Promoters and TRNA constructs

Abstract

Nucleic acids and compositions for improved gene editing are described, comprising hybrid promoters comprising at least one Pol III promoter and at least one tRNA promoter.

Description

PROMOTERS AND TRNA CONSTRUCTSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to United States Provisional Application No. 63 / 613,643, filed December 21, 2023, which is incorporated by reference herein in its entirety for all purposes.SEQUENCE LISTING

[0002] The present application contains a sequence listing that has been submitted electronically in XML format. Said XML copy, created on December 12, 2024, is named “01245-0044-60US.xml” and is 2,704,544 bytes in size. The information m the electronic format of the sequence listing is incorporated herein by reference in its entiretyINTRODUCTION AND SUMMARY

[0003] Genome engineering refers to the strategies and techniques for the targeted, specific modification of the genetic information (genome) of living organisms. Genome engineering is a very active field of research because of the wide range of possible applications, particularly in the areas of human health. For example, genome engineering can be used to alter (e.g., correct or knock-out) a gene carrying a harmful mutation, or to explore the function of a gene. Early technologies developed to knock-out and / or insert a transgene into a living cell were often limited by the random nature of the knock-out or insertion of the new sequence into the genome. Random insertions may result m disrupting normal regulation of neighboring genes leading to severe unwanted effects. Furthermore, technologies that result in random integration offer little reproducibility, as there is no guarantee that the sequence would be inserted at the same place in two different cells.

[0004] CRISPR-based genome editing can provide sequence-specific cleavage of genomic DNA using an endonuclease, such as Cas9, and a guide RNA, such as sgRNA. For example, a nucleic acid encoding the Cas9 enzyme and a nucleic acid encoding an appropriate guide RNA (e.g., sgRNA) can be provided on separate vectors or together on a single vector (if appropriately sized) and administered in vivo or in vitro to knockout or correct (e.g., by altering an aberrant reading frame or when a transgene is also provided), a genetic mutation, for example. The approximately 20 nucleotides at the 5' end of the guide RNA serves as the guide or spacer sequence that can be any sequence complementary to one strand of a genomic target location that has an adjacent protospacer adjacent motif (PAM). The PAM sequence is a short sequence adjacent to the endonuclease cut site and is required for appropriate editing. The nucleotides 3’ of the guide or spacer sequence of the guide RNA serve as a scaffold sequence for interacting with the endonuclease. When a guide RNA and an appropriate endonuclease are expressed, the guide RNA will bind to the endonuclease and direct it to the sequence complementary to the guide sequence, where it will then initiate a double- or singlestranded break (DSB). To repair these breaks, cells typically use an error prone mechanism of non-homologous end joining (NHEJ) which can lead to disruption of function in the target gene through insertions or deletion of codons, shifts in the reading frame, or result in a premature stop codon triggering nonsense-mediated decay. See, e.g., Kumar et ai. (2018) Front. Mol. Neurosci. Vol. 11, Article 413. Moreover, when a transgene (e.g., a heterologous replacement genome section) is provided with the guide RNA / endonuclease, the transgene may be inserted into the cut site to replace a genetic segment, sometimes by a process called homologous recombination.

[0005] The ability to efficiently induce multiple genome edits is a desirable outcome in the field of gene editing. However, current genome editing systems attempting to induce multiple independent gene edits have the challenge of frequently needing multiple transfer vehicles to be administered to accommodate multiple guide RM As and / or endonucleases and vector size limitations. A system that could utilize a single type of endonuclease and a single or reduced number of transfer vehicles would greatly facilitate commercialization of genome editing systems.

[0006] A number of diseases and disorders can benefit from genome editing. Moreover, a number of diseases and disorders can benefit from gene editing that involves making an excision (e.g., removing a segment) in tire genome. For example, repetitive DNA sequences, including trinucleotide repeats and other sequences with self-complementarity, tend to show marked genetic instability' and are recognized as a major cause of neurological and neuromuscular diseases. In particular, trinucleotide repeats (TNRs) in or near various genes are associated with a number of neurological and neuromuscular conditions, including degenerative conditions such as myotonic dystrophy type 1 (DM1), Huntington’s disease, and various types of spinocerebellar ataxia.

[0007] Muscular dystrophies (MD) are a group of more than 30 genetic diseases characterized byprogressive weakness and degeneration of the skeletal muscles that control movement. Duchenne muscular dystrophy (DMD) is one of the most severe forms of MD that affects approximately 1 in 5000 boys and is characterized byrprogressive muscle weakness and premature death. Cardiomyopathy and heart failure are common, incurable, and lethal features of DMD. The disease is caused by mutations in the gene encoding dystrophin (DMD), which result in loss of expression of dystrophin, causing muscle membrane fragility and progressive muscle wasting. Myotonic Dystrophy Ty pe 1 (DM1) is an autosomal dominant muscle disorder caused by the expansion of CTG repeats in the 3’ untranslated region (UTR) of human DMPK gene, w'hich leads to RNA foci and mis-splicmg of genes important for muscle function. Tire disorder affects skeletal and smooth muscle as well as the eye, heart, endocrine system, and central nervous system, and causes muscle weakness, wasting, physical disablement, and shortened lifespan.

[0008] While gene editing strategies using systems (e.g., CRISPR) for treating various diseases and disorders have been previously explored, these strategies have yet to yield a commercially successful strategy, and current multiplex systems utilize multiple guides and endonucleases and require more than one transfer vehicle. Thus, there remains a need for additional, alternative, and effective gene editing strategies, including for multiplexing, for treating diseases and disorders such asdiseases that would benefit from excising portions of genomes, such as DMD and DM1 , Specifically, there remains a need to develop an AAV platform that delivers high-fidelity and balanced sgRNA and dual sgRNA expression.

[0009] Provided herein are novel nucleic acids and compositions comprising engineered promoters, such as hybrid promoters, to deliver sgRNAs expression. Specifically, provided herein are novel nucleic acids comprising hybrid promoters comprising at least one RNA polymerase III (Pol III) promoter and at least one tRNA promoter, and optionally one or more leader sequences and / or trailer sequences (collectively, “hybrid promoters”). Such promoters may drive expression of one or more sgRNAs as disclosed herein.

[0010] Accordingly, the following non-limiting embodiments are provided.

[0011] Embodiment 1 is a nucleic acid comprising a sequence encoding a hybrid promoter, wherein the hybrid promoter comprises (a) a first portion comprising a mini-U6 pol III promoter or Ml 1 pol III promoter, and (b) a second portion comprising a tRNA promoter.

[0012] Embodiment 2 is the nucleic acid of embodiment 1, wherein the nucleic acid is in 5’ to 3’ order, and wherein the sequence encoding the mim-U6 pol III promoter or Ml 1 pol III promoter is 5’ to the tRNA promoter.

[0013] Embodiment 3 is the nucleic acid of embodiment 1 , wherein the nucleic acid is in 3’ to 5’ order, and wherein the sequence encoding the mini-U6 pol III promoter or M l 1 pol III promoter is 3’ to the tRNA promoter.

[0014] Embodiment 4 is the nucleic acid of any one of embodiments 1-3, wherein the mini -U 6 pol III promoter or Ml 1 pol III promoter is a mini-U6 pol III promoter.

[0015] Embodiment 5 is the nucleic acid of embodiment 4, comprising a sequence with about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 9005.

[0016] Embodiment 6 is the nucleic acid of embodiment 4, comprising the sequence of SEQ ID NO: 9005.

[0017] Embodiment 7 is the nucleic acid of any one of embodiments 1-3, wherein the mini-U6 pol III promoter or Mi l pol III promoter is an Ml 1 pol III promoter.

[0018] Embodiment 8 is the nucleic acid of embodiment 7, comprising a sequence with about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 708.

[0019] Embodiment 9 is the nucleic acid of embodiment 7, comprising the sequence of SEQ ID NO: 708.

[0020] Embodiment 10 is the nucleic acid of any one of embodiments 1-9, wherein the tRN A promoter is modified.

[0021] Embodiment 1 1 is the nucleic acid of embodiment 10, wherein the tRNA promoter has been modified so that the tRNA is not functional.

[0022] Embodiment 12 is the nucleic acid of any one of embodiments 1-6 and 10-11, wherein the mmi-U6 pol III promoter is less than about 74 bp in length or greater than about 1 17 bp in length andcomprises, in 5' to 3' order, a distal sequence element (DSE), a proximal sequence element (PSE), and a TATA box.

[0023] Embodiment 13 is the nucleic acid of embodiment 12, wherein the nnni-U6 pol 111 promoter is between about 40 and about 73 bp, between about 50 and about 73 bp, between about 60 and about 73 bp, or between about 70 and about 73 bp in length.

[0024] Embodiment 14 is the nucleic acid of embodiment 12, wherein the mini-U6 pol Ill promoter is greater than about 117 bp, greater than about 120 bp, greater than about 130 bp, greater than about 140 bp, greater than about 150 bp, or greater than about 200 bp in length.

[0025] Embodiment 15 is the nucleic acid of any one of embodiments 1-14, wherein the tRNA comprises at least one of a T-loop, a D-loop, a V-loop, an anticodon loop, a T-stern, a D-stem, tin anticodon stem, and an accepter stem.

[0026] Embodiment 16 is a nucleic acid encoding a tRNA, w herein the tRNA is modified so that it does not produce a functional tRNA.

[0027] Embodiment 17 is the nucleic acid of embodiment 16, wherein the tRNA 1) is modified such that it cannot bind an amino acid; 2) is modified in the D-stem region;, and optionally is modified at position 9, 25, or 26 of the D-stem corresponding to the sequence of SEQ ID NO: 9022 or 9023; 3) is modified at one or more of G9A or G25A corresponding to the sequence of SEQ ID NO: 9022, and / or is modified at one or more of U25C or C26G corresponding to the sequence of SEQ ID NO: 9023; 4) is modified in the anticodon loop; 5) is modified at one or more of positions 33, 34, 35, 36, or 37 of the anticodon loop corresponding to the sequence of any one of SEQ ID NOs: 9019-9027;6) is modified in the anticodon loop and the modification comprises one or more of A33C, C33U, A34U, G34U, G34C, C34U, A35U, C35A, U35C, G35A, U35A, C36A, or U37A corresponding to the sequence of any one of SEQ ID NOs: 9019-9027; 7) is modified in the acceptor stem; 8) is modified at position 70 of the acceptor stem corresponding to the sequence of SEQ ID NO: 9019 and / or is modified at position 73 of the acceptor stem corresponding to the sequence of SEQ ID NO: 9023; and / or 9) is modified at one or more of U70C corresponding to the sequence of SEQ ID NO: 9019 or G73C corresponding to the sequence of SEQ ID NO: 9023.

[0028] Embodiment 18 is the nucleic acid of any one of embodiments 14-17, wherein the tRNA is tRNAGln, tRNAGlu, tRNAGly, tRNApro, tRNAAsp, or tRNAAspMut.

[0029] Embodiment 19 is the nucleic acid of any one of the preceding embodiments, wherein the tRNA is tRNAAspMur.

[0030] Embodiment 20 is the nucleic acid of any one of the preceding embodiments, further comprising a leader sequence, and / or a trailer sequence.

[0031] Embodiment 21 is the nucleic acid sequence of embodiment 20, wherein the leader sequence comprises the sequence of any one of SEQ ID NOs: 8050-8075.

[0032] Embodiment 22 is the nucleic acid sequence of embodiment 20, wherein the leader sequence comprises the sequence of any one of SEQ ID NOs: 8050-8060.

[0033] Embodiment 23 is the nucleic acid sequence of embodiment 20, wherein the leader sequence comprises the sequence of any one of SEQ ID NOs: 8051 or 8053-8060.

[0034] Embodiment 24 is a nucleic acid comprising a sequence encoding a hybrid promoter, wherein the hybrid promoter comprises (a) a first portion comprising a mini-U6 pol III promoter, and (b) a second portion comprising a nucleic acid encoding tRNAAspMu:(SEQ ID NO: 8006).

[0035] Embodiment 25 is the nucleic acid of embodiment 24, wherein the nucleic acid is in 5’ to 3’ order, and wherein tire mini-U6 Pol III promoter is 5’ to the tRNAAsp!v!u!(SEQ ID NO: 8006).

[0036] Embodiment 26 is the nucleic acid of embodiment 24, wherein the nucleic acid is in 3’ to 5’ order, and wherein the mini-U6 Pol III promoter is 3’ to the tRNAAspMu:(SEQ ID NO: 8006).

[0037] Embodiment 27 is the nucleic acid of any one of embodiments 24-26, further comprising a leader sequence, and / or a trailer sequence.

[0038] Embodiment 28 is the nucleic acid sequence of embodiment 27, wherein the leader sequence comprises the sequence of any one of SEQ ID NOs: 8050-8075.

[0039] Embodiment 29 is the nucleic acid sequence of embodiment 27, wherein the leader sequence comprises the sequence of any one of SEQ ID NOs: 8050-8060.

[0040] Embodiment 30 is the nucleic acid sequence of embodiment 27, wherein the leader sequence comprises the sequence of any one of SEQ ID NOs: 8051 or 8053-8060.

[0041] Embodiment 31 is the nucleic acid of embodiment 27, wherein the leader sequence has the sequence of any one of GTGGC (SEQ ID NO: 8050), GTA (SEQ ID NO: 8051), or GGA (SEQ ID NO: 8052).

[0042] Embodiment 32 is the nucleic acid of embodiment 31, wherein the leader sequence is GTGGC (SEQ ID NO: 8050).

[0043] Embodiment 33 is the nucleic acid of embodiment 31, wherein the leader sequence is GTA (SEQ ID NO: 8051).

[0044] Embodiment 34 is the nucleic acid of embodiment 31, wherein the leader sequence is GGA (SEQ ID NO: 8052).

[0045] Embodiment 35 is the nucleic acid of any one of the preceding embodiments, further comprising one or more sgRNAs.

[0046] Embodiment 36 is the nucleic acid of embodiment 35, wherein the one or more sgRNAs targets exon 53.

[0047] Embodiment 37 is the nucleic acid of embodiment 35, wherein the one or more sgRNAs is E53SL320nt(SEQ ID NO: 1080), E53SL7 (SEQ ID NO: 1081), E53SL14 (SEQ ID NO: 1082). E53SL16 (SEQ ID NO: 1083), E53SL23 (SEQ ID NO: 1084), E53SL25 (SEQ ID NO: 1085), or E53SL3 (SEQ ID NO: 1086).

[0048] Embodiment 38 is the nucleic acid of embodiment 35, wherein the one or more sgRNAs targets exon 51.

[0049] Embodiment 39 is the nucleic acid of embodiment 35, wherein the one or more sgRNAs targets exon 2, 3, 6, 9, 44, 45, 47, 48, 50, 51 or 53.

[0050] Embodiment 40 is the nucleic acid of any one of the preceding embodiments, further comprising a trailer sequence,

[0051] Embodiment 41 is the nucleic acid of embodiment 40, wherein the trailer sequence is an adenine (a).

[0052] Embodiment 42 is a nucleic acid comprising a sequence encoding a minimized 7SK promoter comprising SEQ ID NO: 901 1 or 9016.

[0053] Embodiment 43 is a nucleic acid comprising a sequence encoding a minimized U6 promoter comprising SEQ ID NO: 9010 or 9015.

[0054] Embodiment 44 is a nucleic acid comprising a sequence encoding a hy brid promoter, wherein the hybrid promoter comprises (a) a first portion comprising a minimized 7SK promoter and / or a minimized U6 promoter and (b) a second portion comprising a tRNA promoter.

[0055] Embodiment 45 is the nucleic acid of embodiment 44, wherein the first portion comprises a sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 9011 or 9016,

[0056] Embodiment 46 is the nucleic acid of embodiment 44, wherein the first portion comprises the sequence of SEQ ID NO: 9011 or 9016.

[0057] Embodiment 47 is the nucleic acid of embodiment 44, wherein the second portion comprises a sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 9010 or 9015.

[0058] Embodiment 48 is the nucleic acid of embodiment 44, wherein the second portion comprises the sequence of SEQ ID NO: 9010 or 9015.

[0059] Embodiment 49 is the nucleic acid of embodiment 44, comprising a sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 9017 or 9018.

[0060] Embodiment 50 is the nucleic acid of embodiment 44, comprising the sequence of SEQ ID NO: 9017 or 9018.

[0061] Embodiment 51 is the nucleic acid of any one of embodiments 44-50, wherein the tRNA promoter is modified.

[0062] Embodiment 52 is the nucleic acid of embodiment 51, wherein the tRNA promoter is a part of a tRNA that has been modified so that the tRN A is not functional.

[0063] Embodiment 53 is the nucleic acid of any one of embodiments 44-52, wherein the tRNA comprises at least one of a T-loop, a D-loop, a V-loop, an anticodon loop, a T-stem, a D-stem, an anticodon stem, and an accepter stem.

[0064] Embodiment 54 is the nucleic acid of any one of embodiments 44-53, wherein the nucleic acid is in 5’ to 3’ order, and wherein the first portion is 5’ to the second portion.

[0065] Embodiment 55 is the nucleic acid of embodiment 1 , wherein the nucleic acid is in 3 to 5’ order, and wherein the first portion is 3’ to the second portion.

[0066] Embodiment 56 is the nucleic acid of embodiment 52, wherein the tRNA 1) is modified such that it cannot bind an amino acid; 2) is modified in the D-stem region;, and optionally is modified at position 9, 25, or 26 of the D-stem corresponding to the sequence of SEQ ID NO: 9022 or 9023; 3) is modified at one or more of G9A or G25A corresponding to the sequence of SEQ ID NO: 9022, and / or is modified at one or more of U25C or C26G corresponding to the sequence of SEQ ID NO: 9023; 4) is modified in the anticodon loop; 5) is modified at one or more of positions 33, 34, 35, 36, or 37 of the anticodon loop corresponding to the sequence of any one of SEQ ID NOs: 9019-9027; 6) is modified in the anticodon loop and the modification comprises one or more of A33C, C33U, A34U, G34U, G34C, C34U, A35U, C35A, U35C, G35A, U35A, C36A, or U37A corresponding to the sequence of any one of SEQ ID NOs: 9019-9027; 7) is modified in the acceptor stem; 8) is modified at position 70 of the acceptor stem corresponding to the sequence of SEQ ID NO: 9019 and / or is modified at position 73 of the acceptor stem corresponding to the sequence of SEQ ID NO: 9023; and / or 9) is modified at one or more of U70C corresponding to the sequence of SEQ ID NO: 9019 or G73C corresponding to the sequence of SEQ ID NO: 9023.

[0067] Embodiment 57 is the nucleic acid of embodiment 52 or 56, wherein the tRNA is tRNA®”, tRNA®”, tRNA®5', tRNApro, tRNAAsp, or tRNAAspMut.

[0068] Embodiment 58 is the nucleic acid of any one of embodiments 44-56, wherein the tRNA is tRN AAspMut.

[0069] Embodiment 59 is a composition comprising the nucleic acid of any one of the preceding embodiments and optionally further comprising an endonuclease or a nucleic acid encoding an endonuclease.

[0070] Embodiment 60 is the composition of embodiment 59, wherein the nucleic acid of any one of the preceding embodiments and / or the nucleic acid encoding the endonuclease, if presen t, are associated with a viral vector.

[0071] Embodiment 61 is the composition of embodiment 59, wherein the nucleic acid of any one of the preceding embodiments and / or the nucleic acid encoding the endonuclease, if present, are associated with a. viral vector, and wherein the viral vector is an adeno-associated virus vector (AAV), a lenti viral vector, an integrase-deficient lentiviral vector, an adenoviral vector, a vaccinia viral vector, an alphaviral vector, or a herpes simplex viral vector.

[0072] Embodiment 62 is the composition of embodiment 61, wherein the viral vector is an AAV vector.

[0073] Embodiment 63 is the composition of embodiment 62, wherein the AAV vector is anAAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 vector, wherein the number following AAV indicates the AAV serotype.

[0074] Embodiment 64 is the composition of embodiment 63. wherein the AAV vector is anAAV9 vector.

[0075] Embodiment 65 is a vector encoding the nucleic acid of any one of embodiments 1-58.DESCRIPTION OF FIGURES

[0076] Figure 1 show's a simplified schematic of an experimental workflow. HEK293T cells were cotransfected with two plasmids. GFP positive cells were collected by FACS into two parts. One part is used to extract RNA, which is used for sgRNA expression and tRNA cleavage efficiency measurements. The other part is used to extract DNA, which is used for genome editing efficiency measurements.

[0077] Figures 2A-2C show' sgRNA expression (Figure 2A), tRNA cleavage efficiency (Figure 2B), and genome editing efficiency (Figure 2C) of combinations of one of four different type III promoters (Ml 1, miniU6, U6, and 7SK) and one of four different type II promoters (tRNAGln(“Q”), tRNAG,“ (“E’ ’), tRNAGly(“G”), and tRNA‘>ro(“P”)). Hybrid promoters with a miniU6 promoter showed the highest genome editing efficiencies among the hybrid promoters with the same type II promoters.

[0078] Figures 3A-3C show sgRNA expression (Figure 3A), tRN A cleavage efficiency (Figure 3B), and genome editing efficiency (Figure 3C) of 23 different hybrid promoters comprising a miniU6 promoter. Hybrid promoters #28 and #31 showed the highest genome editing efficiency. See Table 14 for further descriptions of the promoters assessed in the experiments of Figures 3A-3C.

[0079] Figure 4 shows the mutations and locations of the mutations of screened hybrid promoters comprising tRNA mutants. The tRNA mutations tested are shown, along with the corresponding promoters comprising the mutations. All listed mutants included an anticodon sequence mutated to bind a stop codon.

[0080] Figures 5A-5C show sgRNA expression (Figure 5A), tRNA cleavage efficiency (Figure 5B), and genome editing efficiency (Figure 5C) of 10 different hybrid promoters with mutations incorporated in the tRNA sequences to potentially inhibit tRNA translational function, function. Three hybrid promoters with mutations incorporated showed better genome editing efficiency than a miniU6-A construct (the miniU6-A construct is construct #31 in Table 14).

[0081] Figures 6A-6B show' a simplified schematic of the method by which tRN A translational activity was measured using a reporter assay to generate the data in, e.g., Figure 7. Figure 6A shows a simplified schematic of the suppressor tRNA reporter assay, including a comparison against a standard (“normal’’) translation event. The reporter has a GFP downstream of the stop codon and can be used to measure the translational activity of the tRNA comprising an anticodon loop against the stop codon. Figure 6B shows a simplified schematic of the workflow' for mutant activity measurement. Hie reporter plasmid was co-transfected w'ith plasmids comprising the hybrid promoter and flow cytometry' was used to measure gene expression.

[0082] Figure 7 shows the suppressor activity for various promoters. Tire results demonstrate that 4 of the 10 tested mutant tRNAs do not have translational activity. The positive control (tRNAAlawith the anticodon modified to bind a TAG stop codon) showed evidence of stop codon readthrough, while the negative control (U6 promoter), which did not contain any tRNA, showed no readthrough,

[0083] Figures 8A-8C show leader sequence optimization results, specifically in sgRNA expression (Figure 8A), tRNA cleavage efficiency (Figure 8B), and editing efficiency (Figure 8C) for the miniU6-Dm hybrid promoter with 11 different leader sequences. The ‘gtggc’ leader sequence showed the highest genome editing efficiency, followed by ‘gta’ and ‘gga’. The ‘gta’ leader sequence showed the highest tRNA cleavage efficiency.

[0084] Figures 9A-9B show' information related to the protospacer dependency analyses depicted in Figures 10 and 11. Figure 9A shows exemplary protospacer sequences. Figure 9B shows six different protospacer constructs tested in the following experiment with five different promoters. One is a U6 promoter and the others are hybrid promoters with one of two different leader sequences ("gtggc’ or ‘gta’). Two of the hybrid promoters comprise an additional "g’ nucleotide between the tRNA and sgRNA.

[0085] Figures 10A-10C show protospacer dependency analysis results, specifically in sgRNA expression (Figure 10A), tRNA cleavage efficiency (Figure 10B), and editing efficiency (Figure 10C) for 30 different testing conditions. In most cases, genome editing efficiency was higher with the hybrid promoters than the U6 promoter. Generally, hybrid promoters comprising the ‘gtggc’ leader sequence showed higher sgRNA expression, and those comprising ‘gta’ showed higher tRNA cleavage efficiency. Moreover, having ’g’ in front of the protospacer had a negative effect on hybrid promoter performance with the exception of guide E53SL320nt(“53.3(20nt)”)> 53.3(20nt), E53.7, E53.14, E53.16, E53.23, and E53.25 correspond to E53SL320nt, E53SL7, E53SL14, E53SL16, E53SL23, and E53SL25, respectively, from Figure 9A.

[0086] Figures 11A-1 ID show' protospacer dependency analysis results, specifically in sgRNA expression (Figure 11 A), tRNA cleavage efficiency (Figure 11B), and editing efficiency (Figure 11C). Each data point represents a different protospacer. For sgRNA expression and genome editing efficiency, the data are expressed as fold change over the U6 promoter. In most cases, genome editing efficiency was higher with the hybrid promoters than the U6 promoter. The hybrid promoter (miniU6- gta-tRNAAspMu:-g) comprising the ‘gta’ leader sequence showed 3.72-fold higher sgRNA expression, 82.9% tRNA cleavage efficiency, and 1.49-fokl higher genome editing efficiency averaged across the six different protospacers, as shown in Figure 11D.

[0087] Figures 12A-12C show the sgRNA expression (Figure 12A), tRNA cleavage efficiency (Figure 12B), and editing efficiency (Figure 12C) for 4 different hybrid promoters including U6, mini-U6-Dm (mini-U6-tRNAAspMut), miniU6.7SK-Dm (minimized U6 / 7SK chimeric promoter- tRNA"spW) and mim7SK.U6-Dm (minimized L6 / 7SK chimeric promoter~tIFNAAspMllt). These figuresshow' that alternate minimized Type III promoters based on chimeric promoter of 7SK and U6 show' comparable activity when used as hybrid promoter.DETAILED DESCRIPTION

[0088] Reference will now be made in detail to certain embodiments of the invention, examples of which are illustrated in the accompanying drawings. While the invention is described in conjunction with the illustrated embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the invention as defined by the appended claims and included embodiments.

[0089] Before describing the present teachings in detail, it is to be understood that the disclosure is not limited to specific compositions or process steps, as such may vary. It should be noted that, as used in this specification and the appended claims, the singular form "‘a”, “an” and “the” include plural references unless the context clearly dictates otherwise. Titus, for example, reference to “a guide” includes a plurality of guides and reference to “a cell” includes a plurality of cells and the like.

[0090] Numeric ranges are inclusive of the numbers defining the range. Measured and measurable values are understood to be approximate, taking into account significant digits and the error associated with the measurement. Also, the use of “comprise”, “comprises”, “comprising”, “’contain”, “contains”, “containing”, “’include”, “includes”, and “including” are not intended to be limiting. It is to be understood that both the foregoing general description and detailed description are exemplary and explanatory' only and are not. restrictive of the teachings.

[0091] Unless specifically noted in the specification, embodiments in the specification that recite “comprising” various components are also contemplated as “consisting of’ or “’consisting essentially of” the recited components; embodiments in the specification that recite “consisting of’ various components are also contemplated as “comprising” or “consisting essentially of’ the recited components; and embodiments in the specification that recite “consisting essentially of’ various components are also contemplated as “consisting of’ or “comprising” the recited components (this interchangeability does not apply to the use of these terms in the claims). The term “or” is used in an inclusive sense, i.e., equivalent to “and / or,” unless the context clearly indicates otherwise.

[0092] The section headings used herein are for organizational purposes only and are not to be construed as limiting the desired subject matter in any v.av. In the event that any material incorporated by reference contradicts any term defined in this specification or any other express content of this specification, this specification controls. While the present teachings are described in conjunction w'ith various embodiments, it is not. intended that the present teachings be limited to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art.I. Definitions

[0093] Unless stated otherwise, the following terms and phrases as used herein are intended to have the foilowing meanings:

[0094] “Polynucleotide,” “nucleic acid,” and “nucleic acid molecule,” are used herein to refer to a multimeric compound comprising nucleosides or nucleoside analogs which have nitrogenous heterocyclic bases or base analogs linked together along a backbone, including conventional RNA, DNA, mixed RNA-DNA, and polymers that are analogs thereof. A nucleic acid “backbone” can be made up of a variety of linkages, including one or more of sugar-phosphodiester linkages, peptidenucleic acid bonds (“’peptide nucleic acids” or PNA; PCT No. WO 95 / 32305), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. Sugar moieties of a nucleic acid can be ribose, deoxyribose, or similar compounds with substitutions, e.g., 2’ methoxy or 2’ halide substitutions. Nitrogenous bases can be conventional bases (A, G, C, T, U), analogs thereof (e.g., modified uridines such as 5-methoxyuridine, pseudouridine, or N 1 -methylpseudouridine, or others); inosine; derivatives of purines or pyrimidines (e.g., N4-methyl deoxyguanosine, deaza- or aza-purines, deaza- or aza-pyrimidines, pyrimidine bases with substituent groups at the 5 or 6 position (e.g., 5- methylcytosine), purine bases with a substituent at the 2, 6, or 8 positions, 2-amino-6- methylaminopurine, 06-methylguariine, 4-thio-pyrimidines, 4-amino-pyrimidines, 4- dimethylhydrazine-pyrimidines, and O4-alkyl-pyrimidines; US Pat. No. 5,378,825 and PCT No. WO 93 / 13121). For general discussion see The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed., Hthed„ 1992).

[0095] The compositions and methods disclosed herein may include a donor nucleic acid, i.e., a “template” nucleic acid. The template nucleic acid may be used as an inserted exogenous nucleic acid sequence (e.g., a gene or portion of a gene) at or near a target site for a Cas nuclease.

[0096] Nucleic acids can include one or more “abasic” residues where the backbone includes no nitrogenous base for position(s) of the polymer (US Pat. No. 5,585,481). A nucleic acid can comprise only conventional RN A or DNA sugars, bases and linkages, or can include both conventional components and substitutions (e.g., conventional bases with 2’ methoxy linkages, or polymers containing both conventional bases and one or more base analogs). Nucleic acid includes “locked nucleic acid” (LNA), an analogue containing one or more LNA nucleotide monomers with a bicyclic furanose unit locked in an RNA mimicking sugar conformation, which enhance hybridization affinity toward complementary RNA and DNA sequences (Vester and Wengel, 2004, Biochemistry 43(42): 13233-41 ). RNA and DNA have different sugar moieties and can differ by the presence of uracil or analogs thereof in RNA and thymine or analogs thereof in DNA.

[0097] “Guide RNA”, “guide RNA”, and simply “guide” are used herein interchangeably to refer to either a crRNA (also known as CRISPR RNA), or the combination of a crRNA and a trRNA (also known as tracrRNA). The crRNA and trRNA may be associated as a single RNA molecule (singleguide RNA, sgRNA) or in two separate RNA molecules (dual guide RNA, dgRNA). “Guide RNA” or “guide RNA” refers to both types. The trRNA may be a naturally-occurring sequence, or a trRNA sequence with modifications or variations compared to naturally-occurring sequences. For clarity, the terms “guide RNA” or “guide” as used herein, and unless specifically stated otherwise, may refer to an RNA molecule (comprising A, C, G, and U nucleotides) or to a DNA molecule encoding such an RNA molecule (comprising A, C, G, and T nucleotides) or complementary sequences thereof. In general, in the case of a DNA nucleic acid construct encoding a guide RNA, the U residues in any of the RNA sequences described herein may be replaced with T residues, and in the case of a guide RNA construct encoded by any of the DNA sequences described herein, the T residues may be replaced with U residues.

[0098] As used herein, a “spacer sequence,” sometimes aiso referred to herein and in the literature as a “spacer,” “protospacer,” “guide sequence,” or “targeting sequence” refers to a sequence within a guide RNA that is complementary to a target sequence and functions to direct a guide RNA to a target sequence for cleavage by an endonuclease, such as, Cas9. For clarity, the terms “spacer sequence”, “spacer,” “protospacer,” “guide sequence,” or “targeting sequence” as used herein, and unless specifically stated otherwise, may refer to an RNA molecule (comprising A, C, G, and U nucleotides) or to a DNA molecule encoding such an RNA molecule (comprising A, C, G, and T nucleotides) or complementary sequences thereof. A guide sequence can be, e.g., 24, 23, 22, 21, 20 or fewer base pairs in length, e.g., in the case of Staphylococcus lugdunensis (i.e., SluCas9) or Staphylococcus aureus (i.e., SaCas9) and related Cas9 homologs / orthologs. In some embodiments, a guide / spacer sequence in the case of SluCas9 or SaCas9 is at least 20 base pairs in length, or more specifically, within 20-25 base pairs in length (see, e.g., Schmidt et al., 2021, Nature Communications, “Improved CRISPR genome editing using small highly active and specific engineered RNA-guided nucleases”). Shorter or longer sequences can also be used as guides, e.g,, 15- , 16-, 17-, 18-, 19-, 20-, 21-, 22-, 23-, 24-, or 25-nucleotides in length. For example, in some embodiments, the guide sequence comprises at least 17, 18, 19, 20, 21, 22, 23, 24, or 25 contiguous nucleotides. In some embodiments, the target sequence is in a gene or on a chromosome, for example, and is complementary to the guide sequence. In some embodiments, the degree of complementarity or identity between a guide sequence and its corresponding target sequence may be about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. For example, in some embodiments, the guide sequence comprises a sequence with about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to at least 17, 18, 19, 20, 21, 2.2, 23, 24, or 25 contiguous nucleotides of a target sequence. In some embodiments, the guide sequence comprises a sequence with about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a target sequence. In some embodiments, the guide sequence and the target region may be 100% complementary or identical. In other embodiments, the guide sequence and the target region may contain at least one mismatch. For example, the guide sequence and the target sequence may contain 1 , 2, 3, or 4 mismatches, where thetotal length of the target sequence is at least 17, 18, 19, 20 or more base pairs. In some embodiments, the guide sequence and the target region may contain 1-4 mismatches where the guide sequence comprises at least 17, 18, 19, 20 or more nucleotides. In some embodiments, the guide sequence and the target region may contain 1, 2, 3, or 4 mismatches where the guide sequence comprises 20 nucleotides. In some embodiments, the guide sequence and the target region do not contain any mismatches.

[0099] As used herein, a ’‘linker sequence” or “linker” is an amino acid sequence to link or connect multiple protein domains. A linker sequence can be “structured” or “unstructured.” A “structured linker” is rigid and functions to prohibit unwanted interactions between the discrete domains. An “unstructured linker” is a flexible linker defined by secondary structure.

[0100] As used herein, a “scaffold sequence,” also referred to as a tracrRNA, refers to a nucleic acid sequence that recruits an endonuclease to a target nucleic acid. Any scaffold sequence that comprises at least one stem loop structure and recruits an endonuclease is encompassed herein. Exemplary scaffold sequences will be evident to one of skill in the art and can be found for example in Jinek, et al. Science (2012.) 337 (6096): 816-821, and Ran, et al. Nature Protocols (2013) 8: 2281- 2308.

[0101] Target sequences for endonucleases, such as Ca.s9s, include both the positive and negative strands of genomic DNA (i.e., the sequence given and the sequence’s reverse complement), as a nucleic acid substrate for a Cas9 is a double stranded nucleic acid. Accordingly, where a guide sequence is said to be “complementary to a target sequence”, it is to be understood that the guide sequence may direct a guide RNA to bind to the reverse complement of a target sequence. Thus, in some embodiments, where the guide sequence binds the reverse complement of a target sequence, the guide sequence is identical to certain nucleotides of tire target sequence (e.g., the target sequence not including the PAM) except for the substitution of U for T in the guide sequence.

[0102] As used herein, “ribonucleoprotein” (RNP) or “RNP complex” refers to a guide RNA together with an endonuclease, such as a Cas9. In some embodiments, the guide RNA guides the endonuclease such as Cas9 to a target sequence, and the guide RNA hybridizes with and the RNP binds to the target sequence, which can be followed by cleaving or nicking (in the context of a modified “nickase” endonuclease).

[0103] As used herein, a first sequence is considered to “comprise a sequence with at least X% identity to” a second sequence if an alignment of the first sequence to the second sequence show's that X% or more of the positions of the second sequence in its entirety are matched by the first sequence. For example, the sequence AAGA comprises a sequence with 100% identity to the sequence AAG because an alignment would give 100% identity in that there are matches to all three positions of the second sequence. The differences between RN A and DNA (generally the exchange of uridine for thymidine or vice versa) and the presence of nucleoside analogs such as modified uridines do not contribute to differences in identity or complementarity among polynucleotides as long as the relevantnucleotides (such as thymidine, uridine, or modified uridine) have the same complement (e.g., adenosine for ail of thymidine, uridine, or modified uridine; another example is cytosine and 5- methylcytosine, both of which have guanosine or modified guanosine as a complement). Thus, for example, the sequence 5’-AXG where X is any modified undine, such as pseudouridine, N1 -methyl pseudouridine, or 5-methoxyuridine, is considered 100% identical to AUG in that both are perfectly complementary to the same sequence (5’-CAU). Exemplary alignment algorithms are the Smith- Waterman and Needleman-Wunsch algorithms, which are well-known m the art. One skilled in the art will understand what choice of algorithm and parameter settings are appropriate for a given pair of sequences to be aligned; for sequences of generally similar length and expected identity >50% for amino acids or >75% for nucleotides, the Needleman-Wunsch algorithm with default settings of the Needleman-Wunsch algorithm interface provided by the EBI at the www.ebrac.uk web server is generally appropriate .

[0104] “mRNA” is used herein to refer to a polynucleotide that is not DNA and comprises an open reading frame that can be translated into a polypeptide (i.e., can serve as a substrate for translation by a ribosome and amino-acylated tRNAs). mRNA can comprise a phosphate-sugar backbone including ribose residues or analogs thereof, e.g., 2 ’-methoxy ribose residues. In some embodiments, the sugars of an mRNA phosphate-sugar backbone consist essentially of ribose residues, 2’-rnethoxy ribose residues, or a combination thereof.

[0105] As used herein, a “target sequence” refers to a sequence of nucleic acid in a target gene that has complementarity to at least a portion of the guide sequence of the guide RNA. The interaction of the target sequence and the guide sequence directs a Cas9 to bind, and potentially nick or cleave (depending on the activity of the agent), within or near the target sequence.

[0106] As used herein, “treatment” when used in the context of a disease or disorder refers to any administration or application of a therapeutic for a disease or disorder in a subject, and includes inhibiting the disease or development of the disease (which may occur before or after the disease is formally diagnosed, e.g., in cases where a subject has a genotype that has the potential or is likely to result in development of the disease), arresting its development, relieving one or more symptoms of the disease, curing the disease, or preventing reoccurrence of one or more symptoms of the disease. For example, treatment of DMD may comprise alleviating symptoms of DMD.

[0107] As used herein, a “tRNA” or “transfer RNA” refers to a molecule with an anticodon that can match with a codon in an mRNA molecule during translation. The tRNA can also be charged with an aminoacyl moiety, which is encoded by the codon to which the tRNA binds. In some embodiments, a tRNA of use herein comprises at least one of a T-loop, a D-loop, a V-loop, an anticodon loop, a T-stem, a D-stem, an anticodon stem, and an accepter stem. Such structural components of tRNAs are understood in the art, such as described in, e.g., Zhang and Ferre-D’Amare, Life (Basel), 2016, 6(1):3; Pak et al.. Transcription, 2017, 8(4):205-219; and Giege and Frugier, Transfer RNA Structure and Identity, In: Madame Curie Bioscience Database, Landes Bioscience(Austin, Texas, USA), 2000-2013. In preferred embodiments, the tRNA comprises one or more intragenic promoters (e.g., the Box A and / or Box B promoters; see, e.g.. Song et al., 2018, PLoS One, 13(8): e0202868).

[0108] As used herein, “ameliorating” refers to any beneficial effect on a phenotype or symptom, such as reducing its severity, slowing or delaying its development, arresting its development, or partially or completely reversing or eliminating it. In the case of quantitative phenotypes such as expression levels, ameliorating encompasses changing the expression level so that it is closer to tire expression level seen in healthy or unaffected cells or individuals,

[0109] A “pharmaceutically acceptable excipient” refers to an agent that is included in a composition (e.g., a pharmaceutical formulation) that is not the active ingredient. Pharmaceutically acceptable excipients may e.g., aid in drug delivery' or support or enhance stability or bioavailability. [001 10] The team “about” or “approximately” means an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined.

[0111] As used herein, '"Streptococcus pyogenes Cas9” may also be referred to as SpCas9, and includes wild type SpCas9 (e.g., SEQ ID NO: 730) and variants thereof. A variant of SpCas9 comprises one or more amino acid changes as compared to SEQ ID NO: 730, including insertion, deletion, or substitution of one or more amino acids, or a chemical modification to one or more amino acids. In some embodiments, the variant includes mutations at D10A or H840A (which creates a single-strand nickase), or mutations at D10A and H840A (which abrogates nuclease activity; this mutant is known as dead Cas9 or dCas9).

[0112] As used herein, “ Staphylococcus aureus Cas9” may also be referred to as SaCas9, and includes wild type SaCas9 (e.g., SEQ ID NO: 711) and variants thereof. A variant of SaCas9 comprises one or more amino acid changes as compared to SEQ ID NO: 711 , including insertion, deletion, or substitution of one or more amino acids, or a chemical modification to one or more amino acids. For clarity, SaCas9KKH is a SaCas9 variant.

[0113] As used herein, “ Staphylococcus lugdunensis Cas9” may also be referred to as SluCas9, and includes wild type SluCas9 (e.g., SEQ ID NO: 712) and variants thereof. A variant of SluCas9 comprises one or more amino acid changes as compared to SEQ ID NO: 712, including insertion, deletion, or substitution of one or more amino acids, or a chemical modification to one or more amino acids.IL Nucleic Acids

[0114] Provided herein is a nucleic acid comprising a sequence encoding a promoter, e.g., any of the hybrid promoters or chimeric promoters disclosed herein. In some embodiments, the hybrid promoter comprises a first portion comprising a pol III promoter (such as a mini-U6 pol III promoter (also referred to herein as a ’‘minimized U6” or “miniU6” promoter) or an Ml 1 pol III promoter) anda second portion comprising a transfer RNA (tRNA) promoter (such as a tRNA comprising a tRNA promoter). In some embodiments, the hybrid promoter comprises a first portion comprising a minimized 7SK promoter and / or a minimized U6 promoter, and a second portion comprising a tRN A promoter. In some embodiments, the hybrid promoter comprises a first portion comprising a minimized 7SK promoter and a minimized U6 promoter, and a second portion comprising a tRNA promoter. In some embodiments, the hybrid promoter comprises one or more additional components, such as a leader sequence and / or a trailer sequence. In some embodiments, tire nucleic acid further encodes one or more additional components, such as one or more protospacer sequences (such as two protospacers, e.g., for dual sgRNA vector expression), a second tRNA (such as a second tRNA that separates a first and a second sgRNA), an endonuclease (such as a Cas9 nuclease, such as a SluCas9 or a SaCas9 nuclease), one or more promoters driving expression of the endonuclease (such as a CK83 promoter), and / or one or more nuclear localization signals. In some embodiments, the hybrid promoter comprises one or more sgRNAs (e.g., two sgRNA s). In some embodiments, the sgRNAs are for use with the same class, type, subtype, and / or species of endonuclease. In some embodiments, the sgRNAs (such as a first sgRNA and a second sgRNA) are for use with a SluCas9 endonuclease. In some embodiments a first sgRNA and a second sgRNA are for use with a SaCas9 endonuclease.The Transfer RNA (tRNA) Component of the Nucleic Acid

[0115] In some embodiments, a sequence encoding a hybrid promoter disclosed herein comprises, as one component, a sequence encoding a transfer RNA (tRNA) promoter, such as a sequence encoding a tRNA comprising a tRNA promoter. In some embodiments, the sequence encoding the hybrid promoter comprises a nucleotide sequence encoding a tRNA. In such embodiments, the nucleotide sequence encoding tire tRNA comprises a nucleotide sequence encoding the tRNA promoter. In such embodiments, the tRNA promoter is “a part of’ the tRNA. In some embodiments, the tRNA is a suppressor tRNA. In some embodiments, the suppressor tRNA comprises an anticodon that hybridizes to a premature stop codon in a target gene (e.g., a mutant dystrophin gene) and that is capable of being aminoacylated with an amino acid.

[0116] In some embodiments, the hybrid promoter comprises one or more tRNA promoters. In some embodiments, the hybrid promoter comprises a tRNA. In some embodiments, the tRNA comprises one or more promoters. In some embodiments, the tRNA comprises one or more intragenic promoters. In some embodiments, the tRNA comprises an A-Box tRNA promoter. In some embodiments, the tRNA comprises a B-Box tRNA promoter. In some embodiments, the tRNA promoter comprises both an A-Box and a B-Box tRNA promoter. In some embodiments, the tRNA comprises an upstream promoter.

[0117] In some embodiments, a hybrid promoter disclosed herein comprises a nucleotide sequence encoding any of the tRNA molecules described in one or more of US2020277607, US2022073933, US202029140I, US2022112489, W02019090154, W02019090169,W02020150608, W02021087401, W02020069199, or WO2018161032, each of which applications is incorporated by reference herein in its entirety. In some embodiments, the tRNA is encoded by any one of TRNAA28, TRNA32, TRNAA34, TRNAM 1. TRNAD10, TRNAM, TRNAI9, TRNAP3, orTRNAW2. Table 1 provides nucleotide sequences for each of these tRNA genes.Table 1.

[0118] In some embodiments, the tRNA comprises a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of any one of SEQ ID Nos: 9019-9027 or 8001-8006. In some embodiments, the tRNA comprises at least 20, 30, 40, 50, 60, 65, or 70 contiguous nucleotides from a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of anyone of SEQ ID Nos: 9019-9027 or 8001-8006. In some embodiments, the tRNA comprises 20-72, SO- 72, 40-72, 50-72, 60-72, 65-72, 70-72, 20-60, 20-50, 20-40, 20-30, 40-60, or 40-50 contiguous nucleotides from a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of any one of SEQ ID Nos: 9019-9027 or 8001-8006. In some embodiments, the tRNA comprises 65-72 or 70-72 contiguous nucleotides from a sequencethat is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of any one of SEQ ID Nos: 9019-9027 or 8001-8006.

[0119] In some embodiments, the tRNA is functional, meaning that it functions as it does in nature. In nature, tRNA assists with translation by providing a physical link between the mRNA and amino acid. In particular, the codon in the mRNA pairs with the anti-codon in the tRNA.

[0120] In some embodiments, the tRNA is non-functional, meaning that it does not function as it does in nature. In some embodiments, the tRNA (such as the nucleotide sequence encoding the tRNA) is modified so that it does not produce a functional tRNA. In some such embodiments, the tRNA is modified so that it does not produce a function tRNA, but the tRNA promoter remains functional (i.e., in its capacity as a promoter). A tRNA may be non-functional, for example, in that it may not recognize the aminoacyl tRN A synthetase enzyme, may not bind to the ribosome, may not bind an amino acid, may not carry or link specific amino acids to nucleic acids, and / or may not properly lead codons in an mRNA molecule. A non-functional tRNA may be lacking other functions that may otherwise be carried out by a functional tRNA.

[0121] In some embodiments, the tRN A has reduced function comprised to a wildtype tRN A.For example, in some embodiments, the tRNA has been altered (e.g., mutated) such that its ability to recognize the aminoacyl tRNA synthetase enzyme, bind to the ribosome, bind an ammo acid, carry or link specific amino acids to nucleic acids, and / or properly lead codons in an mRNA molecule is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% as compared to an unaltered control tRNA. In some embodiments, the tRNA has been altered (e.g., mutated) such that its ability to recognize the aminoacyl tRNA synthetase enzyme, bind to the ribosome, bind an amino acid, carry or link specific amino acids to nucleic acids, and / or properly lead codons in an mRNA molecule is reduced by 1-10%. 10-100%, 10-80%, 10-60%, 10-40%, 10-20%, 20-100%, 20-80%, 20- 60%, 20-40%, 40-100%, 40-80%, 40-60%, 60-100%, 60-80%, 80-100% or 90-100% as compared to an unaltered control tRNA.

[0122] In some embodiments, the tRNA comprises a T-loop, a D-loop, a V-loop, an anticodon loop, a T-stem, a D-stem, an anticodon stem, and / or an accepter stem. Such structural components of tRNAs are understood m the art, such as described in, e.g., Zhang and Ferre -D’Amare, Life (Basel), 2016, 6(1):3; Pak et al., Transcription, 2017, 8(4):205-219; and Giege and Frugier, Transfer RNA Structure and Identity, In: Madame Curie Bioscience Database, Landes Bioscience (Austin, Texas, USA), 2000-2013.

[0123] In some embodiments, the tRNA promoter is modified. In some embodiments, the tRNA comprising the tRNA promoter is modified. In some embodiments, the leader sequence of tire tRNA is modified. In some embodiments, the tRNA is modified so that it is not translated. In some embodiments, the tRNA sequence is modified in the D-stem or at close proximity to the D-stem. In some embodiments, the tRNA sequence is modified at position 9, 25, or 26 of the D-stem with reference to SEQ ID NOs: 9019-9027 as shown in Figure 4. In some embodiments, the tRNAsequence is modified in the D-stem or at close proximity to the D-stem and the modification comprises one or more of G9A, G25A, U25C, or C26G with reference to SEQ ID NOs. 9019-9027 as shown in Figure 4. In some embodiments, the tRNA sequence is modified in the anticodon loop. In some embodiments, the tRNA sequence is modified at positions 33, 34, 35, 36, or 37 of the anticodon loop with reference to SEQ ID NOs: 9019-9027 as shown in Figure 4. In some embodiments, the tRNA sequence is modified in the anticodon loop and the modification comprises one or more of A33C, C33U, A34U, G34U, G34C, C34U, A35U, C35A, U35C, G35A, U35A, C36A, or U37A with reference to SEQ ID NOs: 9019-9027 as shown in Figure 4, In some embodiments, the tRNA sequence is modified in the acceptor stem. In some embodiments, the tRNA sequence is modified at position 70 or 73 of the acceptor stem with reference to SEQ ID NOs: 9019-9027 as shown in Figure 4. In some embodiments, the tRNA sequence is modified in the acceptor stem and the modification comprises one or more of U70C or G73C with reference to SEQ ID NOs: 9019-9027 as shown in Figure 4. In some embodiments, the tRNA comprises more than one of the modifications described above. In some embodiments, the tRNA sequence that is modified is any one of the tRNA sequences of Table 15. In some embodiments, the tRNA sequence that is modified is any one of SEQ ID NOs: 8001-8006.

[0124] In some embodiments, the tRNA comprises the sequence of any one of SEQ ID Nos: 9019-9027 or 8001-8006, but wherein the sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, or 15 nucleotide alterations (e.g., insertions, deletions or substitutions). In some embodiments, the tRNA comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, or 15 nucleotide substitutions as compared to the sequences of any of SEQ ID Nos: 9019-9027 or 8001-8006. In some embodiments, the tRNA comprises 1 or more nucleotide alterations, but retains the ability to drive expression (alone or with another promoter element, e.g., with another Poi III promoter element).

[0125] In particular embodiments, the tRNA 1) is modified such that it cannot bind an amino acid; 2) is modified in the D-stem region, and optionally is modified at position 9, 25, or 26 of the D- stem corresponding to the sequence of SEQ ID NO: 9022 or 9023; 3) is modified at one or more of G9A or G25A corresponding to the sequence of SEQ ID NO: 9022, and / or is modified at one or more of U25C or C26G corresponding to the sequence of SEQ ID NO: 9023; 4) is modified in the anticodon loop; 5) is modified at one or more of positions 33, 34, 35, 36, or 37 of the anticodon loop corresponding to the sequence of any one of SEQ ID NOs: 9019-9027; 6) is modified in the anticodon loop and the modification comprises one or more of A33C, C33U, A34U, G34U, G34C, C34U, A35U, C35A, (J35C, G35A, U35A, C36A, or (J37A corresponding to the sequence of any one of SEQ ID NOs: 9019-902.7; 7) is modified in the acceptor stem; 8) is modified at position 70 of the acceptor stem corresponding to the sequence of SEQ ID NO: 9019 and / or is modified at position 73 of the acceptor stem corresponding to the sequence of SEQ ID NO: 9023; and / or 9) is modified at one or more of U70C corresponding to the sequence of SEQ ID NO: 9019 or G73C corresponding to tire sequence of SEQ ID NO: 9023.

[0126] In some embodiments, the tRNA comprises an anticodon that is complementary to a codon encoding any of the following amino acids: Alanine, Arginine, Asparagine, Aspartic Acid, Cysteine, Glutamine, Glutamic Acid, Glycine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Proline, Serine, Threonine, Tryptophan, Tyrosine, or Valine. In some embodiments, the tRNA binds to any one of the following amino acids: Alanine, Arginine, Asparagine, Aspartic Acid, Cysteine, Glutamine, Glutamic Acid, Glycine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Proline, Serine, Threonine, Tryptophan, Tyrosine, or Valine.

[0127] In some embodiments, the tRNA is tRNA’-jln. tRNA’-jlncarries the ammo acid glutamine (Gin) to the ribosome. In some embodiments, the tRNA is tRNAG1“. tRNAG1“ carries the amino acid glutamic acid (Glu) to the ribosome. In some embodiments, the tRNA is tRNAGly. tRNAGlycarries the amino acid glycine (Gly) to the ribosome. In some embodiments, the tRNA is tRNArt0. tRNA?rocarries the amino acid proline (Pro) to the ribosome. In some embodiments, the tRNA is tRNAAsp. tRNAAspcarries the amino acid aspartic acid (Asp) to the ribosome. In some embodiments, the tRNA is tRNAAsp!y[ut. tRNAAspMutcarries the amino acid aspartate (Asp) to the ribosome, and bears one or more mutations as compared to tRNAAsp.

[0128] In particular embodiments, a nucleic acid composes a sequence encoding a hybrid promoter, and the hybrid promoter comprises (a) a first portion comprising a mini-U6 pol III promoter, and (b) a second portion comprising a nucleic acid encoding tRNAAspMut(SEQ ID NO: 8006). In some embodiments, the nucleic acid is in 5’ to 3’ order, and the mini-116 Pol III promoter is 5’ to the tRNAAspMut(SEQ ID NO: 8006). In other embodiments, the nucleic acid is in 3’ to 5’ order, and the mini-U6 Pol III promoter is 3’ to the tRNAAspMut(SEQ ID NO: 8006).

[0129] In some embodiments, the tRNA is tRNAGlnand comprises a nucleotide sequence that is at least 80%, 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 8001 :GGTTCCATGGTGTAATGGTTAGCACTCTGGACTCTGAATCCAGCGATCCGAGTTCAAATCTCGGTGGAACCT.

[0130] In some embodiments, the tRNA is tRNAGluand comprises a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 8002:TGCCTGGTGtrTCTAGTGGTTAGGATrCGGCGCTCTGAGCGCCGCGGCCGGGGTTCGATTC CCGGl CAGGGAA .

[0131] In some embodiments, the tRN A is tRNA"llyand comprises a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 8003:GCATTGGTGGTTCAGTGGTAGAATTCirGCCTGCCACGCGGGAGGCCCGGGTTCGATTCC CGGCCAATGCA.

[0132] In some embodiments, the tRNA is tRNAProand comprises a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 8004:GGCTCG1 TGG fCTAGGGG 1 A'FGA f'l'C 1 CGC Fl 1XJGG FGCGAG.AGG TCCCGGG 1 FCAA Al C CCGGACGAGCCC.

[0133] In some embodiments, the tRNA is tRNArtspand comprises a nucleotide sequence that is at least 80%, 85%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 8005: TCCTCGTTAGTATAGTGGTTAGTATCCCCGCCTGTCACGCGGGAGACCGGGGTTCAATTC CCCGACGGGGAG.

[0134] In some embodiments, the tRNA is tRNAAspMutand comprises a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 8006 (the mutated sequence is shown in lowercase):TCCTCGTTAGTATAGTGGTTAGTAcgCCCGCCTtea.ACGCGGGAGACCGG<IGTTCAATTCCC CG ACGGGGAc .

[0135] In some embodiments, the tRNA comprises an anticodon mutation to bind the TAG stop codon.

[0136] In some embodiments, the tRNA comprises a leader sequence. In some embodiments, the tRNA comprises a trailer sequence. In some embodiments, the tRNA comprises one or more of a leader sequence and a trailer sequence.

[0137] In particular embodiments, the tRNA promoter is a eukaryotic tRNA promoter. In some embodiments, the tRNA promoter (e.g., a tRNA comprising a tRNA promoter) is a yeast tRNA promoter. In some embodiments, the tRNA promoter is a mammalian (e.g., murine or human) tRNA promoter.Promoters

[0138] In some embodiments, the disclosure provides for novel promoters, such as any of the hybrid or chimeric promoters disclosed herein. In some embodiments, the hybrid promoter comprises, as one component, a pol III promoter that is not the tRNA promoter. In some embodiments, the hybrid promoter comprises, as a second component, a tRNA promoter (such as a tRNA comprising a tRNA promoter). In some embodiments, the pol III promoter is a U6 promoter. In some embodiments, the pol III promoter is a mmi-L!6 promoter. In some embodiments, the pol III promoter is an Ml 1 promoter. In some embodiments, the pol III promoter is a 7SK promoter. In some embodiments, the pol III promoter is a truncated 7SK promoter. In some embodiments, the pol III promoter is a chimeric promoter comprising a 7SK promoter (such as a truncated 7SK promoter) and a mini-U6 promoter. In some embodiments, the chimeric promoter is a mini-U6 / 7SK promoter or a 7SK / mini- U6 promoter as described herein (such as encoded by SEQ ID NOs: 9017 and 9018).

[0139] In particular embodiments, the pol III promoter is a eukaryotic promoter. In some embodiments, the pol III promoter is a yeast promoter. In some embodiments, the pol III promoter is a mammalian (e.g., murine or human) promoter.

[0140] In some embodiments, a nucleic acid disclosed herein, or a vector comprising a nucleic acid disclosed herein, comprises one or more additional promoters, e.g., for expression of one or more vector components, such as an endonuclease as described elsewhere herein. In some embodiments, the vector comprises one or more of a U6, Ml 1, Hl, or 7SK promoter. In some embodiments, the U6 promoter is the human U6 promoter (e.g. , the U6L promoter or U6S promoter). In some embodiments, the U6 promoter is a mini-U6 promoter. In some embodiments, the promoter is the murine U6 promoter.

[0141] In some embodiments, a nucleic acid encoding a U6 promoter (such as a mini-U6 promoter) does not comprise a guanine at the +1 position in the U6 transcriptional start site (i.e., does not comprise a guanine nucleotide (“+1G”) as the last nucleotide of the U6 promoter transcriptional start site). In some embodiments, a nucleic acid encoding the U6 promoter (such as a mini-U6 promoter) does comprise a guanine at the +1 position in the L!6 transcriptional start site (i.e., does comprise a guanine nucleotide (“-rlG”) as the last nucleotide of the U6 promoter transcriptional start site). In some embodiments, the 7SK promoter is a human 7SK promoter. In some embodiments, the 7SK promoter is the 7SKI promoter. In some embodiments, the 7SK promoter is the 7SK2 promoter. In some embodiments, the Hl promoter is a human Hl promoter (e.g,, the H1L promoter or the HIS promoter). In some embodiments, the vector comprises multiple guide sequences, wherein each guide sequence is under the control of a separate promoter.

[0142] At least one module in each promoter functions to position the start site for RNA synthesis. An example of such a module is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.

[0143] In some embodiments, the promoter comprises a distal sequence element (DSE). In some embodiments, the promoter comprises a proximal sequence element (PSE). In some embodiments, the promoter comprises a TATA box. In some embodiments, the promoter comprises, in 5’ to 3' order, a distal sequence element (DSE), a proximal sequence element (PSE) and a TATA box.

[0144] In some embodiments, the U6 promoter comprises a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 702: cgagtccaacacccgtgggaatcccatgggcaccatggcccctcgctccaaaaatgcttcgcgtcgcgcagacactgctcggtagttcggggat cagcgtttgagtaagagcccgcgtctgaaccctccgcgccgccccggccccagtggaaagacgcgcaggcaaaacgcaccacgtgacggagc gtgaccgcgcgccgagcgcgcgccaaggtcgggcaggaagagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattagaattaattgactgta.aacacaaagatattagtacaaaatacgtgacgtagaaa.gtaataatttcttgggtagttgca.gttttaaaatiat gttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaa.

[0145] In some embodiments, the Hl promoter comprises a nucleotide sequence that is at least 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 703: gctcggcgcgcccatatttgcatgtcgcmtgtgttctgggaaatcaccataaacgtgaaatgtctttggatttgggaatcttataagttctgtatgagac cacggta.

[0146] In some embodiments, the 7SK promoter comprises a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 704: tgaCggCgCgCCCtgCagtatttagCatgCCCCaCCCatCtgCaaggCattCtggatagtgtCaaaacagCCggaaatCaagtCCgtttatCtCaaaC tttagcatttgggaataaatgatattgctatgctggttaaattagatttagtaaattcctgctgaagctctagtacgataagtaacttgacctaagtgta aagttgagattccttcaggttatatagcttgtgcgccgcctgggta.

[0147] In some embodiments, die U6 promoter is a hU6c promoter and comprises a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 705:GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAG ATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAG AAAGIAATAATTTCTTGGGTAGITIGCAGTTTTAAAATTATGTTTTAAAATGGACTATCAT ATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGAC GAAACACCg.

[0148] In some embodiments, die U6 promoter is a variant of the hU6c promoter. In some embodiments, die variant of die hL!6c promoter comprises alternative nucleotides as compared to the sequence of SEQ ID NO: 705. In some embodiments, the variant of the hU6c promoter comprises fewer nucleotides as compared to the 249 nucleotides of SEQ ID NO: 705. In some embodiments, the variant of the hU6c promoter has fewer nucleotides in the nucleosome binding sequence of the hU6c promoter of SEQ ID NO: 705. In some embodiments, the variant of die hU6c promoter lacks all of or at least a portion of (e.g., at least 5, 10, 15, 20, 25, or 30 nucleotides) the nucleotides corresponding to nucleotides 96-125 of SEQ ID NO: 705. In some embodiments, the variant of the ill. 6c promoter lacks all of or at least a portion of (e.g., at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 nucleotides) die nucleotides corresponding to nucleotides 81-140 of SEQ ID NO: 705. In some embodiments, die variant of die hL!6c promoter lacks all of or at least a portion of (e.g., at least 10, 20, 30, 40, 50, 60, 65, 70, 75, 80, or 85 nucleotides) the nucleotides corresponding to nucleotides 66- 150 of SEQ ID NO: 705. In some embodiments, the variant of the hU6c promoter lacks all of or at least a portion of (e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, or 120 nucleotides) the nucleotides corresponding to nucleotides 51-170 of SEQ ID NO: 705. In some embodiments, the variant of the hU6c promoter lacks the nucleotides corresponding to nucleotides 96-125 of SEQ IDNO: 705. In some embodiments, the variant of the hU6c promoter comprises 129-219 nucleotides. In some embodiments, the variant of the hU6c promoter comprises 219 nucleotides. In some embodiments, the variant of the hlI6c promoter comprises 189 nucleotides. In some embodiments, the variant of the hU6c promoter comprises 159 nucleotides. In some embodiments, the variant of the hIJ6c promoter comprises 129 nucleotides.

[0149] In some embodiments, the U6 promoter is hU6d30 and comprises a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 9001:GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAG ATAATTGGAATTAATTTGACTGTAAACACAAAGATATAATTTCTTGGGTAGTTTGCAGTT TTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATf TCI FGGCTTTA TATATC TTG FGGAAAGGACGAAACACC.

[0150] In some embodiments, the U6 promoter is hU6d60 and comprises a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 9o%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 9002:GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAG ATAATTGGAATTAATTTGACGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATA TGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTITATATATCTTGTGGAAAGGACG AAACACC.

[0151] In some embodiments, the U6 promoter is hU6d90 and comprises a nucleotide sequence that is at least 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 9003:GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAG ATAATATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATT TCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACC.

[0152] In some embodiments, the U6 promoter is hU6d!20 and comprises a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 9004:GAGGGCCT ATTFCCCA TGA IT CCTTCAT A'FTTGCA I AT ACGATACAAGGCGGAC TA I CAT ATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGAC GAAACACC.

[0153] In some embodiments, the L!6 promoter is a mini-116 promoter and comprises a nucleotide sequence that is at least 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 9005:GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATAGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTAIATATCTIGTGGAAAGGACGAAACACC.

[0154] In some embodiments, the U6 promoter is a mini-U6 promoter and comprises a nucleotide sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 9010: GAGGGCCTATTTCCCATGATTCCTTCATATTTGCAT.

[0155] In some embodiments, the U6 promoter is a mini-U6 promoter and comprises a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 9015: gcttaccg:taacttgaaagtatttcg:atttcttggctttatatatcttgtggaaaggacgaaacacc.

[0156] In some embodiments, the mini-U6 promoter is less than 74 base pairs (“bp”) in length. In some embodiments, the mini-U6 promoter is greater than 117 bp in length. In some embodiments, the promoter is between 40 and 73 bp, between 50 and 73 bp, between 60 and 73 bp, or between 70 and 73 bp in length. In some embodiments, the promoter is greater than 117 bp, greater than 120 bp, greater than 130 bp, greater than 140 bp, greater than 150 bp, or greater than 200 bp in length.

[0157] In some embodiments, the 7SK promoter is a 7SK2 promoter and comprises a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 706:CTGCAGTATTTAGCATGCCCCACCCATCTGCAAGGCATTCTGGATAGTGTCAAAACAGCC GGAAATCAAGTCC'GTTTATCTCAAACTTTAGCATTTTGGGAATAAATGATATTTGCTATG CTGGITAAATIAGATTTTAGITAAATITCCTGC'rGAAGCI'CTAG'rACGATAAGCAACTrG ACCTAAGTGTAAAGTTGAGACTTCCTTCAGGTTTATATAGCTTGTGCGCCGCTTGGGTAC CTC.

[0158] In some embodiments, the 7SK promoter is a variant of the 7SK2 promoter. In some embodiments, tire variant of tire 7SK2 promoter comprises alternative nucleotides as compared to the sequence of SEQ ID NO: 706. In some embodiments, the variant of the 7SK2 promoter e.g., comprises fewer nucleotides as compared to the 243 nucleotides of SEQ ID NO: 706. In some embodiments, the variant of the 7SK2 promoter has fewer nucleotides in the nucleosome binding sequence of the 7SK2 promoter of SEQ ID NO: 706. In some embodiments, the variant of the 7SK2 promoter lacks all of or at least a portion of (e.g., at least 5, 10, 15, 20, 25, or 30 nucleotides) tire nucleotides corresponding to nucleotides 95-12.4 of SEQ ID NO: 706. In some embodiments, the variant of the 7SK2 promoter lacks all of or at least a portion of (e.g., at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 nucleotides) the nucleotides corresponding to nucleotides 81-140 of SEQ ID NO: 706. In some embodiments, the variant of the 7SK2 promoter lacks all of or at least a portion of (e.g., at least 10, 2.0, 30, 40, 50, 60, 65, 70, 75, 80, 85 or 90 nucleotides) the nucleotides corresponding to nucleotides 67-156 of SEQ ID NO: 706. In some embodiments, the variant of the 7SK2 promoter lacks all of or at least a portion of (e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, or 120 nucleotides) the nucleotides corresponding to nucleotides 52-171 of SEQ ID NO: 706. In some embodiments, the variant of the 7SK2 promoter comprises 123-213 nucleotides. In someembodiments, the variant of the 7SK2 promoter comprises 213 nucleotides. In some embodiments, the variant of the 7SK2 promoter comprises 183 nucleotides. In some embodiments, the variant of the 7SK2 promoter comprises 153 nucleotides. In some embodiments, the variant of the 7SK2 promoter comprises 123 nucleotides.

[0159] In some embodiments, the 7SK promoter is 7SKd30 and comprises a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 93%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 9006:CTGCAGTATTTAGCATGCCCCACCCATCTGCAAGGCATTCTGGATAGTGTCAAAACAGCC GGAAATCAAGTCCGTTTATCTCAAACTTTAGCATTTAAATTAGATTTTAGTTAAATTTCCT GCTGAAGCTCTAGTACGATAAGCAACTTGACCTAAGTGTAAAGTTGAGACTTCCTTCAGG TITATATAGCTFGTGCGCCGCTrGGGTACCTC.

[0160] In some embodiments, the 7SK promoter is 7SKd60 and comprises a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 9007:CTGCAGTAm’AGCATGCCCCACCCATCTGCAAGGCATrCTGGATAGTGTCAAAACAGCC GGAAATCAAGTCCGTTTATCTTAAATTTCCTGCTGAAGCTCTAGTACGATAAGCAACTTG ACCTAAGTGTAAAGTTGAGACTTCCTTCAGGTTTATATAGCTTGTGCGCCGCTTGGGTAC CTC.

[0161] In some embodiments, the 7SK promoter is 7SKd90 and comprises a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 9008:CTGCAGTAnTAGCATGCCCCACCCATCTGCAAGGCATTCTGGATAGTGTCAAAACAGCC GGAAATAGCTCTAGTACGAIAAGCAACTTGACCTAAGTGTAAAGTTGAGACTTCCTTCAG GTTTATATAGCTTGTGCGCCGCTTGGGTACCTC.

[0162] In some embodiments, the 7SK promoter is 7SKdl 20 and comprises a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 9009:CTGCAGTATITAGCATGCCCCACCCATCTGCAAGGCAlTCTGGATAGTGTCAGCAACn’G ACCTAAGTGTAAAGTTGAGACTTCCTTCAGGTTTATATAGCTTGTGCGCCGCTTGGGTAC CTC.

[0163] In some embodiments, the 7SK promoter is a truncated 7SK and comprises a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 9011:TTGACCTAAGTGTAAAGTTGAGACTTCCTTCAGGTTTATATAGCTTGTGCGCCGCTTGGGT ACCTC.

[0164] In some embodiments, the 7SK promoter is a truncated 7SK and comprises a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identical to the sequence of SEQ ID NO: 9016:CTGCAGTATTTAGCATGCCCCACCCATCTGCAAGGCATTCTGGATAGTGTCAAAAC.

[0165] In some embodiments, a hybrid promoter encoded by a nucleic acid as disclosed herein comprises a chimeric promoter. In some embodiments, the chimeric promoter comprises a mini-1 J6 promoter and a truncated 7SK promoter. In some embodiments, the chimeric promoter is in 5’ to 3’ order and the mini-U6 promoter is 5’ of the truncated 7SK promoter. In other embodiments, the truncated 7SK promoter is 5’ of the mini-U6 promoter.

[0166] In some embodiments, the chimeric promoter comprises a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 9017:GAGGGCCTATTTCCCATGATTCCTTCATATITGCAITrGACCTAAGTGI'AAAGITGAGACT TCC / ITCAGGTTTATATAGCTTGTGCGCCGCTTGGGTACCTC.

[0167] In some embodiments, the chimeric promoter comprises a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 9018: CTGCAGTATTTAGCATGCCCCACCCATCTGCAAGGCATTCTGGATAGTGTCAAAACgcttaee gtaacttgaaagtatttcgatttcttggctttatatatctgtggaaaggacgaaacacc.

[0168] In some embodiments, the Hl promoter is a Him or mill promoter and comprises a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 707:AATATTTGCATGTCGCTATGTGTTCTGGGAAATCACCATAAACGTGAAATGTCTTTGGAT TTGGGAATCTTATAAGTTCTGTATGAGACCACTCTTTCCC.

[0169] In some embodiments, the promoter is an Ml 1 promoter and comprises a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 708:ATATTTAGCATGTCGCTATGTGTTCTGGGAAACTTGACCTAAGTGTAAAGTTGAGATTTC CTTCAGGTTTATATAGTTCTGTATGAGACCACTCTTTCCC.

[0170] In some embodiments, the disclosure provides for a chimeric Pol III promoter. In some embodiments, the chimeric pol III promoter is a component of any of the hybrid promoters disclosed herein (e.g., the chimeric pol III promoter is a component of a hybrid promoter along with any of the tRNA promoters disclosed herein). In some embodiments, the chimeric pol III promoter is not a component of a hy brid promoter. In some embodiments, the chimeric Pol III promoter comprises a portion of a first pol III promoter (e.g., a U6 promoter) and a portion of a second pol III promoter (e.g., a 7SK promoter). In some embodiments, the chimeric Pol III promoter comprises a portion of a U6 promoter. In some embodiments, the portion of the U6 promoter comprises 1-80, 20-80, 30-80, 20-60, 20-50, 20-40, or 30-40 contiguous nucleotides of a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of77SEQ ID NO: 9005. In some embodiments, the portion of the U6 promoter comprises 1 -40, 10-40, 20- 40, 30-40 of the first 50 nucleotides of a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 9005. In some embodiments, the portion of the U6 promoter comprises 1 -80, 20-80, 30-80, 50-80, 70-80, 50-70, or 60-70 of the last 100 nucleotides of a nucleotide sequence that is at least 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 9005. In some embodiments, the portion of the U6 promoter comprises a nucleotide sequence that is at least 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 9010. In some embodiments, the portion of the U6 promoter comprises a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 9015. In some embodiments, the chimeric Pol III promoter comprises a portion of a 7SK promoter. In some embodiments, the portion of the 7SK promoter comprises 1-80, 20-80, 30-80, 20-60, 20-50, 20-40, or 30-40 contiguous nucleotides of a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93?% 94%, 95%, 96?% 9 / %, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 706. In some embodiments, the portion of the 7SK promoter comprises 1-40, 10-40, 20-40, 30-40, 40-60, 40-65, or 50-60 of the first 70 nucleotides of a nucleotide sequence that is at least 80?% 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 706. In some embodiments, the portion of tire 7SK promoter comprises 1-80, 20-80, 30-80, 50-80, 70-80, 50-70, or 60-70 of the last 100 nucleotides of a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 706. In some embodiments, the portion of the 7SK promoter comprises a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 9011. In some embodiments, the portion of the U6 promoter comprises a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 9016. In some embodiments, the chimeric Pol III promoter comprises a first nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 9010 and comprises a second nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 9011. In some embodiments, the chimeric Pol III promoter comprises a first nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 9016 and comprises a second nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 9015. In some embodiments, the first sequence is 3’ to the second sequence. In some embodiments, the second sequence is 3’ to the first sequence. In some embodiments, the chimeric Pol III promoter comprises a nucleotide sequence that is at. least. 80%, 85 %, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 91%, 98%, 99% or 100% identical tothe sequence of SEQ ID NO: 9017. In some embodiments, the chimeric Pol III promoter comprises a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 9018. sgRNAs

[0171] In some embodiments, a nucleic acid comprising a sequence encoding a hybrid promoter further comprises a sequence or sequences encoding one or more (such as two) single guide RNAs (sgRNAs). The one or more sgRNAs may comprise a targeting sequence (crRNA sequence) and Cas9 nuclease-recruiting sequence (tracrRNA). The one or more sgRN As may be the same sequence, or different sequences. In particular embodiments, the one or more sgRNAs each comprise different sequences. The one or more sgRNAs may be designed to target a specific region of the genome, e.g., a target gene. An sgRNA comprises a spacer sequence, which may be, e.g., any 25-mer, any 24-mer, any 23-mer, any 22-mer, any 21-mer, any 20-mer, any 19-mer, any 18-mer, or any 17-mer (depending on the chosen endonuclease), or any other nucleic acid sequence that is homologous to a region in a gene of interest, (such as DMD, encoding dystrophin) and will direct, an endonuclease to a chosen location.

[0172] In some aspects, the disclosure comprises a nucleic acid comprising a sequence encoding a hybrid promoter and further comprising one or more sequences encoding one or more sgRNAs that target a portion of a genome, such as a DMD exon (e.g., any of exons 2, 3, 6, 9, 44, 45, 47, 48, 50, 51 or 53), or a specific repeat pattern in a genome. In such embodiments, the hybrid promoter may drive expression of the one or more sgRNAs. In some embodiments, one or both of the sgRNAs targets a trinucleotide repeat in a genome. In some embodiments, each sgRNA interacts with the same class, type, subtype, and / or species of endonuclease. In some embodiments, the endonuclease is SpCas9, In some embodiments, the endonuclease is saCas9. In some embodiments, the endonuclease is sluCas9. In some embodiments, the endonuclease is Casl2i2. In some embodiments, the endonuclease is Cpfl. In some embodiments, the endonuclease is Castji. In some embodiments, the spacer component is substantially complementary to a target sequence, such as a DMD exon (e.g., any of exons 2, 3, 6, 9, 44, 45, 47, 48, 50, 51 or 53), wherein the DMD target sequence is adjacent to a 5’-NTTN-3’ PAM sequence as described herein. In the case of a double-stranded target, the sgRNA 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-com piemen tary strand) .

[0173] In some embodiments, one or more sgRNAs targeting exon 53 may be used. In some embodiments, one or more sgRNA targeting exon 51 may be used. In some embodiments, one or more sgRN As targeting one or more of a DMD exon (e.g., any’ of exons 2, 3, 6, 9, 44, 45, 47, 48, 50, 51 or 53) may be used.

[0174] In some embodiments, an sgRNA targeting exon 53 of the dystrophin gene may be used.In some embodiments, the sgRNA is referred to as “ESSSLS20”1” and is: TTTGGATTGCATCTACTGTA (SEQ ID NO: 1080) in 5’ to 3’ orientation.

[0175] In some embodiments, an sgRNA targeting exon 53 of the dystrophin gene may be used. In some embodiments, the sgRNA is referred to as “E53SL7” and is: ACATTTCATTCAACTGTTGCCT (SEQ ID NO: 1081) in 5’ to 3’ orientation.

[0176] In some embodiments, an sgRNA targeting exon 53 of the dystrophin gene may be used.In some embodiments, the sgRNA is referred to as “E53SL14” and is: AGGCCAGAGCCAAGCTTGAGTC (SEQ ID NO: 1082) in 5’ to 3’ orientation.

[0177] In some embodiments, an sgRNA targeting exon 53 of the dystrophin gene may be used. In some embodiments, the sgRNA is referred to as “E53SL16” and is: AAGGAAGAAGCTGAGCAGGTCT (SEQ ID NO: 1083) in 5’ to 3’ orientation,

[0178] In some embodiments, an sgRNA targeting exon 53 of the dystrophin gene may be used.In some embodiments, the sgRNA is referred to as “E53SL23” and is: AAGTACAAGAACACCTTCAGAA (SEQ ID NO: 1084) in 5’ to 3’ orientation.

[0179] In some embodiments, an sgRNA targeting exon 53 of tire dystrophin gene may be used. In some embodiments, the sgRNA is referred to as “E53SL25” and is: TAGTTGAAAGAATTCAGAATCA (SEQ ID NO: 1085) in 5’ to 3’ orientation.

[0180] In some embodiments, an sgRNA targeting exon 53 of the dystrophin gene may be used.In some embodiments, the sgRNA is referred to as “E53SL3” and is: CTTTTGGATTGCATCTACTGTA (SEQ ID NO: 1086) in 5’ to 3’ orientation.

[0181] In some embodiments, a nucleic acid comprising a sequence encoding a hybrid promoter as disclosed herein further comprises a sequence encoding a first sgRNA and a sequence encoding a second sgRNA, wherein the sgRNAs are for use with the same class, type, subtype, and / or species of endonuclease. In some embodiments, the endonuclease is a Cas9 endonuclease. In further embodiments. Staphylococcus aureus (SaCas9) or Staphylococcus lugdunensis (SluCas9).

[0182] In some embodiments, a nucleic acid comprising a sequence encoding a hybrid promoter or a chimeric promoter further comprises a sequence or sequences encoding one or more (such as two) of the guide RN As disclosed in Tables 2 A-2C below. In some embodiments, when the Cas9 nuclease is isolated or derived from SaCas9, the one or more guide RNAs is selected from SEQ ID NOs: 1-35, 1000-1078, and 3000-3069. In some embodiments, when the Cas9 nuclease is isolated or derived from SaCas9, the one or more guide RNAs comprises at least 20 contiguous nucleotides of a guide RNA selected from SEQ ID NOs: 1 -35, 1000-1078, and 3000-3069. In some embodiments, when the Cas9 nuclease is isolated or derived from SaCas9, the one or more guide RNAs comprises a nucleotide sequence having at least 90% sequence identity to a guide RNA selected from SEQ ID NOs: 1-35, 1000-1078, and 3000-3069. In some embodiments, when tire Cas9 nuclease is isolated or derived from SluCas9, the one or more guide sequences selected from SEQ ID NOs: 100-225, 2000-2116, and 4000-4251. In some embodiments, when the Cas9 nuclease is isolated or derived from SluCas9, the one or more guide RNAs comprises at least 20 contiguous nucleotides of a guide RNA selected from SEQ ID NOs: 100-225, 2000-2116, and 4000-4251. In some embodiments, when the Cas9 nuclease is isolated or derived from SluCas9, the one or more guide RNAs comprises a nucleotide sequence having at least 90% sequence identity to a guide RNA selected from SEQ ID NOs: 100-225, 2000-21 16, and 4000-4251.

[0183] In some embodiments, a nucleic acid comprising a sequence encoding a hybrid promoter or a chimeric promoter further comprises a sequence or sequences encoding two of the guide RNAs disclosed in Tables 2A-2C below. In some such embodiments, when the Cas9 nuclease is isolated or derived from SaCas9, a first and a second guide RNA are selected from any one of the following pairs of guide RNAs: SEQ ID NOs: 1020 and 23; 1023 and 23; 1023 and 1037; 1024 and 1055; 1025 and 23; 1025 and 1055; 1026 and 23; 1028 and 1055; 1029 and 1055; 1029 and 1037; 1031 and 1037;1032 and 1037; 1029 and 1027; 1037 and 1048; 1037 and 1051; 1037 and 1053; 20 and 23; 1038 and 23; 21 and 23; 1040 and 23; 1042 and 1037; 1043 and 1037; 1044 and 1037; 1045 and 1037; 1046 and 1037; 24 and 1037; 1047 and 1055; or 1055 and 1022. In other such embodiments, when the Cas9 nuclease is isolated or derived from SluCas9, a first and a second guide RNA are selected from any one of the following pairs of guide RNAs: SEQ ID NOs: 170 and 179; 172 and 179; 179 and 183; 179 and 185; 179 and 187; 179 and 188; 179 and 189; 179 and 193; 179 and 195; 179 and 196; 179 and 197; 200 and 174; or 200 and 176. In some embodiments, when the Cas9 nuclease is isolated or derived from SluCas9, a first and a second guide RNA are selected from any one of the following pairs of guide RNAs: SEQ ID NOs: 117 and 121; 117 and 122; 120 and 121 ; 120 and 123; 120 and 124; 120 and 125; 122 and 126; 122 and 123; 122 and 124; 122 and 125; or 122 and 126; for targeting exon 44. In some embodiments, when the Cas9 nuclease is isolated or derived from SluCas9, a first and a second guide RNA are selected from any one of the following pairs of guide RNAs: SEQ ID NOs: 155 and 156; 155 and 158; 155 and 162; 155 and 163; 162 and 157; 162 and 159; 162 and 164;162 and 166; or 162 and 167; for targeting exon 50. In some embodiments, when the Cas9 nuclease is isolated or derived from SiuCas9, a first and a second guide RNA are selected from any one of the following pairs of guide RNAs: SEQ ID NOs: 211 and 223; 2.11 and 225; 214 and 2.24; 216 and 223; 216 and 225; 220 and 224; 204 and 223; 223 and 224; or 204 and 225; for targeting exon 53. In some embodiments, when the Cas9 nuclease is isolated or derived from SaCas9, a first and a second guide RNA are selected from any one of the following pairs of guide RNAs: SEQ ID NOs: 1068 and 32; 1069 and 32; 1070 and 1075; 1071 and 32; 29 and 1075; 1072 and 27; 1072 and 28; 1072 and 32;1072 and 33; 1073 and 1076; 1073 and 35; 221 and 1077; 1074 and 27; 1074 and 28; 1074 and 33; 32 and 1077; 1075 and 1076; 1075 and 35; 1076 and 26; or 35 and 26; for targeting exon 53. In some embodiments, when the Cas9 nuclease is isolated or derived from SluCas9, a first and a second guide RNA are selected from any one of the following pairs of guide RNAs: SEQ ID NOs: 148 and 134;149 and 135; 150 and 135; 131 and 136; 151 and 136; 139 and 131 ; 139 and 151; 140 and 131; 140and 151; 141 and 148; 144 and 149; 144 and 150; 145 and 131; 145 and 151 ; 146 and 148; 134 and 148; 135 and 149; 135 and 150; 136 and 131; 136 and 151; 131 and 139; 151 and 139; 131 and 140; 151 and 140; 148 and 141; 149 and 144; 150 and 144; 131 and 145; 151 and 145; and 148 and 146. In some embodiments, when the Cas9 nuclease is isolated or derived from SaCas9, a first and a second guide RNA are selected from any one of the following pairs of guide RNAs: 10 and 15; 10 and 16; 12 and 16; 1001 and 1005; 1001 and 15; 1001 and 16; 1003 and 1005; 16 and 1003; 12 and 1010; 12 and 1012; 12 and 1013; 10 and 1016; 1017 and 1005; 1017 and 16; 1018 and 16; 15 and 10; 16 and 10; 16 and 12; 1005 and 1001; 15 and 1001; 16 and 1001 ; 1005 and 1003; 1003 and 16; 1010 and 12; 1012 and 12; 1013 and 12; 1016 and 10; 1005 and 1017; 16 and 1017; and 16 and 1018.

[0184] In some embodiments, any of the nucleic acids disclosed herein (e.g., comprising a sequence encoding any of the hybrid promoters disclosed herein), or composition comprising said nucleic acid, targets a region of the human DMD gene. In some embodiments, the region is an exon. In some embodiments, the region is an intron. In some embodiments, one of the sgRNAs encoded by the nucleic acid targets an exon and one of the sgRNAs encoded by the nucleic acid targets an intron. In some embodiments, the nucleic acid or composition targets exon 45, 51, or 53 of the human DMD gene. In some embodiments, components of a nucleic acid disclosed herein arc capable of excising a DNA fragment from the DMD gene, wherein the DNA fragment is between 5 and 250 nucleotides in length. In particular embodiments, the excised DMD fragment does not comprise an entire exon of the DMD gene.

[0185] In some embodiments, a first sgRNA encoded by the nucleic acid targets a genomic region that is downstream of the genomic region targeted by a second sgRNA encoded by the nucleic acid. In some embodiments, the second sgRNA encoded by the nucleic acid targets a genomic region that is downstream of the genomic region targeted by the first sgRNA encoded by the nucleic acid. In some embodiments, the first gRNA and the second gRNA encoded by the nucleic acid are in the same orientation in the nucleic acid (e.g., both 5’ to 3’ or both 3’ to 5’). In some embodiments, the first gRNA and the second gRNA encoded by the nucleic acid are in opposite orientations.

[0186] In some embodiments, the first sgRNA encoded by the nucleic acid comprises a first scaffold, and the second sgRN A of the nucleic acid or composition comprises a second scaffold, and wherein the first scaffold and the second scaffold are capable of selectively interacting with the same class, type, subtype and / or species of endonuclease. In some embodiments, the first scaffold nucleotide sequence differs from the second scaffold nucleotide sequence by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In some embodiments, the first scaffold nucleotide sequence is identical to the second scaffold nucleotide sequence. In some embodiments, the first scaffold and the second scaffold each comprise the nucleotide sequence of any one of SEQ ID Nos: 501-504, 601, or 900-917. In a preferred embodiment, the first scaffold and the second scaffold each comprise the nucleotide sequence of SEQ ID No: 901.

[0187] In some embodiments, the nucleic acid comprising a sequence encoding a hybrid promoter comprises a sequence encoding a leader sequence and / or a trailer sequence in addition to one or more protospacer sequences. In some embodiments, the hybrid promoter comprises one or more leader sequences and / or one or more trailer sequences.

[0188] In some embodiments, the hybrid promoter comprises: a) a portion comprising a pol III promoter (e.g., a mini-U6, a 7SK, or Mi l promoter) and b) a portion comprising a tRNA promoter (e.g., any of the tRNAs disclosed herein). In some embodiments, the hybrid promoter comprises a leader sequence preceding the portion comprising the pol III promoter. In some embodiments, the hybrid promoter comprises a leader sequence preceding the portion comprising the tRNA promoter. In some embodiments, the hybrid promoter comprises a leader sequence that is 3’ to the portion comprising tire pol III promoter, but 5 ’ to tire portion comprising the tRNA promoter. In some embodiments, the hybrid promoter comprises a leader sequence that is 3’ to the portion comprising the tRNA promoter, but 5’ to the portion comprising the pol III promoter. In some embodiments, the leader sequence comprises one or more guanines. In some embodiments, the first nucleotide in the leader sequence is a guanine . In some embodiments, the leader sequence comprises two or more guanines. In some embodiments, the leader sequence comprises 1, 2, 3, 4, 5, 6, or 7 nucleotides. In some embodiments, the leader sequence is no more than 10, 9, 8, 7, 6, 5, 4, 3 or 2 nucleotides in length. In some embodiments, the leader sequence comprises GTGGC (SEQ ID NO: 8050). In some embodiments, the leader sequence comprises GTA (SEQ ID NO: 8051). In some embodiments, the leader sequence comprises GGA (SEQ ID NO: 8052). In some embodiments, the leader sequence comprises GGC (SEQ ID NO: 8053), GC (SEQ ID NO: 8054), GGGTA (SEQ ID NO: 8055), GGTA (SEQ ID NO: 8056), GTA (SEQ ID NO: 8051), GCTGGA (SEQ ID NO: 8057), GA (SEQ ID NO: 8058), GCCAGA (SEQ ID NO: 8059), or GTCATCC (SEQ ID NO: 8060). In some embodiments, the leader sequence comprises the sequence of any one of SEQ ID NOs: 8050-8075. In some embodiments, the leader sequence comprises the sequence of any one of SEQ ID NOs: 8050-8060. In some embodiments, the leader sequence comprises the sequence of any one of SEQ ID NOs: 8051 or 8053-8060.

[0189] In some embodiments, the nucleic acid comprises a sequence encoding a leader sequence. In some embodiments, the nucleic acid comprises a sequence encoding a tRN A (such as a tRNA comprising a tRNA promoter) preceded by a leader sequence. In some embodiments, the leader sequence is GTGGC (SEQ ID NO: 8050). In some embodiments, the leader sequence is GTA (SEQ ID NO: 8051). In some embodiments, the leader sequence is GGA (SEQ ID NO: 8052). In some embodiments, the nucleic acid comprises a sequence encoding a protospacer that is preceded by a trailer sequence. In some embodiments, the trailer sequence is A (SEQ ID NO: 8049).

[0190] In some embodiments, the nucleic acid comprises a sequence encoding a guide RNA and a sequence encoding a pol III promoter (such as a mini-U6 pol III promoter or an Mi l pol III promoter), and the guide RNA is under the control of the pol III promoter. In some embodiments, thenucleic acid encoding the guide RNA sequence is under the control of a mini-U6 pol III promoter. In some embodiments, the nucleic acid encoding the guide RNA sequence is under the control of an Mi l poi III promoter. In some embodiments, the nucleic acid comprises a sequence encoding a guide RNA and a sequence encoding a tRNA promoter (such as a tRNA sequence encoding a tRNA promoter as described herein), and the guide RNA is under the control of the tRNA promoter. In some embodiments, the nucleic acid comprises a sequence encoding a guide RNA and a sequence encoding a hybrid promoter comprising a pol III promoter (such as a mini-116 pol III promoter or an Ml 1 pol III promoter) and a tRNA promoter, and the guide RNA is under the control of the hybrid promoter. In some embodiments, the nucleic acid comprises a sequence encoding a guide RNA and a sequence encoding a hybrid promoter comprising a chimeric promoter (e.g., a chimeric promoter comprising a mini-U6 pol III promoter and a truncated 7SK pol III promoter) and a tRNA promoter, and the guide RNA is under the control of the hybrid promoter.Table 2A: Exemplary DMD guide sequences (human-hg38.pl2)Table 2B: Exemplary DMD guide sequences (20-uucleotides and 21-uudeotides)Table 2C: Additional DMD Guide Sequences (hum an~hg38.pl 2)Hybrid Promoter Design

[0191] The hybrid promoter may be delivered in vivo or in vitro, e.g., as part of a poiycistronic dual sgRNA vector construct. In some embodiments, tire design of the hybrid promoters described herein can allow for reduced nucleic acid and / or vector size (such as reduced vector size) and / or higher expression of one or more (such as two) sgRNAs, yielding improved genome editing outcomes (such as increased cutting efficiencies), and more balanced expression of two sgRNAs, if present. In some embodiments, the hybrid promoter is part of a poiycistronic dual sgRNA vector arrangement.

[0192] In some embodiments, the nucleic acid encoding the hybrid promoter is in 5’ to 3’ order. In some such embodiments, the sequence encoding the hybrid promoter is 5’ to at least one sequence encoding an sgRNA. In some embodiments, the nucleic acid encoding the hybrid promoter is in 3’ to 5’ order. In some such embodiments, the sequence encoding the hybrid promoter is 3’ to at least one sequence encoding an sgRNA. In some embodiments wherein the nucleic acid is in 5’ to 3’ order, the sequence encoding the pol III promoter (such as the mini-U6 pol III promoter or the Ml 1 pol III promoter) is 5’ to the tRNA promoter. In some such embodiments, a sequence encoding a mini-U6 pol III promoter is 5’ to the tRNA promoter. In other such embodiments, a sequence encoding a Mi l pol III promoter is 5 ’ to the tRNA promoter. In some embodiments wherein the nucleic acid is in 3 ’ to 5’ order, the sequence encoding the pol III promoter (such as the mini-U6 pol III promoter or the Ml 1 pol III promoter) is 3’ to the tRNA promoter. In some such embodiments, a sequence encoding a mini-U6 pol III promoter is 3’ to the tRNA promoter. In other such embodiments, a sequence encoding a Ml 1 pol III promoter is 3’ to the tRNA promoter. In some embodiments wherein the nucleic acid encoding the hybrid promoter is in 5’ to 3’ order, a sequence encoding a chimeric promoter (such as a chimeric promoter comprising a mini-U6 pol III promoter and a 7SK pol III promoter) is 5 ’ to the tRNA promoter. In some embodiments wherein the nucleic acid encoding the hy brid promoter is in 3’ to 5’ order, the sequence encoding the chimeric promoter (such as a chimeric promoter composing a mini-U6 pol III promoter and a 7SK pol III promoter) is 3’ to the tRNA promoter.

[0193] In some embodiments, the hybrid promoter comprises, in 5’ to 3’ orientation: 5’-U6-g- sgRN A-3 ’ .

[0194] In some embodiments, the hybrid promoter comprises, in 5’ to 3’ orientation: 5’-miniU6- gtggc-tRNAAsFMut-sgRNA-3 ’ .

[0195] In some embodiments, the hybrid promoter comprises, in 5’ to 3’ orientation: 5’-miniU6- gta-tRN AAspMut-sgRN A-3 ’ .

[0196] In some embodiments, the hybrid promoter comprises, in 5’ to 3’ orientation: 5’-mmiU6- gtggc-tRNAAspMut-g-sgRN A-3 ’ .

[0197] In some embodiments, the hybrid promoter comprises, in 5’ to 3’ orientation: 5’-miniU6- gte-tRNAAspMur-g-sgRNA-3 ’ .

[0198] In some embodiments, a nucleic acid comprising a sequence encoding a hybrid promoter further comprises a sequence encoding a first sgRNA and a sequence encoding a second sgRNA. In some such embodiments, the sequence encoding the hybrid promoter comprises, as one component, a first tRNA (comprising a first tRNA promoter), and a second tRNA sequence (comprising a second tRN A promoter) separates the first and the second sgRN A in the nucleic acid. In such embodiments, the first tRNA sequence (comprising the first tRN A promoter) may be the same as or different from the second tRNA sequence. In some embodiments, the first tRNA sequence is the same as the second tRNA sequence. In other embodiments, the first tRNA sequence is different from the second tRNA sequence.

[0199] In some embodiments, atRNA sequence of a hybrid promoter is preceded by a leader sequence disclosed herein. Stated differently, in some embodiments, a leader sequence is located 5’ of (such as directly 5’ of) a tRNA sequence in a nucleic acid disclosed herein. In some embodiments, a tRNA sequence of a hybrid promoter is followed by a trailer sequence disclosed herein. Stated differently, in some embodiments, a trailer sequence is located 3’ of (such as directly 3’ of) atRNA sequence in a nucleic acid disclosed herein. In some embodiments, the trailer sequence is followed by an sgRNA sequence in the nucleic acid. Stated differently, in some embodiments, an sgRNA sequence is located 3’ of the trailer sequence m a disclosed nucleic acid.Scaffold Sequences

[0200] A single-molecule guide RNA (sgRNA) can comprise, in the 5’ to 3’ direction, an optional spacer extension sequence, a spacer sequence, a minimum CRISPR repeat sequence, a single-molecule guide linker, a minimum tracrRNA sequence, a 3’ tracrRNA sequence and / or an optional tracrRNA extension sequence. The optional tracrRNA extension can comprise elements that contribute additional functionality' (e.g . stability) to the guide RNA. The single-molecule guide linker can link the minimum CRISPR repeat and the minimum tracrRNA sequence to form a hairpin structure. The optional tracrRNA extension can comprise one or more hairpins. In particular embodiments, the disclosure provides for an sgRN A comprising a spacer sequence and a tracrRNA sequence.

[0201] In some embodiments, each of the guide sequences encoded by a nucleic acid disclosed herein further comprises a scaffold sequence.

[0202] The guide RN A can be considered to comprise a scaffold sequence necessary for endonuclease binding and a spacer sequence required to bind to the genomic target sequence. An exemplary scaffold sequence suitable for use with SaCas9 to follow the guide sequence at its 3’ end is:GTITAAGTACTCTGTGCTGGAAACAGCACAGAATCTACn’AAACAAGGCAAAATGCCGTGTTTATCTCGTCAACTTGTTGGCGAGA (SEQ ID NO: 500) in 5’ to 3’ orientation. In someembodiments, an exemplary' scaffold sequence for use with SaCas9 to follow the 3’ end of the guide sequence is a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 500, or a sequence that differs from SEQ ID NO: 500 by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 nucleotides.

[0203] In some embodiments, a variant of an SaCas9 scaffold sequence may be used. In some embodiments, the SaCas9 scaffold to follow the guide sequence at its 3’ end is referred to as ■‘SaScaffoldVl” and is:GTTTTAGTACTCTGGAAACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCTCGT CAACTTGTTGGCGAGAT (SEQ ID NO: 501 ) in 5’ to 3’ orientation. In some embodiments, an exemplary scaffold sequence for use with SaCas9 to follow the 3’ end of the guide sequence is a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%. 96%, 97%, 98%. 99% or 100% identical to SEQ ID NO: 501 , or a sequence that differs from SEQ ID NO: 501 by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 nucleotides.

[0204] In some embodiments, a variant of an SaCas9 scaffold sequence may be used. In some embodiments, tire SaCas9 scaffold to follow the guide sequence at its 3’ end is referred to as “SaScaffoldV2” and is:GTTTAAGTACTCTGTGCTGGAAACAGCACAGAATCTACTTAAACAAGGCAAAATGCCGT GTTTATCTCGTCAACTTGTTGGCGAGAT (SEQ ID NO: 502) in 5’ to 3’ orientation. In some embodiments, an exemplary scaffold sequence for use with SaCas9 to follow the 3’ end of the guide sequence is a sequence that is at least 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 502, or a sequence that differs from SEQ ID NO: 502 by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 nucleotides.

[0205] In some embodiments, a variant of an SaCas9 scaffold sequence may be used. In some embodiments, the SaCas9 scaffold to follow the guide sequence at its 3’ end is referred to as “SaScaffoldV3” and is:GTTTAAGTACTCTGGAAACAGAATCTACTTAAACAAGGCAAAATGCCGTGTTTATCTCGT CAACTTGTTGGCGAGAT (SEQ ID NO: 503) in 5’ to 3’ orientation. In some embodiments, an exemplary scaffold sequence for use with SaCas9 to follow the 3 ’ end of tire guide sequence is a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 503, or a sequence that differs from SEQ ID NO: 503 by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 nucleotides.

[0206] In some embodiments, a variant of an SaCas9 scaffold sequence may be used. In some embodiments, the SaCas9 scaffold to follow the guide sequence at its 3’ end is referred to as “SaScaffoldV5” and is:GTTTCAGTACTCTGGAAACAGAATCTACTGAAACAAGGCAAAATGCCGTGTTTATCTCGT CAACTTGTTGGCGAGAT (SEQ ID NO: 504) in 5’ to 3’ orientation. In some embodiments, an exemplary scaffold sequence for use with SaCas9 to follow the 3’ end of the guide sequence is asequence that is at least 80%, 85'% 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 504, or a sequence that differs from SEQ ID NO: 504 by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 nucleotides.

[0207] Two exemplary scaffold sequences suitable for use with SluCas9 to follow the guide sequence at its 3' end is:GTTTTAGTACTCTGGAAACAGAATCTACTGAAACAAGACAATATGTCGTGTTTATCCCATCAATTTATTGGTGGGA (SEQ ID NO: 600), andGTTTAAGIACz TCTGTGCTGGAAACAGCACAGAA TCTAC FGAAAC AAGACAATA FGICGT GTTTATCCCATCAATTTATTGGTGGGA (SEQ ID NO: 601) in 5’ to 3’ orientation. In some embodiments, an exemplary sequence for use with SluCas9 to follow the 3’ end of the guide sequence is a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 600 or SEQ ID NO: 601 , or a sequence that differs from SEQ ID NO: 600 or SEQ ID NO: 601 by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 nucleotides.

[0208] Exemplary scaffold sequences suitable for use with SluCas9 to follow the guide sequence at its 3’ end are also shown in Table 3 below in the 5’ to 3’ orientation.Table 3:

[0209] In some embodiments, the scaffold sequence suitable for use with SaCas9 to follow the guide sequence at its 3’ end is selected from any one of SEQ ID NOs: 500-504 in 5’ to 3 orientation. In some embodiments, an exemplary sequence for use with SaCas9 to follow the 3’ end of the guide sequence is a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one off SEQ ID NOs: 500-504, or a sequence that differs from any one of SEQ ID NOs: 500-504 by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 nucleotides.

[0210] In some embodiments, the scaffold sequence suitable for use with SluCas9 to follow the guide sequence at its 3’ end is selected from any one of SEQ ID NOs: 900 or 601 , or 901-917 in 5’ to 3 orientation. In some embodiments, an exemplary sequence for use with SIuCas9 to follow the 3’ end of tire guide sequence is a sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one off SEQ ID NOs: 900 or 601, or 901- 917, or a sequence that differs from any one of SEQ ID NOs: 900 or 601 , or 901-917 by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 nucleotides.[0021 1] In some embodiments, the nucleic acid encoding the hybrid promoter comprises a sequence comprising SEQ ID NO: 500. In some embodiments, the nucleic acid encoding the hybrid promoter comprises a sequence comprising SEQ ID NO: 501. In some embodiments, the nucleic acid encoding the hybrid promoter comprises a sequence comprising SEQ ID NO: 502. In some embodiments, the nucleic acid encoding the hybrid promoter comprises a sequence comprising SEQ ID NO: 503. In some embodiments, the nucleic acid encoding the hybrid promoter comprises a sequence comprising SEQ ID NO: 504. In some embodiments the nucleic acid encoding the hybrid promoter comprises a sequence selected from any one of SEQ ID NOs: 500-504. In some embodiments, both of the sgRNAs comprise a sequence selected from any one of SEQ ID NOs: 500- 504 (i.e., they both comprise the same scaffold sequence). In some embodiments, the nucleotides 3’ of the guide sequence of both sgRNAs are the same sequence. In some embodiments, the nucleotides 3’ of the guide sequence of both sgRNAs are different sequences, but still use with the same class, type, subtype, and / or species of endonuclease.

[0212] In some embodiments, the nucleic acid encoding the hybrid promoter comprises a sequence comprising SEQ ID NO: 900. In some embodiments, the nucleic acid encoding the hybrid promoter comprises a sequence comprising SEQ ID NO: 601 . In some embodiments, the nucleic acid encoding the hybrid promoter comprises a sequence comprising SEQ ID NO: 900. In some embodiments, the nucleic acid encoding the hybrid promoter comprises a sequence comprising SEQ ID NO: 901. In some embodiments, the nucleic acid encoding the hybrid promoter comprises a sequence comprising SEQ ID NO: 902, In some embodiments, the nucleic acid encoding the hybrid promoter comprises a sequence comprising SEQ ID NO: 903. In some embodiments, the nucleic acid encoding the hybrid promoter comprises a sequence comprising SEQ ID NO: 904. In some embodiments, tire nucleic acid encoding the hybrid promoter comprises a sequence comprising SEQ ID NO: 905. In some embodiments, the nucleic acid encoding the hybrid promoter comprises a sequence comprising SEQ ID NO: 906. In some embodiments, the nucleic acid encoding the hybrid promoter comprises a sequence comprising SEQ ID NO: 907. In some embodiments, the nucleic acid encoding the hybrid promoter comprises a sequence comprising SEQ ID NO: 908. In some embodiments, the nucleic acid encoding the hybrid promoter comprises a sequence comprising SEQ ID NO: 909. In some embodiments, the nucleic acid encoding the hybrid promoter comprises a sequence comprising SEQ ID NO: 910. In some embodiments, the nucleic acid encoding the hybrid promoter comprises a sequence comprising SEQ ID NO: 911. In some embodiments, the nucleic acid encoding the hybrid promoter comprises a sequence comprising SEQ ID NO: 912. In some embodiments, the nucleic acid encoding the hybrid promoter comprises a sequence comprising SEQ ID NO: 913. In some embodiments, the nucleic acid encoding the hybrid promoter comprises a sequence comprising SEQ ID NO: 914. In some embodiments, the nucleic acid encoding the hybrid promoter comprises a sequence comprising SEQ ID NO: 915. In some embodiments, the nucleic acid encoding the hybrid promoter comprises a sequence comprising SEQ ID NO: 916. In someembodiments, the nucleic acid encoding the hybrid promoter comprises a sequence comprising SEQ ID NO: 917. In some embodiments, one of the sgRNAs comprises a sequence selected from any one of SEQ ID NOs: 900 or 601, or 901-917, and the other comprises the same or different scaffold sequence, but even if a different sequence is used, the different scaffolds are capable of interacting with the same class, type, subtype, and / or species of endonuclease. In some embodiments, both of the sgRNAs comprise a sequence selected from any one of SEQ ID NOs: 900 or 601, or 901-917. In some embodiments, the nucleotides 3‘ of tire guide sequence of the sgRNAs are the same sequence. In some embodiments, the nucleotides 3’ of the guide sequence of the sgRNAs are different sequences.

[0213] In some embodiments, the scaffold sequence(s) comprises one or more alterations in the stem loop 1 as compared to the stem loop 1 of a wildtype SluCas9 scaffold sequence (e.g., a scaffold comprising the sequence of SEQ ID NO: 900) or a reference SluCas9 scaffold sequence (e.g., a scaffold comprising the sequence of SEQ ID NO: 901 ). In some embodiments, the scaffold sequence comprises one or more alterations in the stem loop 2 as compared to the stem loop 2 of a wildtype SluCas9 scaffold sequence (e.g., a scaffold comprising the sequence of SEQ ID NO: 900) or a reference SluCas9 scaffold sequence (e.g., a scaffold comprising the sequence of SEQ ID NO: 901). In some embodiments, the scaffold sequence comprises one or more alterations in the tetraloop as compared to the tetraloop of a wildtype SluCas9 scaffold sequence (e.g., a scaffold comprising the sequence of SEQ ID NO: 900) or a reference SluCas9 scaffold sequence (e.g., a scaffold comprising the sequence of SEQ ID NO: 901). In some embodiments, the scaffold sequence comprises one or more alterations in the repeat region as compared to the repeat region of a wildtype SluCas9 scaffold sequence (e.g., a scaffold comprising the sequence of SEQ ID NO: 900) or a reference SluCas9 scaffold sequence (e.g., a scaffold comprising the sequence of SEQ ID NO: 901). In some embodiments, the scaffold sequence comprises one or more alterations in the anti-repeat region as compared to the anti-repeat region of a wildtype SluCas9 scaffold sequence (e.g., a scaffold comprising the sequence of SEQ ID NO: 900) or a reference SluCas9 scaffold sequence (e.g., a scaffold comprising the sequence of SEQ ID NO: 901). In some embodiments, the scaffold sequence comprises one or more alterations in the linker region as compared to the linker region of a wildtype SluCas9 scaffold sequence (e.g., a scaffold comprising the sequence of SEQ ID NO: 900) or a reference SluCas9 scaffold sequence (e.g., a scaffold comprising the sequence of SEQ ID NO: 901). See, e.g., Nishimasu et al., 2015, Cell, 162: 1113-1126 for description of regions of a scaffold.

[0214] In some embodiments, an sgRNA comprises (5’ to 3’) at least a spacer sequence, a first complementary’ domain, a linking domain, a second complementary domain, and a proximal domain. A sgRNA or tracrRNA may further comprise a tail domain. Tire linking domain may be hairpinforming. See, e.g., US 2017 / 0007679 for detailed discussion and examples of crRNA and gRNA domains, including second complementarity domains, linking domains, proximal domains, and tail domains.

[0215] The disclosure contemplates RNA equivalents of any of the DNA sequences provided herein (i.e., in which “T’s are replaced with “U”s), or DNA equivalents of any of the RNA sequences provided herein (i.e., in which “U”s are replaced with ‘T’s), as well as complements (including reverse complements) of any of the sequences disclosed herein.Delivery of Guide RNA Compositions; Vectors

[0216] The nucleic acids and compositions disclosed herein may be delivered in vitro or in vivo using any suitable approach for delivering nucleic acids. Exemplary delivery approaches include lipid deliver}' vehicles, nanoparticles, vectors, and electroporation. Accordingly, in some embodiments, a disclosed nucleic acid comprising a sequence encoding a hybrid promoter, and / or a nucleic acid encoding an endonuclease, if present, are associated with a lipid nanoparticle (LNP) or with a vector, such as a viral vector.

[0217] LNPs are a known means for delivery’ of nucleotide and protein cargo, and may be used for delivery of the hybrid promoters, compositions, or pharmaceutical formulations disclosed herein. In some embodiments, the LNPs deliver nucleic acid, protein, or nucleic acid together with protein.

[0218] Electroporation is a well-known means for delivery of cargo, and any electroporation methodology may be used for delivering a nucleic acid comprising a sequence encoding a hybrid promoter disclosed herein.

[0219] In some embodiments, a nucleic acid comprising a sequence encoding a hybrid promoter is delivered in vivo for human therapeutic purposes.

[0220] In some embodiments, a nucleic acid comprising a sequence encoding a hybrid promoter is delivered ex vivo (in vitro) for therapeutic purposes.

[0221] In some embodiments, a nucleic acid comprising a sequence encoding a hybrid promoter is delivered ex vivo (in vitro) for non-therapeutic purposes, e.g., research purposes.

[0222] The nucleic acid encoding the hybrid promoter may be a vector.

[0223] Any suitable type of vector, such as any of those described herein, may be used. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a non -integrating viral vector (i.e., that does not insert sequence from the vector into a host chromosome). In some embodiments, the viral vector is an adeno-associated virus vector (AAV), a lentiviral vector, an integrase-deficient lentiviral vector, an adenoviral vector, a vaccinia viral vector, an alpbaviral vector, or a herpes simplex viral vector. In some embodiments, the vector comprises a muscle-specific promoter. Exemplary muscle -specific promoters include a muscle creatine kinase promoter, a desmin promoter, an MHCK7 promoter, or an SPc5-12 promoter. See US 2004 / 0175727 Al; Wang et al.. Expert Opin Drug Deiiv. (2014) 11, 345-364; Wang et al., Gene Therapy (2008) 15, 1489-1499. In some embodiments, the muscle-specific promoter is a CK8 promoter. In some embodiments, the muscle-specific promoter is a CK8e promoter. In any of the foregoing embodiments, the vector maybe an adeno-associated virus vector (AAV).

[0224] In some embodiments, the viral vector is an adeno-associated virus vector, a lentiviral vector, an integrase-deficient lentiviral vector, an adenoviral vector, a vaccinia viral vector, an alphaviral vector, or a herpes simplex viral vector. In some embodiments, the viral vector is an adeno- associated virus (AAV) vector. In some embodiments, the AAV vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrhl O (see, e.g., SEQ ID NO: 81 of US 9,790,472, which is incorporated by reference herein in its entirety), AAVrh74 (see, e.g., SEQ ID NO: 1 of US 2015 / 0111955, which is incorporated by reference herein in its entirety), or AAV 9 vector, wherein the number following AAV indicates the AAV serotype. In some embodiments, the AAV vector is a single-stranded AAV (ssAAV). In some embodiments, the AAV vector is a double-stranded AAV (dsAAV). Any variant of an AAV vector or serotype thereof, such as a self-complementary AAV (scAAV) vector, is encompassed within the general terms AAV vector, AAV1 vector, etc. See, e.g., McCarty et al.. Gene Ther. 2001 ;8: 1248-54, Naso et al,, BioDrugs 2017; 31:317-334, and references cited therein for detailed discussion of various AAV vectors. In particular embodiments, the vector is an AAV9 vector.

[0225] In some embodiments, tire AAV vector size is measured in length of nucleotides from ITR to ITR, inclusive of both ITRs. In some embodiments, the AAV vector is less than 5 kb in size from ITR to ITR, inclusive of both ITRs. In particular embodiments, the AAV vector is less than 4.9 kb from ITR to ITR in size, inclusive of both ITRs. In further embodiments, the AAV vector is less than 4.85 kb in size from ITR to ITR, inclusive of both ITRs. In further embodiments, the AAV vector is less than 4,8 kb in size from ITR to ITR, inclusive of both ITRs. In further embodiments, the AAV vector is less than 4.75 kb in size from ITR to ITR, inclusive of both ITRs. In further embodiments, the AAV vector is less than 4.7 kb in size from ITR to ITR, inclusive of both ITRs. In some embodiments, the vector is between 3.9-5 kb, 4-5 kb, 4.2-5 kb, 4.4-5 kb, 4.6-5 kb, 4.7-5 kb, 3,9- 4.9 kb, 4.2-4.9 kb, 4.4-4.9 kb, 4.7-4,9 kb, 3.9-4.85 kb, 4.2-4.85 kb, 4.4-4.85 kb, 4.6-4.85 kb, 4.7-4.85 kb, 4.7-4.9 kb, 3.9-4.8 kb, 4.2-4.8 kb, 4.4-4.8 kb or 4.6-4.8 kb from ITR to ITR in size, inclusive of both ITRs. In some embodiments, the vector is between 4.4-4.85 kb in size from ITR to ITR, inclusive of both ITRs.

[0226] In some embodiments, the vector comprises multiple inverted terminal repeats (ITRs). These ITRs may be of an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 serotype. In some embodiments, the ITRs are of an AAV2 serotype. In some embodiments, the 5’ ITR comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 709:GGCCACT CCCTC TCTGCGCGCTCGC I CGC rCACTGAGGCCGGGCGACCAAAGGICGCCCG ACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGG CCAACTCCATCACTAGGGGTTCCT.

[0227] In some embodiments, the 3’ ITR comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 710:AGGAACCCC TAG FGAIGGAGI TGGCCACT CCCT C FC TGCGCGCT CGC FCGC TCACTGAGG CCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAG CGAGCGCGCAG AGAGGGA .

[0228] In some embodiments, in addition to a hybrid promoter sequence, a vector further encodes additional components, such as one or more enhancers, regulatory sequences, and / or additional promoters. In some embodiments, the vector comprises a nucleic acid encoding a Cas9 protein (e.g., an SaCas9 or SluCas9 protein). In some embodiments, the nucleic acid encoding the Cas9 protein is under the control of an additional promoter, such as a CK8e promoter or a CBh promoter.Ribonudeoprotein Complexes

[0229] Compositions are provided herein, comprising (a) a nucleic acid comprising a sequence encoding any of the promoters disclosed herein (e.g., any of the hybrid promoters or chimeric promoters disclosed herein)and (b) one or more endonucleases, such as a Cas9 endonuclease, including any of the Cas9 proteins disclosed herein. In some embodiments, the nucleic acid comprising the sequence encoding the promoter together with a Cas9 is called a ribonucleoprotein complex (RNP).

[0230] In some embodiments, a single sgRNA may be associated with multiple endonucleases (e.g., multiple Cas9 proteins), thereby forming multiple RNPs. In some embodiments, a nucleic acid comprising a sequence encoding a promoter further comprises one or more sequences encoding one or more sgRNAs (such as a first sequence encoding a first sgRNA and a second sequence encoding a second sgRNA). In some such embodiments, expression of the first sequence encoding the first sgRNA and expression of the second sequence encoding the second sgRNA is under the control of the promoter. In some embodiments, a first RNP comprising an endonuclease (e.g., a Cas9 protein) and the first sgRNA that was expressed under the control of the promoter binds to a first target genomic sequence at the same time as a second RNP comprising an endonuclease (e.g., another Cas9 protein) and the second sgRN A that was expressed under the control of the promoter binds to a second target genomic sequence. In some embodiments, a first RNP comprising an endonuclease (e.g., a Cas9 protein) and the first sgRNA cuts at a first target genomic sequence at the same time as a second RNP comprising an endonuclease (e.g., another Cas9 protein) and the second sgRNA cuts at a second target genomic sequence. In some embodiments, a first RNP comprising an endonuclease (e.g,, a Cas9 protein) and the first sgRNA binds to a target genomic sequence at the same time as a second RNP comprising an endonuclease (e.g., another Cas9 protein) and the second sgRNA binds to a target sequence in a separate polynucleotide (e.g., a polynucleotide comprising a donor template). In some embodiments, a first RNP comprising an endonuclease (e.g., a Cas9 protein) and the first sgRNA cutsat a target genomic sequence at the same time as a second RNP comprising an endonuclease (e.g., another Cas9 protein) and the second sgRNA cuts at a target sequence in a separate polynucleotide (e.g., a polynucleotide comprising a donor template). In some embodiments, a second RNP comprising an endonuclease (e.g., a Cas9 protein) and the second sgRNA binds to a target genomic sequence at the same time as a first RNP comprising an endonuclease (e.g., another Cas9 protein) and the first sgRNA binds to a target sequence in a separate polynucleotide (e.g., a polynucleotide comprising a donor template). In some embodiments, a second RNP comprising an endonuclease (e.g,, a Cas9 protein) and the second sgRNA cuts at a target genomic sequence at the same time as a first RNP comprising an endonuclease (e.g., another Cas9 protein) and the first sgRNA cuts at a target sequence in a separate polynucleotide (e.g., a polynucleotide comprising a donor template).

[0231] In some embodiments, the disclosure provides for an RNP complex, wherein each guide RNA (e.g. , a first sgRNA and a second sgRNA that were expressed under the control of a promoter disclosed herein) binds to or is capable of binding to a target sequence in the dystrophin gene.

[0232] In some embodiments, chimeric Cas9 (SaCas9 or SluCas9) nucleases are used, where one domain or region of the protein is replaced by a portion of a different protein. In some embodiments, a Cas9 nuclease domain may be replaced with a domain from a different nuclease such as Fokl. In some embodiments, a Cas9 nuclease may be a modified nuclease.

[0233] In some embodiments, the Cas9 is modified to contain only one functional nuclease domain. For example, the agent protein may be modified such that one of the nuclease domains is mutated or fully or partially deleted to reduce its nucleic acid cleavage activity.

[0234] In some embodiments, a conserved amino acid within a Cas9 protein nuclease domain is substituted to reduce or alter nuclease activity. In some embodiments, a Cas9 nuclease may comprise an amino acid substitution in the RuvC or RuvC-like nuclease domain. Exemplary ammo acid substitutions in the RuvC or RuvC-like nuclease domain include D10A (based on the S. pyogenes Cas9 protein). See, e.g. , Zetsche et al. (2015) Cell Oct 22: 163(3): 759-771. In some embodiments, the Cas9 nuclease may comprise an amino acid substitution in the HNH or HNH-like nuclease domain. Exemplary amino acid substitutions in the HNH or HNH-like nuclease domain include E762A, H840A, N863A, H983A, and D986A (based on the S. pyogenes Cas9 protein, see SEQ ID NO: 730). See, e.g., Zetsche et al. (2015). Further exemplary amino acid substitutions include D917A, E1006A, and DI 255 A (based on the Francisella novicida U1 12 Cpfl (FnCpfl) sequence (UniProtKB - A0Q7Q2 (CPFI FRATN)). Further exemplary amino acid substitutions include DI0A and N580A (based on the 5. aureus Cas9 protein). See, e.g., Friedland et al., 2015, Genome Biol., 16:257.

[0235] In some embodiments, the Cas9 lacks cleavase activity. In some embodiments, the Cas9 comprises a dCas DNA-binding polypeptide. A dCas polypeptide has DNA-binding activity while essentially lacking catalytic (cleavase / nickase) activity. In some embodiments, the dCas polypeptide is a dCas9 polypeptide. In some embodiments, the Cas9 lacking cleavase activity or the dCas DNA- binding polypeptide is a version of a Cas nuclease (e.g., a Cas9 nuclease discussed above) in which itsendonucleolytic active sites are inactivated, e.g., by one or more alterations (e.g., point mutations) in its catalytic domains. See, e.g., US 2014 / 0186958 Al; US 2015 / 0166980 Al.

[0236] In some embodiments, the Cas9 comprises one or more heterologous functional domains (e.g., is or comprises a fusion polypeptide).

[0237] In some embodiments, the heterologous functional domain may facilitate transport of the Cas9 into the nucleus of a cell. For example, the heterologous functional domain may be a nuclear localization signal (NLS). In some embodiments, the Cas9 may be fused with 1-10 NLS(s). In some embodiments, the Cas9 may be fused with 1-5 NLS(s). In some embodiments, the Cas9 may be fused with 1-3 NLS(s). In some embodiments, the Cas9 may be fused with one NLS. Where one NLS is used, the NLS may be attached at the N-terminus or the C-terminus of the Cas9 sequence, and may be directly fused / attached. In some embodiments, where more than one NLS is used, one or more NLS may be attached at the N-terminus and / or one or more NLS may be atached at the C-terminus. In some embodiments, one or more NLSs are directly attached to the Cas9. In some embodiments, one or more NLSs are attached to the Cas9 by means of a linker. In some embodiments, the linker is between 3-25 amino acids in length. In some embodiments, the linker is between 3-6 amino acids in length. In some embodiments, the linker comprises glycine and serine. In some embodiments, the linker comprises the sequence of GSVD (SEQ ID NO: 550) or GSGS (SEQ ID NO: 551 ). It may also be inserted within the Cas9 sequence. In other embodiments, the Cas9 may be fused with more than one NLS. In some embodiments, the Cas9 may be fused with 2, 3, 4, or 5 NLSs. In some embodiments, the Cas9 may be fused with two NLSs. In certain circumstances, the two NLSs may be the same (e.g., two SV40 NLSs) or different. In some embodiments, the Cas9 protein is fused with one or more SV40 NLSs. In some embodiments, the SV40 NLS comprises the amino acid sequence of SEQ ID NO: 713 (PKKKRKV). In some embodiments, tire Cas9 protein (e.g., the SaCas9 or SluCas9 protein) is fused to one or more nucleoplasmin NLSs. In some embodiments, the Cas protein is fused to one or more c-myc NLSs. In some embodiments, the Cas protein is fused to one or more El A NLSs. In some embodiments, the Cas protein is fused to one or more BP (bipartite) NLSs. In some embodiments, the nucleoplasmin NLS comprises the amino acid sequence of SEQ ID NO: 714 (KRPAATKKAGQAKKKK). In some embodiments, the Cas9 protein is fused with a c-Myc NLS. In some embodiments, the c-Myc NLS is SEQ ID NO: 942 (PAAKKKKLD). In some embodiments, the c-Myc NLS is encoded by the nucleic acid sequence of SEQ ID NO: 722 (CCGGCAGCTAAGAAAAAGAAACTGGAT). In some embodiments, the Cas9 is fused to two SV40 NLS sequences linked at tire carboxy terminus. In some embodiments, the Cas9 may be fused with two NLSs, one linked at the N-terminus and one at the C-terminus. In some embodiments, theCas9 may be fused with 3 NLSs. In some embodiments, the Cas9 may be fused with 3 NLSs, two linked at the N-terminus and one linked at the C-terminus. In some embodiments, the Cas9 may be fused with 3 NLSs, one linked at the N-terminus and two linked at the C-terminus. hi some embodiments, the Cas9 may be fused with no NLS. In some embodiments, the Cas9 may be fusedwith one NLS. In some embodiments, the Cas9 may be fused with an NLS on the C-terminus and does not comprise an NLS fused on the N-tenninus. In some embodiments, the Cas9 may be fused with an NLS on the N-terminus and does not comprise an NLS fused on the C-terminus. In some embodiments, the Cas9 protein is fused to an SV40 NLS and to a nucleoplasmin NLS. In some embodiments, the Cas9 protein is fused to an SV40 NLS and to a c-Myc NLS. In some embodiments, the SV40 NLS is fused to the C-terminus of the Cas9, while the nucleoplasmin NLS is fused to the N- terminus of the Cas9 protein. In some embodiments, the SV40 NLS is fused to the C-terminus of the Cas9, while the c-Myc NLS is fused to the N-terminus of the Cas9 protein. In some embodiments, the SV40 NLS is fused to the N-terminus of the Cas9, while the nucleoplasmin NLS is fused to the C- terminus of the Cas9 protein. In some embodiments, the SV40 NLS is fused to the N-terminus of the Cas9, while the c-Myc NLS is fused to the C-terminus of the Cas9 protein. In some embodiments, tire SV40 NLS is fused to the Cas9 protein by means of a linker. In some embodiments, the S V40 NLS and linker is encoded by the nucleic acid sequence of SEQ ID NO: 723 (ATGATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCC). In some embodiments, the nucleoplasmin NLS is fused to the Cas9 protein by means of a linker. In some embodiments, the c-Myc NLS is fused to the Cas9 protein by means of a linker. In some embodiments, an additional domain may be: a) fused to the N- or C-terminus of the Cas protein (e.g., a Cas9 protein), b) fused to the N-tenninus of an NLS fused to the N-terminus of a Cas protein, or c) fused to the C-terminus of an NLS fused to the C-terminus of a Cas protein. In some embodiments, an NLS is fused to the N- and / or C-terminus of the Cas protein by means of a linker. In some embodiments, an NLS is fused to the N-terminus of an N-terminally-fused NLS on a Cas protein by means of a linker, and / or an NLS is fused to the C-terminus of a C-terminally fused NLS on a Cas protein by means of a linker. In some embodiments, the linker is between 3-15, 3-12, 3-10, 3-8, 3-5 ammo acids in length. In some embodiments, the linker comprises glycine. In some embodiments, the linker comprises serine. In some embodiments, the linker is GSVD (SEQ ID NO: 550) or GSGS (SEQ ID NO: 551). In some embodiments, the Cas protein comprises a c-Myc NLS fused to the N- terminus of the Cas protein (or to an N-terminally-fused NLS on the Cas protein), optionally by means of a linker. In some embodiments, the Cas protein comprises an SV40 NLS fused to the C- termmus of the Cas protein (or to a C -terminally-fused NLS on the Cas protein), optionally by means of a linker. In some embodiments, the Cas protein comprises a nucleoplasmin NLS fused to the C- tenninus of the Cas protein (or to a C -terminally-fused NLS on the Cas protein), optionally by means of a linker. In some embodiments, the Cas protein comprises: a) a c-Myc NLS fused to the N- terminus of the Cas protein, optionally by means of a linker, b) an SV40 NLS fused to the C-terminus of the Cas protein, optionally by means of a linker, and c) a nucleoplasmin NLS fused to the C- terminus of the SV40 NLS, optionally by means of a linker. In some embodiments, the Cas protein comprises: a) a c-Myc NLS fused to the N-terminus of the Cas protein, optionally by means of a linker, b) a nucleoplasmin NLS fused to the C-terminus of the Cas protein, optionally by means of alinker, and c) an SV40 NLS fused to the C-terminus of the nucleoplasmin NLS, optionally by means of a linker. In some embodiments, a c-myc NLS is fused to the N-terminus of the Cas9 and an SV40 NLS and / or nucleoplasmin NLS is fused to the C-terminus of tire Cas9. In some embodiments, a c- myc NLS is fused to the N-tenninus of the Cas9 (e.g., by means of a linker such as GSVD), an SV40 NLS is fused to the C-terminus of the Cas9 (e.g., by means of a linker such as GSGS), and a nucleoplasmin NLS is fused to the C-terminus of the SV-40 NLS (e.g., by means of a linker such as GSGS).

[0238] In some embodiments, the heterologous functional domain may be capable of modifying the intracellular half-life of the Cas9. In some embodiments, the half-life of the Cas9 may be increased. In some embodiments, the half-life of the Cas9 may be reduced. In some embodiments, the heterologous functional domain may be capable of increasing the stability of the Cas9. In some embodiments, the heterologous functional domain may be capable of reducing the stability of the Cas9. In some embodiments, the heterologous functional domain may act as a signal peptide for protein degradation. In some embodiments, the protein degradation may be mediated by proteolytic enzymes, such as, for example, proteasomes, lysosomal proteases, or caipain proteases. In some embodiments, the heterologous functional domain may comprise a PEST sequence. In some embodiments, the Cas9 may be modified by addition of ubiquitin or a polyubiquitin chain. In some embodiments, the ubiquitin may be a ubiquitin-like protein (UBL). Non-limiting examples of ubiquitin-like proteins include small ubiquitin-like modifier (SL!MO), ubiquitin cross-reactive protein (UCRP, also known as interferon-stimulated gene-15 (ISG15)), ubiquitin-related modifier-] (URM1), neuronal-precursor-cell-expressed developmentally downregulated protein-8 (NEDD8, also called Rubl in 5. cerevisiae), human leukocyte antigen F-associated (FAT10), autophagy-8 (ATG8) and -12 (ATG12), Fau ubiquitin-like protein (FUB1), membrane-anchored UBL (MUB), ubiquitin foldmodifier-1 (UFM1), and ubiquitin-like protein-5 (UBL5).

[0239] In some embodiments, the heterologous functional domain may be a marker domain. Non-limiting examples of marker domains include fluorescent proteins, purification tags, epitope tags, and reporter gene sequences. In some embodiments, the marker domain may be a fluorescent protein. Non-limiting examples of suitable fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, sfGFP, EGFP, Emerald, Azami Green, Monomeric Azami Green, CopGFP, AceGFP, ZsGreenl), yellow fluorescent proteins (e.g., YFP, EYFP, Citrine, Venus, YPet, PhiYFP, ZsYellowl), blue fluorescent proteins (e.g., EBFP, EBFP2, Azurite, mKaiamal, GFPuv, Sapphire, T-sapplnre,), cyan fluorescent proteins (e.g., ECFP, Cerulean, CyPet, AmCyanl, Midori ishi-Cyan), red fluorescent proteins (e.g., mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFPl, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRedl, AsRed2, eqFP61 1, mRasberry, mStrawberry, Jred), and orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange, niTangerine, tdTomato) or any other suitable fluorescent protein. In other embodiments, the marker domain may be a purification tag and / or an epitope tag. Non-limitingexemplary tags include glutathione-S-transferase (GST), chitin binding protein (CBP), maltose binding protein (MBP), thioredoxin (TRX), poly(NANP), tandem affinity purification (TAP) tag, myc, AcV5, AUl, AU5, E, ECS, E2, FLAG, HA, nus, Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, SI, T7, V5, VSV-G, 6xHis, 8xHis, biotin carboxyl carrier protein (BCCP), poly-His, and calmodulin. Non-limiting exemplary reporter genes include glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, betaglucuronidase, luciferase, or fluorescent proteins.

[0240] In additional embodiments, the heterologous functional domain may target the Cas9 to a specific organelle, cell type, tissue, or organ. In some embodiments, the heterologous functional domain may target the Cas9 to muscle.[002.41] In further embodiments, the heterologous functional domain may be an effector domain. When the Cas9 is directed to its target sequence, e.g., when a Cas9 is directed to a target sequence by a guide RNA, the effector domain may modify or affect the target sequence. In some embodiments, the effector domain may be chosen from a nucleic acid binding domain or a nuclease domain (e.g., a non-Cas nuclease domain). In some embodiments, the heterologous functional domain is a nuclease, such as a FokI nuclease. See, e.g., US Pat. No. 9,023,649.

[0242] In some embodiments, any of the compositions disclosed herein comprising any of the guides and / or endonucleases disclosed herein is sterile and / or substantially pyrogen-free. In particular embodiments, any of the compositions disclosed herein comprise a pharmaceutically acceptable carrier. The phrase “pharmaceutically or pharmacologically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal or human. As used herein “pharmaceutically acceptable carrier” includes any and all solvents (e.g., water), dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible, including pharmaceutically acceptable cell culture media. Pharmaceutically acceptable earners include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In some embodiments, the composition comprises a preservative to prevent the growth of microorganisms.Endonucleases

[0243] In some embodiments, a nucleic acid disclosed herein (such as a nucleic acid comprising a sequence encoding any of the promoters disclosed herein) comprises a sequence encoding an RNA- targeted endonuclease. Also provided herein are compositions comprising a nucleic acid disclosed herein and an endonuclease or a nucleic acid encoding an endonuclease. In some embodiments, the RNA-targeted endonuclease has cleavase activity, which can also be referred to as double-strand endonuclease activity. In some embodiments, the RNA-targeted endonuclease comprises a Cas nuclease. Examples of Cas9 nucleases include those of the type II CRISI’R systems.

[0244] In some embodiments, the Cas protein comprises an amino add sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 730 (designated herein as SpCas9):MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEAT RLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDE VAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQL VQTYNQLFEENP1NASGVDAKAILSARLSKSRRLENL1AQLPGEKKNGLFGNLIALSLGLTPNF KSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKA PLSASMIKRYDEiniQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKP ILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK ILTFR1PYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNE KVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTYKQLKE DYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRE MIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRN FMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQl'VKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYIA'YLQ NGRDMYWDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKK MKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGF1KRQLVETRQITKHVAQILDSRM NIXYDENDKLIREVKVII'LKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKY PKLESEFVYGDYKVYDVRK.MIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIET NGETGEIVWDKGRDFAWRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLWAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKE VKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPED NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHI.FTI, TNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD,

[0245] In some embodiments, the nucleic acid encoding SaCas9 encodes an SaCas9 comprising an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 711:KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRI QRVKKLLFDYNLLTDHSELSGWYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEE DTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQK AYHQLDQSFIDTY1DLLETRRIYYEGPGEGSPFGWKDIKEWYEMLMGHCIYFPEELRSVKYA YNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKGY RVTSTGKPEFTNLKWHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEE IEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDD FILSPVVKRSFIQSIKV1NAIIKKYGLPNDIIIELAREKNSKDAQKM1NEMQKRNRQTNERIEEIIR TTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQ KDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKS1NGGFTSFLRRKWKFKKERNKGYK HHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIF1TPHQIKH1 KDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSP EKLLMYHIIDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGN KLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGWKFVTVKNLDVIKKENYYEVNSKC YEEAKKLKKISNQAEF1ASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDI1YREYLENMN DKRPPRIIKUASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG.

[0246] In some embodiments, the nucleic acid encoding SaCas9 comprises the nucleic acid of SEQ ID NO: 9014:AAGCGCAAITACATCCTGGGCCTGGATATCGGCATCACCTCCGTGGGCTACGGCATCATC GACTATGAGACACGGGA TGTGAT CGACGCCGGCGTGAGAC FGT FCAAGGAGGCCAACG T GGAGAACAATGAGGGCCGGCGGAGCAAGAGGGGAGCAAGGCGCCTGAAGCGGAGAAG GCGCCACAGAATCCAGAGAGTGAAGAAGCTGCTGTTCGATTACAACCTGCTGACCGACC ACTCCGAGCTGTCTGGCATCAATCCTIATGAGGCCCGGGTGAAGGGCCTGTCCCAGAAGC I GTCT GAGGAGGAGT FT FC FGCCG CCC FGCT GC ACC I GGCA AAGAGGAGAGGCG IGCA C AACGTGAATGAGGTGGAGGAGGACACCGGCAACGAGCTGAGCACAAAGGAGCAGATCA GCCGCAATTCCAAGGCCCTGGAGGAGAAGTATGTGGCCGAGCTGCAGCTGGAGCGGCTG AAGAAGGATGGCGAGGTGAGGGGCTCCA I C AATCGCT TCAAGACCT CTGACTACGTGAA GGAGGCCAAGCAGC TGCTGAAGGTGC AGAAGGCCT ACC ACCAGCTGGATCAGAGC FT FA TCGATACATATATCGACCTGCTGGAGACCAGGCGCACATACTATGAGGGACCAGGAGAG GGCTCCCCCTTCGGCTGGAAGGACATCAAGGAGTGGTACGAGATGCTGATGGGCCACTG CACCTATTTTCCAGAGGAGCTGAGATCCGTGAAGTACGCC'FATAACGCCGATCTGTACAA CGCCCTGAATGACCTGAACAACCTGGTCATCACCAGGGATGAGAACGAGAAGCTGGAGT ACTATGAGAAGTTCCAGATCATCGAGAACGTGTTCAAGCAGAAGAAGAAGCCTACACTG AAGCAGATCGCCAAGGAGATCCTGGTGAACGAGGAGGACATCAAGGGCTACCGCGTGA CCAGCACAGGCAAGCCAGAGTTCACCAATCTGAAGGTGTATCACGATATCAAGGACATC ACAGCCCGGAAGGAGATCATCGAGAACGCCGAGCTGCI GGA I CAGATCGCCAAGATCCT GACCA TCT A TCAGAGC FCCGAGGACAT CCAGGAGGAGCTGACCAACC FGAAFAGCGAGC TGACACAGGAGGAGATCGAGCAGATCAGCAATCTGAAGGGCTACACCGGCACACACAA CCTGTCCCTGAAGGCCATCAATCTGATCCTGGATGAGCTGTGGCACACAAACGACAATCA GATCGCCATCTTTAACAGGCTGAAGCTGGI’GCCAAAGAAGGTGGACCTGAGCCAGCAGA AGGAGATCCCA ACCACAC TGG FGGACGA FT FCATCCTG TCCCCCG FGGIGAAGCGGAGC F TCATCCAGAGCATCAAAGTGATCAACGCCATCATCAAGAAGTACGGCCTGCCCAATGAT ATCATCATCGAGCTGGCCAGGGAGAAGAACTCTAAGGACGCCCAGAAGATGATCAATGA GATGCAGAAGAGGAACCGCCAGACCAATGAGCGGATCGAGGAGATCATCAGAACCACA GGCAAGGAGAACGCCAAGTACC I GATCGAGAAGATCAAGC TGCACGAFATGCAGGAGGGCAAGTGTCTGTATAGCCTGGAGGCCATCCCTCTGGAGGACCTGCTGAACAATCCATTCA ACTACGAGGTGGATCACATCATCCCCCGGAGCGTGAGCTTCGACAATTCCTTTAACAATA AGGT GCTGG IGAAGC AGGAGGAGAAC I’CTAAGAAGGGCAA I AGGACCCCTI TCCAGTAC CTGTCTAGCTCCGATTCTAAGATCAGCTACGAGACCTTCAAGAAGCACATCCTGAATCTG GCCAAGGGCAAGGGCCGCATCTCTAAGACCAAGAAGGAGTACCTGCTGGAGGAGCGGG ACATCAACAGATTCAGCGTGCAGAAGGACTTCATCAACCGGAATCTGGTGGACACCAGA TACGCCACACGCGGCCTGATGAATCTGCTGCGGTCCTA'nTCAGAGTGAACAATCTGGAT GTGAAGG FGAAGAGCATC AACGGCGGC TTCACCTCCTTT C TGCGGAGAAAGTGGAAGT F TAAGAAGGAGAGAAACAAGGGCTATAAGCACCACGCCGAGGATGCCCTGATCATCGCCA ATGCCGACTTCATCTTTAAGGAGTGGAAGAAGCTGGACAAGGCCAAGAAAGTGATGGAG AACCAGATGTFCGAGGAGAAGCAGGCCGAGAGCATGCCCGAGATCGAGACCGAGCAGG AGTACAAGGAGATTTTCATCACACCTCACCAGATCAAGCACATCAAGGACTTCAAGGAC TACAAGTATTCCCACAGGGTGGATAAGAAGCCCAACCGCGAGCTGATCAATGACACCCT GTATTCTACAAGGAAGGACGATAAGGGCAATACCCTGATCGTGAACAATCTGAACGGCC IGTACGACAAGGATAATGACAAGCTGAAGAAGCTGATCAACAAGAGCCCCGAGAAGCT GCT GATGTACCACCACGATCCT CAGACATA I CAGAAGCT GAAGCTGA TCAT GGAGCAGT ACGGCGACGAGAAGAACCCACTGTATAAGTACTATGAGGAGACCGGCAACTACCTGACA AAGTATTCCAAGAAGGATAATGGCCCCGTGATCAAGAAGATCAAGTACTATGGCAACAA GCTGAATGCCCACCTGGACATCACCGACGATIACCCCAACAGCCGGAATAAGGTGGTGA AGCTGAGCC FGAAGCCATACAGGT FCGACGTGTACCTGGACAACGGCGTG FATAAGT FI GTGACAGTGAAGAATCTGGATGTGATCAAGAAGGAGAACTACTATGAAGTGAATAGCAA GTGCTACGAGGAGGCCAAGAAGCTGAAGAAGATCAGCAACCAGGCCGAGTTCATCGCCT CTTTTTACAACAATGACCIGATCAAGATCAAIGGCGAGCTGTAIAGAGTGAI'CGGCGTGAACAAIGAICTGCTGAACCGCATCGAAGTGAATATGA TCGACAICACC rACCGGGAGIAT CTGGAGAACATGAATGATAAGAGGCCCCCTCGCATCATCAAGACCATCGCCTCTAAGAC ACAGAGCATCAAGAAGTACTCTACAGACATCCTGGGCAACCTGTATGAGGTGAAGAGCA A.GAAGCACCCTCAGATCATCAAGAA.GGGC.

[0247] In some embodiments comprising a nucleic acid encoding SaCas9, the SaCas9 comprises an amino acid sequence of SEQ ID NO: 711.

[0248] In some embodiments, the SaCas9 is a variant of the amino acid sequence of SEQ ID NO: 711. In some embodiments, the SaCas9 comprises an amino acid other than an E at the position corresponding to position 781 of SEQ ID NO: 711. In some embodiments, the SaCas9 comprises an ammo acid other than an N at the position corresponding to position 967 of SEQ ID NO: 711. In some embodiments, the SaCas9 comprises an amino acid other than an R at the position corresponding to position 1014 of SEQ ID NO: 711. In some embodiments, the SaCas9 comprises a K at the position corresponding to position 781 of SEQ ID NO: 711. In some embodiments, the SaCas9 comprises a K at the position corresponding to position 967 of SEQ ID NO: 711. In someembodiments, the SaCas9 comprises an H at the position corresponding to position 1014 of SEQ ID NO: 711. In some embodiments, the SaCas9 comprises an amino acid other than an E at the position corresponding to position 781 of SEQ ID NO: 711; an amino acid other than an N at the position corresponding to position 967 of SEQ ID NO: 711; and an amino acid other than an R at the position corresponding to position 1014 of SEQ ID NO: 71 1. In some embodiments, the SaCas9 comprises a K at the position corresponding to position 781 of SEQ ID NO: 711 ; a K at the position corresponding to position 967 of SEQ ID NO: 711; and an H at the position corresponding to position 1014 of SEQ ID NO: 711 .

[0249] In some embodiments, the SaCas9 comprises an amino acid other than an R at the position corresponding to position 244 of SEQ ID NO: 711. In some embodiments, the SaCas9 comprises an ammo acid other than an N at the position corresponding to position 412 of SEQ ID NO: 71 1. In some embodiments, the SaCas9 comprises an amino acid other than an N at the position corresponding to position 418 of SEQ ID NO: 71 1. In some embodiments, the SaCas9 comprises an amino acid other than an R at the position corresponding to position 653 of SEQ ID NO: 711. In some embodiments, the SaCas9 comprises an amino acid other than an R at the position corresponding to position 244 of SEQ ID NO: 711; an amino acid other than an N at the position corresponding to position 412 of SEQ ID NO: 71 1 ; an amino acid other than an N at the position corresponding to position 418 of SEQ ID NO: 711; and an amino acid other than an R at the position corresponding to position 653 of SEQ ID NO: 711. In some embodiments, the SaCas9 comprises an A at the position corresponding to position 244 of SEQ ID NO: 711 , In some embodiments, the SaCas9 comprises an A at the position corresponding to position 412 of SEQ ID NO: 71 1. In some embodiments, the SaCas9 comprises an A at the position corresponding to position 418 of SEQ ID NO: 711. In some embodiments, the SaCas9 comprises an A at tire position corresponding to position 653 of SEQ ID NO: 711. In some embodiments, the Sat’ as1-’ comprises an A at the position corresponding to position 244 of SEQ ID NO: 71 1 ; an A at the position corresponding to position 412 of SEQ ID NO: 71 1 ; an A at the position corresponding to position 418 of SEQ ID NO: 71 1 ; and an A at the position corresponding to position 653 of SEQ ID NO: 711.

[0250] In some embodiments, the SaCas9 comprises an amino acid other than an R at the position corresponding to position 244 of SEQ ID NO: 71 1 ; an ammo acid other than an N at the position corresponding to position 412 of SEQ ID NO: 711 ; an amino acid other than an N at the position corresponding to position 418 of SEQ ID NO: 711 ; an amino acid other than an R at the position corresponding to position 653 of SEQ ID NO: 711; an amino acid other than an E at the position corresponding to position 781 of SEQ ID NO: 711; an amino acid other than an N at the position corresponding to position 967 of SEQ ID NO: 711; and an amino acid other than an R at the position corresponding to position 1014 of SEQ ID NO: 711. In some embodiments, the SaCas9 comprises an A at the position corresponding to position 244 of SEQ ID NO: 711; an A at the position corresponding to position 412 of SEQ ID NO: 711; an A at the position corresponding to position 418of SEQ ID NO: 711; an A at the position corresponding to position 653 of SEQ ID NO: 711; a K at the position corresponding to position 781 of SEQ ID NO: 711 ; a K at the position corresponding to position 967 of SEQ ID NO: 711; and an H at the position corresponding to position 1014 of SEQ ID NO: 711.

[0251] In some embodiments, the SaCas9 comprises an amino acid sequence that is at least 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 715 (designated herein as SaCas9-KKH or SACAS9KKH):KRNYILGLDIGITS VGYGHDYETRD VIDA GVRLFKE AN VENNEGRR SKRGARRLKRRRRHRI QRVKKLLFDYNLLTDIISELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEE DTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQK AYHQLDQSFIDIYIDLLEI'RRIYYEGPGEGSPFGWKDIKEWYEMLMGHCIYFPEELRSVKYA YNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEIIANiEEDIKGY RVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEEL'INLNSELTQEE IEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDD FILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIR TTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHnPRSVSFDNSFNNKVL VKQEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQ KDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKS1NGGFTSFLRRKWKFKKERNKGYK HHAEDALIlANADFIFKEWKKLDKAKKVMENQMFEEKQAESxMPEIETEQEYKEIFITPHQIKHI KDFKDYKYSHRVDKKPNRKLINDn.YSTRKDDKGNTUVNNLNGLYDKDNDKLKKLINKSP EKLLMYHIIDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGN KLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGWKFVTVKNLDVIKKENYYEVNSKC YEEAKKLKKISNQAEFIASFYKNDLIKINGELYRVIGVNNDLLNRIEVNMIDIlYREYLENxMN DKRPPHIIKTTASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG.

[0252] In some embodiments, the SaCas9 comprises an amino acid sequence that is at least 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 716 (designated herein as SaCas9-HF):KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRI QRVKKLLFD^'NLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNV'NEVEE DTGNELSTKEQ1SRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQK AYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELASVKYA YNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPI'LKQIAKEILVNEEDIKGY RVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEE lEQISNLKGYTGTflNLSLKAINLILDELWinNDAQIAIFARLKLVPKKVDLSQQKEIPTTLVDD FILSPVVKRSFIQSIKV1NAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIR TTGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVL VKQEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFINRNLVDTRYATAGLMNLLRSYFRVNTS1LDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPE1ETEQEYKEIFITPHQIKHI KDFKDYKYSHRVDKKI’NRELINDI'LYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSP EKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGN KLNAHLDITODYPNSRNKWKLSLKPYRFDVYLDNGWKFVTVKNLDVIKKETsTi'YEVNSKC YEEAKICLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMN DKRPPRIIKTIASKTQSIKKYS1I)ILGNLYEVKSKKHPQIIKKG.

[0253] In some embodiments, the SaCas9 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 717 (designated herein as SaCas9-KKH-HF):KRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRIQRVKKLLFD^'NLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNV'NEVEE DTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQK AYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELASVKYA YNADLYNALNDLNNLVIl'RDENEKLEYYEKFQllENVFKQKKKPTLKQLAKEILVNEEDIKGY RVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEE IEQISNLKGYTGTI-INLSLKAINLILDELWI-ITNDAQIAIFARLKLVPKKVDLSQQKEIPTTLVDD FILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIR TrGKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVL VKQEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQ KDFINRNLVDTRYATAGLMNLLRSYFRVN^LDVKVKSINGGFTSFLRRKWKFKKERNKGYK HIMEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHI KDFKDYKYSHRVDKKPNRKL1NDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSP EKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKWGN KLNAFILDITDDYPNSRNKWKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKC YEEAKKLKKISNQAEFIASFYKNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMN DKRPPHIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG.

[0254] In some embodiments, the nucleic acid encoding SluCas9 encodes a SluCas9 comprising an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 712:NQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENNEGRRSKRGSRRLKRRRIHRLE RVKKLLEDYNLLDQSQIPQSTNPYAIRVKGLSEALSKDELVIALLHIAKRRGIHKIDVIDSNDD VGNELSTKEQLNKNSKLLKDKFVCQIQLERMNEGQVRGEKNRFKTADIIKEIIQLLNVQKNFH QLDENFINKYIELVEMRREYFEGPGKGSPYGWEGDPKAWYETLMGIICTYFPDELRSVKYAY SADLFNALNDLNNLVIQRDGLSKLEYHEKYHIIENVFKQKKKPTLKQIANEINVNPEDIKGYRI I'KSGKI’QFTEFKLYHDLKSVLFDQSILENEDVLDQIAEILTIYQDKDSIKSKLTELDILLNEEDK ENIAQLTGYTGTHRLSLKCIRLVLEEQWYSSRNQMEIFTHLNIKPKKINLTAANKIPKAMIDEFILSPWKR’TTGQAINLINKTIEKYGVPEDIIIELARENNSKDKQKFINEMQKKNENTRKRINEIIG KYGNQNAKRLVEKIRLHDEQEGKCLYSLESIPLEDLLNNPNHYEVDHIIPRSVSFDNSYHNKV LVKQSENSKKSNL1TYQYFNSGKSKLSYNQFKQH1LNLSKSQDRISKKKKEYLLEERDINKFE VQKEFINRNLVDTRYATRELTNYLKAYFSANNMNVKVKTINGSFTDYLRKVWKFKKERNH GYKFniAEDALIIANLADFLFKENKKLKAANlSVLEKPEIETKQLDIQVDSEDNYSEMFIIPKQVQ DIKDFRNFKYSHRVDKICPNRQLINDTLYSTRKKDNSTYIVQTIKDIYAKDNTTLKKQFDKSPE KFLMYQHDPR1TEKLEVIMKQYANEKNPLAKYHEETGEYLTKYSKKNNGPIVKSLKYIGNK LGSHLDVTHQFKSSTKKLVKLSIKPYRFDVYLTDKGYKFmSYLDVLKKDNYYYIPEQKYDK LKLGKAIDKNAKFIASFYKNDLIKLDGEIYKIIGVNSDTRNMIELDLPDIRYKEYCELNNIKGEP RIKKTIGKKWSIEKLTTDVLGNVFTNTQYTKPQLLFKRGN.

[0255] In some embodiments, the SluCas9 is a variant of the ammo acid sequence of SEQ ID NO: 712. In some embodiments, the SluCas9 comprises an amino acid other than an Q at the position corresponding to position 781 of SEQ ID NO: 712. In some embodiments, the SluCas9 comprises an amino acid other than an R at the position corresponding to position 1013 of SEQ ID NO: 712. In some embodiments, the SluCas9 comprises a K at the position corresponding to position 781 of SEQ ID NO: 712. In some embodiments, the ShiCas9 comprises a K at the position corresponding to position 966 of SEQ ID NO: 712. In some embodiments, the SluCas9 comprises an II at the position corresponding to position 1013 of SEQ ID NO: 712. In some embodiments, the SluCas9 comprises an amino acid other than an Q at the position corresponding to position 781 of SEQ ID NO: 712; and an ammo acid other than an R at the position corresponding to position 1013 of SEQ ID NO: 712. In some embodiments, the SluCas9 comprises a K at the position corresponding to position 781 of SEQ ID NO: 712; a K at the position corresponding to position 966 of SEQ ID NO: 712; and an II at the position corresponding to position 1013 of SEQ ID NO: 712.

[0256] In some embodiments, the SluCas9 comprises an ammo acid other than an R at the position corresponding to position 246 of SEQ ID NO: 712. In some embodiments, the SluCas9 comprises an amino acid other than an N at the position corresponding to position 414 of SEQ ID NO: 712. In some embodiments, the SluCas9 comprises an amino acid other than a T at the position corresponding to position 420 of SEQ ID NO: 712. In some embodiments, the SluCas9 comprises an amino acid other than an R at the position corresponding to position 655 of SEQ ID NO: 712. In some embodiments, the SluCas9 comprises an amino acid other than an R at the position corresponding to position 246 of SEQ ID NO: 712; an amino acid other than an N at the position corresponding to position 414 of SEQ ID NO: 712; an amino acid other than a T at the position corresponding to position 420 of SEQ ID NO: 712; and an amino acid other than an R at the position corresponding to position 655 of SEQ ID NO: 712. In some embodiments, the S1uCas9 comprises an A at the position corresponding to position 246 of SEQ ID NO: 712. In some embodiments, the SluCas9 comprises an A at the position corresponding to position 414 of SEQ ID NO: 712. In some embodiments, the SluCas9 comprises an A at the position corresponding to position 420 of SEQ IDNO: 712. In some embodiments, the SluCas9 comprises an A at the position corresponding to position 655 of SEQ ID NO: 712. In some embodiments, the SluCas9 comprises an A at the position corresponding to position 246 of SEQ ID NO: 712; an A at the position corresponding to position 414 of SEQ ID NO: 712; an A at the position corresponding to position 420 of SEQ ID NO: 712; and an A at the position corresponding to position 655 of SEQ ID NO: 712.

[0257] In some embodiments, the SluCas9 comprises an amino acid other than an R at the position corresponding to position 246 of SEQ ID NO: 712; an amino acid other than an N at the position corresponding to position 414 of SEQ ID NO: 712; an amino acid other than a T at the position corresponding to position 420 of SEQ ID NO: 712; an amino acid other than an R at the position corresponding to position 655 of SEQ ID NO: 712; an amino acid other than an Q at the position corresponding to position 781 of SEQ ID NO: 712; a K at the position corresponding to position 966 of SEQ ID NO: 712; and an amino acid other than an R at the position corresponding to position 1013 of SEQ ID NO: 712. In some embodiments, the SluCas9 comprises an A at the position corresponding to position 246 of SEQ ID NO: 712; an A at the position corresponding to position 414 of SEQ ID NO: 712; an A at the position corresponding to position 420 of SEQ ID NO: 712; an A at the position corresponding to position 655 of SEQ ID NO: 712; a K at the position corresponding to position 781 of SEQ ID NO: 712; a K at the position corresponding to position 966 of SEQ ID NO: 712; and an H at the position corresponding to position 1013 of SEQ ID NO: 712.

[0258] In some embodiments, the SluCas9 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 718 (designated herein as SluCas9-KH or SLUCAS9KH):NQKFILGLDIGITSVGYGL1DYETKNIIDAGVRLFPEANVENNEGRRSKRGSRRLKRRRIHRLE RVKKLLEDYNLLDQSQIPQSTNPYAIRVKGLSEALSKDELVIALLHIAKRRGIHKIDVIDSNDD VGNELSTKEQLNKNSKLLKDKFVCQIQLERMNEGQVRGEKNRFKTADIIKEIIQLLNVQKNFH QLDENFINKYIELVEMRREYFEGPGKGSPYGWEGDPKAWYETLMGHCTYFPDELRSVKYAY SADLFNALNDLNNLVIQRDGLSKLEYHEKYiniENVFKQKKKPTLKQIANEINVNPEDIKGYRI TKSGKPQFTEFKLYHDLKSVLFDQS1LENEDVLDQIAE1LTIYQDKDSIKSKLTELDILLNEEDK ENIAQLTGYTGTHRLSLKCIRLVLEEQWYSSRNQMEIFl'HLNIKPKKINLTAANKIPKAMIDEF ILSPWKRTFGQAINLINKIIEKYGVPEDIIIELARENNSKDKQKFINEMQKKNENTRKRINEIIG KYGNQNAKRLVEKIRLHDEQEGKCLYSLESIPLEDLLNNPNHYEVDHIIPRSVSFDNSYHNKV LVKQSENSKKSNLTPYQYFNSGKSKLSYNQFKQHILNLSKSQDR1SKKKKEYLLEERDINKFE VQKEF4NIENLVDTRYATREL1NYLKAYFSANNMNVKVKTINGSFTDYLIIKVWKFKKERNH GYKHHAEDALnANADFLFKENKKLKAVNSVLEKPEIETKQLDIQVDSEDNYSEMFIIPKQVQ DIKDFRNFKYSHRVDKKPNRKLINDTLYSTRKKDNSTY1VQTIKDIYAKDNTTLKKQFDKSPE KFLMYQHDPRTFEKLEV1MKQYANEKNPLAKYHEETGEYLTKYSKKNNGPIVKSLKYIGNKLGSHLDVTHQFKSSl'KKLVKLSIKPYRFDVYLTDKGYKFmSYLDVLKKDNYYYIPEQKYDKLKLGKAIDKNAKFIASFYKNDLIKLDGEIYKIIGVNSDTRNMIELDLPDIRYKEYCELNNIKGEPHIKI<TIGKI<VNSIEI<LTTD VLGNVFTNTQYTKPQLLFKRGN.

[0259] In some embodiments, the SluCas9 comprises an amino acid sequence that is at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 719 (designated herein as S1uCas9-HF):NQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENNEGRRSKRGSRRLKRRRIHRLE RVKKLLEDYNLLDQSQIPQSTNPYAIRVKGLSEALSKDELVIALLHIAKRRGIHKIDVIDSNDD VGNELSTKEQLNKNSKLLKDKFVCQIQLERMNEGQVRGEKNRFKTADIIKEIIQLLNVQKNFH QLDENFINKYIELVEMRREYFEGPGKGSPYGWEGDPKAWYETLMGHCTYFPDELASVKYAY SADLFNALNDLNNLVIQRDGLSKLEYHEKYHIIENVFKQKKKPTLKQIANEINVNPEDIKGYRI I'KSGKI’QFTEFKLYHDLKSVLFDQSILENEDVLDQIAEILTIYQDKDSIKSKLTELDILLNEEDK ENIAQLTGYTGTHRLSLKCIRLVLEEQWYSSRAQMEIFAHLNIKPKKINLTAANKIPKAMIDEF ILSPWKRTFGQAINLINKIIEKYGVPEDIIIELARENNSKDKQKFINEMQKKNENTRKRINEIIG KYGNQNAKRLVEKIRLHDEQEGKCLYSLESIPLEDLLNNPNHYEVDHIIPRSVSFDNSYHNKV LVKQSENSKKSNLTPYQYFNSGKSKLSYNQFKQHILNLSKSQDR1SKKKKEYLLEERDINKFE VQKEFINRNLVDTRYATAELTNYLKAYFSANNMNVKVKTINGSFTDYLRKVWKFKKERNH GYKHIIAEDALITANADFLFKENKKLKAVNSVLEKPEIETKQLDIQVDSEDNYSEMFIIPKQVQ DIKDFRNFKYSHRVDKKPNRQLINDTLYSTRKKDNSTYIVQTIKDIYAKDNTTLKKQFDKSPE 1<FLMYQHDPRTFEKLEVIMKQYANE1<NPLAKYHEEIGEYLTKYSKKNNGPIVKSL1<YIGNK LGSHIXVTHQFKSSTKKLVKLSIKPYRFDVYLTDKGYKFITISYLDVLKKDNYYVTPEQKYDK LKLGKAIDKNAKFIASFYKNDLIKLDGEIYKIIGVNSDTRNMIELDLPDIRYKEYCELNNIKGEP RIKKTIGKKVNSIEKLTTDVLGNVFTNTQYTKPQLLFKRGN.

[0260] In some embodiments, the SluCas9 comprises an ammo acid sequence that is at least80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 720 (designated herein as SluCas9-HF-KH):NQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENNEGRRSKRGSRRLKRRRIHRLE RVKKLLEDYNLLDQSQIPQSTNPYAIRVKGLSEALSKDELVIALLHIAKRRGIHKIDVIDSNDD VGNELS'FKEQLNKNSKLLKDKFVCQIQLERMNEGQVRGEKNRF'KIADIIKEIIQLLNVQKNFH QLDENFINKYIELVEMRREYFEGPGKGSPYGWEGDPKAWYETLMGHCTYFPDELASVKYAY SADLFNALNDLNNLVIQRDGLSKLEYHEKYHIIENVFKQKKKPTLKQIANEINVNPED1KGYR1 TKSGKPQFTEFKLYHDLKSVLFDQSILENEDVLDQIAEILTTYQDKDSIKSKLTELDILLNEEDK ENIAQLTGYTGTHRLSLKCIRLVLEEQWYSSRAQMEIFAHLNIKPKKINLTAANKIPKAMIDEF ILSPVVKRTFGQ AIN LINKIIEKYG VPEDfflEL ARENNSKDKQKFINEMQKKN ENIRKRINEIIG KYGNQNAKRLVEKIRLHDEQEGKCXYSLESIPLEDLLNNPNHYEVDiniPRSVSFDNSYIINKV LVKQSENSKKSNLTPYQYFNSGKSKLSYNQFKQHILNLSKSQDRISKKKKEYLLEERDINKFE VQKEFLNILNLVDTRYATAELTNYXKAYFSANNMNVKVKTINGSFTDYLRKVWKFKKERNH GYKHHAEDALIIANADFLFKENKKLKAVNSVLEKPEIETKQLDIQVDSEDNYSEMFIIPKQVQDIKDFRNFKYSHRVDKKPNRKLINDTLYSTRKKDNSIYIVQTIKDIYAKDNTTLKKQFDKSPE KFLMYQHDPRTFEKLEVIMKQYANEKNPLAKYHEETGEYLTKYSKKNNGPIVKSLKYIGNK LGSHLDVTHQFKSSl'KKLVKLSIKPYRFDVYLTDKGYKFmSYLDVLKKDNYYYIPEQKYDK IXLGKAIDKNAKFIASFYKNDLIKLDGEIYKIIGVNSDTRNMIELDI.PDIRYKEYCELNNIKGEP FIIKKTIGKKVNSIEKLTTDVLGNVFTNYQYTKPQLLFKRGN.

[0261] In some embodiments, die Cas protein is any of the engineered Cas proteins disclosed inSchmidt et al., 2021, Nature Communications, “Improved CRISPR genome editing using small highly active and specific engineered RNA-guided nucleases.”

[0262] In some embodiments, the Cas9 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 7021 (designated herein as sRGNl):MNQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENNEGRRSKRGSRRLKRRRIHRL DRVKFILLAEYDLLDLTNIPKSTNPYQTRVKGLNEKLSKDELVIALLHIAKRRGIHNVDVAAD KEETASDSLSTKDQINKNAKFLESRYVCELQKERLENEGHVRGVENRFLTKDIVREAKKIIDT QMQYYPEIDETFKEKYISLVEI'RREYFEGPGKGSPFGWEGNIKKWFEQMMGHCTYFPEELRS VKYSYSAELFNALNDLNNLVITRDEDAKLNYGEKFQIIENVFKQK.KTPNLKQIAIEIGXrHETEI KGYRVNKSGTPEFTEFKLYHDLKSIVFDKSILENEAILDQIAEILTIYQDEQSIKEELNKLPEILN EQDKAEIAKLIGYNGTHRLSLKCIHLINEELWQTSRNQMEIFNYLNIKPNKVDLSEQNKIPKD MVNDFILSPVVKRTFIQSINVINKVIEKYG1PEDIIIELARENNSDDRKKFINNLQKKNEATRKR1 NEIIGQTGNQNAKRIVEKIRLHDQQEGKCLYSLKDIPLEDLLRNPNNYDIDHnPRSVSFDDSM HNKVLVRREQNAKKNNQTPYQYLTSGYADIKYSVFKQIWLNLAENKDRMTKKKREYLLEE RDINKFEVQKEFINRNLVDTRYATRELTNYLKAYFSANNMNVKVKTINGSFTDYLRKVWKF KKERNHGYKHHAEDALIIANADFLFKENKKLKAVNSVLEKPE1ETKQLDIQVDSEDNYSEMF1 IPKQVQDIKDFRNFKYSHRVDKKPNRQIJNDTLYSTRKKDNSTYIVQTIKDIYAKDNTTL,KKQ FDKSPEKFLMYQFIDPRTFEKLEVIMKQYANEKNPLAKYIIEETGEYLTKYSKKNNGPIVKSLK YIGNKLGSHLDVTHQFKSSTKKLVKLSIKPYRFDWLTDKGYKFITISYLDVLKKDNY^AYPEQKYDKLKLGKAIDKNAKFIASFYKNDLIKLDGEIYKIIGVNSDTRNWELDLPDIRYKEYCELN NIKGEPRIKKTIGKKVNSIEKLTTDVLGNVFTNTQYTKPQLLFKRGN.

[0263] In some embodiments, the Cas9 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to die sequence of SEQ ID NO: 7022 (designated herein as sRGN2):MN QKFILGLDIGITS VGYGL1DYE TKNIIDAGV RLFPEAN VENNEGRRSKRGSRRLKRRRIHRL ERVKSLLSEYKIISGLAPTNNQPYNIRVKGLTEQLTK.DELAVALLHIAKRRGIHKIDVIDSNDD VGNELSTKEQLNKNSKLLKDKFVCQIQLERK4NEGQVRGEKNRFKTADIIKEIIQLLNVQKNFH QLDENFINKYIELVEMRREYFEGPGQGSPFGWNGDLKKWYEMLMGHCTYFPQELRSVKYA YSADLFNALNDLNNLIIQRDNSEKLEYHEKYHIIENVFKQKKKPTLKQIAKEIGVNPEDIKGYR ITKSGTPEFTEFKLYHDLKSVLFDQSILENEDVLDQIAEILTIYQDKDSIKSKLTELDILLNEEDKENIAQLTG'S'NGTHRLSLKCIRLVLEEQWYSSRNQMEIFTHLNIKPKKINLTAANKIPKAMIDEF ILSPVVKRTFIQSINVINKVIEKYGIPEDIIIELARENNSDDRKKFINNLQKKNEATRKRINEIIGQ TGNQNAKRIVEK1RLHDQQEGKCLYSLESIALMDLLNNPQNYEVDHIIPRSVAFDNSIHNKVL VKQIENSKKGNRTPYQYLNSSDAKLS^'NQFKQHILNLSKSKDRISKKKKDYLLEERDINKFEV QKEFINRNLVDTRYATRELTSYLKAYFSANNMDVKVKTINGSFTNHLRKW'RFDKYRNHGY KHHAEDALIIANADFLFKENKKLKAVNSVLEKPEIETKQLDIQVDSEDNYSEMFIIPKQVQDIK DFRNFKYSHRVDKKPNRQLFNDTLYSTRKKDNSIYIVQTIKDFYAKDNTTLKKQFDKSPEKFL WQHDPRTFEKLEVIMKQYANEKNPLAKYHEETGEYLTKYSKKNNGPIVKSLKYIGNKLGS IILDVTHQFKSSTKKLVKLSIKPYRFDVYLTDKGYKFmSYLDVLKKDNYYYIPEQKYDKLKL GKAIDKNAKFIASIYKNDLIKLDGEIYKIIGVNSDTRNMIELDLPDIRYKEYCELNNIKGEPRIK KT1GKKVNSIEKLTTDVLGNVFTNTQYTKPQLLFKRGN.

[0264] In some embodiments, the Cas9 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 7023 (designated herein as sRGN3):MN QKFILGLDIGITS VGYGLIDYE TKNIIDAGV RLFPEAN VENNEGRRSKRGSRRLKRRRIHRL ERVKLLLTEYDLINKEQIPTSNNPYQIRVKGLSEILSKDELAIALLHLAKRRGIHNVDVAADKE ETASDSLSTKDQINKNAKFLESRYVCELQKERLENEGIWRGVENRFLTKDIVREAKKIIDTQM QYYPEIDETFKEKYISLVETRREYFEGPGQGSPFGWNGDLKKWYEMLMGHCTYFPQELRSV KYAYSADLFNALNDLNNLI1QRDNSEKLEYHEKYHI1ENVFKQKKKPTLKQ1AKEIGVNPEDIK GYRITKSGTPEFTSFKLFHDLKKVVKDHAILDDIDLLNQIAEILHYQDKDSIVAELGQLEYLM SEADKQSISELTGYTGTHSLSLKCMNMIIDELWHSSMNQMEVFTYLNMRPKKYELKGYQRIP TDMIDDAILSPVVKRTFIQSINVINKVIEKYGIPEDIIIELARENNSDDRKKFINNLQKKNEATRK RINE11GQTGN QNAKRI VEKIRLHDQQEGKCLY SLESIPLEDLLNNPNHYEVDHI1PRS VSFDN S YHNKVLVKQSENSKKSNLTPYQYFNSGKSKLSYNQFKQHILNLSKSQDRISKKKKEYLLEER DINKFEVQKEFINRNLVD'IRYATRELTNYLKAYFSANNMNVKVK'IINGSF'IDYLRKVWKFK KERNIIGYKHHAEDALIIANADFLFKENKKLKAVNSVLEKPEIETKQLDIQVDSEDNYSEMFII PKQVQDIKDFRNFKYSHRVDKKPNRQLINDTLYSTRKKDNSTYIVQTIKDIYAKDNTTLKKQF DKSPEKF’LMYQHDPRTFEKLEVIMKQYANEKNPLAKYHEETGEYLTKYSKKNNGPIVKSLK YIGNKLGSHLDVTHQFKSSTKKLVKLSIKPYRFDWLTDKGYKFinSYLDVLKKDNYYYIPE QKYDKLKLGKAIDKNAKFIASFYKNDLIKLDGEIYKIIGVNSDTRNMIELDLPDIRYKEYCELN NIKGEPRIKKTIGKKVNSIEKLTTDVLGNVFTNTQYTKPQLL.FKRGN.

[0265] In some embodiments, the Cas9 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 7024 (designated herein as sRGNS.l):MNQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENNEGRRSKRGSRRLKRRRIHRL ERVKLLLTEYDL1NKEQIPTSNNPYQIRVKGLSEILSKDELAIALLHLAKRRGIHNVDVAADKE ETA SDS LSTKI)QINKNAKFL,ESRY VCELQKERLENEGHVRGVENRFLTKDIVREAKKIIDTQMQYYPEIDETFKEKYISLVETRREYFEGPGQGSPFGWNGDLKKWYEMLMGHCTYFPQELRSV KYAYSADLFNALNDLNNLIIQRDNSEKLEYHEKYHIIENVFKQKKKPTLKQIAKEIGVNPEDIK GYRIl'KSGl'PEFTSFKLFHDLKKVVKDHAILDDIDLLNQIAEILTIYQDKDSlVAELGQLEYLM SEADKQSISELTGYTGTHSLSLKCMNMUDELWHSSMNQMEVFTYLNMRPKKYELKGYQRIP TDMIDDAILSPVVKRTFIQSINVINKVIEKYGIPEDIIIELARENNSDDRKKFINNLQKKNEATRK RINE1IGQTGNQNAKRIVEKIRLHDQQEGKCLYSLESIPLEDLLNNPNHYEVDHI1PRSVSFDNS YHNKVLVKQSENSKKSNLTPYQYFNSGKSKLSYNQFKQHILNLSKSQDRISKKKKEYLLEER DINKFEVQKEFTNRNIA'DTRYATRELTNYLKAYFSANNMNVKVKTTNGSFTDYLRKVWKFK KERNIIGYKHHAEDALIIANADFLFKENKKLKAVNSVLEKPEIETKQLDIQVDSEDNYSEMFII PKQVQDIKDFRNFKYSHRVD1<KPNRQL1NDTLYSTRKKDNSTYIVQTIKD1YAI<DNTTL1<KQF DKSPEKF’LMYQHDPRTFEKLEVIMKQYANEKNPLAKYHEETGEYLTKYSKKNNGPIVKSLK YIGNKLGSHLDVTHQFKSSTKKLVKLSIKNYRFDVYLTEKGYKFVTIAYLNVFKKDNYYYIP KDKYQELKEKKKIKDTDQFIASFYKNDLIKLNGDLYKIIGVNSDDRNIIELDYYDIKYKDYCEI NNIKGEPRIKKTIGKKTESIEKFTTDVLGNLYLHSTEKAPQLIFKRGL.

[0266] In some embodiments, the Cas9 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 7025 (designated herein as sRGN3.2):MNQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENNEGRRSKRGSRRLKRRRIHRL ERVKLLLTEYDL1NKEQIPTSNNPYQIRVKGLSEILSKDELAIALLHLAKRRGIHNVDVAADKE ETA SDS LSTKDQINKNAKFLESRYVCELQKERLENEGHVRGVENRFLTKDIVREAKKIIDTQM QYYPEIDETFKEKYISLVEIRREYFEGPGQGSPFGWNGDLKKWYEMLMGHCTYFPQELRSV KYAYSADLFNALNDLNNLIIQRDNSEI<LEYHEI<YHIIENVFKQKKKPTLI<QIAKEIGVNPEDII< GYRITKSGTPEFTSFKLFHDLKKVVKDHAILDDIDLLNQIAEILTIYQDKDSIVAELGQLEYLM SEADKQSISELTGYTGTHSLSLKCMNMIIDELWTJSSMNQMEVFTYLNMRPKKYELKGYQRIP TDMIDDAILSPVVKRTFIQSINVINKVIEKYGIPEDIIIELARENNSDDRKKFINNLQKKNEATRK RINEIIGQTGNQNAKRIVEKIRLHDQQEGKCLYSLESIPLEDLLNNPNIIYEVDHIIPRSVSFDNS YHNKVLVKQSENSKKSNLTPYQYFNSGKSKLSYNQFKQHILNLSKSQDRISKKKKEYLLEER D1NKFEVQKEF1NRNLVDTRYATRELTNYLKAYFSANNMNVKVKTINGSFTDYLRKVWKFK KERNHGYKHHAEDALIIANADFLFKENKKLKAVNSVLEKPEIETKQLDIQVDSEDNYSEMFII PKQVQDIKDFRNFKFSHRVDKKPNRQLINDTLYSTRMKDEHDYIVQTITDIYGKDNTNLKKQ FNKNPEKFLMYQNDPKTFEKLSIIMKQYSDEKNPLAKYYEETGEYLTKYSKKNNGPIVKKIK LLGNKVGNHLDVINKYENSTKKLVKLSIKNYRF'DVYLTEKGYKFVTIAYLNVFKKDNYYYI PKDKYQELKEKKKIKDTDQFTASFYKNDLIKLNGDLYKIIGVNSDDRNIIELDYYDIKYKDYC EINNIKGEPRIKKTIGKKTESIEKFTTDVLGNLYLHSTEKAPQLIFKRGL.

[0267] In some embodiments, the Cas9 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 7026 (designated herein as sRGN3.3):MNQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENNEGRRSKRGSRRLKRRRIHRL ERVKLLLTEYDLINKEQIPTSNNPYQIRVKGLSEILSKDELAIALLHLAKRRGIHNVDVAADKE ETASDSLSTKDQINKNAKFLESRYVCELQKERLENEGHVRGVENRFLl'KDIVREAKKIIDTQM QYYPEIDE / ITKEKYISLVETRREYFEGPGQGSPFGYVNGDL,KKWYEML,MGHCTYFPQELRSV KYAYSADLFNALNDLNNLIIQRDNSEKLEYIIEKYHIIENVFKQKKKPTLKQIAKEIGVNPEDIK GYRITKSGTPEFTSFKLFHDLKKVVKDHAILDDIDLLNQIAEILTIYQDKDSIVAELGQLEYLM SEADKQS1SELTGYTGTHSLSLKCMNMIIDELWHSSMNQMEVF1YLNMRPKKYELKGYQR1P TDMIDDAILSPVYKRTFIQSINVINKVIEKYGIPEDIIIELARENNSDDRKKFINNLQKKNEATRK RINEIIGQTGNQNAKRIVEKIRLFIDQQEGKCLYSLESIPLEDLLNNPNHYEVDHIIPRSVSFDNS YHNKVLVKQSENSKKSNLTPYQYFNSGKSKLSYNQFKQHILNLSKSQDRISKKKKEYLLEER DINKF’EVQKEFINRNLVDl'RYATRELTSYLKAYFSANNMDVKVKnNGSFTNHLRKVWRFD KYRNHGYKHHAEDALIIANADFLFKENKKLQNTNKILEKPTIENNTKKVTVEKEEDYNNVFE TPKLVEDIKQYRDYKFSHRVDKKPNRQLR'JDTLYSTRMKDEHDYIVQTITDIYGKDNTNLKK QFNKNPEKFLMY’QNDPKTFEKLSIIMKQYSDEKNPLAKYA''EETGE¥LTKYSKKNNGPIVKKI KLLGNKVGNHLDVTNKYENSl'KKLVKLSIKNYRF’DVYLTEKGYKFVTLAYLNVFKKDNYYY IPKDKYQELKEKKKIKDTDQFIASFYKNDLIKLNGDLYKIIGVNSDDRNIIELDYYDIKYKDYC EINNIKGEPRIKKTIGKKTESIEKFTTDVLGNLYLHSTEKAPQLIFKRGL.

[0268] In some embodiments, the Cas9 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 7027 (designated herein as sRGN4):MNQKFILGLDIGITSVGYGLIDYETKNIIDAGVRLFPEANVENNEGRRSKRGSRRLKRRRIHRL ERVKKLLEDYNLLDQSQIPQSTNPYAIRVKGLSEALSKDELVIALLHLAKRRGIHNINVSSEDE DASNELSTKEQINRNNKLLKDKYVCEVQLQRLKEGQIRGEKNRF'KTTDILKEIDQLLKVQKD YHNLDIDFINQYKEIVETRREYFEGPGKGSPYGWEGDPKAWYETLMGHCTYFPDELRSVKY AYSADLFNALNDLNNLYIQRDGLSKLEYHEKYHITENVFKQKKKPTLKQIANEINVNPEDIKG YRITKSGKPEFTSFKLFHDLKKVVKDHAILDDIDLLNQIAEILTIYQDKDSI\7AELGQLEYLMS EADKQSISELTGYTGTHSLSLKCMNMIIDELWHSSMNQMEVFTYLNMRPKKYELKGYQRIPT DMIDDAILSPVVKRTFIQSINVINKV1EKYGIPEDIIIELARENNSDDRKKFINNLQKKNEATRKR INEIIGQTGNQNAKRIVEKIRLHDQQEGKCLYSLESIPLEDLLNNPNHYrEVDHIIPRSVSFDNSY HNKVLVKQSENSKKSNLTPYQYFNSGKSKLSYNQFKQHILNLSKSQDRISKKKKEYLLEERDI NKFEVQKEFINRNLVDTR.YATRELTNYLKAYFSANNMNVKVKTINGSFTDYLRKWKFKKE RNHGYKHHAEDALIIANADFLFKENKKLKAVNSVLEKPEIETKQLD1QVDSEDNYSENIFIIPK QVQDIKDFRNFKYSHRVDKKPNRQLINDTLYSTRKKDNSTYIVQTIKDIYAKDNTTLKKQFD KSPEKFLMYQHDPRTFEKLEVIMKQYANEKNPLAKYHEETGEYLTKYSKKNNGPIVKSLKYI GNKLGSHLDVIHQFKSSTKKLVKLSIKPYRFDVYLTDKGYKFITISYLDVLKKDNYYYIPEQK YDKLKLGKAIDKNAKFIASFYKNDLIKLDGEIYKIIGVNSDI'RNMIELDLPDIRYKEYCELNNI KGEPRIKKTIGKKWSIEKLTTDVLGNVFTNTQYTKPQLLFKRGN,

[0269] In some embodiments, the Cas9 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 7028 (designated herein as Staphylococcus hyicus Cas9 or ShyCas9):MNNYILGLDIGITSVGYGIVDSDTREIKDAGVRLFPEANVDNNEGRRSKRGARRLKRRRIHRL DRVKIILLAEYDLLDLTNIPKSTNPYQTRVKGLNEKLSKDELVIALLinAKRRGIHNA^NVMMD DNDSGNELSTKDQLKKNAKALSDKYVCELQLERFEQDYKVRGEKNRFKTEDFVREARKLLE TQSKFFEIDQTFIMRYIEL1ETRREYFEGPGKGSPFGWEGNIKKWFEQMMGHC1YFPEELRSV KYSYSAELFNALNDLNNLVITRDEDAKLNYGEKFQIIENVFKQKKTPNLKQIAIEIGVHETEIK GYRVNKSGKPEFTQFKLYHDLKNIFKDPKYLNDIQLMDNIAEIIITYQDAESIIKELNQLPELLS EREKEKISALSGYSGTHRLSLKCINLLLDDLWESSLNQMELFTKLNLKPKKIDLSQQHKIPSKL VDDFILSPVVKRAF1QSIQVVNAIIDKYGLPEDIIIELARENNSDDRRKFLNQLQKQNEETRKQV EKVLREYGNDNAKRIVQKIKLHNMQEGKCLYSLKDIPLEDLLRNPHHYEVDHIIPRSVAFDNS MHNKVLVRADENSKKGNRTPYQYLNSSESSLSYNEFKQHILNLSKTKDRITKKKREYLLEER DINKFDVQKEFINRNLVDTRYATRELTSLLKAYFSANNLDVKVKTINGSFTNYLRKVWKFDK DIlNKGYKHHi^EDALIlANADFEFKHNKKLRNINKVLDAPSKEVDKKRV'rVQSEDEYNQIFED TQKAQAIKKFEIRKFSHRVDKKPNRQLINDTLYSTRNTOGIEYVVESIKDIYSVNNDKVKTKFK KDPHRLLMYRNDPQTFEKFEKVFKQYESEKNPFAKYYEETGEKIRKFSKTGQGPYINKIKYLR ERLGRHCDVTNKYINSRNKIVQLKIYSYRFDIYQYGNNYKMniSYIDLEQKSNYYYISREKYE QKKKDKQIDDSYKFIGSFYKND11NYNGEMYRVIGVNDSEKNKIQLDMIDISIKDYMELNNIK KTGVIYKTIGKSTTHIEKYTTDILGNLYKAAPPKKPQLIFK,

[0270] In some embodiments, the Cas9 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 7029 (designated herein as Staphylococcus microti Cas9 or Smi Cas9):MEKDYILGLDIGIGSVGYGLIDYDTKSIIDAGVRLFPEANADNNLGRRAKRGARRLKRRRIHR LERVKSLLSEYKIISGLAPTNNQPYNIRVKGLTEQLTKDELAVALLHIAKRRGIHNVDVAADK EETASDSLSTKDQINKNAKFLESRYVCELQKERLENEGHVRGVENRFLTKDIVREAKKIIDTQ MQYYPEIDETFKEKYISLVETRREYYEGPGKGSPYGWDADVKKWYQLMMGHCTYFPVEFRS VKYAYTADLYNALNDLNNLTIARDDNPKLEYHEKYHIIENVFKQKRNPI'LKQIAKEIGVNDI NISGYRVTKSGKPQFTSFKLFHDLKKWKDHAILDDIDLLNQIAEILTrYQDKDSIVAELGQLE YLMSEADKQSISELTGYTGTHSLSLKCMNMIIDELWHSSMNQMEVFTYLNMRPKKYELKGY QRIPTDMIDDAILSPVVKRSFKQAIGVVNAIIKKYGLPKDIIIELARESNSAEKSRYLRAIQKKN EKTRERIEAIIKEYGNENAKGLVQKIKLHDAQEGKCLYSLKDIPLEDLLRNPNNYDIDHIIPRS VSFDDSMHNKVLVRREQNAKKNNQTPYQYLTSGYADIKYSVFKQHVLNLAENKDRMTKKK REYLLEERNINKYDVQKEFINRNLVDTRYTTRELTrLLKTYFITNNLDVKVKTINGSFTDFLR KRWGFKKNRDEGYKHHAEDALIIANADYLFKEHKLLKEIKDVSDLAGDERNSNVKDEDQYE EVFGGYFKIEDIKKYKIKKFSHRVDKKPNRQLINDTIYSI'RVKDDKRYLINTLKNLYDKSNGD LKERMQKDPESLLMYHHDPQTFEKLKIVMSQYENEKNPLAKYFEETGQYLTKYAKHDNGPAniKIKYYGNKLVEIILDITKNYHNPQNKWQLSQKSFRFDVYQTDKGYKFISIAYLTLKNEKN YYAISQEKYDQLKSEKKISNNAVFIGSFYTSDIIEINNEKFRVIGVNSDKNNLIEVDRIDIRQKEF lELEEEKKNNRlKVllGRK'n'NIEKFHTDILGNMYKSKRPKAPQLVFKKG.

[0271] In some embodiments, the Cas9 comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 7030 (designated herein as Staphylococcus pasteuri Cas9 or Spa Cas9):MKEKYILGLDLGITSVGYGIINFETKKIIDAGVRLFPEANVDNNEGRRSKRGSRRLKRRRIHRL ERVKLLLTEYDLINKEQIPTSNNPYQIRVKGLSEILSKDELAIALLHLAKRRGIHNINVSSEDED ASNELSTKEQINRNNKLLKDKYVCEVQLQRLKEGQIRGEKNRFKTTDILKEIDQLLKVQKDY HNLDIDFINQYKEIVETRREYFEGPGQGSPFGWNGDLKKWYEMLMGHCTYFPQELRSVKYA YSADLFNALNDLNNLIIQRDNSEKLEYHEKYHIIENVFKQKKKPTLKQIAKEIGVNPEDIKGYR ITKSGTPQFTEFKIA'HDLKSIVFDKSILENEAILDQIAEILTIYQDEQSIKEELNKLPEILNEQDK AEIAKLIGYNGTHRLSLKCIIILINEELWQTSRNQMEIFm^LNIKPNKVDLSEQNKIPKDMA^ND FILSPVVKRTFIQSINVINKVIEKYGIPEDIIIELARENNSDDRKKFINNLQKKNEATRKR1NEIIG QTGNQNAKRIVEKIRLHDQQEGKCLYSLESLALMDLLNNPQNYEVDHIIPRSVAFDNSIHNKV LVKQIENSKKGNRTPYQYLNSSDAKLS^'NQFKQHILNLSKSKDRISKKKKDYLLEERDINKFE VQKEFINRNLVDTRYATRELTSYLKAYFSANNMDVKVKTINGSFTNFILRKW-RFDKYRNHG YKHHAEDALIIANADFLFKENKKLQNTNKILEKPTIENNTKKVTVEKEEDYNNVFETPKLVED IKQYRDYKFSHRVDKKPNRQLINDTLYSTRMKDEHDYIVQTITDIYGKDNTNLKKQFNKNPE KFLMYQNDPKTFEKLSIIMKQYSDEKNPLAKYYEETGEYLTKYSKKNNGPIVKKIKLLGNKV GNHLDVTNKYENSTKKLVKLSIKNYRFD\'%LTEKGYKFVTIAYLNVFKKDNYhArIPKDKYQ ELKEKKKIKDTDQFIASFYKNDLIKLNGDLYKIIGVNSDDRNIIELDYYDIKYKDYCEINNIKG EPRIKKT1GKKTESIEKFTTD VLGN LYLHSTEKAPQLIFKRGL .

[0272] In some embodiments, the Cas protein comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 7031 (designated herein as Casl2il):MSNKEKNASETRKAYTTKMIPRSHDRMKLLGNFMDYLMDGTPIFFELWNQFGGGIDRDIISG TANKDKISDDLLLAVNWFKVMPINSKPQGVSPSNLANEFQQYSGSEPDIQAQEYFASNFDTE KHQWKDMRVEYERLLAELQLSRSDMHHDLKLMYKEKCIGLSLSTAHYITSVMFGTGAKNN RQTKHQFYSKVIQLLEESTQINSVEQLASIILKAGDCDSYRKLRIRCSRKGATPSILKIVQDYEL GTNHDDEVNVPSLIANLKEKLGRFEYECEWKCMEKIKAFLASKVGPYYLGSYSAMLENALS PIKGMTTKNCKFVLKQIDAKNDIKYENEPFGKIVEGFFDSPYFESDTNVKWVEHPHHIGESNI KTLWEDLNAJHSKYEEDIASLSEDKKEKRIKVYQGDVCQTINTYCEEVGKEAKTPLVQLLRY LYSRKDDIAVDKIIDGITFLSKKHKVEKQKINPVIQKYPSFNFGNNSKLLGKIISPKDKLKHNL KCNRNQVDNYIWIEIKVLNTKTMRWEKHHYALSSTRFLEEVYYPATSENPPDALAARFRTKT NGYEGKPALSAEQIEQIRSAPVGERKVKKRQMRLEAARQQNLLPRYTWGKDFNINICKRGN NFEVTLATKVKKKKEKNYKVVLGYDANIVRKNTYAAIFAHANGDGVIDYNDLPVKPIESGFVTVESQVRDKSYDQLS'S'NGVKLLYCKPHVESRRSFLEKYRNGTMKDNRGNNIQIDFMKDFE AIADDETSLYYFNMKYCKLLQSSIRNHSSQAKEYREEIFELLRDGKLSVLKLSSLSNLSFVMF KVAKSLIGTYFGHLLKKPKNSKSDVKAPPITDEDKQKADPEMFALRLALEEKRLNKVKSKKE VIANKTVAKALELRDKYGPVLIKGENISDTTKKGKKSSTNSFLMDWLARGVANKVKEMVM MHQGLEFVEVNPNFTSHQDPFVHKNPENTFRARYSRCTPSELTEKNRKEILSFLSDKPSKRPT NAYYNEGAMAFLATYGLKKNDVLGVSLEKFKQIMANILHQRSEDQLLFPSRGGMFYLATYK LDADATSVNWNGKQFWVCNADLVAAYNVGLVDIQKDFKKK.

[0273] In some embodiments, the Cas protein comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 7032 (designated herein as Casl2i2):MSSAIKSYKSVLRPNERKNQLLKS1IQCLEDGSAFFFKMLQGLFGGITPEIVRFSTEQEKQQQD IALWCAVNWFRPVSQDSLTHTIASDNLVEKFEEYYGGTASDAIKQYFSASIGESYYWNDCRQ QYYDLCRELGVEVSDLTHDLEILCREKCLAVATESNQNNSIISVLFGTGEKEDRSVKLRITKKI LEAISNLKEIPKNVAPIQEIILNVAKATKETFRQVYAGNLGAPSTLEKFIAKDGQKEFDLKKLQ TDLKKVIRGKSKERDWCCQEELRSYVEQNTIQYDLWAWGEMFNKAHTALKIKSTRNYNFA KQRLEQFKEIQSLNNLLVVKKLNDFFDSEFFSGEETYTICX'HHLGGKDLSKLYKAWEDDPAD PENAIVVLCDDLKNNFKKEPIRNILRYIFTIRQECSAQDILAAAKWQQLDRYKSQKANPSVL GNQGFT^'TNAVILPEKAQRNDRPNSLDLRIWLYLKLRHPDGRWKKHHIPFYDTRFFQEIYAA GNSPVDTCQFRTPRFGYHLPKLTDQTAIRVNKKHVKAAKl'EARIRLAIQQGTLPVSNLKIl'EIS ATINSKGQVRIPVKFDVGRQKGTLQIGDRFCGYDQNQTASHAYSLWEVVKEGQYHKELGCF VRFISSGDIVSITENRGNQFDQLSYEGLAYPQYADWRKKASKFVSLWQITKKNKKKEIVTVE AKEKFDAICKYQPRLYKFNKEYAYLLRDIVRGKSLVELQQIRQEIFRFIEQDCGVTRLGSLSLS TLEIYKAVKGIIYSYFSTALNASKNNPISDEQRKEFDPELFALLEKLELIRI'RKKKQKVERIAN SLIQTCLENNIKFIRGEGDLSTTNNATKKKANSRSMDWIARGVFNKIRQLAPMHNm-FGCGS LYTSHQDPLVIIRNPDKAMKCRWAAIPVKDIGDWLRKLSQNLRAKNIGTGEYYIIQGVKEF LSHYELQDLEEELLKWRSDRKSNIPCWVLQNRLAEKLGNKEAVVYIPVRGGRIYFATHKVAT GAVSIVFDQKQVWVCNADHVAAANIALTVKGIGEQSSDEENPDGSRIKLQLTS.Modified Guide RNAs

[0274] In some embodiments, the guide RNA (e.g., an sgRN A encoded by a nucleic acid disclosed herein) is chemically modified. A guide RNA comprising one or more modified nucleosides or nucleotides is called a “modified” guide RNA or is called a “chemically modified” guide RNA, to describe the presence of one or more non-naturally and / or naturally occurring components or configurations that are used instead of or in addition to the canonical A, G, C, and U residues. In some embodiments, a modified guide RNA is synthesized with a non-canonical nucleoside or nucleotide, is here called “’modified.” Modified nucleosides and nucleotides can include one or more of: (i) alteration, e.g., replacement, of one or both of the non-linking phosphate oxygens and / or of one or more of the linking phosphate oxygens in the phosphodiester backbone linkage (anexemplar^' backbone modification); (ii) alteration, e.g., replacement, of a constituent of the ribose sugar, e.g., of the 2* hydroxyl on the ribose sugar (an exemplary sugar modification); (iii) wholesale replacement of the phosphate moiety with “dephospho” linkers (an exemplary backbone modification); (iv) modification or replacement of a naturally occurring nucleobase, including with a non-canonical nucleobase (an exemplary' base modification); (v) replacement or modification of the ribose-phosphate backbone (an exemplary backbone modification); (vi) modification of the 3' end or 5' end of the oligonucleotide, e.g., removal, modification or replacement of a terminal phosphate group or conjugation of a moiety, cap (such 3' or 5‘ cap modifications may comprise a sugar and / or backbone modification); and (vii) modification or replacement of the sugar (an exemplary sugar modification).[002.75] Chemical modifications such as those listed above can be combined to provide modified guide RNAs comprising nucleosides and nucleotides (collectively “residues”) that can have two, three, four, or more modifications. For example, a modified residue can have a modified sugar and a modified nucleobase, or a modified sugar and a modified phosphodiester. In some embodiments, every' base of a guide RNA is modified, e.g., all bases have a modified phosphate group, such as a phosphorothioate group. In certain embodiments, all, or substantially all, of the phosphate groups of a guide RNA molecule are replaced with phosphorothioate groups. In some embodiments, modified guide RNAs comprise at least one modified residue at or near the 5' end of the RNA. In some embodiments, modified guide RNAs comprise at least one modified residue at or near the 3' end of the RNA.

[0276] In some embodiments, the guide RNA comprises one, two, three or more modified residues. In some embodiments, at least 5% (e.g., at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%) of the positions in a modified guide RNA are modified nucleosides or nucleotides.

[0277] Unmodified nucleic acids can be prone to degradation by, e.g., intracellular nucleases or those found in serum. For example, nucleases can hydrolyze nucleic acid phosphodiester bonds. Accordingly, in one aspect the guide RNAs described herein can contain one or more modified nucleosides or nucleotides, e.g., to introduce stability toward intracellular or serum-based nucleases. In some embodiments, the modified guide RNA molecules described herein can exhibit a reduced innate immune response when introduced into a population of cells, both in vivo and ex vivo. The term “innate immune response” includes a cellular response to exogenous nucleic acids, including single stranded nucleic acids, which involves the induction of cytokine expression and release, particularly the interferons, and cell death.[002.78] In some embodiments of a backbone modification, the phosphate group of a modified residue can be modified by replacing one or more of the oxygens with a different substituent. Further,the modified residue, e.g., modified residue present in a modified nucleic acid, can include the wholesale replacement of an unmodified phosphate moiety with a modified phosphate group as described herein. In some embodiments, the backbone modification of the phosphate backbone can include alterations that result in either an uncharged linker or a charged linker with unsymmetrical charge distribution.

[0279] Examples of modified phosphate groups include, phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters. The phosphorous atom in an unmodified phosphate group is achiral. However, replacement of one of the non-bridging oxygens with one of the above atoms or groups of atoms can render the phosphorous atom chiral. Hie stereogenic phosphorous atom can possess either the “R” configuration (herein Rp) or the “S” configuration (herein Sp). The backbone can also be modified by replacement of a bridging oxygen, (j.e., the oxygen that links the phosphate to the nucleoside), with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates). The replacement can occur at either linking oxygen or at both of the linking oxygens.

[0280] The phosphate group can be replaced by non -phosphorus containing connectors in certain backbone modifications. In some embodiments, the charged phosphate group can be replaced by a neutral moiety. Examples ofrnoieties which can replace the phosphate group can include, without limitation, e.g., methyl phosphonate, hydroxyiamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thiofonnacetal, formacetal, oxime, methyleneimino, methylenemethyl imino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino .

[0281] Scaffolds that can mimic nucleic acids can also be constructed wherein the phosphate linker and ribose sugar are replaced by nuclease resistant nucleoside or nucleotide surrogates. Such modifications may comprise backbone and sugar modifications. In some embodiments, the nucleobases can be tethered by a surrogate backbone. Examples can include, without limitation, the morpholino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates.

[0282] The modified nucleosides and modified nucleotides can include one or more modifications to the sugar group, i.e. a sugar modification. For example, the 2' hydroxyl group (OH) can be modified, e.g. replaced with a number of different ‘ oxy” or “deoxy” substituents. In some embodiments, modifications to the 2' hydroxyl group can enhance the stability of the nucleic acid since the hydroxyl can no longer be deprotonated to form a 2'-alkoxide ion.

[0283] Examples of 2' hydroxyl group modifications can include alkoxy or aryloxy (OR, wherein “R” can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or a sugar); polyethyleneglycols (PEG), O(CH2CH2O)nCH2CH2OR wherein R can be, e.g. , H or optionally substituted alkyl, and n can be aninteger from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10, from 4 to 16, and from 4 to 20). In some embodiments, the 2' hydroxyl group modification can be 2'-0-Me. In some embodiments, the 2' hydroxyl group modification can be a 2'-fluoro modification, which replaces the 2' hydroxyl group with a fluoride. In some embodiments, the 2' hydroxyl group modification can include "‘locked” nucleic acids (LNA) in which the 2' hydroxyl can be connected, e.g., by a Ci-6 alkylene or Ci-e heteroalkylene bridge, to the 4' carbon of the same ribose sugar, where exemplary bridges can include methylene, propylene, ether, or amino budges; 0-amino (w herein amino can be, e.g., NH?; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino) and aminoalkoxy, O(CH?)n-amino, (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino). In some embodiments, the 2' hydroxyl group modification can include "unlocked" nucleic acids (UNA) in which the ribose ring lacks the C2'-C3' bond. In some embodiments, the 2' hydroxyl group modification can include the methoxyethyl group (MOE), (OCH2CH2OCH3, e.g., a PEG derivative).

[0284] “Deoxy” 2' modifications can include hydrogen (i.e. deoxyribose sugars, e.g., at the overhang portions of partially dsRNA); halo (e.g., bromo, chloro, fluoro, or iodo); amino (wherein amino can be, e.g. , NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); NH(CH2CH2NH)nCH2CH2- amino (wherein ammo can be, e.g., as described herein), -NHC(O)R (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionally substituted with e.g., an amino as described herein.

[0285] The sugar modification can comprise a sugar group which may also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Tirus, a modified nucleic acid can include nucleotides containing e.g., arabinose, as the sugar. The modified nucleic acids can also include abasic sugars. These abasic sugars can also be further modified at one or more of the constituent sugar atoms. The modified nucleic acids can also include one or more sugars that are in the L form, e.g. L- nucleosides.

[0286] lire modified nucleosides and modified nucleotides described herein, which can be incorporated into a modified nucleic acid, can include a modified base, also called a nucleobase. Examples of nucleobases include, but are not limited to, adenine (A), guanine (G), cytosine (C), and uracil (U). These nucleobases can be modified or wholly replaced to provide modified residues that can be incorporated into modified nucleic acids. The nucleobase of the nucleotide can be independently selected from a purine, a pyrimidine, a purine analog, or pyrimidine analog. In someembodiments, the nucleobase can include, for example, naturally-occurring and synthetic derivatives of a base.

[0287] In embodiments employing a dual guide RNA, each of the crRNA and the tracr RNA can contain modifications. Such modifications may be at one or both ends of the crRNA and / or tracr RNA. In embodiments comprising sgRNA, one or more residues at one or both ends of the sgRNA may be chemically modified, and / or internal nucleosides may be modified, and / or the entire sgRNA may be chemically modified. Certain embodiments comprise a 5' end modification. Certain embodiments comprise a 3' end modification.

[0288] Modifications of 2’-O-methyl are encompassed.

[0289] Another chemical modification that has been shown to influence nucleotide sugar rings is halogen substitution. For example, 2’-fluoro (2’-F) substitution on nucleotide sugar rings can increase oligonucleotide binding affinity and nuclease stability’. Modifications of 2’-fluoro (2’-F) are encompassed.

[0290] Phosphorothioate (PS) linkage or bond refers to a bond where a sulfur is substituted for one nonbridging phosphate oxygen in a phosphodiester linkage, for example in the bonds between nucleotides bases. When phosphorothioates are used to generate oligonucleotides, the modified oligonucleotides may also be referred to as S-oligos.

[0291] Abasic nucleotides refer to those which lack nitrogenous bases.

[0292] Inverted bases refer to those with linkages that are inverted from the normal 5’ to 3’ linkage (i.e., either a 5’ to 5’ linkage or a 3’ to 3’ linkage).

[0293] An abasic nucleotide can be attached with an inverted linkage. For example, an abasic nucleotide may be attached to the terminal 5’ nucleotide via a 5’ to 5’ linkage, or an abasic nucleotide may be attached to the terminal 3’ nucleotide via a 3’ to 3’ linkage. An inverted abasic nucleotide at either the terminal 5’ or 3’ nucleotide may also be called an inverted abasic end cap.

[0294] In some embodiments, one or more of the first three, four, or five nucleotides at the 5' terminus, and one or more of the last three, four, or five nucleotides at the 3' terminus are modified. In some embodiments, the modification is a 2’-0-Me, 2’-F, inverted abasic nucleotide, PS bond, or other nucleotide modification well known in the art to increase stability and / or performance.

[0295] In some embodiments, the first four nucleotides at the 5' terminus, and the last four nucleotides at the 3' terminus are linked with phosphorothioate (PS) bonds.

[0296] In some embodiments, the first three nucleotides at the 5' terminus, and the last three nucleotides at the 3!terminus comprise a 2'-O-methyl (2'-O-Me) modified nucleotide. In some embodiments, the first three nucleotides at the 5' terminus, and the last three nucleotides at the 3’ terminus comprise a 2'-fluoro (2'-F) modified nucleotide.Determination of Efficacy of Hybrid or Chimeric Promoters and Guide RNAs

[0297] In some embodiments, the efficacy of a hybrid or chimeric promoter is determined when a nucleic acid comprising a sequence encoding the hybrid or chimeric promoter is delivered together with other components that may form an RNP. In some embodiments, a nucleic acid comprising a sequence encoding a hybrid or chimeric promoter is delivered together with an endonuclease (e.g., SaCas9 or SluCas9). In some embodiments, the nucleic acid comprising the sequence encoding the hybrid or chimeric promoter is delivered into a cell that already stably expresses an endonuclease (e.g,, SaCas9 or SluCas9). In some embodiments, the nucleic acid comprising the sequence encoding the hybrid or chimeric promoter is delivered to a cell along with a nucleic acid (e.g., mRNA) comprising a sequence encoding an endonuclease (e.g., SaCas9 or SluCas9).[002.98] In some embodiments, the efficacy (e.g., of driving expression of one or more sgRNAs encoded by the nucleic acid) of a particular hybrid or chimeric promoter is determined based on in vitro models. In some embodiments, the in vitro model is a cell line.

[0299] In some embodiments, the efficacy of a particular hybrid or chimeric promoter (e.g., of driving expression of one or more sgRNAs encoded by the nucleic acid) is determined across multipie in vitro cell models for a hybrid or chimeric promoter selection process. In some embodiments, data from different cell lines comprising nucleic acids comprising sequences encoding different versions of the hybrid or chimeric promoter are compared, e.g., to determine which version of a hybrid or chimeric promoter disclosed herein results in the greatest expression of one or more sgRNAs encoded by the nucleic acid. In some embodiments, cross screening in multiple cell models is performed,

[0300] In some embodiments, the efficacy of particular hybrid or chimeric promoters is determined based on in vivo models. In some embodiments, the in vivo model is a rodent model. In some embodiments, the rodent model is a mouse which expresses, for example, a mutated dystrophin gene. In some embodiments, the in vivo model is a non-human primate, for example cynomolgus monkey.HI. Methods of Gene Editing

[0301] Provided herein are methods of multiplex gene editing using nucleic acids comprising a sequence encoding a hybrid or chimeric promoter and further comprising one or more sequences encoding one or more guide RNAs (e.g,, tandem guide RNAs) and compositions comprising the nucleic acids, to treat or prevent diseases and disorders that would benefit from multiplexing, e.g., from die excision of an exon, intron, or exon-intron j unction. The disclosure provides methods wherein a nucleic acid comprising a sequence encoding a hybrid or chimeric promoter and one or more sequences encoding one or more guide RNAs, when combined with an endonuclease or nucleic acid encoding an endonuclease, is capable of making two or more edits in the genome. For example, the disclosure includes methods of making two cleavages to excise small or large portions of a genome. 'The disclosure includes methods, for example, wherein tandem guide RNAs (e.g., tandem guide RNAs encoded by a nucleic acid disclosed herein, wherein the nucleic acid also comprises asequence encoding a hybrid or chimeric promoter that drives expression of one or both of the tandem guide RNAs), when used with an appropriate endonuclease, function to precisely delete a portion of any of exons 2, 3, 6, 9, 44, 45, 47, 48, 50, 51 or 53 of the DMD gene. However, methods of excising some or all other genomic portions, for treatment of other diseases or other purposes, is contemplated,

[0302] Tire disclosure provides methods that include administration or deliver}' of a nucleic acid comprising a sequence encoding a hybrid or chimeric promoter and one or more sequences encoding one or more guide RNAs capable of genome editing, e.g., excising a portion of a genome. The disclosure provides for methods wherein the sgRNAs, as described herein, are for use with the same class, type, subtype, and / or species of endonuclease, or a composition comprising the nucleic acid and optionally further comprising an endonuclease or a nucleic acid encoding an endonuclease.

[0303] Provided herein are methods of administering or delivering a nucleic acid comprising a sequence encoding a hybrid or chimeric promoter that drives expression of one or both of a first sequence encoding a first spacer and a second sequence encoding a second spacer. In such embodiments, two distinct spacers may target two distinct genomic loci, and the sgRNAs may comprise scaffolds that are the same or different. In some embodiments, the sgRNAs are for use with the same class, type, subtype, and / or species of endonuclease. The present disclosure also provides methods of administering or delivering sgRNAs that may localize a donor template to a Cas-induced double strand break at a sgRN A-specified genomic locus to facilitate gene correction and / or insertion. In one embodiment, a method comprises administration of a nucleic acid disclosed herein, wherein one spacer sequence of the nucleic acid will be designed to target the desired genomic locus and create a double strand break (DSB) while also targeting the donor template with the second spacer. Donor template constructs may be linear DNA with Cas / sgRNA localizing the donor template to the genomic DSB, or donor templates may be circularized with Cas / sgRNA functioning both to localize the donor to the genomic DSB and linearizing the donor template. Donors may have flanking regions of homologous sequences to the targeted genomic locus to enable homology directed repair.Alternatively, donors bearing no homology arms can be inserted into the genomic DSB via non- homologous end joining. Additional embodiments may be administered in a method, including a single sgRN A bridging between genome and donor, or administration of multiple sgRNAs to allow creation of multiple double strand breaks (in genome and / or in donor) and additional bridging interactions between genome and donor,

[0304] lliis disclosure also provides methods of treating a disease or disorder that would benefit from genome editing, such as from the excision of an exon, intron, or exon -intron junction.

[0305] In some embodiments, the disclosure provides for a method of treating a subject (e.g., a subject having DMD), comprising treating the subject with a plurality of nucleic acids comprising a sequence encoding a hybrid or chimeric promoter, wherein the plurality of nucleic acids administered to the subject are processed within the subject to result m separate sgRNA molecules. In someembodiments, the disclosure provides for a method of treating a subject (e.g., a subject having DMD), comprising treating the subject with a plurality of nucleic acids comprising a sequence encoding a hybrid promoter, wherein 1-60%, 1-40%, 1-20%, 1-10%, 1-5%, 5-60%, 5-40%, 5-20%, 5-10%, 10- 60%, 10-40%, 10-20%, 25-60%, 25-40%, or 40-60% of the plurality’ of nucleic acids administered to the subject are processed within the subject to result in separate sgRNA molecules.

[0306] This disclosure provides methods for gene editing and treating Duchenne Muscular Dystrophy (DMD) using any of the hybrid promoters described herein. In some embodiments, any of the compositions described herein may be administered to a subject in need thereof for use in making a double strand break, or excising a portion (e.g., less than about 250 nucleotides) in any of exons 2, 3, 6, 9, 44, 45, 47, 48, 50, 51 or 53 of the dystrophin (DMD) gene, and to treat DMD.

[0307] In some embodiments, the disclosure provides a method of inserting a template DNA into genomic DN A comprising, administering to a cell the nucleic acid comprising a sequence encoding a hybrid promoter and further comprising a first sequence encoding a first sgRNA and a second sequence encoding a second sgRNA, wherein the sgRNAs are in some embodiments for use with the same class, type, subtype, and / or species of endonuclease, or a composition comprising the nucleic acid and optionally further comprising an endonuclease or a nucleic acid encoding an endonuclease, and a template nucleic acid, wherein the template nucleic acid is inserted into the genome.

[0308] In some embodiments, the disclosure provides for a method of inserting a template nucleic acid into genomic DNA comprising administering to a cell (e.g., a cell in a subject): a) a nucleic acid comprising a sequence encoding a hybrid or chimeric promoter and further comprising a first sequence encoding a first sgRNA and a second sequence encoding a second sgRNA, b) a template nucleic acid; and c) an endonuclease or a nucleic acid encoding an endonuclease; wherein the first sgRNA guides the endonuclease to cut the genomic DNA at a specific locus, and wherein the second sgRNA facilitates the insertion of the donor template at the specific locus. In some embodiments, the template nucleic acid is a component of a larger polynucleotide (e.g., a plasmid or vector), and the second sgRNA guides the endonuclease to cut the polynucleotide. In some embodiments, the disclosure provides for a method of inserting a template nucleic acid into genomic DNA comprising administering to a cell (e.g., a cell in a subject): a) a first nucleic acid comprising a sequence encoding a first hybrid or chimeric promoter and further comprising a first sequence encoding a first sgRNA and a second sequence encoding a second sgRNA, b) a second nucleic acid comprising a sequence encoding a second hybrid or chimeric promoter and further comprising a third sequence encoding a third sgRNA and a fourth sequence encoding a fourth sgRNA; c) a template nucleic acid; and c) an endonuclease or a nucleic acid encoding an endonuclease; wherein the first sgRNA guides the endonuclease to cut the genomic DN A at a first locus and wherein the third sgRN A guides the endonuclease to cut the genomic DNA at a second locus, and wherein the second sgRNA and fourth sgRNA facilitate the incorporation of the donor template between the first and second loci.In some embodiments, the sequence encoding the first hybrid or chimeric promoter and the sequence encoding the second hybrid or chimeric promoter are the same. In some embodiments, the sequence encoding the first hybrid or chimeric promoter and the sequence encoding the second hybrid or chim eri c promoter are different. In some embodiments, one end of the template shares homology with a region abutting the first locus and the other end of the template shares homology with a region abutting the second locus. In some embodiments, the template nucleic acid is a component of a larger polynucleotide, and the second sgRNA guides the endonuclease to cut the polynucleotide at a first site and the fourth sgRNA guides the endonuclease to cut the polynucleotide at a second site. In some embodiments, the template nucleic acid is excised from the larger polynucleotide upon cleavage facilitated by the second sgRNA and fourth sgRNA. In some embodiments, a nucleotide sequence is excised from the genomic DNA as a result of the endonuclease cutting the genomic DNA at the first locus and the second locus. In some embodiments, the template is excised from the larger polynucleotide (e.g., plasmid or vector) as a result of the endonuclease cutting the polynucleotide at the first site and at the second site. In some embodiments, the larger polynucleotide is a linear polynucleotide. In some embodiments, the larger polynucleotide is a plasmid. In some embodiments, the larger polynucleotide is a circular plasmid. In some embodiments, the larger polynucleotide is a minicircle nucleic acid. In some embodiments, the larger polynucleotide is a viral nucleic acid.

[0309] In some embodiments, the template or donor nucleic acid for use in an y of the compositions or methods disclosed herein is 1-2.00, 1-150, 1-100, 1-50, 1-2.5, 1-10, 1-5, 5-200, 5-150, 5-100, 5-50, 5-25, 5-10, 10-200, 10-150, 10-100, 10-50, 10-25, 25-2.00, 25-150, 25-100, 25-50, 50- 200, 50-150, 50-100, 100-200, 100-150, or 150-200 nucleotides in length. In some embodiments, any of the methods or compositions disclosed herein excise a portion of genomic DNA that is 200-1000, 200-900, 200-700, 200-500, 200-400, 500-1000, 500-700. 1-200, 1-150, 1-100, 1-50. 1-25, 1-10, 1-5, 5-200, 5-150, 5-100, 5-50, 5-25, 5-10, 10-2.00, 10-150, 10-100, 10-50, 10-25, 25-2.00, 25-150, 25-100, 25-50, 50-200, 50-150, 50-100, 100-200, 100-150, or 150-200 nucleotides in length.

[0310] In some embodiments, nucleic acids comprising sequences encoding hybrid or chimeric promoters described herein, in any of the vector configurations described herein or in association with a lipid nanoparticle, may be administered to a subject in need thereof to make a double-strand break, excise a portion of a gene, and thereby treat diseases such as DMD or DM1. In some embodiments, the disclosure provides a method for treating DMD or DM1 comprising administering a therapeutically effective amount of a nucleic acid comprising a sequence encoding a hybrid or chimeric promoter and further comprising a first sequence encoding a first sgRNA and a second sequence encoding a second sgRNA (wherein, in some embodiments, the sgRNA s are for use with the same class, type, subtype, and / or species of endonuclease), or a composition comprising the nucleic acid and optionally further comprising an endonuclease or a nucleic acid encoding an endonuclease, to a subject having DMD or DM1. In some embodiments, the sgRNAs target the DMPK gene. Inparticular embodiments, the sgRNAs are designed to excise CTG repeats. In particular embodiments, the sgRNAs target the dystrophin gene.

[0311] In some embodiments, the disclosure provides a method for treating a disease or disorder that would benefit from an excision of an exon, intron, or exon-intron junction or portions thereof comprising administering a therapeutically effective amount of a nucleic acid comprising a sequence encoding a hybrid or chimeric promoter and further comprising a first sequence encoding a first sgRNA and a second sequence encoding a second sgRNA (wherein, in some embodiments, the sgRNAs are for use with the same class, type, subtype, and / or species of endonuclease), or a composition comprising the nucleic acid and optionally further comprising an endonuclease or a nucleic acid encoding an endonuclease to a subject in need thereof.

[0312] In some embodiments, the disclosure provides a method for treating a disease or disorder that would benefit from an excision of an exon, intron, or exon-intron junction compri sing administering a therapeutically effective amount of a nucleic acid comprising a sequence encoding a hybrid or chimeric promoter comprising a first sequence encoding a first sgRNA and a second sequence encoding a second sgRNA (wherein, in some embodiments, the sgRNAs are for use with the same class, type, subtype, and / or species of endonuclease), or a composition comprising the nucleic acid and optionally further comprising an endonuclease or a nucleic acid encoding an endonuclease to a subject in need thereof.

[0313] Table 4:

[0314] In some embodiments, trinucleotide repeats or a self-complementary region are excised by a nucleic acid disclosed herein and endonuclease from a locus or gene associated with a disorder, such as a repeat expansion disorder, which may be a trinucleotide repeat expansion disorder. A repeat expansion disorder is one in which unaffected individuals have alleles with a number ofrepeats in a normal range, and individuals having the disorder or at risk for the disorder have one or two aileles with a number of repeats in an elevated range relative to the normal range. Exemplary repeat expansion disorders are listed and described in Table 4. In some embodiments, the repeat expansion disorder is any one of the disorders listed in Table 4. In some embodiments, the repeat expansion disorder is DM1. In some embodiments, the repeat expansion disorder is Huntington’s Disease. In some embodiments, the repeat expansion disorder is Fragile X Syndrome. In some embodiments, tire repeat expansion disorder is a spinocerebellar ataxia. In some embodiments, the repeat expansion disorder is Friedrich’s Ataxia. In some embodiments, the locus or gene from which the trinucleotide repeats are excised is DMPK. In some embodiments, the locus or gene from which the trinucleotide repeats are excised is HTT. In some embodiments, the locus or gene from which the trinucleotide repeats are excised is Frataxin. In some embodiments, the locus or gene from which the trinucleotide repeats are excised is FMRI , In some embodiments, the locus or gene from which the trinucleotide repeats are excised is an A taxin. In some embodiments, the locus or gene from which the trinucleotide repeats are excised is a gene associated with a type of spinocerebellar ataxia.

[0315] 'The number of repeats that is excised may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2.000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000, or in a range bounded by any two of the foregoing numbers.

[0316] For example, where the TNRs are within the DMPK gene, excision of the TNRs may ameliorate one or more phenotypes associated with an expanded-repeat DMPK gene, e.g., one or more of increasing myotonic dystrophy protein kinase activity; increasing phosphorylation of phospholemman, dihydropyridine receptor, myogenin, L-type calcium channel beta subunit, and / or myosin phosphatase targeting subunit; increasing inhibition of myosin phosphatase; and / or ameliorating muscle loss, muscle weakness, hypersomnia, one or more executive function deficiencies, insulin resistance, cataract formation, balding, or male infertility or low fertility.

[0317] Where the TNRs are within the HTT gene, excision of the TNRs may ameliorate one or more phenotypes associated with an expanded-repeat HTT gene, e.g., one or more of striatal neuron loss, involuntary movements, irritability, depression, small involuntary movements, poor coordination, difficulty learning new information or making decisions, difficulty walking, speaking, and / or swallowing, and / or a decline in thinking and / or reasoning abilities.

[0318] Where the TNRs are within the FMRI gene, excision of the TNRs may ameliorate one or more phenotypes associated with an expanded-repeat FMRI gene, e.g., one or more of aberrant FMRI transcript or Fragile X Mental Retardation Protein levels, translational dysregulation of mRNAs normally associated with FMRP, lowered levels of phospho-cofilin (CFL1), increased levels of phospho-cofilin phosphatase PPP2CA, diminished mRNA transport to neuronal synapses, increased expression of HSP27, HSP70, and / or CRY AB, abnormal cellular distribution of lamin A / C isoforms, early-onset menopause such as menopause before age 40 years, defects in ovariandevelopment or function, elevated level of serum gonadotropins (e.g., FSH), progressive intention tremor, parkinsonism, cognitive decline, generalized brain atrophy, impotence, and / or developmental delay.

[0319] Where the TNRs are within the FMR2 gene or adjacent to the 5’ UTR of FMR2, excision of the TNRs may ameliorate one or more phenotypes associated with expanded-repeats in or adjacent to the FMR2 gene, e.g., one or more of aberrant FMR2 expression, developmental delays, poor eye contact, repetitive use of language, and hand -flapping.

[0320] Where the TNRs are within the AR gene, excision of the TNRs may ameliorate one or more phenotypes associated wi th an expanded-repeat AR gene, e.g., one or more of aberrant AR expression; production of a C -terminally truncated fragment of the androgen receptor protein; proteolysis of androgen receptor protein by caspase-3 and / or through the ubiquitin-proteasome pathway; formation of nuclear inclusions comprising CREB-bmding protein; aberrant phosphorylation ofp44 / 42, p38, and / or SAPK / JNK; muscle weakness; muscle wasting; difficulty walking, swallowing, and / or speaking; gynecomastia; and / or male infertility.

[0321] Where the TNRs are within the ATXNl gene, excision of the TNRs may ameliorate one or more phenotypes associated with an expanded-repeat ATXNl gene, e.g., one or more of formation of aggregates comprising ATXNl ; Purkinje cell death; ataxia; muscle stiffness; rapid, involuntary eye movements; limb numbness, tingling, or pain; and / or muscle twitches.

[0322] Where the TNRs are within tire ATXN2 gene, excision of the TNRs may ameliorate one or more phenotypes associated with an expanded-repeat ATXN2 gene, e.g., one or more of aberrant ATXN2 production; Purkinje cell death; ataxia; difficulty speaking or swallowing; loss of sensation and weakness in the limbs; dementia; muscle wasting; uncontrolled muscle tensing; and / or involuntary jerking movements.

[0323] Where the TNRs are within the ATXN3 gene, excision of the TNRs may ameliorate one or more phenotypes associated with an expanded-repeat ATXN3 gene, e.g., one or more of aberrant ATXN3 levels; aberrant beclin-1 levels; inhibition of autophagy; impaired regulation of superoxide dismutase 2; ataxia; difficulty swallowing; loss of sensation and weakness in the limbs; dementia; muscle stiffness; uncontrolled muscle tensing; tremors; restless leg symptoms; and / or muscle cramps.

[0324] Where the TNRs are within the CACNA1A gene, excision of the TNRs may ameliorate one or more phenotypes associated with an expanded-repeat CACNA1 A gene, e.g., one or more of aberrant CaV2.1 voltage-gated calcium channels in CACNAlA-expressing cells; ataxia; difficulty speaking; involuntary eye movements; double vision; loss of arm coordination; tremors; and / or uncontrolled muscle tensing.

[0325] Where the TNRs are within the ATXN7 gene, excision of the TNRs may ameliorate one or more phenotypes associated with an expanded-repeat ATXN7 gene, e.g., one or more of aberrant histone acetylation; aberrant histone deubiquitination; impairm ent of transactivation by CRX;formation of nuclear inclusions comprising ATXN7; ataxia; incoordination of gait; poor coordination of hands, speech and / or eye movements; retinal degeneration; and / or pigmentary macular dystrophy.

[0326] Where the TNRs are within the ATXN8OS gene, excision of the TNRs may ameliorate one or more phenotypes associated with an expanded-repeat ATXN8OS gene, e.g., one or more of formation of ribonuclear inclusions comprising ATXN8OS mRNA; aberrant KLHL1 protein expression; ataxia; difficulty speaking and / or walking; and / or involuntary eye movements.

[0327] Where the TNRs are within the PPP2R2B gene, excision of the TNRs may ameliorate one or more phenotypes associated with an expanded-repeat PPP2R2B gene, e.g., one or more of aberrant PPP2R2B expression; aberrant phosphatase 2 activity; ataxia; cerebellar degeneration; difficulty walking; and / or poor coordination of hands, speech and / or eye movements.

[0328] Where the TNRs are wdthin tire IBP gene, excision of the TNRs may ameliorate one or more phenotypes associated with an expanded-repeat TBP gene, e.g., one or more of aberrant transcription initiation; aberrant TBP protein accumulation (e.g., in cerebellar neurons); aberrant cerebellar neuron cell death; ataxia; difficulty walking; muscle weakness; and / or loss of cognitive abilities.

[0329] Where the TNRs are within the A TNI gene, exci sion of the TNRs may ameliorate one or more phenotypes associated with an expanded-repeat ATN1 gene, e.g., one or more of aberrant transcriptional regulation; aberrant ATN 1 protein accumulation (e.g., in neurons); aberrant neuron cell death; involuntary movements; and / or loss of cognitive abilities.

[0330] In some embodiments, any one or more of the nucleic acids comprising a sequence encoding a hybrid or chimeric promoter, vectors, compositions, or pharmaceutical formulations described herein is for use in a method disclosed herein or in preparing a medicament for treating or preventing a disease or disorder in a subject. In some embodiments, treatment and / or prevention is accomplished with a single dose, e.g,, one-time treatment, of medicament / composition.

[0331] In some embodiments, the disclosure provides a method of treating or preventing a disease or disorder in subject comprising administering any one or more of the nucleic acids comprising a sequence encoding a hybrid or chimeric promoter, vectors, compositions, or pharmaceutical formulations described herein. In some embodiments, the nucleic acids, vectors, compositions, or pharmaceutical formulations described herein are administered as a single dose, e.g., at one time. In some embodiments, the single dose achieves durable treatment and / or prevention. In some embodiments, the method achieves durable treatment and / or prevention. Durable treatment and / or prevention, as used herein, includes treatment and / or prevention that extends at least i) 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, or 15 weeks; ii) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 18, 24, 30, or 36 months; or iii) 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 years. In some embodiments, a single dose of the nucleic acids comprising a sequence encoding a hybrid or chimeric promoter, vectors, compositions, or pharmaceutical formulations described herein is sufficient to treat and / or prevent any of the indications described herein for the duration of the subject’s life.

[0332] In some embodiments, excision of a repeat or self-complementary region ameliorates at least one phenotype or symptom associated with the repeat or self-complementary region or associated with a disorder associated with the repeat or self-complementary region. This may include ameliorating aberrant expression of a gene encompassing or near the repeat or self-complementary’ region, or ameliorating aberrant activity of a gene product (noncoding RNA, mRNA, or polypeptide) encoded by a gene encompassing the repeat or self-complementary region.

[0333] In some embodiments, tire subject is a mammal. In some embodiments, the subject is human.

[0334] For treatment of a subject (e.g., a human), any of the nucleic acids comprising a sequence encoding a hybrid or chimeric promoter, vectors, compositions, or pharmaceutical formulations disclosed herein may be administered m a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The compositions and pharmaceutical formulations may be readily administered in a variety of dosage forms, such as injectable solutions. For parenteral administration in an aqueous solution, for example, the solution will generally be suitably buffered and the liquid diluent first rendered isotonic with, for example, sufficient saline or glucose. Such aqueous solutions may be used, for example, for intravenous, intramuscular, subcutaneous, and / or intraperitoneal administration.Combination Therapy

[0335] In some embodiments, the disclosure comprises combination therapies comprising any of the methods or uses described herein together with an additional therapy suitable for ameliorating any’ disease or disorder that would benefit from an excision of an exon, intron, or exon-intron junction, including DMD and DM1 .EXAMPLES

[0336] The following examples are provided to illustrate certain disclosed embodiments and are not to be construed as limiting the scope of this disclosure in any way.Example 1: Hybrid Promoter DesignA. Materials and Methods

[0337] It was hypothesized that low sgRNA expression level could be a limiting factor in achieving high genome editing efficiency. Using hybrid promoters comprising type III (minimized U6) and type II (tRNA) promoters could further increase sgRNA expression. Because in one embodiment the tRNA is expressed as a 5' fusion to the sgRNA, the tRNA should be cleaved off from the sgRNA. To validate whether this cleavage activity and the resulting genome editing efficiency were dependent on the protospacer sequence, six different protospacers targeting exon 53 were tested with the lead candidate hybrid promoters.B. cRT-PCR

[0338] An RNA ligation reaction was set up as shown in Tables 5-7 in PCJR. tubes, and the tubes were incubated for 4 hours at 25°C in a thermocycler followed by 4°C on hold.Table 5:Table 6:Table 7:

[0339] The samples were purified using RNA Clean & Concentrator-5 following manufacturer’s protocol. Eluted circularized RNA was prepared in PCR tubes as shown in Table 8 and incubated for 5 min at 70°C in thermocycler, followed by 4°C on hold.Table 8:| Total | 10 ul |

[0340] A reverse transcriptase reaction was set up by adding the following reagents of Table 9 in the 10 ul sample from step 3. The reaction was incubated for 1 hour at 42°C in thermocycler, followed by 70°C for 10 min, and 4°C on hold.Table 9:

[0341] PCRwas performed with the following 20 pl reaction shown in Tables 10 and 11 ,Table 10:Table 11

[0342] The PCR product was analyzed by Agilent Tapestation using High Sensitivity D1000 ScreenTape. The intensity of the band around 90 bp was considered a measurement for processed sgRNA, while intensity of the band around 160 bp was considered a measurement for unprocessed sgRNA.

[0343] The results of the assay are shown in Figure 3B,C. ICE Analysis1. Transfection

[0344] 800,000 HEK293T cells per well were subcultured into five 6-well plates, and the plated cells were incubated at 37 °C. On day 2, a transfection was performed using jetOPTIMUS®, modified from the manufacturer’s protocol. Under the transfection protocol, 1 ,550 ng CBh-SluCas9-2A-GFP was diluted into jetOPTIMUS® buffer, reaching total 80 pL, This diluent was prepared as a mastermix for all transfection reactions. Primer Exon53-F, with sequence aaatgtgagataacgtttggaag (SEQ ID NO: 8010) and Exon53-R, with sequence tttcagctttaacgtgattttctg (SEQ ID NO: 8011) were used.

[0345] Next, 50 ng of AAV-U6-hSlu4-sgRNA / CK8e-SluCas9, AAV-mU6-TRNAP-hSlu4- sgRNA / CK8e-SluCas9, and AAV-mU6-TRNAP16-hS1u4-sgRNA / CK8e-SluCas9through AAV- mU6-TRNAY-hSlu4-sgRNA / CK8e-SluCas9 (as shown below in the plate-well matrix) was prepared into 1 .5 ml microcentrifuge tubes, and 80 pL CBh-SluCas9-2A-GFP diluent was added into the 24 tubes. The jetOPTIMUS® reagent was vortexed for 5 seconds and spun down before use. Next, 1 .6 pL of jetOPTIMUS® was diluted into 78.4 pL of jetOPTIMUS® buffer. The solution was vortexed for 1 second and spun down briefly. This diluent was prepared as a master mix for all wells,

[0346] 80 pL of jetOPTIMUS® diluent was added to the plasmid diluent, and the solution was vortexed for 1 second . The resulting solution was incubated for 10 minutes at room temperature, and the transfection mixture was added to each well dropwise, distributing evenly. The plates were gently rocked back-and-forth and side-to-side, and incubated at 37 °C. The plate-well distribution was as follows:00347] The experiment was performed in triplicate to acquire data from three biological replicates.2. FACS

[0348] On day 6, each well was aspirated, and 800 pL TrypLE was added. After 5 min, 800 pL complete medium was added with serum. Tire cells were detached by pipetting and collected into 2mL microcentrifiige tubes. The tubes were centrifuged in 150 x g, 5 min and the supernatant was aspirated and resuspended in 600 pL PBS, 5% FBS. The resuspended cells were filtered through 35 pm cap filter and transfered to 1.5 mL microcentrifuge tube.3. Genomic DNA Extract

[0349] Maxwell® RSC Cultured Cells DNA Kit was prepared following manufacturer’s protocol. The sorted cells were gently pipetted, and 280 pL was transferred to well #1 of the cartridges. The remaining cells (-400,000) w'ere pelleted at 300 x g for 5 min. The supernatant was removed by aspiration and the pellets were frozen in -80°C, Next, the Maxwell® RSC 48 was ran, and tire genomic DNA was quantified using Nanodrop. 500,000 GFP positive cells were collected in 2 mL microcentrifuge tube. Collected cells were in approximately 1,400 pL solution. 500,000 negative cells were also collected from non-transfected well.4. ICE Analysis

[0350] The genomic DNA was diluted to 5 ng / pL, and PCR was performed for 25 samples. pVXHOO 1-024 and a Negative. Tire components are described in Table 12, and the protocol is described in Table 13.Table 12:Table 13:

[0351] A small amount (5pl) of the of the PCR product was analyzed to verify size and appropriate amplification on a E-Gel™ EX Double Comb Agarose Gels, 2%. 10 pl of PCR product was sent out for Sanger Sequencing. Automated ICE analyses were run, using sequencing file from Negative as a mock file for analysis. The following plasmids were tested, as shown in Table 14, Table 15 provides sequences of the plasmid elements.Table 14:Table 15:

[0352] Results for the ICE analysis are shown in Figure 3C.D. sgRNA Quantification1. Materials

[0353] The materials used for sgRNA quantification are listed in Tables 16, 17, and 18, Table 16: Common Reagents and MaterialsTable 17: ReagentsTable 18: Equipment2. RNA Extraction

[0354] Maxwell®1RSC simplyRNA Cells Kit were prepared following the manufacturer’s protocol from the frozen cell pellets. The Maxwell1® RSC 48 was run, and the RNA was quantified using Nanodrop.3. RT-qPCR

[0355] For plate preparation, Composition of Standards (“Standards” or “STDs”) were prepared in TE buffer, as shown in Table 19. Each STD was mixed well using a vortex mixer. Each reaction contains 4 pL of samples.Table 19: Composition of Standards

[0356] The qScnpt XLT One-Step RT-qPCR ToughMix was thawed, and placed Low ROX on wet ice or m a refrigerator set to maintain at 4°C. After thawing, mixture was centrifuged briefly and maintained at 4°C- and was not vortexed. The number of samples was determined and considered STDs. Tw'o additional samples were added to compensate for pipetting error and to ensure sufficient volume of reagent for all samples assayed in triplicate. The appropriate volume of RT-qPCR Master Mix formulation was prepared, as shown in Table 20.Table 20: Composition of Master Mix Formulation

[0357] 16jxL of Master Mix were pipetted into each well of 384-weIl qPCR plate according to plate layout. 4pL of samples and STDs were loaded into each well. The plate was sealed with a MicroAmp Clear Adhesive Film.

[0358] Using the Quant Studio Flex 6, a new experiment was loaded on the Quant Studio software. Each well was assigned on the Quant Studio software with respective sample IDs and dye. STDs were assigned as one target and samples were assigned as another target. The targets were inputted into the thermocycler program and saved as described m Table 21.Table 21 : QuantStudio Real-Time PCR Settings

[0359] After the experiment was run, the auto-set threshold (under the amplification-plot section) for each STD was noted. The threshold was manually edited for samples to be equivalent with STDs. The file was saved to the server, and the Ct values were exported from Quant Studio software into an excel file.

[0360] To analyze the data, a scatter plot was created with the theoretical copy numbers (log) on the x-axis and the corresponding Ct values on the y-axis, and a linear regression model with the quation y=mx +b was generated, where y = Ct value, instalment response; m = slope or PCR efficiency, x = theoretical copy numbers (log); and b = y-intercept.

[0361] The log copy number is calculated using the following formula: Copy Number (log) = (Ct mean - Intercept) / Slope.

[0362] The copy number per well is calculated using the following formula: Copy number (per well) = 10A(Log Copy Number).

[0363] The Copy number per ng of RNA is calculated using the following formula: Copy number (per ng)::::Copy number per well / ng input RNA.E. tRNA Mutants Activity Test1. Materials and Methods

[0364] As discussed above, low sgRNA expression level could be a limiting factor when trying to achieve high genome editing efficiency. Using hybrid promoters comprising type III (minimized U6) and type II (tRN A) promoters could increase sgRNA expression. Using a tRNA promoter to drive sgRNA expression lead to overexpression of the tRNA. However, altering the expression level and ratio of tRNAs may have undesired side effects on a cell. Using a tRNA mutant that may not function as a tRNA in translational machinery could avoid such side effects.

[0365] The purpose of this experiment was to screen different tRNA mutants for translation activity. For easy validation of tRNA activity, all tested mutant tRNAs had an anticodon against the stop codon. Suppressor tRNAs are mutant tRNAs that can charge an amino acid where the stop codon is located, making the ribosome read through the stop codon. A reporter vector that detects suppressor tRNA activity was cotransfected with a vector expressing an sgRNA under each hybrid promoter. The reporter activity was detected by flow cytometry.2. Transfection

[0366] Two-hundred thousand (200,000) HEK293T cells / well were subcultured into two 24-well plates, each containing 0.5 mL complete medium, lire plates were incubated at 37 °C. On day 2, transfection was performed using JetOPTIMUS, modified from the manufacturer’s protocol. 200 ng of reporter was diluted using Plasmid 126, 127, and 128(as described in Table 22) into JetOPTIMUS buffer, reaching 20 uL total. This diluent was prepared as a master mix for each transfection reaction, with the same stop codon tested (See Table 22 below). 200 ng each of plasmids 089 and 1 15-125 was added to separate 1.5 mL microcentrifuge tubes. 20 pL reporter vector diluent was added to each tube. Transfection and flow cytometry were performed m three biological replicates. The reagents were mixed by vortex JetOPTIMUS for 5 seconds and spun down before use. Next, 0.4 pL JetOPTIMUS® was diluted into 19.6 pL JetOPTIMUS buffer, and the mixture was vortexed for 1 second and spun down briefly. This diluent was prepared as a master mix for all wells. 20 pL of JetOPTIMUS® was added as a diluent to the plasmid diluent, and the solution was vortexed for 1 second, and incubated for 10 minutes at room temperature. The transfection mixture was added to each well dropwise and distributed evenly. The plates were gently rocked back and forth and from side to side, and incubated at 37 °C.Table 22:3. Flow Cytometry

[0367] On Day 5 (3 days post-transfection), each well was aspirated, and 200 pl, TrypLE was added. After 6 minutes, 200 pL complete medium with serum was added. Cells were detached bypipetting and collected into 2 mL microcentrifuge tubes. The tubes were centrifuged at 150 x g, 5 min, and tire supernatant was aspirated, then resuspended in 200 pL PBS, 5% FBS. The resuspended cells were filtered through Flowmifilters into 1.5 ml, microcentrifuge tubes. Then, the miRFP670nano signal and mGreenLantem signal were gated using Negative cells (tor no signal), U6 (for only miRFP670nano), and Positive (for both miRFP670nano and mGreenLantem), and percentages were recorded for 100,000 cells and miRFP670nano positi ve cells, as well as the miRFP670nano and mGreenLantem double positive population.4. Analysis

[0368] The percentage of double positive cells in the rniRFP670nano positive population was calculated as an indication of suppressor tRNA activity. Results are shown in Figures 12A-12C.F. Results

[0369] As discussed above with regards to the cRT-PCR analysis, Figure 3B shows tRNA cleavage efficiency data. As shown, hybrid promoters with a miniL!6-tRNAProand modified leader sequence (labeled as 27-29) showed high tRNA cleavage.

[0370] As discussed above with regards to the ICE analysis, Figure 3C shows genome editing efficiency. Hybrid promoters with miniU6 showed the highest genome editing efficiency among the hybrid promoters with the same type II promoters. As shown, hybrid promoters #28 and #31 showed the highest genome editing efficiency. Hybrid promoter #28 comprises a miniU6-tRNAp!0and modified leader sequence. Hybrid promoter #31 comprises a mmiU6 fused to a tRNA other than tRNAGiB, tRNAGiu, tRNAGiy, or tRNA?ro.

[0371] As discussed above with regard to the sgRNA quantification. Figure 3A shows sgRNA expression. Promoters 27-29 are miniU6-tRNAprohybrid promoters with a modified leader sequence. Promoter 30 is a mmiU6-tRNAGlu hybrid promoter with modified leader sequence. Promoters 31-48 are a miniU6 fused to a tRNA other than tRNA61", t.RNAG I“, tRNAGly, or tRNAPro. See Table 14 for further descriptions of the promoters assessed in the experiments of Figures 3A-3C. The hybrid promoters showed high sgRNA expression compared to a standard U6 promoter.

[0372] As discussed above with regard to the tRNA Mutants Activity Testing, Figures 12A-C show the sgRNA expression (Figure 12A), tRNA cleavage efficiency (Figure 12B), and editing efficiency (Figure 12C) for 4 different promoters, including U6, mini-U6-Dm (mini-U6-tRNAAspM’“'), miniU6.7SK~Dm (minimized U6 / 7SK chimeric promoter-tRNAAspMut; SEQ ID NO: 9017) and mini7SK.U6-Dm (minimized 7SK / U6 chimeric promoter-tRNAAspMut; SEQ ID NO: 9018). These promoters included the ‘gta’ leader sequence. These figures show that alternate minimized Type IIIpromoters based on chimeric promoter of 7SK and U6 show comparable activity when used as hybrid promoter. Each of the mini-U6-Dm (mini-L!6dl<NAAspMut), miniU6.7SK-Dm (minimized U6 / 7SK chimeric promoter-tRNAAspMul) and mini7SK.U6-Dm (minimized 7SK / U6 chimeric promoter- tRNAAspMutshowed improved sgRNA expression when compared to U6 (Figure 12A). The three hybrid promoters also showed high tRNA cleavage efficiency (Figure 12B) and editing efficiency(Figure 12C).

Claims

What is claimed is:

1. A nucleic acid comprising a sequence encoding a hybrid promoter, wherein the hybrid promoter comprises (a) a first portion comprising a mini-U6 pol III promoter or Mi l pol III promoter, and (b) a second portion comprising a tRNA promoter.

2. The nucleic acid of claim 1, wherein the nucleic acid is in 5’ to 3’ order, and wherein the sequence encoding the mini-116 poi III promoter or Ml 1 pol III promoter is 5’ to the tRNA promoter.

3. The nucleic acid of claim 1 , wherein the nucleic acid is in 3’ to 5’ order, and wherein the sequence encoding the mini-U6 pol III promoter or Ml 1 pol III promoter is 3’ to the tRNA promoter.

4. The nucleic acid of any one of claims 1-3, wherein the mini-116 pol III promoter or Mi l pol III promoter is a mini-U6 pol III promoter.

5. The nucleic acid of claim 4, comprising a sequence with about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 9005.

6. The nucleic acid of claim 4, comprising the sequence of SEQ ID NO: 9005.

7. The nucleic acid of any one of claims 1-3, wherein the nnni-L!6 pol III promoter or MH pol III promoter is an Ml 1 pol III promoter.

8. The nucleic acid of claim 7, comprising a sequence with about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 708.

9. The nucleic acid of claim 7, comprising the sequence of SEQ ID NO: 708.

10. The nucleic acid of any one of claims 1-9, wherein the tRNA promoter is modified.

11. The nucleic acid of claim 10, wherein the tRNA promoter has been modified so that the tRNA is not functional.

12. The nucleic acid of any one of claims 1-6 and 10-11, wherein the mini-116 pol III promoter is less than about 74 bp in length or greater than about 117 bp in length and comprises, in 5’ to 3' order, a distal sequence element (DSE), a proximal sequence element (PSE), and a TATA box.

13. Tire nucleic acid of claim 12, wherein the mini-U6 pol III promoter is between about 40 and about 73 bp, between about 50 and about 73 bp, between about 60 and about 73 bp, or between about 70 and about 73 bp in length.

14. The nucleic acid of claim 12, wherein the mini-U6 pol III promoter is greater than about 117 bp, greater than about 120 bp, greater than about 130 bp, greater than about 140 bp, greater than about 150 bp, or greater than about 200 bp in length.

15. The nucleic acid of any one of claims 1-14, wherein the tRN A comprises at least one of a T-loop, a D-loop, a V-loop, an anticodon loop, a T-stem, a D-stem, an anticodon stem, and an accepter stem.

16. A nucleic acid encoding a tRNA, wherein the tRNA is modified so that it does not produce a functional tRNA.

17. The nucleic acid of claim 16, wherein the tRNA 1) is modified such that it cannot bind an amino acid; 2) is modified in the D-stem region;, and optionally is modified at position 9, 25, or 26 of the D-stem corresponding to the sequence of SEQ ID NO: 9022 or 9023; 3) is modified at one or more of G9A or G25A corresponding to the sequence of SEQ ID NO: 9022, and / or is modified at one or more of U25C or C26G corresponding to the sequence of SEQ ID NO: 9023; 4) is modified in the anticodon loop; 5) is modified at one or more of positions 33, 34, 35, 36, or 37 of the anticodon loop corresponding to the sequence of any one of SEQ ID NOs: 9019-9027; 6) is modified in the anticodon loop and the modification comprises one or more of A33C, C33U, A34U, G34U, G34C, C34U, A35U, C35A, U35C, G35A, U35A, C36A, or U37A corresponding to the sequence of anyone of SEQ ID NOs: 9019-9027; 7) is modified in the acceptor stem; 8) is modified at position 70 of the acceptor stem corresponding to the sequence of SEQ ID NO: 9019 and / or is modified at position 73 of the acceptor stem corresponding to the sequence of SEQ ID NO: 9023; and / or 9) is modified at one or more of U70C corresponding to the sequence of SEQ ID NO: 9019 or G73C corresponding to the sequence of SEQ ID NO: 9023.

18. The nucleic acid of any one of claims 14-17, wherein tire tRNA is tRNAul“, tRNA',lu, tRNAGly, tRNAPro, tRNAAsp, or tRNAAspMut.

19. The nucleic acid of any one of the preceding claims, wherein the tRNA is tRNAAspMu:.

20. The nucleic acid of any one of the preceding claims, further comprising a leader sequence, and / or a trailer sequence.21 . The nucleic acid sequence of claim 20, wherein the leader sequence compri ses the sequence of anyone of SEQ ID NOs: 8050-8075.

22. The nucleic acid sequence of claim 20, wherein the leader sequence comprises the sequence of any one of SEQ ID NOs: 8050-8060.

23. The nucleic acid sequence of claim 20, wherein the leader sequence comprises the sequence of anyone of SEQ ID NOs: 8051 or 8053-8060.

24. A nucleic acid comprising a sequence encoding a hybrid promoter, wherein the hybrid promoter comprises (a) a first portion comprising a mini-116 pol III promoter, and (b) a second portion comprising a nucleic acid encoding tRNAAsp!v!u!(SEQ ID NO: 8006).

25. Tie nucleic acid of claim 24, wherein the nucleic acid is in 5’ to 3’ order, and wherein the mini-U6 Pol III promoter is 5’ to the tRNAAspMut(SEQ ID NO: 8006).

26. Tie nucleic acid of claim 24, wherein the nucleic acid is in 3’ to 5’ order, and wherein the mini-116 Pol III promoter is 3’ to the tRNAAspMut(SEQ ID NO: 8006).

27. The nucleic acid of any one of claims 24-26, further comprising a leader sequence, and / or a trailer sequence.

28. The nucleic acid sequence of claim 27, wherein the leader sequence comprises the sequence of anyone of SEQ ID NOs: 8050-8075.

29. The nucleic acid sequence of claim 27, wherein the leader sequence comprises the sequence of any one of SEQ ID NOs: 8050-8060.

30. The nucleic acid sequence of claim 27, wherein the leader sequence comprises the sequence of any one of SEQ ID NOs: 8051 or 8053-8060.31 . Hie nucleic acid of claim 27, wherein the leader sequence has the sequence of any one of GTGGC (SEQ ID NO: 8050), GTA (SEQ ID NO: 8051), or GGA (SEQ ID NO: 8052).

32. The nucleic acid of claim 31, wherein the leader sequence is GTGGC (SEQ ID NO: 8050).

33. The nucleic acid of claim 31, wherein the leader sequence is GTA (SEQ ID NO: 8051).

34. The nucleic acid of claim 31, wherein the leader sequence is GGA (SEQ ID NO: 8052).

35. The nucleic acid of any one of the preceding claims, further comprising one or more sgRNAs.

36. Ihe nucleic acid of claim 35, wherein the one or more sgRN As targets exon 53.

37. The nucleic acid of claim 35, wherein the one or more sgRNAs is E53SL320nt(SEQ ID NO: 1080), E53SL7 (SEQ ID NO: 1081 ), E53SL14 (SEQ ID NO: 1082), E53SL16 (SEQ ID NO: 1083), E53SL23 (SEQ ID NO: 1084), E53SL25 (SEQ ID NO: 1085), or E53SL3 (SEQ ID NO: 1086).

38. The nucleic acid of claim 35 , wherein the one or more sgRNAs targets exon 51.

39. The nucleic acid of claim 35, wherein the one or more sgRNAs targets exon 2, 3, 6, 9, 44, 45, 47, 48, 50, 51 or 53.

40. The nucleic acid of any one of the preceding claims, further comprising a trailer sequence.

41. Ihe nucleic acid of claim 40, wherein the trailer sequence is an adenine (a).

42. A nucleic acid comprising a sequence encoding a minimized 7SK promoter comprising SEQ ID NO: 9011 or 9016.

43. A nucleic acid comprising a sequence encoding a minimized U6 promoter comprising SEQ ID NO:9010 or 9015.

44. A nucleic acid comprising a sequence encoding a hybrid promoter, wherein the hybrid promoter comprises (a) a first portion comprising a minimized 7SK promoter and / or a minimized U6 promoter and (b) a second portion comprising a tRNA promoter.

45. Ihe nucleic acid of claim 44, wherein the first portion comprises a sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 9011 or 9016.

46. Tire nucleic add of claim 44, wherein the first portion comprises the sequence of SEQ ID NO:9011 or 9016.

47. The nucleic acid of claim 44, wherein the second portion comprises a sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%. 97%, 98%, 99%, or 100% identity to SEQ ID NO: 9010 or 9015.

48. The nucleic acid of claim 44, wherein the second portion comprises the sequence of SEQ ID NO: 9010 or 9015.

49. The nucleic acid of claim 44, comprising a sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%. 97%, 98%, 99%, or 100% identity to SEQ ID NO: 9017 or 9018.

50. The nucleic acid of claim 44, comprising the sequence of SEQ ID NO: 9017 or 9018.51 . The nucleic acid of any one of claims 44-50, wherein the tRNA promoter is modified.

52. The nucleic acid of claim 51, wherein the tRNA promoter is apart of a tRNA that has been modified so that the tRNA is not functional.

53. The nucleic acid of any one of claims 44-52, wherein the tRNA comprises at least one of a T-loop, a D-loop, a V-loop, an anticodon loop, a T-stem, a D-stem, an anticodon stem, and an accepter stem.

54. The nucleic acid of any one of claims 44-53, wherein the nucleic acid is in 5’ to 3’ order, and wherein the first portion is 5’ to the second portion.

55. The nucleic acid of claim 1, wherein the nucleic acid is in 3' to 5’ order, and w he rein the first portion is 3’ to the second portion.

56. The nucleic acid of claim 52, wherein the tRNA I) is modified such that it cannot bind an amino acid; 2) is modified m the D-stem region;, and optionally is modified at position 9, 25, or 26 of the D-stem corresponding to the sequence of SEQ ID NO: 9022 or 9023; 3) is modified at one or more of G9A or G25A corresponding to the sequence of SEQ ID NO: 9022, and / or is modified at one or more of U25C or C26G corresponding to the sequence of SEQ ID NO: 9023; 4) is modified in the anticodon loop; 5) is modified at one or more of positions 33, 34, 35, 36, or 37 of the anticodon loop corresponding to the sequence of any one of SEQ ID NOs: 9019-9027; 6) is modified in the anticodon loop and the modification comprises one or more of A33C, C33U, A34U, G34U, G34C, C34U, A35U, C35A, U35C, G35A, U35A, C36A, or U37A corresponding to tire sequence of any one of SEQ ID NOs: 9019-9027; 7) is modified in the acceptor stem; 8) is modified at position 70 of the acceptor stem corresponding to the sequence of SEQ ID NO: 9019 and / or is modified at position 73 of the acceptor stem corresponding to the sequence of SEQ ID NO: 9023; and / or 9) is modified at one or more of U70C corresponding to the sequence of SEQ ID NO: 9019 or G73C corresponding to the sequence of SEQ ID NO: 9023.

57. The nucleic acid of claim 52 or 56, wherein the tRNA is t:RNAGin, tRNAOlu, tRNA'jly, tRNAEro, tRNAAsp, or tRNAAspMut.

58. Hie nucleic acid of any one of claims 44-56, wherein tire tRNA is tRNAAspJV[ut.

59. A composition comprising the nucleic acid of any one of the preceding claims and optionally further comprising an endonuclease or a nucleic acid encoding an endonuclease.

60. The composition of claim 59, wherein the nucleic acid of any one of the preceding claims and / or the nucleic acid encoding the endonuclease, if present, are associated with a viral vector.

61. The composition of claim 59, wherein the nucleic acid of any one of the preceding claims and / or the nucleic acid encoding the endonuclease, if present, are associated with a viral vector, and wherein the viral vector is an adeno-associated virus vector (AAV), a lentiviral vector, an integrasedeficient lentiviral vector, an adenoviral vector, a vaccinia viral vector, an alphaviral vector, or a herpes simplex viral vector.

62. The composition of claim 61, wherein the viral vector is an AA V vector.

63. The composition of claim 62, wherein the AAA7vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 vector, wherein the number following AAV indicates the AAV serotype.

64. The composition of claim 63, wherein tire AA V vector is an AAV9 vector.

65. A vector encoding the nucleic acid of any one of claims 1 -58.

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