WO2023081377A2

WO2023081377A2 - Compositions comprising an rna guide targeting ciita and uses thereof

Info

Publication number: WO2023081377A2
Application number: PCT/US2022/048989
Authority: WO
Inventors: Quinton Norman WESSELLS; Tia Marie DITOMMASO; Jeffrey Raymond HASWELL; Noah Michael Jakimo
Original assignee: Arbor Biotechnologies, Inc.
Priority date: 2021-11-05
Filing date: 2022-11-04
Publication date: 2023-05-11
Also published as: TW202334421A; WO2023081377A3

Abstract

The present invention relates to compositions comprising RNA guides targeting CIITA, processes for characterizing the compositions, cells comprising the compositions, and methods of using the compositions.

Description

COMPOSITIONS COMPRISING AN RNA GUIDE TARGETING CIITA AND USES THEREOF

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application 63/276,229, filed November 5, 2021 and U.S. Provisional Application No. 63/294,121, filed December 28, 2021, the contents of each of which are incorporated by reference herein in their entirety.

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 CIITA 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’.

The present disclosure is based, at least in part, on the development of a system for genetic editing of a Class II Major Histocompatibility Complex Transactivator (CIITA) gene. The system may comprise a Cas12i CRISPR nuclease polypeptide (e.g., a Cas12i2 CRISPR nuclease polypeptide) and an RNA guide mediating cleavage at a genetic site within the CIITA gene by the CRISPR nuclease polypeptide. As reported herein, the gene editing system disclosed herein has achieved successful editing of CIITA gene with high editing efficiency.

Accordingly, provided herein are gene editing systems for editing CIITA gene, pharmaceutical compositions or kits comprising such, methods of using the gene editing systems to produce genetically modified cells, and the resultant cells thus produced. Also provided herein are uses of the gene editing systems disclosed herein, the pharmaceutical compositions and kits comprising such, and/or the genetically modified cells thus produced for treating a subject.

In some aspects, the present disclosure features system for genetic editing of a CIITA gene, comprising (i) a Cas12i polypeptide or a first nucleic acid encoding the Cas12i polypeptide, and (ii) an RNA guide or a nucleic acid encoding the RNA guide. The RNA guide comprises a spacer sequence specific to a target sequence within a CIITA gene, the target sequence being adjacent to a protospacer adjacent motif (PAM) comprising the motif of 5’-NTTN-3’, which is located 5’ to the non-target strand of the target sequence.

In some aspects, the present disclosure features a composition comprising an RNA guide, wherein the RNA guide comprises: (i) a spacer sequence that specifically binds a target sequence within a CIITA gene, wherein the target sequence comprises any one of SEQ ID NOS: 1163-1177; and (ii) a direct repeat sequence.

In some aspects, the present disclosure features a composition comprising an RNA guide, wherein the RNA guide comprises: (i) a spacer sequence that specifically binds a target sequence within a CIITA gene, wherein the target sequence comprises any one of SEQ ID NOS: 1163, 1166, 1167, 1173, or 1175; and (ii) a direct repeat sequence.

In some aspects, the present disclosure features a composition comprising an RNA guide, wherein the RNA guide comprises: (i) a spacer sequence that is at least 90% identical to a sequence of any one of SEQ ID NOS: 1207-1221; and (ii) a direct repeat sequence.

In some aspects, the present disclosure features a composition comprising an RNA guide, wherein the RNA guide comprises: (i) a spacer sequence that is at least 90% identical to a sequence of any one of SEQ ID NOS: 1207, 1210, 1211, 1217, or 1219; 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, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon

18, exon 19, exon 20, exon 21, exon 22, intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, intron 7, intron 8, intron 9, intron 10, intron 11, intron 12, intron 13, intron 14, intron 15, intron 16, intron 17, intron 18, intron

19, intron 20, or intron 21 of the CIITA gene.

In another aspect of the composition, the CIITA gene comprises the sequence set forth in www.ncbi.nlm.nih.gov/gene/4261 (which is incorporated herein by reference as November 5, 2021), the reverse complement thereof, a variant thereof, or the reverse complement of a variant thereof.

In some embodiments, the spacer sequence has a length of between 15-30 nucleotides or between 20- 30 nucleotides. In some embodiments, the direct repeat sequence has a length of between 15-40 or 23-36 nucleotides. In some embodiments, the direct repeat sequence is a Cas12il direct repeat sequence, a Cas12i2 direct repeat sequence, a Cas12i3 direct repeat sequence, or a Cas12i4 direct repeat sequence.

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: 472-932 or 1018-1102; 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: 472-932 or 1018-1102; 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: 472-932 or 1018-1102; 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: 472-932 or 1018-1102; 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: 472-932 or 1018-1102; 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: 472-932; 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: 472-932; 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: 472-932; 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: 472-932; 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: 472-932; 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: 472-932; 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: 472-932; 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: 472-932; 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: 472-932; 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: 472-932.

In some embodiments, 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: 752, 815, 802, 821, and 824; 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: 752, 815, 802, 821, and 824; 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: 752, 815, 802, 821, and 824; 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: 752, 815, 802, 821, and 824; or 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: 752, 815, 802, 821, and 824. In some embodiments, the spacer sequence comprises: a. nucleotide 1 through nucleotide 16 of any one of SEQ ID NOs: 752, 815, 802, 821, and 824; b. nucleotide 1 through nucleotide 17 of any one of SEQ ID NOs: 752, 815, 802, 821, and 824; c. nucleotide 1 through nucleotide 18 of any one of SEQ ID NOs: 752, 815, 802, 821, and 824; d. nucleotide 1 through nucleotide 19 of any one of SEQ ID NOs: 752, 815, 802, 821, and 824; or e. nucleotide 1 through nucleotide 20 of any one of SEQ ID NOs: 752, 815, 802, 821, and 824.

In another aspect of the composition, the spacer sequence comprises: a. nucleotide 1 through nucleotide 16 of any one of SEQ ID NOs: 472-932 or 1018-1102; b. nucleotide 1 through nucleotide 17 of any one of SEQ ID NOs: 472-932 or 1018-1102; c. nucleotide 1 through nucleotide 18 of any one of SEQ ID NOs: 472-932 or 1018-1102; d. nucleotide 1 through nucleotide 19 of any one of SEQ ID NOs: 472-932 or 1018-1102; e. nucleotide 1 through nucleotide 20 of any one of SEQ ID NOs: 472-932 or 1018-1102; f. nucleotide 1 through nucleotide 21 of any one of SEQ ID NOs: 472-932; g. nucleotide 1 through nucleotide 22 of any one of SEQ ID NOs: 472-932; h. nucleotide 1 through nucleotide 23 of any one of SEQ ID NOs: 472-932; i. nucleotide 1 through nucleotide 24 of any one of SEQ ID NOs: 472-932; j. nucleotide 1 through nucleotide 25 of any one of SEQ ID NOs: 472-932; k. nucleotide 1 through nucleotide 26 of any one of SEQ ID NOs: 472-932; 1. nucleotide 1 through nucleotide 27 of any one of SEQ ID NOs: 472-932; m. nucleotide 1 through nucleotide 28 of any one of SEQ ID NOs: 472-932; n. nucleotide 1 through nucleotide 29 of any one of SEQ ID NOs: 472-932; or o. nucleotide 1 through nucleotide 30 of any one of SEQ ID NOs: 472-932.

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 a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1133-1151; 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: 1133-1151; 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: 1133-1151; 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: 1133-1151; 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: 1133-1151; 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: 1133-1151; 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: 1133-1151; 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: 1133-1151; 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: 1133-1151; 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: 1133-1151; 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: 1133-1151; 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: 1133-1151; 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: 1133-1151; 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: 1133-1151; or o. a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1133-1151 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: 1133-1151; b. nucleotide 2 through nucleotide 36 of any one of SEQ ID NOs: 1133-1151; c. nucleotide 3 through nucleotide 36 of any one of SEQ ID NOs: 1133-1151; d. nucleotide 4 through nucleotide 36 of any one of SEQ ID NOs: 1133-1151; e. nucleotide 5 through nucleotide 36 of any one of SEQ ID NOs: 1133-1151; f. nucleotide 6 through nucleotide 36 of any one of SEQ ID NOs: 1133-1151; g. nucleotide 7 through nucleotide 36 of any one of SEQ ID NOs: 1133-1151; h. nucleotide 8 through nucleotide 36 of any one of SEQ ID NOs: 1133-1151; i. nucleotide 9 through nucleotide 36 of any one of SEQ ID NOs: 1133-1151; j. nucleotide 10 through nucleotide 36 of any one of SEQ ID NOs: 1133-1151; k. nucleotide 11 through nucleotide 36 of any one of SEQ ID NOs: 1133-1151; 1. nucleotide 12 through nucleotide 36 of any one of SEQ ID NOs: 1133-1151; m. nucleotide 13 through nucleotide 36 of any one of SEQ ID NOs: 1133- 1151; n. nucleotide 14 through nucleotide 36 of any one of SEQ ID NOs: 1133-1151; or o. any one of SEQ ID NOs: 1133-1151 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: 1152; b. nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1152; c. nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1152; d. nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1152; e. nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1152; f. nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1152; g. nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1152; h. nucleotide 8 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1152; i. nucleotide 9 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1152; j. nucleotide 10 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1152; k. nucleotide 11 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1152; 1. nucleotide 12 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1152; m. nucleotide 13 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1152; n. nucleotide 14 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1152; or o. a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1153 or SEQ ID NO: 1154 or a portion thereof.

In another aspect of the composition, the direct repeat comprises: a. nucleotide 1 through nucleotide 36 of SEQ ID NO: 1152; b. nucleotide 2 through nucleotide 36 of SEQ ID NO: 1152; c. nucleotide 3 through nucleotide 36 of SEQ ID NO: 1152; d. nucleotide 4 through nucleotide 36 of SEQ ID NO: 1152; e. nucleotide 5 through nucleotide 36 of SEQ ID NO: 1152; f. nucleotide 6 through nucleotide 36 of SEQ ID NO: 1152; g. nucleotide 7 through nucleotide 36 of SEQ ID NO: 1152; h. nucleotide 8 through nucleotide 36 of SEQ ID NO: 1152; i. nucleotide 9 through nucleotide 36 of SEQ ID NO: 1152; j. nucleotide 10 through nucleotide 36 of SEQ ID NO: 1152; k. nucleotide 11 through nucleotide 36 of SEQ ID NO: 1152; 1. nucleotide 12 through nucleotide 36 of SEQ ID NO: 1152; m. nucleotide 13 through nucleotide 36 of SEQ ID NO: 1152; n. nucleotide 14 through nucleotide 36 of SEQ ID NO: 1152; or o. SEQ ID NO: 1153 or SEQ ID NO: 1154 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: 1155 or SEQ ID NO: 1156; b. nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1155 or SEQ ID NO: 1156; c. nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1155 or SEQ ID NO: 1156; d. nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1155 or SEQ ID NO: 1156; e. nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1155 or SEQ ID NO: 1156; f. nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1155 or SEQ ID NO: 1156; g. nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1155 or SEQ ID NO: 1156; h. nucleotide 8 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1155 or SEQ ID NO: 1156; i. nucleotide 9 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1155 or SEQ ID NO: 1156; j. nucleotide 10 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1155 or SEQ ID NO: 1156; k. nucleotide 11 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1155 or SEQ ID NO: 1156; 1. nucleotide 12 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1155 or SEQ ID NO: 1156; m. nucleotide 13 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1155 or SEQ ID NO: 1156; n. nucleotide 14 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1155 or SEQ ID NO: 1156; o. nucleotide 15 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1155 or SEQ ID NO: 1156; or p. a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1157 or a portion thereof.

In another aspect of the composition, the direct repeat comprises: a. nucleotide 1 through nucleotide 36 of SEQ ID NO: 1155 or SEQ ID NO: 1156; b. nucleotide 2 through nucleotide 36 of SEQ ID NO: 1155 or SEQ ID NO: 1156; c. nucleotide 3 through nucleotide 36 of SEQ ID NO: 1155 or SEQ ID NO: 1156; d. nucleotide 4 through nucleotide 36 of SEQ ID NO: 1155 or SEQ ID NO: 1156; e. nucleotide 5 through nucleotide 36 of SEQ ID NO: 1155 or SEQ ID NO: 1156; f. nucleotide 6 through nucleotide 36 of SEQ ID NO: 1155 or SEQ ID NO: 1156; g. nucleotide 7 through nucleotide 36 of SEQ ID NO: 1155 or SEQ ID NO: 1156; h. nucleotide 8 through nucleotide 36 of SEQ ID NO: 1155 or SEQ ID NO: 1156; i. nucleotide 9 through nucleotide 36 of SEQ ID NO: 1155 or SEQ ID NO: 1156; j. nucleotide 10 through nucleotide 36 of SEQ ID NO: 1155 or SEQ ID NO: 1156; k. nucleotide 11 through nucleotide 36 of SEQ ID NO: 1155 or SEQ ID NO: 1156; 1. nucleotide 12 through nucleotide 36 of SEQ ID NO: 1155 or SEQ ID NO: 1156; m. nucleotide 13 through nucleotide 36 of SEQ ID NO: 1155 or SEQ ID NO: 1156; n. nucleotide 14 through nucleotide 36 of SEQ ID NO: 1155 or SEQ ID NO: 1156; o. nucleotide 15 through nucleotide 36 of SEQ ID NO: 1155 or SEQ ID NO: 1156; or p. SEQ ID NO: 1157 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: 11-471 or 933-1017.

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 some embodiments, the target sequence is adjacent to a protospacer adjacent motif (PAM) comprising the sequence 5 ’ -NTTN-3 ’ , wherein N is any nucleotide . In some embodiments, the target sequence is immediately adjacent to the PAM sequence.

In some embodiments, the PAM comprises the sequence 5’-CTTT-3’ and the target sequence comprises SEQ ID NO: 1163. In some embodiments, the PAM comprises the sequence 5’-CTTT-3’ and the spacer sequence is at least 90% identical to a sequence of SEQ ID NO: 1207. In some embodiments, the PAM comprises the sequence 5’-CTTC-3’ and the target sequence comprises any one of SEQ ID NOS: 1166, 1167, 1173, or 1175. In some embodiments, the PAM comprises the sequence 5’-CTTC-3’ and the spacer sequence is at least 90% identical to a sequence of SEQ ID NO: 1210, 1211, 1217, or 1219.

In another aspect of the composition, the RNA guide has a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1178-1192.

In another aspect of the composition, the RNA guide has the sequence of any one of SEQ ID NOs: 1178-1192.

In another aspect of the composition, the composition further comprises a Cas12i polypeptide or a polyribonucleotide encoding 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: 1104, SEQ ID NO: 1127, SEQ ID NO: 1128, SEQ ID NO: 1129, SEQ ID NO: 1130, or SEQ ID NO: 1131; b. a Cas12i4 polypeptide comprising a sequence that is at least 90% identical to the sequence of SEQ ID NO: 1158, SEQ ID NO: 1159, or SEQ ID NO: 1160; c. a Cas12il polypeptide comprising a sequence that is at least 90% identical to the sequence of SEQ ID NO: 1161; or d. a Cas12i3 polypeptide comprising a sequence that is at least 90% identical to the sequence of SEQ ID NO: 1162.

In another aspect of the composition, the Cas12i polypeptide is: a. a Cas12i2 polypeptide comprising a sequence of SEQ ID NO: 1104, SEQ ID NO: 1127, SEQ ID NO: 1128, SEQ ID NO: 1129, SEQ ID NO: 1130, or SEQ ID NO: 1131; b. a Cas12i4 polypeptide comprising a sequence of SEQ ID NO: 1158, SEQ ID NO: 1159, or SEQ ID NO: 1160; c. a Cas12il polypeptide comprising a sequence of SEQ ID NO: 1161; or d. a Cas12i3 polypeptide comprising a sequence of SEQ ID NO: 1162. In some embodiments, the Cas12i polypeptide comprises one or more mutations relative to SEQ ID NO: 1104. In some embodiments, the Cas12i polypeptide comprises one or more mutations relative to SEQ ID NO: 1104. In some embodiments, the one or more mutations in the Cas12i polypeptide are at positions D581, G624, F626, P868, 1926, V1030, E1035, and/or S1046 of SEQ ID NO: 1104. In some embodiments, the one or more mutations are amino acid substitutions, wherein optionally the amino acid substitutions are chosen from D581R, G624R, F626R, P868T, I926R, V1030G, E1035R, S1046G, or a combination thereof. In some embodiments, the Cas12i polypeptide comprises: (i) mutations at positions D581, D911, 1926, and VI 030 of SEQ ID NO: 1104, which optionally are amino acid substitutions of D581R, D911R, I926R, and V1030G; (ii) mutations at positions D581, 1926, and V1030 of SEQ ID NO: 1104, which optionally are amino acid substitutions of D581R, I926R, and V1030G; (iii) mutations at positions D581, 1926, V1030, and S1046 of SEQ ID NO: 1104, which optionally are amino acid substitutions of D581R, I926R, V1030G, and S1046G;

(iv) mutations at positions D581, G624, F626, 1926, V1030, E1035, and S1046 of SEQ ID NO: 1104, which optionally are amino acid substitutions of D581R, G624R, F626R, I926R, V1030G, E1035R, and S1046G; or

(v) mutations at positions D581, G624, F626, P868, 1926, V1030, E1035, and S1046 of SEQ ID NO: 1104, which optionally are amino acid substitutions of D581R, G624R, F626R, P868T, I926R, V1030G, E1035R, and S1046G.

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

In some embodiments, the composition comprises the polyribonucleotide encoding the Cas12i polypeptide, wherein optionally the polyribonucleotide is a messenger RNA (mRNA).

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 Cas 12i 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.

In some aspects, the present disclosure provides a system comprising: (i) an RNA guide described herein, or a nucleic acid encoding the RNA guide, and (ii) a Cas12i polypeptide, or a nucleic acid encoding the Cas12i polypeptide. In some embodiments of the system, the Cas12i polypeptide is a Cas12i2 polypeptide comprising a sequence that is at least 90% identical to the sequence of SEQ ID NO: 1104, SEQ ID NO: 1127, SEQ ID NO: 1128, SEQ ID NO: 1129, SEQ ID NO: 1130, or SEQ ID NO: 1131. In some embodiments of the system, the Casl2i polypeptide comprises one or more mutations relative to SEQ ID NO: 1104. In some embodiments, the one or more mutations in the Casl2i polypeptide are at positions D581, G624, F626, P868, 1926, V1030, E1035, and/or S1046 of SEQ ID NO: 1104. In some embodiments, the one or more mutations are amino acid substitutions, wherein optionally the amino acid substitutions are chosen from D581R, G624R, F626R, P868T, I926R, V1030G, E1035R, S1046G, or a combination thereof. In some embodiments, the Casl2i polypeptide comprises: (i) mutations at positions D581, D911, 1926, and V1030 of SEQ ID NO: 1104, which optionally are amino acid substitutions of D581R, D911R, I926R, and V1030G; (ii) mutations at positions D581, 1926, and V1030 of SEQ ID NO: 1104, which optionally are amino acid substitutions of D581R, I926R, and VI 030G; (iii) mutations at positions D581, 1926, VI 030, and SI 046 of SEQ ID NO: 1104, which optionally are amino acid substitutions of D581R, I926R, V1030G, and S1046G; (iv) mutations at positions D581, G624, F626, 1926, V1030, E1035, and S1046 of SEQ ID NO: 1104, which optionally are amino acid substitutions of D581R, G624R, F626R, I926R, V1030G, E1035R, and S1046G; or (v) mutations at positions D581, G624, F626, P868, 1926, V1030, E1035, and S1046 of SEQ ID NO: 1104, which optionally are amino acid substitutions of D581R, G624R, F626R, P868T, I926R, V1030G, E1035R, and S1046G. In some embodiments, the Casl2i polypeptide is a Casl2il polypeptide, a Casl2i2 polypeptide, a Casl2i3 polypeptide, or a Casl2i4 polypeptide.

In some aspects, the present disclosure provides a system comprising a pharmaceutical composition comprising a composition or system described herein.

The invention further provides a composition comprising an RNA guide and a Casl2i polypeptide, wherein the RNA guide comprises (i) a spacer sequence that is substantially complementary to a target sequence within a CIITA gene and (ii) a direct repeat sequence.

19, intron 20, or intron 21 of the CIITA gene.

In another aspect of the composition, the CIITA gene comprises the sequence set forth at www.ncbi.nlm.nih.gov/gene/4261, the reverse complement thereof, a variant thereof, or the reverse complement of a variant 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 any one of SEQ ID NOs: 1133-1151; 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: 1133-1151; 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: 1133-1151; 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: 1133-1151; 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: 1133-1151; 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: 1133-1151; 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: 1133-1151; 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: 1133-1151; 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: 1133-1151; 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: 1133-1151; 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: 1133-1151; 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: 1133-1151; 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: 1133-1151; 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: 1133-1151; or o. a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1133-1151 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: 1133-1151; b. nucleotide 2 through nucleotide 36 of any one of SEQ ID NOs: 1133-1151; c. nucleotide 3 through nucleotide 36 of any one of SEQ ID NOs: 1133-1151; d. nucleotide 4 through nucleotide 36 of any one of SEQ ID NOs: 1133-1151; e. nucleotide 5 through nucleotide 36 of any one of SEQ ID NOs: 1133-1151; f. nucleotide 6 through nucleotide 36 of any one of SEQ ID NOs: 1133-1151; g. nucleotide 7 through nucleotide 36 of any one of SEQ ID NOs: 1133-1151; h. nucleotide 8 through nucleotide 36 of any one of SEQ ID NOs: 1133-1151; i. nucleotide 9 through nucleotide 36 of any one of SEQ ID NOs: 1133-1151; j. nucleotide 10 through nucleotide 36 of any one of SEQ ID NOs: 1133-1151; k. nucleotide 11 through nucleotide 36 of any one of SEQ ID NOs: 1133-1151; 1. nucleotide 12 through nucleotide 36 of any one of SEQ ID NOs: 1133-1151; m. nucleotide 13 through nucleotide 36 of any one of SEQ ID NOs: 1133- 1151; n. nucleotide 14 through nucleotide 36 of any one of SEQ ID NOs: 1133-1151; or o. any one of SEQ ID NOs: 1133-1151 or a portion thereof.

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 Cas12i polypeptide is: a. a Cas12i2 polypeptide comprising a sequence of SEQ ID NO: 1104, SEQ ID NO: 1127, SEQ ID NO: 1128, SEQ ID NO: 1129, SEQ ID NO: 1130, or SEQ ID NO: 1131; b. a Cas12i4 polypeptide comprising a sequence of SEQ ID NO: 1158, SEQ ID NO: 1159, or SEQ ID NO: 1160; c. a Cas12il polypeptide comprising a sequence of SEQ ID NO: 1161; or d. a Cas12i3 polypeptide comprising a sequence of SEQ ID NO: 1162.

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

The invention yet further provides an RNA guide comprising (i) a spacer sequence that is substantially complementary to a target sequence within a CIITA 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, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon

19, intron 20, or intron 21 of the CIITA gene. In another aspect of the RNA guide, the CIITA gene comprises the set forth at www.ncbi.nlm.nih.gov/gene/4261, the reverse complement thereof, a variant thereof, or the reverse complement of a variant thereof.

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: 472-932 or 1018-1102; 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: 472-932 or 1018-1102; 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: 472-932 or 1018-1102; 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: 472-932 or 1018-1102; 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: 472-932 or 1018-1102; 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: 472-932; 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: 472-932; 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: 472-932; 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: 472-932; 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: 472-932; 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: 472-932; 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: 472-932; 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: 472-932; 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: 472-932; 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: 472-932.

In another aspect of the RNA guide, the spacer sequence comprises: a. nucleotide 1 through nucleotide 16 of any one of SEQ ID NOs: 472-932 or 1018-1102; b. nucleotide 1 through nucleotide 17 of any one of SEQ ID NOs: 472-932 or 1018-1102; c. nucleotide 1 through nucleotide 18 of any one of SEQ ID NOs: 472-932 or 1018-1102; d. nucleotide 1 through nucleotide 19 of any one of SEQ ID NOs: 472-932 or 1018-1102; e. nucleotide 1 through nucleotide 20 of any one of SEQ ID NOs: 472-932 or 1018-1102; f. nucleotide 1 through nucleotide 21 of any one of SEQ ID NOs: 472-932; g. nucleotide 1 through nucleotide 22 of any one of SEQ ID NOs: 472-932; h. nucleotide 1 through nucleotide 23 of any one of SEQ ID NOs: 472-932; i. nucleotide 1 through nucleotide 24 of any one of SEQ ID NOs: 472-932; j. nucleotide 1 through nucleotide 25 of any one of SEQ ID NOs: 472-932; k. nucleotide 1 through nucleotide 26 of any one of SEQ ID NOs: 472-932; 1. nucleotide 1 through nucleotide 27 of any one of SEQ ID NOs: 472-932; m. nucleotide 1 through nucleotide 28 of any one of SEQ ID NOs: 472-932; n. nucleotide 1 through nucleotide 29 of any one of SEQ ID NOs: 472-932; or o. nucleotide 1 through nucleotide 30 of any one of SEQ ID NOs: 472-932. 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; 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 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; 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 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: 1133-1151; 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: 1133-1151; 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: 1133-1151; 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: 1133-1151; 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: 1133-1151; 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: 1133-1151; 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: 1133-1151; 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: 1133-1151; 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: 1133-1151; 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: 1133-1151; 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: 1133-1151; 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: 1133-1151; 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: 1133-1151; 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: 1133-1151; or o. a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1133-1151 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: 1133-1151; b. nucleotide 2 through nucleotide 36 of any one of SEQ ID NOs: 1133-1151; c. nucleotide 3 through nucleotide 36 of any one of SEQ ID NOs: 1133-1151; d. nucleotide 4 through nucleotide 36 of any one of SEQ ID NOs: 1133-1151; e. nucleotide 5 through nucleotide 36 of any one of SEQ ID NOs: 1133-1151; f. nucleotide 6 through nucleotide 36 of any one of SEQ ID NOs: 1133-1151; g. nucleotide 7 through nucleotide 36 of any one of SEQ ID NOs: 1133-1151; h. nucleotide 8 through nucleotide 36 of any one of SEQ ID NOs: 1133-1151; i. nucleotide 9 through nucleotide 36 of any one of SEQ ID NOs: 1133-1151; j. nucleotide 10 through nucleotide 36 of any one of SEQ ID NOs: 1133-1151; k. nucleotide 11 through nucleotide 36 of any one of SEQ ID NOs: 1133-1151; 1. nucleotide 12 through nucleotide 36 of any one of SEQ ID NOs: 1133-1151; m. nucleotide 13 through nucleotide 36 of any one of SEQ ID NOs: 1133- 1151; n. nucleotide 14 through nucleotide 36 of any one of SEQ ID NOs: 1133-1151; or o. any one of SEQ ID NOs: 1133-1151 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: 1152; b. nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1152; c. nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1152; d. nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1152; e. nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1152; f. nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1152; g. nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1152; h. nucleotide 8 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1152; i. nucleotide 9 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1152; j. nucleotide 10 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1152; k. nucleotide 11 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1152; 1. nucleotide 12 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1152; m. nucleotide 13 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1152; n. nucleotide 14 through nucleotide 36 of a sequence that is at least 90% identical to SEQ ID NO: 1152; or o. a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1153 or SEQ ID NO: 1154 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: 1152; b. nucleotide 2 through nucleotide 36 of SEQ ID NO: 1152; c. nucleotide 3 through nucleotide 36 of SEQ ID NO: 1152; d. nucleotide 4 through nucleotide 36 of SEQ ID NO: 1152; e. nucleotide 5 through nucleotide 36 of SEQ ID NO: 1152; f. nucleotide 6 through nucleotide 36 of SEQ ID NO: 1152; g. nucleotide 7 through nucleotide 36 of SEQ ID NO: 1152; h. nucleotide 8 through nucleotide 36 of SEQ ID NO: 1152; i. nucleotide 9 through nucleotide 36 of SEQ ID NO: 1152; j. nucleotide 10 through nucleotide 36 of SEQ ID NO: 1152; k. nucleotide 11 through nucleotide 36 of SEQ ID NO: 1152; 1. nucleotide 12 through nucleotide 36 of SEQ ID NO: 1152; m. nucleotide 13 through nucleotide 36 of SEQ ID NO: 1152; n. nucleotide 14 through nucleotide 36 of SEQ ID NO: 1152; or o. SEQ ID NO: 1153 or SEQ ID NO: 1154 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: 1155 or SEQ ID NO: 1156; b. nucleotide 2 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1155 or SEQ ID NO: 1156; c. nucleotide 3 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1155 or SEQ ID NO: 1156; d. nucleotide 4 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1155 or SEQ ID NO: 1156; e. nucleotide 5 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1155 or SEQ ID NO: 1156; f. nucleotide 6 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1155 or SEQ ID NO: 1156; g. nucleotide 7 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1155 or SEQ ID NO: 1156; h. nucleotide 8 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1155 or SEQ ID NO: 1156; i. nucleotide 9 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1155 or SEQ ID NO: 1156; j. nucleotide 10 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1155 or SEQ ID NO: 1156; k. nucleotide 11 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1155 or SEQ ID NO: 1156; 1. nucleotide 12 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1155 or SEQ ID NO: 1156; m. nucleotide 13 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1155 or SEQ ID NO: 1156; n. nucleotide 14 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1155 or SEQ ID NO: 1156; o. nucleotide 15 through nucleotide 36 of a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1155 or SEQ ID NO: 1156; or p. a sequence that is at least 90% identical to a sequence of SEQ ID NO: 1157 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: 1155 or SEQ ID NO: 1156; b. nucleotide 2 through nucleotide 36 of SEQ ID NO: 1155 or SEQ ID NO: 1156; c. nucleotide 3 through nucleotide 36 of SEQ ID NO: 1155 or SEQ ID NO: 1156; d. nucleotide 4 through nucleotide 36 of SEQ ID NO: 1155 or SEQ ID NO: 1156; e. nucleotide 5 through nucleotide 36 of SEQ ID NO: 1155 or SEQ ID NO: 1156; f. nucleotide 6 through nucleotide 36 of SEQ ID NO: 1155 or SEQ ID NO: 1156; g. nucleotide 7 through nucleotide 36 of SEQ ID NO: 1155 or SEQ ID NO: 1156; h. nucleotide 8 through nucleotide 36 of SEQ ID NO: 1155 or SEQ ID NO: 1156; i. nucleotide 9 through nucleotide 36 of SEQ ID NO: 1155 or SEQ ID NO: 1156; j. nucleotide 10 through nucleotide 36 of SEQ ID NO: 1155 or SEQ ID NO: 1156; k. nucleotide 11 through nucleotide 36 of SEQ ID NO: 1155 or SEQ ID NO: 1156; 1. nucleotide 12 through nucleotide 36 of SEQ ID NO: 1155 or SEQ ID NO: 1156; m. nucleotide 13 through nucleotide 36 of SEQ ID NO: 1155 or SEQ ID NO: 1156; n. nucleotide 14 through nucleotide 36 of SEQ ID NO: 1155 or SEQ ID NO: 1156; o. nucleotide 15 through nucleotide 36 of SEQ ID NO: 1155 or SEQ ID NO: 1156; or p. SEQ ID NO: 1157 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: 11-471 or 933-1017.

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 has a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1178-1192.

In another aspect of the RNA guide, the RNA guide has the sequence of any one of SEQ ID NOs: 1178-1192.

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.

Also within the scope of the present disclosure is a cell comprising a disrupted CIITA gene, which can be produced by contacting a host cell with the system disclosed herein to genetically edit the CIITA gene in the host cell. Also provided herein is a population of cells, wherein a plurality of host cells in the population comprise a disrupted CIITA gene, which was produced by contacting the population of cells with the system disclosed herein to genetically edit the CIITA gene a plurality of cells in the population. The population of cells may also comprise cells without an edit in the CIITA gene.

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 CIITA sequence, the method comprising contacting a CIITA 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.

The invention yet further provides a method of binding a Cas12i polypeptide and an RNA guide to a target sequence, the method comprising contacting the target sequence with a composition, system, vector, or vector system described herein. In some embodiments, the composition comprises the polyribonucleotide encoding the Cas 12i polypeptide, and the contacting results in production of the Cas 12i polypeptide in the cell.

In one aspect of the method, the CIITA sequence is in a cell. In some embodiments, the cell is cultured in vitro. In some embodiments, the contacting step is performed by administering the system to a subject comprising the host cell. In some embodiments, the the method comprises contacting the cell with the composition, system, or RNA guide as described herein. In some embodiments, the host cell is cultured in vitro. In some embodiments, the contacting step is performed by administering the system for editing the CIITA gene to a subject comprising the host cell.

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

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

In another aspect of the method, the deletion is downstream of the 5’-NTTN-3’ sequence.

In another aspect of the method, the deletion is up to about 40 nucleotides in length. In another aspect of the method, the deletion is up to about 50 nucleotides in length.

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

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

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

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

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

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

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

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

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

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

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

In another aspect of the method, the deletion ends within about 20 nucleotides to about 25 nucleotides of the 5’-NTTN-3’ sequence. In another aspect of the method, the deletion ends within about 25 nucleotides to about 30 nucleotides of the 5’-NTTN-3’ sequence.

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

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

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

In another 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 another 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 another 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 another 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 another 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 another 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 another 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 another 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 another 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 another 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 another aspect of the method, the deletion overlaps with a mutation in the CIITA sequence.

In another aspect of the method, the deletion overlaps with an insertion in the CIITA sequence.

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

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

In another 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: 1178-1192.

The invention yet further provides a method of treating a disease or condition in a subject, the method comprising administering a composition, an RNA guide, or a cell described herein to the subject.

In another aspect of the composition, the RNA guide, the cell, the kit, or the method described herein, the RNA guide and/or the polyribonucleotide encoding the Cas12i polypeptide are comprised within a lipid nanoparticle.

In another aspect of the composition, cell, kit, or method, the RNA guide and the polyribonucleotide encoding the Cas12i polypeptide are comprised within the same lipid nanoparticle.

In another aspect of the composition, cell, kit, or method, the RNA guide and the polyribonucleotide encoding the Cas12i polypeptide are comprised within separate lipid nanoparticles.

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 “CIITA” refers to “Class II Major Histocompatibility Complex Transactivator.” CIITA is a positive regulator of class II major histocompatibility complex gene transcription. Mutations in the CIITA gene are responsible for the bare lymphocyte syndrome, in which the immune system is severely compromised and cannot effectively fight infection. In addition, chromosomal rearrangement of the gene is involved in the pathogenesis of Hodgkin lymphoma and primary mediastinal B cell lymphoma. An example of a CIITA gene sequence is provided at www.ncbi.nlm.nih.gov/gene/4261, which is incorporated herein by reference as of November 5, 2021. It is understood that spacer sequences described herein can target this sequence or the reverse complement thereof, depending upon whether they are indicated as “+” or as set forth in Tables 5 A and 5B. The target sequences listed in Table 5 A and Table 5B are on the non-target strand of the CIITA gene. See also Table 8 herein for cDNA sequences of CIITA isoforms. As used herein, the term “Cas 12i polypeptide” (also referred to herein as Cas 12i) 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. Patent 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 (Cas12il), SEQ ID NO: 5 (Cas12i2), SEQ ID NO: 14 (Cas12i3), or SEQ ID NO: 16 (Cas12i4) of U.S. Patent No. 10,808,245, corresponding to SEQ ID NOs: 1161, 1104, 1162, and 1158 of the present application. In some embodiments, a Cas12i polypeptide of the disclosure is a Cas12il 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 CIITA-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 CIITA target sequence) to which a complex comprising an RNA guide (e.g., a CIITA-targeting RNA guide) and a Cas12i polypeptide binds. For instance, the PAM may be adjacent to the non-target strand of the double stranded target sequence. 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. For instance, the RNA guide may associate with the target strand of the double stranded target sequence. 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 CIITA gene). An RNA guide may be designed to include sequences that are complementary to a specific nucleic acid sequence (e.g., a CIITA 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. For instance, the spacer sequence may be complementary to the target strand of the double stranded 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. For instance, the spacer may bind the target strand of the double stranded 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 CIITA gene sequence, including, but not limited, to the sequence set forth at www.ncbi.nlm.nih.gov/gene/4261 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 the percent (%) of raw indels induced in fifteen CIITA target sequences (T1-T16) using Cas12i2. As detailed further in Example 1, HEK293T cells were transfected with a Cas12i2 plasmid, along with CIITA-targeting RNA guides, resulting in editing of CIITA targets in exon 1, exon 2, exon 3, and exon 4.

FIG. 2 shows the percent (%) of raw indels induced in several CIITA target sequences (Tl, T4, T5, T12, and T14) using a variant Cas12i2 ribonucleoprotein (RNP). As detailed further in Example 2, T cells were transfected with a Cas12i2 RNP targeting CIITA, resulting in editing of CIITA targets in exon 1, exon 2, and exon 3.

DETAILED DESCRIPTION

The present disclosure relates to an RNA guide capable of binding to CIITA 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 CIITA. 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 CIITA target sequence, wherein the CIITA 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 nontarget 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 CIITA. 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 CIITA target sequence. In some embodiments, a complex comprising an RNA guide targeting CIITA and a Cas12i polypeptide binds to a CIITA 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 CIITA target sequence. The RNA guide, the Cas12i polypeptide, and the CIITA target sequence, either alone or together, do not naturally occur. In some embodiments, the RNA guide in the complex comprises a direct repeat and/or a spacer sequence described herein. In some embodiments, the sequence of the RNA guide 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 any one of SEQ ID NOs: 1178-1192. In some embodiments, the RNA guide has a sequence of any one of SEQ ID NOs: 1178-1192.

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, FnCpfl is 1,300 amino acids in length, AsCpfl is 1,307 amino acids in length, and LbCpfl 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 CIITA.

RNA Guide

In some embodiments, the composition described herein comprises an RNA guide targeting a CIITA gene or a portion of CIITA 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 CIITA.

The RNA guide may direct the Cas12i polypeptide as described herein to a CIITA 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) CIITA 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 CIITA target-specific. That is, in some embodiments, an RNA guide binds specifically to one or more CIITA 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 direct repeat sequence followed by a spacer sequence, referring to the sequences in the 5’ to 3’ direction. 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 at least 23 nt in length.

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 CIITA target sequence.

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 CIITA target sequence. See, e.g., Example 1, where indels were measured at multiple CIITA target sequences following delivery of an RNA guide and a Cas12i2 polypeptide of SEQ ID NO: 1128 to HEK293T cells by RNP, and Example 2, where indels were measured in CIITA target sequences following delivery of an RNA guide and a Cas12i2 polypeptide of SEQ ID NO: 1128 to T cells 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

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: 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, or 1151. The direct repeat sequence can comprise nucleotide 2 through nucleotide 36 of any one of SEQ ID NOs: 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, or 1151. The direct repeat sequence can comprise nucleotide 3 through nucleotide 36 of any one of SEQ ID NOs: 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, or 1151. The direct repeat sequence can comprise nucleotide 4 through nucleotide 36 of any one of SEQ ID NOs: 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, or 1151. The direct repeat sequence can comprise nucleotide 5 through nucleotide 36 of any one of SEQ ID NOs: 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, or 1151. The direct repeat sequence can comprise nucleotide 6 through nucleotide 36 of any one of SEQ ID NOs: 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, or 1151. The direct repeat sequence can comprise nucleotide 7 through nucleotide 36 of any one of SEQ ID NOs: 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, or 1151. The direct repeat sequence can comprise nucleotide 8 through nucleotide 36 of any one of SEQ ID NOs: 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, or 1151. The direct repeat sequence can comprise nucleotide 9 through nucleotide 36 of any one of SEQ ID NOs: 1133, 1134, 1135, 1136, 1137, 1138,

1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, or 1151. The direct repeat sequence can comprise nucleotide 10 through nucleotide 36 of any one of SEQ ID NOs: 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, or 1151. The direct repeat sequence can comprise nucleotide 11 through nucleotide 36 of any one of SEQ ID NOs: 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, or 1151. The direct repeat sequence can comprise nucleotide 12 through nucleotide 36 of any one of SEQ ID NOs: 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, or 1151. The direct repeat sequence can comprise nucleotide 13 through nucleotide 36 of any one of SEQ ID NOs:

1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, or 1151. The direct repeat sequence can comprise nucleotide 14 through nucleotide 36 of any one of SEQ ID NOs: 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149,

1150, or 1151.

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: 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, or 1151. 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: 1133, 1134, 1135, 1136, 1137, 1138, 1139,

1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, or 1151. 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: 1133,

1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, or

1151. 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: 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, or 1151. 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: 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, or 1151. 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: 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, or 1151. 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: 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, or 1151. 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: 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, or 1151. 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: 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, or 1151. 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: 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145,

1146, 1147, 1148, 1149, 1150, or 1151. 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: 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, or 1151. 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: 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, or 1151. 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: 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, or 1151.

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: 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, or 1151. 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: 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, or 1151. 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: 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146,

1147, 1148, 1149, 1150, or 1151. 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: 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, or 1151. 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: 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, or 1151. 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: 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, or 1151. 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: 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, or 1151. 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: 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, or 1151. 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: 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, or 1151. 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: 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, or 1151. 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: 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, or 1151. 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: 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, or 1151. 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: 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, or 1151.

In some embodiments, the direct repeat sequence is at least 90% identical to the reverse complement of any one of SEQ ID NOs: 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, or 1151. In some embodiments, the direct repeat sequence is at least 95% identical to the reverse complement of any one of SEQ ID NOs: 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, or 1151. In some embodiments, the direct repeat sequence is the reverse complement of any one of SEQ ID NOs: 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, or 1151.

In some embodiments, the direct repeat sequence is at least 90% identical to any one of SEQ ID NOs: 1133-115 lor a portion of any one of SEQ ID NOs: 1133-1151. In some embodiments, the direct repeat sequence is at least 95% identical to any one of SEQ ID NOs: 1133-1151 or a portion of any one of SEQ ID NOs: 1133-1151. In some embodiments, the direct repeat sequence is 100% identical to any one of SEQ ID NOs: 1133-1151 or a portion of any one of SEQ ID NOs: 1133-1151.

Table 2. Cas12i4 direct repeat sequences

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: 1152-1154. In some embodiments, the direct repeat sequence is at least 95% identical to the reverse complement of any one of SEQ ID NOs: 1152-1154. In some embodiments, the direct repeat sequence is the reverse complement of any one of SEQ ID NOs: 1152-1154.

Table 3. Cas12il direct repeat sequences

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: 1155-1157. In some embodiments, the direct repeat sequence is at least 95% identical to the reverse complement of any one of SEQ ID NOs: 1155-1157. In some embodiments, the direct repeat sequence is the reverse complement of any one of SEQ ID NOs: 1155-1157.

Table 4. Cas12i3 direct repeat sequences

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 Sequence

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 5A or Table 5B, or a portion of a sequence of Table 5 A or Table 5B. The target sequences listed in Table 5 A and Table 5B are on the non-target strand of the CIITA sequence. It should be understood that an indication of SEQ ID NOs: 472- 932 or 1018-1102 should be considered as equivalent to a listing of all of the SEQ ID NOs in the ranges: SEQ ID NOs: 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, 770, 771, 772, 773, 774, 775,776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799,

800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822,

823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844,

845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866,

867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888,

889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910,

911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, or 932, or 1018, 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, or 1102.

The spacer sequence can comprise nucleotide 1 through nucleotide 16 of any one of SEQ ID NOs: 472- 932 or 1018-1102. The spacer sequence can comprise nucleotide 1 through nucleotide 17 of any one of SEQ ID NOs: 472-932 or 1018-1102. The spacer sequence can comprise nucleotide 1 through nucleotide 18 of any one of SEQ ID NOs: 472-932 or 1018-1102. The spacer sequence can comprise nucleotide 1 through nucleotide 19 of any one of SEQ ID NOs: 472-932 or 1018-1102. The spacer sequence can comprise nucleotide 1 through nucleotide 20 of any one of SEQ ID NOs: 472-932 or 1018-1102. The spacer sequence can comprise nucleotide 1 through nucleotide 21 of any one of SEQ ID NOs: 472-932. The spacer sequence can comprise nucleotide 1 through nucleotide 22 of any one of SEQ ID NOs: 472-932. The spacer sequence can comprise nucleotide 1 through nucleotide 23 of any one of SEQ ID NOs: 472-932. The spacer sequence can comprise nucleotide 1 through nucleotide 24 of any one of SEQ ID NOs: 472-932. The spacer sequence can comprise nucleotide 1 through nucleotide 25 of any one of SEQ ID NOs: 472-932. The spacer sequence can comprise nucleotide 1 through nucleotide 26 of any one of SEQ ID NOs: 472-932. The spacer sequence can comprise nucleotide 1 through nucleotide 27 of any one of SEQ ID NOs: 472-932. The spacer sequence can comprise nucleotide 1 through nucleotide 28 of any one of SEQ ID NOs: 472-932. The spacer sequence can comprise nucleotide 1 through nucleotide 29 of any one of SEQ ID NOs: 472-932. The spacer sequence can comprise nucleotide 1 through nucleotide 30 of any one of SEQ ID NOs: 472-932.

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 5A or Table 5B, or a portion of a sequence of Table 5 A or Table 5B. 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: 472-932 or 1018-1102. 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: 472- 932 or 1018-1102. 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: 472-932 or 1018-1102. 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: 472-932 or 1018-1102. 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: 472-932 or 1018-1102. 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: 472-932. 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: 472-932. 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: 472-932. 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: 472-932. 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: 472-932. 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: 472-932. 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: 472-932. 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: 472-932. 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: 472-932. 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: 472-932.

Tables 5A and 5B. Target and spacer sequences

Table 5A- Target and spacer sequences - CIITA exons

The invention includes all combinations of the direct repeats and spacers listed above, consistent with the disclosure herein.

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 A or Table 5B comprises a sequence comprising a thymine in one or more places indicated as uracil in Table 5A or Table 5B.

Exemplary RNA Guides

The invention includes RNA guides that comprise any and all combinations of the direct repeats and spacers described herein (e.g., as set forth in Tables 5A and 5B, above). In some embodiments, the sequence of an RNA guide 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 any one of SEQ ID NOs: 1178-1192. In some embodiments, an RNA guide has a sequence of any one of SEQ ID NOs: 1178-1192.

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 intemucleoside 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 intemucleoside 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 intemucleoside 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 intemucleoside 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 l% to 90%, from l% 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 intemucleoside linkages such as intemucleoside modifications, including modification or replacement of the phosphodiester linkages. Sequences having modified backbones include, among others, those that do not have a phosphoms atom in the backbone. For the purposes of this application, and as sometimes referenced in the art, modified RNAs that do not have a phosphoms atom in their intemucleoside backbone can also be considered to be oligonucleosides. In particular embodiments, a sequence will include ribonucleotides with a phosphoms atom in its intemucleoside 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 ofthese, 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 intemucleoside 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 intemucleoside 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 a-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-(l- thiophosphate)-adenosine, 5 ’ -O-( 1 -thiophosphate)-cytidine (a-thio-cytidine), 5 ’-O-( 1 -thiophosphate)- guanosine, 5’-O-(l-thiophosphate)-uridine, or 5’-O-(l-thiophosphate)-pseudouridine).

Other intemucleoside linkages that may be employed according to the present invention, including intemucleoside 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, l-(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-l-(tetrahydrofuran-2-yl)pyrimidine-2,4(lH,3H)- dione), troxacitabine, tezacitabine, 2 ’-deoxy-2’ -methylidenecytidine (DMDC), and 6-mercaptopurine. Additional examples include fludarabine phosphate, N4-behenoyl-l-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, l-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-l- 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-l -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, l-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 nucleotides (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 AD ARI 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: 1104 and/or encoded by SEQ ID NO: 1103). 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: 1103. 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: 1103. 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 Cas12i polypeptide is a Cas12i2 polypeptide, which comprises an amino acid sequence at least 95% identical to SEQ ID NO: 1104 and comprises one or more mutations relative to SEQ ID NO: 1104. In some embodiments, the one or more mutations in the Cas12i2 polypeptide are at positions D581, G624, F626, P868, 1926, V1030, E1035, and/or S1046 of SEQ ID NO: 1104. In some embodiments, the one or more mutations are amino acid substitutions, which optionally is D581R, G624R, F626R, P868T, I926R, V1030G, E1035R, S1046G, or a combination thereof.

In some embodiments, the Cas12i2 polypeptide comprises mutations at positions D581, D911, 1926, and V1030 (e.g., amino acid substitutions of D581R, D911R, I926R, and V1030G). In some embodiments, the Cas12i2 polypeptide comprises mutations at positions D581, 1926, and V1030 (e.g., amino acid substitutions of D581R, I926R, and V1030G). In some embodiments, the Cas12i2 polypeptide comprises mutations at positions D581, 1926, V1030, and S1046 (e.g., amino acid substitutions of D581R, I926R, V1030G, and S1046G). In some embodiments, the Cas12i2 polypeptide comprises mutations at positions D581, G624, F626, 1926, V1030, E1035, and S1046 (e.g., amino acid substitutions of D581R, G624R, F626R, I926R, V1030G, E1035R, and S1046G). In some embodiments, the Cas12i2 polypeptide comprises mutations at positions D581, G624, F626, P868, 1926, V1030, E1035, and S1046 (e.g., amino acid substitutions of D581R, G624R, F626R, P868T, I926R, V1030G, E1035R, and S1046G).

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: 1103.

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: 1104.

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: 1104. 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: 1104 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: 1127, SEQ ID NO: 1128, SEQ ID NO: 1129, SEQ ID NO: 1130, or SEQ ID NO: 1131.

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: 1127, SEQ ID NO: 1128, SEQ ID NO: 1129, SEQ ID NO: 1130, or SEQ ID NO: 1131. 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: 1127, SEQ ID NO: 1128, SEQ ID NO: 1129, SEQ ID NO: 1130, or SEQ ID NO: 1131 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 Cas12i2 polypeptide of the present invention comprises a polypeptide sequence having at least 95% identity to SEQ ID NO: 1127, SEQ ID NO: 1128, SEQ ID NO: 1129, SEQ ID NO: 1130, or SEQ ID NO: 1131. In some embodiments, a Cas12i2 polypeptide having at least 95% identity to SEQ ID NO: 1127, SEQ ID NO: 1128, SEQ ID NO: 1129, SEQ ID NO: 1130, or SEQ ID NO: 1131 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: 1127, SEQ ID NO: 1128, SEQ ID NO: 1129, SEQ ID NO: 1130, or SEQ ID NO: 1131. 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: 1127, SEQ ID NO: 1128, SEQ ID NO: 1129, SEQ ID NO: 1130, or SEQ ID NO: 1131 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: 1158 and/or encoded by SEQ ID NO: 1132). 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: 1132. 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: 1132. 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: 1132. 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: 1158.

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: 1158. 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: 1158 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: 1159 or SEQ ID NO: 1160.

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: 1159 or SEQ ID NO: 1160. 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: 1159 or SEQ ID NO: 1160 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: 1159 or SEQ ID NO: 1160. 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: 1159 or SEQ ID NO: 1160 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 Cas12il polypeptide described herein (e.g., a polypeptide comprising SEQ ID NO: 1161). In some embodiments, the Cas12il polypeptide comprises at least one RuvC domain.

In some embodiments, the Cas12il 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: 1161.

In some embodiments, the present invention describes a Cas12il 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: 1161. 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 Cas12il 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: 1161 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: 1162). In some embodiments, the Cas12i3 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: 1162.

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: 1162. 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: 1162 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.oqp/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

It is understood that, typically, the target sequence is double stranded, and the spacer sequence can bind to the target sequence by virtue of the spacer sequence being substantially complementary to and hybridizing with a first strand ofthe target sequence (i.e., the target strand orthe spacer-complementary strand). The target sequence can be conveniently described by providing the sequence of the target strand or the non-target strand. More generally, a nucleic acid can bind to a double-standed target sequence by hybridizing with either strand of the double-standed target sequence.

In some embodiments, the target sequence is within a CIITA gene or a locus of a CIITA gene. In some embodiments, the CIITA gene is a mammalian gene. In some embodiments, the CIITA gene is a human gene. For example, in some embodiments, the target sequence is within the sequence of www.ncbi.nlm.nih.gov/gene/4261 or the reverse complement thereof. In some embodiments, the target sequence is within an exon of the CIITA gene of www.ncbi.nlm.nih.gov/gene/4261 (or the reverse complement thereof), e.g., within a sequence of SEQ ID NO: 1105, 1106, 1107, 1108, 1109, 1110, 1111, 1112, 1113, 1114, 1115, 1116, 1117, 1118, 1119, 1120, 1121, 1122, 1123, 1124, 1125, or 1126 (or a reverse complement thereof). Target sequences within an exon of the CIITA gene of www.ncbi.nlm.nih.gov/gene/4261 (and the reverse complement thereof) are set forth in Table 5A. In some embodiments, the target sequence is within an intron of the CIITA gene ofwww.ncbi.nlm.nih.gov/gene/4261 (or the reverse complement thereof). Target sequences within an intron of the CIITA gene of www.ncbi.nlm.nih.gov/gene/4261 (or the reverse complement thereof) are set forth in Table 5B. In some embodiments, the target sequence is within a variant (e.g., a polymorphic variant) of the CIITA gene sequence of www.ncbi.nlm.nih.gov/gene/4261 or the reverse complement thereof. In some embodiments, the CIITA gene sequence is a homolog of the sequence of www.ncbi.nlm.nih.gov/gene/4261 or the reverse complement thereof. For examples, in some embodiments, the CIITA gene sequence is a non-human CIITA sequence. In some embodiments, the CIITA gene sequence is a coding sequence set forth in any one of SEQ ID NOs: 1193-1206. In some embodiments, the CIITA gene sequence is a homolog of a coding sequence set forth in any one of SEQ ID NOs: 1193-1206.

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 ’-TITA-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 CIITA 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, the cell has exactly two copies of the target sequence.

In some embodiments, a CIITA 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 CIITA coding sequence is selected. For example, in some embodiments, an RNA guide is designed to target a sequence in exon 1 (SEQ ID NO: 1105) or exon 2 (SEQ ID NO: 1106). 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 Cas 12i 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 CIITA 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 lOmins, 15mins, 20mins, 25mins, 30mins, 35mins, 40mins, 45mins, 50mins, 55mins, Ihr, 2hr, 3hr, 4hr, or more hours. In some embodiments, the RNA guide and Cas 12i 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, adeno- associated virus (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, a nucleic acid encoding the RNA guide may be located in a viral vector. In some embodiments, a nucleic acid encoding the Cas12i (e.g., Cas12i2) polypeptide may be located in a viral vector. In some embodiments, the viral vector comprises the both the nucleic acid encoding the Cas12i polypeptide and the nucleic acid encoding the RNA guide.

In some embodiments, any of the systems described herein may comprise a nucleic acid encoding the Cas12i polypeptide, which is located in a first vector, and a nucleic acid encoding the RNA guide, which is located on a second vector. In some embodiments, the first and/or second vector is a viral vector. In some specific examples, the first and second vectors are the same type of vector. In other examples, the first and second vectors are different types of vectors.

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 preformed RNA guide/Cas12i polypeptide complex to a cell, where a ternary complex is formed. In some embodiments, an RNA guide and an RNA encoding a Cas12i polypeptide are delivered together in a single composition. In some embodiments, an RNA guide and an RNA encoding a Cas12i polypeptide are delivered in separate compositions. In some embodiments, an RNA guide and an RNA encoding a Cas12i polypeptide delivered in separate compositions are delivered using the same delivery technology. In some embodiments, an RNA guide and an RNA encoding a Cas12i polypeptide delivered in separate compositions are delivered using different delivery technologies. Exemplary intracellular delivery methods, include, but are not limited to: viruses, such as AAV, or virus-like agents; chemical-based transfection methods, such as those using calcium phosphate, dendrimers, liposomes, lipid nanoparticles, 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, a lipid nanoparticle comprises an mRNA encoding a Cas12i polypeptide, an RNA guide, or an mRNA encoding a Cas12i polypeptide and an RNA guide. In some embodiments, the mRNA encoding the Cas12i polypeptide is a transcript of the nucleotide sequence set forth in SEQ ID NO: 1103 or SEQ ID NO: 1132 or a variant thereof. 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.

In some embodiments, any of the systems described herein may comprise one or more LNPs, wherein the LNP comprises the Cas12i polypeptide (e.g., Cas12i2 polypeptide) or the nucleic acid encoding the Cas12i polypeptide, the RNA guide or the nucleic acid encoding the RNA guide, or both. In some embodiments, the system described herein may comprise a LNP, wherein the LNP comprises the Cas12i polypeptide or the nucleic acid encoding the Cas12i polypeptide, and a viral vector comprising the nucleic acid encoding the RNA guide. In some embodiments, the viral vector is an AAV vector. In other embodiments, the system described herein may comprise a LNP, which comprises the RNA guide or the nucleic acid encoding the RNA guide, and a viral vector comprising the nucleic acid encoding the Cas12i polypeptide. In some embodiments, the viral vector is an AAV vector.

In some embodiments, the gene editing system disclosed herein may comprise a Cas12i polypeptide as disclosed herein. In some embodiments, the gene editing system may comprise a nucleic acid encoding the Cas12i polypeptide. For example, the gene editing system may comprise a vector (e.g., a viral vector such as an AAV vector, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO, AAV11 and AAV 12) encoding the Cas12i polypeptide. Alternatively, the gene editing system may comprise a mRNA molecule encoding the Cas12i polypeptide. In some instances, the mRNA molecule may be codon- optimized.

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 fortissue 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 AND THERAPEUTIC APPLICATIONS

The disclosure also provides methods of modifying a target sequence within the CIITA gene. In some embodiments, the methods comprise introducing a CIITA-targeting RNA guide and a Cas12i polypeptide into a cell. The CIITA-targeting RNA guide and Cas12i polypeptide can be introduced as a ribonucleoprotein complex into a cell. The CIITA-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 CIITA gene is of www.ncbi.nlm.nih.gov/gene/4261 (or the reverse complement thereof). In some embodiments, the target sequence is in an exon of a CIITA gene, such as an exon having a sequence set forth in any one of SEQ ID NOs: 1105, 1106, 1107, 1108, 1109, 1110, 1111, 1112, 1113, 1114, 1115, 1116, 1117, 1118, 1119, 1120, 1121, 1122, 1123, 1124, 1125, or 1126 (or the reverse complement thereof). In some embodiments, the target sequence is in an intron of a CIITA gene (e.g., an intron of the sequence of www.ncbi.nlm.nih.gov/gene/4261, or the reverse complement thereof). In other embodiments, the sequence of the CIITA gene is a variant of the sequence of www.ncbi.nlm.nih.gov/gene/4261 (or the reverse complement thereof) or a homolog of the sequence ofwww.ncbi.nlm.nih.gov/gene/4261 (or the reverse complement thereof). For example, in some embodiments, the target sequence is polymorphic variant of the CIITA sequence of www.ncbi.nlm.nih.gov/gene/4261 (or the reverse complement thereof) or a nonhuman form of the CIITA 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 CIITA gene. In some embodiments, the deletion alters function of the CIITA gene. In some embodiments, the deletion inactivates the CIITA gene. In some embodiments, the deletion is a frameshifting deletion. In some embodiments, the deletion is a nonframeshifting 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 ’-TITA-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 ’-TITA-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 ofthe 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 ’-TITA-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) ofthe 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 ’-TITA-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 ’-TITA-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 ofthe 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 ’-TITA-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 ’-TITA-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 ’-TITA-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 ’-TITA-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 ’-TITA-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 ’-TITA-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 ’-TITA-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 ’-TITA-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 ’-TITA-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 ’-TITA-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 ’-TITA-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 ’-TITA-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 ’-TITA-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 ’-TITA-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’ -TITA-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 ’-TITA-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 ’-TITA-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 CIITA gene. In some embodiments, the methods are carried out using a complex comprising a Cas12i enzyme as described herein and an RNA guide comprising a direct repeat and a spacer as described herein. In some embodiments, the sequence of the RNA guide 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 any one of SEQ ID NOs: 1178-1192. In some embodiments, an RNA guide has a sequence of any one of SEQ ID NOs: 1178-1192.

In some embodiments, the RNA guide targeting CIITA is encoded in a plasmid. In some embodiments, the RNA guide targeting CIITA is synthetic or purified RNA. In some embodiments, the Cas12i polypeptide is encoded in a plasmid. In some embodiments, the Cas12i polypeptide is encoded by an RNA that is synthetic or purified.

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 CIITA 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, modified cells produced using any of the gene editing systems disclosed herein may be administered to a subject (e.g., a human patient) in need of treatment. The modified cells may comprise a substitution, insertion, and/or deletion described herein. In some instances, the modified cells may be a heterogenous population comprising cells with different types of gene edits. In some instances, the modified cells may comprise a substantially homogenous cell population, e.g., at least 80% of the cells in the whole population comprising one particular gene edit in the CIITA gene. In some embodiments, the cells can be suspended in a suitable media.

In some aspects, provided herein is a composition comprising the gene editing system or components thereof. Such a composition can be a pharmaceutical composition. A pharmaceutical composition that is useful may be prepared, packaged, or sold in a formulation suitable for oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, intra-lesional, buccal, ophthalmic, intravenous, intra-organ or another route of administration. A pharmaceutical composition of the disclosure may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition (e.g., the gene editing system or components thereof), which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

In some embodiments, a pharmaceutical composition comprising the gene editing system or components thereof as described herein may be administered to a subject in need thereof. In some instances, the gene editing system or components thereof may be delivered to specific cells or tissue, where the gene editing system could function to genetically modify the CIITA gene in such cells.

A formulation of a pharmaceutical composition suitable for parenteral administration may comprise the active agent (e.g., the gene editing system or components thereof or the modified cells) combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such a formulation may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Some injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multidose containers containing a preservative. Some formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained- release or biodegradable formulations. Some formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents.

The pharmaceutical composition may be in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may comprise, in addition to the system or cells, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulation may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or saline. Other acceptable diluents and solvents include, but are not limited to, Ringer’s solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations which that are useful include those which may comprise the cells in a packaged form, in a liposomal preparation, or as a component of a biodegradable polymer system. Some compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

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. In some embodiments, the kit may be useful for research purposes. For example, in some embodiments, the kit may be useful to study gene function.

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

EXAMPLES

Example 1 - Targeting of Mammalian Genes in HEK293T Cells

This Example describes indel assessment on multiple CIITA target sequences using a purified variant Cas12i2 effector and RNA guides introduced into mammalian cells by electroporation. Cas 12i2 RNA guides (crRNAs) were designed and ordered from Integrated DNA Technologies (IDT).

Target sequences were selected by tiling the coding exons of CIITA for 5’-CTTC-3’, 5’-CTTT-3’, and 5’- GTTC-3 ’ PAM sequences, and spacer sequences were designed for the 20-bp target sequences downstream of the PAM sequence.

Table 6. crRNA and Target Sequences. In Table 6, the double stranded target sequence is described by providing the sequence of the non-target strand of the target sequence.

Cas12i2 RNP complexation reactions were made by mixing purified Cas12i2 polypeptide (400 pM) with crRNA (1 mM in 250 mM NaCl) at a 1: 1 (effector: crRNA) volume ratio (2.5: 1 crRNA: effector molar ratio). Complexations were incubated on ice for 30-60 min.

During incubation, HEK293T cells were harvested using TRYPLE™ (recombinant cell-dissociation enzymes; ThermoFisher) and counted. Cells were washed once with PBS and resuspended in SF buffer + supplement (SF CELL LINE 4D-NUCLEOFECTOR™ X KIT S; Lonza #V4XC-2032) at a concentration of 16,480 cells/pL. Resuspended cells were dispensed at 3e5 cells/reaction into Lonza 16-well NUCLEOCUVETTE® strips. Complexed Cas12i2 RNP was added to each reaction at a final concentration of 10 pM (Cas12i2), and transfection enhancer oligos were then added at a final concentration of 4 pM. The final volume of each electroporated reaction was 20 pL. Non-targeting guides were used as negative controls.

The strips were electroporated using an electroporation device (program CM- 130, Lonza 4D- NUCLEOFECTOR™). Immediately following electroporation, 80 pL of pre-warmed DMEM + 10% FBS was added to each well and mixed gently by pipetting. For each technical replicate plate, plated 10 pL (30,000 cells) of diluted nucleofected cells into pre-warmed 96-well plate with wells containing 100 pL DMEM + 10% FBS. Editing plates were incubated for 3 days at 37°C with 5% CO2.

After 3 days, wells were harvested using TRYPLE™ (recombinant cell-dissociation enzymes; ThermoFisher) and transferred to 96-well TWIN. TEC® PCR plates (Eppendorf). Media was flicked off and cells were resuspended in 20 pL QUICKEXTRACT™ (DNA extraction buffer; Lucigen). 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 (Illumina) and run on an NGS instrument (NEXTSEQ™ 550; Illumina). For NGS analysis, the indel mapping function used a sample’s fastq fde, 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 30 nt of each read was required to match the reference and reads where over half of the mapping nucleotides are 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 % indels 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.

As shown in FIG. 1, each of the fifteen tested RNA guides induced indels in CIITA target sequences. Therefore, RNA guides and the variant Cas12i2 of SEQ ID NO: 1128 were able to edit CIITA targets in exon 1, exon 2, exon 3, and exon 4 in HEK293T cells.

Example 2 - Editing of CIITA in T Cells

This Example describes indel assessment on multiple CIITA target sequences using a variant Cas12i2 ribonucleoprotein (RNP) introduced into mammalian cells by electroporation.

Frozen Human Peripheral Blood Mononuclear Cells (PBMCs) from an individual donor were revived and counted using an automated cell counter. CD3+ T cells were isolated using EASYSEP™ Human T Cell Isolation Kit (StemCell Technologies). Following isolation, a sample was collected and stained for CD3 and LIVE/DEAD stain (ThermoFisher) for flow cytometry analysis of surface expression and viability, respectively. Cell density was adjusted to le6 cells/mL and cells were stimulated for 3 days with a cocktail of anti-CD3:CD28 antibodies in IMMUNOCULT™-XF T Cell Expansion Medium (Stemcell Technologies) + IL-2 + L-Glutamine.

Cas12i2 RNP complexation reactions were made by mixing purified variant Cas12i2 (400 pM; SEQ ID NO: 1128) 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). For “effector only” controls, variant Cas12i2 was mixed with 250 mM NaCl at the same volume ratio as the RNA guide. Complexations were incubated on ice for 30-60 min.

Table 7. RNA guide sequences for RNP transfection

During incubation, T cells were removed from the flask and counted. Ie6 cells were stained with CD25 fluorescent antibody and LIVE/DEAD stain to assess activation efficiency and viability, respectively. For electroporation, cells were washed once with PBS and resuspended in P3 buffer + supplement (P3 PRIMARY CELL 4D-NUCLEOFECTOR™ X Kit S; Lonza #V4XP-3032) at a concentration of 10,873 cells/pL. Resuspended cells were dispensed at 2e5 cells/reaction into Lonza 16-well NUCLEOCUVETTE® strips. Complexed Cas12i2 RNP was added to each reaction at a final concentration of 10 pM (Cas12i2). The final volume of each electroporated reaction was 20 pL.

The strips were electroporated using an electroporation device (program EO-115, Lonza 4D- NUCLEOFECTOR™). Immediately following electroporation, 40 pL of pre-warmed IMMUNOCULT™-XF (Stemcell Technologies) + IL-2 + L-Glutamine was added to each well and mixed gently by pipetting. For each technical replicate plate, 15 pL (50,000 cells) of diluted nucleofected cells were plated into a pre-warmed 96- well plate with wells containing IMMUNOCULT™-XF (Stemcell Technologies) + IL-2 + L-Glutamine. Editing plates were incubated for 7 days at 37°C with 5% CO2 with media replacement at 3 days.

After 3 days (during media replacement) or 7 days, wells were transferred to 96-well TWIN. TEC® PCR plates (Eppendorf) and pelleted. Media was flicked off and cells were resuspended in 20 pL DNA extraction buffer (QUICKEXTRACT™; Lucigen). 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 NGS were prepared and analyzed as described in Example 1. As shown in FIG. 2, each of the tested RNA guides induced indels in CIITA target sequences in T cells. Therefore, RNA guides and the variant Cas12i2 of SEQ ID NO: 1128 delivered as RNPs were able to edit CIITA targets in exon 1, exon 2, and exon 3 in T cells.

Claims

WHAT IS CLAIMED IS:

1. A composition comprising an RNA guide, wherein the RNA guide comprises:

(i) a spacer sequence that specifically binds a target sequence within a CIITA gene, wherein the target sequence comprises any one of SEQ ID NOS: 1163, 1166, 1167, 1173, 1175, 1164, 1165, 1168-1172, 1174, 1176, or 1177; and

(ii) a direct repeat sequence.

2. The composition of claim 1, wherein the target sequence comprises any one of SEQ ID NOS: 1163, 1166, 1167, 1173, or 1175.

3. The composition of claim 1 or 2, wherein the spacer sequence is at least 90% identical to a sequence of any one of SEQ ID NOS: 1207-1221.

4. The composition of any of the preceding claims, wherein the spacer sequence is at least 90% identical to a sequence of any one of SEQ ID NOS: 1207, 1210, 1211, 1217, or 1219.

5. The composition of any of the preceding claims, wherein the spacer sequence has a length of between 15-30 nucleotides or between 20-30 nucleotides.

6. The composition of any of the preceding claims, wherein 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: 752, 815, 802, 821, and 824; 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: 752, 815, 802, 821, and 824; 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: 752, 815, 802, 821, and 824; 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: 752, 815, 802, 821, and 824; or 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: 752, 815, 802, 821, and 824.

7. The composition of any of the preceding claims, wherein the spacer sequence comprises: a. nucleotide 1 through nucleotide 16 of any one of SEQ ID NOs: 752, 815, 802, 821, and 824; b. nucleotide 1 through nucleotide 17 of any one of SEQ ID NOs: 752, 815, 802, 821, and 824; c. nucleotide 1 through nucleotide 18 of any one of SEQ ID NOs: 752, 815, 802, 821, and 824; d. nucleotide 1 through nucleotide 19 of any one of SEQ ID NOs: 752, 815, 802, 821, and 824; or e. nucleotide 1 through nucleotide 20 of any one of SEQ ID NOs: 752, 815, 802, 821, and 824. The composition of any of the preceding claims, wherein the direct repeat sequence has a length of between 15-40 or 23-36 nucleotides. The composition of any of the preceding claims, wherein the direct repeat sequence is a Cas12il direct repeat sequence, a Cas12i2 direct repeat sequence, a Cas12i3 direct repeat sequence, or a Cas12i4 direct repeat sequence. The composition of any of the preceding claims, wherein 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.

The composition of any of the preceding claims, wherein 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. The composition of any of the preceding claims, wherein the target sequence is adjacent to a protospacer adjacent motif (PAM) comprising the sequence 5’-NTTN-3’, wherein N is any nucleotide. The composition of claim 12, wherein the target sequence is immediately adjacent to the PAM sequence. The composition of claim 12, wherein the PAM comprises the sequence 5’-ATTA-3’, 5’-ATTT-3’, 5’- ATTG-3’, 5’-ATTC-3’, 5 ’-TITA-3’, 5’-TTTT-3’, 5’-TTTG-3’, 5’-TTTC-3’, 5’-GTTA-3’, 5' -GTTT- S'. 5’-GTTG-3’, 5’-GTTC-3’, 5’-CTTA-3’, 5’-CTTT-3’, 5’-CTTG-3’, or 5’-CTTC-3’. The composition of any of claims 12-14, wherein the PAM comprises the sequence 5’-CTTT-3’ and the target sequence comprises SEQ ID NO: 1163. The composition of any of claims 12-15, wherein the PAM comprises the sequence 5’-CTTT-3’ and the spacer sequence is at least 90% identical to a sequence of SEQ ID NO: 1207. The composition of any of claims 12-16, wherein the PAM comprises the sequence 5’-CTTC-3’ and the target sequence comprises any one of SEQ ID NOS: 1166, 1167, 1173, or 1175. The composition of any of claims 12-14 or 17, wherein the PAM comprises the sequence 5’-CTTC-3’ and the spacer sequence is at least 90% identical to a sequence of SEQ ID NO: 1210, 1211, 1217, or 1219. The composition of any of the preceding claims, wherein the RNA guide has a sequence that is at least 90% identical to a sequence of any one of SEQ ID NOs: 1178-1192. The composition of claim 19, wherein the RNA guide has the sequence of any one of SEQ ID NOs: 1178-1192. The composition of any of the preceding claims, wherein the composition further comprises a Cas12i polypeptide or a polyribonucleotide encoding a Cas12i polypeptide. The composition of claim 21, wherein the Cas12i polypeptide is a Cas12il polypeptide, a Cas12i2 polypeptide, a Cas12i3 polypeptide, or a Cas12i4 polypeptide. The composition of claim 21 or 22, wherein the Cas12i polypeptide is a Cas12i2 polypeptide comprising a sequence that is at least 90% identical to the sequence of SEQ ID NO: 1104, SEQ ID NO: 1127, SEQ ID NO: 1128, SEQ ID NO: 1129, SEQ ID NO: 1130, or SEQ ID NO: 1131. The composition of any of claims 21-23, wherein the Cas12i polypeptide comprises one or more mutations relative to SEQ ID NO: 1104. The composition of claim 24, wherein the one or more mutations in the Cas12i polypeptide are at positions D581, G624, F626, P868, 1926, V1030, E1035, and/or S1046 of SEQ ID NO: 1104. The composition of claim 24 or 25, wherein the one or more mutations are amino acid substitutions, wherein optionally the amino acid substitutions are chosen from D581R, G624R, F626R, P868T, I926R, V1030G, E1035R, S1046G, or a combination thereof. The composition of any of claims 24-26, wherein the Cas12i polypeptide comprises:

(i) mutations at positions D581 , D911 , 1926, and V 1030 of SEQ ID NO: 1104, which optionally are amino acid substitutions of D581R, D911R, I926R, and V1030G;

(ii) mutations at positions D581, 1926, and V1030 of SEQ ID NO: 1104, which optionally are amino acid substitutions of D581R, I926R, and V1030G;

(iii) mutations at positions D581, 1926, V1030, and S1046 of SEQ ID NO: 1104, which optionally are amino acid substitutions of D581R, I926R, V1030G, and S1046G;

(v) mutations at positions D581, G624, F626, P868, 1926, V1030, E1035, and S1046 of SEQ ID NO: 1104, which optionally are amino acid substitutions of D581R, G624R, F626R, P868T, I926R, V1030G, E1035R, and S1046G. The composition of any one of claims 21 to 27, wherein the RNA guide and the Cas12i polypeptide form a ribonucleoprotein complex. The composition of claim 28, wherein the ribonucleoprotein complex binds a target nucleic acid. The composition of any of claims 21-27, which comprises the polyribonucleotide encoding the Cas 12i polypeptide, wherein optionally the polyribonucleotide is a messenger RNA (mRNA). The composition of any of claims 21-30, wherein the composition is present within a cell. The composition of any one of claims 21 to 24, wherein the RNA guide and the Cas12i polypeptide are encoded in a vector, e.g., expression vector. The composition of claim 32, wherein 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. A nucleic acid encoding an RNA guide as defined in any of the preceding claims. A vector comprising the nucleic acid of claim 34. A system comprising: (i) the RNA guide as defined in any of claims 1-33, or a nucleic acid encoding the RNA guide, and (ii) a Cas12i polypeptide, or a nucleic acid encoding the Cas12i polypeptide. A vector system comprising one or more vectors encoding (i) the RNA guide as defined in any of claims 1 to 33 and (ii) a Cas12i polypeptide, optionally wherein the vector system comprises a first vector encoding the RNA guide and a second vector encoding the Cas12i polypeptide. A pharmaceutical composition comprising the composition or system of any of the preceding claims. A cell comprising the composition or an RNA guide as defined in any one of claims 1 to 33, the nucleic acid of claim 34, the vector of claim 35, the system of claim 36, or the vector system of claim 37. The cell of claim 39, wherein 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. A kit comprising the composition or the RNA guide of any one of claims 1 to 33, the nucleic acid of claim 34, the vector of claim 35, the system of claim 36, or the vector system of claim 37. A method of binding a Cas12i polypeptide and an RNA guide to a target sequence, the method comprising contacting the target sequence with the composition of any of claims 1-33, the vector of claim 35, the system of claim 36, or the vector system of claim 47. The method of claim 42, wherein the composition comprises the polyribonucleotide encoding the Cas12i polypeptide, and the contacting results in production of the Cas12i polypeptide in the cell. A method of editing a CIITA sequence, the method comprising contacting a CIITA sequence with a composition or an RNA guide of any one of claims 1 to 33. The method of claim 44, wherein the CIITA sequence is in a cell. The method of claim 45, wherein the cell is cultured in vitro. The method of claim 45 or 46, wherein the contacting step is performed by administering the system to a subject comprising the host cell. The method of any of claims 42-47, wherein the composition or the RNA guide induces a deletion in the CIITA sequence. The method of claim 48, wherein the deletion is adjacent to a 5’-NTTN-3’ sequence, wherein N is any nucleotide. The method of claim 48 or 49, wherein the deletion is downstream of the 5’-NTTN-3’ sequence. The method of any one of claims 48 to 50, wherein the deletion is up to about 50 nucleotides in length. The method of any one of claims 48 to 51, wherein the deletion is up to about 40 nucleotides in length. The method of any one of claims 48 to 52, wherein the deletion is from about 4 nucleotides to 40 nucleotides in length, about 4 nucleotides to 25 nucleotides in length, about 10 nucleotides to 25 nucleotides in length, or about 10 nucleotides to 15 nucleotides in length. The method of any one of claims 48 to 53, wherein the deletion starts within about 5 nucleotides to about 15 nucleotides of the 5’-NTTN-3’ sequence, about 5 nucleotides to about 10 nucleotides of the 5’-NTTN-3’ sequence, about 10 nucleotides to about 15 nucleotides of the 5’-NTTN-3’ sequence, about 5 nucleotides to about 15 nucleotides downstream of the 5’-NTTN-3’ sequence, about 5 nucleotides to about 10 nucleotides downstream of the 5’-NTTN-3’ sequence, or about 10 nucleotides to about 15 nucleotides downstream of the 5’-NTTN-3’ sequence. The method of any one of claims 48 to 54, wherein the deletion ends within about 20 nucleotides to about 30 nucleotides of the 5’-NTTN-3’ sequence, about 20 nucleotides to about 25 nucleotides of the 5’-NTTN-3’ sequence, about 25 nucleotides to about 30 nucleotides of the 5’-NTTN-3’ sequence, about 20 nucleotides to about 30 nucleotides downstream of the 5’-NTTN-3’ sequence, about 20 nucleotides to about 25 nucleotides downstream of the 5’-NTTN-3’ sequence, or about 25 nucleotides to about 30 nucleotides downstream of the 5’-NTTN-3’ sequence.

56. The method of any one of claims 48 to 55, wherein 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, 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, 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, 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, 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, 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, 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, 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 ofthe 5’-NTTN-3’ sequence, or starts within about 10 nucleotides to about 15 nucleotides downstream ofthe 5’-NTTN-3’ sequence and ends within about 25 nucleotides to about 30 nucleotides downstream of the 5’-NTTN-3’ sequence.

57. The method of any one of claims 48 to 56, wherein 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’.

58. The method of any one of claims 48 to 57, wherein the deletion disrupts one or both alleles of the CIITA sequence.

59. A method of treating a disease or condition, in a subject, the method comprising administering the composition or the RNA guide of any one of claims 1 to 33, or the cell of claim 39 or 30 to the subject.

60. 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 CIITA 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’. An RNA guide comprising (i) a spacer sequence that is substantially complementary to a target sequence within a CIITA gene and (ii) a direct repeat sequence.