COMPOSITIONS COMPRISING AN RNA GUIDE TARGETING B2M AND USES THEREOF

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Oct. 29, 2021, is named 51451-014WO3_Sequence_Listing_10_29_21_ST25, and is 369,037 bytes in size.

BACKGROUND

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) genes, collectively known as CRISPR-Cas or CRISPR/Cas systems, are adaptive immune systems in archaea and bacteria that defend particular species against foreign genetic elements.

SUMMARY OF THE INVENTION

It is against the above background that the present invention provides certain advantages and advancements over the prior art. Although this invention disclosed herein is not limited to specific advantages or functionalities, the invention provides a composition comprising an RNA guide, wherein the RNA guide comprises (i) a spacer sequence that is substantially complementary to a target sequence within a B2M gene and (ii) a direct repeat sequence; wherein the target sequence is adjacent to a protospacer adjacent motif (PAM) comprising the sequence 5′-NTTN-3′.

In one aspect of the composition, the target sequence is within exon 1, exon 2, exon 3, exon 4, intron 1, intron 2, or intron 3 of the B2M gene.

In another aspect of the composition, the B2M gene comprises the sequence of SEQ ID NO: 773, the reverse complement of SEQ ID NO: 773, a variant of SEQ ID NO: 773, or the reverse complement of a variant of SEQ ID NO: 773.

In another aspect of the composition, the spacer sequence comprises: a. nucleotide 1 through nucleotide 16 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 391-770 or 1019-1218; b. nucleotide 1 through nucleotide 17 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 391-770 or 1019-1218; c. nucleotide 1 through nucleotide 18 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 391-770 or 1019-1218; d. nucleotide 1 through nucleotide 19 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 391-770 or 1019-1218; e. nucleotide 1 through nucleotide 20 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 391-770 or 1019-1218; f. nucleotide 1 through nucleotide 21 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 391-770; g. nucleotide 1 through nucleotide 22 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 391-552 and 554-770; h. nucleotide 1 through nucleotide 23 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 391-552 and 555-770; i. nucleotide 1 through nucleotide 24 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 391-552 and 556-770; j. nucleotide 1 through nucleotide 25 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 391-552 and 556-770; k. nucleotide 1 through nucleotide 26 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 391-552 and 556-770; 1. nucleotide 1 through nucleotide 27 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 391-552 and 556-770; m. nucleotide 1 through nucleotide 28 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 391-552 and 556-770; n. nucleotide 1 through nucleotide 29 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 391-552 and 556-770; or o. nucleotide 1 through nucleotide 30 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 391-540, 542-552, and 556-770.

In another aspect of the composition, the spacer sequence comprises: a. nucleotide 1 through nucleotide 16 of any one of SEQ ID NOs: 391-770 or 1019-1218; b. nucleotide 1 through nucleotide 17 of any one of SEQ ID NOs: 391-770 or 1019-1218; c. nucleotide 1 through nucleotide 18 of any one of SEQ ID NOs: 391-770 or 1019-1218; d. nucleotide 1 through nucleotide 19 of any one of SEQ ID NOs: 391-770 or 1019-1218; e. nucleotide 1 through nucleotide 20 of any one of SEQ ID NOs: 391-770 or 1019-1218; f. nucleotide 1 through nucleotide 21 of any one of SEQ ID NOs: 391-770; g. nucleotide 1 through nucleotide 22 of any one of SEQ ID NOs: 391-552 and 554-770; h. nucleotide 1 through nucleotide 23 of any one of SEQ ID NOs: 391-552 and 555-770; i. nucleotide 1 through nucleotide 24 of any one of SEQ ID NOs: 391-552 and 556-770; j. nucleotide 1 through nucleotide 25 of any one of SEQ ID NOs: 391-552 and 556-770; k. nucleotide 1 through nucleotide 26 of any one of SEQ ID NOs: 391-552 and 556-770; 1. nucleotide 1 through nucleotide 27 of any one of SEQ ID NOs: 391-552 and 556-770; m. nucleotide 1 through nucleotide 28 of any one of SEQ ID NOs: 391-552 and 556-770; n. nucleotide 1 through nucleotide 29 of any one of SEQ ID NOs: 391-552 and 556-770; or o. nucleotide 1 through nucleotide 30 of any one of SEQ ID NOs: 391-540, 542-552, and 556-770.

In another aspect of the composition, the direct repeat comprises: a. nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; b. nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; c. nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; d. nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; e. nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; f. nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; g. nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; h. nucleotide 8 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; i. nucleotide 9 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; j. nucleotide 10 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; k. nucleotide 11 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; 1. nucleotide 12 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; m. nucleotide 13 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; n. nucleotide 14 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; o. nucleotide 1 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; p. nucleotide 2 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; q. nucleotide 3 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; r. nucleotide 4 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; s. nucleotide 5 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; t. nucleotide 6 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; u. nucleotide 7 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; v. nucleotide 8 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; w. nucleotide 9 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; x. nucleotide 10 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; y. nucleotide 11 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; z. nucleotide 12 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; or aa. a sequence that is at least 90% identical to a sequence of SEQ ID NO: 10 or a portion thereof.

In another aspect of the composition, the direct repeat comprises: a. nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1-8; b. nucleotide 2 through nucleotide 36 of any one of SEQ ID NOs: 1-8; c. nucleotide 3 through nucleotide 36 of any one of SEQ ID NOs: 1-8; d. nucleotide 4 through nucleotide 36 of any one of SEQ ID NOs: 1-8; e. nucleotide 5 through nucleotide 36 of any one of SEQ ID NOs: 1-8; f. nucleotide 6 through nucleotide 36 of any one of SEQ ID NOs: 1-8; g. nucleotide 7 through nucleotide 36 of any one of SEQ ID NOs: 1-8; h. nucleotide 8 through nucleotide 36 of any one of SEQ ID NOs: 1-8; i. nucleotide 9 through nucleotide 36 of any one of SEQ ID NOs: 1-8; j. nucleotide 10 through nucleotide 36 of any one of SEQ ID NOs: 1-8; k. nucleotide 11 through nucleotide 36 of any one of SEQ ID NOs: 1-8; 1. nucleotide 12 through nucleotide 36 of any one of SEQ ID NOs: 1-8; m. nucleotide 13 through nucleotide 36 of any one of SEQ ID NOs: 1-8; n. nucleotide 14 through nucleotide 36 of any one of SEQ ID NOs: 1-8; o. nucleotide 1 through nucleotide 34 of SEQ ID NO: 9; p. nucleotide 2 through nucleotide 34 of SEQ ID NO: 9; q. nucleotide 3 through nucleotide 34 of SEQ ID NO: 9; r. nucleotide 4 through nucleotide 34 of SEQ ID NO: 9; s. nucleotide 5 through nucleotide 34 of SEQ ID NO: 9; t. nucleotide 6 through nucleotide 34 of SEQ ID NO: 9; u. nucleotide 7 through nucleotide 34 of SEQ ID NO: 9; v. nucleotide 8 through nucleotide 34 of SEQ ID NO: 9; w. nucleotide 9 through nucleotide 34 of SEQ ID NO: 9; x. nucleotide 10 through nucleotide 34 of SEQ ID NO: 9; y. nucleotide 11 through nucleotide 34 of SEQ ID NO: 9; z. nucleotide 12 through nucleotide 34 of SEQ ID NO: 9; or aa. SEQ ID NO: 10 or a portion thereof.

In another aspect of the composition, the direct repeat comprises: a. nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 788-805; b. nucleotide 2 through nucleotide 36 of any one of SEQ ID NOs: 788-805; c. nucleotide 3 through nucleotide 36 of any one of SEQ ID NOs: 788-805; d. nucleotide 4 through nucleotide 36 of any one of SEQ ID NOs: 788-805; e. nucleotide 5 through nucleotide 36 of any one of SEQ ID NOs: 788-805; f. nucleotide 6 through nucleotide 36 of any one of SEQ ID NOs: 788-805; g. nucleotide 7 through nucleotide 36 of any one of SEQ ID NOs: 788-805; h. nucleotide 8 through nucleotide 36 of any one of SEQ ID NOs: 788-805; i. nucleotide 9 through nucleotide 36 of any one of SEQ ID NOs: 788-805; j. nucleotide 10 through nucleotide 36 of any one of SEQ ID NOs: 788-805; k. nucleotide 11 through nucleotide 36 of any one of SEQ ID NOs: 788-805; 1. nucleotide 12 through nucleotide 36 of any one of SEQ ID NOs: 788-805; m. nucleotide 13 through nucleotide 36 of any one of SEQ ID NOs: 788-805; n. nucleotide 14 through nucleotide 36 of any one of SEQ ID NOs: 788-805; or o. SEQ ID NO: 806 or a portion thereof.

In another aspect of the composition, the direct repeat comprises: a. nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 807; b. nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 807; c. nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 807; d. nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 807; e. nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 807; f. nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 807; g. nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 807; h. nucleotide 8 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 807; i. nucleotide 9 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 807; j. nucleotide 10 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 807; k. nucleotide 11 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 807; 1. nucleotide 12 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 807; m. nucleotide 13 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 807; n. nucleotide 14 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 807; or o. a sequence that is at least 90% identical to a sequence of SEQ ID NO: 808 or SEQ ID NO: 809 or a portion thereof.

In another aspect of the composition, the direct repeat comprises: a. nucleotide 1 through nucleotide 36 of SEQ ID NO: 807; b. nucleotide 2 through nucleotide 36 of SEQ ID NO: 807; c. nucleotide 3 through nucleotide 36 of SEQ ID NO: 807; d. nucleotide 4 through nucleotide 36 of SEQ ID NO: 807; e. nucleotide 5 through nucleotide 36 of SEQ ID NO: 807; f. nucleotide 6 through nucleotide 36 of SEQ ID NO: 807; g. nucleotide 7 through nucleotide 36 of SEQ ID NO: 807; h. nucleotide 8 through nucleotide 36 of SEQ ID NO: 807; i. nucleotide 9 through nucleotide 36 of SEQ ID NO: 807; j. nucleotide 10 through nucleotide 36 of SEQ ID NO: 807; k. nucleotide 11 through nucleotide 36 of SEQ ID NO: 807; 1. nucleotide 12 through nucleotide 36 of SEQ ID NO: 807; m. nucleotide 13 through nucleotide 36 of SEQ ID NO: 807; n. nucleotide 14 through nucleotide 36 of SEQ ID NO: 807; or o. SEQ ID NO: 808 or SEQ ID NO: 809 or a portion thereof.

In another aspect of the composition, the direct repeat comprises: a. nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 810 or SEQ ID NO: 811; b. nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 810 or SEQ ID NO: 811; c. nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 810 or SEQ ID NO: 811; d. nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 810 or SEQ ID NO: 811; e. nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 810 or SEQ ID NO: 811; f. nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 810 or SEQ ID NO: 811; g. nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 810 or SEQ ID NO: 811; h. nucleotide 8 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 810 or SEQ ID NO: 811; i. nucleotide 9 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 810 or SEQ ID NO: 811; j. nucleotide 10 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 810 or SEQ ID NO: 811; k. nucleotide 11 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 810 or SEQ ID NO: 811; 1. nucleotide 12 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 810 or SEQ ID NO: 811; m. nucleotide 13 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 810 or SEQ ID NO: 811; n. nucleotide 14 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 810 or SEQ ID NO: 811; o. nucleotide 15 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 810 or SEQ ID NO: 811; or p. a sequence that is at least 90% identical to a sequence of SEQ ID NO: 812 or a portion thereof.

In another aspect of the composition, the direct repeat comprises: a. nucleotide 1 through nucleotide 36 of SEQ ID NO: 810 or SEQ ID NO: 811; b. nucleotide 2 through nucleotide 36 of SEQ ID NO: 810 or SEQ ID NO: 811; c. nucleotide 3 through nucleotide 36 of SEQ ID NO: 810 or SEQ ID NO: 811; d. nucleotide 4 through nucleotide 36 of SEQ ID NO: 810 or SEQ ID NO: 811; e. nucleotide 5 through nucleotide 36 of SEQ ID NO: 810 or SEQ ID NO: 811; f. nucleotide 6 through nucleotide 36 of SEQ ID NO: 810 or SEQ ID NO: 811; g. nucleotide 7 through nucleotide 36 of SEQ ID NO: 810 or SEQ ID NO: 811; h. nucleotide 8 through nucleotide 36 of SEQ ID NO: 810 or SEQ ID NO: 811; i. nucleotide 9 through nucleotide 36 of SEQ ID NO: 810 or SEQ ID NO: 811; j. nucleotide 10 through nucleotide 36 of SEQ ID NO: 810 or SEQ ID NO: 811; k. nucleotide 11 through nucleotide 36 of SEQ ID NO: 810 or SEQ ID NO: 811; 1. nucleotide 12 through nucleotide 36 of SEQ ID NO: 810 or SEQ ID NO: 811; m. nucleotide 13 through nucleotide 36 of SEQ ID NO: 810 or SEQ ID NO: 811; n. nucleotide 14 through nucleotide 36 of SEQ ID NO: 810 or SEQ ID NO: 811; o. nucleotide 15 through nucleotide 36 of SEQ ID NO: 810 or SEQ ID NO: 811; or p. SEQ ID NO: 812 or a portion thereof.

In another aspect of the composition, the spacer sequence is substantially complementary to the complement of a sequence of any one of SEQ ID NOs: 11-390 or 819-1018.

In another aspect of the composition, the PAM comprises the sequence 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′.

In another aspect of the composition, the target sequence is immediately adjacent to the PAM sequence.

In another aspect of the composition, the composition further comprises a Cas12i polypeptide.

In another aspect of the composition, the Cas12i polypeptide is: a. a Cas12i2 polypeptide comprising a sequence that is at least 90% identical to the sequence of SEQ ID NO: 772, SEQ ID NO: 782, SEQ ID NO: 783, SEQ ID NO: 784, SEQ ID NO: 785, or SEQ ID NO: 786; b. a Cas12i4 polypeptide comprising a sequence that is at least 90% identical to the sequence of SEQ ID NO: 814, SEQ ID NO: 815, or SEQ ID NO: 816; c. a Cas12i1 polypeptide comprising a sequence that is at least 90% identical to the sequence of SEQ ID NO: 817; or d. a Cas12i3 polypeptide comprising a sequence that is at least 90% identical to the sequence of SEQ ID NO: 818.

In another aspect of the composition, the Cas12i polypeptide is: a. a Cas12i2 polypeptide comprising a sequence of SEQ ID NO: 772, SEQ ID NO: 782, SEQ ID NO: 783, SEQ ID NO: 784, SEQ ID NO: 785, or SEQ ID NO: 786; b. a Cas12i4 polypeptide comprising a sequence of SEQ ID NO: 814, SEQ ID NO: 815, or SEQ ID NO: 816; c. a Cas12i1 polypeptide comprising a sequence of SEQ ID NO: 817; or d. a Cas12i3 polypeptide comprising a sequence of SEQ ID NO: 818.

In another aspect of the composition, the RNA guide and the Cas12i polypeptide form a ribonucleoprotein complex.

In another aspect of the composition, the ribonucleoprotein complex binds a target nucleic acid.

In another aspect of the composition, the composition is present within a cell.

In another aspect of the composition, the RNA guide and the Cas12i polypeptide are encoded in a vector, e.g., expression vector. In another aspect of the composition, the RNA guide and the Cas12i polypeptide are encoded in a single vector or the RNA guide is encoded in a first vector and the Cas12i polypeptide is encoded in a second vector.

The invention further provides a vector system comprising one or more vectors encoding an RNA guide disclosed herein and a Cas12i polypeptide. In an embodiment, the vector system comprises a first vector encoding an RNA guide disclosed herein and a second vector encoding a Cas12i polypeptide. The vectors may be expression vectors.

The invention further provides a composition comprising an RNA guide and a Cas12i polypeptide, wherein the RNA guide comprises (i) a spacer sequence that is substantially complementary to a target sequence within a B2M gene and (ii) a direct repeat sequence.

In one aspect of the composition, the target sequence is within exon 1, exon 2, exon 3, exon 4, intron 1, intron 2, or intron 3 of the B2M gene.

In another aspect of the composition, the B2M gene comprises the sequence of SEQ ID NO: 773, the reverse complement of SEQ ID NO: 773, a variant of the sequence of SEQ ID NO: 773, or the reverse complement of a variant of SEQ ID NO: 773.

In another aspect of the composition, the spacer sequence is substantially complementary to the complement of a sequence of any one of SEQ ID NOs: 11-390 or 819-1018.

In another aspect of the composition, the target sequence is adjacent to a protospacer adjacent motif (PAM) comprising the sequence 5′-NTTN-3′.

In another aspect of the composition, the target sequence is immediately adjacent to the PAM sequence.

In another aspect of the composition, the target sequence is within 1, 2, 3, 4, or 5 nucleotides of the PAM sequence.

In another aspect of the composition, the RNA guide and the Cas12i polypeptide form a ribonucleoprotein complex.

In another aspect of the composition, the ribonucleoprotein complex binds a target nucleic acid.

In another aspect of the composition, the composition is present within a cell.

In another aspect of the composition, the RNA guide does not consist of the sequence of:

(SEQ ID NO: 778)

AGAAAUCCGUCUUUCAUUGACGGAAUGUCGGAUGGAUGAAACC;

(SEQ ID NO: 779)

AGAAAUCCGUCUUUCAUUGACGGCUAUCUCUUGUACUACACUG;

(SEQ ID NO: 780)

AGAAAUCCGUCUUUCAUUGACGGACACGGCAGGCAUACUCAUC;

or

(SEQ ID NO: 781)

AGAAAUCCGUCUUUCAUUGACGGGUCACAGCCCAAGAUAGUUA.

The invention yet further provides an RNA guide comprising (i) a spacer sequence that is substantially complementary to a target sequence within a B2M gene and (ii) a direct repeat sequence.

In one aspect of the RNA guide, the target sequence is within exon 1, exon 2, exon 3, exon 4, intron 1, intron 2, or intron 3 of the B2M gene.

In another aspect of the RNA guide, the B2M gene comprises the sequence of SEQ ID NO: 773, the reverse complement of SEQ ID NO: 773, a variant of the sequence of SEQ ID NO: 773, or the reverse complement of a variant of SEQ ID NO: 773.

In another aspect of the RNA guide, the spacer sequence comprises: a. nucleotide 1 through nucleotide 16 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 391-770 or 1019-1218; b. nucleotide 1 through nucleotide 17 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 391-770 or 1019-1218; c. nucleotide 1 through nucleotide 18 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 391-770 or 1019-1218; d. nucleotide 1 through nucleotide 19 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 391-770 or 1019-1218; e. nucleotide 1 through nucleotide 20 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 391-770 or 1019-1218; f. nucleotide 1 through nucleotide 21 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 391-770; g. nucleotide 1 through nucleotide 22 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 391-552 and 554-770; h. nucleotide 1 through nucleotide 23 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 391-552 and 555-770; i. nucleotide 1 through nucleotide 24 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 391-552 and 556-770; j. nucleotide 1 through nucleotide 25 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 391-552 and 556-770; k. nucleotide 1 through nucleotide 26 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 391-552 and 556-770; 1. nucleotide 1 through nucleotide 27 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 391-552 and 556-770; m. nucleotide 1 through nucleotide 28 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 391-552 and 556-770; n. nucleotide 1 through nucleotide 29 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 391-552 and 556-770; or o. nucleotide 1 through nucleotide 30 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 391-540, 542-552, and 556-770.

In another aspect of the RNA guide, the spacer sequence comprises: a. nucleotide 1 through nucleotide 16 of any one of SEQ ID NOs: 391-770 or 1019-1218; b. nucleotide 1 through nucleotide 17 of any one of SEQ ID NOs: 391-770 or 1019-1218; c. nucleotide 1 through nucleotide 18 of any one of SEQ ID NOs: 391-770 or 1019-1218; d. nucleotide 1 through nucleotide 19 of any one of SEQ ID NOs: 391-770 or 1019-1218; e. nucleotide 1 through nucleotide 20 of any one of SEQ ID NOs: 391-770 or 1019-1218; f. nucleotide 1 through nucleotide 21 of any one of SEQ ID NOs: 391-770; g. nucleotide 1 through nucleotide 22 of any one of SEQ ID NOs: 391-552 and 554-770; h. nucleotide 1 through nucleotide 23 of any one of SEQ ID NOs: 391-552 and 555-770; i. nucleotide 1 through nucleotide 24 of any one of SEQ ID NOs: 391-552 and 556-770; j. nucleotide 1 through nucleotide 25 of any one of SEQ ID NOs: 391-552 and 556-770; k. nucleotide 1 through nucleotide 26 of any one of SEQ ID NOs: 391-552 and 556-770; 1. nucleotide 1 through nucleotide 27 of any one of SEQ ID NOs: 391-552 and 556-770; m. nucleotide 1 through nucleotide 28 of any one of SEQ ID NOs: 391-552 and 556-770; n. nucleotide 1 through nucleotide 29 of any one of SEQ ID NOs: 391-552 and 556-770; or o. nucleotide 1 through nucleotide 30 of any one of SEQ ID NOs: 391-540, 542-552, and 556-770.

In another aspect of the RNA guide, the direct repeat comprises: a. nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; b. nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; c. nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; d. nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; e. nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; f. nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; g. nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; h. nucleotide 8 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; i. nucleotide 9 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; j. nucleotide 10 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; k. nucleotide 11 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; l. nucleotide 12 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; m. nucleotide 13 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; n. nucleotide 14 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1-8; o. nucleotide 1 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; p. nucleotide 2 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; q. nucleotide 3 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; r. nucleotide 4 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; s. nucleotide 5 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; t. nucleotide 6 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; u. nucleotide 7 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; v. nucleotide 8 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; w. nucleotide 9 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; x. nucleotide 10 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; y. nucleotide 11 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; z. nucleotide 12 through nucleotide 34 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 9; or aa. a sequence that is at least 90% identical to a sequence of SEQ ID NO: 10 or a portion thereof.

In another aspect of the RNA guide, the direct repeat comprises: a. nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1-8; b. nucleotide 2 through nucleotide 36 of any one of SEQ ID NOs: 1-8; c. nucleotide 3 through nucleotide 36 of any one of SEQ ID NOs: 1-8; d. nucleotide 4 through nucleotide 36 of any one of SEQ ID NOs: 1-8; e. nucleotide 5 through nucleotide 36 of any one of SEQ ID NOs: 1-8; f. nucleotide 6 through nucleotide 36 of any one of SEQ ID NOs: 1-8; g. nucleotide 7 through nucleotide 36 of any one of SEQ ID NOs: 1-8; h. nucleotide 8 through nucleotide 36 of any one of SEQ ID NOs: 1-8; i. nucleotide 9 through nucleotide 36 of any one of SEQ ID NOs: 1-8; j. nucleotide 10 through nucleotide 36 of any one of SEQ ID NOs: 1-8; k. nucleotide 11 through nucleotide 36 of any one of SEQ ID NOs: 1-8; l. nucleotide 12 through nucleotide 36 of any one of SEQ ID NOs: 1-8; m. nucleotide 13 through nucleotide 36 of any one of SEQ ID NOs: 1-8; n. nucleotide 14 through nucleotide 36 of any one of SEQ ID NOs: 1-8; o. nucleotide 1 through nucleotide 34 of SEQ ID NO: 9; p. nucleotide 2 through nucleotide 34 of SEQ ID NO: 9; q. nucleotide 3 through nucleotide 34 of SEQ ID NO: 9; r. nucleotide 4 through nucleotide 34 of SEQ ID NO: 9; s. nucleotide 5 through nucleotide 34 of SEQ ID NO: 9; t. nucleotide 6 through nucleotide 34 of SEQ ID NO: 9; u. nucleotide 7 through nucleotide 34 of SEQ ID NO: 9; v. nucleotide 8 through nucleotide 34 of SEQ ID NO: 9; w. nucleotide 9 through nucleotide 34 of SEQ ID NO: 9; x. nucleotide 10 through nucleotide 34 of SEQ ID NO: 9; y. nucleotide 11 through nucleotide 34 of SEQ ID NO: 9; z. nucleotide 12 through nucleotide 34 of SEQ ID NO: 9; or aa. SEQ ID NO: 10 or a portion thereof.

In another aspect of the RNA guide, the direct repeat comprises: a. nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 788-805; b. nucleotide 2 through nucleotide 36 of any one of SEQ ID NOs: 788-805; c. nucleotide 3 through nucleotide 36 of any one of SEQ ID NOs: 788-805; d. nucleotide 4 through nucleotide 36 of any one of SEQ ID NOs: 788-805; e. nucleotide 5 through nucleotide 36 of any one of SEQ ID NOs: 788-805; f. nucleotide 6 through nucleotide 36 of any one of SEQ ID NOs: 788-805; g. nucleotide 7 through nucleotide 36 of any one of SEQ ID NOs: 788-805; h. nucleotide 8 through nucleotide 36 of any one of SEQ ID NOs: 788-805; i. nucleotide 9 through nucleotide 36 of any one of SEQ ID NOs: 788-805; j. nucleotide 10 through nucleotide 36 of any one of SEQ ID NOs: 788-805; k. nucleotide 11 through nucleotide 36 of any one of SEQ ID NOs: 788-805; l. nucleotide 12 through nucleotide 36 of any one of SEQ ID NOs: 788-805; m. nucleotide 13 through nucleotide 36 of any one of SEQ ID NOs: 788-805; n. nucleotide 14 through nucleotide 36 of any one of SEQ ID NOs: 788-805; or o. SEQ ID NO: 806 or a portion thereof.

In another aspect of the RNA guide, the direct repeat comprises: a. nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 807; b. nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 807; c. nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 807; d. nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 807; e. nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 807; f. nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 807; g. nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 807; h. nucleotide 8 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 807; i. nucleotide 9 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 807; j. nucleotide 10 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 807; k. nucleotide 11 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 807; l. nucleotide 12 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 807; m. nucleotide 13 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 807; n. nucleotide 14 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 807; or o. a sequence that is at least 90% identical to a sequence of SEQ ID NO: 808 or SEQ ID NO: 809 or a portion thereof.

In another aspect of the RNA guide, the direct repeat comprises: a. nucleotide 1 through nucleotide 36 of SEQ ID NO: 807; b. nucleotide 2 through nucleotide 36 of SEQ ID NO: 807; c. nucleotide 3 through nucleotide 36 of SEQ ID NO: 807; d. nucleotide 4 through nucleotide 36 of SEQ ID NO: 807; e. nucleotide 5 through nucleotide 36 of SEQ ID NO: 807; f. nucleotide 6 through nucleotide 36 of SEQ ID NO: 807; g. nucleotide 7 through nucleotide 36 of SEQ ID NO: 807; h. nucleotide 8 through nucleotide 36 of SEQ ID NO: 807; i. nucleotide 9 through nucleotide 36 of SEQ ID NO: 807; j. nucleotide 10 through nucleotide 36 of SEQ ID NO: 807; k. nucleotide 11 through nucleotide 36 of SEQ ID NO: 807; l. nucleotide 12 through nucleotide 36 of SEQ ID NO: 807; m. nucleotide 13 through nucleotide 36 of SEQ ID NO: 807; n. nucleotide 14 through nucleotide 36 of SEQ ID NO: 807; or o. SEQ ID NO: 808 or SEQ ID NO: 809 or a portion thereof.

In another aspect of the RNA guide, the direct repeat comprises: a. nucleotide 1 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 810 or SEQ ID NO: 811; b. nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 810 or SEQ ID NO: 811; c. nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 810 or SEQ ID NO: 811; d. nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 810 or SEQ ID NO: 811; e. nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 810 or SEQ ID NO: 811; f. nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 810 or SEQ ID NO: 811; g. nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 810 or SEQ ID NO: 811; h. nucleotide 8 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 810 or SEQ ID NO: 811; i. nucleotide 9 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 810 or SEQ ID NO: 811; j. nucleotide 10 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 810 or SEQ ID NO: 811; k. nucleotide 11 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 810 or SEQ ID NO: 811; l. nucleotide 12 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 810 or SEQ ID NO: 811; m. nucleotide 13 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 810 or SEQ ID NO: 811; n. nucleotide 14 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 810 or SEQ ID NO: 811; o. nucleotide 15 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 810 or SEQ ID NO: 811; or p. a sequence that is at least 90% identical to a sequence of SEQ ID NO: 812 or a portion thereof.

In another aspect of the RNA guide, the direct repeat comprises: a. nucleotide 1 through nucleotide 36 of SEQ ID NO: 810 or SEQ ID NO: 811; b. nucleotide 2 through nucleotide 36 of SEQ ID NO: 810 or SEQ ID NO: 811; c. nucleotide 3 through nucleotide 36 of SEQ ID NO: 810 or SEQ ID NO: 811; d. nucleotide 4 through nucleotide 36 of SEQ ID NO: 810 or SEQ ID NO: 811; e. nucleotide 5 through nucleotide 36 of SEQ ID NO: 810 or SEQ ID NO: 811; f. nucleotide 6 through nucleotide 36 of SEQ ID NO: 810 or SEQ ID NO: 811; g. nucleotide 7 through nucleotide 36 of SEQ ID NO: 810 or SEQ ID NO: 811; h. nucleotide 8 through nucleotide 36 of SEQ ID NO: 810 or SEQ ID NO: 811; i. nucleotide 9 through nucleotide 36 of SEQ ID NO: 810 or SEQ ID NO: 811; j. nucleotide 10 through nucleotide 36 of SEQ ID NO: 810 or SEQ ID NO: 811; k. nucleotide 11 through nucleotide 36 of SEQ ID NO: 810 or SEQ ID NO: 811; l. nucleotide 12 through nucleotide 36 of SEQ ID NO: 810 or SEQ ID NO: 811; m. nucleotide 13 through nucleotide 36 of SEQ ID NO: 810 or SEQ ID NO: 811; n. nucleotide 14 through nucleotide 36 of SEQ ID NO: 810 or SEQ ID NO: 811; o. nucleotide 15 through nucleotide 36 of SEQ ID NO: 810 or SEQ ID NO: 811; or p. SEQ ID NO: 812 or a portion thereof.

In another aspect of the RNA guide, the spacer sequence is substantially complementary to the complement of a sequence of any one of SEQ ID NOs: 11-390 or 819-1018.

In another aspect of the RNA guide, the target sequence is adjacent to a protospacer adjacent motif (PAM) comprising the sequence 5′-NTTN-3′, wherein N is any nucleotide.

In another aspect of the RNA guide, the PAM comprises the sequence 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′.

In another aspect of the RNA guide, the target sequence is immediately adjacent to the PAM sequence.

In another aspect of the RNA guide, the target sequence is within 1, 2, 3, 4, or 5 nucleotides of the PAM sequence.

In another aspect of the RNA guide, the RNA guide does not consist of the sequence of:

The invention yet further provides a nucleic acid encoding an RNA guide as described herein.

The invention yet further provides a vector comprising such an RNA guide as described herein.

The invention yet further provides a cell comprising a composition, an RNA guide, a nucleic acid, or a vector as described herein.

In one aspect of the cell, the cell is a eukaryotic cell, an animal cell, a mammalian cell, a human cell, a primary cell, a cell line, a stem cell, or a T cell.

The invention yet further provides a kit comprising a composition, an RNA guide, a nucleic acid, or a vector as described herein.

The invention yet further provides a method of editing a B2M sequence, the method comprising contacting a B2M sequence with a composition or an RNA guide as described herein. In an embodiment, the method is carried out in vitro. In an embodiment, the method is carried out ex vivo.

In one aspect of the method, the B2M sequence is in a cell.

In one aspect of the method, the composition or the RNA guide induces a deletion in the B2M sequence.

In one aspect of the method, the deletion is adjacent to a 5′-NTTN-3′ sequence, wherein N is any nucleotide.

In one aspect of the method, the deletion is downstream of the 5′-NTTN-3′ sequence.

In one aspect of the method, the deletion is up to about 40 nucleotides in length.

In one aspect of the method, the deletion is from about 4 nucleotides to 40 nucleotides in length.

In one aspect of the method, the deletion is from about 4 nucleotides to 25 nucleotides in length.

In one aspect of the method, the deletion is from about 10 nucleotides to 25 nucleotides in length.

In one aspect of the method, the deletion is from about 10 nucleotides to 15 nucleotides in length.

In one aspect of the method, the deletion starts within about 5 nucleotides to about 15 nucleotides of the 5′-NTTN-3′ sequence.

In one aspect of the method, the deletion starts within about 5 nucleotides to about 10 nucleotides of the 5′-NTTN-3′ sequence.

In one aspect of the method, the deletion starts within about 10 nucleotides to about 15 nucleotides of the 5′-NTTN-3′ sequence.

In one aspect of the method, the deletion starts within about 5 nucleotides to about 15 nucleotides downstream of the 5′-NTTN-3′ sequence.

In one aspect of the method, the deletion starts within about 5 nucleotides to about 10 nucleotides downstream of the 5′-NTTN-3′ sequence.

In one aspect of the method, the deletion starts within about 10 nucleotides to about 15 nucleotides downstream of the 5′-NTTN-3′ sequence.

In one aspect of the method, the deletion ends within about 20 nucleotides to about 30 nucleotides of the 5′-NTTN-3′ sequence.

In one aspect of the method, the deletion ends within about 20 nucleotides to about 25 nucleotides of the 5′-NTTN-3′ sequence.

In one aspect of the method, the deletion ends within about 25 nucleotides to about 30 nucleotides of the 5′-NTTN-3′ sequence.

In one aspect of the method, the deletion ends within about 20 nucleotides to about 30 nucleotides downstream of the 5′-NTTN-3′ sequence.

In one aspect of the method, the deletion ends within about 20 nucleotides to about 25 nucleotides downstream of the 5′-NTTN-3′ sequence.

In one aspect of the method, the deletion ends within about 25 nucleotides to about 30 nucleotides downstream of the 5′-NTTN-3′ sequence.

In one aspect of the method, the deletion starts within about 5 nucleotides to about 15 nucleotides downstream of the 5′-NTTN-3′ sequence and ends within about 20 nucleotides to about 30 nucleotides downstream of the 5′-NTTN-3′ sequence.

In one aspect of the method, the deletion starts within about 5 nucleotides to about 15 nucleotides downstream of the 5′-NTTN-3′ sequence and ends within about 20 nucleotides to about 25 nucleotides downstream of the 5′-NTTN-3′ sequence.

In one aspect of the method, the deletion starts within about 5 nucleotides to about 15 nucleotides downstream of the 5′-NTTN-3′ sequence and ends within about 25 nucleotides to about 30 nucleotides downstream of the 5′-NTTN-3′ sequence.

In one aspect of the method, the deletion starts within about 5 nucleotides to about 10 nucleotides downstream of the 5′-NTTN-3′ sequence and ends within about 20 nucleotides to about 30 nucleotides downstream of the 5′-NTTN-3′ sequence.

In one aspect of the method, the deletion starts within about 5 nucleotides to about 10 nucleotides downstream of the 5′-NTTN-3′ sequence and ends within about 20 nucleotides to about 25 nucleotides downstream of the 5′-NTTN-3′ sequence.

In one aspect of the method, the deletion starts within about 5 nucleotides to about 10 nucleotides downstream of the 5′-NTTN-3′ sequence and ends within about 25 nucleotides to about 30 nucleotides downstream of the 5′-NTTN-3′ sequence.

In one aspect of the method, the deletion starts within about 10 nucleotides to about 15 nucleotides downstream of the 5′-NTTN-3′ sequence and ends within about 20 nucleotides to about 30 nucleotides downstream of the 5′-NTTN-3′ sequence.

In one aspect of the method, the deletion starts within about 10 nucleotides to about 15 nucleotides downstream of the 5′-NTTN-3′ sequence and ends within about 20 nucleotides to about 25 nucleotides downstream of the 5′-NTTN-3′ sequence.

In one aspect of the method, the deletion starts within about 10 nucleotides to about 15 nucleotides downstream of the 5′-NTTN-3′ sequence and ends within about 25 nucleotides to about 30 nucleotides downstream of the 5′-NTTN-3′ sequence.

In one aspect of the method, the 5′-NTTN-3′ sequence is 5′-CTTT-3′, 5′-CTTC-3′, 5′-GTTT-3′, 5′-GTTC-3′, 5′-TTTC-3′, 5′-GTTA-3′, or 5′-GTTG-3′.

In one aspect of the method, the deletion overlaps with a mutation in the B2M sequence.

In one aspect of the method, the deletion overlaps with an insertion in the B2M sequence.

In one aspect of the method, the deletion removes a repeat expansion of the B2M sequence or a portion thereof.

In one aspect of the method, the deletion disrupts one or both alleles of the B2M sequence.

In one aspect of the composition, RNA guide, nucleic acid, vector, cell, kit, or method described herein, the RNA guide does not consist of the sequence of:

In one aspect of the composition, RNA guide, nucleic acid, vector, cell, kit, or method described herein, the RNA guide comprises the sequence of any one of SEQ ID NOs: 1222-1230.

Definitions

The present invention will be described with respect to particular, but the invention is not limited thereto but only by the claims. Terms as set forth hereinafter are generally to be understood in their common sense unless indicated otherwise.

As used herein, the term “activity” refers to a biological activity. In some embodiments, activity includes enzymatic activity, e.g., catalytic ability of an effector. For example, activity can include nuclease activity.

As used herein the term “B2M” refers to “02 microglobulin” or “beta-2 microglobulin.” B2M is a component of major histocompatibility complex (MHC) class I molecules, which are found on the surfaces of all nucleated vertebrate cells and function to display peptide fragments of proteins within the cells to cytotoxic T cells. SEQ ID NO: 773 as set forth herein provides an example of a B2M gene sequence. It is understood that spacer sequences described herein can target SEQ ID NO: 773 or the reverse complement thereof, depending upon whether they are indicated as “+” or “−” as set forth in Table 5. The target sequences listed in Table 5 and Table 6 are on the non-target strand of the B2M gene.

As used herein, the term “Cas12i polypeptide” (also referred to herein as Cas12i) refers to a polypeptide that binds to a target sequence on a target nucleic acid specified by an RNA guide, wherein the polypeptide has at least some amino acid sequence homology to a wild-type Cas12i polypeptide. In some embodiments, the Cas12i polypeptide comprises at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with any one of SEQ ID NOs: 1-5 and 11-18 of U.S. Pat. No. 10,808,245, which is incorporated by reference herein in its entirety. In some embodiments, a Cas12i polypeptide comprises at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with any one of SEQ ID NO: 3 (Cas12i1), SEQ ID NO: 5 (Cas12i2), SEQ ID NO: 14 (Cas12i3), or SEQ ID NO: 16 (Cas12i4) of U.S. Pat. No. 10,808,245, corresponding to SEQ ID NOs: 817, 772, 818, and 814 of the present application. In some embodiments, a Cas12i polypeptide of the disclosure is a Cas12i1 polypeptide or Cas12i2 polypeptide as described in PCT/US2021/025257. In some embodiments, the Cas12i polypeptide cleaves a target nucleic acid (e.g., as a nick or a double strand break).

As used herein, the term “complex” refers to a grouping of two or more molecules. In some embodiments, the complex comprises a polypeptide and a nucleic acid molecule interacting with (e.g., binding to, coming into contact with, adhering to) one another. As used herein, the term “complex” can refer to a grouping of an RNA guide and a polypeptide (e.g., a Cas12i polypeptide). As used herein, the term “complex” can refer to a grouping of an RNA guide, a polypeptide, and a target sequence. As used herein, the term “complex” can refer to a grouping of a B2M-targeting RNA guide and a Cas12i polypeptide.

As used herein, the term “protospacer adjacent motif” or “PAM” refers to a DNA sequence adjacent to a target sequence (e.g., a B2M target sequence) to which a complex comprising an RNA guide (e.g., a B2M-targeting RNA guide) and a Cas12i polypeptide binds. In the case of a double-stranded target, the RNA guide binds to a first strand of the target (e.g., the target strand or the spacer-complementary strand), and a PAM sequence as described herein is present in the second, complementary strand (e.g., the non-target strand or the non-spacer-complementary strand). As used herein, the term “adjacent” includes instances in which the RNA guide of a complex comprising an RNA guide and a Cas12i polypeptide specifically binds, interacts, or associates with a target sequence that is immediately adjacent to a PAM. In such instances, there are no nucleotides between the target sequence and the PAM. The term “adjacent” also includes instances in which there are a small number (e.g., 1, 2, 3, 4, or 5) of nucleotides between the target sequence, to which the RNA guide binds, and the PAM. In some embodiments, the PAM sequence as described herein is present in the non-target strand (e.g., the non-spacer-complementary strand). In such a case, the term “adjacent” includes a PAM sequence as described herein as being immediately adjacent to (or within a small number, e.g., 1, 2, 3, 4, or 5 nucleotides of) a sequence in the non-target strand.

As used herein, the term “RNA guide” refers to any RNA molecule that facilitates the targeting of a polypeptide (e.g., a Cas12i polypeptide) described herein to a target sequence (e.g., a sequence of a B2M gene). An RNA guide may be designed to include sequences that are complementary to a specific nucleic acid sequence (e.g., a B2M nucleic acid sequence). An RNA guide may comprise a DNA targeting sequence (i.e., a spacer sequence) and a direct repeat (DR) sequence. The term “crRNA” is also used herein to refer to an RNA guide.

In some embodiments, a spacer sequence is complementary to a target sequence. As used herein, the term “complementary” refers to the ability of nucleobases of a first nucleic acid molecule, such as an RNA guide, to base pair with nucleobases of a second nucleic acid molecule, such as a target sequence. Two complementary nucleic acid molecules are able to non-covalently bind under appropriate temperature and solution ionic strength conditions. In some embodiments, a first nucleic acid molecule (e.g., a spacer sequence of an RNA guide) comprises 100% complementarity to a second nucleic acid (e.g., a target sequence). In some embodiments, a first nucleic acid molecule (e.g., a spacer sequence of an RNA guide) is complementary to a second nucleic acid molecule (e.g., a target sequence) if the first nucleic acid molecule comprises at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementarity to the second nucleic acid. As used herein, the term “substantially complementary” refers to a polynucleotide (e.g., a spacer sequence of an RNA guide) that has a certain level of complementarity to a target sequence. In some embodiments, the level of complementarity is such that the polynucleotide can hybridize to the target sequence with sufficient affinity to permit an effector polypeptide (e.g., Cas12i) that is complexed with the polynucleotide to act (e.g., cleave) on the target sequence. In some embodiments, a spacer sequence that is substantially complementary to a target sequence has less than 100% complementarity to the target sequence. In some embodiments, a spacer sequence that is substantially complementary to a target sequence has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementarity to the target sequence. In some embodiments, an RNA guide with a spacer sequence that is substantially complementary to a target sequence has 100% complementarity to the target sequence.

As used herein, the terms “target” and “target sequence” refer to a nucleic acid sequence to which an RNA guide specifically binds. In some embodiments, the DNA targeting sequence (e.g., spacer) of an RNA guide binds to a target sequence. In the case of a double-stranded target, the RNA guide binds to a first strand of the target (i.e., the target strand or the spacer-complementary strand), and a PAM sequence as described herein is present in the second, complementary strand (i.e., the non-target strand or the non-spacer-complementary strand). In some embodiments, the target strand (i.e., the spacer-complementary strand) comprises a 5′-NAAN-3′ sequence. In some embodiments, the target sequence is a sequence within a B2M gene sequence, including, but not limited, to the sequence set forth in SEQ ID NO: 773 or the reverse complement thereof.

As used herein, the terms “upstream” and “downstream” refer to relative positions within a single nucleic acid (e.g., DNA) sequence in a nucleic acid molecule. “Upstream” and “downstream” relate to the 5′ to 3′ direction, respectively, in which RNA transcription occurs. A first sequence is upstream of a second sequence when the 3′ end of the first sequence occurs before the 5′ end of the second sequence. A first sequence is downstream of a second sequence when the 5′ end of the first sequence occurs after the 3′ end of the second sequence. In some embodiments, the 5′-NTTN-3′ sequence is upstream of an indel described herein, and a Cas12i-induced indel is downstream of the 5′-NTTN-3′ sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows indel activity by variant Cas12i2 of SEQ ID NO: 782 and several individual RNA guides targeting B2M at various concentrations in HEK293T cells.

FIG. 2 shows indel activity by variant Cas12i2 of SEQ ID NO: 783 and several individual RNA guides targeting B2M at various concentrations in primary T cells. Error bars represent standard deviation of the mean of four technical replicates from one representative donor.

FIG. 3 shows B2M expression reduction by variant Cas12i2 of SEQ ID NO: 783 and several individual RNA guides targeting B2M at various concentrations in primary T cells. Error bars represent standard deviation of the mean of four technical replicates from one representative donor.

FIG. 4 shows viability of cells (via DAPI staining) seven days following introduction of variant Cas12i2 ribonucleoproteins (RNPs) targeting B2M at various concentrations in primary T cells. Error bars represent standard deviation of the mean of four technical replicates from one representative donor.

DETAILED DESCRIPTION

The present disclosure relates to an RNA guide capable of binding to B2M and methods of use thereof. In some aspects, a composition comprising an RNA guide having one or more characteristics is described herein. In some aspects, a method of producing the RNA guide is described. In some aspects, a method of delivering a composition comprising the RNA guide is described.

Composition

In some aspects, the invention described herein comprises compositions comprising an RNA guide targeting B2M. In some embodiments, the RNA guide is comprised of a direct repeat component and a spacer component. In some embodiments, the RNA guide binds a Cas12i polypeptide. In some embodiments, the spacer component is substantially complementary to a B2M target sequence, wherein the B2M target sequence is adjacent to a 5′-NTTN-3′ PAM sequence as described herein. In the case of a double-stranded target, the RNA guide binds to a first strand of the target (i.e., the target strand or the spacer-complementary strand) and a PAM sequence as described herein is present in the second, complementary strand (i.e., the non-target strand or the non-spacer-complementary strand).

In some embodiments, the invention described herein comprises compositions comprising a complex, wherein the complex comprises an RNA guide targeting B2M. In some embodiments, the invention comprises a complex comprising an RNA guide and a Cas12i polypeptide. In some embodiments, the RNA guide and the Cas12i polypeptide bind to each other in a molar ratio of about 1:1. In some embodiments, a complex comprising an RNA guide and a Cas12i polypeptide binds to a B2M target sequence. In some embodiments, a complex comprising an RNA guide targeting B2M and a Cas12i polypeptide binds to a B2M target sequence at a molar ratio of about 1:1. In some embodiments, the complex comprises enzymatic activity, such as nuclease activity, that can cleave the B2M target sequence. The RNA guide, the Cas12i polypeptide, and the B2M target sequence, either alone or together, do not naturally occur.

Use of the compositions disclosed herein has advantages over those of other known nuclease systems. Cas12i polypeptides are smaller than other nucleases. For example, Cas12i2 is 1,054 amino acids in length, whereas S. pyogenes Cas9 (SpCas9) is 1,368 amino acids in length, S. thermophilus Cas9 (StCas9) is 1,128 amino acids in length, FnCpf1 is 1,300 amino acids in length, AsCpf1 is 1,307 amino acids in length, and LbCpf1 is 1,246 amino acids in length. Cas12i RNA guides, which do not require a trans-activating CRISPR RNA (tracrRNA), are also smaller than Cas9 RNA guides. The smaller Cas12i polypeptide and RNA guide sizes are beneficial for delivery. Compositions comprising a Cas12i polypeptide also demonstrate decreased off-target activity compared to compositions comprising an SpCas9 polypeptide. See PCT/US2021/025257, which is incorporated by reference in its entirety. Furthermore, indels induced by compositions comprising a Cas12i polypeptide differ from indels induced by compositions comprising an SpCas9 polypeptide. For example, SpCas9 polypeptides primarily induce insertions and deletions of 1 nucleotide in length. However, Cas12i polypeptides induce larger deletions, which can be beneficial in disrupting a larger portion of a gene such as B2M.

RNA Guide

In some embodiments, the composition described herein comprises an RNA guide targeting a B2M gene or a portion of B2M gene. In some embodiments, the composition described herein comprises two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or more) RNA guides targeting B2M.

The RNA guide may direct the Cas12i polypeptide as described herein to a B2M target sequence. Two or more RNA guides may target two or more separate Cas12i polypeptides (e.g., Cas12i polypeptides having the same or different sequence) as described herein to two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or more) B2M target sequences.

Those skilled in the art reading the below examples of particular kinds of RNA guides will understand that, in some embodiments, an RNA guide is B2M target-specific. That is, in some embodiments, an RNA guide binds specifically to one or more B2M target sequences (e.g., within a cell) and not to non-targeted sequences (e.g., non-specific DNA or random sequences within the same cell).

In some embodiments, the RNA guide comprises a spacer sequence followed by a direct repeat sequence, referring to the sequences in the 5′ to 3′ direction. In some embodiments, the RNA guide comprises a first direct repeat sequence followed by a spacer sequence and a second direct repeat sequence, referring to the sequences in the 5′ to 3′ direction. In some embodiments, the first and second direct repeats of such an RNA guide are identical. In some embodiments, the first and second direct repeats of such an RNA guide are different.

In some embodiments, the spacer sequence and the direct repeat sequence(s) of the RNA guide are present within the same RNA molecule. In some embodiments, the spacer and direct repeat sequences are linked directly to one another. In some embodiments, a short linker is present between the spacer and direct repeat sequences, e.g., an RNA linker of 1, 2, or 3 nucleotides in length. In some embodiments, the spacer sequence and the direct repeat sequence(s) of the RNA guide are present in separate molecules, which are joined to one another by base pairing interactions.

Additional information regarding exemplary direct repeat and spacer components of RNA guides is provided as follows.

Direct Repeat

In some embodiments, the RNA guide comprises a direct repeat sequence. In some embodiments, the direct repeat sequence of the RNA guide has a length of between 12-100, 13-75, 14-50, or 15-40 nucleotides (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides).

In some embodiments, the direct repeat sequence is or comprises a sequence of Table 1 or a portion of a sequence of Table 1. The direct repeat sequence can comprise nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 2 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 3 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 4 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 5 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 6 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 7 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 8 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 9 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 10 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 11 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 12 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 13 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 14 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can comprise nucleotide 1 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can comprise nucleotide 2 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can comprise nucleotide 3 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can comprise nucleotide 4 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can comprise nucleotide 5 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can comprise nucleotide 6 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can comprise nucleotide 7 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can comprise nucleotide 8 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can comprise nucleotide 9 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can comprise nucleotide 10 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can comprise nucleotide 11 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can comprise nucleotide 12 through nucleotide 34 of SEQ ID NO: 9. In some embodiments, the direct repeat sequence is set forth in SEQ ID NO: 10. In some embodiments, the direct repeat sequence comprises a portion of the sequence set forth in SEQ ID NO: 10.

In some embodiments, the direct repeat sequence has or comprises a sequence comprising at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity) to a sequence of Table 1 or a portion of a sequence of Table 1. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 2 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 3 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 4 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 5 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 6 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 7 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 8 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 9 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 10 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 11 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 12 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 13 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 14 through nucleotide 36 of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 1 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 2 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 3 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 4 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 5 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 6 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 7 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 8 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 9 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 10 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 11 through nucleotide 34 of SEQ ID NO: 9. The direct repeat sequence can have or comprise a sequence having at least 90% identity to a sequence comprising 12 through nucleotide 34 of SEQ ID NO: 9. In some embodiments, the direct repeat sequence has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity) to SEQ ID NO: 10. In some embodiments, the direct repeat sequence has at least 90% identity to a portion of the sequence set forth in SEQ ID NO: 10.

In some embodiments, compositions comprising a Cas12i2 polypeptide and an RNA guide comprising the direct repeat of SEQ ID NO: 10 and a spacer length of 20 nucleotides are capable of introducing indels into a B2M target sequence. See, e.g., Example 1, where indels were measured at eleven B2M target sequences following transient transfection of an RNA guide and Cas12i2 polypeptide of SEQ ID NO: 782, and Example 2, wherein indels were measured at four B2M target sequences following delivery of an RNA guide and Cas12i2 polypeptide of SEQ ID NO: 783 by RNP.

In some embodiments, the direct repeat sequence is or comprises a sequence that is at least 90% identical to the reverse complement of any one of SEQ ID NOs: 1-10. In some embodiments, the direct repeat sequence is or comprises the reverse complement of any one of SEQ ID NOs: 1-10.

TABLE 1

Cas12i2 direct repeat sequences.

Sequence

identifier
Direct Repeat Sequence

SEQ ID NO: 1
GUUGCAAAACCCAAGAAAUCCGUCUUUCAUUGACGG

SEQ ID NO: 2
AAUAGCGGCCCUAAGAAAUCCGUCUUUCAUUGACGG

SEQ ID NO: 3
AUUGGAACUGGCGAGAAAUCCGUCUUUCAUUGACGG

SEQ ID NO: 4
CCAGCAACACCUAAGAAAUCCGUCUUUCAUUGACGG

SEQ ID NO: 5
CGGCGCUCGAAUAGGAAAUCCGUCUUUCAUUGACGG

SEQ ID NO: 6
GUGGCAACACCUAAGAAAUCCGUCUUUCAUUGACGG

SEQ ID NO: 7
GUUGCAACACCUAAGAAAUCCGUCUUUCAUUGACGG

SEQ ID NO: 8
GUUGCAAUGCCUAAGAAAUCCGUCUUUCAUUGACGG

SEQ ID NO: 9
GCAACACCUAAGAAAUCCGUCUUUCAUUGACGGG

SEQ ID NO: 10
AGAAAUCCGUCUUUCAUUGACGG

In some embodiments, the direct repeat sequence is a sequence of Table 2 or a portion of a sequence of Table 2. The direct repeat sequence can comprise nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, or 805. The direct repeat sequence can comprise nucleotide 2 through nucleotide 36 of any one of SEQ ID NOs: 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, or 805. The direct repeat sequence can comprise nucleotide 3 through nucleotide 36 of any one of SEQ ID NOs: 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, or 805. The direct repeat sequence can comprise nucleotide 4 through nucleotide 36 of any one of SEQ ID NOs: 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, or 805. The direct repeat sequence can comprise nucleotide 5 through nucleotide 36 of any one of SEQ ID NOs: 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, or 805. The direct repeat sequence can comprise nucleotide 6 through nucleotide 36 of any one of SEQ ID NOs: 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, or 805. The direct repeat sequence can comprise nucleotide 7 through nucleotide 36 of any one of SEQ ID NOs: 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, or 805. The direct repeat sequence can comprise nucleotide 8 through nucleotide 36 of any one of SEQ ID NOs: 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, or 805. The direct repeat sequence can comprise nucleotide 9 through nucleotide 36 of any one of SEQ ID NOs: 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, or 805. The direct repeat sequence can comprise nucleotide 10 through nucleotide 36 of any one of SEQ ID NOs: 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, or 805. The direct repeat sequence can comprise nucleotide 11 through nucleotide 36 of any one of SEQ ID NOs: 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, or 805. The direct repeat sequence can comprise nucleotide 12 through nucleotide 36 of any one of SEQ ID NOs: 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, or 805. The direct repeat sequence can comprise nucleotide 13 through nucleotide 36 of any one of SEQ ID NOs: 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, or 805. The direct repeat sequence can comprise nucleotide 14 through nucleotide 36 of any one of SEQ ID NOs: 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, or 805.

In some embodiments, the direct repeat sequence has at least 95% identity (e.g., at least 95%, 96%, 97%, 98% or 99% identity) to a sequence of Table 2 or a portion of a sequence of Table 2. The direct repeat sequence can have at least 95% identity to a sequence comprising nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, or 805. The direct repeat sequence can have at least 95% identity to a sequence comprising 2 through nucleotide 36 of any one of SEQ ID NOs: 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, or 805. The direct repeat sequence can have at least 95% identity to a sequence comprising 3 through nucleotide 36 of any one of SEQ ID NOs: 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, or 805. The direct repeat sequence can have at least 95% identity to a sequence comprising 4 through nucleotide 36 of any one of SEQ ID NOs: 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, or 805. The direct repeat sequence can have at least 95% identity to a sequence comprising 5 through nucleotide 36 of any one of SEQ ID NOs: 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, or 805. The direct repeat sequence can have at least 95% identity to a sequence comprising 6 through nucleotide 36 of any one of SEQ ID NOs: 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, or 805. The direct repeat sequence can have at least 95% identity to a sequence comprising 7 through nucleotide 36 of any one of SEQ ID NOs: 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, or 805. The direct repeat sequence can have at least 95% identity to a sequence comprising 8 through nucleotide 36 of any one of SEQ ID NOs: 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, or 805. The direct repeat sequence can have at least 95% identity to a sequence comprising 9 through nucleotide 36 of any one of SEQ ID NOs: 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, or 805. The direct repeat sequence can have at least 95% identity to a sequence comprising 10 through nucleotide 36 of any one of SEQ ID NOs: 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, or 805. The direct repeat sequence can have at least 95% identity to a sequence comprising 11 through nucleotide 36 of any one of SEQ ID NOs: 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, or 805. The direct repeat sequence can have at least 95% identity to a sequence comprising 12 through nucleotide 36 of any one of SEQ ID NOs: 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, or 805. The direct repeat sequence can have at least 95% identity to a sequence comprising 13 through nucleotide 36 of any one of SEQ ID NOs: 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, or 805.

In some embodiments, the direct repeat sequence has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity) to a sequence of Table 2 or a portion of a sequence of Table 2. The direct repeat sequence can have at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 36 of any one of SEQ ID NOs: 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, or 805. The direct repeat sequence can have at least 90% identity to a sequence comprising 2 through nucleotide 36 of any one of SEQ ID NOs: 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, or 805. The direct repeat sequence can have at least 90% identity to a sequence comprising 3 through nucleotide 36 of any one of SEQ ID NOs: 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, or 805. The direct repeat sequence can have at least 90% identity to a sequence comprising 4 through nucleotide 36 of any one of SEQ ID NOs: 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, or 805. The direct repeat sequence can have at least 90% identity to a sequence comprising 5 through nucleotide 36 of any one of SEQ ID NOs: 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, or 805. The direct repeat sequence can have at least 90% identity to a sequence comprising 6 through nucleotide 36 of any one of SEQ ID NOs: 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, or 805. The direct repeat sequence can have at least 90% identity to a sequence comprising 7 through nucleotide 36 of any one of SEQ ID NOs: 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, or 805. The direct repeat sequence can have at least 90% identity to a sequence comprising 8 through nucleotide 36 of any one of SEQ ID NOs: 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, or 805. The direct repeat sequence can have at least 90% identity to a sequence comprising 9 through nucleotide 36 of any one of SEQ ID NOs: 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, or 805. The direct repeat sequence can have at least 90% identity to a sequence comprising 10 through nucleotide 36 of any one of SEQ ID NOs: 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, or 805. The direct repeat sequence can have at least 90% identity to a sequence comprising 11 through nucleotide 36 of any one of SEQ ID NOs: 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, or 805. The direct repeat sequence can have at least 90% identity to a sequence comprising 12 through nucleotide 36 of any one of SEQ ID NOs: 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, or 805. The direct repeat sequence can have at least 90% identity to a sequence comprising 13 through nucleotide 36 of any one of SEQ ID NOs: 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, or 805.

In some embodiments, the direct repeat sequence is at least 90% identical to the reverse complement of any one of SEQ ID NOs: 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, or 805. In some embodiments, the direct repeat sequence is at least 95% identical to the reverse complement of any one of SEQ ID NOs: 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, or 805. In some embodiments, the direct repeat sequence is the reverse complement of any one of SEQ ID NOs: 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, or 805.

In some embodiments, the direct repeat sequence is at least 90% identical to SEQ ID NO: 806 or a portion of SEQ ID NO: 806. In some embodiments, the direct repeat sequence is at least 95% identical to SEQ ID NO: 806 or a portion of SEQ ID NO: 806. In some embodiments, the direct repeat sequence is 100% identical to SEQ ID NO: 806 or a portion of SEQ ID NO: 806.

TABLE 2

Cas12i4 direct repeat sequences.

Sequence

identifier
Direct Repeat Sequence

SEQ ID NO:
UCUCAACGAUAGUCAGACAUGUGUCCUCAGUGACAC

788

SEQ ID NO:
UUUUAACAACACUCAGGCAUGUGUCCACAGUGACAC

789

SEQ ID NO:
UUGAACGGAUACUCAGACAUGUGUUUCCAGUGACAC

790

SEQ ID NO:
UGCCCUCAAUAGUCAGAUGUGUGUCCACAGUGACAC

791

SEQ ID NO:
UCUCAAUGAUACUUAGAUACGUGUCCUCAGUGACAC

792

SEQ ID NO:
UCUCAAUGAUACUCAGACAUGUGUCCCCAGUGACAC

793

SEQ ID NO:
UCUCAAUGAUACUAAGACAUGUGUCCUCAGUGACAC

794

SEQ ID NO:
UCUCAACUAUACUCAGACAUGUGUCCUCAGUGACAC

795

SEQ ID NO:
UCUCAACGAUACUCAGACAUGUGUCCUCAGUGACAC

796

SEQ ID NO:
UCUCAACGAUACUAAGAUAUGUGUCCUCAGCGACAC

797

SEQ ID NO:
UCUCAACGAUACUAAGAUAUGUGUCCCCAGUGACAC

798

SEQ ID NO:
UCUCAACGAUACUAAGAUAUGUGUCCACAGUGACAC

799

SEQ ID NO:
UCUCAACAAUACUCAGACAUGUGUCCCCAGUGACAC

800

SEQ ID NO:
UCUCAACAAUACUAAGGCAUGUGUCCCCAGUGACCC

801

SEQ ID NO:
UCUCAAAGAUACUCAGACACGUGUCCCCAGUGACAC

802

SEQ ID NO:
UCUCAAAAAUACUCAGACAUGUGUCCUCAGUGACAC

803

SEQ ID NO:
GCGAAACAACAGUCAGACAUGUGUCCCCAGUGACAC

804

SEQ ID NO:
CCUCAACGAUAUUAAGACAUGUGUCCGCAGUGACAC

805

SEQ ID NO:
AGACAUGUGUCCUCAGUGACAC

806

In some embodiments, the direct repeat sequence is a sequence of Table 3 or a portion of a sequence of Table 3. In some embodiments, the direct repeat sequence has at least 95% identity (e.g., at least 95%, 96%, 97%, 98% or 99% identity) to a sequence of Table 3 or a portion of a sequence of Table 3. In some embodiments, the direct repeat sequence has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity) to a sequence of Table 3 or a portion of a sequence of Table 3. In some embodiments, the direct repeat sequence is at least 90% identical to the reverse complement of any one of SEQ ID NOs: 807-809. In some embodiments, the direct repeat sequence is at least 95% identical to the reverse complement of any one of SEQ ID NOs: 807-809. In some embodiments, the direct repeat sequence is the reverse complement of any one of SEQ ID NOs: 807-809.

TABLE 3

Cas12i1 direct repeat sequences.

Sequence

identifier
Direct Repeat Sequence

SEQ ID NO: 807
GUUGGAAUGACUAAUUUUUGUGCC

CACCGUUGGCAC

SEQ ID NO: 808
AAUUUUUGUGCCCAUCGUUGGCAC

SEQ ID NO: 809
AUUUUUGUGCCCAUCGUUGGCAC

In some embodiments, the direct repeat sequence is a sequence of Table 4 or a portion of a sequence of Table 4. In some embodiments, the direct repeat sequence has at least 95% identity (e.g., at least 95%, 96%, 97%, 98% or 99% identity) to a sequence of Table 4 or a portion of a sequence of Table 4. In some embodiments, the direct repeat sequence has at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity) to a sequence of Table 4 or a portion of a sequence of Table 4. In some embodiments, the direct repeat sequence is at least 90% identical to the reverse complement of any one of SEQ ID NOs: 810-812. In some embodiments, the direct repeat sequence is at least 95% identical to the reverse complement of any one of SEQ ID NOs: 810-812. In some embodiments, the direct repeat sequence is the reverse complement of any one of SEQ ID NOs: 810-812.

TABLE 4

Cas12i3 direct repeat sequences.

Sequence

identifier
Direct Repeat Sequence

SEQ ID NO: 810
CUAGCAAUGACCUAAUAGUGUG

UCCUUAGUUGACAU

SEQ ID NO: 811
CCUACAAUACCUAAGAAAUCCG

UCCUAAGUUGACGG

SEQ ID NO: 812
AUAGUGUGUCCUUAGUUGACAU

In some embodiments, a direct repeat sequence described herein comprises a uracil (U). In some embodiments, a direct repeat sequence described herein comprises a thymine (T). In some embodiments, a direct repeat sequence according to Tables 1-4 comprises a sequence comprising a thymine in one or more places indicated as uracil in Tables 1-4.

Spacer

In some embodiments, the RNA guide comprises a DNA targeting or spacer sequence. In some embodiments, the spacer sequence of the RNA guide has a length of between 12-100, 13-75, 14-50, or 15-30 nucleotides (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) and is complementary a specific target sequence. In some embodiments, the spacer sequence is designed to be complementary to a specific DNA strand, e.g., of a genomic locus.

In some embodiments, the RNA guide spacer sequence is substantially identical to a complementary strand of a target sequence. In some embodiments, the RNA guide comprises a sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to a complementary strand of a reference nucleic acid sequence, e.g., target sequence. The percent identity between two such nucleic acids can be determined manually by inspection of the two optimally aligned nucleic acid sequences or by using software programs or algorithms (e.g., BLAST, ALIGN, CLUSTAL) using standard parameters.

In some embodiments, the RNA guide comprises a spacer sequence that has a length of between 12-100, 13-75, 14-50, or 15-30 nucleotides (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) and at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% complementary to a target sequence. In some embodiments, the RNA guide comprises a sequence at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% complementary to a target DNA sequence. In some embodiments, the RNA guide comprises a sequence at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% complementary to a target genomic sequence. In some embodiments, the RNA guide comprises a sequence, e.g., RNA sequence, that is a length of up to 50 and at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% complementary to a target sequence. In some embodiments, the RNA guide comprises a sequence at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% complementary to a target DNA sequence. In some embodiments, the RNA guide comprises a sequence at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% complementary to a target genomic sequence.

In some embodiments, the spacer sequence is or comprises a sequence of Table 5 or a portion of a sequence of Table 5. The target sequences listed in Table 5 and Table 6 are on the non-target strand of the B2M sequence. It should be understood that an indication of SEQ ID NOs: 391-770 or 1019-1218 should be considered as equivalent to a listing of SEQ ID NOs: 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766, 767, 768, 769, and 770, or 1019, 1020, 1021, 1022, 1023, 1024, 1025, 1026, 1027, 1028, 1029, 1030, 1031, 1032, 1033, 1034, 1035, 1036, 1037, 1038, 1039, 1040, 1041, 1042, 1043, 1044, 1045, 1046, 1047, 1048, 1049, 1050, 1051, 1052, 1053, 1054, 1055, 1056, 1057, 1058, 1059, 1060, 1061, 1062, 1063, 1064, 1065, 1066, 1067, 1068, 1069, 1070, 1071, 1072, 1073, 1074, 1075, 1076, 1077, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1085, 1086, 1087, 1088, 1089, 1090, 1091, 1092, 1093, 1094, 1095, 1096, 1097, 1098, 1099, 1100, 1101, 1102, 1103, 1104, 1105, 1106, 1107, 1108, 1109, 1110, 1111, 1112, 1113, 1114, 1115, 1116, 1117, 1118, 1119, 1120, 1121, 1122, 1123, 1124, 1125, 1126, 1127, 1128, 1129, 1130, 1131, 1132, 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, 1151, 1152, 1153, 1154, 1155, 1156, 1157, 1158, 1159, 1160, 1161, 1162, 1163, 1164, 1165, 1166, 1167, 1168, 1169, 1170, 1171, 1172, 1173, 1174, 1175, 1176, 1177, 1178, 1179, 1180, 1181, 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, 1199, 1200, 1201, 1202, 1203, 1204, 1205, 1206, 1207, 1208, 1209, 1210, 1211, 1212, 1213, 1214, 1215, 1216, 1217, and 1218.

The spacer sequence can comprise nucleotide 1 through nucleotide 16 of any one of SEQ ID NOs: 391-770 or 1019-1218. The spacer sequence can comprise nucleotide 1 through nucleotide 17 of any one of SEQ ID NOs: 391-770 or 1019-1218. The spacer sequence can comprise nucleotide 1 through nucleotide 18 of any one of SEQ ID NOs: 391-770 or 1019-1218. The spacer sequence can comprise nucleotide 1 through nucleotide 19 of any one of SEQ ID NOs: 391-770 or 1019-1218. The spacer sequence can comprise nucleotide 1 through nucleotide 20 of any one of SEQ ID NOs: 391-770 or 1019-1218. The spacer sequence can comprise nucleotide 1 through nucleotide 21 of any one of SEQ ID NOs: 391-770. The spacer sequence can comprise nucleotide 1 through nucleotide 22 of any one of SEQ ID NOs: 391-552 and 554-770. The spacer sequence can comprise nucleotide 1 through nucleotide 23 of any one of SEQ ID NOs: 391-552 and 555-770. The spacer sequence can comprise nucleotide 1 through nucleotide 24 of any one of SEQ ID NOs: 391-552 and 556-770. The spacer sequence can comprise nucleotide 1 through nucleotide 25 of any one of SEQ ID NOs: 391-552 and 556-770. The spacer sequence can comprise nucleotide 1 through nucleotide 26 of any one of SEQ ID NOs: 391-552 and 556-770. The spacer sequence can comprise nucleotide 1 through nucleotide 27 of any one of SEQ ID NOs: 391-552 and 556-770. The spacer sequence can comprise nucleotide 1 through nucleotide 28 of any one of SEQ ID NOs: 391-552 and 556-770. The spacer sequence can comprise nucleotide 1 through nucleotide 29 of any one of SEQ ID NOs: 391-552 and 556-770. The spacer sequence can comprise nucleotide 1 through nucleotide 30 of any one of SEQ ID NOs: 391-540, 542-552, and 556-770.

In some embodiments, the spacer sequence has or comprises a sequence having at least 90% identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity) to a sequence of Table 5 or a portion of a sequence of Table 5. The spacer sequence can have or comprise a sequence having at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 16 of any one of SEQ ID NOs: 391-770 or 1019-1218. The spacer sequence can have or comprise a sequence having at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 17 of any one of SEQ ID NOs: 391-770 or 1019-1218. The spacer sequence can have or comprise a sequence having at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 18 of any one of SEQ ID NOs: 391-770 or 1019-1218. The spacer sequence can have or comprise a sequence having at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 19 of any one of SEQ ID NOs: 391-770 or 1019-1218. The spacer sequence can have or comprise a sequence having at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 20 of any one of SEQ ID NOs: 391-770 or 1019-1218. The spacer sequence can have or comprise a sequence having at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 21 of any one of SEQ ID NOs: 391-770. The spacer sequence can have or comprise a sequence having at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 22 of any one of SEQ ID NOs: 391-552 and 554-770. The spacer sequence can have or comprise a sequence having at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 23 of any one of SEQ ID NOs: 391-552 and 555-770. The spacer sequence can have or comprise a sequence having at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 24 of any one of SEQ ID NOs: 391-552 and 556-770. The spacer sequence can have or comprise a sequence having at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 25 of any one of SEQ ID NOs: 391-552 and 556-770. The spacer sequence can have or comprise a sequence having at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 26 of any one of SEQ ID NOs: 391-552 and 556-770. The spacer sequence can have or comprise a sequence having at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 27 of any one of SEQ ID NOs: 391-552 and 556-770. The spacer sequence can have or comprise a sequence having at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 28 of any one of SEQ ID NOs: 391-552 and 556-770. The spacer sequence can have or comprise a sequence having at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 29 of any one of SEQ ID NOs: 391-552 and 556-770. The spacer sequence can have or comprise a sequence having at least 90% identity to a sequence comprising nucleotide 1 through nucleotide 30 of any one of SEQ ID NOs: 391-540, 542-552, and 556-770.

TABLE 5

Target and spacer sequences

B2M exon
strand
PAM
target sequence
spacer sequence

B2M_exon_1
+
TTTA
ATATAAGTGGAGGCGTCGCGCTG
AUAUAAGUGGAGGCGUCGCGCUGGCGGG

GCGGGCA (SEQ ID NO: 11)
CA (SEQ ID NO: 391)

B2M_exon_1
−
CTTA
TATTAAACGCGTGCCCAGCCAAT
UAUUAAACGCGUGCCCAGCCAAUCAGGA

CAGGACA (SEQ ID NO: 12)
CA (SEQ ID NO: 392)

B2M_exon_1
−
CTTC
AGGAATGCCCGCCAGCGCGACGC
AGGAAUGCCCGCCAGCGCGACGCCUCCA

CTCCACT (SEQ ID NO: 13)
CU (SEQ ID NO: 393)

B2M_exon_1
+
TTTC
TGGCCTGGAGGCTATCCAGCGTG
UGGCCUGGAGGCUAUCCAGCGUGAGUCU

AGTCTCT (SEQ ID NO: 14)
CU (SEQ ID NO: 394)

B2M_exon_1
+
CTTT
CTGGCCTGGAGGCTATCCAGCGT
CUGGCCUGGAGGCUAUCCAGCGUGAGUC

GAGTCTC (SEQ ID NO: 15)
UC (SEQ ID NO: 395)

B2M_exon_1
+
CTTA
GCTGTGCTCGCGCTACTCTCTCT
GCUGUGCUCGCGCUACUCUCUCUUUCUG

TTCTGGC (SEQ ID NO: 16)
GC (SEQ ID NO: 396)

B2M_exon_1
+
ATTC
GGGCCGAGATGTCTCGCTCCGTG
GGGCCGAGAUGUCUCGCUCCGUGGCCUU

GCCTTAG (SEQ ID NO: 17)
AG (SEQ ID NO: 397)

B2M_exon_1
+
ATTC
CTGAAGCTGACAGCATTCGGGCC
CUGAAGCUGACAGCAUUCGGGCCGAGAU

GAGATGT (SEQ ID NO: 18)
GU (SEQ ID NO: 398)

B2M_exon_1
+
GTTT
AATATAAGTGGAGGCGTCGCGCT
AAUAUAAGUGGAGGCGUCGCGCUGGCGG

GGCGGGC (SEQ ID NO: 19)
GC (SEQ ID NO: 399)

B2M_exon_1
+
ATTG
GCTGGGCACGCGTTTAATATAAG
GCUGGGCACGCGUUUAAUAUAAGUGGAG

TGGAGGC (SEQ ID NO: 20)
GC (SEQ ID NO: 400)

B2M_exon_2
+
TTTG
TCACAGCCCAAGATAGTTAAGTG
UCACAGCCCAAGAUAGUUAAGUGGGGUA

GGGTAAG (SEQ ID NO: 21)
AG (SEQ ID NO: 401)

B2M_exon_2
+
GTTA
AGTGGGGTAAGTCTTACATTCTT
AGUGGGGUAAGUCUUACAUUCUUUUGUA

TTGTAAG (SEQ ID NO: 22)
AG (SEQ ID NO: 402)

B2M_exon_2
−
TTTC
CATTCTCTGCTGGATGACGTGAG
CAUUCUCUGCUGGAUGACGUGAGUAAAC

TAAACCT (SEQ ID NO: 23)
CU (SEQ ID NO: 403)

B2M_exon_2
+
ATTC
TTTTGTAAGCTGCTGAAAGTTGT
UUUUGUAAGCUGCUGAAAGUUGUGUAUG

GTATGAG (SEQ ID NO: 24)
AG (SEQ ID NO: 404)

B2M_exon_2
+
CTTT
GTCACAGCCCAAGATAGTTAAGT
GUCACAGCCCAAGAUAGUUAAGUGGGGU

GGGGTAA (SEQ ID NO: 25)
AA (SEQ ID NO: 405)

B2M_exon_2
+
CTTA
CATTCTTTTGTAAGCTGCTGAAA
CAUUCUUUUGUAAGCUGCUGAAAGUUGU

GTTGTGT (SEQ ID NO: 26)
GU (SEQ ID NO: 406)

B2M_exon_2
+
ATTC
ACCCCCACTGAAAAAGATGAGTA
ACCCCCACUGAAAAAGAUGAGUAUGCCU

TGCCTGC (SEQ ID NO: 27)
GC (SEQ ID NO: 407)

B2M_exon_2
+
CTTT
CAGCAAGGACTGGTCTTTCTATC
CAGCAAGGACUGGUCUUUCUAUCUCUUG

TCTTGTA (SEQ ID NO: 28)
UA (SEQ ID NO: 408)

B2M_exon_2
+
TTTC
TATCTCTTGTACTACACTGAATT
UAUCUCUUGUACUACACUGAAUUCACCC

CACCCCC (SEQ ID NO: 29)
CC (SEQ ID NO: 409)

B2M_exon_2
+
CTTT
CTATCTCTTGTACTACACTGAAT
CUAUCUCUUGUACUACACUGAAUUCACC

TCACCCC (SEQ ID NO: 30)
CC (SEQ ID NO: 410)

B2M_exon_2
+
TTTC
AGCAAGGACTGGTCTTTCTATCT
AGCAAGGACUGGUCUUUCUAUCUCUUGU

CTTGTAC (SEQ ID NO: 31)
AC (SEQ ID NO: 411)

B2M_exon_2
+
CTTT
TGTAAGCTGCTGAAAGTTGTGTA
UGUAAGCUGCUGAAAGUUGUGUAUGAGU

TGAGTAG (SEQ ID NO: 32)
AG (SEQ ID NO: 412)

B2M_exon_2
+
CTTG
TCTTTCAGCAAGGACTGGTCTTT
UCUUUCAGCAAGGACUGGUCUUUCUAUC

CTATCTC (SEQ ID NO: 33)
UC (SEQ ID NO: 413)

B2M_exon_2
+
CTTG
TACTACACTGAATTCACCCCCAC
UACUACACUGAAUUCACCCCCACUGAAA

TGAAAAA (SEQ ID NO: 34)
AA (SEQ ID NO: 414)

B2M_exon_2
+
TTTT
GTAAGCTGCTGAAAGTTGTGTAT
GUAAGCUGCUGAAAGUUGUGUAUGAGUA

GAGTAGT (SEQ ID NO: 35)
GU (SEQ ID NO: 415)

B2M_exon_2
−
CTTA
CCCCACTTAACTATCTTGGGCTG
CCCCACUUAACUAUCUUGGGCUGUGACA

TGACAAA (SEQ ID NO: 36)
AA (SEQ ID NO: 416)

B2M_exon_2
−
CTTT
CAGCAGCTTACAAAAGAATGTAA
CAGCAGCUUACAAAAGAAUGUAAGACUU

GACTTAC (SEQ ID NO: 37)
AC (SEQ ID NO: 417)

B2M_exon_2
−
TTTC
AGCAGCTTACAAAAGAATGTAAG
AGCAGCUUACAAAAGAAUGUAAGACUUA

ACTTACC (SEQ ID NO: 38)
CC (SEQ ID NO: 418)

B2M_exon_2
−
CTTA
CAAAAGAATGTAAGACTTACCCC
CAAAAGAAUGUAAGACUUACCCCACUUA

ACTTAAC (SEQ ID NO: 39)
AC (SEQ ID NO: 419)

B2M_exon_2
+
ATTC
AGACTTGTCTTTCAGCAAGGACT
AGACUUGUCUUUCAGCAAGGACUGGUCU

GGTCTTT (SEQ ID NO: 40)
UU (SEQ ID NO: 420)

B2M_exon_2
−
CTTA
ACTATCTTGGGCTGTGACAAAGT
ACUAUCUUGGGCUGUGACAAAGUCACAU

CACATGG (SEQ ID NO: 41)
GG (SEQ ID NO: 421)

B2M_exon_2
−
CTTG
GGCTGTGACAAAGTCACATGGTT
GGCUGUGACAAAGUCACAUGGUUCACAC

CACACGG (SEQ ID NO: 42)
GG (SEQ ID NO: 422)

B2M_exon_2
−
GTTC
ACACGGCAGGCATACTCATCTTT
ACACGGCAGGCAUACUCAUCUUUUUCAG

TTCAGTG (SEQ ID NO: 43)
UG (SEQ ID NO: 423)

B2M_exon_2
−
CTTT
TTCAGTGGGGGTGAATTCAGTGT
UUCAGUGGGGGUGAAUUCAGUGUAGUAC

AGTACAA (SEQ ID NO: 44)
AA (SEQ ID NO: 424)

B2M_exon_2
−
TTTT
TCAGTGGGGGTGAATTCAGTGTA
UCAGUGGGGGUGAAUUCAGUGUAGUACA

GTACAAG (SEQ ID NO: 45)
AG (SEQ ID NO: 425)

B2M_exon_2
−
TTTT
CAGTGGGGGTGAATTCAGTGTAG
CAGUGGGGGUGAAUUCAGUGUAGUACAA

TACAAGA (SEQ ID NO: 46)
GA (SEQ ID NO: 426)

B2M_exon_2
−
TTTC
AGTGGGGGTGAATTCAGTGTAGT
AGUGGGGGUGAAUUCAGUGUAGUACAAG

ACAAGAG (SEQ ID NO: 47)
AG (SEQ ID NO: 427)

B2M_exon_2
−
ATTC
AGTGTAGTACAAGAGATAGAAAG
AGUGUAGUACAAGAGAUAGAAAGACCAG

ACCAGTC (SEQ ID NO: 48)
UC (SEQ ID NO: 428)

B2M_exon_2
−
CTTG
CTGAAAGACAAGTCTGAATGCTC
CUGAAAGACAAGUCUGAAUGCUCCACUU

CACTTTT (SEQ ID NO: 49)
UU (SEQ ID NO: 429)

B2M_exon_2
+
TTTG
TAAGCTGCTGAAAGTTGTGTATG
UAAGCUGCUGAAAGUUGUGUAUGAGUAG

AGTAGTC (SEQ ID NO: 50)
UC (SEQ ID NO: 430)

B2M_exon_2
+
ATTG
AAAAAGTGGAGCATTCAGACTTG
AAAAAGUGGAGCAUUCAGACUUGUCUUU

TCTTTCA (SEQ ID NO: 51)
CA (SEQ ID NO: 431)

B2M_exon_2
+
GTTT
CATCCATCCGACATTGAAGTTGA
CAUCCAUCCGACAUUGAAGUUGACUUAC

CTTACTG (SEQ ID NO: 52)
UG (SEQ ID NO: 432)

B2M_exon_2
+
GTTG
ACTTACTGAAGAATGGAGAGAGA
ACUUACUGAAGAAUGGAGAGAGAAUUGA

ATTGAAA (SEQ ID NO: 53)
AA (SEQ ID NO: 433)

B2M_exon_2
−
ATTA
ATATTGCCAGGGTATTTCACTTG
AUAUUGCCAGGGUAUUUCACUUGGGGCU

GGGCTAA (SEQ ID NO: 54)
AA (SEQ ID NO: 434)

B2M_exon_2
−
TTTG
GAGTACCTGAGGAATATCGGGAA
GAGUACCUGAGGAAUAUCGGGAAAAGAC

AAGACAC (SEQ ID NO: 55)
AC (SEQ ID NO: 435)

B2M_exon_2
−
CTTT
GGAGTACCTGAGGAATATCGGGA
GGAGUACCUGAGGAAUAUCGGGAAAAGA

AAAGACA (SEQ ID NO: 56)
CA (SEQ ID NO: 436)

B2M_exon_2
−
ATTC
TCTGCTGGATGACGTGAGTAAAC
UCUGCUGGAUGACGUGAGUAAACCUGAA

CTGAATC (SEQ ID NO: 57)
UC (SEQ ID NO: 437)

B2M_exon_2
−
CTTT
CCATTCTCTGCTGGATGACGTGA
CCAUUCUCUGCUGGAUGACGUGAGUAAA

GTAAACC (SEQ ID NO: 58)
CC (SEQ ID NO: 438)

B2M_exon_2
−
TTTG
ACTTTCCATTCTCTGCTGGATGA
ACUUUCCAUUCUCUGCUGGAUGACGUGA

CGTGAGT (SEQ ID NO: 59)
GU (SEQ ID NO: 439)

B2M_exon_2
−
ATTT
GACTTTCCATTCTCTGCTGGATG
GACUUUCCAUUCUCUGCUGGAUGACGUG

ACGTGAG (SEQ ID NO: 60)
AG (SEQ ID NO: 440)

B2M_exon_2
−
ATTC
AGGAAATTTGACTTTCCATTCTC
AGGAAAUUUGACUUUCCAUUCUCUGCUG

TGCTGGA (SEQ ID NO: 61)
GA (SEQ ID NO: 441)

B2M_exon_2
−
CTTC
AATGTCGGATGGATGAAACCCAG
AAUGUCGGAUGGAUGAAACCCAGACACA

ACACATA (SEQ ID NO: 62)
UA (SEQ ID NO: 442)

B2M_exon_2
−
CTTC
AGTAAGTCAACTTCAATGTCGGA
AGUAAGUCAACUUCAAUGUCGGAUGGAU

TGGATGA (SEQ ID NO: 63)
GA (SEQ ID NO: 443)

B2M_exon_2
−
ATTC
TTCAGTAAGTCAACTTCAATGTC
UUCAGUAAGUCAACUUCAAUGUCGGAUG

GGATGGA (SEQ ID NO: 64)
GA (SEQ ID NO: 444)

B2M_exon_2
−
ATTC
TCTCTCCATTCTTCAGTAAGTCA
UCUCUCCAUUCUUCAGUAAGUCAACUUC

ACTTCAA (SEQ ID NO: 65)
AA (SEQ ID NO: 445)

B2M_exon_2
−
TTTC
AATTCTCTCTCCATTCTTCAGTA
AAUUCUCUCUCCAUUCUUCAGUAAGUCA

AGTCAAC (SEQ ID NO: 66)
AC (SEQ ID NO: 446)

B2M_exon_2
+
CTTA
CTGAAGAATGGAGAGAGAATTGA
CUGAAGAAUGGAGAGAGAAUUGAAAAAG

AAAAGTG (SEQ ID NO: 67)
UG (SEQ ID NO: 447)

B2M_exon_2
−
TTTT
CAATTCTCTCTCCATTCTTCAGT
CAAUUCUCUCUCCAUUCUUCAGUAAGUC

AAGTCAA (SEQ ID NO: 68)
AA (SEQ ID NO: 448)

B2M_exon_2
+
CTTT
TCCCGATATTCCTCAGGTACTCC
UCCCGAUAUUCCUCAGGUACUCCAAAGA

AAAGATT (SEQ ID NO: 69)
UU (SEQ ID NO: 449)

B2M_exon_2
+
TTTT
CCCGATATTCCTCAGGTACTCCA
CCCGAUAUUCCUCAGGUACUCCAAAGAU

AAGATTC (SEQ ID NO: 70)
UC (SEQ ID NO: 450)

B2M_exon_2
+
TTTC
CCGATATTCCTCAGGTACTCCAA
CCGAUAUUCCUCAGGUACUCCAAAGAUU

AGATTCA (SEQ ID NO: 71)
CA (SEQ ID NO: 451)

B2M_exon_2
+
ATTC
CTCAGGTACTCCAAAGATTCAGG
CUCAGGUACUCCAAAGAUUCAGGUUUAC

TTTACTC (SEQ ID NO: 72)
UC (SEQ ID NO: 452)

B2M_exon_2
+
ATTC
AGGTTTACTCACGTCATCCAGCA
AGGUUUACUCACGUCAUCCAGCAGAGAA

GAGAATG (SEQ ID NO: 73)
UG (SEQ ID NO: 453)

B2M_exon_2
+
GTTT
ACTCACGTCATCCAGCAGAGAAT
ACUCACGUCAUCCAGCAGAGAAUGGAAA

GGAAAGT (SEQ ID NO: 74)
GU (SEQ ID NO: 454)

B2M_exon_2
+
TTTA
CTCACGTCATCCAGCAGAGAATG
CUCACGUCAUCCAGCAGAGAAUGGAAAG

GAAAGTC (SEQ ID NO: 75)
UC (SEQ ID NO: 455)

B2M_exon_2
+
ATTT
CCTGAATTGCTATGTGTCTGGGT
CCUGAAUUGCUAUGUGUCUGGGUUUCAU

TTCATCC (SEQ ID NO: 76)
CC (SEQ ID NO: 456)

B2M_exon_2
+
TTTC
CTGAATTGCTATGTGTCTGGGTT
CUGAAUUGCUAUGUGUCUGGGUUUCAUC

TCATCCA (SEQ ID NO: 77)
CA (SEQ ID NO: 457)

B2M_exon_2
+
ATTG
CTATGTGTCTGGGTTTCATCCAT
CUAUGUGUCUGGGUUUCAUCCAUCCGAC

CCGACAT (SEQ ID NO: 78)
AU (SEQ ID NO: 458)

B2M_exon_2
−
CTTT
TTCAATTCTCTCTCCATTCTTCA
UUCAAUUCUCUCUCCAUUCUUCAGUAAG

GTAAGTC (SEQ ID NO: 79)
UC (SEQ ID NO: 459)

B2M_exon_2
+
TTTC
ATCCATCCGACATTGAAGTTGAC
AUCCAUCCGACAUUGAAGUUGACUUACU

TTACTGA (SEQ ID NO: 80)
GA (SEQ ID NO: 460)

B2M_exon_2
+
ATTG
AAGTTGACTTACTGAAGAATGGA
AAGUUGACUUACUGAAGAAUGGAGAGAG

GAGAGAA (SEQ ID NO: 81)
AA (SEQ ID NO: 461)

B2M_exon_2
+
ATTA
ATGTGTCTTTTCCCGATATTCCT
AUGUGUCUUUUCCCGAUAUUCCUCAGGU

CAGGTAC (SEQ ID NO: 82)
AC (SEQ ID NO: 462)

B2M_exon_2
−
TTTT
TCAATTCTCTCTCCATTCTTCAG
UCAAUUCUCUCUCCAUUCUUCAGUAAGU

TAAGTCA (SEQ ID NO: 83)
CA (SEQ ID NO: 463)

B2M_exon_3
+
CTTA
ATGTCTTCCTTTTTTTTCTCCAC
AUGUCUUCCUUUUUUUUCUCCACUGUCU

TGTCTTT (SEQ ID NO: 84)
UU (SEQ ID NO: 464)

B2M_exon_3
−
CTTA
CCTCCATGATGCTGCTTACATGT
CCUCCAUGAUGCUGCUUACAUGUCUCGA

CTCGATC (SEQ ID NO: 85)
UC (SEQ ID NO: 465)

B2M_exon_3
−
CTTA
CATGTCTCGATCTATGAAAAAGA
CAUGUCUCGAUCUAUGAAAAAGACAGUG

CAGTGGA (SEQ ID NO: 86)
GA (SEQ ID NO: 466)

B2M_exon_3
+
CTTC
CTTTTTTTTCTCCACTGTCTTTT
CUUUUUUUUCUCCACUGUCUUUUUCAUA

TCATAGA (SEQ ID NO: 87)
GA (SEQ ID NO: 467)

B2M_exon_3
+
CTTT
TTTTTCTCCACTGTCTTTTTCAT
UUUUUCUCCACUGUCUUUUUCAUAGAUC

AGATCGA (SEQ ID NO: 88)
GA (SEQ ID NO: 468)

B2M_exon_3
+
TTTT
TTTTCTCCACTGTCTTTTTCATA
UUUUCUCCACUGUCUUUUUCAUAGAUCG

GATCGAG (SEQ ID NO: 89)
AG (SEQ ID NO: 469)

B2M_exon_3
+
TTTT
TTTCTCCACTGTCTTTTTCATAG
UUUCUCCACUGUCUUUUUCAUAGAUCGA

ATCGAGA (SEQ ID NO: 90)
GA (SEQ ID NO: 470)

B2M_exon_3
+
TTTT
TCTCCACTGTCTTTTTCATAGAT
UCUCCACUGUCUUUUUCAUAGAUCGAGA

CGAGACA (SEQ ID NO: 91)
CA (SEQ ID NO: 471)

B2M_exon_3
+
TTTT
CTCCACTGTCTTTTTCATAGATC
CUCCACUGUCUUUUUCAUAGAUCGAGAC

GAGACAT (SEQ ID NO: 92)
AU (SEQ ID NO: 472)

B2M_exon_3
+
TTTC
TCCACTGTCTTTTTCATAGATCG
UCCACUGUCUUUUUCAUAGAUCGAGACA

AGACATG (SEQ ID NO: 93)
UG (SEQ ID NO: 473)

B2M_exon_3
+
CTTT
TTCATAGATCGAGACATGTAAGC
UUCAUAGAUCGAGACAUGUAAGCAGCAU

AGCATCA (SEQ ID NO: 94)
CA (SEQ ID NO: 474)

B2M_exon_3
+
TTTT
TCATAGATCGAGACATGTAAGCA
UCAUAGAUCGAGACAUGUAAGCAGCAUC

GCATCAT (SEQ ID NO: 95)
AU (SEQ ID NO: 475)

B2M_exon_3
+
TTTT
CATAGATCGAGACATGTAAGCAG
CAUAGAUCGAGACAUGUAAGCAGCAUCA

CATCATG (SEQ ID NO: 96)
UG (SEQ ID NO: 476)

B2M_exon_3
+
TTTT
TTCTCCACTGTCTTTTTCATAGA
UUCUCCACUGUCUUUUUCAUAGAUCGAG

TCGAGAC (SEQ ID NO: 97)
AC (SEQ ID NO: 477)

B2M_exon_3
+
GTTT
TTGACCTTGAGAAAATGTTTTTG
UUGACCUUGAGAAAAUGUUUUUGUUUCA

TTTCACT (SEQ ID NO: 98)
CU (SEQ ID NO: 478)

B2M_exon_3
+
TTTC
ATAGATCGAGACATGTAAGCAGC
AUAGAUCGAGACAUGUAAGCAGCAUCAU

ATCATGG (SEQ ID NO: 99)
GG (SEQ ID NO: 479)

B2M_exon_3
−
TTTT
CTCAAGGTCAAAAACTTACCTCC
CUCAAGGUCAAAAACUUACCUCCAUGAU

ATGATGC (SEQ ID NO:
GC (SEQ ID NO: 480)

100)

B2M_exon_3
−
ATTT
TCTCAAGGTCAAAAACTTACCTC
UCUCAAGGUCAAAAACUUACCUCCAUGA

CATGATG (SEQ ID NO:
UG (SEQ ID NO: 481)

101)

B2M_exon_3
−
TTTC
TCAAGGTCAAAAACTTACCTCCA
UCAAGGUCAAAAACUUACCUCCAUGAUG

TGATGCT (SEQ ID NO:
CU (SEQ ID NO: 482)

102)

B2M_exon_3
+
GTTT
TTGTTTCACTGTCCTGAGGACTA
UUGUUUCACUGUCCUGAGGACUAUUUAU

TTTATAG (SEQ ID NO:
AG (SEQ ID NO: 483)

103)

B2M_exon_3
+
TTTT
TGTTTCACTGTCCTGAGGACTAT
UGUUUCACUGUCCUGAGGACUAUUUAUA

TTATAGA (SEQ ID NO:
GA (SEQ ID NO: 484)

104)

B2M_exon_3
+
CTTG
AGAAAATGTTTTTGTTTCACTGT
AGAAAAUGUUUUUGUUUCACUGUCCUGA

CCTGAGG (SEQ ID NO:
GG (SEQ ID NO: 485)

105)

B2M_exon_3
+
TTTG
ACCTTGAGAAAATGTTTTTGTTT
ACCUUGAGAAAAUGUUUUUGUUUCACUG

CACTGTC (SEQ ID NO:
UC (SEQ ID NO: 486)

106)

B2M_exon_3
+
TTTT
GACCTTGAGAAAATGTTTTTGTT
GACCUUGAGAAAAUGUUUUUGUUUCACU

TCACTGT (SEQ ID NO:
GU (SEQ ID NO: 487)

107)

B2M_exon_3
+
TTTT
TGACCTTGAGAAAATGTTTTTGT
UGACCUUGAGAAAAUGUUUUUGUUUCAC

TTCACTG (SEQ ID NO:
UG (SEQ ID NO: 488)

108)

B2M_exon_4
−
ATTG
TATTAGGTATTACAAGTAATCTA
UAUUAGGUAUUACAAGUAAUCUAGAAAU

GAAATGA (SEQ ID NO:
GA (SEQ ID NO: 489)

109)

B2M_exon_4
−
ATTT
ACATTGTATTAGGTATTACAAGT
ACAUUGUAUUAGGUAUUACAAGUAAUCU

AATCTAG (SEQ ID NO:
AG (SEQ ID NO: 490)

110)

B2M_exon_4
−
TTTG
CATAGCATTTACATTGTATTAGG
CAUAGCAUUUACAUUGUAUUAGGUAUUA

TATTACA (SEQ ID NO:
CA (SEQ ID NO: 491)

111)

B2M_exon_4
−
ATTT
GCATAGCATTTACATTGTATTAG
GCAUAGCAUUUACAUUGUAUUAGGUAUU

GTATTAC (SEQ ID NO:
AC (SEQ ID NO: 492)

112)

B2M_exon_4
−
TTTA
CATTGTATTAGGTATTACAAGTA
CAUUGUAUUAGGUAUUACAAGUAAUCUA

ATCTAGA (SEQ ID NO:
GA (SEQ ID NO: 493)

113)

B2M_exon_4
−
CTTA
AACAATAACAACTATTTGCATAG
AACAAUAACAACUAUUUGCAUAGCAUUU

CATTTAC (SEQ ID NO:
AC (SEQ ID NO: 494)

114)

B2M_exon_4
−
TTTT
CTTGTCATTATTCCTTAAACAAT
CUUGUCAUUAUUCCUUAAACAAUAACAA

AACAACT (SEQ ID NO:
CU (SEQ ID NO: 495)

115)

B2M_exon_4
−
ATTA
TTCCTTAAACAATAACAACTATT
UUCCUUAAACAAUAACAACUAUUUGCAU

TGCATAG (SEQ ID NO:
AG (SEQ ID NO: 496)

116)

B2M_exon_4
−
CTTG
TCATTATTCCTTAAACAATAACA
UCAUUAUUCCUUAAACAAUAACAACUAU

ACTATTT (SEQ ID NO:
UU (SEQ ID NO: 497)

117)

B2M_exon_4
−
TTTC
TTGTCATTATTCCTTAAACAATA
UUGUCAUUAUUCCUUAAACAAUAACAAC

ACAACTA (SEQ ID NO:
UA (SEQ ID NO: 498)

118)

B2M_exon_4
−
TTTT
TCTTGTCATTATTCCTTAAACAA
UCUUGUCAUUAUUCCUUAAACAAUAACA

TAACAAC (SEQ ID NO:
AC (SEQ ID NO: 499)

119)

B2M_exon_4
−
ATTA
GGTATTACAAGTAATCTAGAAAT
GGUAUUACAAGUAAUCUAGAAAUGAUUU

GATTTAA (SEQ ID NO:
AA (SEQ ID NO: 500)

120)

B2M_exon_4
−
TTTT
TTCTTGTCATTATTCCTTAAACA
UUCUUGUCAUUAUUCCUUAAACAAUAAC

ATAACAA (SEQ ID NO:
AA (SEQ ID NO: 501)

121)

B2M_exon_4
−
ATTC
CTTAAACAATAACAACTATTTGC
CUUAAACAAUAACAACUAUUUGCAUAGC

ATAGCAT (SEQ ID NO:
AU (SEQ ID NO: 502)

122)

B2M_exon_4
−
ATTA
CAAGTAATCTAGAAATGATTTAA
CAAGUAAUCUAGAAAUGAUUUAAAGUAU

AGTATAC (SEQ ID NO:
AC (SEQ ID NO: 503)

123)

B2M_exon_4
−
ATTC
CTTGCTAAAATATTAAATCCTTC
CUUGCUAAAAUAUUAAAUCCUUCAGAUA

AGATACT (SEQ ID NO:
CU (SEQ ID NO: 504)

124)

B2M_exon_4
−
TTTA
AAGTATACAGGAGGATGTGGATA
AAGUAUACAGGAGGAUGUGGAUAGGUUA

GGTTATA (SEQ ID NO:
UA (SEQ ID NO: 505)

125)

B2M_exon_4
−
TTTT
GCTCCCTCTTAGAGTCTGCATAC
GCUCCCUCUUAGAGUCUGCAUACUCCUC

TCCTCAT (SEQ ID NO:
AU (SEQ ID NO: 506)

126)

B2M_exon_4
−
CTTT
TGCTCCCTCTTAGAGTCTGCATA
UGCUCCCUCUUAGAGUCUGCAUACUCCU

CTCCTCA (SEQ ID NO:
CA (SEQ ID NO: 507)

127)

B2M_exon_4
−
CTTC
AGATACTTTTGCTCCCTCTTAGA
AGAUACUUUUGCUCCCUCUUAGAGUCUG

GTCTGCA (SEQ ID NO:
CA (SEQ ID NO: 508)

128)

B2M_exon_4
−
ATTA
AATCCTTCAGATACTTTTGCTCC
AAUCCUUCAGAUACUUUUGCUCCCUCUU

CTCTTAG (SEQ ID NO:
AG (SEQ ID NO: 509)

129)

B2M_exon_4
−
CTTG
CTAAAATATTAAATCCTTCAGAT
CUAAAAUAUUAAAUCCUUCAGAUACUUU

ACTTTTG (SEQ ID NO:
UG (SEQ ID NO: 510)

130)

B2M_exon_4
−
TTTT
TTTCTTGTCATTATTCCTTAAAC
UUUCUUGUCAUUAUUCCUUAAACAAUAA

AATAACA (SEQ ID NO:
CA (SEQ ID NO: 511)

131)

B2M_exon_4
−
ATTT
AAAGTATACAGGAGGATGTGGAT
AAAGUAUACAGGAGGAUGUGGAUAGGUU

AGGTTAT (SEQ ID NO:
AU (SEQ ID NO: 512)

132)

B2M_exon_4
−
ATTG
TATATCTATTCCTTGCTAAAATA
UAUAUCUAUUCCUUGCUAAAAUAUUAAA

TTAAATC (SEQ ID NO:
UC (SEQ ID NO: 513)

133)

B2M_exon_4
−
ATTT
GGTGTCCAAGAAGGGGTCCTGAA
GGUGUCCAAGAAGGGGUCCUGAAACCAA

ACCAATC (SEQ ID NO:
UC (SEQ ID NO: 514)

134)

B2M_exon_4
−
CTTA
AATATCCATAGATTTGGTGTCCA
AAUAUCCAUAGAUUUGGUGUCCAAGAAG

AGAAGGG (SEQ ID NO:
GG (SEQ ID NO: 515)

135)

B2M_exon_4
−
TTTA
TAGAAGGGACTTAAATATCCATA
UAGAAGGGACUUAAAUAUCCAUAGAUUU

GATTTGG (SEQ ID NO:
GG (SEQ ID NO: 516)

136)

B2M_exon_4
−
TTTT
ATAGAAGGGACTTAAATATCCAT
AUAGAAGGGACUUAAAUAUCCAUAGAUU

AGATTTG (SEQ ID NO:
UG (SEQ ID NO: 517)

137)

B2M_exon_4
−
ATTT
TATAGAAGGGACTTAAATATCCA
UAUAGAAGGGACUUAAAUAUCCAUAGAU

TAGATTT (SEQ ID NO:
UU (SEQ ID NO: 518)

138)

B2M_exon_4
−
GTTA
TATGCAAATACTATACCATTTTA
UAUGCAAAUACUAUACCAUUUUAUAGAA

TAGAAGG (SEQ ID NO:
GG (SEQ ID NO: 519)

139)

B2M_exon_4
−
TTTG
GTGTCCAAGAAGGGGTCCTGAAA
GUGUCCAAGAAGGGGUCCUGAAACCAAU

CCAATCC (SEQ ID NO:
CC (SEQ ID NO: 520)

140)

B2M_exon_4
−
TTTT
TTTTCTTGTCATTATTCCTTAAA
UUUUCUUGUCAUUAUUCCUUAAACAAUA

CAATAAC (SEQ ID NO:
AC (SEQ ID NO: 521)

141)

B2M_exon_4
−
TTTT
AAATCTGTTATTTGTCATTCAAA
AAAUCUGUUAUUUGUCAUUCAAAGUACA

GTACAGC (SEQ ID NO:
GC (SEQ ID NO: 522)

142)

B2M_exon_4
−
TTTA
CTGAGCATGTACAGACTTTTTTT
CUGAGCAUGUACAGACUUUUUUUUCUUG

TCTTGTC (SEQ ID NO:
UC (SEQ ID NO: 523)

143)

B2M_exon_4
+
TTTG
CTGAATCCACAGATGTGGAGCCC
CUGAAUCCACAGAUGUGGAGCCCCUGGA

CTGGATA (SEQ ID NO:
UA (SEQ ID NO: 524)

144)

B2M_exon_4
+
GTTT
GCTGAATCCACAGATGTGGAGCC
GCUGAAUCCACAGAUGUGGAGCCCCUGG

CCTGGAT (SEQ ID NO:
AU (SEQ ID NO: 525)

145)

B2M_exon_4
+
TTTG
ATCCATGGTTTGCTGAATCCACA
AUCCAUGGUUUGCUGAAUCCACAGAUGU

GATGTGG (SEQ ID NO:
GG (SEQ ID NO: 526)

146)

B2M_exon_4
+
TTTT
GATCCATGGTTTGCTGAATCCAC
GAUCCAUGGUUUGCUGAAUCCACAGAUG

AGATGTG (SEQ ID NO:
UG (SEQ ID NO: 527)

147)

B2M_exon_4
+
TTTT
TGATCCATGGTTTGCTGAATCCA
UGAUCCAUGGUUUGCUGAAUCCACAGAU

CAGATGT (SEQ ID NO:
GU (SEQ ID NO: 528)

148)

B2M_exon_4
+
GTTT
TTGATCCATGGTTTGCTGAATCC
UUGAUCCAUGGUUUGCUGAAUCCACAGA

ACAGATG (SEQ ID NO:
UG (SEQ ID NO: 529)

149)

B2M_exon_4
+
CTTT
GAATGACAAATAACAGATTTAAA
GAAUGACAAAUAACAGAUUUAAAAUUUU

ATTTTCA (SEQ ID NO:
CA (SEQ ID NO: 530)

150)

B2M_exon_4
+
TTTC
CCCAGTGTTTTTGATCCATGGTT
CCCAGUGUUUUUGAUCCAUGGUUUGCUG

TGCTGAA (SEQ ID NO:
AA (SEQ ID NO: 531)

151)

B2M_exon_4
+
TTTT
TCCCCAGTGTTTTTGATCCATGG
UCCCCAGUGUUUUUGAUCCAUGGUUUGC

TTTGCTG (SEQ ID NO:
UG (SEQ ID NO: 532)

152)

B2M_exon_4
+
TTTT
TTCCCCAGTGTTTTTGATCCATG
UUCCCCAGUGUUUUUGAUCCAUGGUUUG

GTTTGCT (SEQ ID NO:
CU (SEQ ID NO: 533)

153)

B2M_exon_4
+
TTTT
TTTCCCCAGTGTTTTTGATCCAT
UUUCCCCAGUGUUUUUGAUCCAUGGUUU

GGTTTGC (SEQ ID NO:
GC (SEQ ID NO: 534)

154)

B2M_exon_4
+
CTTT
TTTTCCCCAGTGTTTTTGATCCA
UUUUCCCCAGUGUUUUUGAUCCAUGGUU

TGGTTTG (SEQ ID NO:
UG (SEQ ID NO: 535)

155)

B2M_exon_4
+
TTTA
AGGAATAATGACAAGAAAAAAAA
AGGAAUAAUGACAAGAAAAAAAAGUCUG

GTCTGTA (SEQ ID NO:
UA (SEQ ID NO: 536)

156)

B2M_exon_4
−
TTTG
CTCCCTCTTAGAGTCTGCATACT
CUCCCUCUUAGAGUCUGCAUACUCCUCA

CCTCATG (SEQ ID NO:
UG (SEQ ID NO: 537)

157)

B2M_exon_4
+
TTTT
CCCCAGTGTTTTTGATCCATGGT
CCCCAGUGUUUUUGAUCCAUGGUUUGCU

TTGCTGA (SEQ ID NO:
GA (SEQ ID NO: 538)

158)

B2M_exon_4
−
CTTT
TTTTTCTTGTCATTATTCCTTAA
UUUUUCUUGUCAUUAUUCCUUAAACAAU

ACAATAA (SEQ ID NO:
AA (SEQ ID NO: 539)

159)

B2M_exon_4
+
TTTG
AATGACAAATAACAGATTTAAAA
AAUGACAAAUAACAGAUUUAAAAUUUUC

TTTTCAA (SEQ ID NO:
AA (SEQ ID NO: 540)

160)

B2M_exon_4
+
TTTA
AAATTTTCAAGGCATAGTTTTAT
AAAUUUUCAAGGCAUAGUUUUAUACCUG

ACCTGA (SEQ ID NO: 161)
A (SEQ ID NO: 541)

B2M_exon_4
−
CTTT
ACTGAGCATGTACAGACTTTTTT
ACUGAGCAUGUACAGACUUUUUUUUCUU

TTCTTGT (SEQ ID NO:
GU (SEQ ID NO: 542)

162)

B2M_exon_4
−
GTTG
TGTCTTTACTGAGCATGTACAGA
UGUCUUUACUGAGCAUGUACAGACUUUU

CTTTTTT (SEQ ID NO:
UU (SEQ ID NO: 543)

163)

B2M_exon_4
−
ATTC
AGCAAACCATGGATCAAAAACAC
AGCAAACCAUGGAUCAAAAACACUGGGG

TGGGGAA (SEQ ID NO:
AA (SEQ ID NO: 544)

164)

B2M_exon_4
−
CTTC
CGTATCCAGGGGCTCCACATCTG
CGUAUCCAGGGGCUCCACAUCUGUGGAU

TGGATTC (SEQ ID NO:
UC (SEQ ID NO: 545)

165)

B2M_exon_4
−
ATTC
AAAGTACAGCGGGCCTTCCGTAT
AAAGUACAGCGGGCCUUCCGUAUCCAGG

CCAGGGG (SEQ ID NO:
GG (SEQ ID NO: 546)

166)

B2M_exon_4
−
TTTG
TCATTCAAAGTACAGCGGGCCTT
UCAUUCAAAGUACAGCGGGCCUUCCGUA

CCGTATC (SEQ ID NO:
UC (SEQ ID NO: 547)

167)

B2M_exon_4
+
ATTT
AAAATTTTCAAGGCATAGTTTTA
AAAAUUUUCAAGGCAUAGUUUUAUACCU

TACCTGA (SEQ ID NO:
GA (SEQ ID NO: 548)

168)

B2M_exon_4
−
ATTT
GTCATTCAAAGTACAGCGGGCCT
GUCAUUCAAAGUACAGCGGGCCUUCCGU

TCCGTAT (SEQ ID NO:
AU (SEQ ID NO: 549)

169)

B2M_exon_4
−
TTTA
AATCTGTTATTTGTCATTCAAAG
AAUCUGUUAUUUGUCAUUCAAAGUACAG

TACAGCG (SEQ ID NO:
CG (SEQ ID NO: 550)

170)

B2M_exon_4
−
ATTT
TAAATCTGTTATTTGTCATTCAA
UAAAUCUGUUAUUUGUCAUUCAAAGUAC

AGTACAG (SEQ ID NO:
AG (SEQ ID NO: 551)

171)

B2M_exon_4
−
CTTG
AAAATTTTAAATCTGTTATTTGT
AAAAUUUUAAAUCUGUUAUUUGUCAUUC

CATTCAA (SEQ ID NO:
AA (SEQ ID NO: 552)

172)

B2M_exon_4
+
TTTC
AAGGCATAGTTTTATACCTGA
AAGGCAUAGUUUUAUACCUGA (SEQ

(SEQ ID NO: 173)
ID NO: 553)

B2M_exon_4
+
TTTT
CAAGGCATAGTTTTATACCTGA
CAAGGCAUAGUUUUAUACCUGA (SEQ

(SEQ ID NO: 174)
ID NO: 554)

B2M_exon_4
+
ATTT
TCAAGGCATAGTTTTATACCTGA
UCAAGGCAUAGUUUUAUACCUGA (SEQ

(SEQ ID NO: 175)
ID NO: 555)

B2M_exon_4
−
GTTA
TTTGTCATTCAAAGTACAGCGGG
UUUGUCAUUCAAAGUACAGCGGGCCUUC

CCTTCCG (SEQ ID NO:
CG (SEQ ID NO: 556)

176)

B2M_exon_4
−
CTTA
GAGTCTGCATACTCCTCATGACC
GAGUCUGCAUACUCCUCAUGACCUGGCC

TGGCCCG (SEQ ID NO:
CG (SEQ ID NO: 557)

177)

B2M_exon_4
−
CTTA
ACTATCTTAACAAGCTTTGAGTG
ACUAUCUUAACAAGCUUUGAGUGCAAGA

CAAGAGA (SEQ ID NO:
GA (SEQ ID NO: 558)

178)

B2M_exon_4
−
TTTA
ACTTCTTTGAGCATCAGATTCCT
ACUUCUUUGAGCAUCAGAUUCCUAAUCU

AATCIGG (SEQ ID NO:
GG (SEQ ID NO: 559)

179)

B2M_exon_4
−
ATTA
TATTTCTAAATTTTCCCCCAAAT
UAUUUCUAAAUUUUCCCCCAAAUUCUAA

TCTAAGC (SEQ ID NO:
GC (SEQ ID NO: 560)

180)

B2M_exon_4
−
ATTT
CTAAATTTTCCCCCAAATTCTAA
CUAAAUUUUCCCCCAAAUUCUAAGCAGA

GCAGAGT (SEQ ID NO:
GU (SEQ ID NO: 561)

181)

B2M_exon_4
−
TTTC
TAAATTTTCCCCCAAATTCTAAG
UAAAUUUUCCCCCAAAUUCUAAGCAGAG

CAGAGTA (SEQ ID NO:
UA (SEQ ID NO: 562)

182)

B2M_exon_4
−
ATTT
TCCCCCAAATTCTAAGCAGAGTA
UCCCCCAAAUUCUAAGCAGAGUAUGUAA

TGTAAAT (SEQ ID NO:
AU (SEQ ID NO: 563)

183)

B2M_exon_4
−
TTTT
CCCCCAAATTCTAAGCAGAGTAT
CCCCCAAAUUCUAAGCAGAGUAUGUAAA

GTAAATT (SEQ ID NO:
UU (SEQ ID NO: 564)

184)

B2M_exon_4
−
TTTC
CCCCAAATTCTAAGCAGAGTATG
CCCCAAAUUCUAAGCAGAGUAUGUAAAU

TAAATTG (SEQ ID NO:
UG (SEQ ID NO: 565)

185)

B2M_exon_4
−
ATTC
TAAGCAGAGTATGTAAATTGGAA
UAAGCAGAGUAUGUAAAUUGGAAGUUAA

GITAACT (SEQ ID NO:
CU (SEQ ID NO: 566)

186)

B2M_exon_4
−
ATTG
GAAGTTAACTTATGCACGCTTAA
GAAGUUAACUUAUGCACGCUUAACUAUC

CTATCTT (SEQ ID NO:
UU (SEQ ID NO: 567)

187)

B2M_exon_4
−
GTTA
ACTTATGCACGCTTAACTATCTT
ACUUAUGCACGCUUAACUAUCUUAACAA

AACAAGC (SEQ ID NO:
GC (SEQ ID NO: 568)

188)

B2M_exon_4
−
CTTA
TGCACGCTTAACTATCTTAACAA
UGCACGCUUAACUAUCUUAACAAGCUUU

GCTTTGA (SEQ ID NO:
GA (SEQ ID NO: 569)

189)

B2M_exon_4
+
GTTT
AAGGAATAATGACAAGAAAAAAA
AAGGAAUAAUGACAAGAAAAAAAAGUCU

AGTCTGT (SEQ ID NO:
GU (SEQ ID NO: 570)

190)

B2M_exon_4
−
CTTA
ACAAGCTTTGAGTGCAAGAGATT
ACAAGCUUUGAGUGCAAGAGAUUGAAGA

GAAGAGT (SEQ ID NO:
GU (SEQ ID NO: 571)

191)

B2M_exon_4
−
CTTT
GAGTGCAAGAGATTGAAGAGTTC
GAGUGCAAGAGAUUGAAGAGUUCAAAUC

AAATCTG (SEQ ID NO:
UG (SEQ ID NO: 572)

192)

B2M_exon_4
−
TTTG
AGTGCAAGAGATTGAAGAGTTCA
AGUGCAAGAGAUUGAAGAGUUCAAAUCU

AATCTGA (SEQ ID NO:
GA (SEQ ID NO: 573)

193)

B2M_exon_4
−
ATTG
AAGAGTTCAAATCTGACCAAGAT
AAGAGUUCAAAUCUGACCAAGAUGUUGA

GTTGATG (SEQ ID NO:
UG (SEQ ID NO: 574)

194)

B2M_exon_4
−
GTTC
AAATCTGACCAAGATGTTGATGT
AAAUCUGACCAAGAUGUUGAUGUUGGAU

TGGATAA (SEQ ID NO:
AA (SEQ ID NO: 575)

195)

B2M_exon_4
−
GTTG
ATGTTGGATAAGAGAATTCTCTG
AUGUUGGAUAAGAGAAUUCUCUGCUCCC

CTCCCCA (SEQ ID NO:
CA (SEQ ID NO: 576)

196)

B2M_exon_4
−
ATTC
ATCCAATCCAAATGCGGCATCTT
AUCCAAUCCAAAUGCGGCAUCUUCAAAC

CAAACCT (SEQ ID NO:
CU (SEQ ID NO: 577)

197)

B2M_exon_4
−
TTTG
GAATTCATCCAATCCAAATGCGG
GAAUUCAUCCAAUCCAAAUGCGGCAUCU

CATCTTC (SEQ ID NO:
UC (SEQ ID NO: 578)

198)

B2M_exon_4
−
ATTT
GGAATTCATCCAATCCAAATGCG
GGAAUUCAUCCAAUCCAAAUGCGGCAUC

GCATCTT (SEQ ID NO:
UU (SEQ ID NO: 579)

199)

B2M_exon_4
−
ATTA
AAAAGCAAGCAAGCAGAATTTGG
AAAAGCAAGCAAGCAGAAUUUGGAAUUC

AATTCAT (SEQ ID NO:
AU (SEQ ID NO: 580)

200)

B2M_exon_4
−
TTTG
TGCATAAAGTGTAAGTGTATAAG
UGCAUAAAGUGUAAGUGUAUAAGCAUAU

CATATCA (SEQ ID NO:
CA (SEQ ID NO: 581)

201)

B2M_exon_4
−
TTTT
GTGCATAAAGTGTAAGTGTATAA
GUGCAUAAAGUGUAAGUGUAUAAGCAUA

GCATATC (SEQ ID NO:
UC (SEQ ID NO: 582)

202)

B2M_exon_4
−
TTTC
CAATAATCCTGTCAATTATATTT
CAAUAAUCCUGUCAAUUAUAUUUCUAAA

CTAAATT (SEQ ID NO:
UU (SEQ ID NO: 583)

203)

B2M_exon_4
−
ATTT
TGTGCATAAAGTGTAAGTGTATA
UGUGCAUAAAGUGUAAGUGUAUAAGCAU

AGCATAT (SEQ ID NO:
AU (SEQ ID NO: 584)

204)

B2M_exon_4
−
ATTA
TTATAACCCTACATTTTGTGCAT
UUAUAACCCUACAUUUUGUGCAUAAAGU

AAAGTGT (SEQ ID NO:
GU (SEQ ID NO: 585)

205)

B2M_exon_4
−
GTTA
ACATTATTATAACCCTACATTTT
ACAUUAUUAUAACCCUACAUUUUGUGCA

GTGCATA (SEQ ID NO:
UA (SEQ ID NO: 586)

206)

B2M_exon_4
−
ATTA
TAAAGAAGATCATGTCCATGTTA
UAAAGAAGAUCAUGUCCAUGUUAACAUU

ACATTAT (SEQ ID NO:
AU (SEQ ID NO: 587)

207)

B2M_exon_4
−
GTTG
CCAGCCCTCCTAGAGCTACCTGT
CCAGCCCUCCUAGAGCUACCUGUGGAGC

GGAGCAA (SEQ ID NO:
AA (SEQ ID NO: 588)

208)

B2M_exon_4
−
ATTC
TCTGCTCCCCACCTCTAAGTTGC
UCUGCUCCCCACCUCUAAGUUGCCAGCC

CAGCCCT (SEQ ID NO:
CU (SEQ ID NO: 589)

209)

B2M_exon_4
−
GTTG
GATAAGAGAATTCTCTGCTCCCC
GAUAAGAGAAUUCUCUGCUCCCCACCUC

ACCTCTA (SEQ ID NO:
UA (SEQ ID NO: 590)

210)

B2M_exon_4
−
ATTA
TAACCCTACATTTTGTGCATAAA
UAACCCUACAUUUUGUGCAUAAAGUGUA

GTGTAAG (SEQ ID NO:
AG (SEQ ID NO: 591)

211)

B2M_exon_4
−
ATTT
CCAATAATCCTGTCAATTATATT
CCAAUAAUCCUGUCAAUUAUAUUUCUAA

TCTAAAT (SEQ ID NO:
AU (SEQ ID NO: 592)

212)

B2M_exon_4
−
ATTA
TAACAAATTTCCAATAATCCTGT
UAACAAAUUUCCAAUAAUCCUGUCAAUU

CAATTAT (SEQ ID NO:
AU (SEQ ID NO: 593)

213)

B2M_exon_4
−
ATTC
ATTATAACAAATTTCCAATAATC
AUUAUAACAAAUUUCCAAUAAUCCUGUC

CTGTCAA (SEQ ID NO:
AA (SEQ ID NO: 594)

214)

B2M_exon_4
−
GTTT
CCCTGTTTGAAAATAAAGGGGTA
CCCUGUUUGAAAAUAAAGGGGUAAUAGU

ATAGTGG (SEQ ID NO:
GG (SEQ ID NO: 595)

215)

B2M_exon_4
−
CTTG
AAGACTGTTTCCCTGTTTGAAAA
AAGACUGUUUCCCUGUUUGAAAAUAAAG

TAAAGGG (SEQ ID NO:
GG (SEQ ID NO: 596)

216)

B2M_exon_4
−
TTTA
CCAAGTGGAACTTGAAGACTGTT
CCAAGUGGAACUUGAAGACUGUUUCCCU

TCCCTGT (SEQ ID NO:
GU (SEQ ID NO: 597)

217)

B2M_exon_4
−
TTTT
ACCAAGTGGAACTTGAAGACTGT
ACCAAGUGGAACUUGAAGACUGUUUCCC

TTCCCTG (SEQ ID NO:
UG (SEQ ID NO: 598)

218)

B2M_exon_4
−
TTTT
TACCAAGTGGAACTTGAAGACTG
UACCAAGUGGAACUUGAAGACUGUUUCC

TTTCCCT (SEQ ID NO:
CU (SEQ ID NO: 599)

219)

B2M_exon_4
−
TTTT
TTACCAAGTGGAACTTGAAGACT
UUACCAAGUGGAACUUGAAGACUGUUUC

GTTTCCC (SEQ ID NO:
CC (SEQ ID NO: 600)

220)

B2M_exon_4
−
TTTC
CCTGTTTGAAAATAAAGGGGTAA
CCUGUUUGAAAAUAAAGGGGUAAUAGUG

TAGTGGG (SEQ ID NO:
GG (SEQ ID NO: 601)

221)

B2M_exon_4
−
ATTT
TTTACCAAGTGGAACTTGAAGAC
UUUACCAAGUGGAACUUGAAGACUGUUU

TGTTTCC (SEQ ID NO:
CC (SEQ ID NO: 602)

222)

B2M_exon_4
−
TTTA
CACTGTGAGCCAAACTCTATATA
CACUGUGAGCCAAACUCUAUAUACAAGG

CAAGGGG (SEQ ID NO:
GG (SEQ ID NO: 603)

223)

B2M_exon_4
−
CTTT
ACACTGTGAGCCAAACTCTATAT
ACACUGUGAGCCAAACUCUAUAUACAAG

ACAAGGG (SEQ ID NO:
GG (SEQ ID NO: 604)

224)

B2M_exon_4
−
ATTC
CTAATCTGGAAAATGTGAATCAC
CUAAUCUGGAAAAUGUGAAUCACUGAGG

TGAGGCC (SEQ ID NO:
CC (SEQ ID NO: 605)

225)

B2M_exon_4
−
TTTG
AGCATCAGATTCCTAATCTGGAA
AGCAUCAGAUUCCUAAUCUGGAAAAUGU

AATGTGA (SEQ ID NO:
GA (SEQ ID NO: 606)

226)

B2M_exon_4
−
CTTT
GAGCATCAGATTCCTAATCTGGA
GAGCAUCAGAUUCCUAAUCUGGAAAAUG

AAATGTG (SEQ ID NO:
UG (SEQ ID NO: 607)

227)

B2M_exon_4
−
CTTC
TTTGAGCATCAGATTCCTAATCT
UUUGAGCAUCAGAUUCCUAAUCUGGAAA

GGAAAAT (SEQ ID NO:
AU (SEQ ID NO: 608)

228)

B2M_exon_4
−
GTTC
ACATTTTTTACCAAGTGGAACTT
ACAUUUUUUACCAAGUGGAACUUGAAGA

GAAGACT (SEQ ID NO:
CU (SEQ ID NO: 609)

229)

B2M_exon_4
−
ATTT
AACTTCTTTGAGCATCAGATTCC
AACUUCUUUGAGCAUCAGAUUCCUAAUC

TAATCTG (SEQ ID NO:
UG (SEQ ID NO: 610)

230)

B2M_exon_4
−
GTTT
GAAAATAAAGGGGTAATAGTGGG
GAAAAUAAAGGGGUAAUAGUGGGAGUGA

AGTGAGA (SEQ ID NO:
GA (SEQ ID NO: 611)

231)

B2M_exon_4
−
GTTT
TATGATTTATTTAACTTGTGGAA
UAUGAUUUAUUUAACUUGUGGAACAAAA

CAAAAAT (SEQ ID NO:
AU (SEQ ID NO: 612)

232)

B2M_exon_4
−
TTTC
ATTCATTATAACAAATTTCCAAT
AUUCAUUAUAACAAAUUUCCAAUAAUCC

AATCCTG (SEQ ID NO:
UG (SEQ ID NO: 613)

233)

B2M_exon_4
−
GTTT
CATTCATTATAACAAATTTCCAA
CAUUCAUUAUAACAAAUUUCCAAUAAUC

TAATCCT (SEQ ID NO:
CU (SEQ ID NO: 614)

234)

B2M_exon_4
−
CTTA
TATGACAAAATGTTTCATTCATT
UAUGACAAAAUGUUUCAUUCAUUAUAAC

ATAACAA (SEQ ID NO:
AA (SEQ ID NO: 615)

235)

B2M_exon_4
−
TTTA
TCAAATGTATAAGAAGTAAATAT
UCAAAUGUAUAAGAAGUAAAUAUGAAUC

GAATCTT (SEQ ID NO:
UU (SEQ ID NO: 616)

236)

B2M_exon_4
−
CTTT
ATCAAATGTATAAGAAGTAAATA
AUCAAAUGUAUAAGAAGUAAAUAUGAAU

TGAATCT (SEQ ID NO:
CU (SEQ ID NO: 617)

237)

B2M_exon_4
−
CTTA
CTTTATCAAATGTATAAGAAGTA
CUUUAUCAAAUGUAUAAGAAGUAAAUAU

AATATGA (SEQ ID NO:
GA (SEQ ID NO: 618)

238)

B2M_exon_4
−
TTTG
AAAATAAAGGGGTAATAGTGGGA
AAAAUAAAGGGGUAAUAGUGGGAGUGAG

GTGAGAT (SEQ ID NO:
AU (SEQ ID NO: 619)

239)

B2M_exon_4
−
ATTA
ACCACAACCATGCCTTACTTTAT
ACCACAACCAUGCCUUACUUUAUCAAAU

CAAATGT (SEQ ID NO:
GU (SEQ ID NO: 620)

240)

B2M_exon_4
−
TTTA
ACTTGTGGAACAAAAATAAACCA
ACUUGUGGAACAAAAAUAAACCAGAUUA

GATTAAC (SEQ ID NO:
AC (SEQ ID NO: 621)

241)

B2M_exon_4
−
ATTT
AACTTGTGGAACAAAAATAAACC
AACUUGUGGAACAAAAAUAAACCAGAUU

AGATTAA (SEQ ID NO:
AA (SEQ ID NO: 622)

242)

B2M_exon_4
−
TTTA
TTTAACTTGTGGAACAAAAATAA
UUUAACUUGUGGAACAAAAAAAACCAG

ACCAGAT (SEQ ID NO:
AU (SEQ ID NO: 623)

243)

B2M_exon_4
−
ATTT
ATTTAACTTGTGGAACAAAAATA
AUUUAACUUGUGGAACAAAAAUAAACCA

AACCAGA (SEQ ID NO:
GA (SEQ ID NO: 624)

244)

B2M_exon_4
−
TTTA
TGATTTATTTAACTTGTGGAACA
UGAUUUAUUUAACUUGUGGAACAAAAAU

AAAATAA (SEQ ID NO:
AA (SEQ ID NO: 625)

245)

B2M_exon_4
−
TTTT
ATGATTTATTTAACTTGTGGAAC
AUGAUUUAUUUAACUUGUGGAACAAAAA

AAAAATA (SEQ ID NO:
UA (SEQ ID NO: 626)

246)

B2M_exon_4
−
CTTG
TGGAACAAAAATAAACCAGATTA
UGGAACAAAAAUAAACCAGAUUAACCAC

ACCACAA (SEQ ID NO:
AA (SEQ ID NO: 627)

247)

B2M_exon_4
+
ATTG
TTTAAGGAATAATGACAAGAAAA
UUUAAGGAAUAAUGACAAGAAAAAAAAG

AAAAGTC (SEQ ID NO:
UC (SEQ ID NO: 628)

248)

B2M_exon_4
+
CTTT
ATTTTCAAACAGGGAAACAGTCT
AUUUUCAAACAGGGAAACAGUCUUCAAG

TCAAGTT (SEQ ID NO:
UU (SEQ ID NO: 629)

249)

B2M_exon_4
+
GTTG
TTATTGTTTAAGGAATAATGACA
UUAUUGUUUAAGGAAUAAUGACAAGAAA

AGAAAAA (SEQ ID NO:
AA (SEQ ID NO: 630)

250)

B2M_exon_4
+
CTTT
GAGTGCTGTCTCCATGTTTGATG
GAGUGCUGUCUCCAUGUUUGAUGUAUCU

TATCTGA (SEQ ID NO:
GA (SEQ ID NO: 631)

251)

B2M_exon_4
+
TTTG
AGTGCTGTCTCCATGTTTGATGT
AGUGCUGUCUCCAUGUUUGAUGUAUCUG

ATCTGAG (SEQ ID NO:
AG (SEQ ID NO: 632)

252)

B2M_exon_4
+
GTTT
GATGTATCTGAGCAGGTTGCTCC
GAUGUAUCUGAGCAGGUUGCUCCACAGG

ACAGGTA (SEQ ID NO:
UA (SEQ ID NO: 633)

253)

B2M_exon_4
+
TTTG
ATGTATCTGAGCAGGTTGCTCCA
AUGUAUCUGAGCAGGUUGCUCCACAGGU

CAGGTAG (SEQ ID NO:
AG (SEQ ID NO: 634)

254)

B2M_exon_4
+
GTTG
CTCCACAGGTAGCTCTAGGAGGG
CUCCACAGGUAGCUCUAGGAGGGCUGGC

CTGGCAA (SEQ ID NO:
AA (SEQ ID NO: 635)

255)

B2M_exon_4
+
CTTA
GAGGTGGGGAGCAGAGAATTCTC
GAGGUGGGGAGCAGAGAAUUCUCUUAUC

TTATCCA (SEQ ID NO:
CA (SEQ ID NO: 636)

256)

B2M_exon_4
+
ATTC
TCTTATCCAACATCAACATCTTG
UCUUAUCCAACAUCAACAUCUUGGUCAG

GTCAGAT (SEQ ID NO:
AU (SEQ ID NO: 637)

257)

B2M_exon_4
+
CTTA
TCCAACATCAACATCTTGGTCAG
UCCAACAUCAACAUCUUGGUCAGAUUUG

ATTTGAA (SEQ ID NO:
AA (SEQ ID NO: 638)

258)

B2M_exon_4
+
CTTG
GTCAGATTTGAACTCTTCAATCT
GUCAGAUUUGAACUCUUCAAUCUCUUGC

CTTGCAC (SEQ ID NO:
AC (SEQ ID NO: 639)

259)

B2M_exon_4
+
ATTT
GAACTCTTCAATCTCTTGCACTC
GAACUCUUCAAUCUCUUGCACUCAAAGC

AAAGCTT (SEQ ID NO:
UU (SEQ ID NO: 640)

260)

B2M_exon_4
+
TTTG
AACTCTTCAATCTCTTGCACTCA
AACUCUUCAAUCUCUUGCACUCAAAGCU

AAGCTTG (SEQ ID NO:
UG (SEQ ID NO: 641)

261)

B2M_exon_4
+
CTTC
AATCTCTTGCACTCAAAGCTTGT
AAUCUCUUGCACUCAAAGCUUGUUAAGA

TAAGATA (SEQ ID NO:
UA (SEQ ID NO: 642)

262)

B2M_exon_4
+
CTTG
CACTCAAAGCTTGTTAAGATAGT
CACUCAAAGCUUGUUAAGAUAGUUAAGC

TAAGCGT (SEQ ID NO:
GU (SEQ ID NO: 643)

263)

B2M_exon_4
+
CTTG
TTAAGATAGTTAAGCGTGCATAA
UUAAGAUAGUUAAGCGUGCAUAAGUUAA

GTTAACT (SEQ ID NO:
CU (SEQ ID NO: 644)

264)

B2M_exon_4
+
GTTA
AGATAGTTAAGCGTGCATAAGTT
AGAUAGUUAAGCGUGCAUAAGUUAACUU

AACTTCC (SEQ ID NO:
CC (SEQ ID NO: 645)

265)

B2M_exon_4
+
GTTA
AGCGTGCATAAGTTAACTTCCAA
AGCGUGCAUAAGUUAACUUCCAAUUUAC

TTTACAT (SEQ ID NO:
AU (SEQ ID NO: 646)

266)

B2M_exon_4
+
GTTA
ACTTCCAATTTACATACTCTGCT
ACUUCCAAUUUACAUACUCUGCUUAGAA

TAGAATT (SEQ ID NO:
UU (SEQ ID NO: 647)

267)

B2M_exon_4
+
GTTA
TAATGAATGAAACATTTTGTCAT
UAAUGAAUGAAACAUUUUGUCAUAUAAG

ATAAGAT (SEQ ID NO:
AU (SEQ ID NO: 648)

268)

B2M_exon_4
+
TTTG
TTATAATGAATGAAACATTTTGT
UUAUAAUGAAUGAAACAUUUUGUCAUAU

CATATAA (SEQ ID NO:
AA (SEQ ID NO: 649)

269)

B2M_exon_4
+
ATTT
GTTATAATGAATGAAACATTTTG
GUUAUAAUGAAUGAAACAUUUUGUCAUA

TCATATA (SEQ ID NO:
UA (SEQ ID NO: 650)

270)

B2M_exon_4
+
ATTG
GAAATTTGTTATAATGAATGAAA
GAAAUUUGUUAUAAUGAAUGAAACAUUU

CATTTTG (SEQ ID NO:
UG (SEQ ID NO: 651)

271)

B2M_exon_4
+
ATTA
TTGGAAATTTGTTATAATGAATG
UUGGAAAUUUGUUAUAAUGAAUGAAACA

AAACATT (SEQ ID NO:
UU (SEQ ID NO: 652)

272)

B2M_exon_4
+
ATTG
ACAGGATTATTGGAAATTTGTTA
ACAGGAUUAUUGGAAAUUUGUUAUAAUG

TAATGAA (SEQ ID NO:
AA (SEQ ID NO: 653)

273)

B2M_exon_4
+
ATTC
TACTTTGAGTGCTGTCTCCATGT
UACUUUGAGUGCUGUCUCCAUGUUUGAU

TTGATGT (SEQ ID NO:
GU (SEQ ID NO: 654)

274)

B2M_exon_4
+
TTTA
GAAATATAATTGACAGGATTATT
GAAAUAUAAUUGACAGGAUUAUUGGAAA

GGAAATT (SEQ ID NO:
UU (SEQ ID NO: 655)

275)

B2M_exon_4
+
TTTG
GGGGAAAATTTAGAAATATAATT
GGGGAAAAUUUAGAAAUAUAAUUGACAG

GACAGGA (SEQ ID NO:
GA (SEQ ID NO: 656)

276)

B2M_exon_4
+
ATTT
GGGGGAAAATTTAGAAATATAAT
GGGGGAAAAUUUAGAAAUAUAAUUGACA

TGACAGG (SEQ ID NO:
GG (SEQ ID NO: 657)

277)

B2M_exon_4
+
CTTA
GAATTTGGGGGAAAATTTAGAAA
GAAUUUGGGGGAAAAUUUAGAAAUAUAA

TATAATT (SEQ ID NO:
UU (SEQ ID NO: 658)

278)

B2M_exon_4
+
TTTA
CATACTCTGCTTAGAATTTGGGG
CAUACUCUGCUUAGAAUUUGGGGGAAAA

GAAAATT (SEQ ID NO:
UU (SEQ ID NO: 659)

279)

B2M_exon_4
+
ATTT
ACATACTCTGCTTAGAATTTGGG
ACAUACUCUGCUUAGAAUUUGGGGGAAA

GGAAAAT (SEQ ID NO:
AU (SEQ ID NO: 660)

280)

B2M_exon_4
+
CTTC
CAATTTACATACTCTGCTTAGAA
CAAUUUACAUACUCUGCUUAGAAUUUGG

TTTGGGG (SEQ ID NO:
GG (SEQ ID NO: 661)

281)

B2M_exon_4
+
ATTT
AGAAATATAATTGACAGGATTAT
AGAAAUAUAAUUGACAGGAUUAUUGGAA

TGGAAAT (SEQ ID NO:
AU (SEQ ID NO: 662)

282)

B2M_exon_4
+
TTTA
TAATTCTACTTTGAGTGCTGTCT
UAAUUCUACUUUGAGUGCUGUCUCCAUG

CCATGTT (SEQ ID NO:
UU (SEQ ID NO: 663)

283)

B2M_exon_4
+
CTTT
ATAATTCTACTTTGAGTGCTGTC
AUAAUUCUACUUUGAGUGCUGUCUCCAU

TCCATGT (SEQ ID NO:
GU (SEQ ID NO: 664)

284)

B2M_exon_4
+
CTTC
TTTATAATTCTACTTTGAGTGCT
UUUAUAAUUCUACUUUGAGUGCUGUCUC

GTCTCCA (SEQ ID NO:
CA (SEQ ID NO: 665)

285)

B2M_exon_4
+
TTTG
AAGATGCCGCATTTGGATTGGAT
AAGAUGCCGCAUUUGGAUUGGAUGAAUU

GAATTCC (SEQ ID NO:
CC (SEQ ID NO: 666)

286)

B2M_exon_4
+
GTTT
GAAGATGCCGCATTTGGATTGGA
GAAGAUGCCGCAUUUGGAUUGGAUGAAU

TGAATTC (SEQ ID NO:
UC (SEQ ID NO: 667)

287)

B2M_exon_4
+
TTTC
AGGTTTGAAGATGCCGCATTTGG
AGGUUUGAAGAUGCCGCAUUUGGAUUGG

ATTGGAT (SEQ ID NO:
AU (SEQ ID NO: 668)

288)

B2M_exon_4
+
TTTT
CAGGTTTGAAGATGCCGCATTTG
CAGGUUUGAAGAUGCCGCAUUUGGAUUG

GATTGGA (SEQ ID NO:
GA (SEQ ID NO: 669)

289)

B2M_exon_4
+
CTTT
TCAGGTTTGAAGATGCCGCATTT
UCAGGUUUGAAGAUGCCGCAUUUGGAUU

GGATTGG (SEQ ID NO:
GG (SEQ ID NO: 670)

290)

B2M_exon_4
+
TTTC
TTTTCAGGTTTGAAGATGCCGCA
UUUUCAGGUUUGAAGAUGCCGCAUUUGG

TTTGGAT (SEQ ID NO:
AU (SEQ ID NO: 671)

291)

B2M_exon_4
+
ATTT
GGATTGGATGAATTCCAAATTCT
GGAUUGGAUGAAUUCCAAAUUCUGCUUG

GCTTGCT (SEQ ID NO:
CU (SEQ ID NO: 672)

292)

B2M_exon_4
+
TTTT
CTTTTCAGGTTTGAAGATGCCGC
CUUUUCAGGUUUGAAGAUGCCGCAUUUG

ATTTGGA (SEQ ID NO:
GA (SEQ ID NO: 673)

293)

B2M_exon_4
+
TTTC
TTTTCTTTTCAGGTTTGAAGATG
UUUUCUUUUCAGGUUUGAAGAUGCCGCA

CCGCATT (SEQ ID NO:
UU (SEQ ID NO: 674)

294)

B2M_exon_4
+
TTTT
CTTTTCTTTTCAGGTTTGAAGAT
CUUUUCUUUUCAGGUUUGAAGAUGCCGC

GCCGCAT (SEQ ID NO:
AU (SEQ ID NO: 675)

295)

B2M_exon_4
+
TTTT
TCTTTTCTTTTCAGGTTTGAAGA
UCUUUUCUUUUCAGGUUUGAAGAUGCCG

TGCCGCA (SEQ ID NO:
CA (SEQ ID NO: 676)

296)

B2M_exon_4
+
CTTT
TTCTTTTCTTTTCAGGTTTGAAG
UUCUUUUCUUUUCAGGUUUGAAGAUGCC

ATGCCGC (SEQ ID NO:
GC (SEQ ID NO: 677)

297)

B2M_exon_4
+
ATTG
CTAACCTTTTTCTTTTCTTTTCA
CUAACCUUUUUCUUUUCUUUUCAGGUUU

GGTTTGA (SEQ ID NO:
GA (SEQ ID NO: 678)

298)

B2M_exon_4
+
ATTC
ATTGCTAACCTTTTTCTTTTCTT
AUUGCUAACCUUUUUCUUUUUUUUCAG

TTCAGGT (SEQ ID NO:
GU (SEQ ID NO: 679)

299)

B2M_exon_4
+
CTTT
TCTTTTCAGGTTTGAAGATGCCG
UCUUUUCAGGUUUGAAGAUGCCGCAUUU

CATTTGG (SEQ ID NO:
GG (SEQ ID NO: 680)

300)

B2M_exon_4
+
ATTT
TGTCATATAAGATTCATATTTAC
UGUCAUAUAAGAUUCAUAUUUACUUCUU

TTCTTAT (SEQ ID NO:
AU (SEQ ID NO: 681)

301)

B2M_exon_4
+
TTTG
GATTGGATGAATTCCAAATTCTG
GAUUGGAUGAAUUCCAAAUUCUGCUUGC

CTTGCTT (SEQ ID NO:
UU (SEQ ID NO: 682)

302)

B2M_exon_4
+
ATTC
CAAATTCTGCTTGCTTGCTTTTT
CAAAUUCUGCUUGCUUGCUUUUUAAUAU

AATATTG (SEQ ID NO:
UG (SEQ ID NO: 683)

303)

B2M_exon_4
+
GTTA
ACATGGACATGATCTTCTTTATA
ACAUGGACAUGAUCUUCUUUAUAAUUCU

ATTCTAC (SEQ ID NO:
AC (SEQ ID NO: 684)

304)

B2M_exon_4
+
GTTA
TAATAATGTTAACATGGACATGA
UAAUAAUGUUAACAUGGACAUGAUCUUC

TCTTCTT (SEQ ID NO:
UU (SEQ ID NO: 685)

305)

B2M_exon_4
+
TTTA
TGCACAAAATGTAGGGTTATAAT
UGCACAAAAUGUAGGGUUAUAAUAAUGU

AATGTTA (SEQ ID NO:
UA (SEQ ID NO: 686)

306)

B2M_exon_4
+
CTTT
ATGCACAAAATGTAGGGTTATAA
AUGCACAAAAUGUAGGGUUAUAAUAAUG

TAATGTT (SEQ ID NO:
UU (SEQ ID NO: 687)

307)

B2M_exon_4
+
CTTA
CACTTTATGCACAAAATGTAGGG
CACUUUAUGCACAAAAUGUAGGGUUAUA

TTATAAT (SEQ ID NO:
AU (SEQ ID NO: 688)

308)

B2M_exon_4
+
CTTA
TACACTTACACTTTATGCACAAA
UACACUUACACUUUAUGCACAAAAUGUA

ATGTAGG (SEQ ID NO:
GG (SEQ ID NO: 689)

309)

B2M_exon_4
+
ATTG
GATGAATTCCAAATTCTGCTTGC
GAUGAAUUCCAAAUUCUGCUUGCUUGCU

TTGCTTT (SEQ ID NO:
UU (SEQ ID NO: 690)

310)

B2M_exon_4
+
ATTG
ATATGCTTATACACTTACACTTT
AUAUGCUUAUACACUUACACUUUAUGCA

ATGCACA (SEQ ID NO:
CA (SEQ ID NO: 691)

311)

B2M_exon_4
+
TTTT
AATATTGATATGCTTATACACTT
AAUAUUGAUAUGCUUAUACACUUACACU

ACACTTT (SEQ ID NO:
UU (SEQ ID NO: 692)

312)

B2M_exon_4
+
TTTT
TAATATTGATATGCTTATACACT
UAAUAUUGAUAUGCUUAUACACUUACAC

TACACTT (SEQ ID NO:
UU (SEQ ID NO: 693)

313)

B2M_exon_4
+
CTTT
TTAATATTGATATGCTTATACAC
UUAAUAUUGAUAUGCUUAUACACUUACA

TTACACT (SEQ ID NO:
CU (SEQ ID NO: 694)

314)

B2M_exon_4
+
CTTG
CTTTTTAATATTGATATGCTTAT
CUUUUUAAUAUUGAUAUGCUUAUACACU

ACACTTA (SEQ ID NO:
UA (SEQ ID NO: 695)

315)

B2M_exon_4
+
CTTG
CTTGCTTTTTAATATTGATATGC
CUUGCUUUUUAAUAUUGAUAUGCUUAUA

TTATACA (SEQ ID NO:
CA (SEQ ID NO: 696)

316)

B2M_exon_4
+
ATTC
TGCTTGCTTGCTTTTTAATATTG
UGCUUGCUUGCUUUUUAAUAUUGAUAUG

ATATGCT (SEQ ID NO:
CU (SEQ ID NO: 697)

317)

B2M_exon_4
+
TTTA
ATATTGATATGCTTATACACTTA
AUAUUGAUAUGCUUAUACACUUACACUU

CACTTTA (SEQ ID NO:
UA (SEQ ID NO: 698)

318)

B2M_exon_4
+
GTTA
TTGTTTAAGGAATAATGACAAGA
UUGUUUAAGGAAUAAUGACAAGAAAAAA

AAAAAAA (SEQ ID NO:
AA (SEQ ID NO: 699)

319)

B2M_exon_4
+
TTTT
GTCATATAAGATTCATATTTACT
GUCAUAUAAGAUUCAUAUUUACUUCUUA

TCTTATA (SEQ ID NO:
UA (SEQ ID NO: 700)

320)

B2M_exon_4
+
ATTC
ATATTTACTTCTTATACATTTGA
AUAUUUACUUCUUAUACAUUUGAUAAAG

TAAAGTA (SEQ ID NO:
UA (SEQ ID NO: 701)

321)

B2M_exon_4
+
GTTA
AATGGCATAGTTGGGGTGACACA
AAUGGCAUAGUUGGGGUGACACAGCUGU

GCTGTCT (SEQ ID NO:
CU (SEQ ID NO: 702)

322)

B2M_exon_4
+
GTTG
GGGTGACACAGCTGTCTAGTGGG
GGGUGACACAGCUGUCUAGUGGGAGGCC

AGGCCAG (SEQ ID NO:
AG (SEQ ID NO: 703)

323)

B2M_exon_4
+
CTTC
TATATTTTAGCCAGCGTTCTTTC
UAUAUUUUAGCCAGCGUUCUUUCCUGCG

CTGCGGG (SEQ ID NO:
GG (SEQ ID NO: 704)

324)

B2M_exon_4
+
ATTT
TAGCCAGCGTTCTTTCCTGCGGG
UAGCCAGCGUUCUUUCCUGCGGGCCAGG

CCAGGTC (SEQ ID NO:
UC (SEQ ID NO: 705)

325)

B2M_exon_4
+
TTTT
AGCCAGCGTTCTTTCCTGCGGGC
AGCCAGCGUUCUUUCCUGCGGGCCAGGU

CAGGTCA (SEQ ID NO:
CA (SEQ ID NO: 706)

326)

B2M_exon_4
+
TTTA
GCCAGCGTTCTTTCCTGCGGGCC
GCCAGCGUUCUUUCCUGCGGGCCAGGUC

AGGTCAT (SEQ ID NO:
AU (SEQ ID NO: 707)

327)

B2M_exon_4
+
GTTC
TTTCCTGCGGGCCAGGTCATGAG
UUUCCUGCGGGCCAGGUCAUGAGGAGUA

GAGTATG (SEQ ID NO:
UG (SEQ ID NO: 708)

328)

B2M_exon_4
+
CTTT
CCTGCGGGCCAGGTCATGAGGAG
CCUGCGGGCCAGGUCAUGAGGAGUAUGC

TATGCAG (SEQ ID NO:
AG (SEQ ID NO: 709)

329)

B2M_exon_4
+
TTTC
CTGCGGGCCAGGTCATGAGGAGT
CUGCGGGCCAGGUCAUGAGGAGUAUGCA

ATGCAGA (SEQ ID NO:
GA (SEQ ID NO: 710)

330)

B2M_exon_4
+
ATTT
AATATTTTAGCAAGGAATAGATA
AAUAUUUUAGCAAGGAAUAGAUAUACAA

TACAATC (SEQ ID NO:
UC (SEQ ID NO: 711)

331)

B2M_exon_4
+
TTTA
ATATTTTAGCAAGGAATAGATAT
AUAUUUUAGCAAGGAAUAGAUAUACAAU

ACAATCA (SEQ ID NO:
CA (SEQ ID NO: 712)

332)

B2M_exon_4
+
ATTT
TAGCAAGGAATAGATATACAATC
UAGCAAGGAAUAGAUAUACAAUCAUCCC

ATCCCTT (SEQ ID NO:
UU (SEQ ID NO: 713)

333)

B2M_exon_4
+
TTTT
AGCAAGGAATAGATATACAATCA
AGCAAGGAAUAGAUAUACAAUCAUCCCU

TCCCTTG (SEQ ID NO:
UG (SEQ ID NO: 714)

334)

B2M_exon_4
+
TTTA
GCAAGGAATAGATATACAATCAT
GCAAGGAAUAGAUAUACAAUCAUCCCUU

CCCTTGG (SEQ ID NO:
GG (SEQ ID NO: 715)

335)

B2M_exon_4
+
CTTG
GTCTCCCTGGGGGATTGGTTTCA
GUCUCCCUGGGGGAUUGGUUUCAGGACC

GGACCCC (SEQ ID NO:
CC (SEQ ID NO: 716)

336)

B2M_exon_4
+
ATTG
GTTTCAGGACCCCTTCTTGGACA
GUUUCAGGACCCCUUCUUGGACACCAAA

CCAAATC (SEQ ID NO:
UC (SEQ ID NO: 717)

337)

B2M_exon_4
+
GTTT
CAGGACCCCTTCTTGGACACCAA
CAGGACCCCUUCUUGGACACCAAAUCUA

ATCTATG (SEQ ID NO:
UG (SEQ ID NO: 718)

338)

B2M_exon_4
+
CTTG
TAATACCTAATACAATGTAAATG
UAAUACCUAAUACAAUGUAAAUGCUAUG

CTATGCA (SEQ ID NO:
CA (SEQ ID NO: 719)

339)

B2M_exon_4
+
ATTA
CTTGTAATACCTAATACAATGTA
CUUGUAAUACCUAAUACAAUGUAAAUGC

AATGCTA (SEQ ID NO:
UA (SEQ ID NO: 720)

340)

B2M_exon_4
+
TTTC
TAGATTACTTGTAATACCTAATA
UAGAUUACUUGUAAUACCUAAUACAAUG

CAATGTA (SEQ ID NO:
UA (SEQ ID NO: 721)

341)

B2M_exon_4
+
ATTT
CTAGATTACTTGTAATACCTAAT
CUAGAUUACUUGUAAUACCUAAUACAAU

ACAATGT (SEQ ID NO:
GU (SEQ ID NO: 722)

342)

B2M_exon_4
+
TTTA
AATCATTTCTAGATTACTTGTAA
AAUCAUUUCUAGAUUACUUGUAAUACCU

TACCTAA (SEQ ID NO:
AA (SEQ ID NO: 723)

343)

B2M_exon_4
+
CTTT
AAATCATTTCTAGATTACTTGTA
AAAUCAUUUCUAGAUUACUUGUAAUACC

ATACCTA (SEQ ID NO:
UA (SEQ ID NO: 724)

344)

B2M_exon_4
+
ATTA
GGAATCTGATGCTCAAAGAAGTT
GGAAUCUGAUGCUCAAAGAAGUUAAAUG

AAATGGC (SEQ ID NO:
GC (SEQ ID NO: 725)

345)

B2M_exon_4
+
TTTG
CATATAACCTATCCACATCCTCC
CAUAUAACCUAUCCACAUCCUCCUGUAU

TGTATAC (SEQ ID NO:
AC (SEQ ID NO: 726)

346)

B2M_exon_4
+
CTTC
TATAAAATGGTATAGTATTTGCA
UAUAAAAUGGUAUAGUAUUUGCAUAUAA

TATAACC (SEQ ID NO:
CC (SEQ ID NO: 727)

347)

B2M_exon_4
+
TTTA
AGTCCCTTCTATAAAATGGTATA
AGUCCCUUCUAUAAAAUGGUAUAGUAUU

GTATTTG (SEQ ID NO:
UG (SEQ ID NO: 728)

348)

B2M_exon_4
+
ATTT
AAGTCCCTTCTATAAAATGGTAT
AAGUCCCUUCUAUAAAAUGGUAUAGUAU

AGTATTT (SEQ ID NO:
UU (SEQ ID NO: 729)

349)

B2M_exon_4
+
CTTG
GACACCAAATCTATGGATATTTA
GACACCAAAUCUAUGGAUAUUUAAGUCC

AGTCCCT (SEQ ID NO:
CU (SEQ ID NO: 730)

350)

B2M_exon_4
+
CTTC
TTGGACACCAAATCTATGGATAT
UUGGACACCAAAUCUAUGGAUAUUUAAG

TTAAGTC (SEQ ID NO:
UC (SEQ ID NO: 731)

351)

B2M_exon_4
+
TTTC
AGGACCCCTTCTTGGACACCAAA
AGGACCCCUUCUUGGACACCAAAUCUAU

TCTATGG (SEQ ID NO:
GG (SEQ ID NO: 732)

352)

B2M_exon_4
+
ATTT
GCATATAACCTATCCACATCCTC
GCAUAUAACCUAUCCACAUCCUCCUGUA

CTGTATA (SEQ ID NO:
UA (SEQ ID NO: 733)

353)

B2M_exon_4
+
TTTC
CAGATTAGGAATCTGATGCTCAA
CAGAUUAGGAAUCUGAUGCUCAAAGAAG

AGAAGTT (SEQ ID NO:
UU (SEQ ID NO: 734)

354)

B2M_exon_4
+
TTTT
CCAGATTAGGAATCTGATGCTCA
CCAGAUUAGGAAUCUGAUGCUCAAAGAA

AAGAAGT (SEQ ID NO:
GU (SEQ ID NO: 735)

355)

B2M_exon_4
+
ATTT
TCCAGATTAGGAATCTGATGCTC
UCCAGAUUAGGAAUCUGAUGCUCAAAGA

AAAGAAG (SEQ ID NO:
AG (SEQ ID NO: 736)

356)

B2M_exon_4
+
TTTG
TTCCACAAGTTAAATAAATCATA
UUCCACAAGUUAAAUAAAUCAUAAAACU

AAACTTG (SEQ ID NO:
UG (SEQ ID NO: 737)

357)

B2M_exon_4
+
TTTT
GTTCCACAAGTTAAATAAATCAT
GUUCCACAAGUUAAAUAAAUCAUAAAAC

AAAACTT (SEQ ID NO:
UU (SEQ ID NO: 738)

358)

B2M_exon_4
+
TTTT
TGTTCCACAAGTTAAATAAATCA
UGUUCCACAAGUUAAAUAAAUCAUAAAA

TAAAACT (SEQ ID NO:
CU (SEQ ID NO: 739)

359)

B2M_exon_4
+
ATTT
TTGTTCCACAAGTTAAATAAATC
UUGUUCCACAAGUUAAAUAAAUCAUAAA

ATAAAAC (SEQ ID NO:
AC (SEQ ID NO: 740)

360)

B2M_exon_4
+
TTTA
TTTTTGTTCCACAAGTTAAATAA
UUUUUGUUCCACAAGUUAAAUAAAUCAU

ATCATAA (SEQ ID NO:
AA (SEQ ID NO: 741)

361)

B2M_exon_4
+
GTTT
ATTTTTGTTCCACAAGTTAAATA
AUUUUUGUUCCACAAGUUAAAUAAAUCA

AATCATA (SEQ ID NO:
UA (SEQ ID NO: 742)

362)

B2M_exon_4
+
GTTC
CACAAGTTAAATAAATCATAAAA
CACAAGUUAAAUAAAUCAUAAAACUUGA

CTTGATG (SEQ ID NO:
UG (SEQ ID NO: 743)

363)

B2M_exon_4
+
GTTA
ATCTGGTTTATTTTTGTTCCACA
AUCUGGUUUAUUUUUGUUCCACAAGUUA

AGTTAAA (SEQ ID NO:
AA (SEQ ID NO: 744)

364)

B2M_exon_4
+
TTTG
ATAAAGTAAGGCATGGTTGTGGT
AUAAAGUAAGGCAUGGUUGUGGUUAAUC

TAATCTG (SEQ ID NO:
UG (SEQ ID NO: 745)

365)

B2M_exon_4
+
ATTT
GATAAAGTAAGGCATGGTTGTGG
GAUAAAGUAAGGCAUGGUUGUGGUUAAU

TTAATCT (SEQ ID NO:
CU (SEQ ID NO: 746)

366)

B2M_exon_4
+
CTTA
TACATTTGATAAAGTAAGGCATG
UACAUUUGAUAAAGUAAGGCAUGGUUGU

GTTGTGG (SEQ ID NO:
GG (SEQ ID NO: 747)

367)

B2M_exon_4
+
CTTC
TTATACATTTGATAAAGTAAGGC
UUAUACAUUUGAUAAAGUAAGGCAUGGU

ATGGTTG (SEQ ID NO:
UG (SEQ ID NO: 748)

368)

B2M_exon_4
+
TTTA
CTTCTTATACATTTGATAAAGTA
CUUCUUAUACAUUUGAUAAAGUAAGGCA

AGGCATG (SEQ ID NO:
UG (SEQ ID NO: 749)

369)

B2M_exon_4
+
ATTT
ACTTCTTATACATTTGATAAAGT
ACUUCUUAUACAUUUGAUAAAGUAAGGC

AAGGCAT (SEQ ID NO:
AU (SEQ ID NO: 750)

370)

B2M_exon_4
+
GTTG
TGGTTAATCTGGTTTATTTTTGT
UGGUUAAUCUGGUUUAUUUUUGUUCCAC

TCCACAA (SEQ ID NO:
AA (SEQ ID NO: 751)

371)

B2M_exon_4
+
TTTG
TCATATAAGATTCATATTTACTT
UCAUAUAAGAUUCAUAUUUACUUCUUAU

CTTATAC (SEQ ID NO:
AC (SEQ ID NO: 752)

372)

B2M_exon_4
+
GTTA
AATAAATCATAAAACTTGATGTG
AAUAAAUCAUAAAACUUGAUGUGUUAUC

TTATCTC (SEQ ID NO:
UC (SEQ ID NO: 753)

373)

B2M_exon_4
+
GTTA
TCTCTTATATCTCACTCCCACTA
UCUCUUAUAUCUCACUCCCACUAUUACC

TTACCCC (SEQ ID NO:
CC (SEQ ID NO: 754)

374)

B2M_exon_4
+
ATTC
ACATTTTCCAGATTAGGAATCTG
ACAUUUUCCAGAUUAGGAAUCUGAUGCU

ATGCTCA (SEQ ID NO:
CA (SEQ ID NO: 755)

375)

B2M_exon_4
+
TTTG
GCTCACAGTGTAAAGGGCCTCAG
GCUCACAGUGUAAAGGGCCUCAGUGAUU

TGATTCA (SEQ ID NO:
CA (SEQ ID NO: 756)

376)

B2M_exon_4
+
GTTT
GGCTCACAGTGTAAAGGGCCTCA
GGCUCACAGUGUAAAGGGCCUCAGUGAU

GTGATTC (SEQ ID NO:
UC (SEQ ID NO: 757)

377)

B2M_exon_4
+
CTTG
TATATAGAGTTTGGCTCACAGTG
UAUAUAGAGUUUGGCUCACAGUGUAAAG

TAAAGGG (SEQ ID NO:
GG (SEQ ID NO: 758)

378)

B2M_exon_4
+
CTTG
GTAAAAAATGTGAACCCCTTGTA
GUAAAAAAUGUGAACCCCUUGUAUAUAG

TATAGAG (SEQ ID NO:
AG (SEQ ID NO: 759)

379)

B2M_exon_4
+
GTTC
CACTTGGTAAAAAATGTGAACCC
CACUUGGUAAAAAAUGUGAACCCCUUGU

CTTGTAT (SEQ ID NO:
AU (SEQ ID NO: 760)

380)

B2M_exon_4
+
CTTG
ATGTGTTATCTCTTATATCTCAC
AUGUGUUAUCUCUUAUAUCUCACUCCCA

TCCCACT (SEQ ID NO:
CU (SEQ ID NO: 761)

381)

B2M_exon_4
+
CTTC
AAGTTCCACTTGGTAAAAAATGT
AAGUUCCACUUGGUAAAAAAUGUGAACC

GAACCCC (SEQ ID NO:
CC (SEQ ID NO: 762)

382)

B2M_exon_4
+
TTTT
CAAACAGGGAAACAGTCTTCAAG
CAAACAGGGAAACAGUCUUCAAGUUCCA

TTCCACT (SEQ ID NO:
CU (SEQ ID NO: 763)

383)

B2M_exon_4
+
ATTT
TCAAACAGGGAAACAGTCTTCAA
UCAAACAGGGAAACAGUCUUCAAGUUCC

GTTCCAC (SEQ ID NO:
AC (SEQ ID NO: 764)

384)

B2M_exon_4
+
TTTA
TTTTCAAACAGGGAAACAGTCTT
UUUUCAAACAGGGAAACAGUCUUCAAGU

CAAGTTC (SEQ ID NO:
UC (SEQ ID NO: 765)

385)

B2M_exon_4
−
CTTC
AAACCTGAAAAGAAAAGAAAAAG
AAACCUGAAAAGAAAAGAAAAAGGUUAG

GTTAGCA (SEQ ID NO:
CA (SEQ ID NO: 766)

386)

B2M_exon_4
+
ATTA
CCCCTTTATTTTCAAACAGGGAA
CCCCUUUAUUUUCAAACAGGGAAACAGU

ACAGTCT (SEQ ID NO:
CU (SEQ ID NO: 767)

387)

B2M_exon_4
+
CTTA
TATCTCACTCCCACTATTACCCC
UAUCUCACUCCCACUAUUACCCCUUUAU

TTTATTT (SEQ ID NO:
UU (SEQ ID NO: 768)

388)

B2M_exon_4
+
TTTC
AAACAGGGAAACAGTCTTCAAGT
AAACAGGGAAACAGUCUUCAAGUUCCAC

TCCACTT (SEQ ID NO:
UU (SEQ ID NO: 769)

389)

B2M_exon_4
−
GTTA
GCAATGAATTTATTTTATTTGGA
GCAAUGAAUUUAUUUUAUUUGGAUUGCA

TTGCAGA (SEQ ID NO:
GA (SEQ ID NO: 770)

390)

seq

seq

id

id

annotation
strand
pam
no
target_seq
no
spacer

intron
+
ATT
819
CTGAAGCTGACAGCATTCGG
1019
CUGAAGCUGACAGCAUUCGG

C

intron
+
CTT
820
CTGGCCTGGAGGCTATCCAG
1020
CUGGCCUGGAGGCUAUCCAG

T

intron
+
TTT
821
TGGCCTGGAGGCTATCCAGC
1021
UGGCCUGGAGGCUAUCCAGC

C

intron
+
CTT
822
CTCTCCCGCTCTGCACCCTC
1022
CUCUCCCGCUCUGCACCCUC

C

intron
+
CTT
823
CCTTCTCCAAGTTCTCCTTG
1023
CCUUCUCCAAGUUCUCCUUG

C

intron
+
CTT
824
TCCAAGTTCTCCTTGGTGGC
1024
UCCAAGUUCUCCUUGGUGGC

C

intron
+
GTT
825
TCCTTGGTGGCCCGCCGTGG
1025
UCCUUGGUGGCCCGCCGUGG

C

intron
+
CTT
826
GTGGCCCGCCGTGGGGCTAG
1026
GUGGCCCGCCGUGGGGCUAG

G

intron
+
CTT
827
CCCCTTTCGGCGGGGAGCAG
1027
CCCCUUUCGGCGGGGAGCAG

G

intron
+
CTT
828
CGGCGGGGAGCAGGGGAGAC
1028
CGGCGGGGAGCAGGGGAGAC

T

intron
+
TTT
829
GGCGGGGAGCAGGGGAGACC
1029
GGCGGGGAGCAGGGGAGACC

C

intron
+
CTT
830
GGCCTACGGCGACGGGAGGG
1030
GGCCUACGGCGACGGGAGGG

T

intron
+
TTT
831
GCCTACGGCGACGGGAGGGT
1031
GCCUACGGCGACGGGAGGGU

G

intron
+
GTT
832
AGGGCGTCGATAAGCGTCAG
1032
AGGGCGUCGAUAAGCGUCAG

T

intron
+
TTT
833
GGGCGTCGATAAGCGTCAGA
1033
GGGCGUCGAUAAGCGUCAGA

A

intron
+
GTT
834
GGGGAGGGTTTCTCTTCCGC
1034
GGGGAGGGUUUCUCUUCCGC

G

intron
+
GTT
835
CTCTTCCGCTCTTTCGCGGG
1035
CUCUUCCGCUCUUUCGCGGG

T

intron
+
TTT
836
TCTTCCGCTCTTTCGCGGGG
1036
UCUUCCGCUCUUUCGCGGGG

C

intron
+
CTT
837
CGCTCTTTCGCGGGGCCTCT
1037
CGCUCUUUCGCGGGGCCUCU

C

intron
+
CTT
838
CGCGGGGCCTCTGGCTCCCC
1038
CGCGGGGCCUCUGGCUCCCC

T

intron
+
TTT
839
GCGGGGCCTCTGGCTCCCCC
1039
GCGGGGCCUCUGGCUCCCCC

C

intron
+
GTT
840
GTGAACGCGTGGAGGGGCGC
1040
GUGAACGCGUGGAGGGGCGC

T

intron
+
TTT
841
TGAACGCGTGGAGGGGCGCT
1041
UGAACGCGUGGAGGGGCGCU

G

intron
+
CTT
842
GGGTCTGGGGGAGGCGTCGC
1042
GGGUCUGGGGGAGGCGUCGC

G

intron
−
CTT
843
CCCGGGCGACGCCTCCCCCA
1043
CCCGGGCGACGCCUCCCCCA

A

intron
−
GTT
844
ACAAACCTCAGCGCCGCGCC
1044
ACAAACCUCAGCGCCGCGCC

C

intron
−
CTT
845
GGGACGAGCCTACCCGTCCC
1045
GGGACGAGCCUACCCGUCCC

T

intron
−
TTT
846
GGACGAGCCTACCCGTCCCC
1046
GGACGAGCCUACCCGUCCCC

G

intron
−
CTT
847
TCGACGCCCTAAACTTTGTC
1047
UCGACGCCCUAAACUUUGUC

A

intron
−
CTT
848
GTCCCGACCCTCCCGTCGCC
1048
GUCCCGACCCUCCCGUCGCC

T

intron
−
TTT
849
TCCCGACCCTCCCGTCGCCG
1049
UCCCGACCCUCCCGUCGCCG

G

intron
−
CTT
850
CCCACTCCCAGGCCACCCCG
1050
CCCACUCCCAGGCCACCCCG

C

intron
−
CTT
851
CCCGAGATCCAGCCCTGGAC
1051
CCCGAGAUCCAGCCCUGGAC

C

intron
−
CTT
852
GAGAAGGGAAGTCACGGAGC
1052
GAGAAGGGAAGUCACGGAGC

G

intron
−
CTT
853
AGGAATGCCCGCCAGCGCGA
1053
AGGAAUGCCCGCCAGCGCGA

C

intron
+
ATT
854
TGAGGGAAAGATACCAAGTC
1054
UGAGGGAAAGAUACCAAGUC

A

intron
+
GTT
855
ATTCTTCAAAATGGAGGTGG
1055
AUUCUUCAAAAUGGAGGUGG

T

intron
+
TTT
856
TTCTTCAAAATGGAGGTGGC
1056
UUCUUCAAAAUGGAGGUGGC

A

intron
+
ATT
857
TTCAAAATGGAGGTGGCTTG
1057
UUCAAAAUGGAGGUGGCUUG

C

intron
+
CTT
858
AAAATGGAGGTGGCTTGTTG
1058
AAAAUGGAGGUGGCUUGUUG

C

intron
+
CTT
859
TTGGGAAGGTGGAAGCTCAT
1059
UUGGGAAGGUGGAAGCUCAU

G

intron
+
GTT
860
GGAAGGTGGAAGCTCATTTG
1060
GGAAGGUGGAAGCUCAUUUG

G

intron
+
ATT
861
GGCCAGAGTGGAAATGGAAT
1061
GGCCAGAGUGGAAAUGGAAU

T

intron
+
TTT
862
GCCAGAGTGGAAATGGAATT
1062
GCCAGAGUGGAAAUGGAAUU

G

intron
+
ATT
863
GGAGAAATCGATGACCAAAT
1063
GGAGAAAUCGAUGACCAAAU

G

intron
+
CTT
864
GTGCCTGATATAGCTTGACA
1064
GUGCCUGAUAUAGCUUGACA

G

intron
+
CTT
865
ACACCAAGTTAGCCCCAAGT
1065
ACACCAAGUUAGCCCCAAGU

G

intron
+
GTT
866
GCCCCAAGTGAAATACCCTG
1066
GCCCCAAGUGAAAUACCCUG

A

intron
+
ATT
867
ATGTGTCTTTTCCCGATATT
1067
AUGUGUCUUUUCCCGAUAUU

A

intron
+
GTT
868
AGTGGGGTAAGTCTTACATT
1068
AGUGGGGUAAGUCUUACAUU

A

intron
+
CTT
869
CATTCTTTTGTAAGCTGCTG
1069
CAUUCUUUUGUAAGCUGCUG

A

intron
+
ATT
870
TTTTGTAAGCTGCTGAAAGT
1070
UUUUGUAAGCUGCUGAAAGU

C

intron
+
CTT
871
TGTAAGCTGCTGAAAGTTGT
1071
UGUAAGCUGCUGAAAGUUGU

T

intron
+
TTT
872
GTAAGCTGCTGAAAGTTGTG
1072
GUAAGCUGCUGAAAGUUGUG

T

intron
+
TTT
873
TAAGCTGCTGAAAGTTGTGT
1073
UAAGCUGCUGAAAGUUGUGU

G

intron
+
GTT
874
TGTATGAGTAGTCATATCAT
1074
UGUAUGAGUAGUCAUAUCAU

G

intron
+
CTT
875
GATATAAAAAAGGTCTATGG
1075
GAUAUAAAAAAGGUCUAUGG

T

intron
+
TTT
876
ATATAAAAAAGGTCTATGGC
1076
AUAUAAAAAAGGUCUAUGGC

G

intron
+
ATT
877
GGATTGTCAGGGAATGTTCT
1077
GGAUUGUCAGGGAAUGUUCU

G

intron
+
ATT
878
TCAGGGAATGTTCTTAAAGA
1078
UCAGGGAAUGUUCUUAAAGA

G

intron
+
GTT
879
TTAAAGATCAGATTAGTGGC
1079
UUAAAGAUCAGAUUAGUGGC

C

intron
+
CTT
880
AAGATCAGATTAGTGGCACC
1080
AAGAUCAGAUUAGUGGCACC

A

intron
+
ATT
881
GTGGCACCTGCTGAGATACT
1081
GUGGCACCUGCUGAGAUACU

A

intron
+
GTT
882
CTGAACCAGTAGTTTCCCTG
1082
CUGAACCAGUAGUUUCCCUG

T

intron
+
TTT
883
TGAACCAGTAGTTTCCCTGC
1083
UGAACCAGUAGUUUCCCUGC

C

intron
+
GTT
884
CCCTGCAGTTGAGCAGGGAG
1084
CCCUGCAGUUGAGCAGGGAG

T

intron
+
TTT
885
CCTGCAGTTGAGCAGGGAGC
1085
CCUGCAGUUGAGCAGGGAGC

C

intron
+
GTT
886
AGCAGGGAGCAGCAGCAGCA
1086
AGCAGGGAGCAGCAGCAGCA

G

intron
+
CTT
887
CACAAATACATATACACTCT
1087
CACAAAUACAUAUACACUCU

G

intron
+
CTT
888
ACACTTCTTACCTACTGGCT
1088
ACACUUCUUACCUACUGGCU

A

intron
+
CTT
889
TTACCTACTGGCTTCCTCTA
1089
UUACCUACUGGCUUCCUCUA

C

intron
+
CTT
890
CCTACTGGCTTCCTCTAGCT
1090
CCUACUGGCUUCCUCUAGCU

A

intron
+
CTT
891
CTCTAGCTTTTGTGGCAGCT
1091
CUCUAGCUUUUGUGGCAGCU

C

intron
+
CTT
892
TGTGGCAGCTTCAGGTATAT
1092
UGUGGCAGCUUCAGGUAUAU

T

intron
+
TTT
893
GTGGCAGCTTCAGGTATATT
1093
GUGGCAGCUUCAGGUAUAUU

T

intron
+
TTT
894
TGGCAGCTTCAGGTATATTT
1094
UGGCAGCUUCAGGUAUAUUU

G

intron
+
CTT
895
AGGTATATTTAGCACTGAAC
1095
AGGUAUAUUUAGCACUGAAC

C

intron
+
ATT
896
AGCACTGAACGAACATCTCA
1096
AGCACUGAACGAACAUCUCA

T

intron
+
TTT
897
GCACTGAACGAACATCTCAA
1097
GCACUGAACGAACAUCUCAA

A

intron
+
CTT
898
GTTTGTAAGTCCTGCTGTCC
1098
GUUUGUAAGUCCUGCUGUCC

T

intron
+
TTT
899
TTTGTAAGTCCTGCTGTCCT
1099
UUUGUAAGUCCUGCUGUCCU

G

intron
+
GTT
900
GTAAGTCCTGCTGTCCTAGC
1100
GUAAGUCCUGCUGUCCUAGC

T

intron
+
TTT
901
TAAGTCCTGCTGTCCTAGCA
1101
UAAGUCCUGCUGUCCUAGCA

G

intron
+
CTT
902
TCCAGTACTTTCTGGCTGGA
1102
UCCAGUACUUUCUGGCUGGA

C

intron
+
CTT
903
CTGGCTGGATTGGTATCTGA
1103
CUGGCUGGAUUGGUAUCUGA

T

intron
+
TTT
904
TGGCTGGATTGGTATCTGAG
1104
UGGCUGGAUUGGUAUCUGAG

C

intron
+
ATT
905
GTATCTGAGGCTAGTAGGAA
1105
GUAUCUGAGGCUAGUAGGAA

G

intron
+
CTT
906
TTCCTGCTGGGTAGCTCTAA
1106
UUCCUGCUGGGUAGCUCUAA

G

intron
+
GTT
907
CTGCTGGGTAGCTCTAAACA
1107
CUGCUGGGUAGCUCUAAACA

C

intron
+
ATT
908
ATGGGTAGGAACAGCAGCCT
1108
AUGGGUAGGAACAGCAGCCU

C

intron
+
ATT
909
TGCCAGCCTTATTTCTAACC
1109
UGCCAGCCUUAUUUCUAACC

C

intron
+
CTT
910
TTTCTAACCATTTTAGACAT
1110
UUUCUAACCAUUUUAGACAU

A

intron
+
ATT
911
CTAACCATTTTAGACATTTG
1111
CUAACCAUUUUAGACAUUUG

T

intron
+
TTT
912
TAACCATTTTAGACATTTGT
1112
UAACCAUUUUAGACAUUUGU

C

intron
+
ATT
913
TAGACATTTGTTAGTACATG
1113
UAGACAUUUGUUAGUACAUG

T

intron
+
TTT
914
AGACATTTGTTAGTACATGG
1114
AGACAUUUGUUAGUACAUGG

T

intron
+
TTT
915
GACATTTGTTAGTACATGGT
1115
GACAUUUGUUAGUACAUGGU

A

intron
+
ATT
916
GTTAGTACATGGTATTTTAA
1116
GUUAGUACAUGGUAUUUUAA

T

intron
+
TTT
917
TTAGTACATGGTATTTTAAA
1117
UUAGUACAUGGUAUUUUAAA

G

intron
+
GTT
918
GTACATGGTATTTTAAAAGT
1118
GUACAUGGUAUUUUAAAAGU

A

intron
+
ATT
919
TAAAAGTAAAACTTAATGTC
1119
UAAAAGUAAAACUUAAUGUC

T

intron
+
TTT
920
AAAAGTAAAACTTAATGTCT
1120
AAAAGUAAAACUUAAUGUCU

T

intron
+
TTT
921
AAAGTAAAACTTAATGTCTT
1121
AAAGUAAAACUUAAUGUCUU

A

intron
+
CTT
922
ATGTCTTCCTTTTTTTTCTC
1122
AUGUCUUCCUUUUUUUUCUC

A

intron
+
CTT
923
CTTTTTTTTCTCCACTGTCT
1123
CUUUUUUUUCUCCACUGUCU

C

intron
+
CTT
924
TTTTTCTCCACTGTCTTTTT
1124
UUUUUCUCCACUGUCUUUUU

T

intron
+
TTT
925
TTTTCTCCACTGTCTTTTTC
1125
UUUUCUCCACUGUCUUUUUC

T

intron
+
CTT
926
TTCATAGATCGAGACATGTA
1126
UUCAUAGAUCGAGACAUGUA

T

intron
+
TTT
927
TCATAGATCGAGACATGTAA
1127
UCAUAGAUCGAGACAUGUAA

T

intron
+
TTT
928
CATAGATCGAGACATGTAAG
1128
CAUAGAUCGAGACAUGUAAG

T

intron
+
TTT
929
ATAGATCGAGACATGTAAGC
1129
AUAGAUCGAGACAUGUAAGC

C

intron
+
GTT
930
TTGACCTTGAGAAAATGTTT
1130
UUGACCUUGAGAAAAUGUUU

T

intron
+
TTT
931
TGACCTTGAGAAAATGTTTT
1131
UGACCUUGAGAAAAUGUUUU

T

intron
+
TTT
932
GACCTTGAGAAAATGTTTTT
1132
GACCUUGAGAAAAUGUUUUU

T

intron
+
TTT
933
ACCTTGAGAAAATGTTTTTG
1133
ACCUUGAGAAAAUGUUUUUG

G

intron
+
CTT
934
AGAAAATGTTTTTGTTTCAC
1134
AGAAAAUGUUUUUGUUUCAC

G

intron
+
GTT
935
TTGTTTCACTGTCCTGAGGA
1135
UUGUUUCACUGUCCUGAGGA

T

intron
+
TTT
936
TGTTTCACTGTCCTGAGGAC
1136
UGUUUCACUGUCCUGAGGAC

T

intron
+
TTT
937
GTTTCACTGTCCTGAGGACT
1137
GUUUCACUGUCCUGAGGACU

T

intron
+
TTT
938
TTTCACTGTCCTGAGGACTA
1138
UUUCACUGUCCUGAGGACUA

G

intron
+
GTT
939
CACTGTCCTGAGGACTATTT
1139
CACUGUCCUGAGGACUAUUU

T

intron
+
TTT
940
ACTGTCCTGAGGACTATTTA
1140
ACUGUCCUGAGGACUAUUUA

C

intron
+
ATT
941
ATAGACAGCTCTAACATGAT
1141
AUAGACAGCUCUAACAUGAU

T

intron
+
TTT
942
TAGACAGCTCTAACATGATA
1142
UAGACAGCUCUAACAUGAUA

A

intron
−
GTT
943
TCATGTTAGAGCTGTCTATA
1143
UCAUGUUAGAGCUGUCUAUA

A

intron
−
GTT
944
GAGCTGTCTATAAATAGTCC
1144
GAGCUGUCUAUAAAUAGUCC

A

intron
−
ATT
945
TCTCAAGGTCAAAAACTTAC
1145
UCUCAAGGUCAAAAACUUAC

T

intron
−
TTT
946
CTCAAGGTCAAAAACTTACC
1146
CUCAAGGUCAAAAACUUACC

T

intron
−
TTT
947
TCAAGGTCAAAAACTTACCT
1147
UCAAGGUCAAAAACUUACCU

C

intron
−
CTT
948
CATGTCTCGATCTATGAAAA
1148
CAUGUCUCGAUCUAUGAAAA

A

intron
−
ATT
949
AGTTTTACTTTTAAAATACC
1149
AGUUUUACUUUUAAAAUACC

A

intron
−
GTT
950
TACTTTTAAAATACCATGTA
1150
UACUUUUAAAAUACCAUGUA

T

intron
−
TTT
951
ACTTTTAAAATACCATGTAC
1151
ACUUUUAAAAUACCAUGUAC

T

intron
−
TTT
952
CTTTTAAAATACCATGTACT
1152
CUUUUAAAAUACCAUGUACU

A

intron
−
CTT
953
TAAAATACCATGTACTAACA
1153
UAAAAUACCAUGUACUAACA

T

intron
−
TTT
954
AAAATACCATGTACTAACAA
1154
AAAAUACCAUGUACUAACAA

T

intron
−
TTT
955
AAATACCATGTACTAACAAA
1155
AAAUACCAUGUACUAACAAA

A

intron
−
GTT
956
GAAATAAGGCTGGCAGAATA
1156
GAAAUAAGGCUGGCAGAAUA

A

intron
−
GTT
957
CTACCCATGAATACATTGTT
1157
CUACCCAUGAAUACAUUGUU

C

intron
−
ATT
958
TTTAGAGCTACCCAGCAGGA
1158
UUUAGAGCUACCCAGCAGGA

G

intron
−
GTT
959
AGAGCTACCCAGCAGGAACA
1159
AGAGCUACCCAGCAGGAACA

T

intron
−
TTT
960
GAGCTACCCAGCAGGAACAA
1160
GAGCUACCCAGCAGGAACAA

A

intron
−
CTT
961
CTACTAGCCTCAGATACCAA
1161
CUACUAGCCUCAGAUACCAA

C

intron
−
ATT
962
TAGGATGCTAGGACAGCAGG
1162
UAGGAUGCUAGGACAGCAGG

A

intron
−
CTT
963
CAAACAAAGGCCTATACCTT
1163
CAAACAAAGGCCUAUACCUU

A

intron
−
CTT
964
TTGAGATGTTCGTTCAGTGC
1164
UUGAGAUGUUCGUUCAGUGC

C

intron
−
CTT
965
AGATGTTCGTTCAGTGCTAA
1165
AGAUGUUCGUUCAGUGCUAA

G

intron
−
GTT
966
GTTCAGTGCTAAATATACCT
1166
GUUCAGUGCUAAAUAUACCU

C

intron
−
GTT
967
AGTGCTAAATATACCTGAAG
1167
AGUGCUAAAUAUACCUGAAG

C

intron
−
GTT
968
AGAGTGTATATGTATTTGTG
1168
AGAGUGUAUAUGUAUUUGUG

A

intron
−
ATT
969
GTGCAAGTGCTGCTGCTGCT
1169
GUGCAAGUGCUGCUGCUGCU

T

intron
−
TTT
970
TGCAAGTGCTGCTGCTGCTC
1170
UGCAAGUGCUGCUGCUGCUC

G

intron
−
GTT
971
AGAAACCATGCTGTGCATCA
1171
AGAAACCAUGCUGUGCAUCA

C

intron
−
CTT
972
AAGAACATTCCCTGACAATC
1172
AAGAACAUUCCCUGACAAUC

T

intron
−
TTT
973
AGAACATTCCCTGACAATCC
1173
AGAACAUUCCCUGACAAUCC

A

intron
−
ATT
974
CCTGACAATCCCAATATGCA
1174
CCUGACAAUCCCAAUAUGCA

C

intron
−
ATT
975
TTTATATCAGATGGGATGGG
1175
UUUAUAUCAGAUGGGAUGGG

G

intron
−
GTT
976
ATATCAGATGGGATGGGACT
1176
AUAUCAGAUGGGAUGGGACU

T

intron
−
TTT
977
TATCAGATGGGATGGGACTC
1177
UAUCAGAUGGGAUGGGACUC

A

intron
−
ATT
978
AGGGTAGTATGGCCATAGAC
1178
AGGGUAGUAUGGCCAUAGAC

C

intron
−
CTT
979
TTTATATCAAAGCAGCTTTA
1179
UUUAUAUCAAAGCAGCUUUA

T

intron
−
TTT
980
TTATATCAAAGCAGCTTTAT
1180
UUAUAUCAAAGCAGCUUUAU

T

intron
−
TTT
981
TATATCAAAGCAGCTTTATG
1181
UAUAUCAAAGCAGCUUUAUG

T

intron
−
TTT
982
ATATCAAAGCAGCTTTATGA
1182
AUAUCAAAGCAGCUUUAUGA

T

intron
−
TTT
983
TATCAAAGCAGCTTTATGAT
1183
UAUCAAAGCAGCUUUAUGAU

A

intron
−
CTT
984
ATGATATGACTACTCATACA
1184
AUGAUAUGACUACUCAUACA

T

intron
−
TTT
985
TGATATGACTACTCATACAC
1185
UGAUAUGACUACUCAUACAC

A

intron
−
CTT
986
CAGCAGCTTACAAAAGAATG
1186
CAGCAGCUUACAAAAGAAUG

T

intron
−
TTT
987
AGCAGCTTACAAAAGAATGT
1187
AGCAGCUUACAAAAGAAUGU

C

intron
−
CTT
988
GGAGTACCTGAGGAATATCG
1188
GGAGUACCUGAGGAAUAUCG

T

intron
−
TTT
989
GAGTACCTGAGGAATATCGG
1189
GAGUACCUGAGGAAUAUCGG

G

intron
−
ATT
990
ATATTGCCAGGGTATTTCAC
1190
AUAUUGCCAGGGUAUUUCAC

A

intron
−
ATT
991
CCAGGGTATTTCACTTGGGG
1191
CCAGGGUAUUUCACUUGGGG

G

intron
−
ATT
992
CACTTGGGGCTAACTTGGTG
1192
CACUUGGGGCUAACUUGGUG

T

intron
−
TTT
993
ACTTGGGGCTAACTTGGTGT
1193
ACUUGGGGCUAACUUGGUGU

C

intron
−
CTT
994
GGGCTAACTTGGTGTCAAGC
1194
GGGCUAACUUGGUGUCAAGC

G

intron
−
CTT
995
GTGTCAAGCTATATCAGGCA
1195
GUGUCAAGCUAUAUCAGGCA

G

intron
−
GTT
996
ACATTTGGTCATCGATTTCT
1196
ACAUUUGGUCAUCGAUUUCU

T

intron
−
TTT
997
CATTTGGTCATCGATTTCTC
1197
CAUUUGGUCAUCGAUUUCUC

A

intron
−
ATT
998
GGTCATCGATTTCTCCCAAT
1198
GGUCAUCGAUUUCUCCCAAU

T

intron
−
TTT
999
GTCATCGATTTCTCCCAATT
1199
GUCAUCGAUUUCUCCCAAUU

G

intron
−
ATT
1000
CTCCCAATTCCATTTCCACT
1200
CUCCCAAUUCCAUUUCCACU

T

intron
−
TTT
1001
TCCCAATTCCATTTCCACTC
1201
UCCCAAUUCCAUUUCCACUC

C

intron
−
ATT
1002
CATTTCCACTCTGGCCAAAT
1202
CAUUUCCACUCUGGCCAAAU

C

intron
−
ATT
1003
CCACTCTGGCCAAATGAGCT
1203
CCACUCUGGCCAAAUGAGCU

T

intron
−
TTT
1004
CACTCTGGCCAAATGAGCTT
1204
CACUCUGGCCAAAUGAGCUU

C

intron
−
CTT
1005
CACCTTCCCAACAAGCCACC
1205
CACCUUCCCAACAAGCCACC

C

intron
−
CTT
1006
CCAACAAGCCACCTCCATTT
1206
CCAACAAGCCACCUCCAUUU

C

intron
−
ATT
1007
TGAAGAATAAACCGTGACTT
1207
UGAAGAAUAAACCGUGACUU

T

intron
−
TTT
1008
GAAGAATAAACCGTGACTTG
1208
GAAGAAUAAACCGUGACUUG

T

intron
−
TTT
1009
AAGAATAAACCGTGACTTGG
1209
AAGAAUAAACCGUGACUUGG

G

intron
−
CTT
1010
GTATCTTTCCCTCATAATTC
1210
GUAUCUUUCCCUCAUAAUUC

G

intron
−
CTT
1011
CCCTCATAATTCCTCTATAC
1211
CCCUCAUAAUUCCUCUAUAC

T

intron
−
TTT
1012
CCTCATAATTCCTCTATACA
1212
CCUCAUAAUUCCUCUAUACA

C

intron
−
ATT
1013
CTCTATACATGCCTTTTTTG
1213
CUCUAUACAUGCCUUUUUUG

C

intron
−
CTT
1014
TTTGTTTTTTTTCTAGCAGA
1214
UUUGUUUUUUUUCUAGCAGA

T

intron
−
TTT
1015
TTGTTTTTTTTCTAGCAGAT
1215
UUGUUUUUUUUCUAGCAGAU

T

intron
−
TTT
1016
TGTTTTTTTTCTAGCAGATT
1216
UGUUUUUUUUCUAGCAGAUU

T

intron
−
TTT
1017
GTTTTTTTTCTAGCAGATTT
1217
GUUUUUUUUCUAGCAGAUUU

T

intron
−
TTT
1018
TTTTTTTTCTAGCAGATTTC
1218
UUUUUUUUCUAGCAGAUUUC

G

The invention includes all combinations of the direct repeats and spacers listed above, consistent with the disclosure herein. In embodiments, the RNA guide does not consist of the sequence of

- AGAAAUCCGUCUUUCAUUGACGGAAUGUCGGAUGGAUGAAACC (SEQ ID NO: 778);
- AGAAAUCCGUCUUUCAUUGACGGCUAUCUCUUGUACUACACUG (SEQ ID NO: 779);
- AGAAAUCCGUCUUUCAUUGACGGACACGGCAGGCAUACUCAUC (SEQ ID NO: 780); or
- AGAAAUCCGUCUUUCAUUGACGGGUCACAGCCCAAGAUAGUUA (SEQ ID NO: 781).

In some embodiments, a spacer sequence described herein comprises a uracil (U). In some embodiments, a spacer sequence described herein comprises a thymine (T). In some embodiments, a spacer sequence according to Table 5 comprises a sequence comprising a thymine in one or more places indicated as uracil in Table 5.

Modifications

The RNA guide may include one or more covalent modifications with respect to a reference sequence, in particular the parent polyribonucleotide, which are included within the scope of this invention.

Exemplary modifications can include any modification to the sugar, the nucleobase, the internucleoside linkage (e.g. to a linking phosphate/to a phosphodiester linkage/to the phosphodiester backbone), and any combination thereof. Some of the exemplary modifications provided herein are described in detail below.

The RNA guide may include any useful modification, such as to the sugar, the nucleobase, or the internucleoside linkage (e.g. to a linking phosphate/to a phosphodiester linkage/to the phosphodiester backbone). One or more atoms of a pyrimidine nucleobase may be replaced or substituted with optionally substituted amino, optionally substituted thiol, optionally substituted alkyl (e.g., methyl or ethyl), or halo (e.g., chloro or fluoro). In certain embodiments, modifications (e.g., one or more modifications) are present in each of the sugar and the internucleoside linkage. Modifications may be modifications of ribonucleic acids (RNAs) to deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs) or hybrids thereof). Additional modifications are described herein.

In some embodiments, the modification may include a chemical or cellular induced modification. For example, some nonlimiting examples of intracellular RNA modifications are described by Lewis and Pan in “RNA modifications and structures cooperate to guide RNA-protein interactions” from Nat Reviews Mol Cell Biol, 2017, 18:202-210.

Different sugar modifications, nucleotide modifications, and/or internucleoside linkages (e.g., backbone structures) may exist at various positions in the sequence. One of ordinary skill in the art will appreciate that the nucleotide analogs or other modification(s) may be located at any position(s) of the sequence, such that the function of the sequence is not substantially decreased. The sequence may include from about 1% to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e. any one or more of A, G, U or C) or any intervening percentage (e.g., from 1% to 20%>, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100%).

In some embodiments, sugar modifications (e.g., at the 2′ position or 4′ position) or replacement of the sugar at one or more ribonucleotides of the sequence may, as well as backbone modifications, include modification or replacement of the phosphodiester linkages. Specific examples of a sequence include, but are not limited to, sequences including modified backbones or no natural internucleoside linkages such as internucleoside modifications, including modification or replacement of the phosphodiester linkages. Sequences having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this application, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In particular embodiments, a sequence will include ribonucleotides with a phosphorus atom in its internucleoside backbone.

Modified sequence backbones may include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates such as 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates such as 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acid forms are also included. In some embodiments, the sequence may be negatively or positively charged.

The modified nucleotides, which may be incorporated into the sequence, can be modified on the internucleoside linkage (e.g., phosphate backbone). Herein, in the context of the polynucleotide backbone, the phrases “phosphate” and “phosphodiester” are used interchangeably. Backbone phosphate groups can be modified by replacing one or more of the oxygen atoms with a different substituent. Further, the modified nucleosides and nucleotides can include the wholesale replacement of an unmodified phosphate moiety with another internucleoside linkage as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters. Phosphorodithioates have both non-linking oxygens replaced by sulfur. The phosphate linker can also be modified by the replacement of a linking oxygen with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene-phosphonates).

The α-thio substituted phosphate moiety is provided to confer stability to RNA and DNA polymers through the unnatural phosphorothioate backbone linkages. Phosphorothioate DNA and RNA have increased nuclease resistance and subsequently a longer half-life in a cellular environment.

In specific embodiments, a modified nucleoside includes an alpha-thio-nucleoside (e.g., 5′-O-(1-thiophosphate)-adenosine, 5′-O-(1-thiophosphate)-cytidine (a-thio-cytidine), 5′-O-(1-thiophosphate)-guanosine, 5′-O-(1-thiophosphate)-uridine, or 5′-O-(1-thiophosphate)-pseudouridine).

Other internucleoside linkages that may be employed according to the present invention, including internucleoside linkages which do not contain a phosphorous atom, are described herein.

In some embodiments, the sequence may include one or more cytotoxic nucleosides. For example, cytotoxic nucleosides may be incorporated into sequence, such as bifunctional modification. Cytotoxic nucleoside may include, but are not limited to, adenosine arabinoside, 5-azacytidine, 4′-thio-aracytidine, cyclopentenylcytosine, cladribine, clofarabine, cytarabine, cytosine arabinoside, 1-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl)-cytosine, decitabine, 5-fluorouracil, fludarabine, floxuridine, gemcitabine, a combination of tegafur and uracil, tegafur ((RS)-5-fluoro-1-(tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione), troxacitabine, tezacitabine, 2′-deoxy-2′-methylidenecytidine (DMDC), and 6-mercaptopurine. Additional examples include fludarabine phosphate, N4-behenoyl-1-beta-D-arabinofuranosylcytosine, N4-octadecyl-1-beta-D-arabinofuranosylcytosine, N4-palmitoyl-1-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl) cytosine, and P-4055 (cytarabine 5′-elaidic acid ester).

In some embodiments, the sequence includes one or more post-transcriptional modifications (e.g., capping, cleavage, polyadenylation, splicing, poly-A sequence, methylation, acylation, phosphorylation, methylation of lysine and arginine residues, acetylation, and nitrosylation of thiol groups and tyrosine residues, etc). The one or more post-transcriptional modifications can be any post-transcriptional modification, such as any of the more than one hundred different nucleoside modifications that have been identified in RNA (Rozenski, J, Crain, P, and McCloskey, J. (1999). The RNA Modification Database: 1999 update. Nucl Acids Res 27: 196-197) In some embodiments, the first isolated nucleic acid comprises messenger RNA (mRNA). In some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine. In some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine. In some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine. In some embodiments, mRNA comprises at least one nucleoside selected from the group consisting of inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

The sequence may or may not be uniformly modified along the entire length of the molecule. For example, one or more or all types of nucleotide (e.g., naturally-occurring nucleotides, purine or pyrimidine, or any one or more or all of A, G, U, C, I, pU) may or may not be uniformly modified in the sequence, or in a given predetermined sequence region thereof. In some embodiments, the sequence includes a pseudouridine. In some embodiments, the sequence includes an inosine, which may aid in the immune system characterizing the sequence as endogenous versus viral RNAs. The incorporation of inosine may also mediate improved RNA stability/reduced degradation. See for example, Yu, Z. et al. (2015) RNA editing by ADAR1 marks dsRNA as “self”. Cell Res. 25, 1283-1284, which is incorporated by reference in its entirety.

Cas12i Polypeptide

In some embodiments, the composition of the present invention includes a Cas12i polypeptide as described in PCT/US2019/022375.

In some embodiments, the composition of the present invention includes a Cas12i2 polypeptide described herein (e.g., a polypeptide comprising SEQ ID NO: 772 and/or encoded by SEQ ID NO: 771). In some embodiments, the Cas12i2 polypeptide comprises at least one RuvC domain.

A nucleic acid sequence encoding the Cas12i2 polypeptide described herein may be substantially identical to a reference nucleic acid sequence, e.g., SEQ ID NO: 771. In some embodiments, the Cas12i2 polypeptide is encoded by a nucleic acid comprising a sequence having least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to the reference nucleic acid sequence, e.g., SEQ ID NO: 771. The percent identity between two such nucleic acids can be determined manually by inspection of the two optimally aligned nucleic acid sequences or by using software programs or algorithms (e.g., BLAST, ALIGN, CLUSTAL) using standard parameters. One indication that two nucleic acid sequences are substantially identical is that the nucleic acid molecules hybridize to the complementary sequence of the other under stringent conditions of temperature and ionic strength (e.g., within a range of medium to high stringency). See, e.g., Tijssen, “Hybridization with Nucleic Acid Probes. Part I. Theory and Nucleic Acid Preparation” (Laboratory Techniques in Biochemistry and Molecular Biology, Vol 24).

In some embodiments, the Cas12i2 polypeptide is encoded by a nucleic acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more sequence identity, but not 100% sequence identity, to a reference nucleic acid sequence, e.g., SEQ ID NO: 771.

In some embodiments, the present invention describes a Cas12i2 polypeptide having a specified degree of amino acid sequence identity to one or more reference polypeptides, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99%, but not 100%, sequence identity to the amino acid sequence of SEQ ID NO: 772. Homology or identity can be determined by amino acid sequence alignment, e.g., using a program such as BLAST, ALIGN, or CLUSTAL, as described herein.

Also provided is a Cas12i2 polypeptide of the present invention having enzymatic activity, e.g., nuclease or endonuclease activity, and comprising an amino acid sequence which differs from the amino acid sequences of SEQ ID NO: 772 by 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acid residue(s), when aligned using any of the previously described alignment methods.

In some embodiments, the Cas12i2 polypeptide comprises a polypeptide having a sequence of SEQ ID NO: 782, SEQ ID NO: 783, SEQ ID NO: 784, SEQ ID NO: 785, or SEQ ID NO: 786.

In some embodiments, the Cas12i2 polypeptide of the present invention comprises a polypeptide sequence having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 782, SEQ ID NO: 783, SEQ ID NO: 784, SEQ ID NO: 785, or SEQ ID NO: 786. In some embodiments, a Cas12i2 polypeptide having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 782, SEQ ID NO: 783, SEQ ID NO: 784, SEQ ID NO: 785, or SEQ ID NO: 786 maintains the amino acid changes (or at least 1, 2, 3 etc. of these changes) that differentiate the polypeptide from its respective parent/reference sequence.

In some embodiments, the present invention describes a Cas12i2 polypeptide having a specified degree of amino acid sequence identity to one or more reference polypeptides, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99%, but not 100%, sequence identity to the amino acid sequence of SEQ ID NO: 782, SEQ ID NO: 783, SEQ ID NO: 784, SEQ ID NO: 785, or SEQ ID NO: 786. Homology or identity can be determined by amino acid sequence alignment, e.g., using a program such as BLAST, ALIGN, or CLUSTAL, as described herein.

Also provided is a Cas12i2 polypeptide of the present invention having enzymatic activity, e.g., nuclease or endonuclease activity, and comprising an amino acid sequence which differs from the amino acid sequences of SEQ ID NO: 782, SEQ ID NO: 783, SEQ ID NO: 784, SEQ ID NO: 785, or SEQ ID NO: 786 by 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acid residue(s), when aligned using any of the previously described alignment methods.

In some embodiments, the composition of the present invention includes a Cas12i4 polypeptide described herein (e.g., a polypeptide comprising SEQ ID NO: 814 and/or encoded by SEQ ID NO: 787). In some embodiments, the Cas12i4 polypeptide comprises at least one RuvC domain.

A nucleic acid sequence encoding the Cas12i4 polypeptide described herein may be substantially identical to a reference nucleic acid sequence, e.g., SEQ ID NO: 787. In some embodiments, the Cas12i4 polypeptide is encoded by a nucleic acid comprising a sequence having least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity to the reference nucleic acid sequence, e.g., SEQ ID NO: 787. The percent identity between two such nucleic acids can be determined manually by inspection of the two optimally aligned nucleic acid sequences or by using software programs or algorithms (e.g., BLAST, ALIGN, CLUSTAL) using standard parameters. One indication that two nucleic acid sequences are substantially identical is that the nucleic acid molecules hybridize to the complementary sequence of the other under stringent conditions of temperature and ionic strength (e.g., within a range of medium to high stringency).

In some embodiments, the Cas12i4 polypeptide is encoded by a nucleic acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more sequence identity, but not 100% sequence identity, to a reference nucleic acid sequence, e.g., SEQ ID NO: 787.

In some embodiments, the present invention describes a Cas12i4 polypeptide having a specified degree of amino acid sequence identity to one or more reference polypeptides, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99%, but not 100%, sequence identity to the amino acid sequence of SEQ ID NO: 814. Homology or identity can be determined by amino acid sequence alignment, e.g., using a program such as BLAST, ALIGN, or CLUSTAL, as described herein.

Also provided is a Cas12i4 polypeptide of the present invention having enzymatic activity, e.g., nuclease or endonuclease activity, and comprising an amino acid sequence which differs from the amino acid sequences of SEQ ID NO: 814 by 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acid residue(s), when aligned using any of the previously described alignment methods.

In some embodiments, the Cas12i4 polypeptide comprises a polypeptide having a sequence of SEQ ID NO: 815 or SEQ ID NO: 816.

In some embodiments, the Cas12i4 polypeptide of the present invention comprises a polypeptide sequence having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 815 or SEQ ID NO: 816. In some embodiments, a Cas12i4 polypeptide having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 815 or SEQ ID NO: 816 maintains the amino acid changes (or at least 1, 2, 3 etc. of these changes) that differentiate it from its respective parent/reference sequence.

In some embodiments, the present invention describes a Cas12i4 polypeptide having a specified degree of amino acid sequence identity to one or more reference polypeptides, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99%, but not 100%, sequence identity to the amino acid sequence of SEQ ID NO: 815 or SEQ ID NO: 816. Homology or identity can be determined by amino acid sequence alignment, e.g., using a program such as BLAST, ALIGN, or CLUSTAL, as described herein.

Also provided is a Cas12i4 polypeptide of the present invention having enzymatic activity, e.g., nuclease or endonuclease activity, and comprising an amino acid sequence which differs from the amino acid sequences of SEQ ID NO: 815 or SEQ ID NO: 816 by 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acid residue(s), when aligned using any of the previously described alignment methods.

In some embodiments, the composition of the present invention includes a Cas12i1 polypeptide described herein (e.g., a polypeptide comprising SEQ ID NO: 817). In some embodiments, the Cas12i4 polypeptide comprises at least one RuvC domain.

In some embodiments, the Cas12i1 polypeptide of the present invention comprises a polypeptide sequence having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 817.

In some embodiments, the present invention describes a Cas12i1 polypeptide having a specified degree of amino acid sequence identity to one or more reference polypeptides, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99%, but not 100%, sequence identity to the amino acid sequence of SEQ ID NO: 817. Homology or identity can be determined by amino acid sequence alignment, e.g., using a program such as BLAST, ALIGN, or CLUSTAL, as described herein.

Also provided is a Cas12i1 polypeptide of the present invention having enzymatic activity, e.g., nuclease or endonuclease activity, and comprising an amino acid sequence which differs from the amino acid sequences of SEQ ID NO: 817 by 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acid residue(s), when aligned using any of the previously described alignment methods.

In some embodiments, the composition of the present invention includes a Cas12i3 polypeptide described herein (e.g., a polypeptide comprising SEQ ID NO: 818). In some embodiments, the Cas12i4 polypeptide comprises at least one RuvC domain.

In some embodiments, the Cas12i3 polypeptide of the present invention comprises a polypeptide sequence having at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 818.

In some embodiments, the present invention describes a Cas12i3 polypeptide having a specified degree of amino acid sequence identity to one or more reference polypeptides, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99%, but not 100%, sequence identity to the amino acid sequence of SEQ ID NO: 818. Homology or identity can be determined by amino acid sequence alignment, e.g., using a program such as BLAST, ALIGN, or CLUSTAL, as described herein.

Also provided is a Cas12i3 polypeptide of the present invention having enzymatic activity, e.g., nuclease or endonuclease activity, and comprising an amino acid sequence which differs from the amino acid sequences of SEQ ID NO: 818 by 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 amino acid residue(s), when aligned using any of the previously described alignment methods.

Although the changes described herein may be one or more amino acid changes, changes to the Cas12i polypeptide may also be of a substantive nature, such as fusion of polypeptides as amino- and/or carboxyl-terminal extensions. For example, the Cas12i polypeptide may contain additional peptides, e.g., one or more peptides. Examples of additional peptides may include epitope peptides for labelling, such as a polyhistidine tag (His-tag), Myc, and FLAG. In some embodiments, the Cas12i polypeptide described herein can be fused to a detectable moiety such as a fluorescent protein (e.g., green fluorescent protein (GFP) or yellow fluorescent protein (YFP)).

In some embodiments, the Cas12i polypeptide comprises at least one (e.g., two, three, four, five, six, or more) nuclear localization signal (NLS). In some embodiments, the Cas12i polypeptide comprises at least one (e.g., two, three, four, five, six, or more) nuclear export signal (NES). In some embodiments, the Cas12i polypeptide comprises at least one (e.g., two, three, four, five, six, or more) NLS and at least one (e.g., two, three, four, five, six, or more) NES.

In some embodiments, the Cas12i polypeptide described herein can be self-inactivating. See, Epstein et al., “Engineering a Self-Inactivating CRISPR System for AAV Vectors,” Mol. Ther., 24 (2016): S50, which is incorporated by reference in its entirety.

In some embodiments, the nucleotide sequence encoding the Cas12i polypeptide described herein can be codon-optimized for use in a particular host cell or organism. For example, the nucleic acid can be codon-optimized for any non-human eukaryote including mice, rats, rabbits, dogs, livestock, or non-human primates. Codon usage tables are readily available, for example, at the “Codon Usage Database” available at www.kazusa.orjp/codon/and these tables can be adapted in a number of ways. See Nakamura et al. Nucl. Acids Res. 28:292 (2000), which is incorporated herein by reference in its entirety. Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, PA).

Target Sequence

In some embodiments, the target sequence is within a B2M gene or a locus of a B2M gene. In some embodiments, the B2M gene is a mammalian gene. In some embodiments, the B2M gene is a human gene. For example, in some embodiments, the target sequence is within the sequence of SEQ ID NO: 773 or the reverse complement thereof. In some embodiments, the target sequence is within an exon of the B2M gene set forth in SEQ ID NO: 773 (or the reverse complement thereof), e.g., within a sequence of SEQ ID NO: 774, 775, 776, or 777 (or a reverse complement thereof). Target sequences within an exon of the B2M gene of SEQ ID NO: 773 (and the reverse complement thereof) are set forth in Table 5. In some embodiments, the target sequence is within an intron of the B2M gene set forth in SEQ ID NO: 773 (or the reverse complement thereof), e.g., within a sequence of SEQ ID NO: 1219, 1220, or 1221 (or a reverse complement thereof). Target sequences within an intron of the B2M gene of SEQ ID NO: 773 (or the reverse complement thereof) are set forth in Table 5. In some embodiments, the target sequence is within a variant (e.g., a polymorphic variant) of the B2M gene sequence set forth in SEQ ID NO: 773 or the reverse complement thereof. In some embodiments, the B2M gene sequence is a homolog of the sequence set forth in SEQ ID NO: 773 or the reverse complement thereof. For examples, in some embodiments, the B2M gene sequence is a non-human B2M sequence.

In some embodiments, the target sequence is adjacent to a 5′-NTTN-3′ PAM sequence, wherein N is any nucleotide. The 5′-NTTN-3′ sequence may be immediately adjacent to the target sequence or, for example, within a small number (e.g., 1, 2, 3, 4, or 5) of nucleotides of the target sequence. In some embodiments the 5′-NTTN-3′ sequence is 5′-NTTY-3′, 5′-NTTC-3′, 5′-NTTT-3′, 5′-NTTA-3′, 5′-NTTB-3′, 5′-NTTG-3′, 5′-CTTY-3′, 5‘-DTTR’3′, 5′-CTTR-3′, 5′-DTTT-3′, 5′-ATTN-3′, or 5′-GTTN-3′, wherein Y is C or T, B is any nucleotide except for A, D is any nucleotide except for C, and R is A or G. In some embodiments, the 5′-NTTN-3′ sequence is 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′.

In some embodiments, the target sequence is single-stranded (e.g., single-stranded DNA). In some embodiments, the target sequence is double-stranded (e.g., double-stranded DNA). In some embodiments, the target sequence comprises both single-stranded and double-stranded regions. In some embodiments, the target sequence is linear. In some embodiments, the target sequence is circular. In some embodiments, the target sequence comprises one or more modified nucleotides, such as methylated nucleotides, damaged nucleotides, or nucleotides analogs. In some embodiments, the target sequence is not modified. In some embodiments, the RNA guide binds to a first strand of a double-stranded target sequence (e.g., the target strand or the spacer-complementary strand), and the 5′-NTTN-3′ PAM sequence is present in the second, complementary strand (e.g., the non-target strand or the non-spacer-complementary strand). In some embodiments, the RNA guide binds adjacent to a 5′-NAAN-3′ sequence on the target strand (e.g., the spacer-complementary strand).

In some embodiments, the target sequence is present in a cell. In some embodiments, the target sequence is present in the nucleus of the cell. In some embodiments, the target sequence is endogenous to the cell. In some embodiments, the target sequence is a genomic DNA. In some embodiments, the target sequence is a chromosomal DNA. In some embodiments, the target sequence is a protein-coding gene or a functional region thereof, such as a coding region, or a regulatory element, such as a promoter, enhancer, a 5′ or 3′ untranslated region, etc. In some embodiments, the target sequence is a plasmid.

In some embodiments, the target sequence is present in a readily accessible region of the target sequence. In some embodiments, the target sequence is in an exon of a target gene. In some embodiments, the target sequence is across an exon-intron junction of a target gene. In some embodiments, the target sequence is present in a non-coding region, such as a regulatory region of a gene. In some embodiments, wherein the target sequence is exogenous to a cell, the target sequence comprises a sequence that is not found in the genome of the cell.

In some embodiments, the target sequence is exogenous to a cell. In some embodiments, the target sequence is a horizontally transferred plasmid. In some embodiments, the target sequence is integrated in the genome of the cell. In some embodiments, the target sequence is not integrated in the genome of the cell. In some embodiments, the target sequence is a plasmid in the cell. In some embodiments, the target sequence is present in an extrachromosomal array.

In some embodiments, the target sequence is an isolated nucleic acid, such as an isolated DNA or an isolated RNA. In some embodiments, the target sequence is present in a cell-free environment. In some embodiments, the target sequence is an isolated vector, such as a plasmid. In some embodiments, the target sequence is an ultrapure plasmid.

The target sequence is a locus of the B2M gene that hybridizes to the RNA guide. In some embodiments, a cell has only one copy of the target sequence. In some embodiments, a cell has more than one copy, such as at least about any one of 2, 3, 4, 5, 10, 100, or more copies of the target sequence.

In some embodiments, a B2M target sequence is selected to be edited by a Cas12i polypeptide and an RNA guide using one or more of the following criteria. First, in some embodiments, a target sequence near the 5′ end of the B2M coding sequence is selected. For example, in some embodiments, an RNA guide is designed to target a sequence in exon 1 (SEQ ID NO: 774) or exon 2 (SEQ ID NO: 775). Second, in some embodiments, a target sequence adjacent to a 5′-CTTY-3′ PAM sequence is selected. For example, in some embodiments, an RNA guide is designed to target a sequence adjacent to a 5′-CTTT-3′ or 5′-CTTC-3′ sequence. Third, in some embodiments, a target sequence having low sequence similarity to other genomic sequences is selected. For example, for each target sequence, potential non-target sites can be identified by searching for other genomic sequences adjacent to a PAM sequence and calculating the Levenshtein distance between the target sequence and the PAM-adjacent sequences. The Levenshtein distance (e.g., edit distance) corresponds to the minimum number of edits (e.g., insertions, deletions, or substitutions) required to change one sequence into another (e.g., to change the sequence of a potential non-target locus into the sequence of the on-target locus). Following this analysis, RNA guides are designed for target sequences that do not have potential off-target sequences with a Levenshtein distance of 0 or 1.

Production

The present invention includes methods for production of the RNA guide, methods for production of the polypeptide, and methods for complexing the RNA guide and Cas12i polypeptide.

RNA Guide

In some embodiments, the RNA guide is made by in vitro transcription of a DNA template. Thus, for example, in some embodiments, the RNA guide is generated by in vitro transcription of a DNA template encoding the RNA guide using an upstream promoter sequence (e.g., a T7 polymerase promoter sequence). In some embodiments, the DNA template encodes multiple RNA guides or the in vitro transcription reaction includes multiple different DNA templates, each encoding a different RNA guide. In some embodiments, the RNA guide is made using chemical synthetic methods. In some embodiments, the RNA guide is made by expressing the RNA guide sequence in cells transfected with a plasmid including sequences that encode the RNA guide. In some embodiments, the plasmid encodes multiple different RNA guides. In some embodiments, multiple different plasmids, each encoding a different RNA guide, are transfected into the cells. In some embodiments, the RNA guide is expressed from a plasmid that encodes the RNA guide and also encodes a Cas12i polypeptide. In some embodiments, the RNA guide is expressed from a plasmid that expresses the RNA guide but not a Cas12i polypeptide. In some embodiments, the RNA guide is purchased from a commercial vendor. In some embodiments, the RNA guide is synthesized using one or more modified nucleotide, e.g., as described above.

Cas12i Polypeptide

In some embodiments, the Cas12i polypeptide of the present invention can be prepared by (a) culturing bacteria which produce the Cas12i polypeptide of the present invention, isolating the Cas12i polypeptide, optionally, purifying the Cas12i polypeptide, and complexing the Cas12i polypeptide with an RNA guide. The Cas12i polypeptide can be also prepared by (b) a known genetic engineering technique, specifically, by isolating a gene encoding the Cas12i polypeptide of the present invention from bacteria, constructing a recombinant expression vector, and then transferring the vector into an appropriate host cell that expresses the RNA guide for expression of a recombinant protein that complexes with the RNA guide in the host cell. Alternatively, the Cas12i polypeptide can be prepared by (c) an in vitro coupled transcription-translation system and then complexing with an RNA guide.

In some embodiments, a host cell is used to express the Cas12i polypeptide. The host cell is not particularly limited, and various known cells can be preferably used. Specific examples of the host cell include bacteria such as E. coli, yeasts (budding yeast, Saccharomyces cerevisiae, and fission yeast, Schizosaccharomyces pombe), nematodes (Caenorhabditis elegans), Xenopus laevis oocytes, and animal cells (for example, CHO cells, COS cells and HEK293 cells). The method for transferring the expression vector described above into host cells, i.e., the transformation method, is not particularly limited, and known methods such as electroporation, the calcium phosphate method, the liposome method and the DEAE dextran method can be used.

After a host is transformed with the expression vector, the host cells may be cultured, cultivated or bred, for production of the Cas12i polypeptide. After expression of the Cas12i polypeptide, the host cells can be collected and Cas12i polypeptide purified from the cultures etc. according to conventional methods (for example, filtration, centrifugation, cell disruption, gel filtration chromatography, ion exchange chromatography, etc.).

In some embodiments, the methods for Cas12i polypeptide expression comprises translation of at least 5 amino acids, at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 50 amino acids, at least 100 amino acids, at least 150 amino acids, at least 200 amino acids, at least 250 amino acids, at least 300 amino acids, at least 400 amino acids, at least 500 amino acids, at least 600 amino acids, at least 700 amino acids, at least 800 amino acids, at least 900 amino acids, or at least 1000 amino acids of the Cas12i polypeptide. In some embodiments, the methods for protein expression comprises translation of about 5 amino acids, about 10 amino acids, about 15 amino acids, about 20 amino acids, about 50 amino acids, about 100 amino acids, about 150 amino acids, about 200 amino acids, about 250 amino acids, about 300 amino acids, about 400 amino acids, about 500 amino acids, about 600 amino acids, about 700 amino acids, about 800 amino acids, about 900 amino acids, about 1000 amino acids or more of the Cas12i polypeptide.

A variety of methods can be used to determine the level of production of a Cas12i polypeptide in a host cell. Such methods include, but are not limited to, for example, methods that utilize either polyclonal or monoclonal antibodies specific for the Cas12i polypeptide or a labeling tag as described elsewhere herein. Exemplary methods include, but are not limited to, enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (MA), fluorescent immunoassays (FIA), and fluorescent activated cell sorting (FACS). These and other assays are well known in the art (See, e.g., Maddox et al., J. Exp. Med. 158:1211 [1983]).

The present disclosure provides methods of in vivo expression of the Cas12i polypeptide in a cell, comprising providing a polyribonucleotide encoding the Cas12i polypeptide to a host cell wherein the polyribonucleotide encodes the Cas12i polypeptide, expressing the Cas12i polypeptide in the cell, and obtaining the Cas12i polypeptide from the cell.

Complexing

In some embodiments, an RNA guide targeting B2M is complexed with a Cas12i polypeptide to form a ribonucleoprotein. In some embodiments, complexation of the RNA guide and Cas12i polypeptide occurs at a temperature lower than about any one of 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C., 39° C., 40° C., 41° C., 42° C., 43° C., 44° C., 45° C., 50° C., or 55° C. In some embodiments, the RNA guide does not dissociate from the Cas12i polypeptide at about 37° C. over an incubation period of at least about any one of 10 mins, 15 mins, 20 mins, 25 mins, 30 mins, 35 mins, 40 mins, 45 mins, 50 mins, 55 mins, 1 hr, 2 hr, 3 hr, 4 hr, or more hours.

In some embodiments, the RNA guide and Cas12i polypeptide are complexed in a complexation buffer. In some embodiments, the Cas12i polypeptide is stored in a buffer that is replaced with a complexation buffer to form a complex with the RNA guide. In some embodiments, the Cas12i polypeptide is stored in a complexation buffer.

In some embodiments, the complexation buffer has a pH in a range of about 7.3 to 8.6. In one embodiment, the pH of the complexation buffer is about 7.3. In one embodiment, the pH of the complexation buffer is about 7.4. In one embodiment, the pH of the complexation buffer is about 7.5. In one embodiment, the pH of the complexation buffer is about 7.6. In one embodiment, the pH of the complexation buffer is about 7.7. In one embodiment, the pH of the complexation buffer is about 7.8. In one embodiment, the pH of the complexation buffer is about 7.9. In one embodiment, the pH of the complexation buffer is about 8.0. In one embodiment, the pH of the complexation buffer is about 8.1. In one embodiment, the pH of the complexation buffer is about 8.2. In one embodiment, the pH of the complexation buffer is about 8.3. In one embodiment, the pH of the complexation buffer is about 8.4. In one embodiment, the pH of the complexation buffer is about 8.5. In one embodiment, the pH of the complexation buffer is about 8.6.

In some embodiments, the Cas12i polypeptide can be overexpressed and complexed with the RNA guide in a host cell prior to purification as described herein. In some embodiments, mRNA or DNA encoding the Cas12i polypeptide is introduced into a cell so that the Cas12i polypeptide is expressed in the cell. In some embodiments, the RNA guide is also introduced into the cell, whether simultaneously, separately, or sequentially from a single mRNA or DNA construct, such that the ribonucleoprotein complex is formed in the cell.

Delivery

Compositions or complexes described herein may be formulated, for example, including a carrier, such as a carrier and/or a polymeric carrier, e.g., a liposome, and delivered by known methods to a cell (e.g., a prokaryotic, eukaryotic, plant, mammalian, etc.). Such methods include, but not limited to, transfection (e.g., lipid-mediated, cationic polymers, calcium phosphate, dendrimers); electroporation or other methods of membrane disruption (e.g., nucleofection), viral delivery (e.g., lentivirus, retrovirus, adenovirus, AAV), microinjection, microprojectile bombardment (“gene gun”), fugene, direct sonic loading, cell squeezing, optical transfection, protoplast fusion, impalefection, magnetofection, exosome-mediated transfer, lipid nanoparticle-mediated transfer, and any combination thereof.

In some embodiments, the method comprises delivering one or more nucleic acids (e.g., nucleic acids encoding the Cas12i polypeptide, RNA guide, donor DNA, etc.), one or more transcripts thereof, and/or a pre-formed RNA guide/Cas12i polypeptide complex to a cell, where a ternary complex is formed. Exemplary intracellular delivery methods, include, but are not limited to: viruses or virus-like agents; chemical-based transfection methods, such as those using calcium phosphate, dendrimers, liposomes, or cationic polymers (e.g., DEAE-dextran or polyethylenimine); non-chemical methods, such as microinjection, electroporation, cell squeezing, sonoporation, optical transfection, impalefection, protoplast fusion, bacterial conjugation, delivery of plasmids or transposons; particle-based methods, such as using a gene gun, magnectofection or magnet assisted transfection, particle bombardment; and hybrid methods, such as nucleofection. In some embodiments, the present application further provides cells produced by such methods, and organisms (such as animals, plants, or fungi) comprising or produced from such cells.

In some embodiments, the Cas12i component and the RNA guide component are delivered together. For example, in some embodiments, the Cas12i component and the RNA guide component are packaged together in a single AAV particle. In another example, in some embodiments, the Cas12i component and the RNA guide component are delivered together via lipid nanoparticles (LNPs). In some embodiments, the Cas12i component and the RNA guide component are delivered separately. For example, in some embodiments, the Cas12i component and the RNA guide are packaged into separate AAV particles. In another example, in some embodiments, the Cas12i component is delivered by a first delivery mechanism and the RNA guide is delivered by a second delivery mechanism.

Cells

Compositions or complexes described herein can be delivered to a variety of cells. In some embodiments, the cell is an isolated cell. In some embodiments, the cell is in cell culture or a co-culture of two or more cell types. In some embodiments, the cell is ex vivo. In some embodiments, the cell is obtained from a living organism and maintained in a cell culture. In some embodiments, the cell is a single-cellular organism.

In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a bacterial cell or derived from a bacterial cell. In some embodiments, the cell is an archaeal cell or derived from an archaeal cell.

In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a plant cell or derived from a plant cell. In some embodiments, the cell is a fungal cell or derived from a fungal cell. In some embodiments, the cell is an animal cell or derived from an animal cell. In some embodiments, the cell is an invertebrate cell or derived from an invertebrate cell. In some embodiments, the cell is a vertebrate cell or derived from a vertebrate cell. In some embodiments, the cell is a mammalian cell or derived from a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a zebra fish cell. In some embodiments, the cell is a rodent cell. In some embodiments, the cell is synthetically made, sometimes termed an artificial cell.

In some embodiments, the cell is derived from a cell line. A wide variety of cell lines for tissue culture are known in the art. Examples of cell lines include, but are not limited to, 293T, MF7, K562, HeLa, CHO, and transgenic varieties thereof. Cell lines are available from a variety of sources known to those with skill in the art (see, e.g., the American Type Culture Collection (ATCC) (Manassas, Va.)). In some embodiments, the cell is an immortal or immortalized cell.

In some embodiments, the cell is a primary cell. In some embodiments, the cell is a stem cell such as a totipotent stem cell (e.g., omnipotent), a pluripotent stem cell, a multipotent stem cell, an oligopotent stem cell, or an unipotent stem cell. In some embodiments, the cell is an induced pluripotent stem cell (iPSC) or derived from an iPSC. In some embodiments, the cell is a differentiated cell. For example, in some embodiments, the differentiated cell is a muscle cell (e.g., a myocyte), a fat cell (e.g., an adipocyte), a bone cell (e.g., an osteoblast, osteocyte, osteoclast), a blood cell (e.g., a monocyte, a lymphocyte, a neutrophil, an eosinophil, a basophil, a macrophage, a erythrocyte, or a platelet), a nerve cell (e.g., a neuron), an epithelial cell, an immune cell (e.g., a lymphocyte, a neutrophil, a monocyte, or a macrophage), a liver cell (e.g., a hepatocyte), a fibroblast, or a sex cell. In some embodiments, the cell is a terminally differentiated cell. For example, in some embodiments, the terminally differentiated cell is a neuronal cell, an adipocyte, a cardiomyocyte, a skeletal muscle cell, an epidermal cell, or a gut cell. In some embodiments, the cell is an immune cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a B cell. In some embodiments, the immune cell is a Natural Killer (NK) cell. In some embodiments, the immune cell is a Tumor Infiltrating Lymphocyte (TIL). In some embodiments, the cell is a mammalian cell, e.g., a human cell or a murine cell. In some embodiments, the murine cell is derived from a wild-type mouse, an immunosuppressed mouse, or a disease-specific mouse model. In some embodiments, the cell is a cell within a living tissue, organ, or organism.

Methods

The disclosure also provides methods of modifying a target sequence within the B2M gene. In some embodiments, the methods comprise introducing a B2M-targeting RNA guide and a Cas12i polypeptide into a cell. The B2M-targeting RNA guide and Cas12i polypeptide can be introduced as a ribonucleoprotein complex into a cell. The B2M-targeting RNA guide and Cas12i polypeptide can be introduced on a nucleic acid vector. The Cas12i polypeptide can be introduced as an mRNA. The RNA guide can be introduced directly into the cell.

In some embodiments, the sequence of the B2M gene is set forth in SEQ ID NO: 773 (or the reverse complement thereof). In some embodiments, the target sequence is in an exon of a B2M gene, such as an exon having a sequence set forth in any one of SEQ ID NO: 774, SEQ ID NO: 775, SEQ ID NO: 776, or SEQ ID NO: 777 (or the reverse complement thereof). In some embodiments, the target sequence is in an intron of a B2M gene (e.g., an intron of the sequence set forth in SEQ ID NO: 773, or the reverse complement thereof), such as an intron having a sequence set forth in any one of SEQ ID NO: 1219, SEQ ID NO: 1220, or SEQ ID NO: 1221 (or the reverse complement thereof). In other embodiments, the sequence of the B2M gene is a variant of the sequence set forth in SEQ ID NO: 773 (or the reverse complement thereof) or a homolog of the sequence set forth in SEQ ID NO: 773 (or the reverse complement thereof). For example, in some embodiments, the target sequence is polymorphic variant of the B2M sequence set forth in SEQ ID NO: 773 (or the reverse complement thereof) or a non-human form of the B2M gene.

In some embodiments, an RNA guide as disclosed herein is designed to be complementary to a target sequence that is adjacent to a 5′-NTTN-3′ PAM sequence. The 5′-NTTN-3′ sequence may be immediately adjacent to the target sequence or, for example, within a small number (e.g., 1, 2, 3, 4, or 5) of nucleotides of the target sequence. In some embodiments the 5′-NTTN-3′ sequence is 5′-NTTY-3′, 5′-NTTC-3′, 5′-NTTT-3′, 5′-NTTA-3′, 5′-NTTB-3′, 5′-NTTG-3′, 5′-CTTY-3′, 5‘-DTTR’3′, 5′-CTTR-3′, 5′-DTTT-3′, 5′-ATTN-3′, or 5′-GTTN-3′, wherein Y is C or T, B is any nucleotide except for A, D is any nucleotide except for C, and R is A or G. In some embodiments, the 5′-NTTN-3′ sequence is 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′. In some embodiments, the RNA guide is designed to bind to a first strand of a double-stranded target sequence (e.g., the target strand or the spacer-complementary strand), and the 5′-NTTN-3′ PAM sequence is present in the second, complementary strand (e.g., the non-target strand or the non-spacer-complementary strand).

In some embodiments, the RNA guide binds adjacent to a 5′-NAAN-3′ sequence on the target strand (e.g., the spacer-complementary strand).

In some embodiments, the Cas12i polypeptide has enzymatic activity (e.g., nuclease activity). In some embodiments, the Cas12i polypeptide induces one or more DNA double-stranded breaks in the cell. In some embodiments, the Cas12i polypeptide induces one or more DNA single-stranded breaks in the cell.

In some embodiments, the Cas12i polypeptide induces one or more DNA nicks in the cell. In some embodiments, DNA breaks and/or nicks result in formation of one or more indels (e.g., one or more deletions).

In some embodiments, an RNA guide disclosed herein forms a complex with the Cas12i polypeptide and directs the Cas12i polypeptide to a target sequence adjacent to a 5′-NTTN-3′ sequence. In some embodiments, the complex induces a deletion (e.g., a nucleotide deletion or DNA deletion) adjacent to the 5′-NTTN-3′ sequence. In some embodiments, the complex induces a deletion adjacent to a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the complex induces a deletion adjacent to a T/C-rich sequence.

In some embodiments, the deletion is downstream of a 5′-NTTN-3′ sequence. In some embodiments, the deletion is downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion is downstream of a T/C-rich sequence.

In some embodiments, the deletion alters expression of the B2M gene. In some embodiments, the deletion alters function of the B2M gene. In some embodiments, the deletion inactivates the B2M gene. In some embodiments, the deletion is a frameshifting deletion. In some embodiments, the deletion is a non-frameshifting deletion. In some embodiments, the deletion leads to cell toxicity or cell death (e.g., apoptosis).

In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) of a T/C-rich sequence.

In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a T/C-rich sequence.

In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) of a T/C-rich sequence.

In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of a T/C-rich sequence.

In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) of a T/C-rich sequence.

In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a T/C-rich sequence.

In some embodiments, the deletion ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5′-NTTN-3′ sequence. In some embodiments, the deletion ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a T/C-rich sequence.

In some embodiments, the deletion ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-NTTN-3′ sequence. In some embodiments, the deletion ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of a T/C-rich sequence.

In some embodiments, the deletion ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of the 5′-NTTN-3′ sequence. In some embodiments, the deletion ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of a T/C-rich sequence.

In some embodiments, the deletion ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the 5′-NTTN-3′ sequence. In some embodiments, the deletion ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of a T/C-rich sequence.

In some embodiments, the deletion ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5′-NTTN-3′ sequence. In some embodiments, the deletion ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a T/C-rich sequence.

In some embodiments, the deletion ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-NTTN-3′ sequence. In some embodiments, the deletion ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of a T/C-rich sequence.

In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a T/C-rich sequence.

In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5′-NTTN-3′ sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a T/C-rich sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the T/C-rich sequence.

In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of a T/C-rich sequence.

In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5′-NTTN-3′ sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a T/C-rich sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the T/C-rich sequence.

In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a T/C-rich sequence.

In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5′-NTTN-3′ sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 5 to about 15 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a T/C-rich sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the T/C-rich sequence.

In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a T/C-rich sequence.

In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of the 5′-NTTN-3′ sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of a T/C-rich sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the T/C-rich sequence.

In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) of a T/C-rich sequence.

In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of the 5′-NTTN-3′ sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of a T/C-rich sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the T/C-rich sequence.

In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a T/C-rich sequence.

In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of the 5′-NTTN-3′ sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 5 to about 10 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides) downstream of a T/C-rich sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the T/C-rich sequence.

In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a T/C-rich sequence.

In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5′-NTTN-3′ sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a T/C-rich sequence and ends within about 20 to about 30 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the T/C-rich sequence.

In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) of a T/C-rich sequence.

In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5′-NTTN-3′ sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a T/C-rich sequence and ends within about 20 to about 25 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides) downstream of the T/C-rich sequence.

In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) of a T/C-rich sequence.

In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of the 5′-NTTN-3′ sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-NTTN-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the 5′-ATTA-3′, 5′-ATTT-3′, 5′-ATTG-3′, 5′-ATTC-3′, 5′-TTTA-3′, 5′-TTTT-3′, 5′-TTTG-3′, 5′-TTTC-3′, 5′-GTTA-3′, 5′-GTTT-3′, 5′-GTTG-3′, 5′-GTTC-3′, 5′-CTTA-3′, 5′-CTTT-3′, 5′-CTTG-3′, or 5′-CTTC-3′ sequence. In some embodiments, the deletion starts within about 10 to about 15 nucleotides (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides) downstream of a T/C-rich sequence and ends within about 25 to about 30 nucleotides (e.g., about 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 nucleotides) downstream of the T/C-rich sequence.

In some embodiments, the deletion is up to about 50 nucleotides in length (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides). In some embodiments, the deletion is up to about 40 nucleotides in length (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 nucleotides). In some embodiments, the deletion is between about 4 nucleotides and about 40 nucleotides in length (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 nucleotides). In some embodiments, the deletion is between about 4 nucleotides and about 25 nucleotides in length (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides). In some embodiments, the deletion is between about 10 nucleotides and about 25 nucleotides in length (e.g., about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 nucleotides). In some embodiments, the deletion is between about 10 nucleotides and about 15 nucleotides in length (e.g., about 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 nucleotides).

In some embodiments, the methods described herein are used to engineer a cell comprising a deletion as described herein in a B2M gene.

Compositions, vectors, nucleic acids, RNA guides and cells disclosed herein may be used in therapy. Compositions, vectors, nucleic acids, RNA guides and cells disclosed herein may be used in methods of treating a disease or condition in a subject. Any suitable delivery or administration method known in the art may be used to deliver compositions, vectors, nucleic acids, RNA guides and cells disclosed herein. Such methods may involve contacting a target sequence with a composition, vector, nucleic acid, or RNA guide disclosed herein. Such methods may involve a method of editing a B2M sequence as disclosed herein. In some embodiments, a cell engineered using an RNA guide disclosed herein is used for ex vivo gene therapy. In some embodiments, a cell engineered using an RNA guide disclosed herein is used for CAR T-cell therapy.

Kits

The invention also provides kits or systems that can be used, for example, to carry out a method described herein. In some embodiments, the kits or systems include an RNA guide and a Cas12i polypeptide. In some embodiments, the kits or systems include a polynucleotide that encodes such a Cas12i polypeptide, and optionally the polynucleotide is comprised within a vector, e.g., as described herein. In some embodiments, the kits or systems include a polynucleotide that encodes an RNA guide disclosed herein. The Cas12i polypeptide and the RNA guide (e.g., as a ribonucleoprotein) can be packaged within the same or other vessel within a kit or system or can be packaged in separate vials or other vessels, the contents of which can be mixed prior to use. The kits or systems can additionally include, optionally, a buffer and/or instructions for use of the RNA guide and Cas12i polypeptide.

All references and publications cited herein are hereby incorporated by reference.

EXAMPLES

The following examples are provided to further illustrate some embodiments of the present invention but are not intended to limit the scope of the invention; it will be understood by their exemplary nature that other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.

Example 1—Editing of B2M in a Mammalian Cell Via Transfection

This Example describes indel assessment on multiple B2M targets using Cas12i2 and RNA guide compositions introduced into mammalian cells by transient transfection.

Variant Cas12i2 of SEQ ID NO: 782 was cloned with a CMV promoter in a pcda3.1 backbone (Invitrogen). The plasmids were then maxi-prepped and diluted to 1 μg/L. For RNA guide preparation, a dsDNA fragment encoding an RNA guide was derived by ultramers containing the target sequence scaffold, and the U6 promoter. Ultramers were resuspended in 10 mM Tris·HCl at a pH of 7.5 to a final stock concentration of 100 μM. Working stocks were subsequently diluted to 10 μM, again using 10 mM Tris·HCl to serve as the template for the PCR reaction. The amplification of the RNA guide was done in 50 μL reactions with the following components: 0.02 μl of aforementioned template, 2.5 μl forward primer, 2.5 μl reverse primer, 25 μL NEB HiFi Polymerase, and 20 μl water. Cycling conditions were: 1×(30 s at 98° C.), 30×(10 s at 98° C., 15 s at 67° C.), 1× (2 min at 72° C.). PCR products were cleaned up with a 1.8× SPRI treatment and normalized to 25 ng/L. The prepared RNA guide sequences and their corresponding target sequences are shown in Table 6.

TABLE 6

RNA guide and Target Sequences for Transient Transfection.

Target
RNA Guide
Target Sequence

B2M_exon1_target1
AGAAAUCCGUCUUUCAUUGACGGAGGAAUGC
AGGAATGCCCGCCAGCGCGA

CCGCCAGCGCGA(SEQ ID NO: 1222)
(SEQ ID NO: 1231)

B2M_exon1_target2
AGAAAUCCGUCUUUCAUUGACGGCUGGCCUG
CTGGCCTGGAGGCTATCCAG

GAGGCUAUCCAG (SEQ ID NO: 1223)
(SEQ ID NO: 1232)

B2M_exon2_target1
AGAAAUCCGUCUUUCAUUGACGGUCCCGAUA
TCCCGATATTCCTCAGGTAC

UUCCUCAGGUAC (SEQ ID NO: 1224)
(SEQ ID NO: 1233)

B2M_exon2_target2
AGAAAUCCGUCUUUCAUUGACGGGG
GGAGTACCTGAGGAATATCG

AGUACCUGAGGAAUAUCG (SEQ ID NO: 1225)
(SEQ ID NO: 1234)

B2M_exon2_target3
AGAAAUCCGUCUUUCAUUGACGGCC
CCATTCTCTGCTGGATGACG

AUUCUCUGCUGGAUGACG (SEQ ID NO: 1226)
(SEQ ID NO: 1235)

B2M_exon2_target4
AGAAAUCCGUCUUUCAUUGACGGAA
AATGTCGGATGGATGAAACC

UGUCGGAUGGAUGAAACC (SEQ ID NO: 778)
(SEQ ID NO: 1236)

B2M_exon2_target5
AGAAAUCCGUCUUUCAUUGACGGAG
AGTAAGTCAACTTCAATGTC

UAAGUCAACUUCAAUGUC (SEQ ID NO: 1227)
(SEQ ID NO: 1237)

B2M_exon2_target6
AGAAAUCCGUCUUUCAUUGACGGUU
TTCAATTCTCTCTCCATTCT

CAAUUCUCUCUCCAUUCU (SEQ ID NO: 1228)
(SEQ ID NO: 1238)

B2M_exon2_target7
AGAAAUCCGUCUUUCAUUGACGGCA
CAGCAAGGACTGGTCTTTCT

GCAAGGACUGGUCUUUCU (SEQ ID NO: 1229)
(SEQ ID NO: 1239)

B2M_exon2_target8
AGAAAUCCGUCUUUCAUUGACGGCU
CTATCTCTTGTACTACACTG

AUCUCUUGUACUACACUG (SEQ ID NO: 779)
(SEQ ID NO: 1240)

B2M_exon2_target9
AGAAAUCCGUCUUUCAUUGACGGUU
TTCAGTGGGGGTGAATTCAG

CAGUGGGGGUGAAUUCAG (SEQ ID NO: 1230)
(SEQ ID NO: 1241)

Approximately 16 hours prior to transfection, 100 μl of 25,000 HEK293T cells in DMEM/10% FBS+Pen/Strep were plated into each well of a 96-well plate. On the day of transfection, the cells were 70-90% confluent. For each well to be transfected, a mixture of 0.5 μl of Lipofectamine 2000 and 9.5 μl of Opti-MEM was prepared and then incubated at room temperature for 5-20 minutes (Solution 1). After incubation, the lipofectamine:OptiMEM mixture was added to a separate mixture containing 182 ng of effector plasmid and 14 ng of RNA guide and water up to 10 μL (Solution 2). The solution 1 and solution 2 mixtures were mixed by pipetting up and down and then incubated at room temperature for 25 minutes. Following incubation, 20 μL of the Solution 1 and Solution 2 mixture were added dropwise to each well of a 96 well plate containing the cells. 72 hours post transfection, cells are trypsinized by adding 10 μL of TrypLE to the center of each well and incubated for approximately 5 minutes. 100 μL of D10 media was then added to each well and mixed to resuspend cells. The cells were then spun down at 500 g for 10 minutes, and the supernatant was discarded. QuickExtract buffer was added to ⅕ the amount of the original cell suspension volume. Cells were incubated at 65° C. for 15 minutes, 68° C. for 15 minutes, and 98° C. for 10 minutes.

Samples for Next Generation Sequencing were prepared by two rounds of PCR. The first round (PCR1) was used to amplify specific genomic regions depending on the target. PCR1 products were purified by column purification. Round 2 PCR (PCR2) was done to add Illumina adapters and indexes. Reactions were then pooled, loaded onto a 2% E-gel EX for 10 minutes and gel extracted. Sequencing runs were done with a 150 cycle NextSeq v2.5 mid or high output kit.

As shown in FIG. 1, each of the eleven tested RNA guides induced indels in B2M target sequences. Therefore, RNA guides and the variant Cas12i2 of SEQ ID NO: 782 were able to target B2M targets in exon 1 and exon 2 in mammalian cells.

Example 2—Editing of B2M in a Mammalian Cell by RNP Electroporation

This Example describes ribonucleoprotein (RNP) transfection followed by FACS staining and indel assessment on multiple B2M target sequences using a Cas12i polypeptide in mammalian cells.

CD3+ T cells from three individual donors were revived and counted using an automated cell counter. A sample from each donor was collected and stained for CD3ε and DAPI for flow cytometry analysis of surface expression and viability, respectively. Cell density was adjusted to 1e6 cells/mL and cells were stimulated for 3 days with a cocktail of anti-CD3:CD28 antibodies.

Variant Cas12i2 RNP complexation reactions were made by mixing purified variant Cas12i2 (400 μM; SEQ ID NO: 783) with RNA guide (1 mM in 250 mM NaCl; see sequences in Table 7) at a 1:1 (effector:RNA guide) volume ratio (2.5:1 RNA guide:effector molar ratio). SpCas9 RNP complexation reactions were made by mixing purified SpCas9 (Aldevron; 62 μM) with sgRNA (1 mM in water; see sequences in Table 7) at a 6.45:1 (effector:sgRNA) volume ratio (2.5:1 sgRNA:effector molar ratio). For “effector only” controls, variant Cas12i2 or SpCas9 were mixed with Protein Storage Buffer (25 mM Tris, pH 7.5, 250 mM NaCl, 1 mM TCEP, 50% glycerol) at the same volume ratio as the RNA guide or sgRNA, respectively. Additional controls were included: SpCas9 (Aldevron) with either Lethal #1 (transfection control guide), pooled CD3, or ROSA26 sgRNAs and SpCas9 (Horizon) with either Lethal #1, pooled CD3, or ROSA26 sgRNAs. Complexations were incubated at 37° C. for 30-60 min. Following incubation, RNPs were diluted to 20 μM, 50 μM, 100 μM, or 160 μM effector concentration for variant Cas12i2 and 20 μM or 50 μM for SpCas9.

TABLE 7

RNA guide sequences for RNP transfection.

Target

Guide Name
Gene
Effector
PAM
Strand
RNA guide

Cas12i2_B2M
B2M
Cas12i2
CTTC
TS
AGAAAUCCGUCUUUCAUUGACGGAAUGUCGGAU

exon2_target4

GGAUGAAACC (SEQ ID NO: 778)

Cas12i2_B2M
B2M
Cas12i2
CTTT
BS
AGAAAUCCGUCUUUCAUUGACGGCUAUCUCUUG

exon2_target8

UACUACACUG (SEQ ID NO: 779)

Cas12i2_B2M
B2M
Cas12i2
GTTC
TS
AGAAAUCCGUCUUUCAUUGACGGACACGGCAGG

exon2_target10

CAUACUCAUC (SEQ ID NO: 780)

Cas12i2_B2M
B2M
Cas12i2
CTTT
BS
AGAAAUCCGUCUUUCAUUGACGGGUCACAGCCC

exon2_target11

AAGAUAGUUA (SEQ ID NO: 781)

SpCas9_B2M
B2M
SpCas9
TGG
BS
mG*mG*mC*CGAGAUGUCUCGCUCCGGUUUUAG

exon1_target1

AGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUC

CGUUAUCAACUUGAAAAAGUGGCACCGAGUCGG

UGCmU*mU*mU*U (SEQ ID NO: 813)

Diluted complexed reactions were dispensed at 2 μL per well into a 384-well electroporation plate. Cell suspensions were collected and counted using an automated cell counter. Cell density was adjusted to 1.1e7 cells/mL in P3 buffer and was dispensed at 2e5 cells/reaction (18 μL). Final concentration of variant Cas12i2 RNPs was 2 μM, 5 μM, 10 μM, or 16 μM. Final concentration of SpCas9 RNPs was 2 or 5 μM. The following controls were set up: unelectroporated cells only, cells in P3 primary cell buffer (Lonza #VXP-3032) only, cells in Protein Storage Buffer only. The plate was electroporated using an electroporation device (program EO-115-AA, Lonza HT), excluding the unelectroporated conditions. Each well was split into four 96-well editing plates (containing 200 μL total volume) using robotics (StarLab Hamilton). Editing plates were incubated for 7 days at 37° C. with 100 μL media replacement at day 4.

After 7 days, plates were spun down and the supernatant was removed. Pellets were resuspended in 200 μL of PBS. 100 μL of sample was collected and stained with either the antibody panel (anti-B2M) or anti-CD3E antibody (lethal #1, pooled CD3E, ROSA26, Protein Storage Buffer and unelectroporated for Cas9 controls). All cells were stained with DAPI to assess viability. Remaining cell suspension was transferred to a 96-well PCR plate and pelleted at 500×g for 5 min. Supernatants were removed and pellets were frozen at −80° C.

For gDNA extraction, pellets were thawed to room temperature and resuspended in appropriate volume of DNA extraction buffer (QuickExtract) to give final concentration of 1000 cells/μL. Samples were then cycled in PCR machine at 65° C. for 15 min, 68° C. for 15 min, 98° C. for 10 min. Samples were then frozen at −20° C.

Samples for Next Generation Sequencing (NGS) were prepared by rounds of PCR. The first round (PCR I) was used to amplify the genomic regions flanking the target site and add NGS adapters. The second round (PCR II) was used to add NGS indexes. Reactions were then pooled, purified by column purification, and quantified on a fluorometer (Qubit). Sequencing runs were done using a 150 cycle NGS instrument (NextSeq v2.5) mid or high output kit and run on an NGS instrument (NextSeq 550).

For NGS analysis, the indel mapping function used a sample's fastq file, the amplicon reference sequence, and the forward primer sequence. For each read, a kmer-scanning algorithm was used to calculate the edit operations (match, mismatch, insertion, deletion) between the read and the reference sequence. In order to remove small amounts of primer dimer present in some samples, the first 30nt of each read was required to match the reference and reads where over half of the mapping nucleotides were mismatches were filtered out as well. Up to 50,000 reads passing those filters were used for analysis, and reads were counted as an indel read if they contained an insertion or deletion. The indel % was calculated as the number of indel-containing reads divided by the number of reads analyzed (reads passing filters up to 50,000). The QC standard for the minimum number of reads passing filters was 10,000.

These results demonstrated robust indel activity by variant Cas12i2 RNP targeting multiple B2M targets in primary T cells (FIG. 2), with activity peaking at 16 μM. Flow cytometry staining showed significant reduction of B2M protein expression in T cells following variant Cas12i2 RNP (FIG. 3). Cell viability remained high for all conditions seven days post electroporation of the Cas12i2 RNPs targeting B2M (FIG. 4).

This Example thus shows how to measure viability of cells, e.g., T cells, electroporated with the RNA guide/Cas12i polypeptide complexes described herein, expression of B2M in the cells, and activity on B2M target sequences (indel %) in the cells.

This Example further shows that RNA guides and the variant Cas12i2 of SEQ ID NO: 783 were able to target B2M targets in exon 2 in mammalian cells.

Nucleotide
60 atgagcagcg cgatcaaaag ctacaagagc gttctgcgtc cgaacgagcg taagaaccaa

sequence
120 ctgctgaaaa gcaccattca gtgcctggaa gacggtagcg cgttcttttt caagatgctg

encoding
180 caaggcctgt ttggtggcat caccccggag attgttcgtt tcagcaccga acaggagaaa

Cas12i2-
240 cagcaacagg atatcgcgct gtggtgcgcg gttaactggt tccgtccggt gagccaagac

SEQ ID NO: 771
300 agcctgaccc acaccattgc gagcgataac ctggtggaga agtttgagga atactatggt

360 ggcaccgcga gcgacgcgat caaacagtac ttcagcgcga gcattggcga aagctactat

420 tggaacgact gccgtcaaca gtactatgat ctgtgccgtg agctgggtgt tgaggtgagc

480 gacctgaccc atgatctgga gatcctgtgc cgtgaaaagt gcctggcggt tgcgaccgag

540 agcaaccaga acaacagcat cattagcgtt ctgtttggca ccggcgaaaa agaggaccgt

600 agcgtgaaac tgcgtatcac caagaaaatt ctggaggcga tcagcaacct gaaagaaatc

660 ccgaagaacg ttgcgccgat tcaagagatc attctgaacg tggcgaaagc gaccaaggaa

720 accttccgtc aggtgtatgc gggtaacctg ggtgcgccga gcaccctgga gaaatttatc

780 gcgaaggacg gccaaaaaga gttcgatctg aagaaactgc agaccgacct gaagaaaggt

840 attcgtggta aaagcaagga gcgtgattgg tgctgccagg aagagctgcg tagctacgtg

900 gagcaaaaca ccatccagta tgacctgtgg gcgtggggcg aaatgttcaa caaagcgcac

960 accgcgctga aaatcaagag cacccgtaac tacaactttg cgaagcaacg tctggaacag

1020 ttcaaagaga ttcagagcct gaacaacctg ctggttgtga agaagctgaa cgactttttc

1080 gatagcgaat ttttcagcgg cgaggaaacc tacaccatct gcgttcacca tctgggtggc

1140 aaggacctga gcaaactgta taaggcgtgg gaggatgatc cggcggaccc ggaaaacgcg

1200 attgtggttc tgtgcgacga tctgaaaaac aactttaaga aagagccgat ccgtaacatt

1260 ctgcgttaca tcttcaccat tcgtcaagaa tgcagcgcgc aggacatcct ggcggcggcg

1320 aagtacaacc aacagctgga tcgttataaa agccaaaagg cgaacccgag cgttctgggt

1380 aaccagggct ttacctggac caacgcggtg atcctgccgg agaaggcgca gcgtaacgac

1440 cgtccgaaca gcctggatct gcgtatttgg ctgtacctga aactgcgtca cccggacggt

1500 cgttggaaga aacaccatat cccgttctac gatacccgtt tcttccaaga aatttatgcg

1560 gcgggcaaca gcccggttga cacctgccag tttcgtaccc cgcgtttcgg ttatcacctg

1620 ccgaaactga ccgatcagac cgcgatccgt gttaacaaga aacatgtgaa agcggcgaag

1680 ccgaaactga ccgatcagac cgcgatccgt gttaacaaga aacatgtgaa agcggcgaag

1740 atcaccgaaa ttagcgcgac catcaacagc aaaggtcaag tgcgtattcc ggttaagttt

1800 gacgtgggtc gtcaaaaagg caccctgcag atcggtgacc gtttctgcgg ctacgatcaa

1860 aaccagaccg cgagccacgc gtatagcctg tgggaagtgg ttaaagaggg tcaataccat

1920 aaagagctgg gctgctttgt tcgtttcatc agcagcggtg acatcgtgag cattaccgag

1980 aaccgtggca accaatttga tcagctgagc tatgaaggtc tggcgtaccc gcaatatgcg

2040 gactggcgta agaaagcgag caagttcgtg agcctgtggc agatcaccaa gaaaaacaag

2100 aaaaaggaaa tcgtgaccgt tgaagcgaaa gagaagtttg acgcgatctg caagtaccag

2160 ccgcgtctgt ataaattcaa caaggagtac gcgtatctgc tgcgtgatat tgttcgtggc

2220 aaaagcctgg tggaactgca acagattcgt caagagatct ttcgtttcat tgaacaggac

2280 tgcggtgtta cccgtctggg cagcctgagc ctgagcaccc tggaaaccgt gaaagcggtt

2340 aagggtatca tttacagcta ttttagcacc gcgctgaacg cgagcaagaa caacccgatc

2400 agcgacgaac agcgtaaaga gtttgatccg gaactgttcg cgctgctgga aaagctggag

2460 ctgattcgta cccgtaaaaa gaaacaaaaa gtggaacgta tcgcgaacag cctgattcag

2520 acctgcctgg agaacaacat caagttcatt cgtggtgaag gcgacctgag caccaccaac

2580 aacgcgacca agaaaaaggc gaacagccgt agcatggatt ggttggcgcg tggtgttttt

2640 aacaaaatcc gtcaactggc gccgatgcac aacattaccc tgttcggttg cggcagcctg

2700 tacaccagcc accaggaccc gctggtgcat cgtaacccgg ataaagcgat gaagtgccgt

2760 tgggcggcga tcccggttaa ggacattggc gattgggtgc tgcgtaagct gagccaaaac

2820 ctgcgtgcga aaaacatcgg caccggcgag tactatcacc aaggtgttaa agagttcctg

2880 agccattatg aactgcagga cctggaggaa gagctgctga agtggcgtag cgatcgtaaa

2940 agcaacattc cgtgctgggt gctgcagaac cgtctggcgg agaagctggg caacaaagaa

3000 gcggtggttt acatcccggt tcgtggtggc cgtatttatt ttgcgaccca caaggtggcg

3060 accggtgcgg tgagcatcgt tttcgaccaa aaacaagtgt gggtttgcaa cgcggatcat

3120 gttgcggcgg cgaacatcgc gctgaccgtg aagggtattg gcgaacaaag cagcgacgaa

3162 gagaacccgg atggtagccg tatcaaactg cagctgacca gc

Cas12i2
MSSAIKSYKSVLRPNERKNQLLKSTIQCLEDGSAFFFKMLQGLEGGITPEIVRESTEQEK

amino acid
QQQDIALWCAVNWFRPVSQDSLTHTIASDNLVEKFEEYYGGTASDAIKQYFSASIGESYY

sequence-
WNDCRQQYYDLCRELGVEVSDLTHDLEILCREKCLAVATESNQNNSIISVLFGTGEKEDR

SEQ ID NO: 772
SVKLRITKKILEAISNLKEIPKNVAPIQEIILNVAKATKETFRQVYAGNLGAPSTLEKFI

AKDGQKEFDLKKLQTDLKKVIRGKSKERDWCCQEELRSYVEQNTIQYDLWAWGEMENKAH

TALKIKSTRNYNFAKQRLEQFKEIQSLNNLLVVKKLNDFFDSEFFSGEETYTICVHHLGG

KDLSKLYKAWEDDPADPENAIVVLCDDLKNNFKKEPIRNILRYIFTIRQECSAQDILAAA

KYNQQLDRYKSQKANPSVLGNQGFTWTNAVILPEKAQRNDRPNSLDLRIWLYLKLRHPDG

RWKKHHIPFYDTRFFQEIYAAGNSPVDTCQFRTPRFGYHLPKLTDQTAIRVNKKHVKAAK

TEARIRLAIQQGTLPVSNLKITEISATINSKGQVRIPVKFDVGRQKGTLQIGDRFCGYDQ

NQTASHAYSLWEVVKEGQYHKELGCFVRFISSGDIVSITENRGNQFDQLSYEGLAYPQYA

DWRKKASKFVSLWQITKKNKKKEIVTVEAKEKFDAICKYQPRLYKENKEYAYLLRDIVRG

KSLVELQQIRQEIFRFIEQDCGVTRLGSLSLSTLETVKAVKGIIYSYFSTALNASKNNPI

SDEQRKEFDPELFALLEKLELIRTRKKKQKVERIANSLIQTCLENNIKFIRGEGDLSTIN

NATKKKANSRSMDWLARGVENKIRQLAPMHNITLFGCGSLYTSHQDPLVHRNPDKAMKCR

WAAIPVKDIGDWVLRKLSQNLRAKNIGTGEYYHQGVKEFLSHYELQDLEEELLKWRSDRK

SNIPCWVLQNRLAEKLGNKEAVVYIPVRGGRIYFATHKVATGAVSIVFDQKQVWVCNADH

VAAANIALTVKGIGEQSSDEENPDGSRIKLQLTS

B2M-
ACTTAGCATCTCTGGGGCCAGTCTGCAAAGCGAGGGGGCAGCCTTAATGTGCCTCCAGCCTGAAGT

SEQ ID NO: 773
CCTAGAATGAGCGCCCGGTGTCCCAAGCTGGGGCGCGCACCCCAGATCGGAGGGCGCCGATGTACA

GACAGCAAACTCACCCAGTCTAGTGCATGCCTTCTTAAACATCACGAGACTCTAAGAAAAGGAAAC

TGAAAACGGGAAAGTCCCTCTCTCTAACCTGGCACTGCGTCGCTGGCTTGGAGACAGGTGACGGTC

CCTGCGGGCCTTGTCCTGATTGGCTGGGCACGCGTTTAATATAAGTGGAGGCGTCGCGCTGGCGGG

CATTCCTGAAGCTGACAGCATTCGGGCCGAGATGTCTCGCTCCGTGGCCTTAGCTGTGCTCGCGCT

ACTCTCTCTTTCTGGCCTGGAGGCTATCCAGCGTGAGTCTCTCCTACCCTCCCGCTCTGGTCCTTC

CTCTCCCGCTCTGCACCCTCTGTGGCCCTCGCTGTGCTCTCTCGCTCCGTGACTTCCCTTCTCCAA

GTTCTCCTTGGTGGCCCGCCGTGGGGCTAGTCCAGGGCTGGATCTCGGGGAAGCGGCGGGGTGGCC

TGGGAGTGGGGAAGGGGGTGCGCACCCGGGACGCGCGCTACTTGCCCCTTTCGGCGGGGAGCAGGG

GAGACCTTTGGCCTACGGCGACGGGAGGGTCGGGACAAAGTTTAGGGCGTCGATAAGCGTCAGAGC

GCCGAGGTTGGGGGAGGGTTTCTCTTCCGCTCTTTCGCGGGGCCTCTGGCTCCCCCAGCGCAGCTG

GAGTGGGGGACGGGTAGGCTCGTCCCAAAGGCGCGGCGCTGAGGTTTGTGAACGCGTGGAGGGGCG

CTTGGGGTCTGGGGGAGGCGTCGCCCGGGTAAGCCTGTCTGCTGCGGCTCTGCTTCCCTTAGACTG

GAGAGCTGTGGACTTCGTCTAGGCGCCCGCTAAGTTCGCATGTCCTAGCACCTCTGGGTCTATGTG

GGGCCACACCGTGGGGAGGAAACAGCACGCGACGTTTGTAGAATGCTTGGCTGTGATACAAAGCGG

TTTCGAATAATTAACTTATTTGTTCCCATCACATGTCACTTTTAAAAAATTATAAGAACTACCCGT

TATTGACATCTTTCTGTGTGCCAAGGACTTTATGTGCTTTGCGTCATTTAATTTTGAAAACAGTTA

TCTTCCGCCATAGATAACTACTATGGTTATCTTCTGCCTCTCACAGATGAAGAAACTAAGGCACCG

AGATTTTAAGAAACTTAATTACACAGGGGATAAATGGCAGCAATCGAGATTGAAGTCAAGCCTAAC

CAGGGCTTTTGCGGGAGCGCATGCCTTTTGGCTGTAATTCGTGCATTTTTTTTTAAGAAAAACGCC

TGCCTTCTGCGTGAGATTCTCCAGAGCAAACTGGGCGGCATGGGCCCTGTGGTCTTTTCGTACAGA

GGGCTTCCTCTTTGGCTCTTTGCCTGGTTGTTTCCAAGATGTACTGTGCCTCTTACTTTCGGTTTT

GAAAACATGAGGGGGTTGGGCGTGGTAGCTTACGCCTGTAATCCCAGCACTTAGGGAGGCCGAGGC

GGGAGGATGGCTTGAGGTCCGTAGTTGAGACCAGCCTGGCCAACATGGTGAAGCCTGGTCTCTACA

AAAAATAATAACAAAAATTAGCCGGGTGTGGTGGCTCGTGCCTGTGGTCCCAGCTGCTCCGGTGGC

TGAGGCGGGAGGATCTCTTGAGCTTAGGCTTTTGAGCTATCATGGCGCCAGTGCACTCCAGCGTGG

GCAACAGAGCGAGACCCTGTCTCTCAAAAAAGAAAAAAAAAAAAAAAGAAAGAGAAAAGAAAAGAA

AGAAAGAAGTGAAGGTTTGTCAGTCAGGGGAGCTGTAAAACCATTAATAAAGATAATCCAAGATGG

TTACCAAGACTGTTGAGGACGCCAGAGATCTTGAGCACTTTCTAAGTACCTGGCAATACACTAAGC

GCGCTCACCTTTTCCTCTGGCAAAACATGATCGAAAGCAGAATGTTTTGATCATGAGAAAATTGCA

TTTAATTTGAATACAATTTATTTACAACATAAAGGATAATGTATATATCACCACCATTACTGGTAT

TTGCTGGTTATGTTAGATGTCATTTTAAAAAATAACAATCTGATATTTAAAAAAAAATCTTATTTT

GAAAATTTCCAAAGTAATACATGCCATGCATAGACCATTTCTGGAAGATACCACAAGAAACATGTA

ATGATGATTGCCTCTGAAGGTCTATTTTCCTCCTCTGACCTGTGTGTGGGTTTTGTTTTTGTTTTA

CTGTGGGCATAAATTAATTTTTCAGTTAAGTTTTGGAAGCTTAAATAACTCTCCAAAAGTCATAAA

GCCAGTAACTGGTTGAGCCCAAATTCAAACCCAGCCTGTCTGATACTTGTCCTCTTCTTAGAAAAG

ATTACAGTGATGCTCTCACAAAATCTTGCCGCCTTCCCTCAAACAGAGAGTTCCAGGCAGGATGAA

TCTGTGCTCTGATCCCTGAGGCATTTAATATGTTCTTATTATTAGAAGCTCAGATGCAAAGAGCTC

TCTTAGCTTTTAATGTTATGAAAAAAATCAGGTCTTCATTAGATTCCCCAATCCACCTCTTGATGG

GGCTAGTAGCCTTTCCTTAATGATAGGGTGTTTCTAGAGAGATATATCTGGTCAAGGTGGCCTGGT

ACTCCTCCTTCTCCCCACAGCCTCCCAGACAAGGAGGAGTAGCTGCCTTTTAGTGATCATGTACCC

TGAATATAAGTGTATTTAAAAGAATTTTATACACATATATTTAGTGTCAATCTGTATATTTAGTAG

CACTAACACTTCTCTTCATTTTCAATGAAAAATATAGAGTTTATAATATTTTCTTCCCACTTCCCC

ATGGATGGTCTAGTCATGCCTCTCATTTTGGAAAGTACTGTTTCTGAAACATTAGGCAATATATTC

CCAACCTGGCTAGTTTACAGCAATCACCTGTGGATGCTAATTAAAACGCAAATCCCACTGTCACAT

GCATTACTCCATTTGATCATAATGGAAAGTATGTTCTGTCCCATTTGCCATAGTCCTCACCTATCC

CTGTTGTATTTTATCGGGTCCAACTCAACCATTTAAGGTATTTGCCAGCTCTTGTATGCATTTAGG

TTTTGTTTCTTTGTTTTTTAGCTCATGAAATTAGGTACAAAGTCAGAGAGGGGTCTGGCATATAAA

ACCTCAGCAGAAATAAAGAGGTTTTGTTGTTTGGTAAGAACATACCTTGGGTTGGTTGGGCACGGT

GGCTCGTGCCTGTAATCCCAACACTTTGGGAGGCCAAGGCAGGCTGATCACTTGAAGTTGGGAGTT

CAAGACCAGCCTGGCCAACATGGTGAAATCCCGTCTCTACTGAAAATACAAAAATTAACCAGGCAT

GGTGGTGTGTGCCTGTAGTCCCAGGAATCACTTGAACCCAGGAGGCGGAGGTTGCAGTGAGCTGAG

ATCTCACCACTGCACACTGCACTCCAGCCTGGGCAATGGAATGAGATTCCATCCCAAAAAATAAAA

AAATAAAAAAATAAAGAACATACCTTGGGTTGATCCACTTAGGAACCTCAGATAATAACATCTGCC

ACGTATAGAGCAATTGCTATGTCCCAGGCACTCTACTAGACACTTCATACAGTTTAGAAAATCAGA

TGGGTGTAGATCAAGGCAGGAGCAGGAACCAAAAAGAAAGGCATAAACATAAGAAAAAAAATGGAA

GGGGTGGAAACAGAGTACAATAACATGAGTAATTTGATGGGGGCTATTATGAACTGAGAAATGAAC

TTTGAAAAGTATCTTGGGGCCAAATCATGTAGACTCTTGAGTGATGTGTTAAGGAATGCTATGAGT

GCTGAGAGGGCATCAGAAGTCCTTGAGAGCCTCCAGAGAAAGGCTCTTAAAAATGCAGCGCAATCT

CCAGTGACAGAAGATACTGCTAGAAATCTGCTAGAAAAAAAACAAAAAAGGCATGTATAGAGGAAT

TATGAGGGAAAGATACCAAGTCACGGTTTATTCTTCAAAATGGAGGTGGCTTGTTGGGAAGGTGGA

AGCTCATTTGGCCAGAGTGGAAATGGAATTGGGAGAAATCGATGACCAAATGTAAACACTTGGTGC

CTGATATAGCTTGACACCAAGTTAGCCCCAAGTGAAATACCCTGGCAATATTAATGTGTCTTTTCC

CGATATTCCTCAGGTACTCCAAAGATTCAGGTTTACTCACGTCATCCAGCAGAGAATGGAAAGTCA

AATTTCCTGAATTGCTATGTGTCTGGGTTTCATCCATCCGACATTGAAGTTGACTTACTGAAGAAT

GGAGAGAGAATTGAAAAAGTGGAGCATTCAGACTTGTCTTTCAGCAAGGACTGGTCTTTCTATCTC

TTGTACTACACTGAATTCACCCCCACTGAAAAAGATGAGTATGCCTGCCGTGTGAACCATGTGACT

TTGTCACAGCCCAAGATAGTTAAGTGGGGTAAGTCTTACATTCTTTTGTAAGCTGCTGAAAGTTGT

GTATGAGTAGTCATATCATAAAGCTGCTTTGATATAAAAAAGGTCTATGGCCATACTACCCTGAAT

GAGTCCCATCCCATCTGATATAAACAATCTGCATATTGGGATTGTCAGGGAATGTTCTTAAAGATC

AGATTAGTGGCACCTGCTGAGATACTGATGCACAGCATGGTTTCTGAACCAGTAGTTTCCCTGCAG

TTGAGCAGGGAGCAGCAGCAGCACTTGCACAAATACATATACACTCTTAACACTTCTTACCTACTG

GCTTCCTCTAGCTTTTGTGGCAGCTTCAGGTATATTTAGCACTGAACGAACATCTCAAGAAGGTAT

AGGCCTTTGTTTGTAAGTCCTGCTGTCCTAGCATCCTATAATCCTGGACTTCTCCAGTACTTTCTG

GCTGGATTGGTATCTGAGGCTAGTAGGAAGGGCTTGTTCCTGCTGGGTAGCTCTAAACAATGTATT

CATGGGTAGGAACAGCAGCCTATTCTGCCAGCCTTATTTCTAACCATTTTAGACATTTGTTAGTAC

ATGGTATTTTAAAAGTAAAACTTAATGTCTTCCTTTTTTTTCTCCACTGTCTTTTTCATAGATCGA

GACATGTAAGCAGCATCATGGAGGTAAGTTTTTGACCTTGAGAAAATGTTTTTGTTTCACTGTCCT

GAGGACTATTTATAGACAGCTCTAACATGATAACCCTCACTATGTGGAGAACATTGACAGAGTAAC

ATTTTAGCAGGGAAAGAAGAATCCTACAGGGTCATGTTCCCTTCTCCTGTGGAGTGGCATGAAGAA

GGTGTATGGCCCCAGGTATGGCCATATTACTGACCCTCTACAGAGAGGGCAAAGGAACTGCCAGTA

TGGTATTGCAGGATAAAGGCAGGTGGTTACCCACATTACCTGCAAGGCTTTGATCTTTCTTCTGCC

ATTTCCACATTGGACATCTCTGCTGAGGAGAGAAAATGAACCACTCTTTTCCTTTGTATAATGTTG

TTTTATTCTTCAGACAGAAGAGAGGAGTTATACAGCTCTGCAGACATCCCATTCCTGTATGGGGAC

TGTGTTTGCCTCTTAGAGGTTCCCAGGCCACTAGAGGAGATAAAGGGAAACAGATTGTTATAACTT

GATATAATGATACTATAATAGATGTAACTACAAGGAGCTCCAGAAGCAAGAGAGAGGGAGGAACTT

GGACTTCTCTGCATCTTTAGTTGGAGTCCAAAGGCTTTTCAATGAAATTCTACTGCCCAGGGTACA

TTGATGCTGAAACCCCATTCAAATCTCCTGTTATATTCTAGAACAGGGAATTGATTTGGGAGAGCA

TCAGGAAGGTGGATGATCTGCCCAGTCACACTGTTAGTAAATTGTAGAGCCAGGACCTGAACTCTA

ATATAGTCATGTGTTACTTAATGACGGGGACATGTTCTGAGAAATGCTTACACAAACCTAGGTGTT

GTAGCCTACTACACGCATAGGCTACATGGTATAGCCTATTGCTCCTAGACTACAAACCTGTACAGC

CTGTTACTGTACTGAATACTGTGGGCAGTTGTAACACAATGGTAAGTATTTGTGTATCTAAACATA

GAAGTTGCAGTAAAAATATGCTATTTTAATCTTATGAGACCACTGTCATATATACAGTCCATCATT

GACCAAAACATCATATCAGCATTTTTTCTTCTAAGATTTTGGGAGCACCAAAGGGATACACTAACA

GGATATACTCTTTATAATGGGTTTGGAGAACTGTCTGCAGCTACTTCTTTTAAAAAGGTGATCTAC

ACAGTAGAAATTAGACAAGTTTGGTAATGAGATCTGCAATCCAAATAAAATAAATTCATTGCTAAC

CTTTTTCTTTTCTTTTCAGGTTTGAAGATGCCGCATTTGGATTGGATGAATTCCAAATTCTGCTTG

CTTGCTTTTTAATATTGATATGCTTATACACTTACACTTTATGCACAAAATGTAGGGTTATAATAA

TGTTAACATGGACATGATCTTCTTTATAATTCTACTTTGAGTGCTGTCTCCATGTTTGATGTATCT

GAGCAGGTTGCTCCACAGGTAGCTCTAGGAGGGCTGGCAACTTAGAGGTGGGGAGCAGAGAATTCT

CTTATCCAACATCAACATCTTGGTCAGATTTGAACTCTTCAATCTCTTGCACTCAAAGCTTGTTAA

GATAGTTAAGCGTGCATAAGTTAACTTCCAATTTACATACTCTGCTTAGAATTTGGGGGAAAATTT

AGAAATATAATTGACAGGATTATTGGAAATTTGTTATAATGAATGAAACATTTTGTCATATAAGAT

TCATATTTACTTCTTATACATTTGATAAAGTAAGGCATGGTTGTGGTTAATCTGGTTTATTTTTGT

TCCACAAGTTAAATAAATCATAAAACTTGATGTGTTATCTCTTATATCTCACTCCCACTATTACCC

CTTTATTTTCAAACAGGGAAACAGTCTTCAAGTTCCACTTGGTAAAAAATGTGAACCCCTTGTATA

TAGAGTTTGGCTCACAGTGTAAAGGGCCTCAGTGATTCACATTTTCCAGATTAGGAATCTGATGCT

CAAAGAAGTTAAATGGCATAGTTGGGGTGACACAGCTGTCTAGTGGGAGGCCAGCCTTCTATATTT

TAGCCAGCGTTCTTTCCTGCGGGCCAGGTCATGAGGAGTATGCAGACTCTAAGAGGGAGCAAAAGT

ATCTGAAGGATTTAATATTTTAGCAAGGAATAGATATACAATCATCCCTTGGTCTCCCTGGGGGAT

TGGTTTCAGGACCCCTTCTTGGACACCAAATCTATGGATATTTAAGTCCCTTCTATAAAATGGTAT

AGTATTTGCATATAACCTATCCACATCCTCCTGTATACTTTAAATCATTTCTAGATTACTTGTAAT

ACCTAATACAATGTAAATGCTATGCAAATAGTTGTTATTGTTTAAGGAATAATGACAAGAAAAAAA

AGTCTGTACATGCTCAGTAAAGACACAACCATCCCTTTTTTTCCCCAGTGTTTTTGATCCATGGTT

TGCTGAATCCACAGATGTGGAGCCCCTGGATACGGAAGGCCCGCTGTACTTTGAATGACAAATAAC

AGATTTAAAATTTTCAAGGCATAGTTTTATACCTGA

B2M-Exon 1-
GATTGGCTGGGCACGCGTTTAATATAAGTGGAGGCGTCGCGCTGGCGGGCATTCCTGAAGCTGACA

SEQ ID NO: 774
GCATTCGGGCCGAGATGTCTCGCTCCGTGGCCTTAGCTGTGCTCGCGCTACTCTCTCTTTCTGGCC

TGGAGGCTATCCAGCGTGAGTCTCTCCTACCCTCCCGCTCTGGTCCTTCCTCTCCCGCTCTGCAC

B2M-Exon 2-
AAGTGAAATACCCTGGCAATATTAATGTGTCTTTTCCCGATATTCCTCAGGTACTCCAAAGATTCA

SEQ ID NO: 775
GGTTTACTCACGTCATCCAGCAGAGAATGGAAAGTCAAATTTCCTGAATTGCTATGTGTCTGGGTT

TCATCCATCCGACATTGAAGTTGACTTACTGAAGAATGGAGAGAGAATTGAAAAAGTGGAGCATTC

AGACTTGTCTTTCAGCAAGGACTGGTCTTTCTATCTCTTGTACTACACTGAATTCACCCCCACTGA

AAAAGATGAGTATGCCTGCCGTGTGAACCATGTGACTTTGTCACAGCCCAAGATAGTTAAGTGGGG

TAAGTCTTACATTCTTTTGTAAGCTGCTGAAAGTTGTGTATGAGTAGTC

B2M-Exon 3-
AAAGTAAAACTTAATGTCTTCCTTTTTTTTCTCCACTGTCTTTTTCATAGATCGAGACATGTAAGC

SEQ ID NO: 776
AGCATCATGGAGGTAAGTTTTTGACCTTGAGAAAATGTTTTTGTTTCACTGTCCTGAGGACT

B2M-Exon 4-
GCAATCCAAATAAAATAAATTCATTGCTAACCTTTTTCTTTTCTTTTCAGGTTTGAAGATGCCGCA

SEQ ID NO: 777
TTTGGATTGGATGAATTCCAAATTCTGCTTGCTTGCTTTTTAATATTGATATGCTTATACACTTAC

ACTTTATGCACAAAATGTAGGGTTATAATAATGTTAACATGGACATGATCTTCTTTATAATTCTAC

TTTGAGTGCTGTCTCCATGTTTGATGTATCTGAGCAGGTTGCTCCACAGGTAGCTCTAGGAGGGCT

GGCAACTTAGAGGTGGGGAGCAGAGAATTCTCTTATCCAACATCAACATCTTGGTCAGATTTGAAC

TCTTCAATCTCTTGCACTCAAAGCTTGTTAAGATAGTTAAGCGTGCATAAGTTAACTTCCAATTTA

CATACTCTGCTTAGAATTTGGGGGAAAATTTAGAAATATAATTGACAGGATTATTGGAAATTTGTT

ATAATGAATGAAACATTTTGTCATATAAGATTCATATTTACTTCTTATACATTTGATAAAGTAAGG

CATGGTTGTGGTTAATCTGGTTTATTTTTGTTCCACAAGTTAAATAAATCATAAAACTTGATGTGT

TATCTCTTATATCTCACTCCCACTATTACCCCTTTATTTTCAAACAGGGAAACAGTCTTCAAGTTC

CACTTGGTAAAAAATGTGAACCCCTTGTATATAGAGTTTGGCTCACAGTGTAAAGGGCCTCAGTGA

TTCACATTTTCCAGATTAGGAATCTGATGCTCAAAGAAGTTAAATGGCATAGTTGGGGTGACACAG

CTGTCTAGTGGGAGGCCAGCCTTCTATATTTTAGCCAGCGTTCTTTCCTGCGGGCCAGGTCATGAG

GAGTATGCAGACTCTAAGAGGGAGCAAAAGTATCTGAAGGATTTAATATTTTAGCAAGGAATAGAT

ATACAATCATCCCTTGGTCTCCCTGGGGGATTGGTTTCAGGACCCCTTCTTGGACACCAAATCTAT

GGATATTTAAGTCCCTTCTATAAAATGGTATAGTATTTGCATATAACCTATCCACATCCTCCTGTA

TACTTTAAATCATTTCTAGATTACTTGTAATACCTAATACAATGTAAATGCTATGCAAATAGTTGT

TATTGTTTAAGGAATAATGACAAGAAAAAAAAGTCTGTACATGCTCAGTAAAGACACAACCATCCC

TTTTTTTCCCCAGTGTTTTTGATCCATGGTTTGCTGAATCCACAGATGTGGAGCCCCTGGATACGG

AAGGCCCGCTGTACTTTGAATGACAAATAACAGATTTAAAATTTTCAAGGCATAGTTTTATACCTG

A

SEQ ID NO: 782
MSSAIKSYKS VLRPNERKNQ LLKSTIQCLE DGSAFFFKML QGLEGGITPE IVRESTEQEK

(Variant
QQQDIALWCA VNWFRPVSQD SLTHTIASDN LVEKFEEYYG GTASDAIKQY FSASIGESYY

Cas12i2 of
WNDCRQQYYD LCRELGVEVS DLTHDLEILC REKCLAVATE SNQNNSIISV LFGTGEKEDR

SEQ ID NO: 3
SVKLRITKKI LEAISNLKEI PKNVAPIQEI ILNVAKATKE TFRQVYAGNL GAPSTLEKFI

of PCT/US2021/
AKDGQKEFDL KKLQTDLKKV IRGKSKERDW CCQEELRSYV EQNTIQYDLW AWGEMENKAH

025257)
TALKIKSTRN YNFAKQRLEQ FKEIQSLNNL LVVKKLNDFF DSEFFSGEET YTICVHHLGG

KDLSKLYKAW EDDPADPENA IVVLCDDLKN NFKKEPIRNI LRYIFTIRQE CSAQDILAAA

KYNQQLDRYK SQKANPSVLG NQGETWTNAV ILPEKAQRND RPNSLDLRIW LYLKLRHPDG

RWKKHHIPFY DTRFFQEIYA AGNSPVDTCQ FRTPRFGYHL PKLTDQTAIR VNKKHVKAAK

TEARIRLAIQ QGTLPVSNLK ITEISATINS KGQVRIPVKF RVGRQKGTLQ IGDRFCGYDQ

NQTASHAYSL WEVVKEGQYH KELGCFVRFI SSGDIVSITE NRGNQFDQLS YEGLAYPQYA

DWRKKASKFV SLWQITKKNK KKEIVTVEAK EKFDAICKYQ PRLYKENKEY AYLLRDIVRG

KSLVELQQIR QEIFRFIEQD CGVTRLGSLS LSTLETVKAV KGIIYSYFST ALNASKNNPI

SDEQRKEFDP ELFALLEKLE LIRTRKKKQK VERIANSLIQ TCLENNIKFI RGEGDLSTIN

NATKKKANSR SMDWLARGVF NKIRQLAPMH NITLFGCGSL YTSHQDPLVH RNPDKAMKCR

WAAIPVKDIG RWVLRKLSQN LRAKNRGTGE YYHQGVKEFL SHYELQDLEE ELLKWRSDRK

SNIPCWVLQN RLAEKLGNKE AVVYIPVRGG RIYFATHKVA TGAVSIVEDQ KQVWVCNADH

VAAANIALTG KGIGEQSSDE ENPDGSRIKL QLTS

SEQ ID NO: 783
MSSAIKSYKS VLRPNERKNQ LLKSTIQCLE DGSAFFFKML QGLFGGITPE IVRFSTEQEK

(Variant
QQQDIALWCA VNWFRPVSQD SLTHTIASDN LVEKFEEYYG GTASDAIKQY FSASIGESYY

Cas12i2 of
WNDCRQQYYD LCRELGVEVS DLTHDLEILC REKCLAVATE SNQNNSIISV LFGTGEKEDR

SEQ ID NO: 4
SVKLRITKKI LEAISNLKEI PKNVAPIQEI ILNVAKATKE TFRQVYAGNL GAPSTLEKFI

of PCT/US2021/
AKDGQKEFDL KKLQTDLKKV IRGKSKERDW CCQEELRSYV EQNTIQYDLW AWGEMENKAH

025257)
TALKIKSTRN YNFAKQRLEQ FKEIQSLNNL LVVKKLNDFF DSEFFSGEET YTICVHHLGG

KDLSKLYKAW EDDPADPENA IVVLCDDLKN NFKKEPIRNI LRYIFTIRQE CSAQDILAAA

KYNQQLDRYK SQKANPSVLG NQGFTWTNAV ILPEKAQRND RPNSLDLRIW LYLKLRHPDG

RWKKHHIPFY DTRFFQEIYA AGNSPVDTCQ FRTPRFGYHL PKLTDQTAIR VNKKHVKAAK

TEARIRLAIQ QGTLPVSNLK ITEISATINS KGQVRIPVKF RVGRQKGTLQ IGDRFCGYDQ

NQTASHAYSL WEVVKEGQYH KELGCFVRFI SSGDIVSITE NRGNQFDQLS YEGLAYPQYA

DWRKKASKFV SLWQITKKNK KKEIVTVEAK EKFDAICKYQ PRLYKENKEY AYLLRDIVRG

KSLVELQQIR QEIFRFIEQD CGVTRLGSLS LSTLETVKAV KGIIYSYFST ALNASKNNPI

SDEQRKEFDP ELFALLEKLE LIRTRKKKQK VERIANSLIQ TCLENNIKFI RGEGDLSTIN

NATKKKANSR SMDWLARGVF NKIRQLAPMH NITLFGCGSL YTSHQDPLVH RNPDKAMKCR

WAAIPVKDIG DWVLRKLSQN LRAKNRGTGE YYHQGVKEFL SHYELQDLEE ELLKWRSDRK

SNIPCWVLQN RLAEKLGNKE AVVYIPVRGG RIYFATHKVA TGAVSIVEDQ KQVWVCNADH

VAAANIALTG KGIGEQSSDE ENPDGSRIKL QLTS

SEQ ID NO: 784
MSSAIKSYKS VLRPNERKNQ LLKSTIQCLE DGSAFFFKML QGLEGGITPE IVRESTEQEK

(Variant
QQQDIALWCA VNWERPVSQD SLTHTIASDN LVEKFEEYYG GTASDAIKQY FSASIGESYY

Cas12i2 of
WNDCRQQYYD LCRELGVEVS DLTHDLEILC REKCLAVATE SNQNNSIISV LFGTGEKEDR

SEQ ID NO: 5
SVKLRITKKI LEAISNLKEI PKNVAPIQEI ILNVAKATKE TFRQVYAGNL GAPSTLEKFI

of PCT/US2021/
AKDGQKEFDL KKLQTDLKKV IRGKSKERDW CCQEELRSYV EQNTIQYDLW AWGEMENKAH

025257)
TALKIKSTRN YNFAKQRLEQ FKEIQSLNNL LVVKKLNDFF DSEFFSGEET YTICVHHLGG

KDLSKLYKAW EDDPADPENA IVVLCDDLKN NFKKEPIRNI LRYIFTIRQE CSAQDILAAA

KYNQQLDRYK SQKANPSVLG NQGETWTNAV ILPEKAQRND RPNSLDLRIW LYLKLRHPDG

RWKKHHIPFY DTRFFQEIYA AGNSPVDTCQ FRTPRFGYHL PKLTDQTAIR VNKKHVKAAK

TEARIRLAIQ QGTLPVSNLK ITEISATINS KGQVRIPVKF RVGRQKGTLQ IGDRFCGYDQ

NQTASHAYSL WEVVKEGQYH KELGCFVRFI SSGDIVSITE NRGNQFDQLS YEGLAYPQYA

DWRKKASKFV SLWQITKKNK KKEIVTVEAK EKFDAICKYQ PRLYKENKEY AYLLRDIVRG

KSLVELQQIR QEIFRFIEQD CGVTRLGSLS LSTLETVKAV KGIIYSYFST ALNASKNNPI

SDEQRKEFDP ELFALLEKLE LIRTRKKKQK VERIANSLIQ TCLENNIKFI RGEGDLSTIN

NATKKKANSR SMDWLARGVF NKIRQLAPMH NITLFGCGSL YTSHQDPLVH RNPDKAMKCR

WAAIPVKDIG DWVLRKLSQN LRAKNRGTGE YYHQGVKEFL SHYELQDLEE ELLKWRSDRK

SNIPCWVLQN RLAEKLGNKE AVVYIPVRGG RIYFATHKVA TGAVSIVEDQ KQVWVCNADH

VAAANIALTG KGIGEQSSDE ENPDGGRIKL QLTS

SEQ ID NO: 785
MSSAIKSYKS VLRPNERKNQ LLKSTIQCLE DGSAFFFKML QGLEGGITPE IVRESTEQEK

(Variant
QQQDIALWCA VNWFRPVSQD SLTHTIASDN LVEKFEEYYG GTASDAIKQY FSASIGESYY

Cas12i2 of
WNDCRQQYYD LCRELGVEVS DLTHDLEILC REKCLAVATE SNQNNSIISV LFGTGEKEDR

SEQ ID NO: 495 of
SVKLRITKKI LEAISNLKEI PKNVAPIQEI ILNVAKATKE TFRQVYAGNL GAPSTLEKFI

PCT/US2021/
AKDGQKEFDL KKLQTDLKKV IRGKSKERDW CCQEELRSYV EQNTIQYDLW AWGEMENKAH

025257)
TALKIKSTRN YNFAKQRLEQ FKEIQSLNNL LVVKKLNDFF DSEFFSGEET YTICVHHLGG

KDLSKLYKAW EDDPADPENA IVVLCDDLKN NFKKEPIRNI LRYIFTIRQE CSAQDILAAA

KYNQQLDRYK SQKANPSVLG NQGFTWTNAV ILPEKAQRND RPNSLDLRIW LYLKLRHPDG

RWKKHHIPFY DTRFFQEIYA AGNSPVDTCQ FRTPRFGYHL PKLTDQTAIR VNKKHVKAAK

TEARIRLAIQ QGTLPVSNLK ITEISATINS KGQVRIPVKF RVGRQKGTLQ IGDRFCGYDQ

NQTASHAYSL WEVVKEGQYH KELRCRVRFI SSGDIVSITE NRGNQFDQLS YEGLAYPQYA

DWRKKASKFV SLWQITKKNK KKEIVTVEAK EKFDAICKYQ PRLYKENKEY AYLLRDIVRG

KSLVELQQIR QEIFRFIEQD CGVTRLGSLS LSTLETVKAV KGIIYSYEST ALNASKNNPI

SDEQRKEFDP ELFALLEKLE LIRTRKKKQK VERIANSLIQ TCLENNIKFI RGEGDLSTTN

NATKKKANSR SMDWLARGVF NKIRQLAPMH NITLFGCGSL YTSHQDPLVH RNPDKAMKCR

WAAIPVKDIG DWVLRKLSQN LRAKNRGTGE YYHQGVKEFL SHYELQDLEE ELLKWRSDRK

SNIPCWVLQN RLAEKLGNKE AVVYIPVRGG RIYFATHKVA TGAVSIVEDQ KQVWVCNADH

VAAANIALTG KGIGRQSSDE ENPDGGRIKL QLTS

SEQ ID NO: 786
MSSAIKSYKS VLRPNERKNQ LLKSTIQCLE DGSAFFFKML QGLFGGITPE IVRESTEQEK

(Variant
QQQDIALWCA VNWFRPVSQD SLTHTIASDN LVEKFEEYYG GTASDAIKQY FSASIGESYY

Cas12i2 of
WNDCRQQYYD LCRELGVEVS DLTHDLEILC REKCLAVATE SNQNNSIISV LFGTGEKEDR

SEQ ID NO: 496 of
SVKLRITKKI LEAISNLKEI PKNVAPIQEI ILNVAKATKE TFRQVYAGNL GAPSTLEKFI

PCT/US2021/
AKDGQKEFDL KKLQTDLKKV IRGKSKERDW CCQEELRSYV EQNTIQYDLW AWGEMFNKAH

025257)
TALKIKSTRN YNFAKQRLEQ FKEIQSLNNL LVVKKLNDFF DSEFFSGEET YTICVHHLGG

KDLSKLYKAW EDDPADPENA IVVLCDDLKN NFKKEPIRNI LRYIFTIRQE CSAQDILAAA

KYNQQLDRYK SQKANPSVLG NQGFTWTNAV ILPEKAQRND RPNSLDLRIW LYLKLRHPDG

RWKKHHIPFY DTRFFQEIYA AGNSPVDTCQ FRTPRFGYHL PKLTDQTAIR VNKKHVKAAK

TEARIRLAIQ QGTLPVSNLK ITEISATINS KGQVRIPVKF RVGRQKGTLQ IGDRFCGYDQ

NQTASHAYSL WEVVKEGQYH KELRCRVRFI SSGDIVSITE NRGNQFDQLS YEGLAYPQYA

DWRKKASKFV SLWQITKKNK KKEIVTVEAK EKFDAICKYQ PRLYKFNKEY AYLLRDIVRG

KSLVELQQIR QEIFRFIEQD CGVTRLGSLS LSTLETVKAV KGIIYSYFST ALNASKNNPI

SDEQRKEFDP ELFALLEKLE LIRTRKKKQK VERIANSLIQ TCLENNIKFI RGEGDLSTTN

NATKKKANSR SMDWLARGVF NKIRQLATMH NITLFGCGSL YTSHQDPLVH RNPDKAMKCR

WAAIPVKDIG DWVLRKLSQN LRAKNRGTGE YYHQGVKEFL SHYELQDLEE ELLKWRSDRK

SNIPCWVLQN RLAEKLGNKE AVVYIPVRGG RIYFATHKVA TGAVSIVEDQ KQVWVCNADH

VAAANIALTG KGIGRQSSDE ENPDGGRIKL QLTS

SEQ ID NO: 787
ATGGCTTCCATCTCTAGGCCATACGGCACCAAGCTGCGACCGGACGCACGGAAGAAGGAGATGCTC

(Nucleotide
GATAAGTTCTTTAATACACTGACTAAGGGTCAGCGCGTGTTCGCAGACCTGGCCCTGTGCATCTAT

sequence
GGCTCCCTGACCCTGGAGATGGCCAAGTCTCTGGAGCCAGAAAGTGATTCAGAACTGGTGTGCGCT

encoding
ATTGGGTGGTTTCGGCTGGTGGACAAGACCATCTGGTCCAAGGATGGCATCAAGCAGGAGAATCTG

Cas12i4)
GTGAAACAGTACGAAGCCTATTCCGGAAAGGAGGCTTCTGAAGTGGTCAAAACATACCTGAACAGC

CCCAGCTCCGACAAGTACGTGTGGATCGATTGCAGGCAGAAATTCCTGAGGTTTCAGCGCGAGCTC

GGCACTCGCAACCTGTCCGAGGACTTCGAATGTATGCTCTTTGAACAGTACATTAGACTGACCAAG

GGCGAGATCGAAGGGTATGCCGCTATTTCAAATATGTTCGGAAACGGCGAGAAGGAAGACCGGAGC

AAGAAAAGAATGTACGCTACACGGATGAAAGATTGGCTGGAGGCAAACGAAAATATCACTTGGGAG

CAGTATAGAGAGGCCCTGAAGAACCAGCTGAATGCTAAAAACCTGGAGCAGGTTGTGGCCAATTAC

AAGGGGAACGCTGGCGGGGCAGACCCCTTCTTTAAGTATAGCTTCTCCAAAGAGGGAATGGTGAGC

AAGAAAGAACATGCACAGCAGCTCGACAAGTTCAAAACCGTCCTGAAGAACAAAGCCCGGGACCTG

AATTTTCCAAACAAGGAGAAGCTGAAGCAGTACCTGGAGGCCGAAATCGGCATTCCGGTCGACGCT

AACGTGTACTCCCAGATGTTCTCTAACGGGGTGAGTGAGGTCCAGCCTAAGACCACACGGAATATG

TCTTTTAGTAACGAGAAACTGGATCTGCTCACTGAACTGAAGGACCTGAACAAGGGCGATGGGTTC

GAGTACGCCAGAGAAGTGCTGAACGGGTTCTTTGACTCCGAGCTCCACACTACCGAGGATAAGTTT

AATATCACCTCTAGGTACCTGGGAGGCGACAAATCAAACCGCCTGAGCAAACTCTATAAGATCTGG

AAGAAAGAGGGTGTGGACTGCGAGGAAGGCATTCAGCAGTTCTGTGAAGCCGTCAAAGATAAGATG

GGCCAGATCCCCATTCGAAATGTGCTGAAGTACCTGTGGCAGTTCCGGGAGACAGTCAGTGCCGAG

GATTTTGAAGCAGCCGCTAAGGCTAACCATCTGGAGGAAAAGATCAGCCGGGTGAAAGCCCACCCA

ATCGTGATTAGCAATAGGTACTGGGCTTTTGGGACTTCCGCACTGGTGGGAAACATTATGCCCGCA

GACAAGAGGCATCAGGGAGAGTATGCCGGTCAGAATTTCAAAATGTGGCTGGAGGCTGAACTGCAC

TACGATGGCAAGAAAGCAAAGCACCATCTGCCTTTTTATAACGCCCGCTTCTTTGAGGAAGTGTAC

TGCTATCACCCCTCTGTCGCCGAGATCACTCCTTTCAAAACCAAGCAGTTTGGCTGTGAAATCGGG

AAGGACATTCCAGATTACGTGAGCGTCGCTCTGAAGGACAATCCGTATAAGAAAGCAACCAAACGA

ATCCTGCGTGCAATCTACAATCCCGTCGCCAACACAACTGGCGTTGATAAGACCACAAACTGCAGC

TTCATGATCAAACGCGAGAATGACGAATATAAGCTGGTCATCAACCGAAAAATTTCCGTGGATCGG

CCTAAGAGAATCGAAGTGGGCAGGACAATTATGGGGTACGACCGCAATCAGACAGCTAGCGATACT

TATTGGATTGGCCGGCTGGTGCCACCTGGAACCCGGGGCGCATACCGCATCGGAGAGTGGAGCGTC

CAGTATATTAAGTCCGGGCCTGTCCTGTCTAGTACTCAGGGAGTTAACAATTCCACTACCGACCAG

CTGGTGTACAACGGCATGCCATCAAGCTCCGAGCGGTTCAAGGCCTGGAAGAAAGCCAGAATGGCT

TTTATCCGAAAACTCATTCGTCAGCTGAATGACGAGGGACTGGAATCTAAGGGTCAGGATTATATC

CCCGAGAACCCTTCTAGTTTCGATGTGCGGGGCGAAACCCTGTACGTCTTTAACAGTAATTATCTG

AAGGCCCTGGTGAGCAAACACAGAAAGGCCAAGAAACCTGTTGAGGGGATCCTGGACGAGATTGAA

GCCTGGACATCTAAAGACAAGGATTCATGCAGCCTGATGCGGCTGAGCAGCCTGAGCGATGCTTCC

ATGCAGGGAATCGCCAGCCTGAAGAGTCTGATTAACAGCTACTTCAACAAGAATGGCTGTAAAACC

ATCGAGGACAAAGAAAAGTTTAATCCCGTGCTGTATGCCAAGCTGGTTGAGGTGGAACAGCGGAGA

ACAAACAAGCGGTCTGAGAAAGTGGGAAGAATCGCAGGTAGTCTGGAGCAGCTGGCCCTGCTGAAC

GGGGTTGAGGTGGTCATCGGCGAAGCTGACCTGGGGGAGGTCGAAAAAGGAAAGAGTAAGAAACAG

AATTCACGGAACATGGATTGGTGCGCAAAGCAGGTGGCACAGCGGCTGGAGTACAAACTGGCCTTC

CATGGAATCGGTTACTTTGGAGTGAACCCCATGTATACCAGCCACCAGGACCCTTTCGAACATAGG

CGCGTGGCTGATCACATCGTCATGCGAGCACGTTTTGAGGAAGTCAACGTGGAGAACATTGCCGAA

TGGCACGTGCGAAATTTCTCAAACTACCTGCGTGCAGACAGCGGCACTGGGCTGTACTATAAGCAG

GCCACCATGGACTTCCTGAAACATTACGGTCTGGAGGAACACGCTGAGGGCCTGGAAAATAAGAAA

ATCAAGTTCTATGACTTTAGAAAGATCCTGGAGGATAAAAACCTGACAAGCGTGATCATTCCAAAG

AGGGGCGGGCGCATCTACATGGCCACCAACCCAGTGACATCCGACTCTACCCCGATTACATACGCC

GGCAAGACTTATAATAGGTGTAACGCTGATGAGGTGGCAGCCGCTAATATCGTTATTTCTGTGCTG

GCTCCCCGCAGTAAGAAAAACGAGGAACAGGACGATATCCCTCTGATTACCAAGAAAGCCGAGAGT

AAGTCACCACCGAAAGACCGGAAGAGATCAAAAACAAGCCAGCTGCCTCAGAAA

SEQ ID NO: 814
MASISRPYGTKLRPDARKKEMLDKFFNTLTKGQRVFADLALCIYGSLTLEMAKSLEPESDSELVCA

Cas1214
IGWFRLVDKTIWSKDGIKQENLVKQYEAYSGKEASEVVKTYLNSPSSDKYVWIDCRQKFLRFQREL

amino acid
GTRNLSEDFECMLFEQYIRLIKGEIEGYAAISNMFGNGEKEDRSKKRMYATRMKDWLEANENITWE

sequence of
QYREALKNQLNAKNLEQVVANYKGNAGGADPFFKYSFSKEGMVSKKEHAQQLDKFKTVLKNKARDL

SEQ ID NO: 14
NFPNKEKLKQYLEAEIGIPVDANVYSQMFSNGVSEVQPKTTRNMSFSNEKLDLLTELKDLNKGDGF

of U.S. Pat. No.
EYAREVLNGFFDSELHTTEDKFNITSRYLGGDKSNRLSKLYKIWKKEGVDCEEGIQQFCEAVKDKM

10,808,245)
GQIPIRNVLKYLWQFRETVSAEDFEAAAKANHLEEKISRVKAHPIVISNRYWAFGTSALVGNIMPA

DKRHQGEYAGQNFKMWLEAELHYDGKKAKHHLPFYNARFFEEVYCYHPSVAEITPFKTKQFGCEIG

KDIPDYVSVALKDNPYKKATKRILRAIYNPVANTTGVDKTTNCSFMIKRENDEYKLVINRKISVDR

PKRIEVGRTIMGYDRNQTASDTYWIGRLVPPGTRGAYRIGEWSVQYIKSGPVLSSTQGVNNSTTDQ

LVYNGMPSSSERFKAWKKARMAFIRKLIRQLNDEGLESKGQDYIPENPSSFDVRGETLYVENSNYL

KALVSKHRKAKKPVEGILDEIEAWTSKDKDSCSLMRLSSLSDASMQGIASLKSLINSYFNKNGCKT

IEDKEKFNPVLYAKLVEVEQRRTNKRSEKVGRIAGSLEQLALLNGVEVVIGEADLGEVEKGKSKKQ

NSRNMDWCAKQVAQRLEYKLAFHGIGYFGVNPMYTSHQDPFEHRRVADHIVMRARFEEVNVENIAE

WHVRNFSNYLRADSGTGLYYKQATMDFLKHYGLEEHAEGLENKKIKFYDERKILEDKNLTSVIIPK

RGGRIYMATNPVTSDSTPITYAGKTYNRCNADEVAAANIVISVLAPRSKKNEEQDDIPLITKKAES

KSPPKDRKRSKTSQLPQK

SEQ ID NO: 815
MASISRPYGT KLRPDARKKE MLDKFFNTLT KGQRVFADLA LCIYGSLTLE MAKSLEPESD

(Variant Cas12i4)
SELVCAIGWF RLVDKTIWSK DGIKQENLVK QYEAYSGKEA SEVVKTYLNS PSSDKYVWID

CRQKFLRFQR ELGTRNLSED FECMLFEQYI RLTKGEIEGY AAISNMEGNG EKEDRSKKRM

YATRMKDWLE ANENITWEQY REALKNQLNA KNLEQVVANY KGNAGGADPF FKYSFSKEGM

VSKKEHAQQL DKFKTVLKNK ARDLNFPNKE KLKQYLEAEI GIPVDANVYS QMFSNGVSEV

QPKTTRNMSF SNEKLDLLTE LKDLNKGDGF EYAREVLNGF FDSELHTTED KENITSRYLG

GDKSNRLSKL YKIWKKEGVD CEEGIQQFCE AVKDKMGQIP IRNVLKYLWQ FRETVSAEDF

EAAAKANHLE EKISRVKAHP IVISNRYWAF GTSALVGNIM PADKRHQGEY AGQNFKMWLE

AELHYDGKKA KHHLPFYNAR FFEEVYCYHP SVAEITPFKT KQFGCEIGKD IPDYVSVALK

DNPYKKATKR ILRAIYNPVA NTTGVDKTIN CSFMIKREND EYKLVINRKI SRDRPKRIEV

GRTIMGYDRN QTASDTYWIG RLVPPGTRGA YRIGEWSVQY IKSGPVLSST QGVNNSTTDQ

LVYNGMPSSS ERFKAWKKAR MAFIRKLIRQ LNDEGLESKG QDYIPENPSS FDVRGETLYV

FNSNYLKALV SKHRKAKKPV EGILDEIEAW TSKDKDSCSL MRLSSLSDAS MQGIASLKSL

INSYFNKNGC KTIEDKEKFN PVLYAKLVEV EQRRINKRSE KVGRIAGSLE QLALLNGVEV

VIGEADLGEV EKGKSKKQNS RNMDWCAKQV AQRLEYKLAF HGIGYFGVNP MYTSHQDPFE

HRRVADHIVM RARFEEVNVE NIAEWHVRNF SNYLRADSGT GLYYKQATMD FLKHYGLEEH

AEGLENKKIK FYDERKILED KNLISVIIPK RGGRIYMATN PVTSDSTPIT YAGKTYNRCN

ADEVAAANIV ISVLAPRSKK NREQDDIPLI TKKAESKSPP KDRKRSKTSQ LPQK

SEQ ID NO: 816
MASISRPYGT KLRPDARKKE MLDKFFNTLT KGQRVFADLA LCIYGSLTLE MAKSLEPESD

(Variant Cas12i4)
SELVCAIGWF RLVDKTIWSK DGIKQENLVK QYEAYSGKEA SEVVKTYLNS PSSDKYVWID

CRQKFLRFQR ELGTRNLSED FECMLFEQYI RLTKGEIEGY AAISNMEGNG EKEDRSKKRM

YATRMKDWLE ANENITWEQY REALKNQLNA KNLEQVVANY KGNAGGADPF FKYSFSKEGM

VSKKEHAQQL DKFKTVLKNK ARDLNFPNKE KLKQYLEAEI GIPVDANVYS QMFSNGVSEV

QPKTTRNMSF SNEKLDLLTE LKDLNKGDGF EYAREVLNGF FDSELHTTED KENITSRYLG

GDKSNRLSKL YKIWKKEGVD CEEGIQQFCE AVKDKMGQIP IRNVLKYLWQ FRETVSAEDF

EAAAKANHLE EKISRVKAHP IVISNRYWAF GTSALVGNIM PADKRHQGEY AGQNFKMWLR

AELHYDGKKA KHHLPFYNAR FFEEVYCYHP SVAEITPFKT KQFGCEIGKD IPDYVSVALK

DNPYKKATKR ILRAIYNPVA NTTRVDKTTN CSFMIKREND EYKLVINRKI SRDRPKRIEV

GRTIMGYDRN QTASDTYWIG RLVPPGTRGA YRIGEWSVQY IKSGPVLSST QGVNNSTTDQ

LVYNGMPSSS ERFKAWKKAR MAFIRKLIRQ LNDEGLESKG QDYIPENPSS FDVRGETLYV

FNSNYLKALV SKHRKAKKPV EGILDEIEAW TSKDKDSCSL MRLSSLSDAS MQGIASLKSL

INSYFNKNGC KTIEDKEKFN PVLYAKLVEV EQRRINKRSE KVGRIAGSLE QLALLNGVEV

VIGEADLGEV EKGKSKKQNS RNMDWCAKQV AQRLEYKLAF HGIGYFGVNP MYTSHQDPFE

HRRVADHIVM RARFEEVNVE NIAEWHVRNF SNYLRADSGT GLYYKQATMD FLKHYGLEEH

AEGLENKKIK FYDERKILED KNLISVIIPK RGGRIYMATN PVTSDSTPIT YAGKTYNRCN

ADEVAAANIV ISVLAPRSKK NREQDDIPLI TKKAESKSPP KDRKRSKTSQ LPQK

SEQ ID NO: 817
MSNKEKNASETRKAYTTKMIPRSHDRMKLLGNFMDYLMDGTPIFFELWNQFGGGIDRDIISGTANK

(Cas12i1 of
DKISDDLLLAVNWFKVMPINSKPQGVSPSNLANLFQQYSGSEPDIQAQEYFASNEDTEKHQWKDMR

SEQ ID NO: 3
VEYERLLAELQLSRSDMHHDLKLMYKEKCIGLSLSTAHYITSVMFGTGAKNNRQTKHQFYSKVIQL

of U.S. Pat. No.
LEESTQINSVEQLASIILKAGDCDSYRKLRIRCSRKGATPSILKIVQDYELGTNHDDEVNVPSLIA

10,808,245)
NLKEKLGRFEYECEWKCMEKIKAFLASKVGPYYLGSYSAMLENALSPIKGMTTKNCKFVLKQIDAK

NDIKYENEPFGKIVEGFFDSPYFESDTNVKWVLHPHHIGESNIKTLWEDLNAIHSKYEEDIASLSE

DKKEKRIKVYQGDVCQTINTYCEEVGKEAKTPLVQLLRYLYSRKDDIAVDKIIDGITFLSKKHKVE

KQKINPVIQKYPSFNFGNNSKLLGKIISPKDKLKHNLKCNRNQVDNYIWIEIKVLNTKTMRWEKHH

YALSSTRFLEEVYYPATSENPPDALAARFRTKINGYEGKPALSAEQIEQIRSAPVGLRKVKKRQMR

LEAARQQNLLPRYTWGKDFNINICKRGNNFEVTLATKVKKKKEKNYKVVLGYDANIVRKNTYAAIE

AHANGDGVIDYNDLPVKPIESGFVTVESQVRDKSYDQLSYNGVKLLYCKPHVESRRSFLEKYRNGT

MKDNRGNNIQIDEMKDFEAIADDETSLYYFNMKYCKLLQSSIRNHSSQAKEYREEIFELLRDGKLS

VLKLSSLSNLSFVMFKVAKSLIGTYFGHLLKKPKNSKSDVKAPPITDEDKQKADPEMFALRLALEE

KRLNKVKSKKEVIANKIVAKALELRDKYGPVLIKGENISDTTKKGKKSSTNSFLMDWLARGVANKV

KEMVMMHQGLEFVEVNPNFTSHQDPFVHKNPENTFRARYSRCTPSELTEKNRKEILSFLSDKPSKR

PTNAYYNEGAMAFLATYGLKKNDVLGVSLEKFKQIMANILHQRSEDQLLFPSRGGMFYLATYKLDA

DATSVNWNGKQFWVCNADLVAAYNVGLVDIQKDFKKK

SEQ ID NO: 818
MSISNNNILPYNPKLLPDDRKHKMLVDTFNQLDLIRNNLHDMIIALYGALKYDNIKQFASKEKPHI

(Cas12i3 of
SADALCSINWFRLVKINERKPAIESNQIISKFIQYSGHTPDKYALSHITGNHEPSHKWIDCREYAI

SEQ ID NO: 14
NYARIMHLSFSQFQDLATACLNCKILILNGTLTSSWAWGANSALFGGSDKENFSVKAKILNSFIEN

of U.S. Pat. No.
LKDEMNTTKFQVVEKVCQQIGSSDAADLFDLYRSTVKDGNRGPATGRNPKVMNLFSQDGEISSEQR

10,808,245)
EDFIESFQKVMQEKNSKQIIPHLDKLKYHLVKQSGLYDIYSWAAAIKNANSTIVASNSSNLNTILN

KTEKQQTFEELRKDEKIVACSKILLSVNDTLPEDLHYNPSTSNLGKNLDVFFDLLNENSVHTIENK

EEKNKIVKECVNQYMEECKGLNKPPMPVLLTFISDYAHKHQAQDFLSAAKMNFIDLKIKSIKVVPT

VHGSSPYTWISNLSKKNKDGKMIRTPNSSLIGWIIPPEEIHDQKFAGQNPIIWAVLRVYCNNKWEM

HHFPFSDSRFFTEVYAYKPNLPYLPGGENRSKRFGYRHSTNLSNESRQILLDKSKYAKANKSVLRC

MENMTHNVVFDPKTSLNIRIKTDKNNSPVLDDKGRITFVMQINHRILEKYNNTKIEIGDRILAYDQ

NQSENHTYAILQRTEEGSHAHQFNGWYVRVLETGKVTSIVQGLSGPIDQLNYDGMPVTSHKENCWQ

ADRSAFVSQFASLKISETETFDEAYQAINAQGAYTWNLFYLRILRKALRVCHMENINQFREEILAI

SKNRLSPMSLGSLSQNSLKMIRAFKSIINCYMSRMSFVDELQKKEGDLELHTIMRLTDNKLNDKRV

EKINRASSFLINKAHSMGCKMIVGESDLPVADSKTSKKQNVDRMDWCARALSHKVEYACKLMGLAY

RGIPAYMSSHQDPLVHLVESKRSVLRPRFVVADKSDVKQHHLDNLRRMLNSKTKVGTAVYYREAVE

LMCEELGIHKTDMAKGKVSLSDFVDKFIGEKAIFPQRGGRFYMSTKRLTTGAKLICYSGSDVWLSD

ADEIAAINIGMFVVCDQTGAFKKKKKEKLDDEECDILPFRPM

B2M intron 1
GTGAGTCTCTCCTACCCTCCCGCTCTGGTCCTTCCTCTCCCGCTCTGCACCCTCTGTGGCCCTCGC

(SEQ ID NO: 1219)
TGTGCTCTCTCGCTCCGTGACTTCCCTTCTCCAAGTTCTCCTTGGTGGCCCGCCGTGGGGCTAGTC

CAGGGCTGGATCTCGGGGAAGCGGCGGGGTGGCCTGGGAGTGGGGAAGGGGGTGCGCACCCGGGAC

GCGCGCTACTTGCCCCTTTCGGCGGGGAGCAGGGGAGACCTTTGGCCTACGGCGACGGGAGGGTCG

GGACAAAGTTTAGGGCGTCGATAAGCGTCAGAGCGCCGAGGTTGGGGGAGGGTTTCTCTTCCGCTC

TTTCGCGGGGCCTCTGGCTCCCCCAGCGCAGCTGGAGTGGGGGACGGGTAGGCTCGTCCCAAAGGC

GCGGCGCTGAGGTTTGTGAACGCGTGGAGGGGCGCTTGGGGTCTGGGGGAGGCGTCGCCCGGGTAA

GCCTGTCTGCTGCGGCTCTGCTTCCCTTAGACTGGAGAGCTGTGGACTTCGTCTAGGCGCCCGCTA

AGTTCGCATGTCCTAGCACCTCTGGGTCTATGTGGGGCCACACCGTGGGGAGGAAACAGCACGCGA

CGTTTGTAGAATGCTTGGCTGTGATACAAAGCGGTTTCGAATAATTAACTTATTTGTTCCCATCAC

ATGTCACTTTTAAAAAATTATAAGAACTACCCGTTATTGACATCTTTCTGTGTGCCAAGGACTTTA

TGTGCTTTGCGTCATTTAATTTTGAAAACAGTTATCTTCCGCCATAGATAACTACTATGGTTATCT

TCTGCCTCTCACAGATGAAGAAACTAAGGCACCGAGATTTTAAGAAACTTAATTACACAGGGGATA

AATGGCAGCAATCGAGATTGAAGTCAAGCCTAACCAGGGCTTTTGCGGGAGCGCATGCCTTTTGGC

TGTAATTCGTGCATTTTTTTTTAAGAAAAACGCCTGCCTTCTGCGTGAGATTCTCCAGAGCAAACT

GGGCGGCATGGGCCCTGTGGTCTTTTCGTACAGAGGGCTTCCTCTTTGGCTCTTTGCCTGGTTGTT

TCCAAGATGTACTGTGCCTCTTACTTTCGGTTTTGAAAACATGAGGGGGTTGGGCGTGGTAGCTTA

CGCCTGTAATCCCAGCACTTAGGGAGGCCGAGGCGGGAGGATGGCTTGAGGTCCGTAGTTGAGACC

AGCCTGGCCAACATGGTGAAGCCTGGTCTCTACAAAAAATAATAACAAAAATTAGCCGGGTGTGGT

GGCTCGTGCCTGTGGTCCCAGCTGCTCCGGTGGCTGAGGCGGGAGGATCTCTTGAGCTTAGGCTTT

TGAGCTATCATGGCGCCAGTGCACTCCAGCGTGGGCAACAGAGCGAGACCCTGTCTCTCAAAAAAG

AAAAAAAAAAAAAAAGAAAGAGAAAAGAAAAGAAAGAAAGAAGTGAAGGTTTGTCAGTCAGGGGAG

CTGTAAAACCATTAATAAAGATAATCCAAGATGGTTACCAAGACTGTTGAGGACGCCAGAGATCTT

GAGCACTTTCTAAGTACCTGGCAATACACTAAGCGCGCTCACCTTTTCCTCTGGCAAAACATGATC

GAAAGCAGAATGTTTTGATCATGAGAAAATTGCATTTAATTTGAATACAATTTATTTACAACATAA

AGGATAATGTATATATCACCACCATTACTGGTATTTGCTGGTTATGTTAGATGTCATTTTAAAAAA

TAACAATCTGATATTTAAAAAAAAATCTTATTTTGAAAATTTCCAAAGTAATACATGCCATGCATA

GACCATTTCTGGAAGATACCACAAGAAACATGTAATGATGATTGCCTCTGAAGGTCTATTTTCCTC

CTCTGACCTGTGTGTGGGTTTTGTTTTTGTTTTACTGTGGGCATAAATTAATTTTTCAGTTAAGTT

TTGGAAGCTTAAATAACTCTCCAAAAGTCATAAAGCCAGTAACTGGTTGAGCCCAAATTCAAACCC

AGCCTGTCTGATACTTGTCCTCTTCTTAGAAAAGATTACAGTGATGCTCTCACAAAATCTTGCCGC

CTTCCCTCAAACAGAGAGTTCCAGGCAGGATGAATCTGTGCTCTGATCCCTGAGGCATTTAATATG

TTCTTATTATTAGAAGCTCAGATGCAAAGAGCTCTCTTAGCTTTTAATGTTATGAAAAAAATCAGG

TCTTCATTAGATTCCCCAATCCACCTCTTGATGGGGCTAGTAGCCTTTCCTTAATGATAGGGTGTT

TCTAGAGAGATATATCTGGTCAAGGTGGCCTGGTACTCCTCCTTCTCCCCACAGCCTCCCAGACAA

GGAGGAGTAGCTGCCTTTTAGTGATCATGTACCCTGAATATAAGTGTATTTAAAAGAATTTTATAC

ACATATATTTAGTGTCAATCTGTATATTTAGTAGCACTAACACTTCTCTTCATTTTCAATGAAAAA

TATAGAGTTTATAATATTTTCTTCCCACTTCCCCATGGATGGTCTAGTCATGCCTCTCATTTTGGA

AAGTACTGTTTCTGAAACATTAGGCAATATATTCCCAACCTGGCTAGTTTACAGCAATCACCTGTG

GATGCTAATTAAAACGCAAATCCCACTGTCACATGCATTACTCCATTTGATCATAATGGAAAGTAT

GTTCTGTCCCATTTGCCATAGTCCTCACCTATCCCTGTTGTATTTTATCGGGTCCAACTCAACCAT

TTAAGGTATTTGCCAGCTCTTGTATGCATTTAGGTTTTGTTTCTTTGTTTTTTAGCTCATGAAATT

AGGTACAAAGTCAGAGAGGGGTCTGGCATATAAAACCTCAGCAGAAATAAAGAGGTTTTGTTGTTT

GGTAAGAACATACCTTGGGTTGGTTGGGCACGGTGGCTCGTGCCTGTAATCCCAACACTTTGGGAG

GCCAAGGCAGGCTGATCACTTGAAGTTGGGAGTTCAAGACCAGCCTGGCCAACATGGTGAAATCCC

GTCTCTACTGAAAATACAAAAATTAACCAGGCATGGTGGTGTGTGCCTGTAGTCCCAGGAATCACT

TGAACCCAGGAGGCGGAGGTTGCAGTGAGCTGAGATCTCACCACTGCACACTGCACTCCAGCCTGG

GCAATGGAATGAGATTCCATCCCAAAAAATAAAAAAATAAAAAAATAAAGAACATACCTTGGGTTG

ATCCACTTAGGAACCTCAGATAATAACATCTGCCACGTATAGAGCAATTGCTATGTCCCAGGCACT

CTACTAGACACTTCATACAGTTTAGAAAATCAGATGGGTGTAGATCAAGGCAGGAGCAGGAACCAA

AAAGAAAGGCATAAACATAAGAAAAAAAATGGAAGGGGTGGAAACAGAGTACAATAACATGAGTAA

TTTGATGGGGGCTATTATGAACTGAGAAATGAACTTTGAAAAGTATCTTGGGGCCAAATCATGTAG

ACTCTTGAGTGATGTGTTAAGGAATGCTATGAGTGCTGAGAGGGCATCAGAAGTCCTTGAGAGCCT

CCAGAGAAAGGCTCTTAAAAATGCAGCGCAATCTCCAGTGACAGAAGATACTGCTAGAAATCTGCT

AGAAAAAAAACAAAAAAGGCATGTATAGAGGAATTATGAGGGAAAGATACCAAGTCACGGTTTATT

CTTCAAAATGGAGGTGGCTTGTTGGGAAGGTGGAAGCTCATTTGGCCAGAGTGGAAATGGAATTGG

GAGAAATCGATGACCAAATGTAAACACTTGGTGCCTGATATAGCTTGACACCAAGTTAGCCCCAAG

TGAAATACCCTGGCAATATTAATGTGTCTTTTCCCGATATTCCTCAG

B2M intron 2
GTAAGTCTTACATTCTTTTGTAAGCTGCTGAAAGTTGTGTATGAGTAGTCATATCATAAAGCTGCT

(SEQ ID NO: 1220)
TTGATATAAAAAAGGTCTATGGCCATACTACCCTGAATGAGTCCCATCCCATCTGATATAAACAAT

CTGCATATTGGGATTGTCAGGGAATGTTCTTAAAGATCAGATTAGTGGCACCTGCTGAGATACTGA

TGCACAGCATGGTTTCTGAACCAGTAGTTTCCCTGCAGTTGAGCAGGGAGCAGCAGCAGCACTTGC

ACAAATACATATACACTCTTAACACTTCTTACCTACTGGCTTCCTCTAGCTTTTGTGGCAGCTTCA

GGTATATTTAGCACTGAACGAACATCTCAAGAAGGTATAGGCCTTTGTTTGTAAGTCCTGCTGTCC

TAGCATCCTATAATCCTGGACTTCTCCAGTACTTTCTGGCTGGATTGGTATCTGAGGCTAGTAGGA

AGGGCTTGTTCCTGCTGGGTAGCTCTAAACAATGTATTCATGGGTAGGAACAGCAGCCTATTCTGC

CAGCCTTATTTCTAACCATTTTAGACATTTGTTAGTACATGGTATTTTAAAAGTAAAACTTAATGT

CTTCCTTTTTTTTCTCCACTGTCTTTTTCATAG

B2M intron 3
GTAAGTTTTTGACCTTGAGAAAATGTTTTTGTTTCACTGTCCTGAGGACTATTTATAGACAGCTCT

(SEQ ID NO: 1221)
AACATGATAACCCTCACTATGTGGAGAACATTGACAGAGTAACATTTTAGCAGGGAAAGAAGAATC

CTACAGGGTCATGTTCCCTTCTCCTGTGGAGTGGCATGAAGAAGGTGTATGGCCCCAGGTATGGCC

ATATTACTGACCCTCTACAGAGAGGGCAAAGGAACTGCCAGTATGGTATTGCAGGATAAAGGCAGG

TGGTTACCCACATTACCTGCAAGGCTTTGATCTTTCTTCTGCCATTTCCACATTGGACATCTCTGC

TGAGGAGAGAAAATGAACCACTCTTTTCCTTTGTATAATGTTGTTTTATTCTTCAGACAGAAGAGA

GGAGTTATACAGCTCTGCAGACATCCCATTCCTGTATGGGGACTGTGTTTGCCTCTTAGAGGTTCC

CAGGCCACTAGAGGAGATAAAGGGAAACAGATTGTTATAACTTGATATAATGATACTATAATAGAT

GTAACTACAAGGAGCTCCAGAAGCAAGAGAGAGGGAGGAACTTGGACTTCTCTGCATCTTTAGTTG

GAGTCCAAAGGCTTTTCAATGAAATTCTACTGCCCAGGGTACATTGATGCTGAAACCCCATTCAAA

TCTCCTGTTATATTCTAGAACAGGGAATTGATTTGGGAGAGCATCAGGAAGGTGGATGATCTGCCC

AGTCACACTGTTAGTAAATTGTAGAGCCAGGACCTGAACTCTAATATAGTCATGTGTTACTTAATG

ACGGGGACATGTTCTGAGAAATGCTTACACAAACCTAGGTGTTGTAGCCTACTACACGCATAGGCT

ACATGGTATAGCCTATTGCTCCTAGACTACAAACCTGTACAGCCTGTTACTGTACTGAATACTGTG

GGCAGTTGTAACACAATGGTAAGTATTTGTGTATCTAAACATAGAAGTTGCAGTAAAAATATGCTA

TTTTAATCTTATGAGACCACTGTCATATATACAGTCCATCATTGACCAAAACATCATATCAGCATT

TTTTCTTCTAAGATTTTGGGAGCACCAAAGGGATACACTAACAGGATATACTCTTTATAATGGGTT

TGGAGAACTGTCTGCAGCTACTTCTTTTAAAAAGGTGATCTACACAGTAGAAATTAGACAAGTTTG

GTAATGAGATCTGCAATCCAAATAAAATAAATTCATTGCTAACCTTTTTCTTTTCTTTTCAG

	Number	Date	Country
	63252719	Oct 2021	US
	63107869	Oct 2020	US

COMPOSITIONS COMPRISING AN RNA GUIDE TARGETING B2M AND USES THEREOF

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