR i) a guide RNA comprising a spacer comprising a sequence of any one of SEQ ID NOs 28130, 34442, 45906, 26562, 52666, 51322, 46599, 52898, 26546, 7447, 47047, 49986, 51762, 51754, 52290, 52298, 51474, 52306, 50682, 51706, 52098, 50714, 51498, 52498, 50978, 51746, 52106, 51506, 50674, 52082, 52506, 50538, 52066, 52386, 52090, 52266, 52474, 52258, 52434, 50706, 51490, 52458, 51466, 52354, 51914, 51362, 51058, 50170, 51954, 52250, 51930, 51682, 52594, 52610, 51162, 49162, 50898, 49226, 51658, 52554, 52634, 51394, 49034, 52546, 52522, 52618, 52530, 28322, 26530, 26578, 26602, 26634, 26626, 26698, 26746, 26754, 26786, 26882, 27722, 27730, 27738, 27770, 27754, 27762, 27802, 27850, 27842, 27922, 27946, 27986, 28114, 28122, 28146, 28186, 28194, 28338, 28346, 28322, 28378, 28370, 28458, 28506, 28634, 28642, 28650, 34442, or 45906, or a nucleic acid encoding the guide RNA; ii) an RNA-targeted endonuclease or a nucleic acid encoding the RNA-targeted endonuclease; and iii) optionally a DNA-PK inhibitor. Also provided is a method of excising a trinucleotide repeat (TNR) in the 5′ UTR of the FXN gene comprising delivering to a cell that comprises the TNR i) a guide RNA comprising a spacer comprising a sequence of any one of SEQ ID NOs 28130, 34442, 45906, 26562, 52666, 51322, 46599, 52898, 26546, 7447, 47047, 49986, 51762, 51754, 52290, 52298, 51474, 52306, 50682, 51706, 52098, 50714, 51498, 52498, 50978, 51746, 52106, 51506, 50674, 52082, 52506, 50538, 52066, 52386, 52090, 52266, 52474, 52258, 52434, 50706, 51490, 52458, 51466, 52354, 51914, 51362, 51058, 50170, 51954, 52250, 51930, 51682, 52594, 52610, 51162, 49162, 50898, 49226, 51658, 52554, 52634, 51394, 49034, 52546, 52522, 52618, 52530, 28322, 26530, 26578, 26602, 26634, 26626, 26698, 26746, 26754, 26786, 26882, 27722, 27730, 27738, 27770, 27754, 27762, 27802, 27850, 27842, 27922, 27946, 27986, 28114, 28122, 28146, 28186, 28194, 28338, 28346, 28322, 28378, 28370, 28458, 28506, 28634, 28642, 28650, 34442, or 45906, or a nucleic acid encoding the guide RNA; ii) an RNA-targeted endonuclease or a nucleic acid encoding the RNA-targeted endonuclease; and iii) optionally a DNA-PK inhibitor, wherein at least one TNR is excised. In some embodiments, the gRNA comprises a spacer sequence comprising a sequence of any one of SEQ ID NOs: 51706, 51058, 51754, 52090, 52594, 52098, 52298, 52106, 51682, 52066, 52354, 52458, 52290, 52498, 51658, 51930, 51162, 52506, 51762, 51746, 52386, 52258, 52530, 52634, 27850, 28634, 26882, 28650, 28370, 28194, 26626, 26634, 26786, 26754, 27770, 26578, 28130, 27738, 28338, 28642, 26602, 27754, 27730, and 28122. In some embodiments, the gRNA comprises a spacer sequence comprising a sequence of any one of SEQ ID NOs: 47047, 7447, 7463, 46967, 46768, 7680, and 47032. In some embodiments, the gRNA comprises a spacer sequence comprising a sequence of any one of SEQ ID NOs: 47045, 7445, 7461, 46766, 7678, and 47030. In some embodiments, the methods further comprise administering a DNA-PK inhibitor. In some embodiments, the DNA-PK inhibitor is Compound 6. In some embodiments, the DNA-PK inhibitor is Compound 3.
In some embodiments of methods described herein, only one gRNA or vector encoding only one gRNA is provided or delivered, i.e., the method does not involve providing two or more guides that promote cleavage near a TNR or self-complementary region.
In some embodiments, methods are provided for treating a disease or characterized by a trinucleotide repeat (TNR) in the 3′ UTR of the DMPK gene, the method comprising administering only one guide RNA, or a vector encoding the guide RNA. In some embodiments, methods are provided for method of excising a trinucleotide repeat (TNR) in the 3′ UTR of the DMPK gene, the method comprising administering only one guide RNA, or a vector encoding the guide RNA. In some embodiments, methods are provided for administering only one gRNA, wherein a CTG repeat of the 3′ UTR of the DMPK gene is excised. In some embodiments, wherein only one gRNA, and wherein a CTG repeat of the 3′ UTR of the DMPK gene is excised, the gRNA comprises a spacer sequence comprising a sequence selected from SEQ ID NOs: 3746, 3778, 3394, 3386, 3938, 3818, 3722, 3858, 3370, 1706, 2210, 2114, 1538, and 2594. In some embodiments, wherein only one gRNA, and wherein a CTG repeat of the 3′ UTR of the DMPK gene is excised, the gRNA comprises a spacer sequence comprising a sequence selected from SEQ ID NOs: 3330, 3746, 3778, 3394, 4026, 3386, 3938, 3818, 3722, 3802, 3858, 3514, 3770, 3370, 2202, 1706, 2210, 1778, 2114, 1738, 1746, 2322, 1538, 2514, 2458, 2194, and 2594. In some embodiments, wherein only one gRNA, and wherein a CTG repeat of the 3′ UTR of the DMPK gene is excised, the gRNA comprises a spacer sequence comprising a sequence selected from SEQ ID NOs: 3330, 3314, 2658, 2690, 2554, and 2498. In some embodiments, wherein only one gRNA, and wherein a CTG repeat of the 3′ UTR of the DMPK gene is excised, the gRNA comprises a spacer sequence comprising a sequence selected from SEQ ID NOs: 3314, 2690, 2554, and 2498. In some embodiments, wherein only one gRNA, and wherein a CTG repeat of the 3′ UTR of the DMPK gene is excised, the gRNA comprises a spacer sequence comprising a sequence selected from SEQ ID NOs: 3914, 3514, 1778, 2458, 3858, 3418, 1706, and 2258. In some embodiments, wherein only one gRNA, and wherein a CTG repeat of the 3′ UTR of the DMPK gene is excised, the gRNA comprises a spacer sequence comprising a sequence selected from SEQ ID NOs: 3914, 3418, or 3938. In some embodiments, wherein only one gRNA, and wherein a CTG repeat of the 3′ UTR of the DMPK gene is excised, the gRNA comprises a spacer sequence comprising a sequence selected from SEQ ID NOs: 3916, 3420, or 3940. In some embodiments, wherein only one gRNA, and wherein a CTG repeat of the 3′ UTR of the DMPK gene is excised, the gRNA comprises the sequence of SEQ ID NO: 3914. In some embodiments, wherein only one gRNA, and wherein a CTG repeat of the 3′ UTR of the DMPK gene is excised, the gRNA comprises the sequence of SEQ ID NO: 3418. In some embodiments, wherein only one gRNA, and wherein a CTG repeat of the 3′ UTR of the DMPK gene is excised, the gRNA comprises the sequence of SEQ ID NO: 3938. In some embodiments, the methods comprise further administering a DNA-PK inhibitor. In some embodiments, the DNA-PK inhibitor is Compound 6. In some embodiments, the DNA-PK inhibitor is Compound 3.
In some embodiments, methods are provided for treating a disease or characterized by a trinucleotide repeat (TNR) in the 5′ UTR of the FMR1 gene, the method comprising administering only one guide RNA, or a vector encoding the guide RNA. In some embodiments, methods are provided for method of excising a trinucleotide repeat (TNR) in the 5′ UTR of the FMR1 gene, the method comprising administering only one guide RNA, or a vector encoding the guide RNA. In some embodiments, methods are provided for administering only one gRNA, wherein a TNR in the 5′ UTR of the FMR1 gene is excised. In some embodiments, wherein only one gRNA, and wherein a TNR in the 5′ UTR of the FMR1 gene is excised, the gRNA comprises a spacer sequence comprising a sequence selected from SEQ ID NOs: 5830, 6022, 5262, and 5310. In some embodiments, wherein only one gRNA, and wherein a TNR in the 5′ UTR of the FMR1 gene is excised, the gRNA comprises a spacer sequence comprising a sequence selected from SEQ ID NOs: 5262, 5334, and 5830. In some embodiments, wherein only one gRNA, and wherein a TNR in the 5′ UTR of the FMR1 gene is excised, the gRNA comprises a spacer sequence comprising a sequence selected from SEQ ID NOs: 5264, 5336, 5832, 6024, or 5312. In some embodiments, wherein only one gRNA, and wherein a TNR in the 5′ UTR of the FMR1 gene is excised, the gRNA comprises the sequence of SEQ ID NO: 5262. In some embodiments, wherein only one gRNA, and wherein a TNR in the 5′ UTR of the FMR1 gene is excised, the gRNA comprises the sequence of SEQ ID NO: 5264. In some embodiments, the methods comprise further administering a DNA-PK inhibitor. In some embodiments, the DNA-PK inhibitor is Compound 6. In some embodiments, the DNA-PK inhibitor is Compound 3.
In some embodiments, methods are provided for treating a disease or characterized by a trinucleotide repeat (TNR) in the 5′ UTR of the FXN gene, the method comprising administering only one guide RNA, or a vector encoding the guide RNA. In some embodiments, methods are provided for method of excising a trinucleotide repeat (TNR) in the 5′ UTR of the FXN gene, the method comprising administering only one guide RNA, or a vector encoding the guide RNA. In some embodiments, methods are provided for administering only one gRNA, wherein a TNR in the 5′ UTR of the FXN gene is excised. In some embodiments, wherein only one gRNA, and wherein a TNR in the 5′ UTR of the FXN gene is excised, the gRNA comprises a spacer sequence comprising a sequence selected from SEQ ID NOs: 47047, 7447, 7463, 46967, 46768, 7680, and 47032. In some embodiments, wherein only one gRNA, and wherein a TNR in the 5′ UTR of the FXN gene is excised, the gRNA comprises a spacer sequence comprising a sequence selected from SEQ ID NOs: 47045, 7445, 7461, 46766, 7678, and 47030. In some embodiments, the methods comprise further administering a DNA-PK inhibitor. In some embodiments, the DNA-PK inhibitor is Compound 6. In some embodiments, the DNA-PK inhibitor is Compound 3.
In some embodiments of methods described herein, a pair of guide RNAs that comprise a first and second spacer that deliver the RNA-targeted endonuclease to or near the TNR, or one or more nucleic acids encoding the pair of guide RNAs, are provided or delivered to a cell. For example, where the TNR is in the 3′ UTR of the DMPK gene, the first and second spacers may have the sequences of any one of the following pairs of SEQ ID NOs: 2202 and 3418; 2202 and 3370; 2202 and 3514; 2202 and 3658; 2178 and 3418; 2178 and 3370; 2178 and 3514; 2178 and 3658; 2170 and 3418; 2170 and 3370; 2170 and 3514; 2170 and 3658; 2162 and 3418; 2162 and 3370; 2162 and 3514; 2162 and 3658; 2202 and 4010; 2202 and 4026; 2202 and 3914; 2202 and 3938; 2202 and 3858; 2202 and 3818; 2202 and 3794; 2202 and 3802; 2202 and 3746; 2202 and 3778; 2202 and 3770; 2202 and 3722; 2202 and 3690; 2202 and 3682; 2202 and 3330; 2202 and 3354; 2202 and 3394; 2202 and 3386; 2178 and 4010; 2178 and 4026; 2178 and 3914; 2178 and 3938; 2178 and 3858; 2178 and 3818; 2178 and 3794; 2178 and 3802; 2178 and 3746; 2178 and 3778; 2178 and 3770; 2178 and 3722; 2178 and 3690; 2178 and 3682; 2178 and 3330; 2178 and 3354; 2178 and 3394; 2178 and 3386; 2170 and 4010; 2170 and 4026; 2170 and 3914; 2170 and 3938; 2170 and 3858; 2170 and 3818; 2170 and 3794; 2170 and 3802; 2170 and 3746; 2170 and 3778; 2170 and 3770; 2170 and 3722; 2170 and 3690; 2170 and 3682; 2170 and 3330; 2170 and 3354; 2170 and 3394; 2170 and 3386; 2162 and 4010; 2162 and 4026; 2162 and 3914; 2162 and 3938; 2162 and 3858; 2162 and 3818; 2162 and 3794; 2162 and 3802; 2162 and 3746; 2162 and 3778; 2162 and 3770; 2162 and 3722; 2162 and 3690; 2162 and 3682; 2162 and 3330; 2162 and 3354; 2162 and 3394; 2162 and 3386; 1706 and 3418; 1706 and 3370; 1706 and 3514; 1706 and 3658; 1706 and 4010; 1706 and 4026; 1706 and 3914; 1706 and 3938; 1706 and 3858; 1706 and 3818; 1706 and 3794; 1706 and 3802; 1706 and 3746; 1706 and 3778; 1706 and 3770; 1706 and 3722; 1706 and 3690; 1706 and 3682; 1706 and 3330; 1706 and 3354; 1706 and 3394; 1706 and 3386; 2210 and 3418; 2210 and 3370; 2210 and 3514; 2210 and 3658; 2210 and 4010; 2210 and 4026; 2210 and 3914; 2210 and 3938; 2210 and 3858; 2210 and 3818; 2210 and 3794; 2210 and 3802; 2210 and 3746; 2210 and 3778; 2210 and 3770; 2210 and 3722; 2210 and 3690; 2210 and 3682; 2210 and 3330; 2210 and 3354; 2210 and 3394; 2210 and 3386; 1778 and 3418; 1778 and 3370; 1778 and 3514; 1778 and 3658; 1778 and 4010; 1778 and 4026; 1778 and 3914; 1778 and 3938; 1778 and 3858; 1778 and 3818; 1778 and 3794; 1778 and 3802; 1778 and 3746; 1778 and 3778; 1778 and 3770; 1778 and 3722; 1778 and 3690; 1778 and 3682; 1778 and 3330; 1778 and 3354; 1778 and 3394; 1778 and 3386; 2258 and 3418; 2258 and 3370; 2258 and 3514; 2258 and 3658; 2258 and 4010; 2258 and 4026; 2258 and 3914; 2258 and 3938; 2258 and 3858; 2258 and 3818; 2258 and 3794; 2258 and 3802; 2258 and 3746; 2258 and 3778; 2258 and 3770; 2258 and 3722; 2258 and 3690; 2258 and 3682; 2258 and 3330; 2258 and 3354; 2258 and 3394; 2258 and 3386; 2114 and 3418; 2114 and 3370; 2114 and 3514; 2114 and 3658; 2114 and 4010; 2114 and 4026; 2114 and 3914; 2114 and 3938; 2114 and 3858; 2114 and 3818; 2114 and 3794; 2114 and 3802; 2114 and 3746; 2114 and 3778; 2114 and 3770; 2114 and 3722; 2114 and 3690; 2114 and 3682; 2114 and 3330; 2114 and 3354; 2114 and 3394; 2114 and 3386; 1642 and 3418; 1642 and 3370; 1642 and 3514; 1642 and 3658; 1642 and 4010; 1642 and 4026; 1642 and 3914; 1642 and 3938; 1642 and 3858; 1642 and 3818; 1642 and 3794; 1642 and 3802; 1642 and 3746; 1642 and 3778; 1642 and 3770; 1642 and 3722; 1642 and 3690; 1642 and 3682; 1642 and 3330; 1642 and 3354; 1642 and 3394; 1642 and 3386; 1738 and 3418; 1738 and 3370; 1738 and 3514; 1738 and 3658; 1738 and 4010; 1738 and 4026; 1738 and 3914; 1738 and 3938; 1738 and 3858; 1738 and 3818; 1738 and 3794; 1738 and 3802; 1738 and 3746; 1738 and 3778; 1738 and 3770; 1738 and 3722; 1738 and 3690; 1738 and 3682; 1738 and 3330; 1738 and 3354; 1738 and 3394; 1738 and 3386; 2258 and 3418; 2258 and 3370; 2258 and 3514; 2258 and 3658; 2258 and 4010; 2258 and 4026; 2258 and 3914; 2258 and 3938; 2258 and 3858; 2258 and 3818; 2258 and 3794; 2258 and 3802; 2258 and 3746; 2258 and 3778; 2258 and 3770; 2258 and 3722; 2258 and 3690; 2258 and 3682; 2258 and 3330; 2258 and 3354; 2258 and 3394; 2258 and 3386; 2114 and 3418; 2114 and 3370; 2114 and 3514; 2114 and 3658; 2114 and 4010; 2114 and 4026; 2114 and 3914; 2114 and 3938; 2114 and 3858; 2114 and 3818; 2114 and 3794; 2114 and 3802; 2114 and 3746; 2114 and 3778; 2114 and 3770; 2114 and 3722; 2114 and 3690; 2114 and 3682; 2114 and 3330; 2114 and 3354; 2114 and 3394; 1706 and 3386; 1642 and 3418; 1642 and 3370; 1642 and 3514; 1642 and 3658; 1642 and 4010; 1642 and 4026; 1642 and 3914; 1642 and 3938; 1642 and 3858; 1642 and 3818; 1642 and 3794; 1642 and 3802; 1642 and 3746; 1642 and 3778; 1642 and 3770; 1642 and 3722; 1642 and 3690; 1642 and 3682; 1642 and 3330; 1642 and 3354; 1642 and 3394; 1642 and 3386; 1738 and 3418; 1738 and 3370; 1738 and 3514; 1738 and 3658; 1738 and 4010; 1738 and 4026; 1738 and 3914; 1738 and 3938; 1738 and 3858; 1738 and 3818; 1738 and 3794; 1738 and 3802; 1738 and 3746; 1738 and 3778; 1738 and 3770; 1738 and 3722; 1738 and 3690; 1738 and 3682; 1738 and 3330; 1738 and 3354; 1738 and 3394; 1738 and 3386; 1746 and 3418; 1746 and 3370; 1746 and 3514; 1746 and 3658; 1746 and 4010; 1746 and 4026; 1746 and 3914; 1746 and 3938; 1746 and 3858; 1746 and 3818; 1746 and 3794; 1746 and 3802; 1746 and 3746; 1746 and 3778; 1746 and 3770; 1746 and 3722; 1746 and 3690; 1746 and 3682; 1746 and 3330; 1746 and 3354; 1746 and 3394; 1746 and 3386; 2322 and 3418; 2322 and 3370; 2322 and 3514; 2322 and 3658; 2322 and 4010; 2322 and 4026; 2322 and 3914; 2322 and 3938; 2322 and 3858; 2322 and 3818; 2322 and 3794; 2322 and 3802; 2322 and 3746; 2322 and 3778; 2322 and 3770; 2322 and 3722; 2322 and 3690; 2322 and 3682; 2322 and 3330; 2322 and 3354; 2322 and 3394; 2322 and 3386; 1770 and 3418; 1770 and 3370; 1770 and 3514; 1770 and 3658; 1770 and 4010; 1770 and 4026; 1770 and 3914; 1770 and 3938; 1770 and 3858; 1770 and 3818; 1770 and 3794; 1770 and 3802; 1770 and 3746; 1770 and 3778; 1770 and 3770; 1770 and 3722; 1770 and 3690; 1770 and 3682; 1770 and 3330; 1770 and 3354; 1770 and 3394; 1770 and 3386; 1538 and 3418; 1538 and 3370; 1538 and 3514; 1538 and 3658; 1538 and 4010; 1538 and 4026; 1538 and 3914; 1538 and 3938; 1538 and 3858; 1538 and 3818; 1538 and 3794; 1538 and 3802; 1538 and 3746; 1538 and 3778; 1538 and 3770; 1538 and 3722; 1538 and 3690; 1538 and 3682; 1538 and 3330; 1538 and 3354; 1538 and 3394; 1538 and 3386; 2514 and 3418; 2514 and 3370; 2514 and 3514; 2514 and 3658; 2514 and 4010; 2514 and 4026; 2514 and 3914; 2514 and 3938; 2514 and 3858; 2514 and 3818; 2514 and 3794; 2514 and 3802; 2514 and 3746; 2514 and 3778; 2514 and 3770; 2514 and 3722; 2514 and 3690; 2514 and 3682; 2514 and 3330; 2514 and 3354; 2514 and 3394; 2514 and 3386; 2458 and 3418; 2458 and 3370; 2458 and 3514; 2458 and 3658; 2458 and 4010; 2458 and 4026; 2458 and 3914; 2458 and 3938; 2458 and 3858; 2458 and 3818; 2458 and 3794; 2458 and 3802; 2458 and 3746; 2458 and 3778; 2458 and 3770; 2458 and 3722; 2458 and 3690; 2458 and 3682; 2458 and 3330; 2458 and 3354; 2458 and 3394; 2458 and 3386; 2194 and 3418; 2194 and 3370; 2194 and 3514; 2194 and 3658; 2194 and 4010; 2194 and 4026; 2194 and 3914; 2194 and 3938; 2194 and 3858; 2194 and 3818; 2194 and 3794; 2194 and 3802; 2194 and 3746; 2194 and 3778; 2194 and 3770; 2194 and 3722; 2194 and 3690; 2194 and 3682; 2194 and 3330; 2194 and 3354; 2194 and 3394; 2194 and 3386; 2594 and 3418; 2594 and 3370; 2594 and 3514; 2594 and 3658; 2594 and 4010; 2594 and 4026; 2594 and 3914; 2594 and 3938; 2594 and 3858; 2594 and 3818; 2594 and 3794; 2594 and 3802; 2594 and 3746; 2594 and 3778; 2594 and 3770; 2594 and 3722; 2594 and 3690; 2594 and 3682; 2594 and 3330; 2594 and 3354; 2594 and 3394; 2594 and 3386; 2618 and 3418; 2618 and 3370; 2618 and 3514; 2618 and 3658; 2618 and 4010; 2618 and 4026; 2618 and 3914; 2618 and 3938; 2618 and 3858; 2618 and 3818; 2618 and 3794; 2618 and 3802; 2618 and 3746; 2618 and 3778; 2618 and 3770; 2618 and 3722; 2618 and 3690; 2618 and 3682; 2618 and 3330; 2618 and 3354; 2618 and 3394; and 2618 and 3386. In some embodiments, the methods comprise further administering a DNA-PK inhibitor. In some embodiments, the DNA-PK inhibitor is Compound 6. In some embodiments, the DNA-PK inhibitor is Compound 3.
In a further example, where the TNR is in the 5′ UTR of the FMR1 gene, the first and second spacers may have the sequences of any one of the following pairs of SEQ ID NOs: 5782 and 5262; 5830 and 5262; 5926 and 5262; 5950 and 5262; and 5998 and 5262. In some embodiments, the methods comprise further administering a DNA-PK inhibitor. In some embodiments, the DNA-PK inhibitor is Compound 6. In some embodiments, the DNA-PK inhibitor is Compound 3.
In a further example, where the TNR is in an intron of the FXN gene, the first and second spacers may have the sequences of any one of the following pairs of SEQ ID NOs: 47047 and 7447; 7463 and 46967; 46768 and 7680; 47032 and 7447. In some embodiments, the methods comprise further administering a DNA-PK inhibitor. In some embodiments, the DNA-PK inhibitor is Compound 6. In some embodiments, the DNA-PK inhibitor is Compound 3.
In some embodiments, methods are provided for treating a disease or characterized by a trinucleotide repeat (TNR) in the 3′ UTR of the DMPK gene, the method comprising administering a composition comprising a pair of guide RNAs comprising a first and second spacer sequence, or one or more nucleic acids encoding the pair of guide RNAs. In some embodiments, methods are provided for methods of excising a trinucleotide repeat (TNR) in the 3′ UTR of the DMPK gene, the method comprising administering a composition comprising a pair of guide RNAs comprising a first and second spacer sequence, or one or more nucleic acids encoding the pair of guide RNAs. In some embodiments, the pair of guide RNAs comprise a first and second spacer sequence selected from SEQ ID NOs: 2202 and 3418; 2202 and 3370; 2202 and 3514; 2202 and 3658; 2178 and 3418; 2178 and 3370; 2178 and 3514; 2178 and 3658; 2170 and 3418; 2170 and 3370; 2170 and 3514; 2170 and 3658; 2162 and 3418; 2162 and 3370; 2162 and 3514; 2162 and 3658; 2202 and 4010; 2202 and 4026; 2202 and 3914; 2202 and 3938; 2202 and 3858; 2202 and 3818; 2202 and 3794; 2202 and 3802; 2202 and 3746; 2202 and 3778; 2202 and 3770; 2202 and 3722; 2202 and 3690; 2202 and 3682; 2202 and 3330; 2202 and 3354; 2202 and 3394; 2202 and 3386; 2178 and 4010; 2178 and 4026; 2178 and 3914; 2178 and 3938; 2178 and 3858; 2178 and 3818; 2178 and 3794; 2178 and 3802; 2178 and 3746; 2178 and 3778; 2178 and 3770; 2178 and 3722; 2178 and 3690; 2178 and 3682; 2178 and 3330; 2178 and 3354; 2178 and 3394; 2178 and 3386; 2170 and 4010; 2170 and 4026; 2170 and 3914; 2170 and 3938; 2170 and 3858; 2170 and 3818; 2170 and 3794; 2170 and 3802; 2170 and 3746; 2170 and 3778; 2170 and 3770; 2170 and 3722; 2170 and 3690; 2170 and 3682; 2170 and 3330; 2170 and 3354; 2170 and 3394; 2170 and 3386; 2162 and 4010; 2162 and 4026; 2162 and 3914; 2162 and 3938; 2162 and 3858; 2162 and 3818; 2162 and 3794; 2162 and 3802; 2162 and 3746; 2162 and 3778; 2162 and 3770; 2162 and 3722; 2162 and 3690; 2162 and 3682; 2162 and 3330; 2162 and 3354; 2162 and 3394; 2162 and 3386; 1706 and 3418; 1706 and 3370; 1706 and 3514; 1706 and 3658; 1706 and 4010; 1706 and 4026; 1706 and 3914; 1706 and 3938; 1706 and 3858; 1706 and 3818; 1706 and 3794; 1706 and 3802; 1706 and 3746; 1706 and 3778; 1706 and 3770; 1706 and 3722; 1706 and 3690; 1706 and 3682; 1706 and 3330; 1706 and 3354; 1706 and 3394; 1706 and 3386; 2210 and 3418; 2210 and 3370; 2210 and 3514; 2210 and 3658; 2210 and 4010; 2210 and 4026; 2210 and 3914; 2210 and 3938; 2210 and 3858; 2210 and 3818; 2210 and 3794; 2210 and 3802; 2210 and 3746; 2210 and 3778; 2210 and 3770; 2210 and 3722; 2210 and 3690; 2210 and 3682; 2210 and 3330; 2210 and 3354; 2210 and 3394; 2210 and 3386; 1778 and 3418; 1778 and 3370; 1778 and 3514; 1778 and 3658; 1778 and 4010; 1778 and 4026; 1778 and 3914; 1778 and 3938; 1778 and 3858; 1778 and 3818; 1778 and 3794; 1778 and 3802; 1778 and 3746; 1778 and 3778; 1778 and 3770; 1778 and 3722; 1778 and 3690; 1778 and 3682; 1778 and 3330; 1778 and 3354; 1778 and 3394; 1778 and 3386; 2258 and 3418; 2258 and 3370; 2258 and 3514; 2258 and 3658; 2258 and 4010; 2258 and 4026; 2258 and 3914; 2258 and 3938; 2258 and 3858; 2258 and 3818; 2258 and 3794; 2258 and 3802; 2258 and 3746; 2258 and 3778; 2258 and 3770; 2258 and 3722; 2258 and 3690; 2258 and 3682; 2258 and 3330; 2258 and 3354; 2258 and 3394; 2258 and 3386; 2114 and 3418; 2114 and 3370; 2114 and 3514; 2114 and 3658; 2114 and 4010; 2114 and 4026; 2114 and 3914; 2114 and 3938; 2114 and 3858; 2114 and 3818; 2114 and 3794; 2114 and 3802; 2114 and 3746; 2114 and 3778; 2114 and 3770; 2114 and 3722; 2114 and 3690; 2114 and 3682; 2114 and 3330; 2114 and 3354; 2114 and 3394; 2114 and 3386; 1642 and 3418; 1642 and 3370; 1642 and 3514; 1642 and 3658; 1642 and 4010; 1642 and 4026; 1642 and 3914; 1642 and 3938; 1642 and 3858; 1642 and 3818; 1642 and 3794; 1642 and 3802; 1642 and 3746; 1642 and 3778; 1642 and 3770; 1642 and 3722; 1642 and 3690; 1642 and 3682; 1642 and 3330; 1642 and 3354; 1642 and 3394; 1642 and 3386; 1738 and 3418; 1738 and 3370; 1738 and 3514; 1738 and 3658; 1738 and 4010; 1738 and 4026; 1738 and 3914; 1738 and 3938; 1738 and 3858; 1738 and 3818; 1738 and 3794; 1738 and 3802; 1738 and 3746; 1738 and 3778; 1738 and 3770; 1738 and 3722; 1738 and 3690; 1738 and 3682; 1738 and 3330; 1738 and 3354; 1738 and 3394; 1738 and 3386; 2258 and 3418; 2258 and 3370; 2258 and 3514; 2258 and 3658; 2258 and 4010; 2258 and 4026; 2258 and 3914; 2258 and 3938; 2258 and 3858; 2258 and 3818; 2258 and 3794; 2258 and 3802; 2258 and 3746; 2258 and 3778; 2258 and 3770; 2258 and 3722; 2258 and 3690; 2258 and 3682; 2258 and 3330; 2258 and 3354; 2258 and 3394; 2258 and 3386; 2114 and 3418; 2114 and 3370; 2114 and 3514; 2114 and 3658; 2114 and 4010; 2114 and 4026; 2114 and 3914; 2114 and 3938; 2114 and 3858; 2114 and 3818; 2114 and 3794; 2114 and 3802; 2114 and 3746; 2114 and 3778; 2114 and 3770; 2114 and 3722; 2114 and 3690; 2114 and 3682; 2114 and 3330; 2114 and 3354; 2114 and 3394; 1706 and 3386; 1642 and 3418; 1642 and 3370; 1642 and 3514; 1642 and 3658; 1642 and 4010; 1642 and 4026; 1642 and 3914; 1642 and 3938; 1642 and 3858; 1642 and 3818; 1642 and 3794; 1642 and 3802; 1642 and 3746; 1642 and 3778; 1642 and 3770; 1642 and 3722; 1642 and 3690; 1642 and 3682; 1642 and 3330; 1642 and 3354; 1642 and 3394; 1642 and 3386; 1738 and 3418; 1738 and 3370; 1738 and 3514; 1738 and 3658; 1738 and 4010; 1738 and 4026; 1738 and 3914; 1738 and 3938; 1738 and 3858; 1738 and 3818; 1738 and 3794; 1738 and 3802; 1738 and 3746; 1738 and 3778; 1738 and 3770; 1738 and 3722; 1738 and 3690; 1738 and 3682; 1738 and 3330; 1738 and 3354; 1738 and 3394; 1738 and 3386; 1746 and 3418; 1746 and 3370; 1746 and 3514; 1746 and 3658; 1746 and 4010; 1746 and 4026; 1746 and 3914; 1746 and 3938; 1746 and 3858; 1746 and 3818; 1746 and 3794; 1746 and 3802; 1746 and 3746; 1746 and 3778; 1746 and 3770; 1746 and 3722; 1746 and 3690; 1746 and 3682; 1746 and 3330; 1746 and 3354; 1746 and 3394; 1746 and 3386; 2322 and 3418; 2322 and 3370; 2322 and 3514; 2322 and 3658; 2322 and 4010; 2322 and 4026; 2322 and 3914; 2322 and 3938; 2322 and 3858; 2322 and 3818; 2322 and 3794; 2322 and 3802; 2322 and 3746; 2322 and 3778; 2322 and 3770; 2322 and 3722; 2322 and 3690; 2322 and 3682; 2322 and 3330; 2322 and 3354; 2322 and 3394; 2322 and 3386; 1770 and 3418; 1770 and 3370; 1770 and 3514; 1770 and 3658; 1770 and 4010; 1770 and 4026; 1770 and 3914; 1770 and 3938; 1770 and 3858; 1770 and 3818; 1770 and 3794; 1770 and 3802; 1770 and 3746; 1770 and 3778; 1770 and 3770; 1770 and 3722; 1770 and 3690; 1770 and 3682; 1770 and 3330; 1770 and 3354; 1770 and 3394; 1770 and 3386; 1538 and 3418; 1538 and 3370; 1538 and 3514; 1538 and 3658; 1538 and 4010; 1538 and 4026; 1538 and 3914; 1538 and 3938; 1538 and 3858; 1538 and 3818; 1538 and 3794; 1538 and 3802; 1538 and 3746; 1538 and 3778; 1538 and 3770; 1538 and 3722; 1538 and 3690; 1538 and 3682; 1538 and 3330; 1538 and 3354; 1538 and 3394; 1538 and 3386; 2514 and 3418; 2514 and 3370; 2514 and 3514; 2514 and 3658; 2514 and 4010; 2514 and 4026; 2514 and 3914; 2514 and 3938; 2514 and 3858; 2514 and 3818; 2514 and 3794; 2514 and 3802; 2514 and 3746; 2514 and 3778; 2514 and 3770; 2514 and 3722; 2514 and 3690; 2514 and 3682; 2514 and 3330; 2514 and 3354; 2514 and 3394; 2514 and 3386; 2458 and 3418; 2458 and 3370; 2458 and 3514; 2458 and 3658; 2458 and 4010; 2458 and 4026; 2458 and 3914; 2458 and 3938; 2458 and 3858; 2458 and 3818; 2458 and 3794; 2458 and 3802; 2458 and 3746; 2458 and 3778; 2458 and 3770; 2458 and 3722; 2458 and 3690; 2458 and 3682; 2458 and 3330; 2458 and 3354; 2458 and 3394; 2458 and 3386; 2194 and 3418; 2194 and 3370; 2194 and 3514; 2194 and 3658; 2194 and 4010; 2194 and 4026; 2194 and 3914; 2194 and 3938; 2194 and 3858; 2194 and 3818; 2194 and 3794; 2194 and 3802; 2194 and 3746; 2194 and 3778; 2194 and 3770; 2194 and 3722; 2194 and 3690; 2194 and 3682; 2194 and 3330; 2194 and 3354; 2194 and 3394; 2194 and 3386; 2594 and 3418; 2594 and 3370; 2594 and 3514; 2594 and 3658; 2594 and 4010; 2594 and 4026; 2594 and 3914; 2594 and 3938; 2594 and 3858; 2594 and 3818; 2594 and 3794; 2594 and 3802; 2594 and 3746; 2594 and 3778; 2594 and 3770; 2594 and 3722; 2594 and 3690; 2594 and 3682; 2594 and 3330; 2594 and 3354; 2594 and 3394; 2594 and 3386; 2618 and 3418; 2618 and 3370; 2618 and 3514; 2618 and 3658; 2618 and 4010; 2618 and 4026; 2618 and 3914; 2618 and 3938; 2618 and 3858; 2618 and 3818; 2618 and 3794; 2618 and 3802; 2618 and 3746; 2618 and 3778; 2618 and 3770; 2618 and 3722; 2618 and 3690; 2618 and 3682; 2618 and 3330; 2618 and 3354; 2618 and 3394; and 2618 and 3386. In some embodiments, the pair of guide RNAs comprise a first and second spacer sequence selected from SEQ ID NOs: 2202 and 3418; 2202 and 3370; 2202 and 3514; 2202 and 3658; 2178 and 3418; 2178 and 3370; 2178 and 3514; 2178 and 3658; 2170 and 3418; 2170 and 3370; 2170 and 3514; 2170 and 3658; 2162 and 3418; 2162 and 3370; 2162 and 3514; 2162 and 3658; 2202 and 4010; 2202 and 4026; 2202 and 3914; 2202 and 3938; 2202 and 3858; 2202 and 3818; 2202 and 3794; 2202 and 3802; 2202 and 3746; 2202 and 3778; 2202 and 3770; 2202 and 3722; 2202 and 3690; 2202 and 3682; 2202 and 3330; 2202 and 3354; 2202 and 3394; 2202 and 3386; 2178 and 4010; 2178 and 4026; 2178 and 3914; 2178 and 3938; 2178 and 3858; 2178 and 3818; 2178 and 3794; 2178 and 3802; 2178 and 3746; 2178 and 3778; 2178 and 3770; 2178 and 3722; 2178 and 3690; 2178 and 3682; 2178 and 3330; 2178 and 3354; 2178 and 3394; 2178 and 3386; 2170 and 4010; 2170 and 4026; 2170 and 3914; 2170 and 3938; 2170 and 3858; 2170 and 3818; 2170 and 3794; 2170 and 3802; 2170 and 3746; 2170 and 3778; 2170 and 3770; 2170 and 3722; 2170 and 3690; 2170 and 3682; 2170 and 3330; 2170 and 3354; 2170 and 3394; 2170 and 3386; 2162 and 4010; 2162 and 4026; 2162 and 3914; 2162 and 3938; 2162 and 3858; 2162 and 3818; 2162 and 3794; 2162 and 3802; 2162 and 3746; 2162 and 3778; 2162 and 3770; 2162 and 3722; 2162 and 3690; 2162 and 3682; 2162 and 3330; 2162 and 3354; 2162 and 3394; 2162 and 3386. In some embodiments, the pair of guide RNAs comprise a first and second spacer sequence selected from SEQ ID NOs: 2202 and 3418; 2202 and 3370; 2202 and 3514; 2202 and 3658; 2178 and 3418; 2178 and 3370; 2178 and 3514; 2178 and 3658; 2170 and 3418; 2170 and 3370; 2170 and 3514; 2170 and 3658; 2162 and 3418; 2162 and 3370; 2162 and 3514; and 2162 and 3658. In some embodiments, the pair of guide RNAs comprise a first and second spacer sequence selected from SEQ ID NOs: 3778 and 2514; 3778 and 2258; 3778 and 2210; 3386 and 2514; 3386 and 2258; 3386 and 2210; 3354 and 2514; 3354 and 2258; and 3354 and 2210. In some embodiments, the pair of guide RNAs comprise a first and second spacer sequence selected from SEQ ID NOs: 3778 and 2258; 3778 and 2210; 3386 and 2258; 3386 and 2210; and 3354 and 2514. In some embodiments, the pair of guide RNAs comprise a first and second spacer sequence selected from SEQ ID NOs: 3346 and 2554; 3346 and 2498; 3330 and 2554; 3330 and 2498; 3330 and 2506; and 3330 and 2546. In some embodiments, the pair of guide RNAs comprise a first and second spacer sequence selected from SEQ ID NOs: 3346 and 2554; 3346 and 2498; 3330 and 2554; 3330 and 2498; 3354 and 2546; 3354 and 2506; 3378 and 2546; 3378 and 2506. In some embodiments, the pair of guide RNAs comprise a first and second spacer sequence selected from SEQ ID NOs: 3346 and 2554; 3346 and 2498; 3330 and 2554; and 3330 and 2498. In some embodiments, the pair of guide RNAs comprise a first and second spacer comprising SEQ ID NOs: 1153 and 1129. In some embodiments, the pair of guide RNAs comprise a first and second spacer sequence, wherein the pair of spacer sequences comprise a first spacer sequence selected from SEQ ID NOs: 2856, 2864, 2880, 2896, 2904, 2912, 2936, 2944, 2960, 2992, 3016, 3024, 3064, 3096, 3112, 3128, 3136, 3144, 3160, 3168, 3192, 3200, 3208, 3216, 3224, 3232, 3240, 3248, 3256, 3264, 3314, 3330, 3346, 3354, 3370, 3378, 3386, 3394, 3410, 3418, 3426, 3434, 3442, 3450, 3458, 3474, 3482, 3490, 3498, 3506, 3514, 3522, 3530, 3538, 3546, 3554, 3570, 3578, 3586, 3602, 3610, 3618, 3634, 3642, 3658, 3674, 3682, 3690, 3698, 3706, 3722, 3746, 3762, 3770, 3778, 3794, 3802, 3818, 3826, 3834, 3850, 3858, 3890, 3898, 3906, 3914, 3922, 3930, 3938, 3946, 3994, 4010, 4018, 4026, 4034, 4042, 4208, or 4506, and a second spacer sequence selected from SEQ ID NOs: 560, 584, 608, 616, 656, 672, 688, 696, 712, 744, 752, 760, 840, 864, 960, 976, 984, 1008, 1056, 1128, 1136, 1152, 1224, 1240, 1272, 1338, 1346, 1370, 1378, 1386, 1394, 1402, 1410, 1418, 1426, 1434, 1442, 1458, 1474, 1482, 1490, 1498, 1514, 1538, 1546, 1554, 1562, 1578, 1586, 1594, 1602, 1610, 1626, 1634, 1642, 1650, 1658, 1690, 1706, 1714, 1738, 1746, 1770, 1778, 1786, 1802, 1810, 1818, 1826, 1834, 1842, 1850, 1890, 1914, 1930, 1938, 1946, 1962, 1970, 1978, 1986, 1994, 2010, 2018, 2026, 2042, 2050, 2058, 2090, 2114, 2130, 2162, 2170, 2178, 2202, 2210, 2226, 2242, 2258, 2266, 2274, 2282, 2298, 2314, 2322, 2330, 2338, 2346, 2354, 2370, 2378, 2394, 2418, 2434, 2442, 2458, 2466, 2474, 2498, 2506, 2514, 2522, 2546, 2554, 2570, 2586, 2658, 4989, 4990, 4991, or 4992. In some embodiments, the pair of guide RNAs comprise a first and second spacer sequence, wherein the pair of spacer sequences comprise a first spacer sequence selected from SEQ ID NOs: 3778, 4026, 3794, 4010, 3906 and 3746, and a second spacer sequence selected from SEQ ID NOs: 1778, 1746, 1770, 1586, 1914, and 2210. In some embodiments, the pair of guide RNAs comprise a first and second spacer sequence, wherein the pair of spacer sequences comprise a first and second spacer sequence selected from SEQ ID NOs: 3778 and 1778; 3778 and 1746; 3778 and 1770; 3778 and 1586; 3778 and 1914; 3778 and 2210; 4026 and 1778; 4026 and 1746; 4026 and 1770; 4026 and 1586; 4026 and 1914; 4026 and 2210; 3794 and 1778; 3794 and 1746; 3794 and 1770; 3794 and 1586; 3794 and 1586; 3794 and 1914; 3794 and 2210; 4010 and 1778; 4010 and 1770; 4010 and 1746; 4010 and 1586; 4010 and 1914; 4010 and 2210; 3906 and 1778; 3906 and 1778; 3906 and 1746; 3906 and 1770; 3906 and 1586; 3906 and 1914; 3906 and 2210; 3746 and 1778; 3746 and 1746; 3746 and 1770; 3746 and 1586; 3746 and 1914; and 3746 and 2210. In some embodiments, the pair of guide RNAs comprise a first and second spacer sequence, wherein the pair of spacer sequences comprise a first spacer sequence selected from SEQ ID NOs: 3256, 2896, 3136, and 3224, and a second spacer sequence selected from SEQ ID NOs: 4989, 560, 672, 976, 760, 984, and 616. In some embodiments, the pair of guide RNAs comprise a first and second spacer sequence, wherein the pair of spacer sequences comprise a first and second spacer sequence selected from SEQ ID NOs: 3256 and 4989; 3256 and 984; 3256 and 616; 2896 and 4989; 2896 and 672; 2896 and 760; 3136 and 4989; 3136 and 560; 3224 and 4989; 3224 and 976; and 3224 and 760. In some embodiments, the methods comprise further administering a DNA-PK inhibitor. In some embodiments, the DNA-PK inhibitor is Compound 6. In some embodiments, the DNA-PK inhibitor is Compound 3.
In some embodiments, methods are provided for treating a disease or characterized by a trinucleotide repeat (TNR) in the 5′ UTR of the FMR1 gene, the method comprising administering a composition comprising a pair of guide RNAs comprising a first and second spacer sequence, or one or more nucleic acids encoding the pair of guide RNAs. In some embodiments, methods are provided for method of excising a trinucleotide repeat (TNR) in the 5′ UTR of the FMR1 gene, the method comprising administering a composition comprising a pair of guide RNAs comprising a first and second spacer sequence, or one or more nucleic acids encoding the pair of guide RNAs. In some embodiments, the pair of guide RNAs comprise a first and second spacer sequence selected from SEQ ID NOs: 5782 and 5262; 5830 and 5262; 5926 and 5262; 5950 and 5262; and 5998 and 5262. In some embodiments, the pair of guide RNAs comprise a first and second spacer sequence selected from SEQ ID NOs: 5830 and 5262; and 6022 and 5310. In some embodiments, the pair of guide RNAs comprise a first and second spacer sequence comprising SEQ ID NOs: 5334 and 5830. In some embodiments, the methods comprise further administering a DNA-PK inhibitor. In some embodiments, the DNA-PK inhibitor is Compound 6. In some embodiments, the DNA-PK inhibitor is Compound 3.
In some embodiments, methods are provided for treating a disease or characterized by a trinucleotide repeat (TNR) in the 5′ UTR of the FXN gene, the method comprising administering a composition comprising a pair of guide RNAs comprising a first and second spacer sequence, or one or more nucleic acids encoding the pair of guide RNAs. In some embodiments, methods are provided for method of excising a trinucleotide repeat (TNR) in the 5′ UTR of the FXN gene, the method comprising administering a composition comprising a pair of guide RNAs comprising a first and second spacer sequence, or one or more nucleic acids encoding the pair of guide RNAs. In some embodiments, the pair of guide RNAs comprise a first and second spacer sequence selected from SEQ ID NOs: 47047 and 7447; 7463 and 46967; 46768 and 7680; 47032 and 7447. In some embodiments, the pair of guide RNAs comprise a first and second spacer sequence comprising SEQ ID NOs: 47047 and 7447. In some embodiments, the pair of guide RNAs comprise a first and second spacer sequence comprising SEQ ID NOs: 52898 and 36546. In some embodiments, the methods comprise further administering a DNA-PK inhibitor. In some embodiments, the DNA-PK inhibitor is Compound 6. In some embodiments, the DNA-PK inhibitor is Compound 3.
In some embodiments, methods are provided for excising a trinucleotide repeat (TNR) in the 3′ UTR of the DMPK gene, the method comprising administering a pair of guide RNAs comprising a pair of spacer sequences, wherein the first spacer sequence directs a RNA-guided DNA nuclease to any nucleotide within a first stretch of sequence, wherein the first stretch starts 1 nucleotide from the DMPK-U29 cut site with spCas9 and continues through the repeat. In some embodiments, the first stretch starts 1 nucleotide from the DMPK-U30 cut site with spCas9 and continues through 1 nucleotide before the DMPK-U56 cut site. In some embodiments, the first stretch starts 1 nucleotide from the DMPK-U30 cut site with spCas9 and continues through 1 nucleotide before the DMPK-U52 cut site. In some embodiments, the first stretch is SEQ ID NO: 53413. In some embodiments, the first stretch is SEQ ID NO: 53414. In some embodiments, the first stretch is SEQ ID NO: 53415.
In some embodiments, methods are provided for excising a trinucleotide repeat (TNR) in the 3′ UTR of the DMPK gene, the method comprising administering a pair of guide RNAs comprising a pair of spacer sequences, wherein the second spacer sequence directs a RNA-guided DNA nuclease to any nucleotide within a second stretch of sequence, wherein the second stretch starts 1 nucleotide in from the DMPK-D15 cut site with spCas9 and continues until 1 nucleotide before the DMPK-D51 cut site. In some embodiments, the second stretch starts 1 nucleotide from the DMPK-D35 cut site with spCas9 and continues until 1 nucleotide before the DMPK-D51 cut site. In some embodiments, the second stretch is SEQ ID NO: 53416. In some embodiments, the second stretch is SEQ ID NO: 53417.
In some embodiments, methods are provided for excising a trinucleotide repeat (TNR) in the 3′ UTR of the DMPK gene, the method comprising administering a pair of guide RNAs comprising a pair of spacer sequences, wherein the first spacer sequence directs a RNA-guided DNA nuclease to any nucleotide within a first stretch of sequence, and wherein the second spacer sequence directs a RNA-guided DNA nuclease to any nucleotide within a second stretch of sequence. In some embodiments, the first stretch starts 1 nucleotide from the DMPK-U29 cut site with spCas9 and continues through the repeat. In some embodiments, the first stretch starts 1 nucleotide from the DMPK-U30 cut site with spCas9 and continues through 1 nucleotide before the DMPK-U56 cut site. In some embodiments, the first stretch starts 1 nucleotide from the DMPK-U30 cut site with spCas9 and continues through 1 nucleotide before the DMPK-U52 cut site. In some embodiments, the first stretch is SEQ ID NO: 53413. In some embodiments, the first stretch is SEQ ID NO: 53414. In some embodiments, the first stretch is SEQ ID NO: 53415. In some embodiments, the second stretch starts 1 nucleotide in from the DMPK-D15 cut site with spCas9 and continues until 1 nucleotide before the DMPK-D51 cut site. In some embodiments, the second stretch starts 1 nucleotide from the DMPK-D35 cut site with spCas9 and continues until 1 nucleotide before the DMPK-D51 cut site. In some embodiments, the second stretch is SEQ ID NO: 53416. In some embodiments, the second stretch is SEQ ID NO: 53417. In some embodiments, the methods comprise further administering a DNA-PK inhibitor. In some embodiments, the DNA-PK inhibitor is Compound 6. In some embodiments, the DNA-PK inhibitor is Compound 3.
In some embodiments, the methods further comprise administering an RNA-targeted endonuclease, or a nucleic acid encoding the RNA-targeted endonuclease. In some embodiments, the RNA-targeted endonuclease is a Cas nuclease. In some embodiments, the Cas nuclease is Cas9. In some embodiments, the Cas9 nuclease is from Streptococcus pyogenes (spCas9). In some embodiments, the Cas9 nuclease is from Staphylococcus aureus. In some embodiments, the Cas nuclease is Cpf1.
Any of the foregoing methods and any other method described herein may be combined to the extent feasible with any of the additional features described herein, including in the sections above, the following discussion, and the examples.
In some embodiments, the one or more gRNAs direct the RNA-targeted endonuclease to a site in or near a TNR or self-complementary region. For example, the RNA-targeted endonuclease may be directed to cut within 10, 20, 30, 40, or 50 nucleotides of the TNR or self-complementary region.
In some embodiments, at least a pair of gRNAs are provided which direct the RNA-targeted endonuclease to a pair of sites flanking (i.e., on opposite sides of) a TNR or self-complementary region. For example, the pair of sites flanking a TNR or self-complementary region may each be within 10, 20, 30, 40, or 50 nucleotides of the TNR or self-complementary region but on opposite sides thereof
Where a DNA-PK inhibitor is used in a method disclosed herein, it may be any DNA-PK inhibitor known in the art. DNA-PK inhibitors are discussed in detail, for example, in WO2014/159690; WO2013/163190; WO2018/013840; WO 2019/143675; WO 2019/143677; WO 2019/143678; and Robert et al., Genome Medicine (2015) 7:93, each of which are incorporated by reference herein. In some embodiments, the DNA-PK inhibitor is NU7441, KU-0060648, or any one of Compounds 1, 2, 3, 4, 5, or 6 (structures shown below), each of which is also described in at least one of the foregoing citations. In some embodiments, the DNA-PK inhibitor is Compound 6. In some embodiments, the DNA-PK inhibitor is Compound 3. Structures for exemplary DNA-PK inhibitors are as follows in Table 1A. Unless otherwise indicated, reference to a DNA-PK inhibitor by name or structure encompasses pharmaceutically acceptable salts thereof.
In any of the foregoing embodiments where a DNA-PK inhibitor is used, it may be used in combination with only one gRNA or vector encoding only one gRNA to promote excision, i.e., the method does not always involve providing two or more guides that promote cleavage near a TNR or self-complementary region.
In some embodiments, trinucleotide repeats or a self-complementary region is excised from a locus or gene associated with a disorder, such as a repeat expansion disorder, which may be a trinucleotide repeat expansion disorder. A repeat expansion disorder is one in which unaffected individuals have alleles with a number of repeats in a normal range, and individuals having the disorder or at risk for the disorder have one or two alleles with a number of repeats in an elevated range relative to the normal range. Exemplary repeat expansion disorders are listed and described in Table 1. In some embodiments, the repeat expansion disorder is any one of the disorders listed in Table 1. In some embodiments, the repeat expansion disorder is DM1. In some embodiments, the repeat expansion disorder is HD. In some embodiments, the repeat expansion disorder is FXS. In some embodiments, the repeat expansion disorder is a spinocerebellar ataxia. In some embodiments, the locus or gene from which the trinucleotide repeats are excised is a gene listed in Table 1. In some embodiments, the locus or gene from which the trinucleotide repeats are excised is DMPK. In some embodiments, the locus or gene from which the trinucleotide repeats are excised is HTT. In some embodiments, the locus or gene from which the trinucleotide repeats are excised is Frataxin. In some embodiments, the locus or gene from which the trinucleotide repeats are excised is FMR1. In some embodiments, the locus or gene from which the trinucleotide repeats are excised is an Ataxin. In some embodiments, the locus or gene from which the trinucleotide repeats are excised is a gene associated with a type of spinocerebellar ataxia.
The number of repeats that is excised may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000, or in a range bounded by any two of the foregoing numbers, inclusive, or in any of the ranges listed in the Summary above. In some embodiments, the number of repeats that is excised is in a range listed in Table 1, e.g., as a pathological, premutation, at-risk, or intermediate range.
In some embodiments, excision of a repeat or self-complementary region ameliorates at least one phenotype or symptom associated with the repeat or self-complementary region or associated with a disorder associated with the repeat or self-complementary region. This may include ameliorating aberrant expression of a gene encompassing or near the repeat or self-complementary region, or ameliorating aberrant activity of a gene product (noncoding RNA, mRNA, or polypeptide) encoded by a gene encompassing the repeat or self-complementary region.
For example, where the TNRs are within the DMPK gene, excision of the TNRs may ameliorate one or more phenotypes associated with an expanded-repeat DMPK gene, e.g., one or more of increasing myotonic dystrophy protein kinase activity; increasing phosphorylation of phospholemman, dihydropyridine receptor, myogenin, L-type calcium channel beta subunit, and/or myosin phosphatase targeting subunit; increasing inhibition of myosin phosphatase; and/or ameliorating muscle loss, muscle weakness, hypersomnia, one or more executive function deficiencies, insulin resistance, cataract formation, balding, or male infertility or low fertility.
Where the TNRs are within the HTT gene, excision of the TNRs may ameliorate one or more phenotypes associated with an expanded-repeat HTT gene, e.g., one or more of striatal neuron loss, involuntary movements, irritability, depression, small involuntary movements, poor coordination, difficulty learning new information or making decisions, difficulty walking, speaking, and/or swallowing, and/or a decline in thinking and/or reasoning abilities.
Where the TNRs are within the FMR1 gene, excision of the TNRs may ameliorate one or more phenotypes associated with an expanded-repeat FMR1 gene, e.g., one or more of aberrant FMR1 transcript or Fragile X Mental Retardation Protein levels, translational dysregulation of mRNAs normally associated with FMRP, lowered levels of phospho-cofilin (CFL1), increased levels of phospho-cofilin phosphatase PPP2CA, diminished mRNA transport to neuronal synapses, increased expression of HSP27, HSP70, and/or CRYAB, abnormal cellular distribution of lamin A/C isoforms, early-onset menopause such as menopause before age 40 years, defects in ovarian development or function, elevated level of serum gonadotropins (e.g., FSH), progressive intention tremor, parkinsonism, cognitive decline, generalized brain atrophy, impotence, and/or developmental delay.
Where the TNRs are within the FMR2 gene or adjacent to the 5′ UTR of FMR2, excision of the TNRs may ameliorate one or more phenotypes associated with expanded-repeats in or adjacent to the FMR2 gene, e.g., one or more of aberrant FMR2 expression, developmental delays, poor eye contact, repetitive use of language, and hand-flapping.
Where the TNRs are within the AR gene, excision of the TNRs may ameliorate one or more phenotypes associated with an expanded-repeat AR gene, e.g., one or more of aberrant AR expression; production of a C-terminally truncated fragment of the androgen receptor protein; proteolysis of androgen receptor protein by caspase-3 and/or through the ubiquitin-proteasome pathway; formation of nuclear inclusions comprising CREB-binding protein; aberrant phosphorylation of p44/42, p38, and/or SAPK/JNK; muscle weakness; muscle wasting; difficulty walking, swallowing, and/or speaking; gynecomastia; and/or male infertility.
Where the TNRs are within the ATXN1 gene, excision of the TNRs may ameliorate one or more phenotypes associated with an expanded-repeat ATXN1 gene, e.g., one or more of formation of aggregates comprising ATXN1; Purkinje cell death; ataxia; muscle stiffness; rapid, involuntary eye movements; limb numbness, tingling, or pain; and/or muscle twitches.
Where the TNRs are within the ATXN2 gene, excision of the TNRs may ameliorate one or more phenotypes associated with an expanded-repeat ATXN2 gene, e.g., one or more of aberrant ATXN2 production; Purkinje cell death; ataxia; difficulty speaking or swallowing; loss of sensation and weakness in the limbs; dementia; muscle wasting; uncontrolled muscle tensing; and/or involuntary jerking movements.
Where the TNRs are within the ATXN3 gene, excision of the TNRs may ameliorate one or more phenotypes associated with an expanded-repeat ATXN3 gene, e.g., one or more of aberrant ATXN3 levels; aberrant beclin-1 levels; inhibition of autophagy; impaired regulation of superoxide dismutase 2; ataxia; difficulty swallowing; loss of sensation and weakness in the limbs; dementia; muscle stiffness; uncontrolled muscle tensing; tremors; restless leg symptoms; and/or muscle cramps.
Where the TNRs are within the CACNA1A gene, excision of the TNRs may ameliorate one or more phenotypes associated with an expanded-repeat CACNA1A gene, e.g., one or more of aberrant CaV2.1 voltage-gated calcium channels in CACNA1A-expressing cells; ataxia; difficulty speaking; involuntary eye movements; double vision; loss of arm coordination; tremors; and/or uncontrolled muscle tensing.
Where the TNRs are within the ATXN7 gene, excision of the TNRs may ameliorate one or more phenotypes associated with an expanded-repeat ATXN7 gene, e.g., one or more of aberrant histone acetylation; aberrant histone deubiquitination; impairment of transactivation by CRX; formation of nuclear inclusions comprising ATXN7; ataxia; incoordination of gait; poor coordination of hands, speech and/or eye movements; retinal degeneration; and/or pigmentary macular dystrophy.
Where the TNRs are within the ATXN8OS gene, excision of the TNRs may ameliorate one or more phenotypes associated with an expanded-repeat ATXN8OS gene, e.g., one or more of formation of ribonuclear inclusions comprising ATXN8OS mRNA; aberrant KLHL1 protein expression; ataxia; difficulty speaking and/or walking; and/or involuntary eye movements.
Where the TNRs are within the PPP2R2B gene, excision of the TNRs may ameliorate one or more phenotypes associated with an expanded-repeat PPP2R2B gene, e.g., one or more of aberrant PPP2R2B expression; aberrant phosphatase 2 activity; ataxia; cerebellar degeneration; difficulty walking; and/or poor coordination of hands, speech and/or eye movements.
Where the TNRs are within the TBP gene, excision of the TNRs may ameliorate one or more phenotypes associated with an expanded-repeat TBP gene, e.g., one or more of aberrant transcription initiation; aberrant TBP protein accumulation (e.g., in cerebellar neurons); aberrant cerebellar neuron cell death; ataxia; difficulty walking; muscle weakness; and/or loss of cognitive abilities.
Where the TNRs are within the ATN1 gene, excision of the TNRs may ameliorate one or more phenotypes associated with an expanded-repeat ATN1 gene, e.g., one or more of aberrant transcriptional regulation; aberrant ATN1 protein accumulation (e.g., in neurons); aberrant neuron cell death; involuntary movements; and/or loss of cognitive abilities.
In some embodiments, any one or more of the gRNAs, vectors, DNA-PK inhibitors, compositions, or pharmaceutical formulations described herein is for use in a method disclosed herein or in preparing a medicament for treating or preventing a disease or disorder in a subject. In some embodiments, treatment and/or prevention is accomplished with a single dose, e.g., one-time treatment, of medicament/composition.
In some embodiments, the invention comprises a method of treating or preventing a disease or disorder in subject comprising administering any one or more of the gRNAs, vectors, compositions, or pharmaceutical formulations described herein. In some embodiments, the gRNAs, vectors, compositions, or pharmaceutical formulations described herein are administered as a single dose, e.g., at one time. In some embodiments, the single dose achieves durable treatment and/or prevention. In some embodiments, the method achieves durable treatment and/or prevention. Durable treatment and/or prevention, as used herein, includes treatment and/or prevention that extends at least i) 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 weeks; ii) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, or 36 months; or iii) 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years. In some embodiments, a single dose of the gRNAs, vectors, compositions, or pharmaceutical formulations described herein is sufficient to treat and/or prevent any of the indications described herein for the duration of the subject's life.
In some embodiments, a method of excising a TNR is provided comprising administering a composition comprising a guide RNA, or a vector encoding a guide RNA, comprising any one or more guide sequences of SEQ ID Nos: 101-4988, 5001-7264, or 7301-53372. In some embodiments, gRNAs comprising any one or more of the guide sequences of SEQ ID NOs: 101-4988, 5001-7264, or 7301-53372 are administered to excise a TNR. The guide RNAs may be administered together with an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9) or an mRNA or vector encoding an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9). Any of these methods may further comprise administering a DNA-PK inhibitor, such as any of those described herein.
In some embodiments, a method of treating a TNR-associated disease or disorder is provided comprising administering a composition comprising a guide RNA comprising any one or more of the guide sequences of SEQ ID NOs: 101-4988, 5001-7264, or 7301-53372. The guide RNAs may be administered together with an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9) or an mRNA or vector encoding an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9). Any of these methods may further comprise administering a DNA-PK inhibitor, such as any of those described herein.
In some embodiments, a method of decreasing or eliminating production of an mRNA comprising an expanded trinucleotide repeat is provided comprising administering a guide RNA comprising any one or more of the guide sequences of 101-4988, 5001-7264, or 7301-53372. The guide RNAs may be administered together with an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9) or an mRNA or vector encoding an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9). Any of these methods may further comprise administering a DNA-PK inhibitor, such as any of those described herein.
In some embodiments, a method of decreasing or eliminating production of a protein comprising an expanded amino acid repeat is provided comprising administering a guide RNA comprising any one or more of the guide sequences of 101-4988, 5001-7264, or 7301-53372. The guide RNAs may be administered together with an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9) or an mRNA or vector encoding an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9). Any of these methods may further comprise administering a DNA-PK inhibitor, such as any of those described herein.
In some embodiments, gRNAs comprising any one or more of the guide sequences of SEQ ID NOs: 101-4988, 5001-7264, or 7301-53372 are administered to reduce expression of a polypeptide comprising an expanded amino acid repeat. The gRNAs may be administered together with an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9) or an mRNA or vector encoding an RNA-guided DNA nuclease such as a Cas nuclease (e.g., Cas9). Any of these methods may further comprise administering a DNA-PK inhibitor, such as any of those described herein.
In some embodiments, the gRNAs comprising the guide sequences of Table 2 or of the Sequence Listing together with an RNA-guided DNA nuclease such as a Cas nuclease and a DNA-PK inhibitor induce DSBs, and microhomology-mediated end joining (MMEJ) during repair leads to a mutation in the targeted gene. In some embodiments, MMEJ leads to excision of trinucleotide repeats or a self-complementary sequence.
In some embodiments, the subject is mammalian In some embodiments, the subject is human. In some embodiments, the subject is cow, pig, monkey, sheep, dog, cat, fish, or poultry.
In some embodiments, the use of a guide RNAs comprising any one or more of the guide sequences in Table 2 and/or the Sequence Listing (e.g., in a composition provided herein) is provided for the preparation of a medicament for treating a human subject having a disorder listed in Table 1, such as DM1. Such use may be in combination with administering a DNA-PK inhibitor, such as any of those described herein.
In some embodiments, the guide RNAs, compositions, and formulations are administered intravenously. In some embodiments, the guide RNAs, compositions, and formulations are administered intramuscularly. In some embodiments, the guide RNAs, compositions, and formulations are administered intracranially. In some embodiments, the guide RNAs, compositions, and formulations are administered to cells ex vivo. Where a DNA-PK inhibitor is administered, it may be administered in the same composition as or a different composition from the composition comprising the guide RNA, and may be administered by the same or a different route as the guide RNA. In some embodiments, the DNA-PK inhibitor may be administered intravenously. In some embodiments, the DNA-PK inhibitor may be administered orally.
In some embodiments, the guide RNAs, compositions, and formulations are administered concomitantly with the DNA-PK inhibitor. In some embodiments, DNA-PK inhibitor is administered accordingly to its own dosing schedule.
In some embodiments, a single administration of a composition comprising a guide RNA provided herein is sufficient to excise TNRs or a self-complementary region. In other embodiments, more than one administration of a composition comprising a guide RNA provided herein may be beneficial to maximize therapeutic effects.
In some embodiments, the invention comprises combination therapies comprising any of the methods described herein (e.g., one or more of the gRNAs comprising any one or more of the guide sequences disclosed in Table 2 and/or the Sequence Listing (e.g., in a composition provided herein) together with an additional therapy suitable for ameliorating a disorder associated with the targeted gene and/or one or more symptoms thereof, as described above. Suitable additional therapies for use in ameliorating various disorders, such as those listed in Table 1, and/or one or more symptoms thereof are known in the art.
Delivery of gRNA Compositions
The methods and uses disclosed herein may use any suitable approach for delivering the gRNAs and compositions described herein. Exemplary delivery approaches include vectors, such as viral vectors; lipid nanoparticles; transfection; and electroporation. In some embodiments, vectors or LNPs associated with the gRNAs disclosed herein are for use in preparing a medicament for treating a disease or disorder.
Where a vector is used, it may be a viral vector, such as a non-integrating viral vector. In some embodiments, viral vector is an adeno-associated virus vector, a lentiviral vector, an integrase-deficient lentiviral vector, an adenoviral vector, a vaccinia viral vector, an alphaviral vector, or a herpes simplex viral vector. In some embodiments, the viral vector is an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh10 (see, e.g., SEQ ID NO: 81 of US 9,790,472, which is incorporated by reference herein in its entirety), AAVrh74 (see, e.g., SEQ ID NO: 1 of US 2015/0111955, which is incorporated by reference herein in its entirety), or AAV9 vector, wherein the number following AAV indicates the AAV serotype. Any variant of an AAV vector or serotype thereof, such as a self-complementary AAV (scAAV) vector, is encompassed within the general terms AAV vector, AAV1 vector, etc. See, e.g., McCarty et al., Gene Ther. 2001;8:1248-54, Naso et al., BioDrugs 2017; 31:317-334, and references cited therein for detailed discussion of various AAV vectors.
In some embodiments, the vector (e.g., viral vector, such as an adeno-associated viral vector) comprises a tissue-specific (e.g., muscle-specific) promoter, e.g., which is operatively linked to a sequence encoding the gRNA. In some embodiments, the muscle-specific promoter is a muscle creatine kinase promoter, a desmin promoter, an MHCK7 promoter, or an SPc5-12 promoter. In some embodiments, the muscle-specific promoter is a CK8 promoter. In some embodiments, the muscle-specific promoter is a CK8e promoter. Muscle-specific promoters are described in detail, e.g., in US2004/0175727 A1; Wang et al., Expert Opin Drug Deliv. (2014) 11, 345-364; Wang et al., Gene Therapy (2008) 15, 1489-1499. In some embodiments, the tissue-specific promoter is a neuron-specific promoter, such as an enolase promoter. See, e.g., Naso et al., BioDrugs 2017; 31:317-334; Dashkoff et al., Mol Ther Methods Clin Dev. 2016;3:16081, and references cited therein for detailed discussion of tissue-specific promoters including neuron-specific promoters.
In some embodiments, in addition to guide RNA sequences, the vectors further comprise nucleic acids that do not encode guide RNAs. Nucleic acids that do not encode guide RNA include, but are not limited to, promoters, enhancers, regulatory sequences, and nucleic acids encoding an RNA-guided DNA nuclease, which can be a nuclease such as Cas9. In some embodiments, the vector comprises one or more nucleotide sequence(s) encoding a crRNA, a trRNA, or a crRNA and trRNA. In some embodiments, the vector comprises one or more nucleotide sequence(s) encoding a sgRNA and an mRNA encoding an RNA-guided DNA nuclease, which can be a Cas nuclease, such as Cas9 or Cpf1. In some embodiments, the vector comprises one or more nucleotide sequence(s) encoding a crRNA, a trRNA, and an mRNA encoding an RNA-guided DNA nuclease, which can be a Cas protein, such as, Cas9. In one embodiment, the Cas9 is from Streptococcus pyogenes (i.e., Spy Cas9 or SpCas9). In some embodiments, the nucleotide sequence encoding the crRNA, trRNA, or crRNA and trRNA (which may be a sgRNA) comprises or consists of a guide sequence flanked by all or a portion of a repeat sequence from a naturally-occurring CRISPR/Cas system. The nucleic acid comprising or consisting of the crRNA, trRNA, or crRNA and trRNA may further comprise a vector sequence wherein the vector sequence comprises or consists of nucleic acids that are not naturally found together with the crRNA, trRNA, or crRNA and trRNA.
Lipid nanoparticles (LNPs) are a known means for delivery of nucleotide and protein cargo, and may be used for delivery of the guide RNAs, compositions, or pharmaceutical formulations disclosed herein. In some embodiments, the LNPs deliver nucleic acid, protein, or nucleic acid together with protein.
In some embodiments, the invention comprises a method for delivering any one of the gRNAs disclosed herein to a subject, wherein the gRNA is associated with an LNP. In some embodiments, the gRNA/LNP is also associated with a Cas9 or an mRNA encoding Cas9.
In some embodiments, the invention comprises a composition comprising any one of the gRNAs disclosed and an LNP. In some embodiments, the composition further comprises a Cas9 or an mRNA encoding Cas9.
Electroporation is a well-known means for delivery of cargo, and any electroporation methodology may be used for delivery of any one of the gRNAs disclosed herein. In some embodiments, electroporation may be used to deliver any one of the gRNAs disclosed herein and Cas9 or an mRNA encoding Cas9.
In some embodiments, the invention comprises a method for delivering any one of the gRNAs disclosed herein to an ex vivo cell, wherein the gRNA is encoded by a vector, associated with an LNP, or in aqueous solution. In some embodiments, the gRNA/LNP or gRNA is also associated with a Cas9 or sequence encoding Cas9 (e.g., in the same vector, LNP, or solution).
Screening of gRNA Compositions with a DNA-PK Inhibitor
In some embodiments, methods are provided for screening for a guide RNA that is capable of excising a TNR or self-complementary region, the method comprising: a) contacting a cell with a guide RNA, a RNA-targeted endonuclease, and a DNA-PK inhibitor; b) repeating step a) without a DNA-PK inhibitor; c) comparing the excision of the TNR or self-complementary region from the cell contacted in steps a) as compared to the cell contacted in step b); and d) selecting a guide RNA wherein the excision is improved in the presence of the DNA-PK inhibitor as compared to without the DNA-PK inhibitor.
In some embodiments, methods are provided for screening for a guide RNA that is capable of excising a TNR or self-complementary region in DNA, the method comprising: a) contacting: i) a cell (e.g., a myoblast) with a guide RNA, an RNA-targeted endonuclease, and a DNA-PK inhibitor; and ii) the same type of cell as used in i) with a guide RNA, an RNA-targeted endonuclease but without a DNA-PK inhibitor; b) comparing the excision of the TNR or self-complementary region in DNA from the cell contacted in steps a) i) as compared to the cell contacted in step a) ii); and c) selecting a guide RNA wherein the excision is improved in the presence of the DNA-PK inhibitor as compared to without the DNA-PK inhibitor.
In some embodiments, methods are provided for screening for a pair of guide RNAs that is capable of excising a TNR or self-complementary region in DNA, the method comprising: a) contacting a cell with a pair of guide RNAs, a RNA-targeted endonuclease, and a DNA-PK inhibitor; b) repeating step a) without a DNA-PK inhibitor; c) comparing the excision of the TNR or self-complementary region in DNA from the cell contacted in steps a) as compared to the cell contacted in step b); and d) selecting a pair of guide RNAs wherein the excision is improved in the presence of the DNA-PK inhibitor as compared to without the DNA-PK inhibitor. In some embodiments, methods are provided for screening for a pair of guide RNAs that is capable of excising a TNR or self-complementary region in DNA, the method comprising: a) contacting: i) a cell (e.g., a myoblast) with a pair of guide RNAs, an RNA-targeted endonuclease, and a DNA-PK inhibitor, and ii) the same type of cell as used in a), i) with a pair of guide RNAs, an RNA-targeted endonuclease but without a DNA-PK inhibitor; b) comparing the excision of the TNR or self-complementary region in DNA from the cell contacted in steps a), i) as compared to the cell contacted in step a), ii); and c) selecting a pair of guide RNAs wherein the excision is improved in the presence of the DNA-PK inhibitor as compared to without the DNA-PK inhibitor.
As used herein, “excision is improved” or “improved excision” may refer to a greater amount of excision of a TNR or self-complementary region in DNA, and/or a more desirable excision product (e.g., based on the size or location of the deletion). In some embodiments, determining whether a guide RNA or pair of guide RNAs has improved excision of a TNR or self-complementary region in DNA from DNA of a cell may be done by PCR of genomic DNA of the cell using primers designed to amplify a region of DNA surrounding the TNR or self-complementary region in DNA. PCR products may be evaluated by DNA gel electrophoresis and analyzed for excision of a TNR or self-complementary region in DNA. In some embodiments, excision of the TNR or self-complementary region in DNA may evaluated by sequencing methods (e.g., Sanger sequencing, PacBio sequencing). In some embodiments, percent deletion of the TNR or self-complementary region in DNA may be determined using a ddPCR assay (see e.g.
In some embodiments, the guide RNA or pair of guide RNAs directs the RNA-targeted endonuclease to the 3′ UTR of the DMPK gene. In some embodiments, the guide RNA or pair of guide RNAs directs the RNA-targeted endonuclease to the 5′ UTR of the FMR1 gene. In some embodiments, the guide RNA or pair of guide RNAs directs the RNA-targeted endonuclease to the 5′ UTR of the FXN gene.
In some embodiments, the DNA-PK inhibitor is Compound 6 or Compound 3. In some embodiments, the cell is a wildtype cell, e.g., a wildtype iPSC cell. In some embodiments, the cell is a disease cell, e.g., a cell derived from a patient, e.g., a DM1 iPSC cell, DM1 myoblast, DM1 fibroblast. The screen may include adding DNA-PK inhibitor in increasing doses to evaluate the enhancement of DNA-PK inhibition on single guide excision. The screen may include adding DNA-PK inhibitor in increasing doses to evaluate the enhancement of DNA-PK inhibition on paired guide excision.
Compositions Comprising Guide RNA (gRNAs)
Provided herein are compositions useful for treating diseases and disorders associated with trinucleotide repeats (TNRs) or self-complementary regions of DNA (e.g., the diseases and disorders of Table 1) and for excising trinucleotide repeats or self-complementary regions from DNA, e.g., using one or more guide RNAs or a nucleic encoding the one or more guide RNAs, with an RNA-targeted endonuclease (e.g., a CRISPR/Cas system). The compositions may comprise the guide RNA(s) or a vector(s) encoding the guide RNA(s) and may be administered to subjects having or suspected of having a disease associated with the trinucleotide repeats or self-complementary regions, and may further comprise or be administered in combination with a DNA-PK inhibitor, such as any of those described herein. Exemplary guide sequences are shown in the Table 2 and in the Sequence Listing at SEQ ID NOs: 101-4988, 5001-7264, or 7301-53372.
In some embodiments, the one or more gRNAs direct the RNA-targeted endonuclease to a site in or near a TNR or self-complementary region. For example, the RNA-targeted endonuclease may be directed to cut within 10, 20, 30, 40, or 50 nucleotides of the TNR or self-complementary region.
In some embodiments, at least a pair of gRNAs are provided which direct the RNA-targeted endonuclease to a pair of sites flanking (i.e., on opposite sides of) a TNR or self-complementary region. For example, the pair of sites flanking a TNR or self-complementary region may each be within 10, 20, 30, 40, or 50 nucleotides of the TNR or self-complementary region but on opposite sides thereof. In some embodiments, a pair of gRNAs is provided that comprise guide sequences from Table 2 and/or the Sequence Listing and direct the RNA-targeted endonuclease to a pair of sites according to any of the foregoing embodiments.
Each of the guide sequences shown in Table 2 and in the Sequence Listing at SEQ ID NOs: 101-4988, 5001-7264, or 7301-53372 may further comprise additional nucleotides to form or encode a crRNA, e.g., using any known sequence appropriate for the RNA-targeted endonuclease being used. In some embodiments, the crRNA comprises (5′ to 3′) at least a spacer sequence and a first complementarity domain. The first complementary domain is sufficiently complementary to a second complementarity domain, which may be part of the same molecule in the case of an sgRNA or in a tracrRNA in the case of a dual or modular gRNA, to form a duplex. See, e.g., US 2017/0007679 for detailed discussion of crRNA and gRNA domains, including first and second complementarity domains. For example, an exemplary sequence suitable for use with SpCas9 to follow the guide sequence at its 3′ end is: GUUUUAGAGCUAUGCUGUUUUG (SEQ ID NO: 99) in 5′ to 3′ orientation. In some embodiments, an exemplary sequence for use with SpCas9 to follow the 3′ end of the guide sequence is a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 99, or a sequence that differs from SEQ ID NO: 99 by no more than 1, 2, 3, 4 or 5 nucleotides. Where a tracrRNA is used, in some embodiments, it comprises (5′ to 3′) a second complementary domain and a proximal domain. In the case of a sgRNA, the above guide sequences may further comprise additional nucleotides to form or encode a sgRNA, e.g., using any known sequence appropriate for the RNA-targeted endonuclease being used. In some embodiments, an sgRNA comprises (5′ to 3′) at least a spacer sequence, a first complementary domain, a linking domain, a second complementary domain, and a proximal domain. A sgRNA or tracrRNA may further comprise a tail domain. The linking domain may be hairpin-forming. See, e.g., US 2017/0007679 for detailed discussion and examples of crRNA and gRNA domains, including second complementarity domains, linking domains, proximal domains, and tail domains. For example, an exemplary sequence suitable for use with SpCas9 to follow the 3′ end of the guide sequence is: GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAA AGUGGCACCGAGUCGGUGC (SEQ ID NO:100) in 5′ to 3′ orientation. In some embodiments, an exemplary sequence for use with SpCas9 to follow the 3′ end of the guide sequence is a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 100, or a sequence that differs from SEQ ID NO: 100 by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.
In general, in the case of a DNA vector encoding a gRNA, the U residues in any of the RNA sequences described herein may be replaced with T residues.
SID means SEQ ID NO. In Table 2, the descriptions have the following meaning. The target locus is indicated first, followed by a 5 or 3 to indicate whether the guide directs cleavage 5′ or 3′ of the repeat region (in the orientation of the forward strand) or an 0 to indicate that the guide falls within the repeat region or outside of the segment (e.g., UTR or intron) where the repeats occur, followed by “forward” or “reverse” to indicate the strand to which the sequence corresponds, followed by the genomic coordinates of the sequence (version GRCh38 of the human genome). Thus, for example, for SEQ ID NO: 101, the designation “DMPK 3 forward 19:45769716-45769738” means that the guide directs cleavage 3′ of the repeat region of DMPK and corresponds to the sequence of the forward strand of chromosome 19 positions 45769716-45769738. As/LbCpf1 is sometimes referred to herein as Cpf1. Where a combination of guides is to be used to direct cleavage 5′ and 3′ of a repeat region, one skilled in the art can select a combination of a 5′ guide disclosed herein and a 3′ guide disclosed herein for a given target such as DMPK, FMR1, or FXN.
Provided herein are compositions comprising one or more guide RNAs or one or more nucleic acids encoding one or more guide RNAs. Such compositions may comprise any one or more of the spacer sequences disclosed herein (see, e.g., Table 2 and the Sequence Listing).
The following are guide sequences directed to DMPK: SEQ ID NOs 101-4988. In some embodiments, a composition comprising a guide RNA or a nucleic acid encoding a guide RNA is provided, wherein the guide RNA comprises a spacer sequence comprising any one of SEQ ID NOs 101-4988. A composition comprising a guide RNA or a nucleic acid encoding a guide RNA is provided, wherein the guide RNA comprises a spacer sequence of any one of SEQ ID NOs 101-4988. The following are guide sequences directed to FMR1: SEQ ID NOs 5001-7264. In some embodiments, a composition comprising a guide RNA or a nucleic acid encoding a guide RNA is provided, wherein the guide RNA comprises a spacer sequence comprising any one of SEQ ID NOs 5001-7264. In some embodiments, a composition comprising a guide RNA or a nucleic acid encoding a guide RNA is provided, wherein the guide RNA comprises a spacer sequence of any one of SEQ ID NOs 5001-7264. The following are guide sequences directed to FXN: SEQ ID NOs 7301-53372. In some embodiments, a composition comprising a guide RNA or a nucleic acid encoding a guide RNA is provided, wherein the guide RNA comprises a spacer sequence comprising any one of SEQ ID NOs 7301-53372. In some embodiments, a composition comprising a guide RNA or a nucleic acid encoding a guide RNA is provided, wherein the guide RNA comprises a spacer sequence of any one of SEQ ID NOs 7301-53372.
In some embodiments, a composition comprising one or more guide RNAs (gRNAs), or one or more nucleic acids encoding one or more guide RNAs, is provided, wherein the guide RNAs comprise guide sequences that direct an RNA-targeted endonuclease (e.g., a Cas nuclease such as Cas9), to a target DNA sequence in or near the CTG repeat region in the myotonic dystrophy protein kinase gene (DMPK) associated with myotonic dystrophy type 1. In some embodiments, a composition is provided comprising a gRNA, or nucleic acid encoding a gRNA, wherein the gRNA comprises a DMPK guide sequence shown in Table 2 or the Sequence Listing at SEQ ID NOs: 101-4988. In some embodiments, a composition is provided comprising a gRNA, or nucleic acid encoding a gRNA, wherein the gRNA comprises 17, 18, 19, or 20 contiguous nucleotides of a DMPK guide sequence shown in Table 2 or the Sequence Listing at SEQ ID NOs: 101-4988.
In some embodiments, a composition is provided comprising a gRNA, or nucleic acid encoding a gRNA, wherein the gRNA comprises 17, 18, 19, or 20 contiguous nucleotides of any one of SEQ ID NOs: 4018, 4010, 4002, 4042, 4034, 4026, 3954, 3946, 3994, 3914, 3978, 3906, 3898, 3938, 3922, 3858, 3850, 3882, 3826, 3818, 3842, 3794, 3786, 3762, 3810, 3746, 3778, 3738, 3770, 3722, 3754, 3690, 3666, 3658, 3634, 3586, 3546, 3530, 3642, 3514, 3506, 3490, 3618, 3610, 3602, 3578, 3442, 3522, 3410, 3378, 3434, 3370, 3426, 3418, 3394, 3386, 3330, 3354, 3346, 3314, 3930, 3890, 3834, 3802, 3706, 3698, 3682, 3674, 3570, 3554, 3538, 3498, 3482, 3458, 3474, 3450, 2667, 2666, 2650, 2642, 2626, 2618, 2706, 2690, 2682, 2610, 2674, 2658, 2602, 2594, 2634, 2554, 2546, 2586, 2538, 2578, 2570, 2522, 2498, 2490, 2466, 2458, 2450, 2514, 2506, 2418, 2482, 2474, 2394, 2442, 2434, 2370, 2378, 2354, 2346, 2338, 2314, 2298, 2282, 2274, 2266, 2330, 2258, 2322, 2242, 2234, 2290, 2250, 2218, 2226, 2210, 2194, 2146, 2138, 2122, 2106, 2098, 2090, 2130, 2114, 2034, 2026, 2058, 2050, 2042, 1914, 1786, 1778, 1770, 1842, 1738, 1706, 1690, 1746, 1714, 1650, 1642, 1610, 1586, 1562, 1546, 1578, 1538, 1378, 1370, 1922, 1898, 1906, 1794, 1762, 1698, 1674, 1722, 1362, 1450, 2202, 2178, 2170, 2162, 2018, 2010, 1890, 1962, 1946, 1850, 1818, 1658, 1634, 1602, 1554, 1434, 1426, 1338, 1346, 1978, 1994, 1986, 1970, 1938, 1930, 1810, 1834, 1826, 1802, 1626, 1594, 1514, 1498, 1490, 1482, 1474, 1458, 1442, 1418, 1410, 1402, 1394, or 1386.
In some embodiments, a composition is provided comprising a gRNA, or nucleic acid encoding a gRNA, wherein the gRNA comprises a sequence with about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to at least 17, 18, 19, or 20 contiguous nucleotides of a DMPK guide sequence shown in Table 2 or the Sequence Listing. In some embodiments, a composition is provided comprising a gRNA, or nucleic acid encoding a gRNA, wherein the gRNA comprises a sequence with about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a guide sequence shown in Table 2. In some embodiments, a composition is provided comprising a gRNA, or nucleic acid encoding a gRNA, wherein the gRNA further comprises a trRNA. In each composition and method embodiment described herein, the crRNA (comprising the spacer sequence) and trRNA may be associated as a single RNA (sgRNA) or may be on separate RNAs (dgRNA). In the context of sgRNAs, the crRNA and trRNA components may be covalently linked, e.g., via a phosphodiester bond or other covalent bond.
In some embodiments, a composition is provided comprising a gRNA, or nucleic acid encoding a gRNA, wherein the gRNA comprises a spacer sequence selected from SEQ ID NOs: 4018, 4010, 4002, 4042, 4034, 4026, 3954, 3946, 3994, 3914, 3978, 3906, 3898, 3938, 3922, 3858, 3850, 3882, 3826, 3818, 3842, 3794, 3786, 3762, 3810, 3746, 3778, 3738, 3770, 3722, 3754, 3690, 3666, 3658, 3634, 3586, 3546, 3530, 3642, 3514, 3506, 3490, 3618, 3610, 3602, 3578, 3442, 3522, 3410, 3378, 3434, 3370, 3426, 3418, 3394, 3386, 3330, 3354, 3346, 3314, 3930, 3890, 3834, 3802, 3706, 3698, 3682, 3674, 3570, 3554, 3538, 3498, 3482, 3458, 3474, 3450, 2667, 2666, 2650, 2642, 2626, 2618, 2706, 2690, 2682, 2610, 2674, 2658, 2602, 2594, 2634, 2554, 2546, 2586, 2538, 2578, 2570, 2522, 2498, 2490, 2466, 2458, 2450, 2514, 2506, 2418, 2482, 2474, 2394, 2442, 2434, 2370, 2378, 2354, 2346, 2338, 2314, 2298, 2282, 2274, 2266, 2330, 2258, 2322, 2242, 2234, 2290, 2250, 2218, 2226, 2210, 2194, 2146, 2138, 2122, 2106, 2098, 2090, 2130, 2114, 2034, 2026, 2058, 2050, 2042, 1914, 1786, 1778, 1770, 1842, 1738, 1706, 1690, 1746, 1714, 1650, 1642, 1610, 1586, 1562, 1546, 1578, 1538, 1378, 1370, 1922, 1898, 1906, 1794, 1762, 1698, 1674, 1722, 1362, 1450, 2202, 2178, 2170, 2162, 2018, 2010, 1890, 1962, 1946, 1850, 1818, 1658, 1634, 1602, 1554, 1434, 1426, 1338, 1346, 1978, 1994, 1986, 1970, 1938, 1930, 1810, 1834, 1826, 1802, 1626, 1594, 1514, 1498, 1490, 1482, 1474, 1458, 1442, 1418, 1410, 1402, 1394, or 1386. In some embodiments, a composition is provided comprising a gRNA, or nucleic acid encoding a gRNA, wherein the gRNA comprises a spacer sequence selected from SEQ ID NOs: 3330, 3914, 3418, 3746, 3778, 3394, 4026, 3690, 3794, 3386, 3938, 3682, 3818, 3658, 3722, 3802, 3858, 3514, 3770, 3370, 3354, 4010, 2202, 1706, 2210, 2170, 1778, 2258, 2114, 2178, 1642, 1738, 1746, 2322, 1770, 1538, 2514, 2458, 2194, 2594, 2162, and 2618. In some embodiments, a composition is provided comprising a gRNA, or nucleic acid encoding a gRNA, wherein the gRNA comprises a spacer sequence selected from SEQ ID NOs: 3746, 3778, 3394, 3386, 3938, 3818, 3722, 3858, 3370, 1706, 2210, 2114, 1538, and 2594. In some embodiments, a composition is provided comprising a gRNA, or nucleic acid encoding a gRNA, wherein the gRNA comprises a spacer sequence selected from SEQ ID NOs: 3330, 3746, 3778, 3394, 4026, 3386, 3938, 3818, 3722, 3802, 3858, 3514, 3770, 3370, 2202, 1706, 2210, 1778, 2114, 1738, 1746, 2322, 1538, 2514, 2458, 2194, and 2594. In some embodiments, a composition is provided comprising a gRNA, or nucleic acid encoding a gRNA, wherein the gRNA comprises a spacer sequence selected from SEQ ID NOs: 3330, 3914, 3418, 3746, 3778, 3394, 4026, 3690, 3794, 3386, 3938, 3682, 3818, 3658, and 3722. In some embodiments, a composition is provided comprising a gRNA, or nucleic acid encoding a gRNA, wherein the gRNA comprises a spacer sequence selected from SEQ ID NOs: 2202, 1706, 2210, 2170, 1778, 2258, 2114, 2178, 1642, 1738, 1746, and 2322. In some embodiments, a composition is provided comprising a gRNA, or nucleic acid encoding a gRNA, wherein the gRNA comprises a spacer sequence selected from SEQ ID NOs: 3778, 4026, 3794, 4010, 3906, 3746, 1778, 1746, 1770, 1586, 1914, and 2210. In some embodiments, a composition is provided comprising a gRNA, or nucleic acid encoding a gRNA, wherein the gRNA comprises a spacer sequence selected from SEQ ID NOs: 3378, 3354, 3346, 3330, 3314, 2658, 2690, 2546, 2554, 2498, and 2506. In some embodiments, a composition is provided comprising a gRNA, or nucleic acid encoding a gRNA, wherein the gRNA comprises a spacer sequence selected from SEQ ID NOs: 3330, 3314, 2658, 2690, 2554, and 2498. In some embodiments, a composition is provided comprising a gRNA, or nucleic acid encoding a gRNA, wherein the gRNA comprises a spacer sequence selected from SEQ ID NOs: 3314, 2690, 2554, and 2498. In some embodiments, a composition is provided comprising a gRNA, or nucleic acid encoding a gRNA, wherein the gRNA comprises a spacer sequence selected from SEQ ID NOs: 3914, 3514, 1778, 2458, 3858, 3418, 1706, and 2258. In some embodiments, a composition is provided comprising a gRNA, or nucleic acid encoding a gRNA, wherein the gRNA comprises a spacer sequence selected from SEQ ID NOs: 3914, 2114, 2618, and 3418. In some embodiments, a composition is provided comprising a gRNA, or nucleic acid encoding a gRNA, wherein the gRNA comprises a spacer sequence selected from SEQ ID NOs: 3916, 3420, and 3940. In some embodiments, a composition is provided comprising a gRNA, or nucleic acid encoding a gRNA, wherein the gRNA comprises a spacer sequence selected from SEQ ID NOs: 3914 and 3418. In some embodiments, a composition is provided comprising a gRNA, or nucleic acid encoding a gRNA, wherein the gRNA comprises SEQ ID NO: 3938.
In some embodiments, a composition is provided comprising a gRNA, or nucleic acid encoding a gRNA, wherein the gRNA comprises 17, 18, 19, or 20 contiguous nucleotides of a FXN guide sequence selected from SEQ ID NOs: 4018, 4010, 4002, 4042, 4034, 4026, 3954, 3946, 3994, 3914, 3978, 3906, 3898, 3938, 3922, 3858, 3850, 3882, 3826, 3818, 3842, 3794, 3786, 3762, 3810, 3746, 3778, 3738, 3770, 3722, 3754, 3690, 3666, 3658, 3634, 3586, 3546, 3530, 3642, 3514, 3506, 3490, 3618, 3610, 3602, 3578, 3442, 3522, 3410, 3378, 3434, 3370, 3426, 3418, 3394, 3386, 3330, 3354, 3346, 3314, 3930, 3890, 3834, 3802, 3706, 3698, 3682, 3674, 3570, 3554, 3538, 3498, 3482, 3458, 3474, 3450, 2667, 2666, 2650, 2642, 2626, 2618, 2706, 2690, 2682, 2610, 2674, 2658, 2602, 2594, 2634, 2554, 2546, 2586, 2538, 2578, 2570, 2522, 2498, 2490, 2466, 2458, 2450, 2514, 2506, 2418, 2482, 2474, 2394, 2442, 2434, 2370, 2378, 2354, 2346, 2338, 2314, 2298, 2282, 2274, 2266, 2330, 2258, 2322, 2242, 2234, 2290, 2250, 2218, 2226, 2210, 2194, 2146, 2138, 2122, 2106, 2098, 2090, 2130, 2114, 2034, 2026, 2058, 2050, 2042, 1914, 1786, 1778, 1770, 1842, 1738, 1706, 1690, 1746, 1714, 1650, 1642, 1610, 1586, 1562, 1546, 1578, 1538, 1378, 1370, 1922, 1898, 1906, 1794, 1762, 1698, 1674, 1722, 1362, 1450, 2202, 2178, 2170, 2162, 2018, 2010, 1890, 1962, 1946, 1850, 1818, 1658, 1634, 1602, 1554, 1434, 1426, 1338, 1346, 1978, 1994, 1986, 1970, 1938, 1930, 1810, 1834, 1826, 1802, 1626, 1594, 1514, 1498, 1490, 1482, 1474, 1458, 1442, 1418, 1410, 1402, 1394, or 1386.
In some embodiments a gRNA is useful for single cut excision of a TNR from the DMPK gene with DNA-PK inhibition. In some embodiments, the DNA-PK inhibitor enhances the single cut excision. In some embodiments, a composition is provided comprising a gRNA, or nucleic acid encoding a gRNA, wherein the gRNA comprises a spacer sequence comprising the sequence of SEQ ID NOs: 3914. In some embodiments, a composition is provided comprising a gRNA, or nucleic acid encoding a gRNA, wherein the gRNA comprises the sequence of SEQ ID NOs: 3418. In some embodiments, a composition is provided comprising a gRNA, or nucleic acid encoding a gRNA, wherein the gRNA comprises the sequence of SEQ ID NOs: 3938. In some embodiments, a composition is provided comprising a gRNA, or nucleic acid encoding a gRNA, wherein the gRNA comprises the sequence of SEQ ID NOs: 3916. In some embodiments, a composition is provided comprising a gRNA, or nucleic acid encoding a gRNA, wherein the gRNA comprises the sequence of SEQ ID NOs: 3420. In some embodiments, a composition is provided comprising a gRNA, or nucleic acid encoding a gRNA, wherein the gRNA comprises the sequence of SEQ ID NOs: 3940.
In some embodiments, a pair of guide RNAs or one or more nucleic acids encoding a pair of guide RNAs is provided as one or more compositions, wherein the pair of guide RNAs comprise a first and second spacer sequence selected from: SEQ ID NOs: 2202 and 3418; 2202 and 3370; 2202 and 3514; 2202 and 3658; 2178 and 3418; 2178 and 3370; 2178 and 3514; 2178 and 3658; 2170 and 3418; 2170 and 3370; 2170 and 3514; 2170 and 3658; 2162 and 3418; 2162 and 3370; 2162 and 3514; 2162 and 3658; 2202 and 4010; 2202 and 4026; 2202 and 3914; 2202 and 3938; 2202 and 3858; 2202 and 3818; 2202 and 3794; 2202 and 3802; 2202 and 3746; 2202 and 3778; 2202 and 3770; 2202 and 3722; 2202 and 3690; 2202 and 3682; 2202 and 3330; 2202 and 3354; 2202 and 3394; 2202 and 3386; 2178 and 4010; 2178 and 4026; 2178 and 3914; 2178 and 3938; 2178 and 3858; 2178 and 3818; 2178 and 3794; 2178 and 3802; 2178 and 3746; 2178 and 3778; 2178 and 3770; 2178 and 3722; 2178 and 3690; 2178 and 3682; 2178 and 3330; 2178 and 3354; 2178 and 3394; 2178 and 3386; 2170 and 4010; 2170 and 4026; 2170 and 3914; 2170 and 3938; 2170 and 3858; 2170 and 3818; 2170 and 3794; 2170 and 3802; 2170 and 3746; 2170 and 3778; 2170 and 3770; 2170 and 3722; 2170 and 3690; 2170 and 3682; 2170 and 3330; 2170 and 3354; 2170 and 3394; 2170 and 3386; 2162 and 4010; 2162 and 4026; 2162 and 3914; 2162 and 3938; 2162 and 3858; 2162 and 3818; 2162 and 3794; 2162 and 3802; 2162 and 3746; 2162 and 3778; 2162 and 3770; 2162 and 3722; 2162 and 3690; 2162 and 3682; 2162 and 3330; 2162 and 3354; 2162 and 3394; 2162 and 3386; 1706 and 3418; 1706 and 3370; 1706 and 3514; 1706 and 3658; 1706 and 4010; 1706 and 4026; 1706 and 3914; 1706 and 3938; 1706 and 3858; 1706 and 3818; 1706 and 3794; 1706 and 3802; 1706 and 3746; 1706 and 3778; 1706 and 3770; 1706 and 3722; 1706 and 3690; 1706 and 3682; 1706 and 3330; 1706 and 3354; 1706 and 3394; 1706 and 3386; 2210 and 3418; 2210 and 3370; 2210 and 3514; 2210 and 3658; 2210 and 4010; 2210 and 4026; 2210 and 3914; 2210 and 3938; 2210 and 3858; 2210 and 3818; 2210 and 3794; 2210 and 3802; 2210 and 3746; 2210 and 3778; 2210 and 3770; 2210 and 3722; 2210 and 3690; 2210 and 3682; 2210 and 3330; 2210 and 3354; 2210 and 3394; 2210 and 3386; 1778 and 3418; 1778 and 3370; 1778 and 3514; 1778 and 3658; 1778 and 4010; 1778 and 4026; 1778 and 3914; 1778 and 3938; 1778 and 3858; 1778 and 3818; 1778 and 3794; 1778 and 3802; 1778 and 3746; 1778 and 3778; 1778 and 3770; 1778 and 3722; 1778 and 3690; 1778 and 3682; 1778 and 3330; 1778 and 3354; 1778 and 3394; 1778 and 3386; 2258 and 3418; 2258 and 3370; 2258 and 3514; 2258 and 3658; 2258 and 4010; 2258 and 4026; 2258 and 3914; 2258 and 3938; 2258 and 3858; 2258 and 3818; 2258 and 3794; 2258 and 3802; 2258 and 3746; 2258 and 3778; 2258 and 3770; 2258 and 3722; 2258 and 3690; 2258 and 3682; 2258 and 3330; 2258 and 3354; 2258 and 3394; 2258 and 3386; 2114 and 3418; 2114 and 3370; 2114 and 3514; 2114 and 3658; 2114 and 4010; 2114 and 4026; 2114 and 3914; 2114 and 3938; 2114 and 3858; 2114 and 3818; 2114 and 3794; 2114 and 3802; 2114 and 3746; 2114 and 3778; 2114 and 3770; 2114 and 3722; 2114 and 3690; 2114 and 3682; 2114 and 3330; 2114 and 3354; 2114 and 3394; 2114 and 3386; 1642 and 3418; 1642 and 3370; 1642 and 3514; 1642 and 3658; 1642 and 4010; 1642 and 4026; 1642 and 3914; 1642 and 3938; 1642 and 3858; 1642 and 3818; 1642 and 3794; 1642 and 3802; 1642 and 3746; 1642 and 3778; 1642 and 3770; 1642 and 3722; 1642 and 3690; 1642 and 3682; 1642 and 3330; 1642 and 3354; 1642 and 3394; 1642 and 3386; 1738 and 3418; 1738 and 3370; 1738 and 3514; 1738 and 3658; 1738 and 4010; 1738 and 4026; 1738 and 3914; 1738 and 3938; 1738 and 3858; 1738 and 3818; 1738 and 3794; 1738 and 3802; 1738 and 3746; 1738 and 3778; 1738 and 3770; 1738 and 3722; 1738 and 3690; 1738 and 3682; 1738 and 3330; 1738 and 3354; 1738 and 3394; 1738 and 3386; 2258 and 3418; 2258 and 3370; 2258 and 3514; 2258 and 3658; 2258 and 4010; 2258 and 4026; 2258 and 3914; 2258 and 3938; 2258 and 3858; 2258 and 3818; 2258 and 3794; 2258 and 3802; 2258 and 3746; 2258 and 3778; 2258 and 3770; 2258 and 3722; 2258 and 3690; 2258 and 3682; 2258 and 3330; 2258 and 3354; 2258 and 3394; 2258 and 3386; 2114 and 3418; 2114 and 3370; 2114 and 3514; 2114 and 3658; 2114 and 4010; 2114 and 4026; 2114 and 3914; 2114 and 3938; 2114 and 3858; 2114 and 3818; 2114 and 3794; 2114 and 3802; 2114 and 3746; 2114 and 3778; 2114 and 3770; 2114 and 3722; 2114 and 3690; 2114 and 3682; 2114 and 3330; 2114 and 3354; 2114 and 3394; 1706 and 3386; 1642 and 3418; 1642 and 3370; 1642 and 3514; 1642 and 3658; 1642 and 4010; 1642 and 4026; 1642 and 3914; 1642 and 3938; 1642 and 3858; 1642 and 3818; 1642 and 3794; 1642 and 3802; 1642 and 3746; 1642 and 3778; 1642 and 3770; 1642 and 3722; 1642 and 3690; 1642 and 3682; 1642 and 3330; 1642 and 3354; 1642 and 3394; 1642 and 3386; 1738 and 3418; 1738 and 3370; 1738 and 3514; 1738 and 3658; 1738 and 4010; 1738 and 4026; 1738 and 3914; 1738 and 3938; 1738 and 3858; 1738 and 3818; 1738 and 3794; 1738 and 3802; 1738 and 3746; 1738 and 3778; 1738 and 3770; 1738 and 3722; 1738 and 3690; 1738 and 3682; 1738 and 3330; 1738 and 3354; 1738 and 3394; 1738 and 3386; 1746 and 3418; 1746 and 3370; 1746 and 3514; 1746 and 3658; 1746 and 4010; 1746 and 4026; 1746 and 3914; 1746 and 3938; 1746 and 3858; 1746 and 3818; 1746 and 3794; 1746 and 3802; 1746 and 3746; 1746 and 3778; 1746 and 3770; 1746 and 3722; 1746 and 3690; 1746 and 3682; 1746 and 3330; 1746 and 3354; 1746 and 3394; 1746 and 3386; 2322 and 3418; 2322 and 3370; 2322 and 3514; 2322 and 3658; 2322 and 4010; 2322 and 4026; 2322 and 3914; 2322 and 3938; 2322 and 3858; 2322 and 3818; 2322 and 3794; 2322 and 3802; 2322 and 3746; 2322 and 3778; 2322 and 3770; 2322 and 3722; 2322 and 3690; 2322 and 3682; 2322 and 3330; 2322 and 3354; 2322 and 3394; 2322 and 3386; 1770 and 3418; 1770 and 3370; 1770 and 3514; 1770 and 3658; 1770 and 4010; 1770 and 4026; 1770 and 3914; 1770 and 3938; 1770 and 3858; 1770 and 3818; 1770 and 3794; 1770 and 3802; 1770 and 3746; 1770 and 3778; 1770 and 3770; 1770 and 3722; 1770 and 3690; 1770 and 3682; 1770 and 3330; 1770 and 3354; 1770 and 3394; 1770 and 3386; 1538 and 3418; 1538 and 3370; 1538 and 3514; 1538 and 3658; 1538 and 4010; 1538 and 4026; 1538 and 3914; 1538 and 3938; 1538 and 3858; 1538 and 3818; 1538 and 3794; 1538 and 3802; 1538 and 3746; 1538 and 3778; 1538 and 3770; 1538 and 3722; 1538 and 3690; 1538 and 3682; 1538 and 3330; 1538 and 3354; 1538 and 3394; 1538 and 3386; 2514 and 3418; 2514 and 3370; 2514 and 3514; 2514 and 3658; 2514 and 4010; 2514 and 4026; 2514 and 3914; 2514 and 3938; 2514 and 3858; 2514 and 3818; 2514 and 3794; 2514 and 3802; 2514 and 3746; 2514 and 3778; 2514 and 3770; 2514 and 3722; 2514 and 3690; 2514 and 3682; 2514 and 3330; 2514 and 3354; 2514 and 3394; 2514 and 3386; 2458 and 3418; 2458 and 3370; 2458 and 3514; 2458 and 3658; 2458 and 4010; 2458 and 4026; 2458 and 3914; 2458 and 3938; 2458 and 3858; 2458 and 3818; 2458 and 3794; 2458 and 3802; 2458 and 3746; 2458 and 3778; 2458 and 3770; 2458 and 3722; 2458 and 3690; 2458 and 3682; 2458 and 3330; 2458 and 3354; 2458 and 3394; 2458 and 3386; 2194 and 3418; 2194 and 3370; 2194 and 3514; 2194 and 3658; 2194 and 4010; 2194 and 4026; 2194 and 3914; 2194 and 3938; 2194 and 3858; 2194 and 3818; 2194 and 3794; 2194 and 3802; 2194 and 3746; 2194 and 3778; 2194 and 3770; 2194 and 3722; 2194 and 3690; 2194 and 3682; 2194 and 3330; 2194 and 3354; 2194 and 3394; 2194 and 3386; 2594 and 3418; 2594 and 3370; 2594 and 3514; 2594 and 3658; 2594 and 4010; 2594 and 4026; 2594 and 3914; 2594 and 3938; 2594 and 3858; 2594 and 3818; 2594 and 3794; 2594 and 3802; 2594 and 3746; 2594 and 3778; 2594 and 3770; 2594 and 3722; 2594 and 3690; 2594 and 3682; 2594 and 3330; 2594 and 3354; 2594 and 3394; 2594 and 3386; 2618 and 3418; 2618 and 3370; 2618 and 3514; 2618 and 3658; 2618 and 4010; 2618 and 4026; 2618 and 3914; 2618 and 3938; 2618 and 3858; 2618 and 3818; 2618 and 3794; 2618 and 3802; 2618 and 3746; 2618 and 3778; 2618 and 3770; 2618 and 3722; 2618 and 3690; 2618 and 3682; 2618 and 3330; 2618 and 3354; 2618 and 3394; and 2618 and 3386.
In some embodiments, a pair of guide RNAs or one or more nucleic acids encoding a pair of guide RNAs is provided as one or more compositions, wherein the pair of guide RNAs comprise a first and second spacer sequence comprising a first and second spacer sequence selected from SEQ ID NOs: 2202 and 3418; 2202 and 3370; 2202 and 3514; 2202 and 3658; 2178 and 3418; 2178 and 3370; 2178 and 3514; 2178 and 3658; 2170 and 3418; 2170 and 3370; 2170 and 3514; 2170 and 3658; 2162 and 3418; 2162 and 3370; 2162 and 3514; 2162 and 3658; 2202 and 4010; 2202 and 4026; 2202 and 3914; 2202 and 3938; 2202 and 3858; 2202 and 3818; 2202 and 3794; 2202 and 3802; 2202 and 3746; 2202 and 3778; 2202 and 3770; 2202 and 3722; 2202 and 3690; 2202 and 3682; 2202 and 3330; 2202 and 3354; 2202 and 3394; 2202 and 3386; 2178 and 4010; 2178 and 4026; 2178 and 3914; 2178 and 3938; 2178 and 3858; 2178 and 3818; 2178 and 3794; 2178 and 3802; 2178 and 3746; 2178 and 3778; 2178 and 3770; 2178 and 3722; 2178 and 3690; 2178 and 3682; 2178 and 3330; 2178 and 3354; 2178 and 3394; 2178 and 3386; 2170 and 4010; 2170 and 4026; 2170 and 3914; 2170 and 3938; 2170 and 3858; 2170 and 3818; 2170 and 3794; 2170 and 3802; 2170 and 3746; 2170 and 3778; 2170 and 3770; 2170 and 3722; 2170 and 3690; 2170 and 3682; 2170 and 3330; 2170 and 3354; 2170 and 3394; 2170 and 3386; 2162 and 4010; 2162 and 4026; 2162 and 3914; 2162 and 3938; 2162 and 3858; 2162 and 3818; 2162 and 3794; 2162 and 3802; 2162 and 3746; 2162 and 3778; 2162 and 3770; 2162 and 3722; 2162 and 3690; 2162 and 3682; 2162 and 3330; 2162 and 3354; 2162 and 3394; 2162 and 3386. In some embodiments, a pair of guide RNAs or one or more nucleic acids encoding a pair of guide RNAs is provided as one or more compositions, wherein the pair of guide RNAs comprise a first and second spacer sequence comprising a first and second spacer sequence selected from SEQ ID NOs: 2202 and 3418; 2202 and 3370; 2202 and 3514; 2202 and 3658; 2178 and 3418; 2178 and 3370; 2178 and 3514; 2178 and 3658; 2170 and 3418; 2170 and 3370; 2170 and 3514; 2170 and 3658; 2162 and 3418; 2162 and 3370; 2162 and 3514; and 2162 and 3658. In some embodiments, a pair of guide RNAs or one or more nucleic acids encoding a pair of guide RNAs is provided as one or more compositions, wherein the pair of guide RNAs comprise a first and second spacer sequence comprising a first and second spacer sequence selected from SEQ ID NOs: 3778 and 2514; 3778 and 2258; 3778 and 2210; 3386 and 2514; 3386 and 2258; 3386 and 2210; 3354 and 2514; 3354 and 2258; and 3354 and 2210. In some embodiments, a pair of guide RNAs or one or more nucleic acids encoding a pair of guide RNAs is provided as one or more compositions, wherein the pair of guide RNAs comprise a first and second spacer sequence comprising a first and second spacer sequence selected from SEQ ID NOs: 3778 and 2258; 3778 and 2210; 3386 and 2258; 3386 and 2210; and 3354 and 2514. In some embodiments, a pair of guide RNAs or one or more nucleic acids encoding a pair of guide RNAs is provided as one or more compositions, wherein the pair of guide RNAs comprise a first and second spacer sequence comprising a first and second spacer sequence selected from SEQ ID NOs: 3346 and 2554; 3346 and 2498; 3330 and 2554; 3330 and 2498; 3330 and 2506; and 3330 and 2546. In some embodiments, a pair of guide RNAs or one or more nucleic acids encoding a pair of guide RNAs is provided as one or more compositions, wherein the pair of guide RNAs comprise a first and second spacer sequence comprising a first and second spacer sequence selected from SEQ ID NOs: 3346 and 2554; 3346 and 2498; 3330 and 2554; 3330 and 2498; 3354 and 2546; 3354 and 2506; 3378 and 2546; 3378 and 2506. In some embodiments, a pair of guide RNAs or one or more nucleic acids encoding a pair of guide RNAs is provided as one or more compositions, wherein the pair of guide RNAs comprise a first and second spacer sequence comprising a first and second spacer sequence selected from SEQ ID NOs: 3346 and 2554; 3346 and 2498; 3330 and 2554; and 3330 and 2498. In some embodiments, a pair of guide RNAs or one or more nucleic acids encoding a pair of guide RNAs is provided as one or more compositions, wherein the pair of guide RNAs comprise a first and second spacer sequence comprising SEQ ID NOs: 1153 and 1129.
In some embodiments, a pair of guide RNAs or one or more nucleic acids encoding a pair of guide RNAs is provided as one or more compositions, wherein the pair of guide RNAs comprise a first and second spacer sequence comprising a first spacer sequence selected from SEQ ID NOs: 2856, 2864, 2880, 2896, 2904, 2912, 2936, 2944, 2960, 2992, 3016, 3024, 3064, 3096, 3112, 3128, 3136, 3144, 3160, 3168, 3192, 3200, 3208, 3216, 3224, 3232, 3240, 3248, 3256, 3264, 3314, 3330, 3346, 3354, 3370, 3378, 3386, 3394, 3410, 3418, 3426, 3434, 3442, 3450, 3458, 3474, 3482, 3490, 3498, 3506, 3514, 3522, 3530, 3538, 3546, 3554, 3570, 3578, 3586, 3602, 3610, 3618, 3634, 3642, 3658, 3674, 3682, 3690, 3698, 3706, 3722, 3746, 3762, 3770, 3778, 3794, 3802, 3818, 3826, 3834, 3850, 3858, 3890, 3898, 3906, 3914, 3922, 3930, 3938, 3946, 3994, 4010, 4018, 4026, 4034, 4042, 4208, and 4506, and a second spacer sequence selected from SEQ ID NOs: 560, 584, 608, 616, 656, 672, 688, 696, 712, 744, 752, 760, 840, 864, 960, 976, 984, 1008, 1056, 1128, 1136, 1152, 1224, 1240, 1272, 1338, 1346, 1370, 1378, 1386, 1394, 1402, 1410, 1418, 1426, 1434, 1442, 1458, 1474, 1482, 1490, 1498, 1514, 1538, 1546, 1554, 1562, 1578, 1586, 1594, 1602, 1610, 1626, 1634, 1642, 1650, 1658, 1690, 1706, 1714, 1738, 1746, 1770, 1778, 1786, 1802, 1810, 1818, 1826, 1834, 1842, 1850, 1890, 1914, 1930, 1938, 1946, 1962, 1970, 1978, 1986, 1994, 2010, 2018, 2026, 2042, 2050, 2058, 2090, 2114, 2130, 2162, 2170, 2178, 2202, 2210, 2226, 2242, 2258, 2266, 2274, 2282, 2298, 2314, 2322, 2330, 2338, 2346, 2354, 2370, 2378, 2394, 2418, 2434, 2442, 2458, 2466, 2474, 2498, 2506, 2514, 2522, 2546, 2554, 2570, 2586, 2658, 4989, 4990, 4991, and 4992. In some embodiments, a pair of guide RNAs or one or more nucleic acids encoding a pair of guide RNAs is provided as one or more compositions, wherein the pair of guide RNAs comprise a first and second spacer sequence comprising a first spacer sequence selected from SEQ ID NOs: 3778, 4026, 3794, 4010, 3906 and 3746, and a second spacer sequence selected from SEQ ID NOs: 1778, 1746, 1770, 1586, 1914, and 2210.
In some embodiments, a pair of guide RNAs or one or more nucleic acids encoding a pair of guide RNAs is provided as one or more compositions, wherein the pair of guide RNAs comprise a first and second spacer sequence comprising a first and second spacer sequence selected from SEQ ID NOs: 3778 and 1778; 3778 and 1746; 3778 and 1770; 3778 and 1586; 3778 and 1914; 3778 and 2210; 4026 and 1778; 4026 and 1746; 4026 and 1770; 4026 and 1586; 4026 and 1914; 4026 and 2210; 3794 and 1778; 3794 and 1746; 3794 and 1770; 3794 and 1586; 3794 and 1586; 3794 and 1914; 3794 and 2210; 4010 and 1778; 4010 and 1770; 4010 and 1746; 4010 and 1586; 4010 and 1914; 4010 and 2210; 3906 and 1778; 3906 and 1778; 3906 and 1746; 3906 and 1770; 3906 and 1586; 3906 and 1914; 3906 and 2210; 3746 and 1778; 3746 and 1746; 3746 and 1770; 3746 and 1586; 3746 and 1914; and 3746 and 2210. In some embodiments, a pair of guide RNAs or one or more nucleic acids encoding a pair of guide RNAs is provided as one or more compositions, wherein the pair of guide RNAs comprise a first and second spacer sequence comprising a first and second spacer sequence selected from first spacer sequence selected from SEQ ID NOs: 3256, 2896, 3136, and 3224, and a second spacer sequence selected from SEQ ID NOs: 4989, 560, 672, 976, 760, 984, and 616. In some embodiments, a pair of guide RNAs or one or more nucleic acids encoding a pair of guide RNAs is provided as one or more compositions, wherein the pair of guide RNAs comprise a first and second spacer sequence comprising a first and second spacer sequence selected from SEQ ID NOs: 3256 and 4989; 3256 and 984; 3256 and 616; 2896 and 4989; 2896 and 672; 2896 and 760; 3136 and 4989; 3136 and 560; 3224 and 4989; 3224 and 976; and 3224 and 760.
In some embodiments, a composition is provided comprising a guide RNA or a nucleic acid encoding a guide RNA is provided, wherein the guide RNA comprises a spacer sequence, wherein the spacer sequence directs a RNA-guided DNA nuclease to any nucleotide within a stretch of sequence, wherein the stretch starts 1 nucleotide from the DMPK-U29 cut site and continues through the repeat.
In some embodiments, a composition comprising a guide RNA or a nucleic acid encoding a guide RNA is provided, wherein the guide RNA comprises a spacer sequence, wherein the spacer sequence directs a RNA-guided DNA nuclease to any nucleotide within a stretch of sequence, wherein the stretch is SEQ ID NO: 53413:
In some embodiments, a composition comprising a guide RNA or a nucleic acid encoding a guide RNA is provided, wherein the guide RNA comprises a spacer sequence, wherein the spacer sequence directs a RNA-guided DNA nuclease to any nucleotide within a stretch of sequence, wherein the stretch starts 1 nucleotide from the DMPK-U30 cut site and continues through 1 nucleotide before the DMPK-U56 cut site.
In some embodiments, a composition comprising a guide RNA or a nucleic acid encoding a guide RNA is provided, wherein the guide RNA comprises a spacer sequence, wherein the spacer sequence directs a RNA-guided DNA nuclease to any nucleotide within a stretch of sequence, wherein the stretch is SEQ ID NO: 53414:
In some embodiments, a composition comprising a guide RNA or a nucleic acid encoding a guide RNA is provided, wherein the guide RNA comprises a spacer sequence, wherein the spacer sequence directs a RNA-guided DNA nuclease to any nucleotide within a stretch of sequence, wherein the stretch starts 1 nucleotide from the DMPK-U30 cut site and continues through 1 nucleotide before the DMPK-U52 cut site.
In some embodiments, a composition comprising a guide RNA or a nucleic acid encoding a guide RNA is provided, wherein the guide RNA comprises a spacer sequence, wherein the spacer sequence directs a RNA-guided DNA nuclease to any nucleotide within a stretch of sequence, wherein the stretch is SEQ ID NO: 53415:
In some embodiments, a composition comprising a guide RNA or a nucleic acid encoding a guide RNA is provided, wherein the guide RNA comprises a spacer sequence, wherein the spacer sequence directs a RNA-guided DNA nuclease to any nucleotide within a stretch of sequence, wherein the stretch starts 1 nucleotide from the DMPK-D15 cut site and continues through 1 nucleotide before the DMPK-D51 cut site.
In some embodiments, a composition comprising a guide RNA or a nucleic acid encoding a guide RNA is provided, wherein the guide RNA comprises a spacer sequence, wherein the spacer sequence directs a RNA-guided DNA nuclease to any nucleotide within a stretch of sequence, wherein the stretch is SEQ ID NO: 53416:
In some embodiments, the stretch starts 1 nucleotide from the DMPK-D35 cut site and continues through 1 nucleotide before the DMPK-D51 cut site.
In some embodiments, a composition comprising a guide RNA or a nucleic acid encoding a guide RNA is provided, wherein the guide RNA comprises a spacer sequence, wherein the spacer sequence directs a RNA-guided DNA nuclease to any nucleotide within a stretch of sequence, wherein the stretch is SEQ ID NO: 53417:
The U29 cut site is: chr19: between nucleotides 45,770,383 and 45,770,384 (using Hg38 coordinates), which corresponds to * in the following sequence: ttcacaaccgctccgag*cgtggg.
The U30 cut site is: chr19: between 45,770,385 and 45,770,386 (using Hg38 coordinates), which corresponds to * in the following sequence: gctgggcggagacccac*gctcgg.
The D15 cut site is: chr19: between 45,770,154 and 45,770,155 (using Hg38 coordinates), which corresponds to * in the following sequence: ggctgaggccctgacgt*ggatgg.
The D35 cut site is: chr19: between 45,770,078 and 45,770,079 (using Hg38 coordinates), which corresponds to * in the following sequence: cacgcacccccacctat*cgttgg.
In some embodiments, a composition is provided comprising one or more guide RNAs (gRNA) or one or more nucleic acids encoding one or more guide RNAs, wherein the one or more guide RNAs comprise guide sequences that direct an RNA-targeted endonuclease (e.g., a Cas nuclease such as Cas9), to a target DNA sequence in or near the CTG repeat region in the myotonic dystrophy protein kinase gene (FXN) associated with myotonic dystrophy type 1. In some embodiments, a composition is provided comprising a gRNA, or nucleic acid encoding a gRNA, wherein the gRNA comprises a crRNA comprising a FXN guide sequence shown in Table 2 or the Sequence Listing. In some embodiments, a composition is provided comprising a gRNA, or nucleic acid encoding a gRNA, wherein the gRNA comprises a crRNA comprising 17, 18, 19, or 20 contiguous nucleotides of a FXN guide sequence shown in Table 2 or the Sequence Listing. In some embodiments, a composition is provided comprising a gRNA, or nucleic acid encoding a gRNA, wherein the gRNA comprises a crRNA comprising a sequence with about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to at least 17, 18, 19, or 20 contiguous nucleotides of a FXN guide sequence shown in Table 2 or the Sequence Listing. In some embodiments, a composition is provided comprising a gRNA, or nucleic acid encoding a gRNA, wherein the gRNA comprises a crRNA comprising a sequence with about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a guide sequence shown in Table 2 or the Sequence Listing. In some embodiments, a composition is provided comprising a gRNA, or nucleic acid encoding a gRNA, wherein the gRNA further comprises a trRNA. In each composition and method embodiment described herein, the crRNA and trRNA may be associated as a single RNA (sgRNA) or may be on separate RNAs (dgRNA). In the context of sgRNAs, the crRNA and trRNA components may be covalently linked, e.g., via a phosphodiester bond or other covalent bond.
In some embodiments, a composition is provided comprising a gRNA, or nucleic acid encoding a gRNA, wherein the gRNA comprises a spacer sequence comprising a spacer sequence selected from SEQ ID NOs: 28130, 34442, 45906, 26562, 52666, 51322, 46599, 52898, 26546, 7447, 47047, 49986, 51762, 51754, 52290, 52298, 51474, 52306, 50682, 51706, 52098, 50714, 51498, 52498, 50978, 51746, 52106, 51506, 50674, 52082, 52506, 50538, 52066, 52386, 52090, 52266, 52474, 52258, 52434, 50706, 51490, 52458, 51466, 52354, 51914, 51362, 51058, 50170, 51954, 52250, 51930, 51682, 52594, 52610, 51162, 49162, 50898, 49226, 51658, 52554, 52634, 51394, 49034, 52546, 52522, 52618, 52530, 28322, 26530, 26578, 26602, 26634, 26626, 26698, 26746, 26754, 26786, 26882, 27722, 27730, 27738, 27770, 27754, 27762, 27802, 27850, 27842, 27922, 27946, 27986, 28114, 28122, 28146, 28186, 28194, 28338, 28346, 28322, 28378, 28370, 28458, 28506, 28634, 28642, 28650, 34442, or 45906.
In some embodiments, a composition is provided comprising a gRNA, or nucleic acid encoding a gRNA, wherein the gRNA comprises 17, 18, 19, or 20 contiguous nucleotides of a FXN guide sequence selected from SEQ ID NOs: 28130, 34442, 45906, 26562, 52666, 51322, 46599, 52898, 26546, 7447, 47047, 49986, 51762, 51754, 52290, 52298, 51474, 52306, 50682, 51706, 52098, 50714, 51498, 52498, 50978, 51746, 52106, 51506, 50674, 52082, 52506, 50538, 52066, 52386, 52090, 52266, 52474, 52258, 52434, 50706, 51490, 52458, 51466, 52354, 51914, 51362, 51058, 50170, 51954, 52250, 51930, 51682, 52594, 52610, 51162, 49162, 50898, 49226, 51658, 52554, 52634, 51394, 49034, 52546, 52522, 52618, 52530, 28322, 26530, 26578, 26602, 26634, 26626, 26698, 26746, 26754, 26786, 26882, 27722, 27730, 27738, 27770, 27754, 27762, 27802, 27850, 27842, 27922, 27946, 27986, 28114, 28122, 28146, 28186, 28194, 28338, 28346, 28322, 28378, 28370, 28458, 28506, 28634, 28642, 28650, 34442, or 45906.
In some embodiments, a composition is provided comprising a gRNA, or nucleic acid encoding a gRNA, wherein the gRNA comprises a spacer sequence selected from SEQ ID NOs: 51706, 51058, 51754, 52090, 52594, 52098, 52298, 52106, 51682, 52066, 52354, 52458, 52290, 52498, 51658, 51930, 51162, 52506, 51762, 51746, 52386, 52258, 52530, 52634, 27850, 28634, 26882, 28650, 28370, 28194, 26626, 26634, 26786, 26754, 27770, 26578, 28130, 27738, 28338, 28642, 26602, 27754, 27730, and 28122. In some embodiments, a composition is provided comprising a gRNA, or nucleic acid encoding a gRNA, wherein the gRNA comprises a spacer sequence selected from SEQ ID NOs: 47047, 7447, 7463, 46967, 46768, 7680, and 47032. In some embodiments, a composition is provided comprising a gRNA, or nucleic acid encoding a gRNA, wherein the gRNA comprises a spacer sequence selected from SEQ ID NOs: 47045, 7445, 7461, 46766, 7678, and 47030.
In some embodiments, a pair of guide RNAs or one or more nucleic acids encoding a pair of guide RNAs is provided as one or more compositions, wherein the pair of guide RNAs comprise a first and second spacer sequence selected from SEQ ID NOs: 47047 and 7447; 7463 and 46967; 46768 and 7680; 47032 and 7447. In some embodiments, a pair of guide RNAs or one or more nucleic acids encoding a pair of guide RNAs is provided as one or more compositions, wherein the pair of guide RNAs comprise SEQ ID NOs: 47047 and 7447. In some embodiments, a pair of guide RNAs or one or more nucleic acids encoding a pair of guide RNAs is provided as one or more compositions, wherein the pair of guide RNAs comprise SEQ ID NOs: 52898 and 26546.
In some embodiments, a composition is provided comprising one or more guide RNAs (gRNA) or one or more nucleic acids encoding one or more guide RNAs, wherein the one or more guide RNAs comprise guide sequences that direct an RNA-targeted endonuclease (e.g., a Cas nuclease such as Cas9), to a target DNA sequence in or near the CTG repeat region in the myotonic dystrophy protein kinase gene (FMR1) associated with myotonic dystrophy type 1. In some embodiments, a composition is provided comprising a gRNA, or nucleic acid encoding a gRNA, wherein the gRNA comprises a crRNA comprising a FMR1 guide sequence shown in Table 2 or the Sequence Listing. In some embodiments, a composition is provided comprising a gRNA, or nucleic acid encoding a gRNA, wherein the gRNA comprises a crRNA comprising 17, 18, 19, or 20 contiguous nucleotides of a FMR1 guide sequence shown in Table 2 or the Sequence Listing. In some embodiments, a composition is provided comprising a gRNA, or nucleic acid encoding a gRNA, wherein the gRNA comprises a crRNA comprising a sequence with about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to at least 17, 18, 19, or 20 contiguous nucleotides of a FMR1 guide sequence shown in Table 2 or the Sequence Listing. In some embodiments, a composition is provided comprising a gRNA, or nucleic acid encoding a gRNA, wherein the gRNA comprises a sequence with about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a guide sequence shown in Table 2 or the Sequence Listing. In some embodiments, a composition is provided comprising a gRNA, or nucleic acid encoding a gRNA, wherein the gRNA further comprises a trRNA. In each composition and method embodiment described herein, the crRNA and trRNA may be associated as a single RNA (sgRNA) or may be on separate RNAs (dgRNA). In the context of sgRNAs, the crRNA and trRNA components may be covalently linked, e.g., via a phosphodiester bond or other covalent bond.
In some embodiments, a composition is provided comprising a gRNA, or nucleic acid encoding a gRNA, wherein the gRNA comprises a spacer sequence comprising a spacer sequence selected from SEQ ID NOs: 5262, 5782, 5830, 5926, 5950, 5998, 6022, 5310, and 5334.
In some embodiments, a composition is provided comprising a gRNA, or nucleic acid encoding a gRNA, wherein the gRNA comprises a spacer sequence comprising a spacer sequence selected from SEQ ID NOs: 5830, 6022, 5262, and 5310. In some embodiments, a composition is provided comprising a gRNA, or nucleic acid encoding a gRNA, wherein the gRNA comprises a spacer sequence comprising a spacer sequence selected from SEQ ID NOs: 5262, 5334, and 5830. In some embodiments, a composition is provided comprising a gRNA, or nucleic acid encoding a gRNA, wherein the gRNA comprises a spacer sequence selected from SEQ ID NOs: 5264, 5336, 5832, 6024, and 5312. In some embodiments, a composition is provided comprising a gRNA, or nucleic acid encoding a gRNA, wherein the gRNA comprises a spacer sequence comprising SEQ ID NO: 5262. In some embodiments, a composition is provided comprising a gRNA, or nucleic acid encoding a gRNA, wherein the gRNA comprises a spacer sequence comprising a spacer sequence selected from SEQ ID NOs: 5264.
In some embodiments, a composition is provided comprising a gRNA, or nucleic acid encoding a gRNA, wherein the gRNA comprises 17, 18, 19, or 20 contiguous nucleotides of a FMR1 guide sequence selected from SEQ ID NOs: 5262, 5782, 5830, 5926, 5950, 5998, 6022, 5310, or 5334.
In some embodiments, a pair of guide RNAs or one or more nucleic acids encoding a pair of guide RNAs is provided as one or more compositions, wherein the pair of guide RNAs comprise a first and second spacer sequence selected from SEQ ID NOs: 5782 and 5262; 5830 and 5262; 5926 and 5262; 5950 and 5262; and 5998 and 5262. In some embodiments, a pair of guide RNAs or one or more nucleic acids encoding a pair of guide RNAs is provided as one or more compositions, wherein the pair of guide RNAs comprise a first and second spacer sequence selected from SEQ ID NOs: 5830 and 5262; and 6022 and 5310. In some embodiments, a pair of guide RNAs or one or more nucleic acids encoding a pair of guide RNAs is provided as one or more compositions, wherein the pair of guide RNAs comprise SEQ ID NOs: 5334 and 5830.
In some embodiments, a composition is provided comprising one or more guide RNAs (gRNA) or one or more nucleic acids encoding one or more guide RNAs, wherein the one or more guide RNAs comprise guide sequences that direct an RNA-targeted endonuclease (e.g., a Cas nuclease such as Cas9), to a target DNA sequence in or near the repeat region in the huntingtin (HTT) gene associated with Huntington's disease.
In some embodiments, a composition is provided comprising one or more guide RNAs (gRNA) or one or more nucleic acids encoding one or more guide RNAs, wherein the one or more guide RNAs comprise guide sequences that direct an RNA-targeted endonuclease (e.g., a Cas nuclease such as Cas9), to a target DNA sequence in or near the repeat region in or adjacent to the Fragile X Mental Retardation 2 (FMR2) gene associated with Fragile XE syndrome.
In some embodiments, a composition is provided comprising one or more guide RNAs (gRNA) or one or more nucleic acids encoding one or more guide RNAs, wherein the one or more guide RNAs comprise guide sequences that direct an RNA-targeted endonuclease (e.g., a Cas nuclease such as Cas9), to a target DNA sequence in or near the repeat region in the androgen receptor (AR) gene associated with X-linked spinal and bulbar muscular atrophy (Kennedy disease).
In some embodiments, a composition is provided comprising one or more guide RNAs (gRNA) or one or more nucleic acids encoding one or more guide RNAs, wherein the one or more guide RNAs comprise guide sequences that direct an RNA-targeted endonuclease (e.g., a Cas nuclease such as Cas9), to a target DNA sequence in or near the repeat region in the aristaless related homeobox (ARX) gene associated with ARX-associated infantile epileptic encephalopathy, Early infantile epileptic encephalopathy 1, Ohtahara syndrome, Partington syndrome, or West syndrome.
In some embodiments, a composition is provided comprising one or more guide RNAs (gRNA) or one or more nucleic acids encoding one or more guide RNAs, wherein the one or more guide RNAs comprise guide sequences that direct an RNA-targeted endonuclease (e.g., a Cas nuclease such as Cas9), to a target DNA sequence in or near the repeat region in the Ataxin 1 (ATXN1), Ataxin 2 (ATXN2), Ataxin 3 (ATXN3), Calcium voltage-gated channel subunit alpha 1 A (CACNA1A), Ataxin 7 (ATXN7), ATXN8 opposite strand lncRNA (ATXN80S/SCA8), Serine/threonine-protein phosphatase 2A 55 kDa regulatory subunit B beta isoform (PPP2R2B), or TATA binding protein (TBP) gene associated with a form of spinocerebellar ataxia.
In some embodiments, a composition is provided comprising one or more guide RNAs (gRNA) or one or more nucleic acids encoding one or more guide RNAs, wherein the one or more guide RNAs comprise guide sequences that direct an RNA-targeted endonuclease (e.g., a Cas nuclease such as Cas9), to a target DNA sequence in or near the repeat region in the Atrophin-1 (ATN1) gene associated with Dentatorubropallidoluysian atrophy (DRPLA).
In each of the composition, use, and method embodiments described herein, the guide RNA may comprise two RNA molecules as a “dual guide RNA” or “dgRNA.” The dgRNA comprises a first RNA molecule comprising a crRNA comprising, e.g., a guide sequence shown in Table 2 and the Sequence Listing, and a second RNA molecule comprising a trRNA. The first and second RNA molecules may not be covalently linked, but may form an RNA duplex via the base pairing between portions of the crRNA and the trRNA.
In each of the composition, use, and method embodiments described herein, the guide RNA may comprise a single RNA molecule as a “single guide RNA” or “sgRNA”. The sgRNA may comprise a crRNA (or a portion thereof) comprising a guide sequence shown in Table 2 covalently linked to a trRNA. The sgRNA may comprise 17, 18, 19, or 20 contiguous nucleotides of a guide sequence shown in Table 2 and the Sequence Listing. In some embodiments, the crRNA and the trRNA are covalently linked via a linker. In some embodiments, the sgRNA forms a stem-loop structure via the base pairing between portions of the crRNA and the trRNA. In some embodiments, the crRNA and the trRNA are covalently linked via one or more bonds that are not a phosphodiester bond.
In some embodiments, the trRNA may comprise all or a portion of a trRNA sequence derived from a naturally-occurring CRISPR/Cas system. In some embodiments, the trRNA comprises a truncated or modified wild type trRNA. The length of the trRNA depends on the CRISPR/Cas system used. In some embodiments, the trRNA comprises or consists of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 nucleotides. In some embodiments, the trRNA may comprise certain secondary structures, such as, for example, one or more hairpin or stem-loop structures, or one or more bulge structures.
In some embodiments, a composition is provided comprising one or more guide RNAs (or one or more nucleic acids encoding one or more guide RNAs) wherein the one or more gRNAs comprise a guide sequence of any one of SEQ ID NOs: 101-4988, 5001-7264, or 7301-53372.
In one aspect, a composition is provided comprising a gRNA or a vector encoding a gRNA that comprises a guide sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to any of the nucleic acids of SEQ ID NOs: 101-4988, 5001-7264, or 7301-53372.
In other embodiments, the composition comprises at least one, e.g., at least two gRNAs, or one or more nucleic acids encoding at least one, e.g., at least two gRNAs, wherein the gRNAs comprise guide sequences selected from any two or more of the guide sequences of SEQ ID NOs: 101-4988, 5001-7264, or 7301-53372. In some embodiments, the composition comprises at least two gRNAs that each comprise a guide sequence at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to any of the nucleic acids of SEQ ID NOs: 101-4988, 5001-7264, or 7301-53372.
In some embodiments, a composition is provided comprising a nucleic acid encoding a guide RNA, wherein the nucleic acid encoding the guide RNA is a vector. In some embodiments, a composition is provided comprising one or more nucleic acids encoding one or more guide RNAs, wherein the one or more nucleic acids encoding one or more guide RNAs is one or more vectors.
Any type of vector, such as any of those described herein, may be used. In some embodiments, the composition comprises one or more nucleic acids encoding one or more gRNAs described herein. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a non-integrating viral vector (i.e., that does not insert sequence from the vector into a host chromosome). In some embodiments, the viral vector is an adeno-associated virus vector, a lentiviral vector, an integrase-deficient lentiviral vector, an adenoviral vector, a vaccinia viral vector, an alphaviral vector, or a herpes simplex viral vector. In some embodiments, the vector comprises a muscle-specific promoter. Exemplary muscle-specific promoters include a muscle creatine kinase promoter, a desmin promoter, an MHCK7 promoter, or an SPc5-12 promoter. See US 2004/0175727 A1; Wang et al., Expert Opin Drug Deliv. (2014) 11, 345-364; Wang et al., Gene Therapy (2008) 15, 1489-1499. In some embodiments, the muscle-specific promoter is a CK8 promoter. In some embodiments, the muscle-specific promoter is a CK8e promoter. In any of the foregoing embodiments, the vector may be an adeno-associated virus vector.
The guide RNA compositions disclosed herein are designed to recognize (e.g., hybridize to) a target sequence in or near a trinucleotide repeat or self-complementary region, such as a trinucleotide repeat or self-complementary region in the DIVIPK gene. For example, the target sequence may be recognized and cleaved by a provided Cas cleavase comprising a guide RNA. In some embodiments, an RNA-targeted endonuclease, such as a Cas cleavase, may be directed by a guide RNA to the target sequence, where the guide sequence of the guide RNA hybridizes with the target sequence and the RNA-targeted endonuclease, such as a Cas cleavase, cleaves the target sequence.
In some embodiments, the selection of the one or more guide RNAs is determined based on target sequences within a gene of interest, such as any gene associated with a trinucleotide repeat expansion disease. Exemplary genes of interest are listed in Table 1.
Without being bound by any particular theory, mutations (e.g., excision resulting from repair of a nuclease-mediated DSB) may be provided more efficiently and/or better tolerated when cleavage occurs in certain regions of the gene, thus the location of a DSB is an important factor in the post-excision allele that may result.
In some embodiments, the guide sequence is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to a target sequence present in the human gene of interest. In some embodiments, the target sequence may be complementary to the guide sequence of the guide RNA. In some embodiments, the degree of complementarity or identity between a guide sequence of a guide RNA and its corresponding target sequence may be at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the target sequence and the guide sequence of the gRNA may be 100% complementary or identical. In other embodiments, the target sequence and the guide sequence of the gRNA may contain at least one mismatch. For example, the target sequence and the guide sequence of the gRNA may contain 1, 2, 3, or 4 mismatches, where the total length of the guide sequence is 20. In some embodiments, the target sequence and the guide sequence of the gRNA may contain 1-4 mismatches where the guide sequence is 20 nucleotides.
In some embodiments, a composition or formulation disclosed herein comprises an mRNA comprising an open reading frame (ORF) encoding an RNA-targeted endonuclease, such as a Cas nuclease as described herein. In some embodiments, an mRNA comprising an ORF encoding an RNA-targeted endonuclease, such as a Cas nuclease, is provided, used, or administered.
Modified gRNAs
In some embodiments, the gRNA is chemically modified. A gRNA comprising one or more modified nucleosides or nucleotides is called a “modified” gRNA or “chemically modified” gRNA, to describe the presence of one or more non-naturally and/or naturally occurring components or configurations that are used instead of or in addition to the canonical A, G, C, and U residues. In some embodiments, a modified gRNA is synthesized with a non-canonical nucleoside or nucleotide, is here called “modified.” Modified nucleosides and nucleotides can include one or more of: (i) alteration, e.g., replacement, of one or both of the non-linking phosphate oxygens and/or of one or more of the linking phosphate oxygens in the phosphodiester backbone linkage (an exemplary backbone modification); (ii) alteration, e.g., replacement, of a constituent of the ribose sugar, e.g., of the 2′ hydroxyl on the ribose sugar (an exemplary sugar modification); (iii) wholesale replacement of the phosphate moiety with “dephospho” linkers (an exemplary backbone modification); (iv) modification or replacement of a naturally occurring nucleobase, including with a non-canonical nucleobase (an exemplary base modification); (v) replacement or modification of the ribose-phosphate backbone (an exemplary backbone modification); (vi) modification of the 3′ end or 5′ end of the oligonucleotide, e.g., removal, modification or replacement of a terminal phosphate group or conjugation of a moiety, cap or linker (such 3′ or 5′ cap modifications may comprise a sugar and/or backbone modification); and (vii) modification or replacement of the sugar (an exemplary sugar modification).
Chemical modifications such as those listed above can be combined to provide modified gRNAs comprising nucleosides and nucleotides (collectively “residues”) that can have two, three, four, or more modifications. For example, a modified residue can have a modified sugar and a modified nucleobase, or a modified sugar and a modified phosphodiester. In some embodiments, every base of a gRNA is modified, e.g., all bases have a modified phosphate group, such as a phosphorothioate group. In certain embodiments, all, or substantially all, of the phosphate groups of an gRNA molecule are replaced with phosphorothioate groups. In some embodiments, modified gRNAs comprise at least one modified residue at or near the 5′ end of the RNA. In some embodiments, modified gRNAs comprise at least one modified residue at or near the 3′ end of the RNA.
In some embodiments, the gRNA comprises one, two, three or more modified residues. In some embodiments, at least 5% (e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) of the positions in a modified gRNA are modified nucleosides or nucleotides.
Unmodified nucleic acids can be prone to degradation by, e.g., intracellular nucleases or those found in serum. For example, nucleases can hydrolyze nucleic acid phosphodiester bonds. Accordingly, in one aspect the gRNAs described herein can contain one or more modified nucleosides or nucleotides, e.g., to introduce stability toward intracellular or serum-based nucleases. In some embodiments, the modified gRNA molecules described herein can exhibit a reduced innate immune response when introduced into a population of cells, both in vivo and ex vivo. The term “innate immune response” includes a cellular response to exogenous nucleic acids, including single stranded nucleic acids, which involves the induction of cytokine expression and release, particularly the interferons, and cell death.
In some embodiments of a backbone modification, the phosphate group of a modified residue can be modified by replacing one or more of the oxygens with a different substituent. Further, the modified residue, e.g., modified residue present in a modified nucleic acid, can include the wholesale replacement of an unmodified phosphate moiety with a modified phosphate group as described herein. In some embodiments, the backbone modification of the phosphate backbone can include alterations that result in either an uncharged linker or a charged linker with unsymmetrical charge distribution.
Examples of modified phosphate groups include, phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters. The phosphorous atom in an unmodified phosphate group is achiral. However, replacement of one of the non-bridging oxygens with one of the above atoms or groups of atoms can render the phosphorous atom chiral. The stereogenic phosphorous atom can possess either the “R” configuration (herein Rp) or the “S” configuration (herein Sp). The backbone can also be modified by replacement of a bridging oxygen, (i.e., the oxygen that links the phosphate to the nucleoside), with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates). The replacement can occur at either linking oxygen or at both of the linking oxygens.
The phosphate group can be replaced by non-phosphorus containing connectors in certain backbone modifications. In some embodiments, the charged phosphate group can be replaced by a neutral moiety. Examples of moieties which can replace the phosphate group can include, without limitation, e.g., methyl phosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino.
Scaffolds that can mimic nucleic acids can also be constructed wherein the phosphate linker and ribose sugar are replaced by nuclease resistant nucleoside or nucleotide surrogates. Such modifications may comprise backbone and sugar modifications. In some embodiments, the nucleobases can be tethered by a surrogate backbone. Examples can include, without limitation, the morpholino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates.
The modified nucleosides and modified nucleotides can include one or more modifications to the sugar group, i.e. at sugar modification. For example, the 2′ hydroxyl group (OH) can be modified, e.g. replaced with a number of different “oxy” or “deoxy” substituents. In some embodiments, modifications to the 2′ hydroxyl group can enhance the stability of the nucleic acid since the hydroxyl can no longer be deprotonated to form a 2′-alkoxide ion.
Examples of 2′ hydroxyl group modifications can include alkoxy or aryloxy (OR, wherein “R” can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or a sugar); polyethyleneglycols (PEG), O(CH2CH2O).CH2CH2OR wherein R can be, e.g., H or optionally substituted alkyl, and n can be an integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10, from 4 to 16, and from 4 to 20). In some embodiments, the 2′ hydroxyl group modification can be 2′-O—Me. In some embodiments, the 2′ hydroxyl group modification can be a 2′-fluoro modification, which replaces the 2′ hydroxyl group with a fluoride. In some embodiments, the 2′ hydroxyl group modification can include “locked” nucleic acids (LNA) in which the 2′ hydroxyl can be connected, e.g., by a C1-6 alkylene or C1-6 heteroalkylene bridge, to the 4′ carbon of the same ribose sugar, where exemplary bridges can include methylene, propylene, ether, or amino bridges; 0-amino (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino) and aminoalkoxy, 0(CH2).-amino, (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino) In some embodiments, the 2′ hydroxyl group modification can include “unlocked” nucleic acids (UNA) in which the ribose ring lacks the C2′-C3′ bond. In some embodiments, the 2′ hydroxyl group modification can include the methoxyethyl group (MOE), (OCH2CH2OCH3, e.g., a PEG derivative).
“Deoxy” 2′ modifications can include hydrogen (i.e. deoxyribose sugars, e.g., at the overhang portions of partially dsRNA); halo (e.g., bromo, chloro, fluoro, or iodo); amino (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); NH(CH2CH2NH).CH2CH2-amino (wherein amino can be, e.g., as described herein), —NHC(O)R (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionally substituted with e.g., an amino as described herein.
The sugar modification can comprise a sugar group which may also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a modified nucleic acid can include nucleotides containing e.g., arabinose, as the sugar. The modified nucleic acids can also include abasic sugars. These abasic sugars can also be further modified at one or more of the constituent sugar atoms. The modified nucleic acids can also include one or more sugars that are in the L form, e.g. L- nucleosides.
The modified nucleosides and modified nucleotides described herein, which can be incorporated into a modified nucleic acid, can include a modified base, also called a nucleobase. Examples of nucleobases include, but are not limited to, adenine (A), guanine (G), cytosine (C), and uracil (U). These nucleobases can be modified or wholly replaced to provide modified residues that can be incorporated into modified nucleic acids. The nucleobase of the nucleotide can be independently selected from a purine, a pyrimidine, a purine analog, or pyrimidine analog. In some embodiments, the nucleobase can include, for example, naturally-occurring and synthetic derivatives of a base.
In embodiments employing a dual guide RNA, each of the crRNA and the tracr RNA can contain modifications. Such modifications may be at one or both ends of the crRNA and/or tracr RNA. In embodiments comprising an sgRNA, one or more residues at one or both ends of the sgRNA may be chemically modified, and/or internal nucleosides may be modified, and/or the entire sgRNA may be chemically modified. Certain embodiments comprise a 5′ end modification. Certain embodiments comprise a 3′ end modification.
Modifications of 2′-O-methyl are encompassed.
Another chemical modification that has been shown to influence nucleotide sugar rings is halogen substitution. For example, 2′-fluoro (2′-F) substitution on nucleotide sugar rings can increase oligonucleotide binding affinity and nuclease stability. Modifications of 2′-fluoro (2′-F) are encompassed.
Phosphorothioate (PS) linkage or bond refers to a bond where a sulfur is substituted for one nonbridging phosphate oxygen in a phosphodiester linkage, for example in the bonds between nucleotides bases. When phosphorothioates are used to generate oligonucleotides, the modified oligonucleotides may also be referred to as S-oligos.
Abasic nucleotides refer to those which lack nitrogenous bases.
Inverted bases refer to those with linkages that are inverted from the normal 5′ to 3′ linkage (i.e., either a 5′ to 5′ linkage or a 3′ to 3′ linkage).
An abasic nucleotide can be attached with an inverted linkage. For example, an abasic nucleotide may be attached to the terminal 5′ nucleotide via a 5′ to 5′ linkage, or an abasic nucleotide may be attached to the terminal 3′ nucleotide via a 3′ to 3′ linkage. An inverted abasic nucleotide at either the terminal 5′ or 3′ nucleotide may also be called an inverted abasic end cap.
In some embodiments, one or more of the first three, four, or five nucleotides at the 5′ terminus, and one or more of the last three, four, or five nucleotides at the 3′ terminus are modified. In some embodiments, the modification is a 2′-O—Me, 2′-F, inverted abasic nucleotide, PS bond, or other nucleotide modification well known in the art to increase stability and/or performance.
In some embodiments, the first four nucleotides at the 5′ terminus, and the last four nucleotides at the 3′ terminus are linked with phosphorothioate (PS) bonds.
In some embodiments, the first three nucleotides at the 5′ terminus, and the last three nucleotides at the 3′ terminus comprise a 2′-O-methyl (2′-O—Me) modified nucleotide. In some embodiments, the first three nucleotides at the 5′ terminus, and the last three nucleotides at the 3′ terminus comprise a 2′-fluoro (2′-F) modified nucleotide.
In some embodiments, a composition is encompassed comprising one or more gRNAs comprising one or more guide sequences from Table 2 or the Sequence Listing and an RNA-targeted endonuclease, e.g., a nuclease, such as a Cas nuclease, such as Cas9. In some embodiments, the RNA-targeted endonuclease has cleavase activity, which can also be referred to as double-strand endonuclease activity. In some embodiments, the RNA-targeted endonuclease comprises a Cas nuclease. Examples of Cas9 nucleases include those of the type II CRISPR systems of S. pyogenes, S. aureus, and other prokaryotes (see, e.g., the list in the next paragraph), and modified (e.g., engineered or mutant) versions thereof. See, e.g., US2016/0312198 A1; US 2016/0312199 A1. Other examples of Cas nucleases include a Csm or Cmr complex of a type III CRISPR system or the Cas10, Csm 1, or Cmr2 subunit thereof; and a Cascade complex of a type I CRISPR system, or the Cas3 subunit thereof. In some embodiments, the Cas nuclease may be from a Type-IIA, Type-IIB, or Type-IIC system. For discussion of various CRISPR systems and Cas nucleases see, e.g., Makarova et al., NAT. REV. MICROBIOL. 9:467-477 (2011); Makarova et al., NAT. REV. MICROBIOL, 13: 722-36 (2015); Shmakov et al., MOLECULAR CELL, 60:385-397 (2015).
Non-limiting exemplary species that the Cas nuclease can be derived from include Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Listeria innocua, Lactobacillus gasseri, Francisella novicida, Wolinella succinogenes, Sutterella wadsworthensis, Gammaproteobacterium, Neisseria meningitidis, Campylobacter jejuni, Pasteurella multocida, Fibrobacter succinogene, Rhodospirillum rubrum, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Lactobacillus buchneri, Treponema denficola, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaficum, 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, Streptococcus pasteurianus, Neisseria cinerea, Campylobacter lari, Parvibaculum lavamentivorans, Corynebacterium diphtheria, Acidaminococcus sp., Lachnospiraceae bacterium ND2006, and Acaryochloris marina.
In some embodiments, the Cas nuclease is the Cas9 nuclease from Streptococcus pyogenes. In some embodiments, the Cas nuclease is the Cas9 nuclease from Streptococcus thermophilus. In some embodiments, the Cas nuclease is the Cas9 nuclease from Neisseria meningitidis. In some embodiments, the Cas nuclease is the Cas9 nuclease is from Staphylococcus aureus. In some embodiments, the Cas nuclease is the Cpf1 nuclease from Francisella novicida. In some embodiments, the Cas nuclease is the Cpf1 nuclease from Acidaminococcus sp. In some embodiments, the Cas nuclease is the Cpf1 nuclease from Lachnospiraceae bacterium ND2006. In further embodiments, the Cas nuclease is the Cpf1 nuclease from Francisella tularensis, Lachnospiraceae bacterium, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium, Parcubacteria bacterium, Smithella, Acidaminococcus, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi, Leptospira inadai, Porphyromonas crevioricanis, Prevotella disiens, or Porphyromonas macacae. In certain embodiments, the Cas nuclease is a Cpf1 nuclease from an Acidaminococcus or Lachnospiraceae.
In some embodiments, the gRNA together with an RNA-targeted endonuclease is called a ribonucleoprotein complex (RNP). In some embodiments, the RNA-targeted endonuclease is a Cas nuclease. In some embodiments, the gRNA together with a Cas nuclease is called a Cas RNP. In some embodiments, the RNP comprises Type-I, Type-II, Type-III, Type-IV, or Type-V components. In some embodiments, the Cas nuclease may be from a Type-V system, such as Cas12, or Cas12a (previously known as Cpf1). In some embodiments, the Cas nuclease is the Cas9 protein from the Type-II CRISPR/Cas system. In some embodiment, the gRNA together with Cas9 is called a Cas9 RNP.
Wild type Cas9 has two nuclease domains: RuvC and HNH. The RuvC domain cleaves the non-target DNA strand, and the HNH domain cleaves the target strand of DNA. In some embodiments, the Cas9 protein comprises more than one RuvC domain and/or more than one HNH domain. In some embodiments, the Cas9 protein is a wild type Cas9. In each of the composition, use, and method embodiments, the Cas induces a double strand break in target DNA.
In some embodiments, chimeric Cas nucleases are used, where one domain or region of the protein is replaced by a portion of a different protein. In some embodiments, a Cas nuclease domain may be replaced with a domain from a different nuclease such as Fok 1. In some embodiments, a Cas nuclease may be a modified nuclease.
In other embodiments, the Cas nuclease may be from a Type-I CRISPR/Cas system. In some embodiments, the Cas nuclease may be a component of the Cascade complex of a Type-I CRISPR/Cas system. In some embodiments, the Cas nuclease may be a Cas3 protein. In some embodiments, the Cas nuclease may be from a Type-III CRISPR/Cas system. In some embodiments, the Cas nuclease may have an RNA cleavage activity.
In some embodiments, the RNA-targeted endonuclease has single-strand nickase activity, i.e., can cut one DNA strand to produce a single-strand break, also known as a “nick.” In some embodiments, the RNA-targeted endonuclease comprises a Cas nickase. A nickase is an enzyme that creates a nick in dsDNA, i.e., cuts one strand but not the other of the DNA double helix. In some embodiments, a Cas nickase is a version of a Cas nuclease (e.g., a Cas nuclease discussed above) in which an endonucleolytic active site is inactivated, e.g., by one or more alterations (e.g., point mutations) in a catalytic domain. See, e.g., U.S. Pat. No. 8,889,356 for discussion of Cas nickases and exemplary catalytic domain alterations. In some embodiments, a Cas nickase such as a Cas9 nickase has an inactivated RuvC or HNH domain.
In some embodiments, the RNA-targeted endonuclease is modified to contain only one functional nuclease domain. For example, the agent protein may be modified such that one of the nuclease domains is mutated or fully or partially deleted to reduce its nucleic acid cleavage activity. In some embodiments, a nickase is used having a RuvC domain with reduced activity. In some embodiments, a nickase is used having an inactive RuvC domain. In some embodiments, a nickase is used having an HNH domain with reduced activity. In some embodiments, a nickase is used having an inactive HNH domain.
In some embodiments, a conserved amino acid within a Cas protein nuclease domain is substituted to reduce or alter nuclease activity. In some embodiments, a Cas nuclease may comprise an amino acid substitution in the RuvC or RuvC-like nuclease domain. Exemplary amino acid substitutions in the RuvC or RuvC-like nuclease domain include DlOA (based on the S. pyogenes Cas9 protein). See, e.g., Zetsche et al. (2015) Cell Oct 22:163(3): 759-771. In some embodiments, the Cas nuclease may comprise an amino acid substitution in the HNH or HNH-like nuclease domain. Exemplary amino acid substitutions in the HNH or HNH-like nuclease domain include E762A, H840A, N863A, H983A, and D986A (based on the S. pyogenes Cas9 protein). See, e.g., Zetsche et al. (2015). Further exemplary amino acid substitutions include D917A, E1006A, and D1255A (based on the Francisella novicida U112 Cpf1 (FnCpf1) sequence (UniProtKB-A0Q7Q2 (CPFl_FRATN)).
In some embodiments, an mRNA encoding a nickase is provided in combination with a pair of guide RNAs that are complementary to the sense and antisense strands of the target sequence, respectively. In this embodiment, the guide RNAs direct the nickase to a target sequence and introduce a DSB by generating a nick on opposite strands of the target sequence (i.e., double nicking). In some embodiments, use of double nicking may improve specificity and reduce off-target effects. In some embodiments, a nickase is used together with two separate guide RNAs targeting opposite strands of DNA to produce a double nick in the target DNA. In some embodiments, a nickase is used together with two separate guide RNAs that are selected to be in close proximity to produce a double nick in the target DNA.
In some embodiments, the RNA-targeted endonuclease lacks cleavase and nickase activity. In some embodiments, the RNA-targeted endonuclease comprises a dCas DNA-binding polypeptide. A dCas polypeptide has DNA-binding activity while essentially lacking catalytic (cleavase/nickase) activity. In some embodiments, the dCas polypeptide is a dCas9 polypeptide. In some embodiments, the RNA-targeted endonuclease lacking cleavase and nickase activity or the dCas DNA-binding polypeptide is a version of a Cas nuclease (e.g., a Cas nuclease discussed above) in which its endonucleolytic active sites are inactivated, e.g., by one or more alterations (e.g., point mutations) in its catalytic domains. See, e.g., US 2014/0186958 A1; US 2015/0166980 A1.
In some embodiments, the RNA-targeted endonuclease comprises one or more heterologous functional domains (e.g., is or comprises a fusion polypeptide).
In some embodiments, the heterologous functional domain may facilitate transport of the RNA-targeted endonuclease into the nucleus of a cell. For example, the heterologous functional domain may be a nuclear localization signal (NLS). In some embodiments, the RNA-targeted endonuclease may be fused with 1-10 NLS(s). In some embodiments, the RNA-targeted endonuclease may be fused with 1-5 NLS(s). In some embodiments, the RNA-targeted endonuclease may be fused with one NLS. Where one NLS is used, the NLS may be linked at the N-terminus or the C-terminus of the RNA-targeted endonuclease sequence. It may also be inserted within the RNA-targeted endonuclease sequence. In other embodiments, the RNA-targeted endonuclease may be fused with more than one NLS. In some embodiments, the RNA-targeted endonuclease may be fused with 2, 3, 4, or 5 NLSs. In some embodiments, the RNA-targeted endonuclease may be fused with two NLSs. In certain circumstances, the two NLSs may be the same (e.g., two SV40 NLSs) or different. In some embodiments, the RNA-targeted endonuclease is fused to two SV40 NLS sequences linked at the carboxy terminus. In some embodiments, the RNA-targeted endonuclease may be fused with two NLSs, one linked at the N-terminus and one at the C-terminus. In some embodiments, the RNA-targeted endonuclease may be fused with 3 NLSs. In some embodiments, the RNA-targeted endonuclease may be fused with no NLS.
In some embodiments, the heterologous functional domain may be capable of modifying the intracellular half-life of the RNA-targeted endonuclease. In some embodiments, the half-life of the RNA-targeted endonuclease may be increased. In some embodiments, the half-life of the RNA-targeted endonuclease may be reduced. In some embodiments, the heterologous functional domain may be capable of increasing the stability of the RNA-targeted endonuclease. In some embodiments, the heterologous functional domain may be capable of reducing the stability of the RNA-targeted endonuclease. In some embodiments, the heterologous functional domain may act as a signal peptide for protein degradation. In some embodiments, the protein degradation may be mediated by proteolytic enzymes, such as, for example, proteasomes, lysosomal proteases, or calpain proteases. In some embodiments, the heterologous functional domain may comprise a PEST sequence. In some embodiments, the RNA-targeted endonuclease may be modified by addition of ubiquitin or a polyubiquitin chain In some embodiments, the ubiquitin may be a ubiquitin-like protein (UBL). Non-limiting examples of ubiquitin-like proteins include small ubiquitin-like modifier (SUMO), ubiquitin cross-reactive protein (UCRP, also known as interferon-stimulated gene-15 (ISG15)), ubiquitin-related modifier-1 (URM1), neuronal-precursor-cell-expressed developmentally downregulated protein-8 (NEDD8, also called Rubl in S. cerevisiae), human leukocyte antigen F-associated (FAT10), autophagy-8 (ATG8) and -12 (ATG12), Fau ubiquitin-like protein (FUB1), membrane-anchored UBL (MUB), ubiquitin fold-modifier-1 (UFM1), and ubiquitin-like protein-5 (UBLS).
In some embodiments, the heterologous functional domain may be a marker domain. Non-limiting examples of marker domains include fluorescent proteins, purification tags, epitope tags, and reporter gene sequences. In some embodiments, the marker domain may be a fluorescent protein. Non-limiting examples of suitable fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, sfGFP, EGFP, Emerald, Azami Green, Monomeric Azami Green, CopGFP, AceGFP, ZsGreen1), yellow fluorescent proteins (e.g., YFP, EYFP, Citrine, Venus, YPet, PhiYFP, ZsYellow 1), blue fluorescent proteins (e.g., EBFP, EBFP2, Azurite, mKalamal, GFPuv, Sapphire, T-sapphire,), cyan fluorescent proteins (e.g., ECFP, Cerulean, CyPet, AmCyanl, Midoriishi-Cyan), red fluorescent proteins (e.g., mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRedl, AsRed2, eqFP611, mRasberry, mStrawberry, Jred), and orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine, tdTomato) or any other suitable fluorescent protein. In other embodiments, the marker domain may be a purification tag and/or an epitope tag. Non-limiting exemplary tags include glutathione-S-transferase (GST), chitin binding protein (CBP), maltose binding protein (MBP), thioredoxin (TRX), poly(NANP), tandem affinity purification (TAP) tag, myc, AcV5, AU1, AUS, E, ECS, E2, FLAG, HA, nus, Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, 51, T7, V5, VSV-G, 6xHis, 8xHis, biotin carboxyl carrier protein (BCCP), poly-His, and calmodulin. Non-limiting exemplary reporter genes include glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, or fluorescent proteins.
In additional embodiments, the heterologous functional domain may target the RNA-targeted endonuclease to a specific organelle, cell type, tissue, or organ. In some embodiments, the heterologous functional domain may target the RNA-targeted endonuclease to muscle.
In further embodiments, the heterologous functional domain may be an effector domain. When the RNA-targeted endonuclease is directed to its target sequence, e.g., when a Cas nuclease is directed to a target sequence by a gRNA, the effector domain may modify or affect the target sequence. In some embodiments, the effector domain may be chosen from a nucleic acid binding domain or a nuclease domain (e.g., a non-Cas nuclease domain) In some embodiments, the heterologous functional domain is a nuclease, such as a FokI nuclease. See, e.g., U.S. Pat. No. 9,023,649.
Determination of Efficacy of gRNAs
In some embodiments, the efficacy of a gRNA is determined when delivered or expressed together with other components forming an RNP. In some embodiments, the gRNA is expressed together with an RNA-targeted endonuclease, such as a Cas protein, e.g., Cas9. In some embodiments, the gRNA is delivered to or expressed in a cell line that already stably expresses an RNA-guided DNA nuclease, such as a Cas nuclease or nickase, e.g., Cas9 nuclease or nickase. In some embodiments the gRNA is delivered to a cell as part of a RNP. In some embodiments, the gRNA is delivered to a cell along with a mRNA encoding an RNA-guided DNA nuclease, such as a Cas nuclease or nickase, e.g., Cas9 nuclease or nickase.
As described herein, use of an RNA-guided DNA nuclease and one or more guide RNAs disclosed herein can lead to double-stranded breaks in the DNA which can produce excision of a trinucleotide repeat or self-complementary region upon repair by cellular machinery, e.g., in the presence of a DNA-PK inhibitor.
In some embodiments, the efficacy of particular gRNAs is determined based on in vitro models. In some embodiments, the in vitro model is a cell line containing a target trinucleotide repeat or self-complementary region, such as any such cell line described in the Example section below.
In some embodiments, the efficacy of particular gRNAs is determined across multiple in vitro cell models for a gRNA selection process. In some embodiments, a cell line comparison of data with selected gRNAs is performed. In some embodiments, cross screening in multiple cell models is performed.
In some embodiments, the efficacy of particular gRNAs is determined based on in vivo models. In some embodiments, the in vivo model is a rodent model. In some embodiments, the rodent model is a mouse which expresses a gene comprising an expanded trinucleotide repeat or a self-complementary region. The gene may be the human version or a rodent (e.g., murine) homolog of any of the genes listed in Table 1. In some embodiments, the gene is human DMPK. In some embodiments, the gene is a rodent (e.g., murine) homolog of DMPK In some embodiments, the in vivo model is a non-human primate, for example cynomolgus monkey.
In some embodiments, the efficacy of a guide RNA is measured by an amount of excision of a trinucleotide repeat of interest. The amount of excision may be determined by any appropriate method, e.g., quantitative sequencing; quantitative PCR; quantitative analysis of a Southern blot; etc.
Additional embodiments are provided:
Embodiment 1A is a method of treating a disease or disorder characterized by a trinucleotide repeat (TNR) in DNA, the method comprising delivering to a cell that comprises a TNR i) a guide RNA comprising a spacer that directs an RNA-targeted endonuclease to or near the TNR, or a nucleic acid encoding the guide RNA; ii) an RNA-targeted endonuclease or a nucleic acid encoding the RNA-targeted endonuclease; and iii) optionally a DNA-PK inhibitor.
RNAs that comprise a first and second spacer that deliver the RNA-targeted endonuclease to or near a TNR or self-complementary region, or one or more nucleic acids encoding the pair of guide RNAs, are delivered to the cell.
The following examples are provided to illustrate certain disclosed embodiments and are not to be construed as limiting the scope of this disclosure in any way.
1. Materials and Methods
Guide RNA and Primer sequences. Primer sequences are shown in the Table of Additional Sequences. Cas9 Guide RNAs were used as a dual guide (dgRNA) format unless otherwise indicated as the single guide format (sgRNA). The crRNA contained the spacer sequence listed in the Table of Additional Sequences and was obtained from IDT as AltR-crRNA. The tracrRNA used with SpCas9 was AltR-tracrRNA (IDT Cat. No. 1072534).
Fibroblast immortalization. 2×105 fibroblasts (GM04033 and GM07492, Cone11 Institute) were seeded in 6 well plates. The following day fibroblasts were transduced at MOI 5 with hTERT-neo lentivirus with 10 ug/mL polybrene. Media was changed 24 hours post-transduction. Cells were selected with 0.5mg/ml G418 48 hrs post-transduction in MEM+15% FBS+NEAA.
Immortalized fibroblast electroporation & DNA-PK inhibitor treatment (paired guides). 200 uM crRNA (resuspended in IDTE, IDT Cat. No. 11-01-02-05) and 200 uM tracrRNA (resuspended in IDT duplex buffer, Cat. No. 11-01-03-01) were mixed 1:1 and pre-annealed (incubated 5min at 95° C., then cooled to room temperature). Unless otherwise indicated, RNP assembly was performed using 2 μL of 100 μM pre-annealed 5′ guide, 2 μL of 100 μM pre-annealed 3′ guide, and 2 μL of nuclease where a pair of guides were used, or 4 μL of 100 μM pre-annealed guide and 2 μL of nuclease where only one guide was used. Each RNP was assembled in triplicate. The SpCas9 (IDT) stock solution had a concentration of 10 ug/ul.
Guide (100 uM pre-annealed) and SpCas9 protein (10 ug/ul IDT) were mixed for each nucleofection in 1.7 ml Eppendorf tubes and incubated at room temperature ˜10 minutes to pre-assemble RNPs. (Note: experiments were generally carried out in biological triplicates for each condition.)
20 ul of P2 nucleofection solution (Lonza, Cat. No. V4XP-2032; pre-warmed to room temperature and prepared by adding the included supplement) was added to each RNP mixture.
Cell preparation: 04033 hTert-transformed DM1 patient fibroblasts and 7492 hTert-transformed heathy control fibroblasts were expanded in a T175 flask until confluent. Cells were washed 1× with PBS-, treated with 5 ml of 1× TrypLE Express for 7 minutes, and washed off in 25 ml of serum-containing media (MEM with GlutaMAX, 15% FBS, 1xNEAA).
Cells were spun down for 5 minutes at 500 g and re-suspended in fresh media. Suspensions were filtered through 44 uM filter to ensure a single cell suspension. Cells were counted and aliquoted at ˜300K per electroporation condition in a 15 ml conical tube. All the aliquots were pelleted for 5 minutes at 500 g and media removed just prior to nucleofection.
Nucleofection: 20 ul of the RNP/P2 mixture was used to resuspend the 300K cell pellet and resulting suspension was moved to a 16 well electroporation cuvette. Nucleofection was carried out on the Lonza X-unit (Lonza Bioscience) with the following settings: solution P2 and pulse code EN150.
Plating: Each nucleofected well (˜300K cells in 20 ul) was split into 2 wells of 12-well plates (8 ul per well) containing lml pre-warmed (1) plain medium or (2) medium supplemented with 10 uM Compound 6. Media was changed to plain medium (without Compound 6) in all wells 24 hours after plating. Cells were expanded for 10 days with media changes every 3 days until most wells were nearing confluence.
Harvesting: On day 10 after nucleofection, cells were washed 1× with PBS-, treated with 200 ul of 1X TrypLE Express for 7 minutes, and washed off in 2 ml of serum-containing media. Cells were pelleted for 5 min at 500 g and re-suspended in lml of fresh medium.
CUG foci FISH assay: cells were counted and plated in 384 well high content imaging plates in quadruplicate at 5K cells per well. Cells were allowed to attach overnight before fixation.
Preparation of samples for genotyping: 100 ul of cell suspension was pelleted in 1.7 ml tubes for 5 minutes at 500 g. Cell pellets were re-suspended in 100 ul of Lucigen QuickExtract buffer and lysed at 65° C. for 15 minutes followed by heat inactivation at 98° C. for 2 minutes. Extracts were stored at -80° C.
Preparation of samples for splicing assays: all remaining cells were pelleted in 1.7 ml tubes for 5 min at 500 g. Media was removed, and pellets were frozen until RNA processing.
Genotyping: A PCR mastermix was prepared as follows for 20 ul reactions: 10 ul Phusion 2× Master Mix, 1 ul 10 uM DMPK-nest-F primer, 1 ul 10 uM DMPK-nest-R primer, 7 ul of water. 3 ul of sample in QuickExtract DNA extraction buffer was added to 17 ul of master mix for each reaction. Cycling was performed as a touchdown program: 98° C. for 30 s, followed by 8 cycles of melting at 98° C. for 10 sec, annealing at 72° C. for lOs (decreasing by 0.5C per cycle), extension 72° C. for 30 s. Followed by 27 cycles of 98° C. for 10 s, 68° C. for 10 s, 72° C. for 30 S. Final extension at 72° C. for 10 minutes. Products were analyzed by electrophoresis on 2% agarose gels.
Electroporation & DNAPK-I Treatment (Individual Guides)
The protocol was as described above for the paired guide protocol, except as indicated herein. Electroporations were performed using P3 solution and pulse code CA137 and grown in 24 well plate with or without 10 uM Compound 6. RNP assembly was performed using 4 μL pre-annealed 100 μM guide and 2 μL Cas9 as described above. Harvesting: 48 hrs after nucleofection, cells were washed 1× with PBS-, treated with 200 ul of 1× TrypLE Express for 7 min, and washed off in 2 ml of serum-containing media. Cells were pelleted for 5 min at 500 g and re-suspended in lml fresh media.
For genotyping 50 ul of cell suspension was pelleted in 1.7 ml tubes for 5 min at 500 g. Cell pellets were re-suspended in 100 ul of Lucigen QuickExtract buffer and lysed at 65° C. for 15 min followed by heat inactivation at 98° C. for 2 min. Extracts were stored at −80° C.
Fluorescence In Situ Hybridization (FISH)/IF Co-Staining
MBNL1/(CUG)n foci imaging was used as an orthogonal method to evaluate CTG repeat excision with DMPK guide RNAs in DM1 fibroblasts.
Cells were fixed for 15 min at RT with 4% PFA and washed 5 times for 10 min in 1× PBS at RT. Cells were stored at 4° C. if not probed immediately.
For the fluorescence in situ hybridization (FISH) procedure, cells were permeabilized with 0.5% triton X-100, in 1× PBS at RT for 5 min.
Cells were prewashed with 30% formamide, 2× SSC for 10 min at RT. Cells were then probed for 2 hrs at 37° C., with a 1 ng/uL of Alexa546-(CAG)io probe in 30% formamide, 2× SSC, 2 ug/mL BSA, 66 ug/mL yeast tRNA, 2 mM vanadyl complex.
Cells were then washed for 30 min in 30% formamide, 2× SSC at 42° C., and then in 30% formamide, 2× SSC for 30 min at 37° C., then in 1× SSC for 10 min at RT, and last in 1× PBS for 10 min at RT. Cells were next probed overnight, at 4° C. with anti-MBNL1 antibody (1:1000 dilution, santacruz, 3A4) in 1× PBS +1%BSA. Cells were washed 2 times for 10 min at RT with 1× PBS. Cells were incubated with goat anti-rabbit Alexa 647 in 1× PBS +1%BSA (1:500 dilution) for 1 h at RT. Cells were washed 2 times, for 10 min at RT with 1× PBS. Cells were stained with Hoechst solution (0.1mg/m1) for 5 min, and then washed with 1× PBS once for 5 min.
PBS was aspirated and fresh PBS (100 ul) was added per well. Imaging plates were sealed with adhesive aluminum foils and imaged using MetaXpress (Molecular Devices).
Electroporation of iPS Cells
SpCas9 RNPs for electroporation into iPS cells were prepared as follows. SpCas9 crRNAs were resuspended at 200 μM in IDTE and tracrRNA was resuspended at 200 μM in duplex buffer. Equal amounts of 200 uM crRNA and 200 uM tracrRNA were mixed in a PCR tube, heated to 95° C., and allowed to cool to room temperature, giving guide complex at 100 μM.
Cpf1 guides were resuspended at 100 mM in IDTE.
RNP complexes for experiments corresponding to
RNP complexes for experiments corresponding to
Cell pellets were resuspended in 100 ul of pre-mixed P3 nucleofection solution and transferred to the tube containing pre-assembled RNP. 100 ul of RNP/Cell mixture was transferred to a nucleofection cuvette. Nucleofection was performed using a Lonza X-unit set for solution P3 and pulse code CA137. The cells were promptly moved from the cuvette to the pre-warmed media in the Laminin-coated plate splitting each nucleofection between pain medium or medium supplemented with 3 uM Compound 6 (for experiments corresponding to
Cells were detached using ReLeSR at 37° C. for 6 min and washed off with 2 ml StemFlex. 200 μ1 were passaged into a new 6-well dish with 2 ml StemFlex +4 ul Lamin 411 for further culturing and clonal isolation. The rest was split into 1.7 ml tubes as follows: 600 ul for protein; and 100 ul for DNA extraction.
DNA was extracted using Qiagen Blood and Tissue Kit following the manufacturer's protocol. Genotyping was performed as a nested PCR:
PCR1:
Cycling conditions:
Following completion, the PCR was diluted 1:10 and 2 ul was used as input in the next reaction (PCR2):
Cycling conditions:
Products were analyzed on a 2% agarose gel.
Cardiomyocyte differentiation protocol
Cardiomyocytes were prepared as follows. A culture of iPSCs was purified of differentiated cells by aspiration, then treated with accutase. Cells were plated at 0.133×106 cells per cm2 in StemFlex with ROCKi (10 uM final conc.) and were fed with StemFlex for 2 more days. Then (on “day 0”) media was changed to RPMI/B27 -insulin with small molecule CHIR99021 (StemCell Tech. Cat. no. 72052) (concentration depends on line). For days 1-3, media was changed to RPMI/B27 -insulin. For days 3-5, media was changed to RPMI/B27-insulin with small molecule IWP2 (Tocris Cat no. 3533) (5 uM). For days 5-7, media was changed to RPMI/B27 -insulin. For days 7-11, media was changed to RPMI/B27 +insulin. For days 11-15, media was changed to CDM3L:
Cardiomyocyte Nucleofection Protocol
Plates were prepared as follows. lmg/ml Fibronectin was diluted 1:100 in PBS and 200 ul was added per well in s 24-well plate. Plates were left at room temp for 2 hours. Fibronectin was removed and 500 ul of iCell Cardiomyocytes Maintenance Medium was added to each well and pre-warmed at 37° C.
RNPs were prepared essentially according to procedures described above for fibroblast experiments. Following RNP complex assembly, 20 ul of P3 solution (with supplement added) was added to each RNP and lul of electroporation enhancer (IDT) was added to each RNP mixture.
To prepare cells, media was aspirated from iPSC-derived cardiomyocytes grown in a 6-well dish and cells were washed 1× with 2 ml PBS per well. lml of TrypLE™ Select Enzyme (10X×) was added per well and cells were incubated for 10 min at 37° C.
Cells were gently pipetted and added to a 15mL tube with lml FBS +8 ml PBS per well in 6 well plate to inactivate TrypLE enzymes. Cells were spun down at 1000 RPM for 5 min, PBS was aspirated and cells were resuspended in fresh iCell Cardiomyocytes Maintenance Medium. Cells were passed through a 100um filter to 50mL tube, and slowly pipetted the resuspended cells through. Cells were counted and aliquoted ˜100K cardiomyocytes per nucleofection in 15 ml tubes. Cells were pelleted at 1000RPM for 5 minutes, and media was removed prior to nucleofection.
20 ul of the RNP/P3 mixture was used to resuspend the -100k cell pellet and the suspension was transferred to a 16 well electroporation cuvette. Nucleofection was carried out on the Lonza X-unit, with solution set to P3 and pulse code CA137. After nucleofections cells were plated in prepared 24 well plates and recovered for 48 hours prior to harvesting.
Media was removed and 100 ul of QuickExtract DNA extraction buffer was added to each well and pipetted up and down to remove all cells, then transferred to PCR tubes. Lysis was performed for 15 minutes at 65° C. followed by inactivation for 2 minutes at 98° C. Lysates were stored at −80° C.
Preparation of Neural Progenitor Cells
Basal media was prepared as follows:
The following media were also used. Media 2: Basal media +1 μM LDN 193189 +10 μM SB431542. Media 3: Basal media +1 μM LDN 193189 +10 μM SB431542 +1 μM Cyclopamine +10 ng/mL FGF2. Media 4: Basal media +1 μM Cyclopamine +10 ng/mL FGF2. Media 5: Basal media +10 ng/mL FGF2. NPC Maturation Seeding Media: Basal media +1:100 laminin +1:1,000 Y-27632 ROCK inhibitor. BrainPhys Maturation Media:
To seed iPSCs for neural re-patterning, human iPSCs were subcultured using StemFlex media supplemented at seeding with Laminin5-1-1 (1:400) in 6-well plates to approximately 80% confluence. Monthly mycoplasma analyses and regular karyotyping (5-10 passages) were generally performed to prevent culture artifacts from propagating.
On the day of seeding for differentiation (defined as Day 0), iPSCs were inspected for aberrant spontaneous differentiation. Generally, less than 10% of cultures should exhibit differentiated or loose morphology. Culture media was aspirated and cells were rinsed once with 3 mL Dulbecco's PBS (DPBS, divalent cation-free, Thermo Fisher # 14190144). DPBS was aspirated and 1 mL of warmed (25-35° C.) Accutase solution (Thermo Fisher # A1110501) was immediately dispensed. The plate was gently swirled to ensure even and complete dissociation, then incubated in a 3TC incubator for 10 minutes. The plate was firmly taped every 3-5 minutes to encourage iPSC colonies to dissociate from the plate.
Accutase was neutralized with at least 2 mL of warmed (25-35° C.) culture medium, typically StemFlex (StemCell Tech # 85850) or StemFlex (Thermo Fisher # A3349401). The cell solution was gently triturated to further dissociate any clumped cells.
The cell solution was transferred to a clean 50 mL conical tube and cells were pelleted by centrifugation at ˜150 RCF for 5 minutes.
After aspirating supernatant, the cell pellet was broken up by adding 1 mL of warmed StemFlex supplemented with Y-27632 ROCK inhibitor (1:1000 v/v) and gently tapping tube against the back of the hand. An additional 9 mL of culture media was added, and gently inverted to mix. A viable cell count was obtained using a ViCell Cell Viability Analyzer or equivalent device. 6E6 viable cells were diluted into a total of 12 mL iPSC culture media supplemented with Y-27632 ROCK inhibitor (1:1000) followed by dispensing 2 mL of the cell solution to each well of a matrigel-coated 6-well plate (1E6 cells per well seeding density), then rocking the plate perpendicularly 3-4 times in each direction (left-to-right, front-to-back) to evenly distribute cells in each well. Culture was maintained in a 3TC, 5% CO2, 85% RH incubator. The plates were then left undisturbed for at least 3 hours after seeding. Each day, the media was fully aspirated and replaced according to the following media schedule (see below regarding day 12). For each 6-well plate, prepare and warm at least 12-13 mL of media (2 mL per well). Cultures were inspected for morphological heterogeneity (should be low after first week) or matrigel layer breakdown. Media schedule:
Passaging Re-Ppatterned NPCs
On Day 12, after inspecting the cultures for morphological heterogeneity, culture media was aspirated and cells were rinsed once with 3 mL Dulbecco's PBS (DPBS, divalent cation-free, Thermo Fisher # 14190144). DPBS was then aspirated followed by immediately dispensing 1 mL of warmed (25-35° C.) Accutase solution (Thermo Fisher # A1110501). The plate was gently swirled to ensure even and complete dissociation, then incubated in a 3TC incubator for 10 minutes. The plate was firmly tapped every 3-5 minutes to encourage iPSC colonies to dissociate from the plate. Accutase was neutralized with at least 2 mL of warmed (25-35° C.) Medium 4. Gently triturate cell solution to further dissociated any clumped cells.
Transfer cell solution to a clean 50 mL conical tube. Pellet cells by centrifugation at 300 RCF for 5 minutes. Supernatant was aspirated and the cell pellet was broken up by adding 1 mL of warmed culture media supplemented with Y-27632 ROCK inhibitor (1:1000 v/v) and gently tapping tube against the back of the hand. An additional 9 mL of culture media was added and the tube was gently inverted to mix cell solution. Cells were counted and 9E6 viable cells were diluted into a total of 12 mL iPSC culture media supplemented with Y-27632 ROCK inhibitor (1:1000). 2 mL of the cell solution was dispensed to each well of a matrigel-coated 6-well plate (1.5E6 cells per well maintenance density). The plate was rocked perpendicularly 3-4 times in each direction (left-to-right, front-to-back) to evenly distribute cells in each well. The culture was maintained in a 3TC, 5% CO2, 85% RH incubator. Plates were left undisturbed for at least 3 hours after seeding.
Each day, media was fully aspirated and replaced according to the above media schedule (2 mL media per well).
NPCs were passaged once per week and passaged twice prior to FACS sorting definitive NPCs (takes place during Passage 3).
NPC Flow Cytometry Labeling Protocol
A single-cell suspension was generated and it was confirmed that NPCs are highly dense (seeded at 9E6/6-well plate, allowed to propagate for 5-7 days) and morphologically homogeneous. Culture media was aspirated and cells were washed once with divalent cation-free Dulbecco's PBS (Thermo Fisher, # 14190250), then aspirated, and 1 mL of warmed (25-35° C.) Accutase (Thermo Fisher, # A1110501) was added followed by incubation at 37° C. for 10-15 minutes. The plate was tapped firmly to dislodge adherent NPCs.
Accutase was neutralized by adding 2 mL of warmed (˜35° C.) DMEM-F12 (Thermo Fisher, # 11320033). Cells were pelleted by centrifugation at 300 ×g for 5 minutes at 22° C. Supernatant was aspirated and NPCs resuspended in 5 mL warmed DMEM-F12. A cell count was generated using a ViCell Cell Counting system.
To immunolabel NPCs, the following procedure was used: dispense 2-5E7 cells into 50 mL conical tubes; pellet cells by centrifugation at 300 ×g for 5 minutes at room temperature; aspirate supernatant, taking care not to disturb the cell pellet; wash the cells once in cation-free DPBS; gently triturate the cells to break up clumps; pellet cells by centrifugation at 300 ×g for 5 minutes at room temperature; aspirate supernatant. Label live/dead cells using the fixable dye Zombie Aqua (BioLegend, # 423102) by dispensing 100 μL of diluted (1:250) dye to each well (except autofluorescence controls or fluorescence minus one controls). Foil and incubate cells at 4° C. for 15-30 minutes; pellet cells by centrifugation at 300 ×g for 5 minutes at room temperature; aspirate supernatant; wash the cells once in cation-free DPBS. Gently triturate the cells to break up clumps; pellet cells by centrifugation at 300 ×g for 5 minutes at room temperature; aspirate supernatant. Block non-specific labeling using cold (4° C.) Cell Staining Buffer (BioLegend, # 420201) for 30 minutes at 4C (foiled). After dispensing, gently triturate the cells to break up clumps. Pellet cells by centrifugation at 300 ×g for 5 minutes at room temperature; aspirate supernatant. Dispense 100 μL of antibodies (see table below) per 5E6 total cells diluted in Cell Staining Buffer to each sample (except autofluorescence controls or fluorescence minus one controls). After dispensing, gently triturate the cells to break up clumps. Foil samples and incubate for 30 minutes at 4C. Note: Single-stained compensation controls can be produced using either water-lysed cells (Zombie Aqua L/D) or antibody capture beads (Thermo Fisher, # A10497)
After incubation, wash cells with 5 mL Cell Staining Buffer. Gently triturate the cells to break up clumps and evenly wash. Pellet cells by centrifugation at 300 ×g for 5 minutes at room temperature. Aspirate supernatant. Wash the cells once more in cold Cell Staining Buffer. Gently triturate the cells to break up clumps. Pellet cells by centrifugation at 300 ×g for 5 minutes at room temperature. Aspirate supernatant.
Resuspend cells in Pre-Sort Buffer (BD Bioscience, # 563503) supplemented with normocin (1:500) to a final concentration of 7-10E6 cells per mL. Foil and store at 4C until sorting on BD FACSAria Fusion (within 1-2 hours). Sort into chilled, 15 mL conical tubes pre-coated and filled with 7 mL Media 5 supplemented with Y-27632 ROCK inhibitor (1:1000), normocin (1:500), and 15 mMolar HEPES (Thermo Fisher # 15630080, 1:67 dilution of 1 M stock).
NPC Flow Cytometry Sorting and Analysis
The following procedure was used for NPC sorting and analysis. Set up instrument (BD FACSAria Fusion) using standard settings for a 100-micron nozzle (100 μm-20 psi) with 300 RPM sample agitation and 4° C. sample storage. Run CS&T using beads (BD Biosciences, # 655051; 1 drop in 350 μL DPBS). Do not modify voltages from the CS&T settings. Run Accudrop calibration (BD Biosciences, # 345249; 1 drop in 500 μL DPBS). Left deflector plate position should be set to 32 for calibration, 58-60 for sorting. Verify droplets hit the center of a 15 mL conical tube filled to 7 mL with 70% ethanol. For each sample, collect 10,000 pre-sort events with P1 scatter gate as the stop gate. Set gates as shown below. Collect: FSC-A/SSC-A P1 ->SSC-H/SSC-W P2 ->FSC-H/FSC-W P3->L/D Zombie Aqua (−) (live cells) ->CD184 (+) ->Tra-1-60 (−)/SSEA4 (−) (non-iPSCs)->CD44 (−)/CD271 (−) (non-glia, non-neural crest) ->CD24 (+)/CD15 (lo/mid) (NPC). Sort 1.5-2E6 cells from each line. Keep all samples chilled before and after sort. Seed 1.5E6 viable NPCs suspended in 2 mL of Media 5 supplemented with Y-27632 ROCK inhibitor (1:1000) into a matrigel-coated 6-well plate.
NPC Scale Up
The following procedure was used to scale up NPCs: Passage NPCs once per week. For passage 4 (first passage post-FACS sorting): Confirm NPCs are highly dense (seeded at 9E6/6-well plate, allowed to propagate for 5-7 days) and morphologically homogeneous. Aspirate culture media, wash once with divalent cation-free Dulbecco's PBS (Thermo Fisher, # 14190250). Aspirate the DPBS, and dispense 1 mL of warmed (25-35° C.) Accutase (Thermo Fisher, # A1110501). Incubate at 37C for 8-10 minutes. Tap firmly to dislodge adherent NPCs. Neutralize Accutase by adding 1 mL of warmed (˜35C) Media 5 supplemented with Y-27632 ROCK inhibitor (1:1,000). Pellet cells by centrifugation at 300 ×g for 5 minutes at 22° C. Aspirate supernatant and resuspend NPCs in 5 mL warmed Media 5 supplemented with Y-27632 ROCK inhibitor (1:1,000). Generate a cell count using a ViCell Cell Counting system. Resuspend 9E6 viable NPCs in 12 mL of Media 5 supplemented with Y-27632 ROCK inhibitor (1:1,000). Dispense 2 mL into each well of a matrigel-coated 6-well plate. For passage 5, repeat the above procedure, but scale up to seed 12.5E6 NPCs in 15 mL of Media 5 supplemented with Y-27632 ROCKi (1:1,000). Dispense all cells into a matrigel coated T75 flask.
NPC Banking/Cryopreservation
The following procedure was used for banking/cryopreservation of NPCs: Confirm NPCs are highly dense (seeded at 12.5E6/plate in T75 format, allowed to propagate for 5-7 days) and morphologically homogeneous. Aspirate culture media, wash once with divalent cation-free Dulbecco's PBS (Thermo Fisher, # 14190250). Aspirate the DPBS, and dispense 1 mL of warmed (25-35° C.) Accutase (Thermo Fisher, # A1110501). Incubate at 37C for 10-15 minutes. Tap firmly to dislodge adherent NPCs. Neutralize Accutase by adding 2 mL of warmed (˜35C) Basal Media supplemented with Y-27632 ROCK inhibitor (1:1,000).
Pellet cells by centrifugation at 300 ×g for 5 minutes at 22° C. Aspirate supernatant and resuspend NPCs in 5 mL warmed Basal Media supplemented with Y-27632 ROCK inhibitor (1:1,000). Generate a cell count using a ViCell Cell Counting system. NPCs are banked at 12.5E6/mL in 1 mL CryoStorlO (StemCell Technologies, # 07930). Calculate the number of cells needed to fill the desired number of banked aliquots, then dispense the required volume of the NPC-containing Basal Media supplemented with Y-27632 ROCK inhibitor (1:1,000) into a new 50 mL conical tube. Pellet cells by centrifugation at 300 ×g for 5 minutes at 22° C. Resuspend NPCs in required volume of CryoStor10 (1 mL per desired aliquot), and dispense into 2 mL cryovials (Corning, # 430659). Quickly transfer filled cryovials to a Mr. Frosty freezing container (Thermo, # 5100-0001). Store at −80° C. for at least 24 hr, then transfer to long-term storage in liquid nitrogen.
Neuronal Maturation
The following procedure was used to prepare polyethyleneimine-coated plates: To 474 mL of sterile distilled water, add 25 mL of Borate Buffer pH 8.2 (20X; Sigma, # 08059) and 1 mL of polyethyleneimine (50%; Sigma, # 03880). Swirl the PEI with a Stripette. Sterile filter and store at 4° C. for <1 month. Dispense 0.1% PEI into cell culture plates and incubate at RT for 1 hour. Aspirate PEI. Wash four times with sterile distilled water. Aspirate to dry. Air-dry in a cell culture hood overnight. Store at 4° C. for <2 weeks.
For neuronal maturation, the following procedure was used: On the day of reseeding, confirm NPCs are highly dense (seeded at 12.5E6/T75 flask, allowed to propagate for 5-7 days) and morphologically homogeneous. Aspirate culture media, wash once with divalent cation-free Dulbecco's PBS (Thermo Fisher, # 14190250). Aspirate the DPBS, and dispense 1 mL of warmed (25-35° C.) Accutase (Thermo Fisher, # A1110501). Incubate at 37C for 8-10 minutes. Tap firmly to dislodge adherent NPCs. Neutralize Accutase by adding 2 mL of warmed (˜35C) Basal Media supplemented with Y-27632 ROCK inhibitor (1:1,000). Pellet cells by centrifugation at 300 ×g for 5 minutes at 22° C. Aspirate supernatant and resuspend NPCs in 5 mL warmed Basal Media supplemented with Y-27632 ROCK inhibitor (1:1,000). Generate a cell count using a ViCell Cell Counting system (or equivalent).
Resuspend required number of viable NPCs in Basal Media supplemented with laminin (1:100) and Y-27632 ROCK inhibitor (1:1,000). Dispense cell solution into a polyethyleneimine-coated vessel. The following day (DIV1) perform a full media change of Basal Media with laminin (1:1,000). On DIV 2, perform a full media change of a 50:50 mix of Basal Media with laminin (1:1,000), and BrainPhys supplemented with PD 0332991 (1:5,000), DAPT (1:2,500), laminin (1:1,000). From DIV 3-5, perform daily full media changes with BrainPhys supplemented with PD 0332991 (1:5,000), DAPT (1:2,500), laminin (1:1,000). From DIV7+perform 1/2 media changes with BrainPhys supplemented with PD 0332991 (1:5,000), DAPT (1:2,500), laminin (1:1,000) 2-3 times per week.
NPC Nucleofection
RNP complexes were prepared essentially as described above for fibroblast experiments.
The following procedure was used to prepare the cells. For Basal Media preparation: Combine 500 mL of Neurobasal with 500 mL of Advanced DMEM/F12, then add 20 mL of SM1 supplement (without VitA), 10 mL N2-B supplement, 10 mL GlutaMax, and 2 mL Normocin.
To coat cell culture vessel: Thaw Matrigel on ice at 4C overnight. Dilute 5 mL Matrigel into 495 mL of cold DMEM (1% vol/vol) and stored at 4C. Dispense 0.5 mL per well of a 12 well plate and incubated for 1 hour at RT. Aspirate Matrigel solution immediately prior to cell plating.
To prepare the cells: Aspirate culture media, wash once with divalent cation-free Dulbecco's PBS. Aspirate the DPBS, and dispense 1 mL of warmed (25-35° C.) Accutase. Incubate at 37C for 10-15 minutes. Dislodge adherent NPCs by tapping flask. Neutralize Accutase by adding 2 mL of warmed (˜35C) Basal Media (as above). Pellet cells by centrifugation at 300 ×g for 5 minutes at 22° C. Aspirate supernatant and resuspend NPCs in 5 mL warmed Basal Media (as above), pass through 40um cell strainer, and count. Aliquot cells in 15 ml tubes at 2.5E6 per nucleofection.
To nucleofect: resuspend cell pellets in 100 ul of pre-mixed P3 nucleofection solution and transfer to the tube containing pre-assembled RNP. Transfer 100 ul of RNP/Cell mixture to a nucleofection cuvette. Nucleofect using Lonza X-unit. Set solution to P3 and used pulse code CA137. Wash cells 1× in DPBS. Promptly move the cells from the cuvette to a 12 well pre-coated dish with pre-warmed media containing Rock inhibitor. For recovery, the next day, change the media to Basal Media supplemented with l0ng/mL FGF-2. Continue to culture for total of 5 days, with daily media change supplemented with 1 Ong/mL FGF-2, as above. For harvesting: detach cells using Accutase at 37C for 10 min. Wash 1× with DPBS, pelleted cells, removed PBS and froze pellets at −80C.
DNA was extracted using Qiagen Blood and Tissue Kit following manufacturer's protocol. DNA was digested with HindIII and sized by PCR/agarose electrophoresis using standard techniques. PCR primers:
Neuron Nucleofection
Neurons (e.g., differentiated from NPCs as described above) were nucleofected as follows. RNPs were prepared essentially as described above for fibroblast experiments.
The enclosed supplement was added to AD1 nucleofection solution and 350 ul of solution was added to each RNP complex tube. 7.5 ul of 100 uM electroporation enhancer was added to each RNP tube just prior to nucleofection.
Media was removed from cells one well at a time and replaced with 350 ul of RNP-containing nucleofection solution. Once all wells were replaced, the electrode was gently inserted into well, avoiding bubbles. Cells were nucleofected using Lonza Y-unit nucleofector set to solution AD1 and pulse code EH-158. After nucleofection, the RNP solution was gently removed and replaced with fresh pre-warmed Brainphys media (described in maturation protocol). Cells were allowed to recover for 72 hours at 37° C. prior to harvesting. To harvest media was removed and cells were re-suspended in 500 ul of PBS, pelleted, PBS removed and pellets frozen.
DNA extraction and genotyping was performed as described above for NPC nucleofection.
Western Blot Protocol
Cell Pellets were resuspended in 1× MSD lysis buffer supplemented with protease and phosphatase inhibitors. 50 μl lysis buffer was used for 200K cells.
Lysates were vortexed and sonicated briefly (5-10 sec) at 20 Amp (using a Cole Parmer ultrasonic sonicator) before clearing by centrifugation at 21000× g for 10min at 4° C. Supernatants obtained can be used for protein estimation (BCA assay).
4× LDS buffer was added to the cleared supernatants to obtain a final concentration of lx LDS followed by boiling at 100° C. for 5min.
5-15 μg of cell lysate was run on a 4-12% NuPage Bis-tris gel with 1× MES SDS running buffer, followed by transfer onto a 0.204 Nitrocellulose membrane using the Transblot Turbo system (Instruction manual for catalog # 1704150). The blot was blocked for 1 hour at room temperature with LiCoR PBS blocking buffer (catalog # 927-40000). After one hour, overnight incubation was performed in primary antibody in LiCor PBS blocking buffer with 0.2% Tween-20 at 4° C. with rocking. Concentrations varied with the primary antibody efficiency.
The next day, the membrane was washed 3x times with PBS-T (0.1% tween-20), followed by a 1 hour incubation in secondary antibody (LiCor IRdye 800 or 680) at 1:10000 dilution in LiCoR PBS blocking buffer with 0.1% tween-20. The membrane was washed 3 times with PBS-T (0.1% tween-20), and proceed to signal detection of LiCor fluorescence using Odyssey CLx detector. Antibodies Used (Abcam): Vinculin: ab129002 (1:5000); Frataxin: ab110328 (1:250).
RNA Extraction & qRT-PCR
Mis-splicing correction was used as a functional readout of CTG repeat excision by dual DMPK guide RNAs in DM1 fibroblasts. Total RNA was extracted using Quick-RNA 96 kit in a volume of 20 ul (ZYMO Research). 10 ul of RNA was used to generate first strand cDNA by mixing with 10 ul of 2× RT mastermix from the high capacity cDNA RT kit (Thermo Fisher 4368814). Reaction mixes were spun down to remove air bubbles and loaded into a thermal cycler.
Reverse transcription was performed using a 3-step program, which consisted of 10 minutes at 25° C., 120 minutes at 37° C. , and 5 minutes at 85° C., followed by holding at 4° C.
Splicing was evaluated by qRT-PCR using the PowerUp SYBR green mastermix (Thermo Fisher A25742) in a Quantstudio 12K Flex Real-time PCR system. The composition of 10 ul of a 1× reaction is shown in Table 3 below. Primer sequences are listed in the Table of Additional Sequences.
Source of Materials
The materials listed in Table 4 were obtained from the indicated vendors.
2. TNR Excision of DMPK in Cardiomyocytes and Fibroblasts using paired gRNAs
Analysis of Excision by PCR and gel electrophoresis. Cardiomyocytes were treated with RNP comprising spCas9 and a pair of gRNAs targeting sites flanking the CTG repeat locus of DMPK1 via electroporation as described above. The gRNA pair was one of pairs A-H as indicated in Tables 5 and 6.
Pairs of guides comprising the following 18-mer spacer sequences were tested: SEQ ID NOs: 3348 and 2556; SEQ ID NOs: 3348 and 2500; SEQ ID NOs: 3332 and 2556; SEQ ID NOs: 3332 and 2500; SEQ ID NOs: 3356 and 2548; SEQ ID NOs: 3356 and 2508; SEQ ID NOs: 3380 and 2548; SEQ ID NOs: 3380 and 2508. More specifically, the tested guides were the tested 20-mer guide pairs in
The treatment resulted in excision of the CTG repeat locus to the extent indicated in
Wild-type and heterozygous DM1 patient cardiomyocytes were prepared from iPSCs and treated with RNP comprising spCas9 and a pair of gRNAs targeting sites flanking the CTG repeat locus of DMPK1 via electroporation as described above. The gRNA pair was one of pairs 1 or 2 (as shown in
Wild-type and heterozygous DM1 patient fibroblasts were treated with RNP comprising spCas9 and a pair of gRNAs targeting sites flanking the CTG repeat locus of DMPK1 via electroporation as described above. The gRNA pair was one of pairs 1 or 2 (as shown in
Excision of the CTG repeat locus was confirmed by Sanger sequencing for a representative product (
Analysis of excision by FISH for CUG foci and immunofluorescence for MBNL1 foci. Primary DM1 and wild-type fibroblasts were treated with RNP comprising spCas9 and a pair of gRNAs targeting sites flanking the CTG repeat locus of DMPK1 via electroporation as described above, or no gRNA (negative control). The gRNA pair was one of pairs A-D as indicated in Table 6. Samples of treated cells were assayed by FISH using the Alexa546-(CAG)io probe (custom-ordered from IDT) for the CUG repeat region of DMPK1 mRNA as described above. Samples of treated cells were also assayed by immunofluorescence for MBNL1 protein foci.
The number of CUG foci per nucleus was determined and is shown in
The number of MBNL1 foci per nucleus was determined and is shown in
Analysis of RNA splicing. Primary DM1 fibroblasts were treated with RNP containing gRNA pair 7 (identical to pair C in Table 6) or mock-treated without gRNA as described above, or not treated. Splicing was assayed in MBNL1 (
3. TNR Excision of DMPK with Inhibition of DNA-PK
hTert-transformed DM1 fibroblasts were treated as described above with or without 10 uM of the DNA-PK inhibitor Compound 6 and with RNP containing one of the DMPK gRNA pairs A-D (see Table 6). The treatment resulted in excision of the CTG repeat locus to the extent indicated in
gRNAs comprising the 18-mer spacer sequences of SEQ ID NOs: 3332, 3316, 2660, 2692, 2556, and 2500 were tested. More specifically, the tested guides were the 20-mer guides as shown in Table 5 and Table 6.
hTert-transformed DM1 fibroblasts were treated as described above with or without 10 uM of the DNA-PK inhibitor Compound 6 and with RNP containing one of the following DMPK gRNAs: DMPK-U57 (SEQ ID NO: 3330) (gRNA # 4), DMPK-U60 (SEQ ID NO: 3314) (gRNA # 5), DMPK-R12 (SEQ ID NO: 2658) (gRNA # 6), DMPK-R08 (SEQ ID NO: 2690) (gRNA# 7), DMPK-D03 (SEQ ID NO: 2554) (gRNA # 9), or DMPK-D10 (SEQ ID NO: 2498) (gRNA # 10) (see Table 5,
hTert-transformed DM1 fibroblasts were treated as described above with or without 10 uM of the DNA-PK inhibitor Compound 6 and with RNP containing one of the following DMPK gRNA pairs: A, B, C, or D (see Table 6). Cells were assayed for CUG foci per nucleus by FISH as described above.
hTert-transformed DM1 fibroblasts were treated as described above with or without 10 uM of the DNA-PK inhibitor Compound 6 and with RNP containing one of the following DMPK gRNA pairs: A, B, C, or D. Pair A =guides DMPK-U59 and DMPK-D03; pair B =guides DMPK-U59 and DMPK-D10; pair C =guides DMPK-U57 and DMPK-D03; pair D =guides DMPK-U57 and DMPK-D10 ((sequences shown above, Table 5, and the sequence listing). Mock-treated (M) and cells treated with a control guide targeting AAVS1 (NT) (spacer sequence: accccacagtggggccacta, SEQ ID NO: 31) were also analyzed. The percentages of mis-spliced transcripts were determined for MBNL1 (
4. Excision of Repeats of FMR1 Using Guide Pairs that Overlap Trinucleotide Repeats
M28 CHOC2 and mosaic CHOC1 neuronal precursor cells (NPC) were treated with a combination of 5′ and 3′ FMR1 gRNAs and SpCas9 via electroporation. Locations in FMR1 targeted by various guides are indicated in
5. Excision of CGG Repeats of FMR1 in CHOC1 Cells and in CHOC2 Cells
a. Excision of CGG Repeats of FMR1 in CHOC1 Cells
CHOC1 cells were genotyped using PCR and electrophoresis of the targeted locus (
Nonetheless, treatment of CHOC1 cells with one gRNA targeting a site 3′ of the CGG repeat region of FMR1, paired with a 5′ guide that targeted a sequence in the deleted region and therefore should have been ineffective, still resulted in repeat excision, indicating that one effective guide can be used to excise the repeats. Sequences from clones that underwent such excision with a single guide RNA (SEQ ID NO: 5262) are shown in
b. Excision of CGG Repeats of FMR1 in CHOC2 Cells
CGG repeat excision was evaluated using single or paired gRNAs in differentiated, post-mitotic CHOC2 neurons after SpCas9 RNP electroporation. CHOC2 post-mitotic neurons were treated with RNP comprising spCas9 and guides as indicated below in Table 7a without DNA-PK inhibition. SEQ ID NOs are provided for the spacer region sequences. See Table 2 and/or the Sequence Listing for sequences.
Excision of CGG repeats was analyzed by PCR (
Excision of CGG repeats of FMR1 was further evaluated with treatment of a DNA-PK inhibitor. CHOC2 neuronal precursor cells (NPCs) were treated with RNPs comprising spCas9 and guides as indicated below in Table 7b. SEQ ID NOs are provided for the spacer region sequences. See Table 2 and/or the Sequence Listing for sequences. Following electroporation, CHOC2 NPCs were treated with DMSO or 304 DNA-PK inhibitor (compound 6) as indicated below in Table 7b.
Excision of CGG repeats was analyzed by amplifying FMR1 DNA by PCR and separating the PCR products by electrophoresis using Agilent's 2200 TapeStation (
gRNAs comprising the 18-mer spacer sequences of SEQ ID NOs: 5264, 5336, 5832, 6024, and 5312 were tested. More specifically, the tested guides were the 20-mer guides as shown in Tables 7a and 7b.
6. Excision of GAA Repeats at the Frataxin Locus of FXN
iPS cells (wild-type, 4670, or 68FA) were treated with an RNA-targeted endonuclease (Cpf1 or Cas9) and Frataxin gRNAs as follows, which flank the GAA repeats in the Frataxin locus, with or without 1μM Compound 3. Cpf1 FXN gRNA 1 and 2: SEQ ID NOs: 47047 and 7447, respectively; SpCas9 FXN gRNAs 1 and 2: SEQ ID NOs: 52898 and 26546. Repeat excision was analyzed by PCR and electrophoresis (
Excision of repeats in the FXN locus resulted in elevated FXN levels (
7. Model for MMEJ-based CGG-repeat excision at the Fragile-X locus of FMR1
8. sgRNA screening in the 3′ UTR of DMPK
a. Materials and Methods
sgRNA selection. The 3′ untranslated region (UTR) of the DMPK gene was scanned for NGG or NAG SpCas9 protospacer adjacent motif (PAM) on either the sense or antisense strand, and 20-nucleotide sgRNA spacer sequences adjacent to the PAMs were identified. 172 sgRNAs with NGG PAM and 46 sgRNAs with NAG PAM were selected for evaluation of editing efficiency in HEK293T cells (Table 8).
Plasmids. An all-in-one expression vector pU6-sgRNA-Cbh-SpCas9-2A-EGFP that expresses sgRNA, SpCas9, and EGFP was used to subclone individual sgRNAs. The top and bottom strand oligos for each sgRNA were annealed and then subcloned into the Bbsl restriction sites of the pU6-sgRNA-Cbh-SpCas9-2A-EGFP vector as previously described (Ran, F.A. et al. (2013) Nat. Protoc. 8:2281-2308; PMID: 24157548).
Transfection and PCR amplification. pU6-sgRNA-Cbh-SpCas9-2A-EGFP vectors containing individual sgRNAs were transfected into HEK293T cells seeded in CELLSTAR black 96-well plates (Greiner) using either Lipofectamine 3000 (and 72 hr transfection time) or Lipofectamine 2000 (and 48 hr transfection time) as the transfection reagent (Thermo Fisher Scientific) following manufacturer's protocol. Post transfection, genomic DNA was isolated using DirectPCR lysis reagent (Viagen) supplemented with 0.5 mg/ml of proteinase K (Viagen), and used as template for subsequent PCR. The DMPK 3′ UTR region was amplified using GoTaq Green Master Mix (Promega) and PCR primers flanking the 3′ UTR region (SEQ ID NOs: 32 and 33) (Table of Additional Sequences). Amplification was conducted using the following cycling parameters: 1 cycle at 95° C. for 2 min; 40 cycles of 95° C. for 30 sec, 63° C. for 30 sec, and 72° C. for 90 sec; 1 cycle at 72° C. for 5 min.
Sanger sequencing and TIDE analysis. PCR products were sent to GeneWiz for purification and Sanger sequencing. Sequencing primer UTRsF3 (SEQ ID NO: 34) was used for sgRNAs upstream of the CTG repeat, while the reverse PCR primer (SEQ ID NO: 33) was used for downstream sgRNAs and 13 sgRNAs overlapping the CTG repeat region. The sgRNAs (DMPK-D75, DMPK-D76, DMPK-D85, DMPK-D86, DMPK-D102, DMPK-D103, DMPK-D104, DMPK-D105, DMPK-D119, DMPK-D120, DMPK-D121, DMPK-D122, DMPK-D123, DMPK-D124, DMPK-D125, DMPK-D126, DMPK-D127, DMPK-D128, DMPKD129) that were located close to the reverse PCR primer (SEQ ID NO: 33) were sequenced using sequencing primer UTRsF2 (SEQ ID NO: 35). Indel values were estimated using the TIDE analysis algorithm (DeskGen/Vertex) with the electrophoretograms obtained from Sanger sequencing. TIDE is a method based on the recovery of indels' spectrum from the sequencing electrophoretograms to quantify the proportion of template-mediated editing events (Brinkman, E. A. et al. (2014) Nucleic Acids Res. 42: e168; PMID: 25300484).
Off-target scoring of s2RNAs. Off-target sites were computationally predicted for each sgRNA based on sequence similarity to the hg38 human reference genome, specifically, any site that was identified to have up to 3 mismatches, or up to 2 mismatches and 1 DNA/RNA bulge, relative to the protospacer sequence as well as a protospacer adjacent motif (PAM) sequence of either NGG or NAG. An off-target score was then calculated for each sgRNA based on these computationally predicted off-target sites.
Specifically, each off-target site was given a weight representing the probability of it being edited, based on the site's degree of sequence similarity to the target site and its PAM sequence: (i) weighting based on the number of mismatches was calculated from the published metanalysis of empirical data at Haeussler, M. et. Al. (2016) Genome Biol., 17(148); PMID: 27380939 (if a DNA/RNA bulge was present at the off-target site, the bulge was counted as 2 additional mismatches, based on empirical data that off-target editing at sites with DNA/RNA bulges is observed less frequently than mismatches); and (ii) weighting based on the PAM sequence used the Cutting Frequency Determination model from Doench, J. G. et. Al. (2016) Nat Biotechnol., 34 (2): 184-191; PMID: 26780180. The weight for each off-target site was calculated by multiplying the site's weight based on number of mismatches with the site's weight based on PAM sequence. The overall off-target score for each sgRNA was calculated as the sum of weights for all associated predicted off-target sites. Overall, the off-target score for the sgRNA corresponds to the expected value of the number of off-target sites for that sgRNA. Higher off-target scores correspond with sgRNAs that are more likely to have off-target editing.
b. Results
Two hundred eighteen sgRNAs flanking the CTG repeat expansion of the DMPK gene (Table 8) were selected for editing the CTG repeat expansion. To avoid interference with the DIVIPK coding sequence and mRNA maturation, all selected sgRNAs were located within the 3′UTR of the DMPK gene between the stop codon and the end of the last exon. Among these 218 sgRNAs, 76 (DMPK-U01-DMPK-U76) are located upstream of the CTG repeat expansion (between the stop codon and the CTG repeat expansion), 129 sgRNAs (DMPK-D01-DMPK-D129) are located downstream of the CTG repeat expansion (between the CTG repeat expansion and the end of the last exon of DMPK), and 13 sgRNAs (DMPK-R01-DMPK-R13) are completely or partially overlapping the CTG repeat expansion.
Guides comprising the 18-mer spacer sequence of SEQ ID NOs: 4020, 4012, 4004, 4044, 4036, 4028, 3956, 3948, 3996, 3916, 3980, 3908, 3900, 3940, 3852, 3884, 2828, 3820, 3844, 3796, 3788, 3764, 3812, 3748, 3780, 3740, 3772, 3724, 3756, 3692, 3668, 3660, 3636, 3588, 3548, 3532, 3644, 3516, 3508, 3492, 3620, 3612, 3604, 3580, 3444, 3524, 3412, 3380, 3436, 3372, 3428, 3420, 3396, 3388, 3332, 3356, 3348, 3316, 3932, 3892, 3836, 3804, 3708, 3700, 3684, 3676, 3572, 3556, 3540, 3500, 3484, 3460, 3476, 3452, 2669, 2668, 2652, 2644, 2628, 2620, 2708, 2692, 2684, 2612, 2676, 2660, 2604, 2596, 2636, 2556, 2548, 2588, 2540, 2580, 2572, 2524, 2500, 2492, 2468, 2460, 2452, 2516, 2508, 2420, 2484, 2476, 2696, 2444, 2436, 2372, 2380, 2356, 2348, 2340, 2316, 2300, 2284, 2276, 2268, 2332, 2260, 2324, 2244, 2236, 2292, 2252, 2220, 2228, 2212, 2196, 2148, 2140, 2124, 2108, 2100, 2092, 2132, 2116, 2036, 2028, 2060, 2052, 2044, 1916, 1788, 1780, 1772, 1844, 1740, 1708, 1692, 1748, 1716, 1652, 1644, 1612, 1588, 1564, 1548, 1580, 1540, 1380, 1372, 1924, 1900, 1908, 1796, 1764, 1700, 1676, 1724, 1364, 1452, 2204, 2180, 2172, 2164, 2020, 2012, 1892, 1964, 1948, 1852, 1820, 1660, 1636, 1604, 1556, 1436, 1428, 1340, 1348, 1980, 1996, 1988, 1972, 1940, 1932, 1812, 1836, 1828, 1804, 1628, 1596, 1516, 1500, 1492, 1484, 1476, 1460, 1444, 1420, 1412, 1404, 1396, and 1388 were tested. More specifically, the exemplified guides were 20-mer guides as shown in Table 8.
To assess editing efficiencies, individual sgRNAs were subcloned into the pU6-sgRNA-Cbh-SpCas9-2A-EGFP vector, and transfected into HEK293T cells which contain 5 CTG repeats in the DMPK gene on both alleles. Genomic DNA was extracted 48 hr (for Lipofectamine 2000) or 72 hr (for Lipofectamine 3000) post transfection, and a 1174 bp sequence covering the CTG repeat expansion and the sgRNAs target sites was amplified by PCR. Sanger sequencing and TIDE analysis were then used to quantify the frequency of indels generated by each sgRNA. Results are shown from transfection with Lipofectamine 3000 for upstream guides (
9. CTG Repeat Excision of DMPK with and Without DNA-PK Inhibition
a. Materials and Methods
Preparation of DM1 myoblasts and myotubes. Healthy human myoblast (P01431-18F) and DM1 patient myoblast (03001-32F) were obtained from Cook myosite. Primary human myoblast were cultured in growth medium consisting of Myotonic™ Basal Medium (Cook myosite, MB-2222) plus MyoTonic™ Growth Supplement (Cook myosite, MS-3333). Myoblast differentiation was induced by changing culture medium to MYOTONIC DIFFERENTIATION MEDIA (Cook myoite, MD-5555). Myotubes were formed after changing to differentiation medium, and myotube samples were collected 7 days post differentiation induction. Primary human myoblasts were further purified with EasySep Human CD56 Positive Selection Kit II (StemCell Tech 17855) following manufacturer's protocol 3 days before Nucleofection and maintain in growth medium until nucleofection of RNPs.
sgRNA selection. 42 sgRNAs were selected from the DMPK 3′ UTR screen in HEK293 T cells (Example 8) for further evaluation in DM1 myoblasts. The sgRNAs were selected based on editing efficiency in HEK293 T cells, in silico off-target score, and coverage of regions flanking the CTG repeat region. Of the 42 sgRNAs, 22 upstream and 20 downstream sgRNAs were selected (Table 9).
Preparation of RNPs. RNPs containing Cas9 and sgRNA were prepared at a ratio of 1:6 (single-cut screen) and 1:3 (double-cut screen) Cas:sgRNA. For single-cut screening, RNP complexes were assembled with 30, 20 or 10 pmole of Cas9 and 180,120 or 60 pmole of sgRNA respectively in 10 uL of electroporation buffer. After incubation at room temperature for 20 minutes, 10 uL of this solution was mixed with 3×105 primary myoblasts in 10 uL nucleofection buffer. For Double-cut screen, RNP complexes were first assembled for individual sgRNA with 10 pmole Cas9 and 30 pmole sgRNA in 5 uL electroporation buffer. After incubation at room temperature for 20 minutes, two RNPs were mixed at 1:1 ratio and then with 2×105 primary myoblasts in 10 uL electroporation buffer, so that final RNPs in each reaction contained 20 pmole cas9 +30 pmole sgRNA1 +30 pmole sgRNA2.
Delivery of RNPs to DM1 myoblasts. DM1 myoblasts (Cook myosite 03001-32F; 3×105 cells per reaction for single-cut screen; 2×105 cells per reaction for double-cut screen) were nucleofected with Cas9/sgRNA RNPs. The Lonza Nucleofector 96-well shuttle system was used to deliver Cas9 (Aldevron) and chemically modified sgRNAs (Synthego). In the single-cut screen, three doses of Cas9 (10, 20, or 30 pmols) were evaluated. In the double-cut screen, 20 pmol Cas9 was used. Following electroporation, myoblasts from each well of nucleofection shuttle device were split into 6 identical wells of the 96-well cell culture plate. 24 hours post electroporation, fresh medium were changed. These myoblasts were cultured until 72 hours post electroporation at 37° C/5% CO2, and then harvested for DNA extraction and fluorescent in situ hybridization (FISH) staining, or induced for myotube differentiation by replacing the culture medium with MYOTONIC DIFFERENTIATION MEDIA (Cook myoite, MD-5555) for additional 7 days. DM1 myotubes were then fixed for FISH or harvest for RNA extraction.
PCR Amplification. On day 3 post nucleofection, genomic DNA of DM1 myoblasts was isolated and amplified as described in Example 8.
Sanger sequencing and TIDE analysis. PCR products were analyzed as described in Example 8.
PacBio sequencing. PacBio long read sequencing was used to investigate the impact of guide and DNA PK inhibitor treatment on Cas9 gene editing near the DMPK CTG repeat. Long read sequencing was chosen over Illumina short read sequencing (<<300NT reads) to capture the full complexity of edits in our -1.2 kb amplicons. Gene specific primers CGCTAGGAAGCAGCCAATGA (SEQ ID NO: 53374) and TAGCTCCTCCCAGACCTTCG (SEQ ID NO: 53375), which amplify a 1219 NT amplicon centered on the CTG repeat of the DMPK gene, were appended with PacBio specific 16 NT indexes. The final format for the forward and reverse primers was /5Phos/GGGT(16NT_index) CGCTAGGAAGCAGCCAATGA (SEQ ID NO: 53376) and /5Phos/CAGT(16NT index) TAGCTCCTCCCAGACCTTCG (SEQ ID NO: 53377). The 5′ phosphorylation promotes ligation of the SMRTBell adaptor and the GGGT or CAGT bases added to the forward or reverse primers help to normalize ligation efficiency as well as to facilitate demultiplexing.
To generate the PacBio libraries, WT or DM1 cells were treated with guide and/or compound in 96 well plates. DNA was recovered using the DirectPCR Lysis Reagent (Viagen Bio, 301-C) according to the manufacturer's directions and frozen for future use. 2μl of this lysate was used in 25 μl PCR's with NEB's 2XQ5 PCR mix (New England Biolabs, M0491). Indexed primers were included at 250nM each. All primers and indexes used are shown below. A gradient was used to identify an optimal annealing temperature of 69° C. and a total of 30 cycles were used to generate sufficient amplicon for SMRTBell ligation while minimizing unnecessary amplification that could skew editing distributions. The cycling parameters used are below.
PCR's were diluted 1:10 in Molecular Biology grade water and run on an Agilent 4200 TapeStation (Agilent, G2991AA) using high sensitivity D5000 tapes (Agilent, 5067-5592). Prominent peaks 1200 nucleotides (NT) were detected as well as several smaller bands in some samples, indicative of deletions. Samples were pooled and purified with 2 sequential 0.7 X ratio AmpureXP beads steps (Beckman Coulter, A63880). Serial elution was performed with 100 μl and 25 μl TE according to the manufacture's protocol. Samples were ligated to SMRTBell adaptor and sequenced on a PacBio Sequel II (Fornax Biosciences) using an 8M SMRTCell for 10 hr data collection. Sequence demultiplexing, adapter removal and processing of subreads into circular consensus sequences were performed by Fornax Biosciences. PacBio barcode primers- Indexes (IDT Technologies) are shown in Table 9.
CGCGGCTAGGAAGCAGCCAATGA
TGCGAGCTCCTCCCAGACCTTCG
PacBio data was processed using the PacBio SMRT Tools command line program. Circular consensus sequences were called and demultiplexed using the ccs and lima tools, respectively. Then, reads were aligned to the amplicon using pbmm2 (a wrapper for mimimap2). For alignment, the RNA sequencing presets in pbmm2 were used, on the assumption that these settings would allow detection of large deletions more accurately (because RNA sequencing alignment is already set up to detect introns).
For quality control, all reads were removed that did not map to the reference amplicon with a mapping score (MAPQ) of at least 30. Reads that were less than 400 or more than 1500 base pairs long were also removed. In addition, reads that were split across multiple alignments, reads with more than 20 soft-clipped bases at the beginning or end of the alignment, and reads which were not within at least 10 bp of spanning the entire CIGAR string were removed.
The CIGAR strings were parsed to call all variants observed in each read. Short indels in homopolymer regions were flagged as likely to be spurious, as PacBio sequencing is known to have a relatively high error rate in such areas. Pileups were generated with the bedtools genomecov tool.
Droplet digital PCR (ddPCR). ddPCR primer and probe sequences were designed with Primer3Plus (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi). The Target primer/probe set was used to detect CTG repeat excision, and the Reference primer/probe set was used as a control to amplify a region located in Exon 1 of DMPK gene. The primer and probe sequences are listed in Table 10 below.
The 24 uL of ddPCR reaction consisted of 12 μL of Supermix for Probes (no dUTP) (Bio-Rad Laboratories), 1 μL of reference primers mix (21.6 μM), 1 μL of reference probe (6 μM), 1 IaL of target primers mix (21.6 μM), 1 μL of target probe (6 μM), and 8 μL of sample genomic DNA. Droplets were generated using probe oil with the QX200 Droplet Generator (Bio-Rad Laboratories). Droplets were transferred to a 96-well PCR plate, sealed and cycled in a C1000 deep well Thermocycler (Bio-Rad Laboratories) under the following cycling protocol: 95° C. for 10 min, followed by 40 cycles of 94° C. for 30 seconds (denaturation) and 58° C. for 1 min (annealing) followed by post-cycling steps of 98° C. for 10 min (enzyme inactivation) and an infinite 4° C. hold. The cycled plate was then transferred and read in the FAM and HEX channels using the Bio-Rad QX200 Droplet Reader run on a C1000 Thermal Cycler with a deep-well block (Bio-Rad Laboratories). All ddPCR reactions were run under the following thermal cycling conditions: 1) 95 ° C. for 10 min; 2) 94 ° C. for 30 sec; 3) 58 ° C. for 1 min; 4) steps 2 and 3 repeated 39 times; 5) 98 ° C. for 10 min. ddPCR analysis was performed by the Bio-Rad QuantaSoft Pro Software.
Fluorescence In Situ Hybridization (FISH).
MBNL1/(CUG)n foci imaging was used as an orthogonal method to evaluate CTG repeat excision with DMPK sgRNAs in DM1 myoblasts. Myogenin antibody were used to identify myonuclei in the myotubes differentiated from myoblasts.
Cells were fixed for 15 min at RT with 4% PFA and washed 5 times for 10 min each in lx PBS at RT. Cells were stored at 4° C. if not probed immediately.
For the FISH procedure, cells were permeabilized with 0.5% triton X-100, in 1× PBS at RT for 5 min.
Cells were prewashed with 30% formamide, 2x SSC for 10 min at RT. Cells were then probed for 15 minutes at 80° C., with a 1 ng/μL of Cy3-PNA(CAG)5 probe (PNA Bio, F5001) in 30% formamide, 2× SSC, 2 μg/mL BSA, 66 μg/mL yeast tRNA, 2 mM vanadyl complex.
Cells were then washed for 30 min in 30% formamide, 2x SSC at 42° C., and then in 30% formamide, 2× SSC for 30 min at 37° C., then in 1× SSC for 10 min at RT, and last in 1× PBS for 10 min at RT. Cells were next probed overnight, at 4° C. with anti-MBNL1 antibody (1:1000 dilution, Santacruz, 3A4) anti-Myogenin antibody (1:500 dilution, Abcam-only for Myotube samples) in lx PBS +1% BSA. Cells were washed 2 times for 10 min each at RT with 1× PBS. Cells were incubated with goat anti-rabbit Alexa 647 and goat anti-rabbit Alexa 488 (only for Myotubes) in 1× PBS +1% BSA (1:500 dilution) for 1 hour at RT. Cells were washed 2 times, for 10 min each at RT with lx PBS. Cells were stained with Hoechst solution (0.1 mg/ml) for 5 min, and then washed with 1× PBS once for 5 min.
PBS was aspirated and fresh PBS (100 p.1) was added per well. Imaging plates were sealed with adhesive aluminum foils and imaged using MetaXpress (Molecular Devices).
RNA Extraction and uRT-PCR. Mis-splicing correction was used as a functional readout of CTG repeat excision by pairs of sgRNAs in DM1 myotubes. RNA was extracted with TaqMan® Gene Expression Cells-to-CTTM Kit (Thermal Fisher, AM1728) according to manufacturer's protocol and analyzed by qRT-PCR as described in Example 1.
Primer sequences are listed in the Table of Additional Sequences.
b. Screening of sgRNAs for Editing Efficiency of DMPK in DM1 Myoblasts
Forty two sgRNAs flanking the CTG repeat expansion of the DMPK gene were selected for editing the CTG repeat expansion. Among these 42 sgRNAs, 22 were located upstream of the CTG repeat expansion (between the stop codon and the CTG repeat expansion) and 20 were located downstream of the CTG repeat expansion (between the CTG repeat expansion and the end of the last exon of DMPK or are partially overlapping the CTG repeat expansion).
gRNA comprising the 18-mer spacer sequence of SEQ ID NOs: 3332, 3916, 3420, 3748, 3780, 3396, 4028, 3692, 3796, 3388, 3940, 3684, 3820, 3660, 3724, 3804, 3860, 3516, 3772, 3372, 3356, 4012, 2204, 1708, 2212, 2172, 1780, 2260, 2116, 2180, 1644, 1740, 1748, 2324, 1772, 1540, 2516, 2460, 2196, 2596, 2164, or 2620 were tested. More specifically, the tested guides were the exemplified 20-mer guides as shown in Table 11.
To assess editing efficiencies, individual sgRNAs were prepared as RNPs with spCas9 and delivered to DM1 myoblasts. Genomic DNA was isolated from the cells and amplified by PCR. Sanger sequencing and TIDE analysis were used to quantify the frequency of indels generated by each sgRNA. Results are shown for upstream and downstream guides at three concentrations spCas9 (10, 20, or 30 pmols) as % editing efficiency by TIDE analysis (
The editing efficiencies in DM1 myoblasts were compared to those obtained in HEK293T cells using a Spearman correlation (see Example 8 for HEK293 T cell data used in the analysis).
To visualize the editing efficiencies of individual sgRNAs targeting the 3′ UTR of DMPK, the PCR products from the genomic DNA of treated DM1 myoblasts were separated by DNA gel electrophoresis (
Based on the TIDE scores in DM1 myoblasts (e.g., >30% editing efficiency, Table 11), 15 upstream sgRNAs (DMPK-U57, DMPK-U10, DMPK-U54, DMPK-U26, DMPK-U27, DMPK-U55, DMPK-U6, DMPK-U32, DMPK-U22, DMPK-U56, DMPK-U14, DMPK-U67, DMPK-U20, DMPK-U34, DMPK-U30) and 11 downstream sgRNAs (DMPK-D87, DMPK-D63, DMPK-D42, DMPK-D89, DMPK-D59, DMPK-D34, DMPK-D51, DMPK-D88, DMPK-D68, DMPK-D62, DMPK-D35) were identified for screening as pairs in DM1 myoblasts.
c. CTG repeat excision of DMPK with exemplary guide pairs in DM1 myoblasts
Pairs of sgRNAs were selected and tested for efficiency of CTG repeat excision in DM1 myoblasts, including 3 upstream sgRNAs (SEQ ID NOs: 3778, 3386, 3354) and 3 downstream sgRNAs (SEQ ID NOs: 2514, 2258, 2210). Each sgRNA was tested individually, and the following sgRNAs were tested as pairs (SEQ ID NOs: 3778 and 2258 (pair 1); 3778 and 2210 (pair 2); 3386 and 2258 (pair 3); 3386 and 2210 (pair 4); 3354 and 2514 (pair 5)).
To assess CTG repeat excision efficiencies, pairs of sgRNAs were prepared as RNPs with spCas9 (20 pmol) and delivered to DM1 myoblasts by nucleofection. CTG repeat excision was evaluated by PCR of the wildtype allele (schematic in
CTG repeat excision was further measured using a loss-of-signal ddPCR assay (schematic in
CTG repeat excision was further evaluated by measuring the reduction of (CUG). RNA foci by FISH following treatment with sgRNA pairs or individual sgRNAs in DM1 myoblasts (
The accumulation of CUG repeat RNA can disrupt the function of proteins that normally regulate splicing, resulting in expression of mis-spliced mRNA products of other genes. The effect of CTG repeat excision in DMPK on splicing of other genes was evaluated in DM1 myotubes using the sgRNA pair (SEQ ID NO: 3386/2210). Results showed showing partial restoration of RNA splicing in BIN1 (
d. CTG Repeat Excision of DMPK in DM1 Myoblasts with DNA-PK Inhibition
Individual guide RNAs from the screen for editing efficiency in DM1 myoblasts were further analyzed for CTG repeat excision with and without DNA-PK inhibition. Specifically, DM1 myoblasts were treated with RNPs containing spCas9 and guide RNAs (DMPK-U10 (SEQ ID NO: 3914), DMPK-U40 (SEQ ID NO: 3514), DMPK-D59 (SEQ ID NO: 1778), DMPK-D13 (SEQ ID NO: 2458), DMPK-U16 (SEQ ID NO: 3858), DMPK-U54 (SEQ ID NO: 3418), DMPK-D63 (SEQ ID NO: 1706), or DMPK-D34 (SEQ ID NO: 2258)) with 304 Compound 6 or DMSO. Samples were processed by PCR and TapeStation electrophoresis. More prominent bands in Compound 6 treated samples indicate enhanced excision rates compared to the DMSO control (
Mis-splicing correction was also evaluated in DM1 myoblasts after dual gRNA CTG repeat excision with and without DNA-PK inhibition. DM1 myoblasts were treated with RNPs containing spCas9 and guide RNAs (SEQ ID NO: 3330 also referred to as DMPK-U57 and GDG_Cas9_Dmpk3; and SEQ ID NO: 2554 also referred to as DMPK-D03 and GDG_Cas9_Dmpk_6), with or without 3μM Compound 6. Mis-splicing correction was evaluated for genes GFTP1, BIN1, MBNL2, DMD, NFIX, GOLGA4, and KIF13A in cells treated with the pair of gRNAs (
e. Dose Response of DNA-PK Inhibitor with Exemplary Guide Pairs
The dose response of DNA-PK inhibition on CTG repeat excision of DMPK was evaluated in DM1 patient fibroblasts (cells described above in Example 1). Cells were treated with RNPs containing spCas9 and guide pairs (SEQ ID NO: 3330 (GDG_DMPK3) and SEQ ID NO: 2506 (CRISPR-3); or SEQ ID NO: 3330 (GDG_DMPK3) and SEQ ID NO: 2546 (CRISPR-4)) and an increasing dose of Compound 6 (30nM, 300nM, 3 μM, and 10 μM), or DMSO. A stronger band corresponding to the excised product was observed for both pairs with increasing dose of DNA-PKi (
f. CTG repeat excision of DMPK with SaCas9 and with a DNA-PK inhibitor
Single guide excision was evaluated in DM1 patient fibroblasts (cells described above in Example 1) with and without DNA-PK inhibitor (Compound 6) using saCas9. Cells were treated with RNPs containing saCas9 and individual guides (
g. Screening of CTG repeat excision with individual sgRNAs with DNA-PK inhibition
A screen of the 42 individual SpCas9 sgRNAs targeting the 3′ UTR of DMPK (Table 11) was performed in DM1 myoblasts with DMSO or 3 uM Compound 6. After electroporation cells were incubated with DMSO or 3 uM Compound 6 for 24 hours.
h. Screening of CTG repeat excision with guide pairs with DNA-PK inhibition
A screen of all pairwise combinations of the 42 SpCas9 sgRNAs targeting the 3′ UTR of DMPK gene (Table 11, 22 sgRNAs upstream of the CTG repeat and 20 downstream) was performed in DM1 patient fibroblasts (cells described above in Example 1). After electroporation with RNPs pre-loaded with each guide pair cells were incubated with DMSO or 3 uM Compound 6 for 24 hours.
10. Screen of Individual Frataxin sgRNAs
a. Materials and Methods
sgRNA Selection. A selected region containing the GAA repeat within intron 1 of the FXN gene was scanned for NGG SpCas9 protospacer adjacent motif (PAM) on either sense (+1) or antisense strand (−1), and guide sequences were generated based on the 20-nucleotide sgRNA spacer sequences adjacent to the PAMs. 218 sgRNAs were identified within the region upstream of the GAA repeat (chr9: 69 035 950-69 037 295), and 173 sgRNAs within the region downstream of the GAA repeat (chr9: 69 037 307-69 038 600) (Table 13). Computational off target prediction using an in-house algorithm was performed for each sgRNA in both upstream and downstream regions. Of the total 391 sgRNAs, a subset of 96 sgRNAs was selected to move forward into a screen evaluating editing efficacy in two patient cell lines of long repeat length and at two RNP (ribonucleoprotein) complex concentrations (see
The selection criteria included high editing efficacy across the conditions tested, genomic location and the presence of SNPs (single nucleotide polymorphisms).
Electroporation of RNP Complexes into FA Patient Cells. The Lonza Nucleofector 96-well shuttle system was used to deliver Cas9 (Aldevron) and chemically modified sgRNAs (Synthego) into two cell lines, derived from two patients with long GAA repeats: GM14518 (a lymphoblastoid cell line) and GM03665 (a fibroblast cell line) (Coriell Institute). RNP complexes were first assembled, comprising 36 pmol of Cas9 and 108 pmol sgRNA, in a volume of 12 uL of electroporation buffer. After incubation at room temperature for 30 minutes, this solution was mixed with cells in two dilutions, such that for each cell line two concentrations of RNPs were delivered: one with 15 pmol Cas9 +45 pmol sgRNA (“High”) and another with 7.5 pmol Cas9 +22.5 pmol sgRNA (“Low”). Following electroporation, cells were cultured for 72 hours at 37° C/5% CO2, and then harvested for DNA extraction.
Sanger sequencing and ICE analysis. The relevant loci for each guide were amplified by PCR and the products were sent to GeneWiz for Sanger sequencing. Due to the length and complexity of the locus being analyzed, the sequencing primer was customized for each sgRNA. The primer sequences used for amplification and sequencing of the appropriate locus are shown in the Table of Additional Sequences (SEQ ID NOs: 36-54). Indel values were estimated using ICE (inference of CRISPR edits) analysis algorithm (Synthego). ICE analysis is a method that quantifies the identity and prevalence of indels using Sanger sequencing data (Hsiau, T. et al. (2018) bioRxiv haps://www.biorxiv.org/content/10.1101/251082v3).
b. Results of Single-Cut Screen of Frataxin sgRNAs
A set encompassing 96 sgRNAs flanking the GAA repeat of the FXN gene was selected for editing efficacy evaluation. Among these, 56 sgRNAs were located upstream of the GAA repeat and 40 sgRNAs were positioned downstream of the GAA repeat. To evaluate editing efficacy, RNP complexes containing a chemically modified sgRNA and Cas9 protein were delivered to patient cell lines by nucleofection. Two RNP concentrations were used to obtain a comprehensive overview of editing efficiencies and differentiate the leading sgRNAs with highest cutting efficacy. Additionally, the consistency of indel efficacy between different cell types/donors was assessed for each sgRNA. These cell types consisted of patient lymphoblasts and fibroblasts of long repeat length.
11. GAA Repeat Excision at the Frataxin Locus of FXN in Cardiomyocytes with DNA-PK Inhibition
FA post-mitotic cardiomyocytes were prepared from a culture of iPSCs as described in Example 1.
Cells were treated with spCas9 and a guide pair flanking the GAA repeat (SEQ ID NOs 52666 and 26562) and Compound 6 (3ttM) for 24 hours or DMSO. The rate of repeat excision was evaluated on day 7 and day 14 by ddPCR assay (
12. GAA Repeat Excision at the Frataxin Locus of FXN in FA iPSCs
GAA repeat excision was evaluated with Cpf1 (Cas12a) and SpCas9 in wildtype (WT) and FA iPSCs (4670) using RNP electroporation. DNA gel-electrophoresis showed excised DNA bands after GAA repeat excision with Cpf1 (boxes,
13. GAA Repeat Excision at the Frataxin Locus of FXN in Cortical Neurons with Cpf1
Additional Cpf1 guide pairs were selected for GAA repeat excision in iPSC-derived cortical neurons as shown in Table 15 below.
gRNAs comprising the 18-mer spacer sequences of SEQ ID NOs: 47045, 7445, 7461, 46766, 7678, and 47030 were tested. More specifically, the tested guides were the tested 20-mer guides as shown in Table 15.
Pairs of gRNAs were tested with Cpf1 (Cas12a) in the iPSC-derived cortical neurons. The following guide pairs were used: Guides 1&2 (SEQ ID NOs: 47047 and 7447); Guides 3&4 (SEQ ID NOs: 7463 and 46967); Guides 5&6 (SEQ ID NOs: 46768 and 7680); Guides 7&2 (SEQ ID NOs: 47032 and 7447). DNA gel electrophoresis of PCR products showed excised DNA bands after GAA repeat excision (
GAA repeat excision was further confirmed in single cell nuclei of wildtype iPSC-derived cortical neurons using Cpf1 and gRNAs (SEQ ID NOs 47047 and 7447). Cell nuclei were prepared using the Nuclei Isolation Kit: Nuclei EZ prep (Sigma, NUC101) according to the manufacture's protocol. For nuclei isolation from mouse brain, tissue samples were dounced 2×25x in 2 ml lysis buffer with pestle A and pestle B (Sigma), respectively. Lysate was then transferred into a lml falcon tube on ice for 5min. Lysate was spin down at 500 ×g for 5min and pellet was resuspended in lml lysis buffer, additional 3 ml lysis buffer were added and kept on ice for 5min. Lysate was spin down at 500 ×g for 5min and pellet was resuspended in lml resuspension buffer. Vybrant DyeCycle Ruby Stain (Thermo Fisher, V10309, 1:800) or Hoechst (Invitrogen, H3570, 1:10,000) was added for fluorescent labeling of nuclei. Isolated nuclei were then sorted using a BD FACSMelody Cell Sorter (BD Biosciences) into QuickExtract DNA Extraction Solution (Lucigen, QE9050). Sequencing results showed 8/10 nuclei with a homogenous GAA repeat excision and 2/10 nuclei had a heterogenous GAA excision.
14. In Vivo GAA repeat excision at the frataxin locus of FXN in adult mouse brain
An AAV vector was designed for targeting neurons in adult YG8+/− mice (
A dual guide excision experiment was performed with AsCpf1 (Cas12a) in a mouse model of Friedreich's Ataxis with dual AAV delivery (1:1 ratio) into stratum of adult YG8+/− mice.
Heterozygous adult male FXNem2.1LutzyTg(FXN)YG8Pook/J mice (Jackson laboratory, 030930) were anesthetized and craniotomy was performed according to IACUC approved procedures. lul of mixed AAV (1:1) were injected into striatum (0.5mm Bregma, 1.5 mm lateral, 2.5mm deep). To prevent leakage, the pipette was held in place for 3min before retraction. The incision was sutured and post-operative analgesics were administered and mice were euthanized 2 weeks after AAV injection according to IACUC approved protocols and AVMA Guidelines for Euthanasia of Animals. Brain samples were fixed in 4% PFA for vibratome sectioning and fluorescent imaging of mCherry-KASH labeled striatal neurons. For nuclei isolation and FACS, striatum was dissected and shock frozen. Following AAV1 vectors have been used: a) hSyn-Cas12a and b) Cas12a sgRNA (Sap1) hSyn_mCh-KASH (SignaGen, ˜2.5×10^6 Vg/ml) (see Table 16 below and SEQ ID NOs 53411 and 53412, respectively).
All AAV constructs were synthesized by Genescript. Cas12a and gRNA array sequences have been published elsewhere (Zetsche et al., Nat Biotech, 2017). gRNA array DNA oligos were cloned using one-directional annealing and using a sticky-end design and Sapl restriction of the Cas12a sgRNA vector as described elsewhere (Zetsche et al., Nat Biotech, 2017).
The following fw oligo for cloning the dual Cas12a sgRNA array has been used:
agaTACCATGTTGGCCAGGTTAGTCTAATTTCTACTCTTGTAGATCCA
GCATCTCTGGAAAAATAG (SEQ ID NO: 53410) and
Bold: spacers, aga: SapI cloning overhang).
Results showed successful excision of the GAA repeat in neurons in vivo with dual Cas12a sgRNAs. Histology of the brain 2 weeks after stereotactic injection showed mCherry positive striatum (
15. CTG Repeat Excision with Guide Pairs in DMPK
a. Materials and Methods
Guide and Primer sequences. Primer sequences are shown in the Table of Additional Sequences (SEQ ID NOs: 55-62). The crRNA and tracrRNA used for gRNAs with SpCas9 was GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAA AGUGGCACCGAGUCGGUGCUUUU (SEQ ID NO: 98). The crRNA and tracrRNA used for gRNAs with SaCas9 was GUUUUAGUACUCUGGAAACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCU CGUCAACUUGUUGGCGAGAU (SEQ ID NO: 97).
Preparation and Transfection of SpCas9-Expressing HEK293 T Cells. A cell line stably expressing the CRISPR Cas9 nuclease was purchased from Genecopoeia. Cas9 is integrated at the human AAVS1 Safe Harbor locus (also known as PPP1R2C). This cell line also expresses copGFP and the puromycin resistance gene. In combination with separately transfected or transduced single guide RNAs (sgRNAs), this cell line will sustain double-strand DNA breaks (DSBs) at targeted genome sites. Cas9 expressing HEK 293 T cells were transfected with individual IVT gRNAs using MessengerMax lipofectamine-based delivery. Genomic DNA was isolated from the cells and amplified by PCR. Sanger sequencing and TIDE analysis were used to quantify the frequency of indels generated by each sgRNA.
Preparation and Electroporation of DM1 iPSC Cell Lines. SBI Cell Line: Cells were isolated from peripheral blood mononuclear cells from an adult female DM1 patient (source of cells from Nicholas E. Johnson (Virginia Commonwealth University)) and reprogrammed with the CytoTune®-iPS Sendai reprogramming kit. Individual iPSC clones were isolated, including clone SB1. The SB1 cell line had a pluripotency signature consistent with an iPSC cell line by Nanostring assay. High resolution aCGH karyotyping revealed no gross genomic abnormalities. Southern analysis confirmed that the SB1 cell line contains a pathogenic CTG repeat expansion (˜300 CTG repeats) (
4033-4 Cell Line: A parent fibroblast line derived from an adult DM1 male (GM04033, Coriell Institute) was reprogrammed using CytoTune®-iPS 2.0 Sendai Reprogramming Kit. Individual iPSC clones were isolated, including clone 4033-4. Southern blot analysis confirmed that the 4033-4 cell line contains a pathogenic CTG repeat expansion (3000 CTG repeats).
Electroporation of DM1 iPSC cells: DM1 iPSC cells (200,000 per reaction) were mixed with RNPs prepared as follows.
Broadly, RNP complexes for experiments corresponding to
RNP complexes for experiments corresponding to
Cells were electroporated with a Lonza Nucleofector (CA-137 setting) and harvested 72 hours post electroporation. Genomic DNA was isolated and used as template for subsequent PCR for TIDE analysis and ddPCR deletion analysis.
Sequencing and TIDE Analysis. PCR was Performed on Genomic DNA as Follows.
PCR Sample:
PCR Conditions:
PCR products were cleaned up using AMPure bead-based PCR purification (Beckman Coulter). The AMPure bead bottle was vortexed and aliquoted into a falcon tube. Following incubation for 30 minutes at room temperature, 85 μL of beads were added to each well of PCR products, pipetted up and down 10 times and incubated for 10 minutes. The bead mixture was then placed on a magnet for 5 minutes. Liquid was aspirated, and beads were washed twice with 70% EtOH while keeping the plate on the magnet. The plate was then removed from the magnet and 20 μL of dH2O was added to the beads and pipetted up and down to mix. Following incubation for 5-10 minutes, the plate was placed on the magnet for 1 minute. The dH2O containing the DNA was removed and PCR concentrations were analyzed on by nanodrop.
PCR products were sent for sequenced using Forward Primer (SEQ ID NO: 57) and Reverse Primer (SEQ ID NO: 58). Indel values were estimated using the TIDE analysis algorithm. TIDE is a method based on the recovery of indels' spectrum from the sequencing electrophoretograms to quantify the proportion of template-mediated editing events (Brinkman, E A et al. (2014) Nucleic Acids Res. 42: e168; PMID: 25300484).
Two Loss-of-Signal (LOS) Droplet Digital PCR (ddPCR) Assay. The loss-of-signal ddPCR assay amplifies a region of the 3′ UTR of DMPK that is 5′ of the CTG repeat region or 3′ of the CTG region and detects the loss-of-signal of a probe targeting the amplified region as a result of successful deletion of the CTG repeat region (see
For the 5′ LOS ddPCR assay, Forward Primer (SEQ ID NO: 59), Reverse Primer (SEQ ID NO: 60), and Probe (SEQ ID NO: 61) were used.
For the 3′ LOS ddPCR assay, Forward Primer (SEQ ID NO: 62), Reverse Primer (SEQ ID NO: 63), and Probe (SEQ ID NO: 64) were used.
The ddPCR samples were setup at room temperature. DNA samples were diluted to a concentration of 10-20 ng/μL Diluted DNA (4 μL) was added to 21 μL of ddPCR mix.
dd PCR mix:
The plate was sealed with a heat seal and mixed by vortexing, and then centrifuged briefly. The final volume was 25 μL.
The samples were transferred to a 96 well plate for auto digital generation. Droplets (40 laL) were generated and the plate was transferred to the PCR machine.
A three-step cycling protocol was run:
The reference gene used for 5′ and 3′ loss-of-signal (LOS) ddPCRs was RPP30.
Differentiation Protocol for DM1 Cardiomyocytes. DM1 cardiomyocytes were prepared from the DM1 iSPC cell line SB1. Cells were activated with Wnt (4 l uM CHIR) for 2 days, followed by Wnt inactivation (4 μM WNT-059) for 2 days. Cells were rested for a recovery period in CDM3 media for 6 days. Cells were then transferred to CDM3-no glucose media for metabolic selection for 1 day.
DM1 cardiomyocytes (250,000 per reaction) were mixed with RNPs prepared as follows. Individual chemically synthesized guide RNAs were diluted to 1.5 μg/μl and Cas9 nucleases were diluted to 3 μg/μl and 1 μl of each component was combined together and complexed together for a minimum of 10 minutes at room temperature.
RNP complexes for experiments corresponding to
Cells were electroporated a with Lonza Nucleofector (CA-137 setting) and incubated in iCell Maintenance Media. Cells were harvested 72 hours post electroporation. Genomic DNA was isolated and used as template for subsequent PCR for TIDE analysis and ddPCR deletion analysis.
Off-Target Analysis and Hybrid Capture Assay. Homology-dependent off-target site nomination. Off-target sites were computationally predicted for each sgRNA based on sequence similarity to the hg38 human reference genome and the presence of a protospacer adjacent motif (PAM) sequence using three prediction algorithms; CCTop, CRISPOR and COSMID. CCTop and CRISPOR were used to nominate potential off-target sites with up to 3 mismatches relative to the sgRNA sequence. The COSMID algorithm can nominate off-targets sites with gaps and was used to nominate potential off-target sites with up to 3 mismatches with no gaps or up to 2 mismatches with 1 gap relative to the sgRNA sequence. All three algorithms nominated potential off-target sites with the optimal SpCas9 NGG PAM. Alternate PAMs were also included in the search using COSMID (NAG) and CCTop (NAG, NGA, NAA, NCG, NGC, NTG, and NGT). Predicted off-target sites were filtered to exclude sites overlapping low-complexity regions since these regions are subject to promiscuous probe enrichment and sequencing errors that result in incorrect read mapping and indel calling. A total of 577 potential off-target sites were nominated across the 12 sgRNAs (SEQ ID NOs: 3778, 4026, 3794, 4010, 3906, 3746, 1778, 1746, 1770, 1586, 1914, and 2210).
Hybrid capture probe library design. Percent editing at the on-target site and off-target sites were measured using a hybrid capture assay. Hybrid capture probes were generated to enrich regions of the genome containing the on-target sites and predicted off-targets. For each site, 100 bp flanking region was added both upstream and downstream of the site, and then 120 bp probes were tiled across the site including both flanking regions. Multiple probes were designed per site for all predicted off-target sites as well as on-target sites. Hybrid capture probes from all 12 sgRNAs were pooled together and one Agilent SureSelect probe set was ordered. The total target region of the hybrid capture library was 124.85 kilobases.
Generation of edited and control samples. Hybrid capture assay samples were generated by electroporating two WT donor iPSC lines (1000,000 cells per reaction) with RNPs prepared by assembling 10 gg sgRNA and 10 gg of the SpCas9 nuclease. Cells were electroporated with a Lonza Nucleofector (CA-137 setting) and harvested 72 hours post electroporation. Samples were generated for 12 sgRNAs (SEQ ID NOs: 3778, 4026, 3794, 4010, 3906, 3746, 1778, 1746, 1770, 1586, 1914, and 2210). Control samples electroporated with only 10 gg of the SpCas9 nuclease were also generated. Genomic DNA was isolated (QlAamp UCP Micro Kit) for hybrid capture followed by sequencing. Only one donor was available for the sgRNA SEQ ID NO: 2210.
Hybrid capture library preparation. Hybrid capture enrichment of on-target and off-target regions using hybrid capture probes was performed as per sample preparation described for 200 ng input genomic DNA samples in the Agilent SureSelectXT HS manufacturer's protocol (Agilent Technologies, Santa Clara, Calif., USA).). Briefly, the genomic DNA was fragmented by acoustic shearing with a Covaris LE220 instrument. DNA fragments were end repaired and then adenylated at the 3′ ends. 5′ and 3′ specific adapters were ligated to the DNA fragments, and adapter-ligated DNA fragments were amplified and indexed with indexing primers. Adapter-ligated DNA fragments were validated using the Agilent D1000 ScreenTape assay on the Agilent 4200 TapeStation, and quantified using a Qubit 3.0 Fluorometer with the Qubit dsDNA BR Assay Kit. 1000 ng adapter-ligated DNA fragments were hybridized with biotinylated RNA baits using a pre-programmed thermocycler for 1.5 hours following the manufacturing recommendations. The hybridized DNAs were captured by streptavidin-coated magnetic beads (Dynabeads MyOne Streptavidin T1). After extensive washes, the captured DNA fragments were enriched with limited cycle PCR. Post-captured DNA libraries were validated using the Agilent High Sensitivity D1000 ScreenTape assay on the Agilent 4200 Tape Station and quantified using Qubit 3.0 Fluorometer with the Qubit dsDNA HS Assay Kit. The libraries were subpooled at a concentration of 50 ng/library, with 4-5 libraries per subpool. The subpools were diluted 1:10 in 10 mM Tris-HCl pH 8.0 and quantitated by qPCR using the KAPA Library Quantification kit-Universal. The subpools were normalized to 4 nM and combined equally to create the final sequencing pool.
Hybrid capture library sequencing and analysis. The final sequencing pool was loaded onto the Illumina NextSeq machine (Illumina, San Diego, Calif., USA) at a final concentration of 1.8 μM with 5% PhiX spiked in and sequenced using a Illumina high output v2.5 reagent kit with the following configuration: 150×8×8×150 to achieve 3000X coverage.
Illumina basecalls were converted to FASTQ format and de-multiplexed by sample-specific barcode using bcl2fastq Conversation Software. Sequencing data was aligned with the BWA MEM algorithm using default parameters to human genome build hg38. De-duplication of the aligned reads was completed with SAMtools. For each on-target site and predicted off-target site, primary read alignments that covered the site and an additional 20 bases on each end were considered for indel quantification. The sum of all reads containing indels within 10 bp of the potential SpCas9 cleavage site was divided by the total number of reads aligned to the cleavage site that passed the filtering criterion, giving the indel frequency at that candidate cut site. Sites with at least 0.2% indel frequency difference between at least one pair of edited and control samples were subject to statistical testing to identify sites that may show significant CRISPR/Cas9 editing. For such sites, a one- tailed paired Student's t-test was performed to test for significantly more editing in edited samples relative to controls. If the test result was significant with P <0.05, the site was considered a confirmed off-target. Since only two donors were available for 11 sgRNA and only one donor was available for the 12th sgRNA (SEQ ID NO: 2210), sites that failed the statistical test were manually inspected and if necessary annotated as “potential off-target sites”, and can be further investigated with more donors and higher sequencing depth.
Hybrid capture assay samples were prepared as shown below.
b. Screening of gRNAs in HEK293 T Cells with SpCas9
To assess editing efficiency of individual gRNAs, 169 gRNAs flanking the CTG repeat region of the DMPK gene were selected for screening in HEK293 T cells expressing SpCas9. Cells were transfected with individual gRNAs using lipofectamine-based delivery. Genomic DNA was isolated from the cells and amplified by PCR. Sanger sequencing and TIDE analysis were used to quantify the frequency of indels generated by each sgRNA. Results are shown as % editing efficiency by TIDE analysis (Table 17).
c. Screening of gRNAs in DM1 iPSC Cell Lines with SpCas9
Guide RNAs were selected for screening in two DM1 iPSC cell lines (SB1 and 4033-4). Both cell lines contain a pathogenic CTG repeat region.
Six upstream gRNAs (5′ side of the CTG repeat region) (SEQ ID NOs: 3778, 4026, 3794, 4010, 3906, and 3746) and six downstream gRNAs (3′ side of the CTG repeat region) (SEQ ID NOs: 1778, 1746, 1770, 1586, 1914, and 2210) (see
The same gRNAs were further evaluated for the ability to delete the CTG repeat region of the DMPK gene either alone or in pairs in SB1 cells. Thirty six pair combinations were evaluated for CTG repeat region deletion. A two loss-of-signal ddPCR assay was used to detect repeat deletion (see
Guide RNAs were selected for further testing with SpCas9 in another DM1 iPSC cell line (4033-4). Five upstream gRNAs (SEQ ID NOs: 3778, 4026, 3794, 3906, and 3746) and five downstream gRNAs (SEQ ID NOs: 1778, 1746, 1770, 1586, and 2210) were selected (see
Similar CTG repeat deletion was observed between the DM1 iPSC cell line SB1 (
d. Screening of gRNA Pairs in DM1 Cardiomyocytes with SpCas9
Guide RNAs were selected for further testing in DM1 cardiomyocytes with SpCas9. Five upstream gRNAs (SEQ ID NOs: 3778, 4026, 3794, 3906, and 3746) and five downstream gRNAs (SEQ ID NOs: 1778, 1746, 1770, 1586, and 2210) of the CTG repeat in the 3′ UTR of DMPK (see
Three pairs of gRNAs (SEQ ID NOs: 3746 and 2210; 4026 and 1586; 3778 and 1778) were tested for CTG repeat deletion in DM1 cardiomyocytes and showed similar % deletion as obtained with DM1 iPSC SB1 cells by 5′ LOS ddPCR and 3′ LOS ddPCR (
e. Off-Target Analysis
Twelve guide RNAs were tested for off-target activity with SpCas9 using a hybrid capture assay (SEQ ID NOs: 3778, 4026, 3794, 4010, 3906, 3746, 1778, 1746, 1770, 1586, 1914, and 2210). Results of editing at on-target site and maximum off-target editing across sites and 2 donors are shown in Table 19:
Based on the off-target data, pairs of gRNAs identified as “clean,” “off-target <1%,” or “off-target >1%.” Multiple “clean” gRNAs pairs with SpCas9 were identified that also had greater than 25% CTG repeat deletion in SB1 cells (
f. Screening of gRNAs with SaCas9
Thirty upstream gRNAs and thirty downstream gRNAs of the CTG repeat in the 3′ UTR of DMPK were selected (see
Four upstream gRNAs (SEQ ID NOs: 3256, 2896, 3136, and 3224) and six downstream gRNAs (SEQ ID NOs: 4989, 560, 672, 976, 760, 984, and 616) were selected for evaluation of CTG repeat region deletion in DM1 iPSC SB1 cells with saCas9 (see
This description and exemplary embodiments should not be taken as limiting. For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing quantities, percentages, or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about,” to the extent they are not already so modified. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
It is noted that, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the,” and any singular use of any word, include plural referents unless expressly and unequivocally limited to one referent. As used herein, the term “include” and its grammatical variants are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items.
agaTACCATGTTGGCCAGGTTAGTCTAATTTCTACTCTT
Primers are indicated as forward or reverse primers using F and R, respectively. qPCR primers for amplifying a product specific for a given form of an mRNA have descriptions including text such as “Ex5in,” which indicates that the primers give product in the presence of exon 5 of the indicated mRNA. qPCR primers for amplifying a product from all expected forms of an mRNA have descriptions including “Total.”
This application is a continuation of International Application No, PCT/US2020/048000, filed Aug. 26, 2020; which claims the benefit of priority to U.S. Provisional Application No. 62/892,445, filed Aug. 27, 2019; U.S. Provisional Application No. 62/993,616, filed Mar. 23, 2020; and U.S. Provisional Application No. 63/067,489, filed Aug. 19, 2020; all of which are incorporated by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 62993616 | Mar 2020 | US | |
| 62892445 | Aug 2019 | US | |
| 63067489 | Aug 2020 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2020/048000 | Aug 2020 | US |
| Child | 17681138 | US |
