AAV vectors for gene editing

Abstract

Provided herein polynucleotides configured for incorporation into recombinant adeno-associated virus (AAV) vectors. The polynucleotides encode for CRISPR proteins, gRNA, and ancillary components of AAV vectors useful in the modification of target nucleic acids. The systems are also useful for introduction into cells, for example eukaryotic cells having mutations in the target nucleic acid of a gene. Also provided are methods of using such AAV vectors to modify cells having such mutations.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. provisional patent application Nos. 63 / 123,112, filed on December 9, 2020, and 63 / 235,638, filed on August 20, 2021, the contents of which are incorporated by reference in their entirety herein. INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The sequence listing paragraph application contains a Sequence Listing which has been submitted in ASCII format via EFS-WEB and is hereby incorporated by reference in its entirety. Said ASCII copy, created on December 9, 2021 is named SCRB-028_02WO_SeqList_ST25.txt and is 13 MB in size. BACKGROUND

[0003] Gene editing holds great promise for treating or preventing many genetic diseases. However, safe and targeted delivery of CRISPR gene editing machinery into the desired cells is necessary to achieve therapeutic benefit. There remains a need in the art for compositions and methods for delivering CRISPR gene editing machinery to cells in vitro and / or in vivo. SUMMARY

[0004] The present disclosure relates to AAV vectors for the delivery of CRISPR nucleases to cells for the modification of target nucleic acids.

[0005] In some embodiments, the present disclosure provides polynucleotides useful for production of AAV transgenes (transgene plasmids for example), as well as for the production of recombinant adeno-associated virus (AAV) vectors. In some embodiments, the disclosure provides polynucleotides encoding a first adeno-associated virus (AAV) 5’ inverted terminal repeat (ITR) sequence, a second AAV 3’ ITR sequence, a CRISPR nuclease, a first guide RNA (gRNA), one or more promoters and, optionally, accessory elements; all encompassed in a single expression cassette capable of being incorporated into a single AAV particle. In other embodiments, the polynucleotides comprise sequences encoding a first 5’ AAV ITR sequence, a second 3’ AAV ITR sequence, a CRISPR nuclease, a first gRNA, a first promoter, a second promoter, and, optionally, one or more accessory elements. In still other embodiments, the polynucleotides comprise sequences encoding a first 5’ AAV ITR sequence, a second 3’ AAV ITR sequence, a CRISPR nuclease, a first gRNA, a second gRNA, a first promoter, a second promoter, a third promoter, and, optionally, one or more accessory elements.

[0006] In some embodiments, the sequence encoding the CRISPR protein and the gRNA sequence is less than about 3100, less than about 3090, less than about 3080, less than about 3070, less than about 3060, less than about 3050, or less than about 3040 nucleotides in combined length. In other embodiments, the polynucleotide encoding the CRISPR protein sequence and the gRNA sequence are less than about 3040 to about 3100 nucleotides in combined length.

[0007] In some embodiments, the polynucleotide sequences of the first promoter and the at least one accessory element are greater than at least about 1300, at least about 1350, at least about 1360, at least about 1370, at least about 1380, at least about 1390, at least about 1400, at least about 1500, at least about 1600 nucleotides, at least 1650, at least about 1700, at least about 1750, at least about 1800, at least about 1850, or at least about 1900 nucleotides in combined length. In other embodiments, the polynucleotide sequences of the first promoter, the second promoter, and two or more accessory elements are greater than at least about 1300 to at least about 1900 nucleotides in combined length. In some embodiments, the polynucleotide sequences of the first promoter, the second promoter, and the two or more accessory elements are greater than 1314 nucleotides in combined length. In other embodiments, the polynucleotide sequences of the first promoter, the second promoter, and the two or more accessory elements are greater than 1381 nucleotides in combined length. In one embodiment, the polynucleotide sequences of the first promoter, the second promoter, and the two or more accessory elements comprise at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or at least 35% or more of the total polynucleotide sequence length.

[0008] In some embodiments, the accessory element of the polynucleotide is selected from the group consisting of a poly(A) signal, a gene enhancer element, an intron, a posttranscriphonal regulatory element, a nuclear localization signal (NLS), a deaminase, a DNA glycosylase inhibitor, a stimulator of CRISPR-mediated homology-directed repair, and an activator or repressor of transcription. In some embodiments, the accessory elements enhance the expression, binding, activity, or performance of the CRISPR protein as compared to the CRISPR protein in the absence of said accessory element. In particular embodiments, the enhanced performance is an increase in editing of a target nucleic acid upon expression of the CRISPR components in an in vitro assay of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 1500%, at least about 200%, or at least about 300%.

[0009] In some embodiments, the present disclosure provides a polynucleotide encoding a CRISPR protein that is a Class 2, Type V CRISPR protein. In some embodiments, the Class 2, Type V CRISPR protein is a CasX. In some embodiments, the CasX comprises a sequence selected from the group consisting of SEQ ID NOS: 1-3 and the sequences of SEQ ID NOS: 49160, 40208-40369 and 40828-40912, or a sequence having at least 85%, at least 90%, at least 95%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. In some embodiments, the present disclosure provides a polynucleotide encoding a Class 2, Type V CRISPR protein wherein the encoded CRISPR protein comprises the sequence of SEQ ID NO: 145 comprising at least one modification in one or more domains, wherein the one or more modifications are selected from the group consisting of the modifications set forth in Tables 3033, wherein the one or more modifications results in an improved characteristic relative to the CRISPR protein of SEQ ID NO: 145.

[0010] In some embodiments, the polynucleotide encodes a first and a second gRNA wherein the encoded gRNA each comprise a sequence selected from the group of sequences of SEQ ID NOS: 2101-2285, 39981-40026, 40913-40958, and 41817 as set forth in Table 2, or a sequence having at least 85%, at least 90%, at least 95%, at least 95%, at least 96%, at least 97%, at least 98% identity thereto. In some embodiments, the encoded first and second gRNA comprise a scaffold sequence having one or more modifications relative to SEQ ID NO: 2238, wherein the one or more modifications result in an improved characteristic in the expressed first and second gRNA, wherein the one or more modifications comprise one or more nucleotide substitutions, insertions, and / or deletions as set forth in Table 28, wherein the one or more modifications result in an improved characteristic in the expressed first and second gRNA. In another embodiment, the encoded first and second gRNA comprise a scaffold sequence having one or more modifications relative to SEQ ID NO: 2239, wherein the one or more modifications result in an improved characteristic in the expressed first and second gRNA, wherein the one or more modifications comprise one or more nucleotide substitutions, insertions, and / or deletions as set forth in Table 28, wherein the one or more modifications result in an improved characteristic in the expressed first and second gRNA.

[0011] In some embodiments, the polynucleotide comprises 5' and 3' ITRs, wherein the ITRs are derived from serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV 44.9, AAV-Rh74, or AAVRhlO.

[0012] In some embodiments, the polynucleotide comprises one or more sequences selected from the group consisting of the sequences of Tables 8-10, 12, 13, and 17-22 and 24-27, or a sequence having at least 85%, at least 90%, at least 95%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0013] In other embodiments, the present disclosure provides a recombinant adeno-associated virus (rAAV) comprising an AAV capsid protein, and the polynucleotide of any one of the embodiments disclosed herein. In some embodiments, the AAV capsid protein is derived from serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV 44.9, AAV-Rh74, or AAVRhlO.

[0014] In some embodiments, the present disclosure provides a method of making a recombinant AAV vector, comprising providing a population of cells, and transfecting the population of cells with a vector comprising the polynucleotide of any of the embodiments disclosed herein. In some embodiments, the population of cells expresses the AAV rep and cap proteins.

[0015] In some embodiments, the present disclosure provides AAV vectors wherein one or more component sequences are selected from the group consisting of 5' ITR, 3' ITR, pol III promoter, pol II promoter, encoding sequence for CRISPR nuclease, encoding sequence for gRNA, accessory element, and poly(A) are substantially depleted of CpG dinucleotides, wherein the component sequences retain their functional characteristics (e.g., the ability to drive expression or the ability to retain editing potential for a target nucleic acid). In some embodiments, the AAV vectors that are substantially depleted of CpG dinucleotides exhibit reduced immunogenic properties (e.g., reduced ability to elicit inflammatory cytokines or antibodies to components of the AAV), e.g. when administered.

[0016] In some embodiments, the present disclosure provides a method for modifying a target nucleic acid in a population of mammalian cells, comprising contacting a plurality of the cells with an effective amount of the rAAV of any of the embodiments disclosed herein, wherein the target nucleic acid of a gene of the cells targeted by the expressed gRNA is modified by the expressed CRISPR protein.

[0017] In some embodiments, the present disclosure provides a method for treating a disease in a subject (e.g. a human) caused by one or more mutations in a gene of the subject, comprising administering a therapeutically effective dose of the rAAV of any of the embodiments disclosed herein.

[0018] In some embodiments, the present disclosure provides a method of reducing the immunogenicity of an rAAV, comprising deleting all or a portion of the CpG dinucleotides of the sequences of the AAV components selected from the group consisting of 5' ITR, 3' ITR, pol III promoter, pol II promoter, encoding sequence for CRISPR nuclease, encoding sequence for gRNA, accessory element, and poly(A). INCORPORATION BY REFERENCE

[0019] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0020] The contents of WO / 2020 / 247882, published December 10, 2020 and PCT / US2021 / 061673, filed December 2, 2021, are incorporated by reference in their entireties herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0022] FIG. 1 shows a schematic of the AAV construct described in Example 1.

[0023] FIG. 2 shows results of an editing assay using AAV transgene plasmids nucleofected into mNPCs, as described in Example 1, demonstrating that the CasX and targeting guide in three different vectors (constructs 1, 2, and 3) edits on target (tdTomato) with high efficiency compared to non-targeting control (NT). Editing was assessed by FACS 5 days posttransfection. Data are presented as mean ± SEM for n= 3 replicates.

[0024] FIG. 3 shows results of an editing assay using AAV transgene plasmids nucleofected into mNPCs at four different dose levels, as described in Example 1. CasX delivered as an AAV transgene plasmid to mNPCs edits on target with high efficiency in a dose-dependent manner, compared to non-targeting control (NT). CasX variant 491 with scaffold variant 174 and spacer targeting tdTomato in three different vectors (constructs 1, 2, and 3) were nucleofected in mNPCs, and editing was assessed by FACS 5 days post-transfection. Data are presented as mean ± SEM for n= 3 replicates.

[0025] FIG. 4 shows results of an editing assay using AAV vector construct 3 transduced into mNPCs at 3-fold dilutions, assessed by FACS five days post-transduction, as described in Example 1. Data are presented as mean ± SEM for n= 3 replicates. MOI: multiplicity of infection.

[0026] FIG. 5 is a scanning transmission micrograph showing AAV particles with packaged CasX variant 438, gRNA scaffold 174 and spacer 12.7, as described in Example 2. AAV were negatively stained with 1% uranyl acetate. Empty particles are identified by a dark electron dense circle at the center of the capsid.

[0027] FIG. 6 shows results of an immunohistochemistry staining of mouse coronal brain sections, as described in Example 3. Mice received an ICV injection of 1 x 1011 AAV packaged with CasX 491, gRNA scaffold 174 with spacer 12.7 (top panel), which were able to edit the tdTom locus in the Ai9 mice (edited cells appear white). The bottom panel shows that CasX 491 and scaffold 174 with a non-targeting spacer administered as an AAV ICV injection did not edit at the tdTom locus. Tissues were processed for immunohistochemical analysis 1 month postinjection.

[0028] FIG. 7 shows the results of an editing assay of the tdTom locus in mNPCs using AAV transgene plasmids of constructs having variations in the CasX promoters, as described in Example 4. Editing was assessed by FACS 5 days post-transfection. Data are presented as mean ± SEM for n= 3 replicates.

[0029] FIG. 8 shows the results of an editing assay of the tdTom locus in mNPCs using AAV transgene plasmids of constructs having variations in the CasX promoters, as described in Example 4. Editing was assessed by FACS 5 days post-transfection. Data are presented as mean ± SEM for n = 3 replicates.

[0030] FIG. 9 shows the results of an editing assay of the tdTom locus in mNPCs using AAV transgene plasmids of constructs having variations in the CasX promoters and transgene size (see table insert), as described in Example 4. Editing was assessed by FACS 5 days posttransfection. Data are presented as mean ± SEM for n = 3 replicates.

[0031] FIG. 10 shows the results of an editing assay of the tdTom locus in mNPCs using AAV vectors incorporating the same promoters as shown in FIG. 9, as described in Example 4. The graph on the left are results testing 3-fold dilutions of the constructs, while the graph on the right are results of editing using an MOI of 2 x 105 vg / cell. Editing was assessed by FACS 5 days post-transfection. Data are presented as mean ± SEM for n = 3 replicates.

[0032] FIG. 11 shows the results of an editing assay of the tdTom locus in mNPCs using AAV vectors with protein promoter variants designed to reduce transgene size, compared to AAV with the top 4 protein promoter variants identified previously (AAV.3, AAV.4, AAV.5 and AAV.6), as described in Example 4. Editing was assessed by FACS 5 days post-transfection. Data are presented as mean ± SEM for n = 3 replicates. The dashed line shows editing levels of AAV.4, the AAV construct that in this experiment was used as a baseline for comparison across the variants.

[0033] FIG. 12 is a graph of percent editing versus transgene size for all constructs having varying promoters tested in this study. Constructs circled with dashes were identified as having above average editing while minimizing transgene size. The dashed line shows editing levels of AAV.4, the AAV construct that in this experiment was used as a baseline for comparison across variants.

[0034] FIG. 13 shows the results of an editing assay of mNPCs using AAV transgene plasmids having variations in gRNA promoter strength, as described in Example 5. Editing was assessed by FACS 5 days post-transfection. Data are presented as mean ± SEM for n= 3 replicates.

[0035] FIG. 14 shows the results of an editing assay of mNPCs using three different AAV vectors having variations in gRNA promoter strength, as described in Example 5. The graph on the left are results testing 3-fold dilutions of the constructs ranging from 1 x 104 to 5 x 105 vg / cell, while the graph on the right are results of editing using an MOI of 3 x 105 vg / cell. Editing was assessed by FACS 5 days post-transfection. Data are presented as mean ± SEM for n= 3 replicates.

[0036] FIG. 15 is a bar graph that shows percent editing of the tdTom locus in mNPCs in an experiment to assess use of truncated U6 RNA promoters in constructs when delivered in AAV transgene plasmids designed to minimize the footprint of the Pol III promoter in the delivered transgene, as described in Example 5. Editing was assessed by FACS 5 days post-transfection. Data are presented as mean ± SEM for n= 3 replicates.

[0037] FIG. 16 is a bar graph that shows percent editing of the tdTom locus in mNPCs comparing base construct 53 to construct 85, when delivered as AAV vector designed to minimize the footprint of the Pol III promoter in the delivered transgene, as described in Example 5.

[0038] FIG. 17 is a bar graph that shows editing results of the tdTom locus in an experiment to assess the effects of constructs having engineered U6 RNA promoters when delivered to mNPCs in an AAV vector designed to minimize the footprint of the Pol III promoter in the AAV transgene, as described in Example 5. Editing was assessed by FACS 5 days post-transfection. Data are presented as mean ± SEM for n= 3 replicates.

[0039] FIG. 18 is a scatter plot depicting transgene size of all AAV variants tested having engineered U6 RNA promoters on the X-axis vs. percent of mNPCs edited on the Y-axis, as described in Example 5. The dashed line indicates construct 53, having the largest promoter tested, while the dotted line indicates construct 89, having the smallest promoter tested.

[0040] FIG. 19 shows the results of an editing assay of the tdTom locus in mNPCs in an experiment to assess the effects of constructs having engineered Pol III RNA promoters when delivered in an AAV vector designed to minimize the footprint of the Pol III promoter in the AAV transgene, as described in Example 5. Editing was assessed by FACS 5 days posttransfection. Data are presented as mean ± SEM for n= 3 replicates.

[0041] FIG. 20 is a bar graph showing AAV-mediated editing level in mNPCs at an MOI of 3.0E+5 vg / cell using the indicated constructs, as described in Example 5.

[0042] FIG. 21 is a scatter plot depicting the transgene size of all variants tested on the X-axis vs. the percent of mNPCs edited on the Y-axis, as described in Example 5.

[0043] FIG. 22 shows the results of an editing assay of the tdTom locus in mNPCs using AAV transgene plasmids having variations in poly(A) signals, as described in Example 6. Data are presented as mean ± SEM for n= 3 replicates.

[0044] FIG. 23 shows the results of an editing assay of the tdTom locus in mNPCs using two AAV vectors having the top poly(A) signals, as described in Example 6. Editing was assessed by FACS 5 days post-transfection. Data are presented as mean ± SEM for n= 3 replicates.

[0045] FIG. 24 are schematics of AAV plasmid constructs containing guide RNA transcriptional units (gRNA scaffold-spacer stack driven by a U6 promoter) in different orientations in regards to the protein promoter transcriptional unit, as described in Example 7. The tapered points depicts the orientation of the transcriptional unit for protein or guide RNA.

[0046] FIG. 25 shows the results of an editing assay of the tdTom locus in mNPCs using AAV transgene plasmids having differences in regulatory element orientation, as described in Example 7. Editing was assessed by FACS 5 days post-transfection. Data are presented as mean ± SEM for n= 3 replicates.

[0047] FIG. 26 shows the results of an editing assay of NPCs using AAV vectors containing guide RNA transcriptional units (gRNA scaffold-spacer stack driven by a U6 promoter) in different orientations in relation to the protein promoter transcriptional unit, as described in Example 7. The graph on the left shows results testing 3-fold dilutions of the constructs ranging from 1 x 104 to 2 x 106 vg / cell. The bar graph on the right shows AAV-mediated percent editing in mNPCs at an MOI of 3.0E+5 vg / cell. Editing was assessed by FACS 5 days post-transfection. Data are presented as mean ± SEM for n= 3 replicates.

[0048] FIG. 27 is a bar graph of results of an editing assay of the tdTom locus in mNPCs using AAV transgene plasmid constructs having different post-transcriptional regulatory elements compared to constructs not having post-transcriptional regulatory elements, as described in Example 8. Editing was assessed by FACS 5 days post-transfection. Data are presented as mean ± SEM for n= 3 replicates.

[0049] FIG. 28 is bar graph showing AAV-mediated editing levels (grey bars) of mNPCs at a viral MOI of 3.0E+5 compared to nucleofection editing using 150 ng of AAV-cis plasmids (dark bars) expressing the CasX protein 491 under the control of top promoters without (constructs 4, 5, 6) or in combination with different post-transcriptional regulatory element sequences (constructs 35-37 for base plasmid 4, constructs 38-39 for base plasmid 5, and constructs 42-43 for base plasmid 6)., as described in Example 8. Editing was assessed by FACS 5 days posttransfection. Data are presented as mean ± SEM for n= 3 replicates.

[0050] FIG. 29 is a bar graph showing AAV-mediated editing levels of mNPCs at a viral MOI of 3.0E+5 for constructs under promoters without (constructs 58, 59, 53) or in combination of different post-transcriptional regulatory element sequences (respectively constructs 72-74 for base plasmid 58 containing Jet promoter, constructs 75-77 for base plasmid 59 containing Jet+USP promoter, and constructs 80-81 for base plasmid 53 containing UbC promoter), as described in Example 8. Editing was assessed by FACS 5 days post-transfection. Data (n=3) are presented as mean ± SEM.

[0051] FIG. 30 is a scatterplot comparing the transgene size of each construct evaluated (from ITR to ITR, in bp) to AAV-mediated editing levels in mNPCs at a MOI of 3.0e+5 vg / cell, as described in Example 8. The circled data points represent the top identified constructs in terms of editing levels of select transgene size. The horizontal grey line shows the editing level of the benchmark vector AAV.53 for comparative purposes. The vertical grey line delimits vectors that are over or under a 4.9kb transgene size.

[0052] FIG. 31 is a violin plot displaying AAV-mediated fold-improvement from the inclusion of the indicated PTRE element in the transgene plasmid, relative to its base (transgene with same promoter but no PTRE, indicated by gray dashed line), as described in Example 8.

[0053] FIG. 32 is a bar chart showing editing results of constructs with different neuronal enhancers delivered as AAV transgene plasmids to mNPCs, as described in Example 8. The gray lines show editing levels of reference plasmid 64, harboring CMV enhancer + core promoter. Editing was assessed by FACS 5 days post-transfection. Data are presented as mean ± SEM for n= 3 replicates.

[0054] FIG. 33 shows schematics of AAV constructs with alternative gRNA configurations for constructs having multiple gRNA, as described in Example 9. The top schematic is architecture 1, while the bottom is architecture 2. The tapered points depict the orientation of the transcriptional unit for protein or guide RNA.

[0055] FIG. 34 shows schematics of AAV constructs with alternative gRNA configurations for constructs having multiple gRNA, as described in Example 9. The tapered points depicts the orientation of the transcriptional unit for protein or guide RNA.

[0056] FIG. 35 shows schematics of guide RNA stack (Pol III promoter, scaffold, spacer) architectures tested with nucleofection and AAV transduction, as described in Example 9. Transgene harbors dual stacks in different orientations, with spacer 12.7, 12.2 and non-target spacer NT. The tapered points depict the orientation of the transcriptional unit for protein or guide RNA.

[0057] FIG. 36 shows the results of an editing assay for constructs having guide RNA stacks delivered via plasmid transfection to mNPCs, showing constructs with RNA stacks edit with enhanced potency compared to non-targeting control (NT), as described in Example 9. Editing was assessed by FACS 5 days post-transfection. Data are presented as mean ± SEM for n= 3 replicates.

[0058] FIG. 37 shows the results of an editing assay of mNPCs using AAV transgene plasmid constructs having multiple gRNA in different architectures and with different combinations of spacers (see FIG. 35) compared to construct 3 having a single gRNA and to a non-targeting construct, as described in Example 9. Editing was assessed by FACS 5 days post-transfection. Data are presented as mean ± SEM for n= 3 replicates.

[0059] FIG. 38 shows the results of an editing assay of mNPCs using AAV vector constructs 45-48 having multiple gRNA in different architectures and with different combinations of spacers (see FIG. 35) compared to construct 3, as described in Example 9. The left panel shows editing results using 3-fold MOI dilutions ranging from 1 x 104 to 3 x 105 vg / cell, while the right panel shows editing results at an MOI of 3 x 105 vg / cell. Editing was assessed by FACS 5 days post-transfection. Data are presented as mean ± SEM for n= 3 replicates.

[0060] FIG. 39 is a bar graph of percent editing in mNPCs using AAV transgene plasmid constructs with varying 5’ NLS combinations (2, 7, and 9 in Table 15) with 3’ NLS 1, 8 and 9 in mNPCs, as described in Example 10.

[0061] FIG. 40 is a bar graph of percent editing in mNPCs using AAV vectors with varying 5’ NLS combinations with 3’ NLS 1, 8 and 9 in mNPCs, as described in Example 10.

[0062] FIG. 41 is a bar graph of percent editing in mNPCs using AAV vectors with varying NLS combinations when delivered in a vector designed to minimize the footprint of Pol III promoter in the transgene.

[0063] FIG. 42 is a schematic showing the organization of the components of an exemplary AAV transgene between the 5’ and 3’ ITRs, as described in Example 12.

[0064] FIG. 43 A show results of editing assays in mNPCs nucleofected with 1000 of AAV-cis plasmids expressing CasX protein 491 expression of CMV and guide variants 174, 229-237 with spacer 11.30 targeting the mouse RHO exon 1 locus demonstrating improved activity at mouse RHO exon 1 in a dose-dependent manner, as described in Example 12. Triplicate wells were pooled together for gDNA extraction and therefore treated as n=l.

[0065] FIG. 43B is a bar graph showing fold-change in editing levels for each engineered scaffolds (229-237) relative to guide 174 with spacer 11.30 (set to a value of 1.0) across two plasmid nucleofection doses 1000 and 500ng of AAV-cis plasmids, as described in Example 12. Triplicate wells were pooled together for gDNA extraction and therefore treated as n=l.

[0066] FIG. 44A show editing results of engineered guide 235 compared to 174 with spacer 11.1 targeting RHO at the exogenous RHO-GFP locus (with GFP as the reporter), under the expression of Pol III hU6 promote in ARPE-19 cells, demonstrating improved activity by the 235 variant at the human RHO locus, with increased on-target activity at WT exogenous RHO without off-target cleavage at the mutant RHO reporter gene, as described in Example 12. Data (n=3) are presented as mean ± SD.

[0067] FIG. 44B is a bar graph displaying fold-change in editing levels of engineered guide 235 compared to 174 at the human RHO locus, with p59.491.235.11.1 normalized to benchmark p59.491.174.11.1 levels (set to value 1.0) in cells nucleofected with 1000 ng of each plasmid, as described in Example 12. Data (n=3) are presented as mean ± SD.

[0068] FIG. 45A shows editing levels in mNPCs by AAV-mediated expression of CasX molecule and engineered guide variant 235 compared to guide scaffold 174 with spacer 11.30 at 3 different MOI levels, confirming increased editing levels at the endogenous mouse Rho exon 1 locus with no off-target locus, as described in Example 12.

[0069] FIG. 45B is a bar graph displaying fold-change in editing levels in mNPCs by AAV-mediated expression of CasX molecule and engineered guide variant 235 compared to guide scaffold 174 with spacer 11.30 in cells infected at a 5.0e+5 MOI, as described in Example 12. Data are presented as the mean of n =3.

[0070] FIG. 46A shows editing results at the human RHO locus in mNPCs nucleofected with 1000 and 500 ng of AAV-cis plasmids expressing CasX protein 491 and sgRNA-scaffold 174 with on-target spacers of varying length, demonstrating improved on-target editing at the mouse RHO locus, as described in Example 12. Spacers variants are: 11.30 (20 nt WT RHO), 11.38 (18 nt WT RHO), and 11.39 (19 nt WT RHO), respectively. A control spacer, no-target (NT), designed to not recognize any sequence across the mouse and human genomes, was also tested as a negative control to ensure no unspecific targeting resulting from the expression of the CasX protein alone. Triplicate wells were pooled together for gDNA extraction and therefore treated as n=l.

[0071] FIGS. 46B is a bar graph showing editing levels at the human RHO locus in nucleofected mNPCs with 1000 ng of AAV-cis plasmids expressing CasX protein 491 and sgRNA-scaffold 174 with the indicated off-target spacers, as described in Example 12.

[0072] FIG. 46C is a bar graph displaying fold-change in editing levels at the human RHO locus in nucleofected mNPCs for each sgRNA-scaffold 174 with spacer variants 11.38 and 11.39 normalized to levels of parental sgRNA-scaffold-spacer 174.11.30, as described in Example 12. Data shows means + SD across 3 different biological replicates.

[0073] FIG. 47A is a Whisker box graph showing editing results of RHO in a mouse model comparing AAV-mediated delivery of sgRNA scaffold variants and optimized spacers compared to benchmark construct, as described in Example 13. Each dot represents one retina (n=8-16). One-way ANOVA statistical test was performed, *** = p <0.001.

[0074] FIG. 47B is a Whisker box graph showing the relative fold-change in editing of RHO in a mouse model comparing AAV-mediated delivery of sgRNA scaffold variants 174 and 235 and optimized spacers compared to benchmark construct, as described in Example 13. Values are relative to the benchmark vector AAV.RHO. 174.11.30 (set to a value of 1). Each dot represents one retina (n=8-16).

[0075] FIG. 48A is a bar graph showing CTC-PAM editing levels (indel rates) at the mouse RHO locus in mNPCs nucleofected with 1000 and 500 ng of AAV-cis plasmids expressing the CasX protein variant 491, 515 ,527, 528, 535, 536 or 537, respectively, and sgRNA-scaffold 235.11.37 (on target), as described in Example 14. A control spacer, no-target (NT), designed to not recognize any sequence across the mouse and human genomes, was also tested as a negative control to ensure no unspecific targeting resulting from the expression of the CasX protein alone. Triplicate wells were pooled together for gDNA extraction and therefore treated as n=l.

[0076] FIG. 48B is a bar graph showing CTC-PAM editing levels (indel rates) at the mouse RHO locus in mNPCs nucleofected with AAV-cis plasmids expressing the CasX protein variant 491, 515, 527, 528, 535, 536 or 537, respectively, and sgRNA-scaffold 235.11.39 (off-target), as described in Example 14.

[0077] FIG. 48C shows a bar graph displaying fold-change in editing levels for each indicated CasX protein variant with guide 235 and spacer 11.39, with results normalized to levels of the parental CasX protein 491, as described in Example 14.

[0078] FIG. 49A shows a bar graph showing editing levels in ARPE-19 mNPC nucleofected with 1000 ng of AAV-cis plasmids expressing CasX protein variant 491, 515, 527, 528, 535, 536 or 537and guide variant 235 with spacer 11.41 or 11.43, as described in Example 14. Data (n=3) are presented as mean ± SD.

[0079] FIG. 49B shows a bar graph displaying fold-change in editing levels in ARPE-19 mNPC nucleofected with 1000 ng of AAV-cis plasmids expressing CasX protein variant 515, 527, 528, 535, 536 or 537 and guide variant 235 with spacer 11.41 or 11.43 relative to benchmark p59.491.235.11.41 levels (set to a value of 1.0), as described in Example 14. Data (n=3) are presented as mean ± SD.

[0080] FIG. 50A shows a bar graph of AAV-mediated editing levels in mNPCs at the endogenous mouse Rho exon 1 locus, as described in Example 14. mNPCs were infected using a 3.0e+5 and 1.0e+5 vg / cell MOI with AAV vectors expressing the indicated CasX protein 491, 515, 527, 528, 535, or 537 and sgRNA-scaffold variant 235.11.39, as described in Example 14. Data (n=3) are presented as the mean.

[0081] FIG. 50B is a bar graph displaying fold-change in editing levels for the indicated CasX variant with guide scaffold 235 relative to guide 174 with spacer 11.39in cells infected with the indicated MOI, as described in Example 14.

[0082] FIG. 51 is an illustration of reference mRHO exon 1 locus and target amino acid residue P23 (CCC) sequence (highlighted in bold), showing spacer 11.30 target sequence and expected CasX-mediated cleavage, as described in Example 15. The most common predicted edits quantified in CRISPResso edits (substitution / deletions) are displayed under the reference genome).

[0083] FIG. 52A shows results of in vivo AAV CasX-mediated editing of the mRHO P23 locus in retinae in C57BL6J mice (n=6-8; quantification in percent of total indels detected by NGS), as described in Example 15.

[0084] FIG. 52B shows the fraction (%) of AAV CasX-mediated frame-shift edits of the mRHO P23 locus in the retinae in C57BL6J (n=6-8) mice (n=6-8; quantification in percent of total indels detected by NGS), as described in Example 15.

[0085] FIGS. 53A-53F show representative fluorescence imaging of retinas from AAV-CasX treated mice or negative controls and stained, as described in Example 15. Cell nuclei were counterstained with DAPI (top row; FIGS. 53A-C) to visualized retinal layers and stained with HA-tag (bottom row, FIGS. 53D-F) antibody to detect CasX expression in photoreceptors (ONL) and other retinal layers (INL;GCL). Legends: ONL= Outer nuclear layer; INL= Inner nuclear layer, GCL= Ganglion cell layer.

[0086] FIG. 54A is a box plot showing median, minimal and highest editing values using AAV-mediated expression of CasX 491 detected by NGS 3 weeks post-injection in wild-type retinae injected with 5.0e+9 vg / eye of AAV.X.491.174.11.30 vectors, in which the 491 protein is driven by promoter variants designed to selectively express in rod photoreceptors (X=RP1-RP5) or a ubiquitous promoter (X=CMV) , as described in Example 16. The grey line is placed at the editing levels achieved by AAV.RP1.491.174.11.30 to compare to other viral vectors tested.

[0087] FIG. 54B is a plot displaying levels of editing achieved by AAV vectors in wild-type retinae injected with 5.0e+9 vg / eye of AAV.X.491.174.11.30 vectors, compared to total transgene size (bp), as described in Example 16. The grey line delimitates transgenes below or above 4.9kb size.

[0088] FIG. 55 shows in vivo editing results that AAV-mediated expression of CasX 491 and sgRNA spacer 174.4.76 in rod photoreceptors led to detectable levels of editing levels at integrated Nrl-GFP locus in a dose-dependent manner, as described in Example 16. The bar graph shows editing levels detected by NGS at the integrated GFP locus 4-weeks and 12-weeks post-injection in heterozygous Nrl-GFP mice injected with the indicated doses of AAV.RP1.491.174.4.76 vectors in one eye, and the vehicle control in the contralateral eye).

[0089] FIG. 56A shows a western blot of retinal lysates from positive (Cl, uninjected homozygous Nrl-GFP retinae) and negative (N, uninjected C57BL / 6J retinae) controls, vehicle groups (V, AAV formulation buffer injected retinae) and AAV-CasX 491, sgRNA 174 and spacer 4.76 treated retinae with the medium dose 1.9e+9 (M) or high dose 1.0e+10 vg (H arm. Blots display the respective bands for the HA protein (CasX protein, top), GFP protein (middle) and GAPDH (bottom panels) used as a loading control, as described in Example 16. Levels of percent editing in the retinae detected by NGS are displayed under the blot for each sample.

[0090] FIG. 56B is a scatter boxplot representing levels of GFP protein detected in the western blots of FIG. 56A (ratios of densitometric values of the GFP band for total amount of proteins, normalized to the vehicle group levels) , as described in Example 16. One-way ANOVA statistical analysis was performed (* =p<0.5).

[0091] FIG. 56C is a plot correlating GFP protein fraction to levels of editing achieved in mouse retinae of the AAV-treated mice, for both the 1.0e+9 and 1.0e+10 dose groups, as described in Example 16.

[0092] FIG. 57A is a bar graph representing the ratio of GFP fluorescence levels (superior to inferior retina mean grey values) detected by fundus imaging at 4-weeks compared to 12-weeks post-injection in mice injected with two dose levels of AAV constructs, as described in Example 16.

[0093] FIG. 57B displays representative images of fluorescence fundus imaging of GFP in retina from mice injected with 1.0e+9 vg (#13) or l.Oe+lOvg (#34) with the AAV constructs at 4-weeks and (left panel) or 12-weeks (right panel), as described in Example 16.

[0094] FIGS. 58A- 58L present histology images or retinae of mice stained with various immunochemistry reagents, as described in Example 16, confirming efficient knock-down of GFP in photoreceptor cells in an AAV-dose dependent manner. The images are representative confocal images of cross-sectioned retinae injected with vehicle (FIGS. 58A, 58B, 58C, 58D), AAV-CasX at a 1.0e+9 vg dose (FIGS. 58E, 58F, 58G, and 58H) and l.OE+lOvg dose (FIGS. 581, 58J, 58K, and 58L). Structural imaging shows GFP expression by rod photoreceptors in the outer segment (images in FIGS. 58A, 58E, 581 and images FIGS. 58C, 58G, and 58K for 20X and 40X magnifications, respectively). Cell nuclei were counterstained with Hoechst (FIGS. 58B, 58F, and 58J) and cells stained with anti-HA to correlate levels of HA (CasX transgene levels; FIGS. 58D, 58H, and 58L; 40X magnification) and GFP expressed in photoreceptors. White box outlines in B and F indicate retinal regions analyzed at 40X magnification in FIGS. 58C and 58G. Legend: RPE=retinal pigment epithelium, OS=outer segment, ONL=outer nuclear layer, INL=inner nuclear layer, GCL= ganglion.

[0095] FIG. 59A shows results of an immunohistochemistry staining of a mouse liver section showing that CasX 491 and scaffold 174 with spacer 12.7 administered as an AAV IV injection was able to edit the tdTom locus in vivo in Ai9 mice, as described in Example 3. The images are representative of n=3 animals.

[0096] FIG. 59B shows results of an immunohistochemistry staining of a mouse heart section showing that CasX 491 and scaffold 174 with spacer 12.7 administered as an AAV IV injection was able to edit the tdTom locus in vivo in Ai9 mice, as described in Example 3. The images are representative of n=3 animals.

[0097] FIG. 60 is a graph of the quantification of percent editing at the B2M locus 5 days post-transduction of AAVs into human NPCs in a series of three-fold dilution of MOI, as described in Example 17. Editing levels were determined by NGS as indel rate and by flow cytometry as population of cells that do not express the HLA protein due to successful editing at the B2M locus.

[0098] FIG. 61 shows the results of an editing assay measured as indel rate detected by NGS at the human AAVS1 locus in human induced neurons (iNs) using the three indicated AAVs, each containing CasX 491 and gRNA with a specific spacer targeting AAVS1, as described in Example 17.

[0099] FIG. 62 is a bar graph exhibiting percent editing at the B2M locus in human iNs 14 days post-transduction of AAVs expressing CasX 491 driven by various protein promoters at an MOI of 2E4 or 6.67E3, as described in Example 17.

[0100] FIG. 63 shows the results of an editing assay using AAV transgene plasmids nucleofected into hNPCs, as described in Example 18, demonstrating that CpG reduction or depletion within the Ula promoter (construct ID 178 and 179), U6 promoter (construct ID 180 and 181), or bGH poly(A) (construct ID 182) did not significantly reduce CasX-mediated editing at the B2M locus compared to the editing achieved with the original CpG+ AAV vector (construct ID 177). The controls used in this experiment were the non-targeting (NT) spacer and no treatment (NTx).

[0101] FIG. 64 is a bar graph showing editing results of the tdTomato locus in an experiment to assess the effects of AAV constructs having engineered Pol III promoter hybrid variants when delivered to mNPCs in an AAV vector, as described in Example 5. Editing was assessed by FACS five days post-nucleofection.

[0102] FIG. 65 is a schematic of the regions and domains of a guide RNA used to design a scaffold library, as described in Example 20.

[0103] FIG. 66 is a pie chart of the relative distribution and design of the scaffold library with both unbiased (double and single mutations) and targeted mutations (towards the triplex, scaffold stem bubble, pseudoknot, and extended stem and loop) indicated, as described in Example 20.

[0104] FIG. 67 is a schematic of the triplex mutagenesis designed to specifically incorporate alternate triplex-forming base pairs into the triplex, as described in Example 20. Solid lines indicate the Watson-Crick pair in the triplex; the third strand nucleotide is indicated as a dotted line representing the non-canonical interaction with the purine of the duplex. In the library, each of the 5 locations indicated was replaced with all possible triplex motifs (G:GC, TAT, G:GC) = 243 sequences. Sequence of ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCANNNAUCAAAG (SEQ ID NO: 41829).

[0105] FIG. 68 is a bar chart with results of the enrichment values of reference guide scaffolds 174 and 175 in each screen, as described in Example 20.

[0106] FIG. 69 are scatterplots showing the log2 enrichment value for each measured single nucleotide substitution, deletion, or insertion, as measured in each of two independent screens of the mutant libraries for guide scaffolds 174 and 175, as described in Example 20.

[0107] FIG. 70 are heat maps for single mutants in guide scaffolds 174 and 175 showing specific mutable regions in the scaffold across the sequences, as described in Example 20. Yellow shades reflect values with similar enrichment to the reference scaffolds; red shades indicate an increase in enrichment, and thus activity, relative to the reference scaffold; blue shades indicate a loss of activity relative to the wildtype scaffold; white indicates missing data (or a substitution that would result in wildtype sequence.

[0108] FIG. 71 is a scatterplot that compares the log2 enrichment of single nucleotide mutations on reference guide scaffolds 174 and 175, as described in Example 20. Only those mutations to positions that were analogous between 174 and 175 are shown. Results suggest that, overall, guide scaffold 174 is more tolerant to changes than 175.

[0109] FIG. 72 is a bar chart showing the average (and 95% confidence interval) log2 enrichment values for a set of scaffolds in which the pseudoknot pairs have been shuffled, such that each new pseudoknot has the same composition of base pairs, but in a different order within the stem, as described in Example 20. Each bar represents a set of scaffolds with the G: A (or A:G) pair location indicated (see diagram at right). 291 pseudoknot stems were tested; numbers above bars indicate the number of stems with the G: A (or A:G) pair at each position.

[0110] FIG. 73 is a schematic of the pseudoknot sequence of FIGS. 55 and 56, given 5’ to 3’, with the two strand sequences separated by an underscore.

[0111] FIG. 74 is a bar chart showing the average (and 95% confidence interval) log2 enrichment values for scaffolds, divided by the predicted secondary structure stability of the pseudoknot stem region, as described in Example 20. Scaffolds with very stable stems (e.g., AG < -7 kcal / mol) had high enrichment values on average, whereas scaffolds with destabilized stems (AG > -5 kcal / mol) had low enrichment values on average.

[0112] FIG. 75 is a heat map of all double mutants of positions 7 and 29 in scaffold 175, as described in Example 20. The pseudoknot sequence is given 5’ to 3’, on the right.

[0113] FIG. 76 is a graph of a survival assay to determine the selective stringency of the CcdB selection to different spacers when targeted by CasX protein 515 and Scaffold 174, as described in Example 21. DETAILED DESCRIPTION

[0114] While exemplary embodiments have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the inventions claimed herein. It should be understood that various alternatives to the embodiments described herein may be employed in practicing the embodiments of the disclosure. It is intended that the claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0115] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present embodiments, suitable methods and materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Definitions

[0116] The terms "polynucleotide" and "nucleic acid," used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, terms "polynucleotide" and "nucleic acid" encompass single-stranded DNA; doublestranded DNA; multi-stranded DNA; single-stranded RNA; double-stranded RNA; multistranded RNA; genomic DNA; cDNA; DNA-RNA hybrids; and a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0117] "Hybridizable" or "complementary" are used interchangeably to mean that a nucleic acid (e.g., RNA, DNA) comprises a sequence of nucleotides that enables it to non-covalently bind, i.e., form Watson-Crick base pairs and / or G / U base pairs, "anneal", or "hybridize," to another nucleic acid in a sequence-specific, antiparallel, manner (i.e., a nucleic acid specifically binds to a complementary nucleic acid) under the appropriate in vitro and / or in vivo conditions of temperature and solution ionic strength. It is understood that the sequence of a polynucleotide need not be 100% complementary to that of its target nucleic acid sequence to be specifically hybridizable; it can have at least about 70%, at least about 80%, or at least about 90%, or at least about 95% sequence identity and still hybridize to the target nucleic acid sequence. Moreover, a polynucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure, a 'bulge', ‘bubble’ and the like).

[0118] A “gene,” for the purposes of the present disclosure, includes a DNA region encoding a gene product (e.g., a protein, RNA), as well as all DNA regions which regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and / or transcribed sequences. Accordingly, a gene may include accessory element sequences including, but not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites and locus control regions. Coding sequences encode a gene product upon transcription or transcription and translation; the coding sequences of the disclosure may comprise fragments and need not contain a full-length open reading frame. A gene can include both the strand that is transcribed as well as the complementary strand containing the anticodons.

[0119] The term "downstream" refers to a nucleotide sequence that is located 3' to a reference nucleotide sequence. In certain embodiments, downstream nucleotide sequences relate to sequences that follow the starting point of transcription. For example, the translation initiation codon of a gene is located downstream of the start site of transcription.

[0120] The term "upstream" refers to a nucleotide sequence that is located 5' to a reference nucleotide sequence. In certain embodiments, upstream nucleotide sequences relate to sequences that are located on the 5' side of a coding region or starting point of transcription. For example, most promoters are located upstream of the start site of transcription.

[0121] The term “adjacent to” with respect to polynucleotide or amino acid sequences refers to sequences that are next to, or adjoining each other in a polynucleotide or polypeptide. The skilled artisan will appreciate that two sequences can be considered to be adjacent to each other and still encompass a limited amount of intervening sequence, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides or amino acids.

[0122] The term “accessory element” is used interchangeably herein with the term “accessory sequence,” and is intended to include, inter alia, polyadenylation signals (poly(A) signal), enhancer elements, introns, posttranscriptional regulatory elements (PTREs), nuclear localization signals (NLS), deaminases, DNA glycosylase inhibitors, additional promoters, factors that stimulate CRISPR-mediated homology-directed repair (e.g. in cis or in trans), activators or repressors of transcription, self-cleaving sequences, and fusion domains, for example a fusion domain fused to a CRISPR protein. It will be understood that the choice of the appropriate accessory element or elements will depend on the encoded component to be expressed (e.g., protein or RNA) or whether the nucleic acid comprises multiple components that require different polymerases or are not intended to be expressed as a fusion protein.

[0123] The term "promoter" refers to a DNA sequence that contains a transcription start site and additional sequences to facilitate polymerase binding and transcription. Exemplary eukaryotic promoters include elements such as a TATA box, and / or B recognition element (BRE) and assists or promotes the transcription and expression of an associated transcribable polynucleotide sequence and / or gene (or transgene). A promoter can be synthetically produced or can be derived from a known or naturally occurring promoter sequence or another promoter sequence. A promoter can be proximal or distal to the gene to be transcribed. A promoter can also include a chimeric promoter comprising a combination of two or more heterologous sequences to confer certain properties. A promoter of the present disclosure can include variants of promoter sequences that are similar in composition, but not identical to, other promoter sequence(s) known or provided herein. A promoter can be classified according to criteria relating to the pattern of expression of an associated coding or transcribable sequence or gene operably linked to the promoter, such as constitutive, developmental, tissue-specific, inducible, etc. A promoter can also be classified according to its strength. As used in the context of a promoter, “strength” refers to the rate of transcription of the gene controlled by the promoter. A “strong” promoter means the rate of transcription is high, while a “weak” promoter means the rate of transcription is relatively low.

[0124] A promoter of the disclosure can be a Polymerase II (Pol II) promoter. Polymerase II transcribes all protein coding and many non-coding genes. A representative Pol II promoter includes a core promoter, which is a sequence of about 100 base pairs surrounding the transcription start site, and serves as a binding platform for the Pol II polymerase and associated general transcription factors. The promoter may contain one or more core promoter elements such as the TATA box, BRE, Initiator (INR), motif ten element (MTE), downstream core promoter element (DPE), downstream core element (DCE), although core promoters lacking these elements are known in the art.

[0125] A promoter of the disclosure can be a Polymerase III (Pol III) promoter. Pol III transcribes DNA to synthesize small ribosomal RNAs such as the 5S rRNA, tRNAs, and other small RNAs. Representative Pol III promoters use internal control sequences (sequences within the transcribed section of the gene) to support transcription, although upstream elements such as the TATA box are also sometimes used. All Pol III promoters are envisaged as within the scope of the instant disclosure.

[0126] The term “enhancer” refers to regulatory DNA sequences that, when bound by specific proteins called transcription factors, regulate the expression of an associated gene. Enhancers may be located in the intron of the gene, or 5’ or 3’ of the coding sequence of the gene. Enhancers may be proximal to the gene (i.e., within a few tens or hundreds of base pairs (bp) of the promoter), or may be located distal to the gene (i.e., thousands of bp, hundreds of thousands of bp, or even millions of bp away from the promoter). A single gene may be regulated by more than one enhancer, all of which are envisaged as within the scope of the instant disclosure.

[0127] As used herein, a “post-transcriptional regulatory element (PRE),” such as a hepatitis PRE, refers to a DNA sequence that, when transcribed creates a tertiary structure capable of exhibiting post-transcriptional activity to enhance or promote expression of an associated gene operably linked thereto.

[0128] As used herein, a “post-transcriptional regulatory element (PTRE),” such as a hepatitis PTRE, refers to a DNA sequence that, when transcribed creates a tertiary structure capable of exhibiting post-transcriptional activity to enhance or promote expression of an associated gene operably linked thereto.

[0129] “Recombinant,” as used herein, means that a particular nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction, and / or ligation steps resulting in a construct having a structural coding or non-coding sequence distinguishable from endogenous nucleic acids found in natural systems. Generally, DNA sequences encoding the structural coding sequence can be assembled from cDNA fragments and short oligonucleotide linkers, or from a series of synthetic oligonucleotides, to provide a synthetic nucleic acid which is capable of being expressed from a recombinant transcriptional unit contained in a cell or in a cell-free transcription and translation system. Such sequences can be provided in the form of an open reading frame uninterrupted by internal non-translated sequences, or introns, which are typically present in eukaryotic genes. Genomic DNA comprising the relevant sequences can also be used in the formation of a recombinant gene or transcriptional unit. Sequences of non-translated DNA may be present 5’ or 3’ from the open reading frame, where such sequences do not interfere with manipulation or expression of the coding regions, and may indeed act to modulate production of a desired product by various mechanisms (see “enhancers” and “promoters”, above).

[0130] The term “recombinant polynucleotide” or “recombinant nucleic acid” refers to one which is not naturally occurring, e.g., is made by the artificial combination of two otherwise separated segments of sequence through human intervention. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. Such is usually done to replace a codon with a redundant codon encoding the same or a conservative amino acid, while typically introducing or removing a sequence recognition site. Alternatively, it is performed to join together nucleic acid segments of desired functions to generate a desired combination of functions. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques.

[0131] Similarly, the term “recombinant polypeptide” or “recombinant protein” refers to a polypeptide or protein which is not naturally occurring, e.g., is made by the artificial combination of two otherwise separated segments of amino sequence through human intervention. Thus, e.g., a protein that comprises a heterologous amino acid sequence is recombinant.

[0132] As used herein, the term “contacting” means establishing a physical connection between two or more entities. For example, contacting a target nucleic acid with a guide nucleic acid means that the target nucleic acid and the guide nucleic acid are made to share a physical connection; e.g., can hybridize if the sequences share sequence similarity.

[0133] “Dissociation constant”, or “Ka”, are used interchangeably and mean the affinity between a ligand “L” and a protein “P”; i.e., how tightly a ligand binds to a particular protein. It can be calculated using the formula Ka=[L] [P] / [LP], where [P], [L] and [LP] represent molar concentrations of the protein, ligand and complex, respectively.

[0134] The disclosure provides systems and methods useful for editing a target nucleic acid sequence. As used herein “editing” is used interchangeably with “modifying” and includes but is not limited to cleaving, nicking, deleting, knocking in, knocking out, and the like.

[0135] By “cleavage” it is meant the breakage of the covalent backbone of a target nucleic acid molecule (e.g., RNA, DNA). Cleavage can be initiated by a variety of methods including, but not limited to, enzymatic or chemical hydrolysis of a phosphodiester bond. Both single-stranded cleavage and double-stranded cleavage are possible, and double-stranded cleavage can occur as a result of two distinct single-stranded cleavage events.

[0136] The term "knock-out" refers to the elimination of a gene or the expression of a gene. For example, a gene can be knocked out by either a deletion or an addition of a nucleotide sequence that leads to a disruption of the reading frame. As another example, a gene may be knocked out by replacing a part of the gene with an irrelevant sequence. The term "knock-down" as used herein refers to reduction in the expression of a gene or its gene product(s). As a result of a gene knock-down, the protein activity or function may be attenuated or the protein levels may be reduced or eliminated.

[0137] As used herein, "homology-directed repair" (HDR) refers to the form of DNA repair that takes place during repair of double-strand breaks in cells. This process requires nucleotide sequence homology, and uses a donor template to repair or knock-out a target DNA, and leads to the transfer of genetic information from the donor to the target. Homology-directed repair can result in an alteration of the sequence of the target sequence by insertion, deletion, or mutation if the donor template differs from the target DNA sequence and part or all of the sequence of the donor template is incorporated into the target DNA.

[0138] As used herein, "non-homologous end joining" (NHEJ) refers to the repair of doublestrand breaks in DNA by direct ligation of the break ends to one another without the need for a homologous template (in contrast to homology-directed repair, which requires a homologous sequence to guide repair). NHEJ often results in the loss (deletion) of nucleotide sequence near the site of the double- strand break.

[0139] As used herein "micro-homology mediated end joining" (MMEJ) refers to a mutagenic DSB repair mechanism, which always associates with deletions flanking the break sites without the need for a homologous template (in contrast to homology-directed repair, which requires a homologous sequence to guide repair). MMEJ often results in the loss (deletion) of nucleotide sequence near the site of the double- strand break. A polynucleotide or polypeptide has a certain percent "sequence similarity" or "sequence identity" to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same, and in the same relative position, when comparing the two sequences. Sequence similarity (sometimes referred to as percent similarity, percent identity, or homology) can be determined in a number of different manners. To determine sequence similarity, sequences can be aligned using the methods and computer programs that are known in the art, including BLAST, available over the world wide web at ncbi.nlm.nih.gov / BLAST. Percent complementarity between particular stretches of nucleic acid sequences within nucleic acids can be determined using any convenient method. Example methods include BLAST programs (basic local alignment search tools) and PowerBLAST programs (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656) or by using the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), e.g., using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489).

[0140] The terms “polypeptide,” and “protein” are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence.

[0141] A “vector” or “expression vector” is a replicon, such as plasmid, phage, virus, virus-like particle or cosmid, to which another DNA segment, i.e., an “insert”, may be attached so as to bring about the replication or expression of the attached segment in a cell.

[0142] The term “naturally-occurring” or “unmodified” or “wild type” as used herein as applied to a nucleic acid, a polypeptide, a cell, or an organism, refers to a nucleic acid, polypeptide, cell, or organism that is found in nature.

[0143] As used herein, a “mutation” refers to an insertion, deletion, substitution, duplication, or inversion of one or more amino acids or nucleotides as compared to a wild-type or reference amino acid sequence or to a wild-type or reference nucleotide sequence.

[0144] As used herein the term "isolated" is meant to describe a polynucleotide, a polypeptide, or a cell that is in an environment different from that in which the polynucleotide, the polypeptide, or the cell naturally occurs. An isolated genetically modified host cell may be present in a mixed population of genetically modified host cells.

[0145] A "host cell," as used herein, denotes a eukaryotic cell, a prokaryotic cell, or a cell from a multicellular organism (e.g., in a cell line), which eukaryotic or prokaryotic cells are used as recipients for a nucleic acid (e.g., an expression vector), and include the progeny of the original cell which has been genetically modified by the nucleic acid. It is understood that the progeny of a single cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation. A "recombinant host cell" (also referred to as a "genetically modified host cell") is a host cell into which has been introduced a heterologous nucleic acid, e.g., an expression vector.

[0146] A “target cell marker” refers to a molecule expressed by a target cell including but not limited to cell-surface receptors, cytokine receptors, antigens, tumor-associated antigens, glycoproteins, oligonucleotides, enzymatic substrates, antigenic determinants, or binding sites that may be present in the on the surface of a target tissue or cell that may serve as ligands for an antibody fragment or glycoprotein tropism factor.

[0147] The term "conservative amino acid substitution" refers to the interchangeability in proteins of amino acid residues having similar side chains. For example, a group of amino acids having aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains consists of serine and threonine; a group of amino acids having amide-containing side chains consists of asparagine and glutamine; a group of amino acids having aromatic side chains consists of phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains consists of lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains consists of cysteine and methionine. Exemplary conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.

[0148] The term “antibody,” as used herein, encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), nanobodies, single domain antibodies such as VHH antibodies, and antibody fragments so long as they exhibit the desired antigen-binding activity or immunological activity. Antibodies represent a large family of molecules that include several types of molecules, such as IgD, IgG, IgA, IgM and IgE.

[0149] An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody and that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, single chain diabodies, linear antibodies, a single domain antibody, a single domain camelid antibody, single-chain variable fragment (scFv) antibody molecules, and multispecific antibodies formed from antibody fragments.

[0150] As used herein, “treatment” or “treating,” are used interchangeably herein and refer to an approach for obtaining beneficial or desired results, including but not limited to a therapeutic benefit and / or a prophylactic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder or disease being treated. A therapeutic benefit can also be achieved with the eradication or amelioration of one or more of the symptoms or an improvement in one or more clinical parameters associated with the underlying disease such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder.

[0151] The terms “therapeutically effective amount” and “therapeutically effective dose”, as used herein, refer to an amount of a drug or a biologic, alone or as a part of a composition, that is capable of having any detectable, beneficial effect on any symptom, aspect, measured parameter or characteristics of a disease state or condition when administered in one or repeated doses to a subject such as a human or an experimental animal. Such effect need not be absolute to be beneficial.

[0152] As used herein, “administering” means a method of giving a dosage of a compound (e.g., a composition of the disclosure) or a composition (e.g., a pharmaceutical composition) to a subject.

[0153] A “subject” is a mammal. Mammals include, but are not limited to, domesticated animals, non-human primates, humans, dogs, rabbits, mice, rats and other rodents.

[0154] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. I. General Methods

[0155] The practice of the present invention employs, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, which can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference.

[0156] Where a range of values is provided, it is understood that endpoints are included and that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included.

[0157] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0158] It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0159] It will be appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. In other cases, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. It is intended that all combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein. II. AAV Vectors

[0160] In a first aspect, the present disclosure relates to AAV vectors optimized for the expression and delivery of CRISPR nucleases to target cells and / or tissues for genetic editing.

[0161] Wild-type AAV is a small, single-stranded DNA virus belonging to the parvovirus family. The wild-type AAV genome is made up of two genes that encode four replication proteins and three capsid proteins, respectively, and is flanked on either side by inverted terminal repeats (ITRs) having 130-145 nucleotides that fold into a hairpin shape important for replication. The virion is composed of three capsid proteins, Vpl, Vp2, and Vp3, produced in a 1:1:10 ratio from the same open reading frame but from differential splicing (Vpl) and alternative translational start sites (Vp2 and Vp3, respectively). The cap gene produces an additional, non-structural protein called the Assembly-Activating Protein (AAP). This protein is produced from ORF2 and is essential for the capsid-assembly process. The capsid forms a supramolecular assembly of approximately 60 individual capsid protein subunits into a nonenveloped, T-l icosahedral lattice capable of protecting the AAV genome.

[0162] Being naturally replication-defective and capable of transducing nearly every cell type in the human body, AAV represents a suitable vector for therapeutic use in gene therapy or vaccine delivery. Typically, when producing a recombinant AAV vector, the sequence between the two ITRs is replaced with one or more sequences of interest (e.g., a transgene), and the Rep and Cap sequences are provided in trans, making the ITRs the only viral DNA that remains in the vector. The resulting recombinant AAV vector genome construct comprises two cis-acting 130 to 145-nucleotide ITRs flanking an expression cassette encoding the transgene sequences of interest, providing at least 4.7 kb or more for packaging of foreign DNA that can include a transgene, one or more promoters and accessory elements, such that the total size of the vector is below 5 to 5.2 kb, which is compatible with packaging within the AAV capsid (it being understood that as the size of the construct exceeds this threshold, the packaging efficiency of the vector decreases). The transgene may be used to correct or ameliorate gene deficiencies in the cells of a subject. In the context of CRISPR-mediated gene editing, however, the size limitation of the expression cassette is a challenge for most CRISPR systems, given the large size of the nucleases.

[0163] The present disclosure provides polynucleotides for production of AAV transgene plasmids as well as for the production of AAV viral vectors. In some embodiments, the polynucleotides comprise sequences encoding a first adeno-associated virus (AAV) 5’ inverted terminal repeat (ITR) sequence, a second AAV 3’ ITR sequence, a CRISPR nuclease, a first guide RNA (gRNA), one or more promoters and, optionally, accessory elements; all encompassed in a single expression cassette encoded by a single polynucleotide capable of being incorporated into a single AAV viral particle. In other embodiments, the polynucleotides comprise sequences encoding a first 5’ AAV ITR sequence, a second 3’ AAV ITR sequence, a CRISPR nuclease, a first gRNA, a first promoter, a second promoter, and, optionally, one or more accessory elements.

[0164] The promoter and accessory elements can be operably linked to a transgene, e.g. the CRISPR protein and / or gRNA, in a manner which permits its transcription, translation and / or expression in a cell transfected with the AAV vector of the embodiments. As used herein, “operably linked” sequences include both accessory element sequences that are contiguous with the gene of interest and accessory element sequences that are at a distance to control the gene of interest. In some embodiments, the CRISPR protein and the first gRNA are under the control of, and operably linked to, a first promoter. In other embodiments, the CRISPR protein is under the control of and operably linked to a first promoter and the first gRNA is under the control of and operably linked to a second promoter.

[0165] In some embodiments, the disclosure provides accessory elements for inclusion in the AAV vector that include, but are not limited to sequences that control transcription initiation, termination, promoters, enhancer elements, RNA processing signal sequences, enhancer elements, sequences that stabilize cytoplasmic mRNA, sequences that enhance translation efficiency (i.e., Kozak consensus sequence), an intron, a post-transcriptional regulatory element (PTRE), a nuclear localization signal (NLS), a deaminase, a DNA glycosylase inhibitor, a second guide RNA, a stimulator of CRISPR-mediated homology-directed repair, and an activator or repressor of transcription.

[0166] By “adeno-associated virus inverted terminal repeats” or “AAV ITRs” is meant the art recognized regions found at each end of the AAV genome which function together in cis as origins of DNA replication and as packaging signals for the virus. AAV ITRs, together with the AAV rep coding region, provide for the efficient excision and rescue from, and integration of a nucleotide sequence interposed between two flanking ITRs into a mammalian cell genome.

[0167] The nucleotide sequences of AAV ITR regions are known. See, for example Kotin, R. M. (1994) Human Gene Therapy 5:793-801; Berns, K. I. “Parvoviridae and their Replication” in Fundamental Virology, 2nd Edition, (B. N. Fields and D. M. Knipe, eds.). As used herein, an AAV ITR need not have the wild-type nucleotide sequence depicted, but may be altered, e.g., by the insertion, deletion or substitution of nucleotides. Additionally, the AAV ITR may be derived from any of several AAV serotypes, including without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV 44.9, AAV-Rh74, and AAVRhlO, and modified capsids of these serotypes. Furthermore, 5' and 3' ITRs which flank a selected nucleotide sequence in an AAV vector need not necessarily be identical or derived from the same AAV serotype or isolate, so long as they function as intended, i.e., to allow for excision and rescue of the sequence of interest from a host cell genome or vector, and to allow integration of the heterologous sequence into the recipient cell genome when AAV Rep gene products are present in the cell. Use of AAV serotypes for integration of heterologous sequences into a host cell is known in the art (see, e.g., WO2018195555A1 and US20180258424A1, incorporated by reference herein). In one particular embodiment, the ITRs are derived from serotype AAV1. In another particular embodiment, the ITRs are derived from serotype AAV2, including the 5’ ITR having sequence CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGAC CTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACT CCATCACTAGGGGTTCCT (SEQ ID NO: 40557) and the 3’ ITR having sequence AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTG AGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTG AGCGAGCGAGCGCGCAGCTGCCTGCAGG (SEQ ID NO: 40576).

[0168] By “AAV rep coding region” is meant the region of the AAV genome which encodes the replication proteins Rep 78, Rep 68, Rep 52 and Rep 40. These Rep expression products have been shown to possess many functions, including recognition, binding and nicking of the AAV origin of DNA replication, DNA helicase activity and modulation of transcription from AAV (or other heterologous) promoters. The Rep expression products are collectively required for replicating the AAV genome.

[0169] By “AAV cap coding region” is meant the region of the AAV genome which encodes the capsid proteins VP1, VP2, and VP3, or functional homologues thereof. These Cap expression products supply the packaging functions which are collectively required for packaging the viral genome.

[0170] In some embodiments, the AAV vector is of serotype 9 or of serotype 6, which have been demonstrated to effectively deliver polynucleotides to motor neurons and glia throughout the spinal cord in preclinical models of Amyotrophic lateral sclerosis (ALS) (Foust, KD. et al. Therapeutic AAV9-mediated suppression of mutant RHO slows disease progression and extends survival in models of inherited ALS. Mol Ther. 21(12):2148 (2013)). In some embodiments, the methods provide use of AAV9 or AAV6 for targeting of neurons via intraparenchymal brain injection. In some embodiments, the methods provide use of AAV9 for intravenous administering of the vector wherein the AAV9 has the ability to penetrate the blood-brain barrier and drive gene expression in the nervous system via both neuronal and glial tropism of the vector. In other embodiments, the AAV vector is of serotype 8, which have been demonstrated to effectively deliver polynucleotides to retinal cells.

[0171] In some embodiments, the one or more accessory elements are selected from the group consisting of a poly(A) signal, a gene enhancer element, an intron, a posttranscriptional regulatory element (PTRE), a nuclear localization signal (NLS), a deaminase, a DNA glycosylase inhibitor, a third promoter, a second guide RNA (targeting a different or overlapping segment of the target nucleic acid), a stimulator of CRISPR-mediated homology-directed repair, and an activator or repressor of transcription. In some cases, the PTRE is selected from the group consisting of cytomegalovirus immediate / early intronA, hepatitis B virus PRE (HPRE), Woodchuck Hepatitis virus PRE (WPRE), and 5’ untranslated region (UTR) of human heat shock protein 70 mRNA (Hsp70).

[0172] In the foregoing, the one or more accessory elements are operably linked to the CRISPR protein. It has been discovered that the inclusion of the accessory element(s) in the polynucleotide of the AAV construct can enhance the expression, binding, activity, or performance of the CRISPR protein as compared to the CRISPR protein in the absence of said accessory element in an AAV construct. In one embodiment, the inclusion of the one or more accessory elements results in an increase in editing of a target nucleic acid by the CRISPR protein in an in vitro assay of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 1500%, at least about 200%, or at least about 300% as compared to the CRISPR protein in the absence of said accessory element in an AAV construct.

[0173] In a feature of the AAV vectors of the present disclosure, it has been discovered that utilization of certain Class 2 CRISPR systems of smaller size permit the inclusion of additional sequence space in the polynucleotides used in the making of the AAV vectors that can be utilized for the remaining components of the transgene, as described herein. In some embodiments, the Class 2 CRISPR system comprises a Type V protein selected from the group consisting of Casl2a, Casl2b, Casl2c, Casl2d (CasY), Casl2j and CasX, and the associated guide RNA of the respective system. In a particular embodiment, the CRISPR protein is a CasX, wherein the CasX comprises a sequence selected from the group consisting of SEQ ID NOS: 1-3 and SEQ ID NOS: 49-160, 40208-40369 and 40828-40912 as listed in Table 3, or a sequence having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto. In a particular embodiment, the CRISPR protein is a CasX, wherein the CasX comprises a sequence selected from the group consisting of the sequences of SEQ ID NOS: 1-3 and SEQ ID NOS: 49-160 and 40208-40369 and 40828-40912 as listed in Table 3. In some embodiments, the gRNA comprises a scaffold sequence selected from the group consisting of SEQ ID NOS: 2101-2285, 39981-40026, 40913-40958, and 41817 as set forth in Table 2, or a sequence having at least 85%, at least 90%, at least 95%, at least 95%, at least 96%, at least 97%, at least 98% identity thereto. In a particular embodiment, the gRNA comprises a sequence selected from the group of sequences of SEQ ID NOS: 2101-2285, 39981-40026, 40913-40958, and 41817 as set forth in Table 2. In the foregoing embodiments, the gRNA further comprises a targeting sequence complementary to a target nucleic acid to be modified, wherein the targeting sequence has at least 15 to 20 nucleotides. The CasX protein and gRNA component embodiments contemplated for incorporation into the AAV vectors of the disclosure are described more fully, below.

[0174] As described, supra, the smaller size of the Class 2, Type V proteins and gRNA contemplated for inclusion in the AAV constructs permit inclusion of additional or larger components that can be packaged into a single AAV particle. In some embodiments, the polynucleotide encoding the CRISPR protein sequence and the gRNA sequence are less than about 3100, about 3090, about 3080, about 3070, about 3060, about 3050, or less than about 3040 nucleotides in length. In other embodiments, the polynucleotide encoding the CRISPR protein sequence and the gRNA sequence are less than about 3040 to about 3100 nucleotides in combined length. Thus, in light of the total length of the expression cassette that can be packaged into an AAV particle, in some embodiments, the polynucleotide sequences of the first promoter and the at least one accessory element have greater than at least about 1300, at least about 1350, at least about 1360, at least about 1370, at least about 1380, at least about 1390, at least about 1400, at least about 1500, at least about 1600 nucleotides, at least 1650, at least about 1700, at least about 1750, at least about 1800, at least about 1850, or at least about 1900 nucleotides in combined length. In other embodiments, the polynucleotide sequences of the first promoter and the at least one accessory element have greater than at least about 1300 to at least about 1900 nucleotides in combined length. In one embodiment, the polynucleotide sequences of the first promoter and the at least one accessory element have greater than 1314 nucleotides in combined length. In another embodiment, the polynucleotide sequences of the first promoter and the at least one accessory element have greater than 1381 nucleotides in combined length. In other embodiments, the polynucleotide sequences of the first promoter, the second promoter and the at least one accessory element have greater than at least about 1300, at least about 1350, at least about 1360, at least about 1370, at least about 1380, at least about 1390, at least about 1400, at least about 1500, at least about 1600 nucleotides, at least 1650, at least about 1700, at least about 1750, at least about 1800, at least about 1850, or at least about 1900 nucleotides in combined length. In other embodiments, the polynucleotide sequences of the first promoter, the second promoter and the at least one accessory element have greater than at least about 1300 to at least about 1900 nucleotides in combined length. In one embodiment, the polynucleotide sequences of the first promoter, the second promoter, and the at least one accessory element have greater than 1314 nucleotides in combined length. In other embodiments, the polynucleotide sequences of the first promoter, the second promoter, and the at least one accessory element have greater than 1381 nucleotides in combined length. In still other embodiments, the polynucleotide sequences of the first promoter, the second promoter, and the two or more accessory elements have greater than at least about 1300, at least about 1350, at least about 1360, at least about 1370, at least about 1380, at least about 1390, at least about 1400, at least about 1500, at least about 1600 nucleotides, at least 1650, at least about 1700, at least about 1750, at least about 1800, at least about 1850, or at least about 1900 nucleotides in combined length. In other embodiments, the polynucleotide sequences of the first promoter, the second promoter, and the two or more accessory elements have greater than at least about 1300 to at least about 1900 nucleotides in combined length. In one embodiment, the polynucleotide sequences of the first promoter, the second promoter, and the two or more accessory elements have greater than 1314 nucleotides in combined length. In another embodiment, the polynucleotide sequences of the first promoter, the second promoter, and the two or more accessory elements have greater than 1381 nucleotides in combined length.

[0175] In some embodiments, the present disclosure provides a polynucleotide comprising a first adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence, a second AAV ITR sequence, a first promoter sequence, a sequence encoding a CRISPR protein, a second promoter sequence, a sequence encoding at least a first guide RNA (gRNA), and one or more accessory element sequences, wherein at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35% or more of the nucleotides of the polynucleotide sequence comprise the first and second promoters and the one or more accessory element sequences in combined length. In other embodiments, the present disclosure provides a polynucleotide comprising a first adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence, a second AAV ITR sequence, a first promoter sequence, a sequence encoding a CRISPR protein, a second promoter sequence, a sequence encoding a first guide RNA (gRNA), a third promoter sequence, a sequence encoding a second gRNA, and one or more accessory element sequences, wherein at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, or 35% or more of the nucleotides of the polynucleotide sequence comprise the first, second, and third promoters and the one or more accessory element sequences in combined length. As detailed in the Examples, it has been discovered that the ability to devote more of the total polynucleotide of the expression cassette of an AAV transgene to the promoters, a second gRNA, and / or the accessory elements results in enhanced expression of and / or performance of the CRISPR protein and gRNA, when expressed in the target host cell; either in an in vitro assay or in vivo in a subject. In some embodiments, the use of alternative or longer promoters and / or accessory elements (e.g., poly(A) signal, a gene enhancer element, an intron, a posttranscriptional regulatory element (PTRE), a nuclear localization signal (NLS), a deaminase, a DNA glycosylase inhibitor, a stimulator of CRISPR-mediated homology-directed repair, and an activator or repressor of transcription) in the AAV polynucleotides and resulting AAV vectors results in an increase in editing of a target nucleic acid of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 1500%, at least about 200%, or at least about 300% when the AAV is assessed in an in vitro assay compared to a construct not having the alternative or longer promoters and / or accessory elements. In one embodiment, the first promoter sequence of the polynucleotide has at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, or at least about 800 nucleotides. In another embodiment, the second promoter sequence of the polynucleotide has at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, or at least about 800 nucleotides. Embodiments of the promoters are described more fully, below.

[0176] In some embodiments, the present disclosure provides a polynucleotide, wherein the polynucleotide comprises one or more sequences selected from the group of sequences set forth in Tables 8-10, 12, 13, 17-22 and 24-27, or a sequence having at least 85%, at least 90%, at least 95%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. In another embodiment, the present disclosure provides a polynucleotide, wherein the polynucleotide comprises a sequence selected from the group of sequences set forth in Tables 810, 12, 13, and 17-23 and 24-27. In some embodiments, the polynucleotide sequence differs from those set forth in Tables 8-10, 12, 13, and 17-22 and 24-26 only in the selection of the targeting sequences of the gRNA or gRNAs encoded by the polynucleotide, wherein the targeting sequence is a sequence having 15 to 30 nucleotides capable of hybridizing with the sequence of a target nucleic acid. In a particular embodiment of the foregoing, the targeting sequence is selected from the group of sequences set forth in Table 27. In some embodiments, the present disclosure provides a polynucleotide of any of the embodiments described herein, wherein the polynucleotide has the configuration of a construct of FIG. 24, FIGS. 33-35, or FIG. 42.

[0177] In some embodiments, the present disclosure provides a polynucleotide for use in the making of an AAV vector, wherein the polynucleotide comprises one or more sequences selected from the group of sequences set forth in Tables 8-10, 12, 13, and 17-22 and 24-27, or a sequence having at least 85%, at least 90%, at least 95%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. In another embodiment, the present disclosure provides a polynucleotide for use in the making of an AAV vector, wherein the polynucleotide comprises a sequence selected from the group of sequences set forth in Tables 810, 12, 13, 17-22 and 24-27. In some embodiments, the polynucleotide sequence differs from those set forth in Tables 8-10, 12, 13, 17-22 and 24-26 only in the selection of the targeting sequences of the gRNA or gRNAs encoded by the polynucleotide, wherein the targeting sequence is a sequence having 15 to 30 nucleotides and is capable of hybridizing with the sequence of a target nucleic acid to be modified. In a particular embodiment of the foregoing, the targeting sequence is selected from the group of sequences set forth in Table 27. In some embodiments, the present disclosure provides a polynucleotide of any of the embodiments described herein for use in the making of an AAV vector, wherein the polynucleotide has the configuration of a construct of FIG. 24, FIGS. 33-35, or FIG. 42. III. Guide Nucleic Acids of the AAV systems

[0178] In some embodiments, the disclosure relates to specifically-designed guide ribonucleic acids (gRNA) utilized in the AAV systems that have utility in genome editing of a target nucleic acid in a cell. The present disclosure provides specifically-designed gRNAs with targeting sequences that are complementary to (and are therefore able to hybridize with) the target nucleic acid as a component of the gene editing AAV systems. It is envisioned that in some embodiments, multiple gRNAs (e.g., multiple gRNAs) are delivered in the AAV system for the modification of a target nucleic acid. For example, a pair of gRNAs with targeting sequences to different or overlapping regions of the target nucleic acid sequence can be used, when each is complexed with a CRISPR nuclease, in order to bind and cleave at two different or overlapping sites within the gene, which is then edited by non-homologous end joining (NHEJ), homology- directed repair (HDR), homology-independent targeted integration (HITI), micro-homology mediated end joining (MMEJ), single strand annealing (SSA) or base excision repair (BER).

[0179] In some embodiments, the disclosure provides gRNAs utilized in the systems that have utility in genome editing a gene in a eukaryotic cell. In a particular embodiment, the gRNA of the systems are capable of forming a complex with a CRISPR nuclease; a ribonucleoprotein (RNP) complex, described more fully, below. a. Reference gRNA and gRNA variants

[0180] In some embodiments, a gRNA of the present disclosure comprises a sequence of a naturally-occurring guide RNA (a “reference gRNA”). In some embodiments, a reference gRNA of the disclosure may be subjected to one or more mutagenesis methods, such as the mutagenesis methods described herein, which may include Deep Mutational Evolution (DME), deep mutational scanning (DMS), error prone PCR, cassette mutagenesis, random mutagenesis, staggered extension PCR, gene shuffling, or domain swapping (as described herein, as well as in WO2020247883A2, incorporated by reference herein), in order to generate one or more variants (referred to herein as "gRNA variant") with enhanced or varied properties relative to the reference gRNA. gRNA variants also include variants comprising one or more exogenous sequences, for example fused to either the 5' or 3' end, or inserted internally. The activity of reference gRNAs or the variant from which it was derived may be used as a benchmark against which the activity of gRNA variants are compared, thereby measuring improvements in function or other characteristics of the gRNA variants. In other embodiments, a reference gRNA or a gRNA variant may be subjected to one or more deliberate, specifically-targeted mutations in order to produce a gRNA variant; for example a rationally designed variant.

[0181] In some embodiments, the guide is a ribonucleic acid molecule (“gRNA”), and in other embodiments, the guide is a chimera, and comprises both DNA and RNA.

[0182] The gRNAs of the disclosure comprise two segments; a targeting sequence and a proteinbinding segment. The targeting segment of a gRNA includes a nucleotide sequence (referred to interchangeably as a guide sequence, a spacer, a targeting sequence, or a targeting region) that is complementary to, and therefore can hybridize with, a specific sequence (a target site) within the target nucleic acid (e.g., a target ssRNA, a target ssDNA, the complementary strand of a double stranded target DNA, etc.), described more fully below. The targeting sequence of a gRNA is capable of binding to a target nucleic acid sequence, including a coding sequence, a complement of a coding sequence, a non-coding sequence, and to accessory elements. The protein-binding segment (or “protein-binding sequence”) interacts with (e.g., binds to) a CasX protein as a complex, forming an RNP (described more fully, below). The protein-binding segment is alternatively referred to herein as a "scaffold", which is comprised of several regions, described more fully, below.

[0183] In the case of a dual guide RNA (dgRNA), the targeter and the activator portions each have a duplex-forming segment, where the duplex forming segment of the targeter and the duplex-forming segment of the activator have complementarity with one another and hybridize to one another to form a double stranded duplex (dsRNA duplex for a gRNA). When the gRNA is a gRNA, the term “targeter” or “targeter RNA” is used herein to refer to a crRNA-like molecule (crRNA: "CRISPR RNA") of a CasX dual guide RNA (and therefore of a CasX single guide RNA when the “activator" and the "targeter” are linked together, e.g., by intervening nucleotides). The crRNA has a 5' region that anneals with the tracrRNA followed by the nucleotides of the targeting sequence. Thus, for example, a guide RNA (dgRNA or sgRNA) comprises a guide sequence and a duplex-forming segment of a crRNA, which can also be referred to as a crRNA repeat. A corresponding tracrRNA-like molecule (activator) also comprises a duplex-forming stretch of nucleotides that forms the other half of the dsRNA duplex of the protein-binding segment of the guide RNA. Thus, a targeter and an activator, as a corresponding pair, hybridize to form a dual guide RNA, referred to herein as a “dual-molecule gRNA”, a “dgRNA”, a “double-molecule guide RNA”, or a “two-molecule guide RNA”. Sitespecific binding and / or cleavage of a target nucleic acid sequence (e.g., genomic DNA) by the CasX protein can occur at one or more locations (e.g., a sequence of a target nucleic acid) determined by base-pairing complementarity between the targeting sequence of the gRNA and the target nucleic acid sequence. Thus, for example, the gRNA of the disclosure have sequences complementarity to and therefore can hybridize with the target nucleic acid that is adjacent to a sequence complementary to a TC PAM motif or a PAM sequence, such as ATC, CTC, GTC, or TTC. Because the targeting sequence of a guide sequence hybridizes with a sequence of a target nucleic acid sequence, a targeter can be modified by a user to hybridize with a specific target nucleic acid sequence, so long as the location of the PAM sequence is considered. Thus, in some cases, the sequence of a targeter may be the complement to a non-naturally occurring sequence. In other cases, the sequence of a targeter may be a naturally-occurring sequence, derived from the complement to the gene sequence to be edited. In other embodiments, the activator and targeter of the gRNA are covalently linked to one another (rather than hybridizing to one another) and comprise a single molecule, referred to herein as a “single-molecule gRNA,” “single guide RNA”, a “single-molecule guide RNA,” a “one-molecule guide RNA”, or a “sgRNA”. In some embodiments, the sgRNA includes an “activator” or a “targeter” and thus can be an “activator-RNA” and a “targeter-RNA,” respectively. In some embodiments, the gRNA is a ribonucleic acid molecule (“gRNA”), and in other embodiments, the gRNA is a chimera, and comprises both DNA and RNA. As used herein, the term gRNA cover naturally-occurring molecules, as well as sequence variants (e.g. non-naturally occurring modified nucleotides).

[0184] Collectively, the assembled gRNAs of the disclosure comprise four distinct regions, or domains: the RNA triplex, the scaffold stem, the extended stem, and the targeting sequence that, in the embodiments of the disclosure, is specific for a target nucleic acid and is located on the 3’end of the gRNA. The RNA triplex, the scaffold stem, and the extended stem, together, are referred to as the “scaffold” of the gRNA (gRNA scaffold). The gRNA scaffolds of the disclosure can comprise RNA, or RNA and DNA. b. RNA triplex

[0185] In some embodiments of the guide NAs provided herein (including reference sgRNAs), there is a RNA triplex, and the RNA triplex comprises the sequence of a UUU—nX(~4-15)— UUU (SEQ ID NO: 19) stem loop that ends with an AAAG (SEQ ID NO: 40786) after 2 intervening stem loops (the scaffold stem loop and the extended stem loop), forming a pseudoknot that may also extend past the triplex into a duplex pseudoknot. The UU-UUU-AAA (SEQ ID NO: 40787) sequence of the triplex forms as a nexus between the targeting sequence, scaffold stem, and extended stem. In exemplary CasX sgRNAs, the UUU-loop-UUU region is coded for first, then the scaffold stem loop, and then the extended stem loop, which is linked by the tetraloop, and then an AAAG (SEQ ID NO: 40786) closes off the triplex before becoming the targeting sequence. c. Scaffold Stem Loop

[0186] In some embodiments of CasX sgRNAs of the disclosure, the triplex region is followed by the scaffold stem loop. The scaffold stem loop is a region of the gRNA that is bound by CasX protein (such as a CasX variant protein). In some embodiments, the scaffold stem loop is a fairly short and stable stem loop. In some cases, the scaffold stem loop does not tolerate many changes, and requires some form of an RNA bubble. In some embodiments, the scaffold stem is necessary for CasX sgRNA function. While it is perhaps analogous to the nexus stem of Cas9 as being a critical stem loop, the scaffold stem of a CasX sgRNA, in some embodiments, has a necessary bulge (RNA bubble) that is different from many other stem loops found in CRISPR / Cas systems. In some embodiments, the presence of this bulge is conserved across sgRNA that interact with different CasX proteins. An exemplary sequence of a scaffold stem loop sequence of a gRNA comprises the sequence CCAGCGACUAUGUCGUAUGG (SEQ ID NO: 14). d. Extended Stem Loop

[0187] In some embodiments of the CasX sgRNAs of the disclosure, the scaffold stem loop is followed by the extended stem loop. In some embodiments, the extended stem comprises a synthetic tracr and crRNA fusion that is largely unbound by the CasX protein. In some embodiments, the extended stem loop can be highly malleable. In some embodiments, a single guide gRNA is made with a GAAA (SEQ ID NO: 40788) tetraloop linker or a GAGAAA (SEQ ID NO: 40789) linker between the tracr and crRNA in the extended stem loop. In some cases, the targeter and activator of a CasX sgRNA are linked to one another by intervening nucleotides and the linker can have a length of from 3 to 20 nucleotides. In some embodiments of the CasX sgRNAs of the disclosure, the extended stem is a large 32-bp loop that sits outside of the CasX protein in the ribonucleoprotein complex. An exemplary sequence of an extended stem loop sequence of a sgRNA comprises the sequence GCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGC (SEQ ID NO: 15). e. Targeting Sequence

[0188] In some embodiments of the gRNAs of the disclosure, the extended stem loop is followed by a region that forms part of the triplex, and then the targeting sequence (or "spacer") at the 3' end of the gRNA. The targeting sequence targets the CasX ribonucleoprotein holo complex to a specific region of the target nucleic acid sequence of the gene to be modified. Thus, for example, gRNA targeting sequences of the disclosure have sequences complementarity to, and therefore can hybridize to, a portion of a gene in a target nucleic acid in a eukaryotic cell (e.g., a eukaryotic chromosome, chromosomal sequence, etc.) as a component of the RNP when the TC PAM motif or any one of the PAM sequences TTC, ATC, GTC, or CTC is located 1 nucleotide 5' to the non-target strand sequence complementary to the target sequence. The targeting sequence of a gRNA can be modified so that the gRNA can target a desired sequence of any desired target nucleic acid sequence, so long as the PAM sequence location is taken into consideration. In some embodiments, the gRNA scaffold is 5' of the targeting sequence, with the targeting sequence on the 3' end of the gRNA. In some embodiments, the PAM motif sequence recognized by the nuclease of the RNP is TC. In other embodiments, the PAM sequence recognized by the nuclease of the RNP is NTC; i.e., ATC, CTC, GTC, or TTC.

[0189] In some embodiments, the disclosure provides a gRNA wherein the targeting sequence of the gRNA is complementary to a target nucleic acid sequence of a gene to be modified. In some embodiments, the targeting sequence of the gRNA is complementary to a target nucleic acid sequence of a gene comprising one or more mutations compared to a wild-type gene sequence for purposes of editing the sequence comprising the mutations with the CasX:gRNA systems of the disclosure. In such cases, the modification effected by the CasX:gRNA system can either correct or compensate for the mutation or can knock down or knock out expression of the mutant gene product. In other embodiments, the targeting sequence of the gRNA is complementary to a target nucleic acid sequence of a wild-type gene for purposes of editing the sequence to introduce a mutation with the CasX:gRNA systems of the disclosure in order to knock-down or knock-out the gene. In some embodiments, the targeting sequence of a gRNA is designed to be specific for an exon of the gene of the target nucleic acid. In other embodiments, the targeting sequence of a gRNA is designed to be specific for an intron of the gene of the target nucleic acid. In other embodiments, the targeting sequence of the gRNA is designed to be specific for an intron-exon junction of the gene of the target nucleic acid. In other embodiments, the targeting sequence of the gRNA is designed to be specific for a regulatory element of the gene of the target nucleic acid. In some embodiments, the targeting sequence of the gRNA is designed to be complementary to a sequence comprising one or more single nucleotide polymorphisms (SNPs) in a gene of the target nucleic acid. SNPs that are within the coding sequence or within noncoding sequences are both within the scope of the instant disclosure. In other embodiments, the targeting sequence of the gRNA is designed to be complementary to a sequence of an intergenic region of the gene of the target nucleic acid.

[0190] In some embodiments, the targeting sequence is specific for a regulatory element that regulates expression of the gene product. Such regulatory elements include, but are not limited to promoter regions, enhancer regions, intergenic regions, 5' untranslated regions (5' UTR), 3' untranslated regions (3' UTR), conserved elements, and regions comprising cis-regulatoiy elements. The promoter region is intended to encompass nucleotides within 5 kb of the initiation point of the encoding sequence or, in the case of gene enhancer elements or conserved elements, can be thousands of bp, hundreds of thousands of bp, or even millions of bp away from the encoding sequence of the gene of the target nucleic acid. In the foregoing, the targets are those in which the encoding gene of the target is intended to be knocked out or knocked down such that the gene product is not expressed or is expressed at a lower level in a cell.

[0191] In some embodiments, the targeting sequence of a gRNA incorporated into the AAV of any of the embodiments described herein has between 14 and 35 consecutive nucleotides. In some embodiments, the targeting sequence of a gRNA has between 10 and 30 consecutive nucleotides. In some embodiments, the targeting sequence has 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive nucleotides. In some embodiments, the targeting sequence of the gRNA consists of 20 consecutive nucleotides. In some embodiments, the targeting sequence consists of 19 consecutive nucleotides. In some embodiments, the targeting sequence consists of 18 consecutive nucleotides. In some embodiments, the targeting sequence consists of 17 consecutive nucleotides. In some embodiments, the targeting sequence consists of 16 consecutive nucleotides. In some embodiments, the targeting sequence consists of 15 consecutive nucleotides. In some embodiments, the targeting sequence has 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive nucleotides and the targeting sequence can comprise 0 to 5, 0 to 4, 0 to 3, or 0 to 2 mismatches relative to the target nucleic acid sequence and retain sufficient binding specificity such that the RNP comprising the gRNA comprising the targeting sequence can form a complementary bond with respect to the target nucleic acid to be modified. In some embodiments, the targeting sequence of a gRNA incorporated into the AAV of any of the embodiments described herein comprises a sequence selected from the group consisting of the sequences of SEQ ID NO: 41056-41776, as set forth in Table 27, or a sequence having at least about 80%, or at least 90%, or at least 95% thereto. In some embodiments, the targeting sequence of a gRNA incorporated into the AAV of any of the embodiments described herein consists of a sequence selected from the group consisting of the sequences of SEQ ID NO: 41056-41776, as set forth in Table 27.

[0192] In some embodiments, the CasX:gRNA system comprises a first gRNA and further comprises a second (and optionally a third, fourth, fifth, or more) gRNA, wherein the second gRNA or additional gRNA has a targeting sequence complementary to a different or overlapping portion of the target nucleic acid sequence compared to the targeting sequence of the first gRNA such that multiple points in the target nucleic acid are targeted, and for example, multiple breaks are introduced in the target nucleic acid by the CasX. It will be understood that in such cases, the second or additional gRNA is complexed with an additional copy of the CasX protein. By selection of the targeting sequences of the gRNA, defined regions of the target nucleic acid sequence bracketing a mutation can be modified or edited using the CasX:gRNA systems described herein, including facilitating the insertion of a donor template or the excision of the DNA between the cleavage sites in cases, for example, where mutant repeats occur or where removal of an exon comprising mutations nevertheless results in expression of a functional gene product. f. gRNA scaffolds

[0193] With the exception of the targeting sequence domain, the remaining components of the gRNA are referred to herein as the scaffold. In some embodiments, the gRNA scaffolds are derived from naturally occurring sequences, described below as reference gRNA. In other embodiments, the gRNA scaffolds are variants of reference gRNA wherein mutations, insertions, deletions or domain substitutions are introduced to confer desirable properties on the gRNA.

[0194] In some embodiments, a CasX reference gRNA comprises a sequence isolated or derived from Deltaproteobacter. In some embodiments, the sequence is a CasX tracrRNA sequence. Exemplary CasX reference tracrRNA sequences isolated or derived from Deltaproteobacter may include: ACAUCUGGCGCGUUUAUUCCAUUACUUUGGAGCCAGUCCCAGCGACUAUGUCGU AUGGACGAAGCGCUUAUUUAUCGGAGA (SEQ ID NO: 22) and ACAUCUGGCGCGUUUAUUCCAUUACUUUGGAGCCAGUCCCAGCGACUAUGUCGU AUGGACGAAGCGCUUAUUUAUCGG (SEQ ID NO: 23). Exemplary crRNA sequences isolated or derived from Deltaproteobacter may comprise a sequence of CCGAUAAGUAAAACGCAUCAAAG (SEQ ID NO: 24). In some embodiments, a CasX reference gRNA comprises a sequence identical to a sequence isolated or derived from Deltaproteobacter.

[0195] In some embodiments, a CasX reference guide RNA comprises a sequence isolated or derived from Planctomycetes. In some embodiments, the sequence is a CasX tracrRNA sequence. Exemplary CasX reference tracrRNA sequences isolated or derived from Planctomycetes may include: UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUA UGGGUAAAGCGCUUAUUUAUCGGAGA (SEQ ID NO: 25) and UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUA UGGGUAAAGCGCUUAUUUAUCGG (SEQ ID NO: 26). Exemplary crRNA sequences isolated or derived from Planctomycetes may comprise a sequence of UCUCCGAUAAAUAAGAAGCAUCAAAG (SEQ ID NO: 27). In some embodiments, a CasX reference gRNA comprises a sequence identical to a sequence isolated or derived from Planctomycetes.

[0196] In some embodiments, a CasX reference gRNA comprises a sequence isolated or derived from Candidatus Sungbacteria. In some embodiments, the sequence is a CasX tracrRNA sequence. Exemplary CasX reference tracrRNA sequences isolated or derived from Candidatus Sungbacteria may comprise sequences of: GUUUACACACUCCCUCUCAUAGGGU (SEQ ID NO: 28), GUUUACACACUCCCUCUCAUGAGGU (SEQ ID NO: 11), UUUUACAUACCCCCUCUCAUGGGAU (SEQ ID NO: 12) and GUUUACACACUCCCUCUCAUGGGGG (SEQ ID NO: 13). In some embodiments, a CasX reference guide RNA comprises a sequence identical to a sequence isolated or derived from Candidatus Sungbacteria.

[0197] Table 1 provides the sequences of reference gRNA tracr, cr and scaffold sequences. In some embodiments, the disclosure provides gRNA variant sequences wherein the gRNA has a scaffold comprising a sequence having at least one nucleotide modification relative to a reference gRNA sequence having a sequence of any one of SEQ ID NOS: 4-16 of Table 1. It will be understood that in those embodiments wherein a vector comprises a DNA encoding sequence for a gRNA, or where a gRNA is a chimera of RNA and DNA, that thymine (T) bases can be substituted for the uracil (U) bases of any of the gRNA sequence embodiments described herein. Table 1. Reference gRNA tracr and scaffold sequences SEQ ID NO. Nucleotide Sequence 4 ACAUCUGGCGCGUUUAUUCCAUUACUUUGGAGCCAGUCCCAGCGACUAUGUCGUAUGGACGAAG C GCUUAUUUAU C GGAGAGAAAC C GAUAAGUAAAAC GCAU CAAAG 5 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGGGUAAAGC GCUUAUUUAU C GGAGAGAAAU C C GAUAAAUAAGAAGCAU CAAAG 6 ACAUCUGGCGCGUUUAUUCCAUUACUUUGGAGCCAGUCCCAGCGACUAUGUCGUAUGGACGAAG CGCUUAUUUAUCGGAGA 7 ACAUCUGGCGCGUUUAUUCCAUUACUUUGGAGCCAGUCCCAGCGACUAUGUCGUAUGGACGAAG CGCUUAUUUAUCGG 8 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGGGUAAAGC GCUUAUUUAUCGGAGA 9 UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGGGUAAAGC GCUUAUUUAUCGG 10 GUUUACACACU C C CU CU CAUAGGGU 11 GUUUACACACU C C CU CU CAU GAGGU 12 UUUUACAUAC C C C CU CU CAU GGGAU 13 GUUUACACACUCCCUCUCAUGGGGG 14 CCAGCGACUAUGUCGUAUGG 15 GC GCUUAUUUAU C GGAGAGAAAU C C GAUAAAUAAGAAGC 16 GGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGGGUAAAGCGCUU AUUUAUCGGA g. gRNA variants

[0198] In another aspect, the disclosure relates to gRNA variants, which comprise one or more modifications relative to a reference gRNA scaffold or are derived from another gRNA variant. As used herein, “scaffold” refers to all parts to the gRNA necessary for gRNA function with the exception of the spacer sequence.

[0199] In some embodiments, a gRNA variant comprises one or more nucleotide substitutions, insertions, deletions, or swapped or replaced regions relative to a reference gRNA sequence of the disclosure. In some embodiments, a mutation can occur in any region of a reference gRNA scaffold to produce a gRNA variant. In some embodiments, the scaffold of the gRNA variant sequence has at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70%, at least 80%, at least 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the sequence of SEQ ID NO: 4 or SEQ ID NO: 5. In other embodiments, a gRNA variant comprises one or more nucleotide substitutions, insertions, deletions, or swapped or replaced regions relative to a gRNA variant sequence of the disclosure. In some embodiments, the scaffold of the gRNA variant sequence has at least 50%, at least 60%, or at least 70%, at least 80%, at least 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the sequence of SEQ ID NO: 2238 or SEQ ID NO: 2239.

[0200] In some embodiments, a gRNA variant comprises one or more nucleotide changes within one or more regions of the reference gRNA scaffold that improve a characteristic of the reference gRNA. Exemplary regions include the RNA triplex, the pseudoknot, the scaffold stem loop, and the extended stem loop. In some cases, the variant scaffold stem further comprises a bubble. In other cases, the variant scaffold further comprises a triplex loop region. In still other cases, the variant scaffold further comprises a 5' unstructured region. In some embodiments, the gRNA variant scaffold comprises a scaffold stem loop having at least 60% sequence identity, at least 70% sequence identity, at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity to SEQ ID NO: 14. In some embodiments, the gRNA variant scaffold comprises a scaffold stem loop having at least 60% sequence identity to SEQ ID NO: 14. In other embodiments, the gRNA variant comprises a scaffold stem loop having the sequence of CCAGCGACUAUGUCGUAGUGG (SEQ ID NO: 32). In other embodiments, the disclosure provides a gRNA scaffold comprising, relative to SEQ ID NO: 5, a C18G substitution, a G55 insertion, a UI deletion, and a modified extended stem loop in which the original 6 nt loop and 13 most-loop-proximal base pairs (32 nucleotides total) are replaced by a Uvsx hairpin (4 nt loop and 5 loop-proximal base pairs; 14 nucleotides total) and the loop-distal base of the extended stem was converted to a fully base-paired stem contiguous with the new Uvsx hairpin by deletion of the A99 and substitution of G65U. In the foregoing embodiment, the gRNA scaffold 174 comprises the sequence ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG (SEQ ID NO: 2238).

[0201] All gRNA variants that have one or more improved characteristics, or add one or more new functions when the variant gRNA is compared to a reference gRNA described herein, are envisaged as within the scope of the disclosure. A representative example of such a gRNA variant is guide 174 (SEQ ID NO: 2238), the design of which is described in the Examples, and guide 235 (SEQ ID NO: 39987). In some embodiments, the gRNA variant adds a new function to the RNP comprising the gRNA variant. In some embodiments, the gRNA variant has an improved characteristic selected from: increased stability; increased transcription of the gRNA; increased resistance to nuclease activity; increased folding rate of the gRNA; decreased side product formation during folding; increased productive folding; increased binding affinity to a CasX protein; increased binding affinity to a target nucleic acid when complexed with a CasX protein; increased gene editing when complexed with a CasX protein; increased specificity of editing of the target nucleic acid when complexed with a CasX protein; decreased off-target editing when complexed with a CasX protein; and increased ability to utilize a greater spectrum of one or more PAM sequences, including ATC, CTC, GTC, or TTC, in the editing of target nucleic acid when complexed with a CasX protein, and any combination thereof. In some cases, the one or more of the improved characteristics of the gRNA variant is at least about 1.1 to about 100,000-fold increased relative to the reference gRNA of SEQ ID NO: 4 or SEQ ID NO: 5, or to gRNA variant 174 or 175. In other cases, the one or more improved characteristics of the gRNA variant is at least about 1.1, at least about 10, at least about 100, at least about 1000, at least about 10,000, at least about 100,000-fold or more increased relative to the reference gRNA of SEQ ID NO: 4 or SEQ ID NO: 5, or to gRNA variant 174 or 175. In other cases, the one or more of the improved characteristics of the gRNA variant is about 1.1 to 100,00-fold, about 1.1 to 10,00-fold, about 1.1 to 1,000-fold, about 1.1 to 500-fold, about 1.1 to 100-fold, about 1.1 to 50fold, about 1.1 to 20-fold, about 10 to 100,00-fold, about 10 to 10,00-fold, about 10 to 1,000fold, about 10 to 500-fold, about 10 to 100-fold, about 10 to 50-fold, about 10 to 20-fold, about 2 to 70-fold, about 2 to 50-fold, about 2 to 30-fold, about 2 to 20-fold, about 2 to 10-fold, about 5 to 50-fold, about 5 to 30-fold, about 5 to 10-fold, about 100 to 100,00-fold, about 100 to 10,00fold, about 100 to 1,000-fold, about 100 to 500-fold, about 500 to 100,00-fold, about 500 to 10,00-fold, about 500 to 1,000-fold, about 500 to 750-fold, about 1,000 to 100,00-fold, about 10,000 to 100,00-fold, about 20 to 500-fold, about 20 to 250-fold, about 20 to 200-fold, about 20 to 100-fold, about 20 to 50-fold, about 50 to 10,000-fold, about 50 to 1,000-fold, about 50 to 500-fold, about 50 to 200-fold, or about 50 to 100-fold, increased relative to the reference gRNA of SEQ ID NO: 4 or SEQ ID NO: 5, or to gRNA variant 174 or 175. In other cases, the one or more improved characteristics of the gRNA variant is about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20fold, 25-fold, 30-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100fold, 110-fold, 120-fold, 130-fold, 140-fold, 150-fold, 160-fold, 170-fold, 180-fold, 190-fold, 200-fold, 210-fold, 220-fold, 230-fold, 240-fold, 250-fold, 260-fold, 270-fold, 280-fold, 290fold, 300-fold, 310-fold, 320-fold, 330-fold, 340-fold, 350-fold, 360-fold, 370-fold, 380-fold, 390-fold, 400-fold, 425-fold, 450-fold, 475-fold, or 500-fold increased relative to the reference gRNA of SEQ ID NO: 4 or SEQ ID NO: 5, or to gRNA variant 174 or 175.

[0202] In some embodiments, a gRNA variant can be created by subjecting a reference gRNA or a gRNA variant to a one or more mutagenesis methods, such as the mutagenesis methods described herein, below, which may include Deep Mutational Evolution (DME), deep mutational scanning (DMS), error prone PCR, cassette mutagenesis, random mutagenesis, staggered extension PCR, gene shuffling, or domain swapping, in order to generate the gRNA variants of the disclosure. The activity of reference gRNA or gRNA variant may be used as a benchmark against which the activity of gRNA variants are compared, thereby measuring improvements in function of gRNA variants. In other embodiments, a reference gRNA or gRNA variant may be subjected to one or more deliberate, targeted mutations, substitutions, or domain swaps in order to produce a gRNA variant, for example a rationally designed variant. Exemplary gRNA variants produced by such methods are described in the Examples and representative sequences of gRNA scaffolds are presented in Table 2.

[0203] In some embodiments, the gRNA variant comprises one or more modifications compared to a reference guide nucleic acid scaffold sequence or a gRNA variant scaffold sequence, wherein the one or more modification is selected from: at least one nucleotide substitution in a region of the gRNA, at least one nucleotide deletion in a region of the gRNA; at least one nucleotide insertion in a region of the gRNA ; a substitution of all or a portion of a region of the gRNA; a deletion of all or a portion of a region of the gRNA; or any combination of the foregoing. In some cases, the modification is a substitution of 1 to 15 consecutive or non-consecutive nucleotides in the gRNA in one or more regions. In other cases, the modification is a deletion of 1 to 10 consecutive or non-consecutive nucleotides in the gRNA in one or more regions. In other cases, the modification is an insertion of 1 to 10 consecutive or non-consecutive nucleotides in the gRNA in one or more regions. In other cases, the modification is a substitution of the scaffold stem loop or the extended stem loop with an RNA stem loop sequence from a heterologous RNA source with proximal 5' and 3' ends. In some cases, a gRNA variant of the disclosure comprises two or more modifications in one region relative to a gRNA. In other cases, a gRNA variant of the disclosure comprises modifications in two or more regions. In other cases, a gRNA variant comprises any combination of the foregoing modifications described in this paragraph. In some embodiments, exemplary modifications of gRNA of the disclosure include the modifications of Table 2.

[0204] In some embodiments, a 5' G is added to a gRNA variant sequence, relative to a reference gRNA, for expression in vivo, as transcription from a U6 promoter is more efficient and more consistent with regard to the start site when the +1 nucleotide is a G. In other embodiments, two 5' Gs are added to generate a gRNA variant sequence for in vitro transcription to increase production efficiency, as T7 polymerase strongly prefers a G in the +1 position and a purine in the +2 position. In some cases, the 5’ G bases are added to the reference scaffolds of Table 1. In other cases, the 5’ G bases are added to the variant scaffolds of Table 2.

[0205] Table 2 provides exemplary gRNA variant scaffold sequences. In some embodiments, the gRNA variant scaffold comprises any one of the sequences SEQ ID NOS: 2101-2285, 39981-40026, 40913-40958, or 41817 as listed in Table 2, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity thereto. In some embodiments, the gRNA variant scaffold comprises any one of the sequences SEQ ID NOS: 2238-2285, 39981-40026, 40913-40958, or 41817, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity thereto. In some embodiments, the gRNA variant scaffold comprises any one of the sequences SEQ ID NOS: 2281-2285, 39981-40026, 40913-40958, or 41817, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity thereto. It will be understood that in those embodiments wherein a vector comprises a DNA encoding sequence for a gRNA, or where a gRNA is a chimera of RNA and DNA, that thymine (T) bases can be substituted for the uracil (U) bases of any of the gRNA sequence embodiments described herein. Table 2. Exemplary gRNA Scaffold Sequences SEQ ID NO: Name NUCLEOTIDE SEQUENCE OR DESCRIPTION OF MODIFICATION 2101 ND phage replication stable 2102 ND Kissing loopbl 2103 ND Kissing loopa 2104 ND 32: uvsX hairpin 2105 ND PP7 2106 ND 64: trip mut, extended stem truncation 2107 ND hyperstable tetraloop 2108 ND C18G 2109 ND U17G 2110 ND CUUCGG loop 2111 ND MS2 2112 ND -1, A2G, -78, G77U 2113 ND QB 2114 ND 45,44 hairpin 2115 ND U1A 2116 ND A14C, U17G 2117 ND CUUCGG loop modified 2118 ND Kissing loop_b2 2119 ND -76:78, -83:87 2120 ND -4 2121 ND extended stem truncation 2122 ND C55 2123 ND trip mut 2124 ND -76:78 SEQ ID NO: Name NUCLEOTIDE SEQUENCE OR DESCRIPTION OF MODIFICATION 2125 ND -1:5 2126 ND -83:87 2127 ND =+G28, A82U, -84, 2128 ND =+51U 2129 ND -1:4, +G5A, +G86, 2130 ND =+A94 2131 ND =+G72 2132 ND shorten front, CUUCGG loop modified, extend extended 2133 ND A14C 2134 ND -1:3, +G3 2135 ND =+C45, +U46 2136 ND CUUCGG loop modified, fun start 2137 ND -93:94 2138 ND =+U45 2139 ND -69, -94 2140 ND -94 2141 ND modified CUUCGG, minus U in 1st triplex 2142 ND -1:4, +C4, A14C, U17G, +G72, -76:78, -83:87 2143 ND U1C, -73 2144 ND Scaffold uuCG, stem uuCG. Stem swap, t shorten 2145 ND Scaffold uuCG, stem uuCG. Stem swap 2146 ND =+G60 2147 ND no stem Scaffold uuCG 2148 ND no stem Scaffold uuCG, fun start 2149 ND Scaffold uuCG, stem uuCG, fun start 2150 ND Pseudoknots 2151 ND Scaffold uuCG, stem uuCG 2152 ND Scaffold uuCG, stem uuCG, no start 2153 ND Scaffold uuCG 2154 ND =+GCUC36 2155 ND G quadriplex telomere basket+ ends 2156 ND G quadriplex M3q 2157 ND G quadriplex telomere basket no ends 2158 ND 45,44 hairpin (old version) 2159 ND Sarcin-ricin loop 2160 ND uvsX, C18G 2161 ND truncated stem loop, C18G, trip mut (U10C) 2162 ND short phage rep, C18G 2163 ND phage rep loop, C18G 2164 ND =+G18, stacked onto 64 2165 ND truncated stem loop, C18G, -1 A2G 2166 ND phage rep loop, C18G, trip mut (U10C) 2167 ND short phage rep, C18G, trip mut (U10C) 2168 ND uvsX, trip mut (U10C) 2169 ND truncated stem loop SEQ ID NO: Name NUCLEOTIDE SEQUENCE OR DESCRIPTION OF MODIFICATION 2170 ND =+A17, stacked onto 64 2171 ND 3' HDV genomic ribozyme 2172 ND phage rep loop, trip mut (U10C) 2173 ND -79:80 2174 ND short phage rep, trip mut (U10C) 2175 ND extra truncated stem loop 2176 ND U17G, C18G 2177 ND short phage rep 2178 ND uvsX, C18G, -1 A2G 2179 ND uvsX, C18G, trip mut (U10C), -1 A2G, HDV -99 G65U 2180 ND 3' HDV antigenomic ribozyme 2181 ND uvsX, C18G, trip mut (U10C), -1 A2G, HDV AA(98:99)C 2182 ND 3' HDV ribozyme (Lior Nissim, Timothy Lu) 2183 ND TAC(1:3)GA, stacked onto 64 2184 ND uvsX, -1 A2G 2185 ND truncated stem loop, C18G, trip mut (U10C), -1 A2G, HDV -99 G65U 2186 ND short phage rep, C18G, trip mut (U10C), -1 A2G, HDV -99 G65U 2187 ND 3' sTRSV WT viral Hammerhead ribozyme 2188 ND short phage rep, C18G, -1 A2G 2189 ND short phage rep, C18G, trip mut (U10C), -1 A2G, 3' genomic HDV 2190 ND phage rep loop, C18G, trip mut (U10C), -1 A2G, HDV -99 G65U 2191 ND 3' HDV ribozyme (Owen Ryan, Jamie Cate) 2192 ND phage rep loop, C18G, -1 A2G 2193 ND 0.14 2194 ND -78, G77U 2195 ND ND 2196 ND short phage rep, -1 A2G 2197 ND truncated stem loop, C18G, trip mut (U10C), -1 A2G 2198 ND -1, A2G 2199 ND truncated stem loop, trip mut (U10C), -1 A2G 2200 ND uvsX, C18G, trip mut (U10C), -1 A2G 2201 ND phage rep loop, -1 A2G 2202 ND phage rep loop, trip mut (U10C), -1 A2G 2203 ND phage rep loop, C18G, trip mut (U10C), -1 A2G 2204 ND truncated stem loop, C18G 2205 ND uvsX, trip mut (U10C), -1 A2G 2206 ND truncated stem loop, -1 A2G 2207 ND short phage rep, trip mut (U10C), -1 A2G 2208 ND 5'HDV ribozyme (Owen Ryan, Jamie Cate) 2209 ND 5'HDV genomic ribozyme 2210 ND truncated stem loop, C18G, trip mut (U10C), -1 A2G, HDV AA(98:99)C 2211 ND 5'env25 pistol ribozyme (with an added CUUCGG loop) 2212 ND 5'HDV antigenomic ribozyme 2213 ND 3' Hammerhead ribozyme (Lior Nissim, Timothy Lu) guide scaffold scar 2214 ND =+A27, stacked onto 64 SEQ ID NO: Name NUCLEOTIDE SEQUENCE OR DESCRIPTION OF MODIFICATION 2215 ND 5'Hammerhead ribozyme (Lior Nissim, Timothy Lu) smaller scar 2216 ND phage rep loop, C18G, trip mut (U10C), -1 A2G, HDV AA(98:99)C 2217 ND -27, stacked onto 64 2218 ND 3' Hatchet 2219 ND 3' Hammerhead ribozyme (Lior Nissim, Timothy Lu) 2220 ND 5' Hatchet 2221 ND 5' HDV ribozyme (Lior Nissim, Timothy Lu) 2222 ND 5' Hammerhead ribozyme (Lior Nissim, Timothy Lu) 2223 ND 3' HH15 Minimal Hammerhead ribozyme 2224 ND 5' RBMX recruiting motif 2225 ND 3' Hammerhead ribozyme (Lior Nissim, Timothy Lu) smaller scar 2226 ND 3' env25 pistol ribozyme (with an added CUUCGG loop) 2227 ND 3' Env-9 Twister 2228 ND =+AUUAUCUCAUUACU25 2229 ND 5' Env-9 Twister 2230 ND 3' Twisted Sister 1 2231 ND no stem 2232 ND 5' HH15 Minimal Hammerhead ribozyme 2233 ND 5' Hammerhead ribozyme (Lior Nissim, Timothy Lu) guide scaffold scar 2234 ND 5' Twisted Sister 1 2235 ND 5' sTRSV WT viral Hammerhead ribozyme 2236 ND 148: =+G55, stacked onto 64 2237 ND 158: 103+148(+G55) -99, G65U 2238 174 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG 2239 175 ACUGGCGCCUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAU GGGUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG 2240 176 GCUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG 2241 177 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAU GGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG 2242 181 ACUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAU GGGUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG 2243 182 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAU GGGUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG 2244 183 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG 2245 184 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAU UGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG 2246 185 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAU UGGGUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG 2247 186 ACUGGCGCCUUUAUCAUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUA UGGGUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG 2248 187 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCGCCCUCUUCGGAGGGAAGCAUCAAAG 2249 188 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCACAUGAGGAUCACCCAUGUGAGCAUCAAAG SEQ ID NO: Name NUCLEOTIDE SEQUENCE OR DESCRIPTION OF MODIFICATION 2250 189 ACUGGCACUUUUACCUGAUUACUUUGAGAGCCAACACCAGCGACUAUGUCGUAG UGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG 2251 190 ACUGGCACUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG 2252 191 ACUGGCCCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG 2253 192 ACUGGCGCUUUUACCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG 2254 193 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAACACCAGCGACUAUGUCGUAG UGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG 2255 195 ACUGGCACCUUUACCUGAUUACUUUGAGAGCCAACACCAGCGACUAUGUCGUAU GGGUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG 2256 196 ACUGGCACCUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAU GGGUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG 2257 197 ACUGGCCCCUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAU GGGUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG 2258 198 ACUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAACACCAGCGACUAUGUCGUAU GGGUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAAAG 2259 199 GCUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG 2260 200 GACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUA GUGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG 2261 201 ACUGGCGCCUUUAUCUGAUUACUUUGGAGAGCCAUCACCAGCGACUAUGUCGUA GUGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG 2262 202 ACUGGCGCAUUUAUCUGAUUACUUUGUGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG 2263 203 ACUGGCGCCUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG 2264 204 ACUGGCGCUUUUAUCUGAUUACUUUGGAGAGCCAUCACCAGCGACUAUGUCGUA GUGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG 2265 205 ACUGGCGCAUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG 2266 206 ACUGGCGCUUUUAUCUGAUUACUUUGUGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG 2267 207 ACUGGCGCUUUUAUUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUA GUGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG 2268 208 ACGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAGU GGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG 2269 209 ACUGGCGCUUUUAUAUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG 2270 210 ACUGGCGCUUUUAUCUUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUA GUGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG 2271 211 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAGCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG 2272 212 ACUGGCGCUGUUAUCUGAUUACUUCGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCGAAG 2273 213 ACUGGCGCUCUUAUCUGAUUACUUCGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCGAAG 2274 214 ACUGGCGCUUGUAUCUGAUUACUCUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAGAG SEQ ID NO: Name NUCLEOTIDE SEQUENCE OR DESCRIPTION OF MODIFICATION 2275 215 ACUGGCGCUUCUAUCUGAUUACUCUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAGAG 2276 216 ACUGGCGCUUUGAUCUGAUUACCUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAGG 2277 217 ACUGGCGCUUUCAUCUGAUUACCUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAGG 2278 218 ACUGGCGCUGUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG 2279 219 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCGAAG 2280 220 ACUGGCGCUUUUAUCUGAUUACUUCGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG 2281 221 ACUGGCACUUCUAUCUGAUUACUCUGAGAGCCAUCACCAGCGACUAUGUCGUAU GGGUAAAGCCGCUUACGGACUUCGGUCCGUAAGAGGCAUCAGAG 2282 222 ACUGGCACUUCUAUCUGAUUACUCUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAGAG 2283 223 ACUGGCACCUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAU GGGUAAAGCCGCUUACGGACUUCGGUCCGUAAGAGGCAUCAAAG 2284 224 ACUGGCACUUGUAUCUGAUUACUCUGAGAGCCAUCACCAGCGACUAUGUCGUAU GGGUAAAGCCGCUUACGGACUUCGGUCCGUAAGAGGCAUCAGAG 2285 225 ACUGGCACUUGUAUCUGAUUACUCUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAGAG 41817 226 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUGCACUAUGGGCGCAGCGUCAAUGACGCUGACGGUACAGGCCAG ACAAUUAUUGUCUGGUAUAGUGCAGCAUCAAAG 39981 229 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCCCCGUACACCAUUAGGGUACGGGGAGCAUCAAAGCGAGACGU AAUUACGUCUCGUUUUUUUU 39982 230 ACUGGCACUUCUAUCUGAUUACUCUGAGAGCCAUCACCAGCGACUAUGUCGUAU GGGUAAAGCGCUUACGGACUUCGGUCCGUAAGAAGCAUCAGAG 39983 231 ACUGGCGCUUCUAUCUGAUUACUCUGAGAGCCAUCACCAGCGACUAUGUCGUAU GGGUAAAGCCGCUUACGGACUUCGGUCCGUAAGAGGCAUCAGAG 39984 232 ACUGGCACUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAU GGGUAAAGCCGCUUACGGACUUCGGUCCGUAAGAGGCAUCAGAG 39985 233 ACUGGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAU GGGUAAAGCCGCUUACGGACUUCGGUCCGUAAGAGGCAUCAGAG 39986 234 ACUGGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAU GGGUAAAGCGCCUUACGGACUUCGGUCCGUAAGGAGCAUCAGAG 39987 235 ACUGGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCCGCUUACGGACUUCGGUCCGUAAGAGGCAUCAGAG 39988 236 ACGGGACUUUCUAUCUGAUUACUCUGAAGUCCCUCACCAGCGACUAUGUCGUAU GGGUAAAGCCGCUUACGGACUUCGGUCCGUAAGAGGCAUCAGAG 39989 237 ACCUGUAGUUCUAUCUGAUUACUCUGACUACAGUCACCAGCGACUAUGUCGUAU GGGUAAAGCCGCUUACGGACUUCGGUCCGUAAGAGGCAUCAGAG 39990 238 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUGCACGGUGGGCGCAGCUUCGGCUGACGGUACACCGUGCAGCAU CAAAG 39991 239 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUGCACGGUGGGCGCAGCUUCGGCUGACGGUACACCGGUGGGCGC AGCUUCGGCUGACGGUACACCGUGCAGCAUCAAAG SEQ ID NO: Name NUCLEOTIDE SEQUENCE OR DESCRIPTION OF MODIFICATION 39992 240 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUGCACGGUGGGCGCAGCUUCGGCUGACGGUACACCGGUGGGCGC AGCUUCGGCUGACGGUACACCGGUGGGCGCAGCUUCGGCUGACGGUACACCGUG CAGCAUCAAAG 39993 241 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUGCACGGUGGGCGCAGCUUCGGCUGACGGUACACCGGUGGGCGC AGCUUCGGCUGACGGUACACCGGUGGGCGCAGCUUCGGCUGACGGUACACCGGU GGGCGCAGCUUCGGCUGACGGUACACCGUGCAGCAUCAAAG 39994 242 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUGCACGGUGGGCGCAGCUUCGGCUGACGGUACACCGGUGGGCGC AGCUUCGGCUGACGGUACACCGGUGGGCGCAGCUUCGGCUGACGGUACACCGGU GGGCGCAGCUUCGGCUGACGGUACACCGGUGGGCGCAGCUUCGGCUGACGGUAC ACCGUGCAGCAUCAAAG 39995 243 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUGCACCUAGCGGAGGCUAGGUGCAGCAUCAAAG 39996 244 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUGCACCUCGGCUUGCUGAAGCGCGCACGGCAAGAGGCGAGGUGC AGCAUCAAAG 39997 245 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUGCACCUCUCUCGACGCAGGACUCGGCUUGCUGAAGCGCGCACG GCAAGAGGCGAGGGGCGGCGACUGGUGAGUACGCCAAAAAUUUUGACUAGCGGA GGCUAGAAGGAGAGAGGUGCAGCAUCAAAG 39998 246 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUGCACGGUGCCCGUCUGUUGUGUCGAGAGACGCCAAAAAUUUUG ACUAGCGGAGGCUAGAAGGAGAGAGAUGGGUGCCGUGCAGCAUCAAAG 39999 247 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUGCACAUGGAGAGGAGAUGUGCAGCAUCAAAG 40000 248 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUGCACAUGGAGAUGUGCAGCAUCAAAG 40001 249 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUUGGGCGCAGCGUCAAUGACGCUGACGGUACAAGCAUCAAAG 40002 250 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUGCACUAUGGGCGCAGCGUCAAUGACGCUGACGGUACAGGCCAC AUGAGGAUCACCCAUGUGGUAUAGUGCAGCAUCAAAG 40003 251 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUGCACUAUGGGCGCAGCUCAUGAGGAUCACCCAUGAGCUGACGG UACAGGCCACAUGAGGAUCACCCAUGUGGUAUAGUGCAGCAUCAAAG 40004 252 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUGCACUAUGGGCGCAGCGUCAAUGACGCUGACGGUACAGGCCAC AUGGCAGUCGUAACGACGCGGGUGGUAUAGUGCAGCAUCAAAG 40005 253 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUGCACUAUGGGCGCAGCAAACAUGGCAGUCCUAAGGACGCGGGU UUUGCUGACGGUACAGGCCACAUGGCAGUCGUAACGACGCGGGUGGUAUAGUGC AGCAUCAAAG 40006 254 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUGCACUAUGGGCGCAGACAUGGCAGUCGUAACGACGCGGGUCUG ACGGUACAGGCCACAUGAGGAUCACCCAUGUGGUAUAGUGCAGCAUCAAAG 40007 255 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUGCACUAAGGAGUUUAUAUGGAAACCCUUAGUGCAGCAUCAAAG 40008 256 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCAGGAAGCACUAUGGGCGCAGCGUCAAUGACGCUGACGGUACA SEQ ID NO: Name NUCLEOTIDE SEQUENCE OR DESCRIPTION OF MODIFICATION GGCCAGACAAUUAUUGUCUGGUAUAGUGCAGCAGCAGAACAAUUUGCUGAGGGC UAUUGAGGCGCAACAGCAUCUGUUGCAACUCACAGUCUGGGGCAUCAAGCAGCU CCAGGCAAGAAUCCUGAGCAUCAAAG 40009 257 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUGCACGCCCUGAAGAAGGGCGUGCAGCAUCAAAG 40010 258 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUGCACGGCUCGUGUAGCUCAUUAGCUCCGAGCCGUGCAGCAUCA AAG 40011 259 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUGCACCCGUGUGCAUCCGCAGUGUCGGAUCCACGGGUGCAGCAU CAAAG 40012 260 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUGCACGGAAUCCAUUGCACUCCGGAUUUCACUAGGUGCAGCAUC AAAG 40013 261 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUGCACAUGCAUGUCUAAGACAGCAUGUGCAGCAUCAAAG 40014 262 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUGCACAAAACAUAAGGAAAACCUAUGUUGUGCAGCAUCAAAG 40015 263 ACUGGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCCGCUUACGGACUAUGGGCGCAGCGUCAAUGACGCUGACGGUACA GGCCAGACAAUUAUUGUCUGGUAUAGUCCGUAAGAGGCAUCAGAG 40016 264 ACUGGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCCGCUUACGGGUGGGCGCAGCGUCAAUGACGCUGACGGUACAGGC CAGACAAUUAUUGUCUGGUACCCGUAAGAGGCAUCAGAG 40017 265 ACUGGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCCGCUUACGGUAUGGGCGCAGCGUCAAUGACGCUGACGGUACAGG CCACAUGAGGAUCACCCAUGUGGUAUACCGUAAGAGGCAUCAGAG 40018 266 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCCCUAUGGGCGCAGCGUCAAUGACGCUGACGGUACAGGCCACA UGAGGAUCACCCAUGUGGUAUAGGGAGCAUCAAAG 40019 267 ACUGGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCCGCUUACGGUAUGGGCGCAGCUCAUGAGGAUCACCCAUGAGCUG ACGGUACAGGCCACAUGAGGAUCACCCAUGUGGUAUACCGUAAGAGGCAUCAGA G 40020 268 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCCCUAUGGGCGCAGCUCAUGAGGAUCACCCAUGAGCUGACGGU ACAGGCCACAUGAGGAUCACCCAUGUGGUAUAGGGAGCAUCAAAG 40021 269 ACUGGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCCGCUUACGGUAUGGGCGCAGCGUCAAUGACGCUGACGGUACAGG CCACAUGGCAGUCGUAACGACGCGGGUGGUAUACCGUAAGAGGCAUCAGAG 40022 270 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCCCUAUGGGCGCAGCGUCAAUGACGCUGACGGUACAGGCCACA UGGCAGUCGUAACGACGCGGGUGGUAUAGGGAGCAUCAAAG 40023 271 ACUGGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCCGCUUACGGUAUGGGCGCAGCAAACAUGGCAGUCCUAAGGACGC GGGUUUUGCUGACGGUACAGGCCACAUGGCAGUCGUAACGACGCGGGUGGUAUA CCGUAAGAGGCAUCAGAG 40024 272 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCCCUAUGGGCGCAGCAAACAUGGCAGUCCUAAGGACGCGGGUU UUGCUGACGGUACAGGCCACAUGGCAGUCGUAACGACGCGGGUGGUAUAGGGAG CAUCAAAG SEQ ID NO: Name NUCLEOTIDE SEQUENCE OR DESCRIPTION OF MODIFICATION 40025 273 ACUGGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCCGCUUACGGUAUGGGCGCAGACAUGGCAGUCGUAACGACGCGGG UCUGACGGUACAGGCCACAUGAGGAUCACCCAUGUGGUAUACCGUAAGAGGCAU CAGAG 40026 274 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCCCUAUGGGCGCAGACAUGGCAGUCGUAACGACGCGGGUCUGA CGGUACAGGCCACAUGAGGAUCACCCAUGUGGUAUAGGGAGCAUCAAAG 40913 275 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGU CGUAGUGGGUAAAGCUGCACUAUGGGCGCAGCACCUGAGGAUCACCCAG GUGCUGACGGUACAGGCCACCUGAGGAUCACCCAGGUGGUAUAGUGCAG CAUCAAAG 40914 276 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGU CGUAGUGGGUAAAGCUGCACUAUGGGCGCAGCGCAUGAGGAUCACCCAU GCGCUGACGGUACAGGCCGCAUGAGGAUCACCCAUGCGGUAUAGUGCAG CAUCAAAG 40915 277 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGU CGUAGUGGGUAAAGCUGCACUAUGGGCGCAGCGCCUGAGGAUCACCCAG GCGCUGACGGUACAGGCCGCCUGAGGAUCACCCAGGCGGUAUAGUGCAG CAUCAAAG 40916 278 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGU CGUAGUGGGUAAAGCUGCACUAUGGGCGCAGCGCCUGAGCAUCAGCCAG GCGCUGACGGUACAGGCCGCCUGAGCAUCAGCCAGGCGGUAUAGUGCAG CAUCAAAG 40917 279 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGU CGUAGUGGGUAAAGCUGCACUAUGGGCGCAGCACAUGAGCAUCAGCCAU GUGCUGACGGUACAGGCCACAUGAGCAUCAGCCAUGUGGUAUAGUGCAG CAUCAAAG 40918 280 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGU CGUAGUGGGUAAAGCUGCACUAUGGGCGCAGCACAUGAGUAUCAACCAU GUGCUGACGGUACAGGCCACAUGAGUAUCAACCAUGUGGUAUAGUGCAG CAUCAAAG 40919 281 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGU CGUAGUGGGUAAAGCUGCACUAUGGGCGCAGCACAUGAGAAUCAGCCAU GUGCUGACGGUACAGGCCACAUGAGAAUCAGCCAUGUGGUAUAGUGCAG CAUCAAAG 40920 282 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGU CGUAGUGGGUAAAGCUGCACUAUGGGCGCAGCCCUUGAGGAUCACCCAU GUGCUGACGGUACAGGCCCCUUGAGGAUCACCCAUGUGGUAUAGUGCAG CAUCAAAG 40921 283 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGU CGUAGUGGGUAAAGCUGCACUAUGGGCGCAGCACUUGAGGAUCACCCAU GUGCUGACGGUACAGGCCACUUGAGGAUCACCCAUGUGGUAUAGUGCAG CAUCAAAG 40922 284 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGU CGUAGUGGGUAAAGCUGCACUAUGGGCGCAGCACCUGAGGAUCACCCAU GUGCUGACGGUACAGGCCACCUGAGGAUCACCCAUGUGGUAUAGUGCAG CAUCAAAG SEQ ID NO: Name NUCLEOTIDE SEQUENCE OR DESCRIPTION OF MODIFICATION 40923 285 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUGCACUAUGGGCGCAGCACAUGAGGAUCACCUAUGUGCUGACGG UACAGGCCACAUGAGGAUCACCUAUGUGGUAUAGUGCAGCAUCAAAG 40924 286 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUGCACUAUGGGCGCAGCACAUUAGGAUCACCAAUGUGCUGACGG UACAGGCCACAUUAGGAUCACCAAUGUGGUAUAGUGCAGCAUCAAAG 40925 287 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUGCACUAUGGGCGCAGCACAUUAGGAUCACCGAUGUGCUGACGG UACAGGCCACAUUAGGAUCACCGAUGUGGUAUAGUGCAGCAUCAAAG 40926 288 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUGCACUAUGGGCGCAGCACAUUAGGAUCACCUAUGUGCUGACGG UACAGGCCACAUUAGGAUCACCUAUGUGGUAUAGUGCAGCAUCAAAG 40927 289 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUGCACUAUGGGCGCAGCACAUGAGGAUUACCCAUGUGCUGACGG UACAGGCCACAUGAGGAUUACCCAUGUGGUAUAGUGCAGCAUCAAAG 40928 290 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUGCACUAUGGGCGCAGCACAUGAGGAUAACCCAUGUGCUGACGG UACAGGCCACAUGAGGAUAACCCAUGUGGUAUAGUGCAGCAUCAAAG 40929 291 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUGCACUAUGGGCGCAGCACAUGAGGAUGACCCAUGUGCUGACGG UACAGGCCACAUGAGGAUGACCCAUGUGGUAUAGUGCAGCAUCAAAG 40930 292 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUGCACUAUGGGCGCAGCACAUGAGGACCACCCAUGUGCUGACGG UACAGGCCACAUGAGGACCACCCAUGUGGUAUAGUGCAGCAUCAAAG 40931 293 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUGCACUAUGGGCGCAGCAGAUGAGGAUCACCCAUGGGCUGACGG UACAGGCCAGAUGAGGAUCACCCAUGGGGUAUAGUGCAGCAUCAAAG 40932 294 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUGCACUAUGGGCGCAGCACAUGGGGAUCACCCAUGUGCUGACGG UACAGGCCACAUGGGGAUCACCCAUGUGGUAUAGUGCAGCAUCAAAG 40933 295 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUGCACUAUGGGCGCAGCACAUGAGGAUCACCCAUGUGCUGACGG UACAGGCCACAUGAGGAUCACCCAUGUGGUAUAGUGCAGCAUCAAAG 40934 296 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCACCUGAGGAUCACCCAGGUGAGCAUCAAAG 40935 297 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCGCAUGAGGAUCACCCAUGCGAGCAUCAAAG 40936 298 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCGCCUGAGGAUCACCCAGGCGAGCAUCAAAG 40937 299 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCGCCUGAGCAUCAGCCAGGCGAGCAUCAAAG 40938 300 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCACAUGAGCAUCAGCCAUGUGAGCAUCAAAG 40939 301 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCACAUGAGUAUCAACCAUGUGAGCAUCAAAG 40940 302 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCACAUGAGAAUCAGCCAUGUGAGCAUCAAAG 40941 303 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCCCUUGAGGAUCACCCAUGUGAGCAUCAAAG SEQ ID NO: Name NUCLEOTIDE SEQUENCE OR DESCRIPTION OF MODIFICATION 40942 304 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCACUUGAGGAUCACCCAUGUGAGCAUCAAAG 40943 305 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCACCUGAGGAUCACCCAUGUGAGCAUCAAAG 40944 306 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCACAUGAGGAUCACCUAUGUGAGCAUCAAAG 40945 307 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCACAUUAGGAUCACCAAUGUGAGCAUCAAAG 40946 308 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCACAUUAGGAUCACCGAUGUGAGCAUCAAAG 40947 309 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCACAUUAGGAUCACCUAUGUGAGCAUCAAAG 40948 310 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCACAUGAGGAUUACCCAUGUGAGCAUCAAAG 40949 311 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCACAUGAGGAUAACCCAUGUGAGCAUCAAAG 40950 312 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCACAUGAGGAUGACCCAUGUGAGCAUCAAAG 40951 313 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCACAUGAGGACCACCCAUGUGAGCAUCAAAG 40952 314 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCAGAUGAGGAUCACCCAUGGGAGCAUCAAAG 40953 315 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCACAUGGGGAUCACCCAUGUGAGCAUCAAAG 40954 317 ACUGGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUCACAUGAGGAUCACCCAUGUGAGCAUCAGAG 40955 318 ACUGGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUGCACUAUGGGCGCAGCGUCAAUGACGCUGACGGUACAGGCCAC AUGAGGAUCACCCAUGUGGUAUAGUGCAGCAUCAGAG 40956 319 ACUGGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUGCACUAUGGGCGCAGCUCAUGAGGAUCACCCAUGAGCUGACGG UACAGGCCACAUGAGGAUCACCCAUGUGGUAUAGUGCAGCAUCAGAG 40957 320 ACUGGCGCUUCUAUCUGAUUACUCUGAGCGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUGCACUAUGGGCGCAGACAUGGCAGUCGUAACGACGCGGGUCUG ACGGUACAGGCCACAUGAGGAUCACCCAUGUGGUAUAGUGCAGCAUCAGAG 40958 321 ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAG UGGGUAAAGCUGCACUAUGGGGCCACAUGAGGAUCACCCAUGUGGUGUACAGCG CAGCGUCAAUGACGCUGACGAUAGUGCAGCAUCAAAG

[0206] In some embodiments, a sgRNA variant comprises one or more additional modifications to a sequence of SEQ ID NO:2238, SEQ ID NO:2239, SEQ ID NO:2240, SEQ ID NO:2241, SEQ ID NO:2243, SEQ ID NO:2256, SEQ ID NO:2274, SEQ ID NO:2275, SEQ ID NO:2279, SEQ ID NO:2281, SEQ ID NO: 2285, SEQ ID NO: 39984, SEQ ID NO: 39987, or SEQ ID NO: 40003 of Table 2.

[0207] In some embodiments of the gRNA variants of the disclosure, the gRNA variant comprises at least one modification compared to the reference guide scaffold of SEQ ID NO:5, 59 wherein the at least one modification is selected from one or more of (a) a C18G substitution in the triplex loop; (b) a G55 insertion in the stem bubble; (c) a UI deletion; (d) a modification of the extended stem loop wherein (i) a 6 nt loop and 13 loop-proximal base pairs are replaced by a Uvsx hairpin; and (ii) a deletion of A99 and a substitution of G65U that results in a loop-distal base that is fully base-paired.

[0208] In some embodiments, a gRNA variant comprises an exogenous stem loop having a long non-coding RNA (IncRNA). As used herein, a IncRNA refers to a non-coding RNA that is longer than approximately 200 bp in length. In some embodiments, the 5' and 3' ends of the exogenous stem loop are base paired; i.e., interact to form a region of duplex RNA. In some embodiments, the 5' and 3' ends of the exogenous stem loop are base paired, and one or more regions between the 5' and 3' ends of the exogenous stem loop are not base paired, forming the loop.

[0209] In some embodiments, the disclosure provide gRNA variants with nucleotide modifications relative to reference gRNA having: (a) substitution of 1 to 15 consecutive or non-consecutive nucleotides in the gRNA variant in one or more regions; (b) a deletion of 1 to 10 consecutive or non-consecutive nucleotides in the gRNA variant in one or more regions; (c) an insertion of 1 to 10 consecutive or non-consecutive nucleotides in the gRNA variant in one or more regions; (d) a substitution of the scaffold stem loop or the extended stem loop with an RNA stem loop sequence from a heterologous RNA source with proximal 5' and 3' ends; or any combination of (a)-(d). Any of the substitutions, insertions and deletions described herein can be combined to generate a gRNA variant of the disclosure. For example, a gRNA variant can comprise at least one substitution and at least one deletion relative to a reference gRNA, at least one substitution and at least one insertion relative to a reference gRNA, at least one insertion and at least one deletion relative to a reference gRNA, or at least one substitution, one insertion and one deletion relative to a reference gRNA.

[0210] In some embodiments, a sgRNA variant of the disclosure comprises one or more modifications to the sequence of a previously generated variant, the previously generated variant itself serving as the sequence to be modified. In some cases, one or modifications are introduced to the pseudoknot region of the scaffold. In other cases, one or modifications are introduced to the triplex region of the scaffold. In other cases, one or modifications are introduced to the scaffold bubble. In other cases, one or modifications are introduced to the extended stem region of the scaffold. In still other cases, one of modifications are introduced into two or more of the foregoing regions. Such modifications can comprise an insertion, deletion, or substitution of one or more nucleotides in the foregoing regions, or any combination thereof. Exemplary methods to generate and assess the modifications are described in Example 20.

[0211] In some embodiments, a sgRNA variant comprises one or more modifications to a sequence of SEQ ID NO: 2238, SEQ ID NO: 2239, SEQ ID NO: 2240, SEQ ID NO: 2241, SEQ ID NO:2241, SEQ ID NO:2274, SEQ ID NO:2275, SEQ ID NO: 2279, or SEQ ID NO: 2285, SEQ ID NO: 39984, SEQ ID NO: 39987, or SEQ ID NO: 40003.

[0212] In exemplary embodiments, a gRNA variant comprises one or more modifications relative to gRNA scaffold variant 174 (SEQ ID NO:2238), wherein the resulting gRNA variant exhibits a improved functional characteristic compared to the parent 174, when assessed in an in vitro or in vivo assay under comparable conditions. In other exemplary embodiments, a gRNA variant comprises one or more modifications relative to gRNA scaffold variant 175 (SEQ ID NO:2239), wherein the resulting gRNA variant exhibits a improved functional characteristic compared to the parent 175, when assessed in an in vitro or in vivo assay under comparable conditions. For example, variants with modifications to the triplex loop of gRNA variant 175 show high enrichment relative to the 175 scaffold, particularly mutations to C15 or C17. Additionally, changes to either member of the predicted pair in the pseudoknot stem between G7 and A29 are both highly enriched relative to the 175 scaffold, with converting A29 to a C or a T to form a canonical Watson-Crick pairing (G7:C29), and the second of which would form a GU wobble pair (G7:U29), both of which may be expected to increase stability of the helix relative to the G:A pair. In addition, the insertion of a C at position 54 in guide scaffold 175 results in an enriched modification.

[0213] In some embodiments, the disclosure provides gRNA variants comprising one or more modifications to the gRNA scaffold variant 174 (SEQ ID NO: 2238) selected from the group consisting of the modifications of Table 28, wherein the resulting gRNA variant exhibits an improved functional characteristic compared to the parent 174, when assessed in an in vitro or in vivo assay under comparable conditions. In some embodiments, the improved functional characteristic is one or more functional properties selected from the group consisting of increased editing activity, increased pseudoknot stem stability, increased triplex region stability, increased scaffold stem stability, extended stem stability, reduced off-target folding intermediates, and increased binding affinity to a Class 2, Type V CRISPR protein. In the foregoing embodiments, the gRNA comprising one or more modifications to the gRNA scaffold variant 174 selected from the group consisting of the modifications of Table 28 (with a linked targeting sequence and complexed with a Class 2, Type V CRISPR protein) exhibits an improved enrichment score (log2) of at least about 2.0, at least about 2.5, at least about 3, or at least about 3.5 greater compared to the score of the gRNA scaffold of SEQ ID NO: 2238 in an in vitro assay.

[0214] In some embodiments, the disclosure provides gRNA variants comprising one or more modifications to the gRNA scaffold variant 175 (SEQ ID NO: 2239) selected from the group consisting of the modifications of Table 29, wherein the resulting gRNA variant exhibits an improved functional characteristic compared to the parent 175, when assessed in an in vitro or in vivo assay under comparable conditions. In some embodiments, the improved functional characteristic is one or more functional properties selected from the group consisting of increased editing activity, increased pseudoknot stem stability, increased triplex region stability, increased scaffold stem stability, extended stem stability, reduced off-target folding intermediates, and increased binding affinity to a Class 2, Type V CRISPR protein. In the foregoing embodiments, the gRNA comprising one or more modifications to the gRNA scaffold variant 175 selected from the group consisting of the modifications of Table 29 (with a linked targeting sequence and complexed with a Class 2, Type V CRISPR protein) exhibits an improved enrichment score (log2) of at least about 1.2, at least about 1.5, at least about 2.0, at least about 2.5, at least about 3, or at least about 3.5 greater compared to the score of the gRNA scaffold of SEQ ID NO: 2239 in an in vitro assay.

[0215] In a particular embodiment, the one or more modifications of gRNA scaffold variant 174 are selected from the group consisting of nucleotide positions UI 1, U24, A29, U65, C66, C68, A69, U76, G77, A79, and A87. In a particular embodiment, the modifications of gRNA scaffold variant 174 are UI IC, U24C, A29C, U65C, C66G, C68U, an insertion of ACGGA at position 69, an insertion of UCCGU at position 76, G77A, an insertion of GA at position 79, A87G. In another particular embodiment, the modifications of gRNA scaffold variant 175 are selected from the group consisting of nucleotide positions C9, UI 1, Cl7, U24, A29, G54, C65, A89, and A96. In a particular embodiment, the modifications of gRNA scaffold variant 174 are C9U, UI IC, C17G, U24C, A29C, an insertion of G at position 54, an insertion of C at position 65, A89G, and A96G.

[0216] In exemplary embodiments, a gRNA variant comprises one or more modifications relative to gRNA scaffold variant 215 (SEQ ID NO:2275), wherein the resulting gRNA variant exhibits an improved functional characteristic compared to the parent 215, when assessed in an in vitro or in vivo assay under comparable conditions.

[0217] In exemplary embodiments, a gRNA variant comprises one or more modifications relative to gRNA scaffold variant 221 (SEQ ID NO: 2281), wherein the resulting gRNA variant exhibits an improved functional characteristic compared to the parent 221, when assessed in an in vitro or in vivo assay under comparable conditions.

[0218] In exemplary embodiments, a gRNA variant comprises one or more modifications relative to gRNA scaffold variant 225 (SEQ ID NO: 2285), wherein the resulting gRNA variant exhibits an improved functional characteristic compared to the parent 225, when assessed in an in vitro or in vivo assay under comparable conditions.

[0219] In exemplary embodiments, a gRNA variant comprises one or more modifications relative to gRNA scaffold variant 235 (SEQ ID NO: 39987), wherein the resulting gRNA variant exhibits an improved functional characteristic compared to the parent 225, when assessed in an in vitro or in vivo assay under comparable conditions.

[0220] In exemplary embodiments, a gRNA variant comprises one or more modifications relative to gRNA scaffold variant 251 (SEQ ID NO: 40003), wherein the resulting gRNA variant exhibits an improved functional characteristic compared to the parent 251, when assessed in an in vitro or in vivo assay under comparable conditions.

[0221] In the foregoing embodiments, the improved functional characteristic includes, but is not limited to one or more of increased stability, increased transcription of the gRNA, increased resistance to nuclease activity, increased folding rate of the gRNA, decreased side product formation during folding, increased productive folding, increased binding affinity to a CasX protein, increased binding affinity to a target nucleic acid when complexed with the CasX protein, increased gene editing when complexed with the CasX protein, increased specificity of editing when complexed with the CasX protein, decreased off-target editing when complexed with the CasX protein, and increased ability to utilize a greater spectrum of one or more PAM sequences, including ATC, CTC, GTC, or TTC, in the modifying of target nucleic acid when complexed with the CasX protein. In some cases, the one or more of the improved characteristics of the gRNA variant is at least about 1.1 to about 100,000-fold improved relative to the gRNA from which it was derived. In other cases, the one or more improved characteristics of the gRNA variant is at least about 1.1, at least about 10, at least about 100, at least about 1000, at least about 10,000, at least about 100,000-fold or more improved relative to the gRNA from which it was derived. In other cases, the one or more of the improved characteristics of the gRNA variant is about 1.1 to 100,00-fold, about 1.1 to 10,00-fold, about 1.1 to 1,000-fold, about 1.1 to 500-fold, about 1.1 to 100-fold, about 1.1 to 50-fold, about 1.1 to 20-fold, about 10 to 100,00-fold, about 10 to 10,00-fold, about 10 to 1,000-fold, about 10 to 500-fold, about 10 to 100-fold, about 10 to 50-fold, about 10 to 20-fold, about 2 to 70-fold, about 2 to 50-fold, about 2 to 30-fold, about 2 to 20-fold, about 2 to 10-fold, about 5 to 50-fold, about 5 to 30-fold, about 5 to 10-fold, about 100 to 100,00-fold, about 100 to 10,00-fold, about 100 to 1,000-fold, about 100 to 500-fold, about 500 to 100,00-fold, about 500 to 10,00-fold, about 500 to 1,000-fold, about 500 to 750-fold, about 1,000 to 100,00-fold, about 10,000 to 100,00-fold, about 20 to 500-fold, about 20 to 250-fold, about 20 to 200-fold, about 20 to 100-fold, about 20 to 50-fold, about 50 to 10,000-fold, about 50 to 1,000-fold, about 50 to 500-fold, about 50 to 200-fold, or about 50 to 100-fold, improved relative to the gRNA from which it was derived. In other cases, the one or more improved characteristics of the gRNA variant is about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20fold, 25-fold, 30-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100fold, 110-fold, 120-fold, 130-fold, 140-fold, 150-fold, 160-fold, 170-fold, 180-fold, 190-fold, 200-fold, 210-fold, 220-fold, 230-fold, 240-fold, 250-fold, 260-fold, 270-fold, 280-fold, 290fold, 300-fold, 310-fold, 320-fold, 330-fold, 340-fold, 350-fold, 360-fold, 370-fold, 380-fold, 390-fold, 400-fold, 425-fold, 450-fold, 475-fold, or 500-fold improved relative to the gRNA from which it was derived.

[0222] In some embodiments, the gRNA variant comprises an exogenous extended stem loop, with such differences from a reference gRNA described as follows. In some embodiments, an exogenous extended stem loop has little or no identity to the reference stem loop regions disclosed herein (e.g., SEQ ID NO: 15). In some embodiments, an exogenous stem loop is at least 10 bp, at least 20 bp, at least 30 bp, at least 40 bp, at least 50 bp, at least 60 bp, at least 70 bp, at least 80 bp, at least 90 bp, at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 600 bp, at least 700 bp, at least 800 bp, at least 900 bp, at least 1,000 bp, at least 2,000 bp, at least 3,000 bp, at least 4,000 bp, at least 5,000 bp, at least 6,000 bp, at least 7,000 bp, at least 8,000 bp, at least 9,000 bp, at least 10,000 bp, at least 12,000 bp, at least 15,000 bp or at least 20,000 bp. In some embodiments, the gRNA variant comprises an extended stem loop region comprising at least 10, at least 100, at least 500, at least 1000, or at least 10,000 nucleotides. In some embodiments, the heterologous stem loop increases the stability of the gRNA. In some embodiments, the heterologous RNA stem loop is capable of binding a protein, an RNA structure, a DNA sequence, or a small molecule. In some embodiments, an exogenous stem loop region replacing the stem loop comprises an RNA stem loop or hairpin in which the resulting gRNA has increased stability and, depending on the choice of loop, can interact with certain cellular proteins or RNA. Such exogenous extended stem loops can comprise, for example a thermostable RNA such as MS2 hairpin (ACAUGAGGAUCACCCAUGU (SEQ ID NO: 35)), Qp hairpin (UGCAUGUCUAAGACAGCA (SEQ ID NO: 36)), UI hairpin II (AAUCCAUUGCACUCCGGAUU (SEQ ID NO: 37)), Uvsx (CCUCUUCGGAGG (SEQ ID NO: 38)), PP7 hairpin (AGGAGUUUCUAUGGAAACCCU (SEQ ID NO: 39)), Phage replication loop (AGGUGGGACGACCUCUCGGUCGUCCUAUCU (SEQ ID NO: 40)), Kissing loop a (UGCUCGCUCCGUUCGAGCA (SEQ ID NO: 41)), Kissing loop bl (UGCUCGACGCGUCCUCGAGCA (SEQ ID NO: 42)), Kissing loop_b2 (UGCUCGUUUGCGGCUACGAGCA (SEQ ID NO: 43)), G quadriplex M3q (AGGGAGGGAGGGAGAGG (SEQ ID NO: 44)), G quadriplex telomere basket (GGUUAGGGUUAGGGUUAGG (SEQ ID NO: 45)), Sarcin-ricin loop (CUGCUCAGUACGAGAGGAACCGCAG (SEQ ID NO: 46)) or Pseudoknots (UACACUGGGAUCGCUGAAUUAGAGAUCGGCGUCCUUUCAUUCUAUAUACUUUGG AGUUUUAAAAUGUCUCUAAGUACA (SEQ ID NO: 47)). In some embodiments, the extended stem loop comprises UGGGCGCAGCGUCAAUGACGCUGACGGUACA (Stem IIB; SEQ ID NO: 41843), GCACUAUGGGCGCAGCGUCAAUGACGCUGACGGUACAGGCCAGACAAUUAUUGU CUGGUAUAGUGC (Stem II; SEQ ID NO: 41844), CAGGAAGCACUAUGGGCGCAGCGUCAAUGACGCUGACGGUACAGGCCAGACAAU UAUUGUCUGGUAUAGUGCAGCAGCAGAACAAUUUGCUGAGGGCUAUUGAGGCGC AACAGCAUCUGUUGCAACUCACAGUCUGGGGCAUCAAGCAGCUCCAGGCAAGAA UCCUG (Stem II-V SEQ ID NO: 41845), GCUGACGGUACAGGC (RBE; SEQ ID NO: 41846), and AGGAGCUUUGUUCCUUGGGUUCUUGGGAGCAGCAGGAAGCACUAUGGGCGCAGC GUCAAUGACGCUGACGGUACAGGCCAGACAAUUAUUGUCUGGUAUAGUGCAGCA GCAGAACAAUUUGCUGAGGGCUAUUGAGGCGCAACAGCAUCUGUUGCAACUCAC AGUCUGGGGCAUCAAGCAGCUCCAGGCAAGAAUCCUGGCUGUGGAAAGAUACCU AAAGGAUCAACAGCUCCU (full-length RRE; SEQ ID NO: 41847).

[0223] In some embodiments, a gRNA variant comprises a terminal fusion partner. The term gRNA variant is inclusive of variants that include exogenous sequences such as terminal fusions, or internal insertions. Exemplary terminal fusions may include fusion of the gRNA to a selfcleaving ribozyme or protein binding motif. As used herein, a “ribozyme” refers to an RNA or segment thereof with one or more catalytic activities similar to a protein enzyme. Exemplary ribozyme catalytic activities may include, for example, cleavage and / or ligation of RNA, cleavage and / or ligation of DNA, or peptide bond formation. In some embodiments, such fusions could either improve scaffold folding or recruit DNA repair machinery. For example, a gRNA may in some embodiments be fused to a hepatitis delta virus (HDV) antigenomic ribozyme, HDV genomic ribozyme, hatchet ribozyme (from metagenomic data), env25 pistol ribozyme (representative from Aliistipes putredinis), HH15 Minimal Hammerhead ribozyme, tobacco ringspot virus (TRSV) ribozyme, WT viral Hammerhead ribozyme (and rational variants), or Twisted Sister 1 or RBMX recruiting motif. Hammerhead ribozymes are RNA motifs that catalyze reversible cleavage and ligation reactions at a specific site within an RNA molecule. Hammerhead ribozymes include type I, type II and type III hammerhead ribozymes. The HDV, pistol, and hatchet ribozymes have self-cleaving activities. gRNA variants comprising one or more ribozymes may allow for expanded gRNA function as compared to a gRNA reference. For example, gRNAs comprising self-cleaving ribozymes can, in some embodiments, be transcribed and processed into mature gRNAs as part of polycistronic transcripts. Such fusions may occur at either the 5’ or the 3’ end of the gRNA. In some embodiments, a gRNA variant comprises a fusion at both the 5’ and the 3’ end, wherein each fusion is independently as described herein.

[0224] In the embodiments of the gRNA variants, the gRNA variant further comprises a spacer (or targeting sequence) region located at the 3’ end of the gRNA, capable of hybridizing with a target nucleic acid which comprises at least 14 to about 35 nucleotides wherein the spacer is designed with a sequence that is complementary to a target nucleic acid. In some embodiments, the encoded gRNA variant comprises a targeting sequence of at least 10 to 20 nucleotides complementary to a target nucleic acid. In some embodiments, the targeting sequence has 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the encoded gRNA variant comprises a targeting sequence having 20 nucleotides. In some embodiments, the targeting sequence has 25 nucleotides. In some embodiments, the targeting sequence has 24 nucleotides. In some embodiments, the targeting sequence has 23 nucleotides. In some embodiments, the targeting sequence has 22 nucleotides. In some embodiments, the targeting sequence has 21 nucleotides. In some embodiments, the targeting sequence has 20 nucleotides. In some embodiments, the targeting sequence has 19 nucleotides. In some embodiments, the targeting sequence has 18 nucleotides. In some embodiments, the targeting sequence has 17 nucleotides. In some embodiments, the targeting sequence has 16 nucleotides. In some embodiments, the targeting sequence has 15 nucleotides. In some embodiments, the targeting sequence has 14 nucleotides. h. Complex Formation with CasX Protein

[0225] In some embodiments, a gRNA variant has an improved ability to form an RNP complex with a Class 2, Type V protein, including CasX variant proteins comprising any one of the sequences SEQ ID NOS: 49-160, 40208-40369, or 40828-40912 of Table 3, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In some embodiments, upon expression, the gRNA variant is complexed as an RNP with a CasX variant protein comprising any one of the sequences SEQ ID NOS: 49-160, 40208-40369, or 40828-40912 of Table 3, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. In some embodiments, upon expression, the gRNA variant is complexed as an RNP with a CasX variant protein comprising any one of the sequences SEQ ID NOS: 85-160, 40208-40369, or 40828-40912, or a sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto.

[0226] In some embodiments, a gRNA variant has an improved ability to form a complex with a CasX variant protein when compared to a reference gRNA, thereby improving its ability to form a cleavage-competent ribonucleoprotein (RNP) complex with the CasX protein, as described in the Examples. Improving ribonucleoprotein complex formation may, in some embodiments, improve the efficiency with which functional RNPs are assembled. In some embodiments, greater than 90%, greater than 93%, greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% of RNPs comprising a gRNA variant and its targeting sequence are competent for gene editing of a target nucleic acid.

[0227] Exemplary nucleotide changes that can improve the ability of gRNA variants to form a complex with CasX protein may, in some embodiments, include replacing the scaffold stem with a thermostable stem loop. Without wishing to be bound by any theory, replacing the scaffold stem with a thermostable stem loop could increase the overall binding stability of the gRNA variant with the CasX protein. Alternatively, or in addition, removing a large section of the stem loop could change the gRNA variant folding kinetics and make a functional folded gRNA easier and quicker to structurally-assemble, for example by lessening the degree to which the gRNA variant can get “tangled” in itself. In some embodiments, choice of scaffold stem loop sequence could change with different spacers that are utilized for the gRNA. In some embodiments, scaffold sequence can be tailored to the spacer and therefore the target sequence. Biochemical assays can be used to evaluate the binding affinity of CasX protein for the gRNA variant to form the RNP, including the assays of the Examples. For example, a person of ordinary skill can measure changes in the amount of a fluorescently tagged gRNA that is bound to an immobilized CasX protein, as a response to increasing concentrations of an additional unlabeled “cold competitor” gRNA. Alternatively, or in addition, fluorescence signal can be monitored to or seeing how it changes as different amounts of fluorescently labeled gRNA are flowed over immobilized CasX protein. Alternatively, the ability to form an RNP can be assessed using in vitro cleavage assays against a defined target nucleic acid sequence. IV. CRISPR Proteins of the AAV Systems

[0228] The present disclosure provides AAV systems encoding a CRISPR nuclease that have utility in genome editing of eukaryotic cells, as well as being an integral component of the selfinactivating feature of the construct. In some embodiments, the CRISPR nuclease employed in the genome editing systems is a Class 2, Type V nuclease. Although members of Class 2, Type V CRISPR-Cas systems have differences, they share some common characteristics that distinguish them from the Cas9 systems. Firstly, the Class 2, Type V nucleases possess a single RNA-guided RuvC domain-containing effector but no HNH domain, and they recognize T-rich PAM 5' upstream to the target region on the non-targeted strand, which is different from Cas9 systems which rely on G-rich PAM at 3' side of target sequences. Type V nucleases generate staggered double-stranded breaks distal to the PAM sequence, unlike Cas9, which generates a blunt end in the proximal site close to the PAM. In addition, Type V nucleases degrade ssDNA in trans when activated by target dsDNA or ssDNA binding in cis. In some embodiments, the Type V nucleases of the embodiments recognize a 5'-TC PAM motif and produce staggered ends cleaved solely by the RuvC domain. In some embodiments, the Type V nuclease is selected from the group consisting of Casl2a, Casl2b, Casl2c, Casl2d (CasY), Casl2j, Casl2k, CasPhi, C2c4, C2c8, C2c5, C2cl0, C2c9, CasZ and CasX. In some embodiments, the present disclosure provides AAV systems encoding a CasX variant protein and one or more gRNA acids that upon expression in a transfected cell are able to form an RNP complex and are specifically designed to modify a target nucleic acid sequence in eukaryotic cells, as well as cleave the self-inactivating segments incorporated into the polynucleotide comprising the transgene of the AAV construct.

[0229] The term “CasX protein”, as used herein, refers to a family of proteins, and encompasses all naturally occurring CasX proteins, proteins that share at least 50% identity to naturally occurring CasX proteins, as well as CasX variants possessing one or more improved characteristics relative to a naturally-occurring reference CasX protein, described more fully, below.

[0230] CasX proteins of the disclosure comprise at least one of the following domains: a nontarget strand binding (NTSB) domain, a target strand loading (TSL) domain, a helical I domain (which is further divided into helical I-I and I-II subdomains), a helical II domain, an oligonucleotide binding domain (OBD, which is further divided into OBD-I and OBD-II subdomains), and a RuvC DNA cleavage domain (which is further divided into RuvC-I and II subdomains). The RuvC domain may be modified or deleted in a catalytically-dead CasX variant, described more fully, below.

[0231] In some embodiments, a CasX variant protein can bind and / or modify (e.g., nick, catalyze a double-strand break, methylate, demethylate, etc.) a target nucleic acid at a specific sequence targeted by an associated gRNA, which hybridizes to a sequence within the target nucleic acid sequence. a. Reference CasX Proteins

[0232] The disclosure provides naturally-occurring CasX proteins (referred to herein as a "reference CasX protein"), which were subsequently modified to create the CasX variants of the disclosure. For example, reference CasX proteins can be isolated from naturally occurring prokaryotes, such as Deltaproteobacteria, Planctomycetes, or Candidates Sungbacteria species. A reference CasX protein is a type II CRISPR / Cas endonuclease belonging to the CasX (interchangeably referred to as Casl2e) family of proteins that interacts with a guide RNA to form a ribonucleoprotein (RNP) complex.

[0233] In some cases, a reference CasX protein is isolated or derived from Deltaproteobacter. In some embodiments, a reference CasX protein comprises a sequence identical to a sequence of: 1 MEKRINKIRK KLSADNATKP VSRSGPMKTL LVRVMTDDLK KRLEKRRKKP EVMPQVISNN 61 AANNLRMLLD DYTKMKEAIL QVYWQEFKDD HVGLMCKFAQ PASKKIDQNK LKPEMDEKGN 121 LTTAGFACSQ CGQPLFVYKL EQVSEKGKAY TNYFGRCNVA EHEKLILLAQ LKPEKDSDEA 181 VTYSLGKFGQ RALDFYSIHV TKESTHPVKP LAQIAGNRYA SGPVGKALSD ACMGTIASFL 241 SKYQDIIIEH QKWKGNQKR LESLRELAGK ENLEYPSVTL PPQPHTKEGV DAYNEVIARV 301 RMWVNLNLWQ KLKLSRDDAK PLLRLKGFPS FPWERRENE VDWWNTINEV KKLIDAKRDM 361 GRVFWSGVTA EKRNTILEGY NYLPNENDHK KREGSLENPK KPAKRQFGDL LLYLEKKYAG 421 DWGKVFDEAW ERIDKKIAGL TSHIEREEAR NAEDAQSKAV LTDWLRAKAS FVLERLKEMD 481 EKEFYACEIQ LQKWYGDLRG NPFAVEAENR WDISGFSIG SDGHSIQYRN LLAWKYLENG 541 KREFYLLMNY GKKGRIRFTD GTDIKKSGKW QGLLYGGGKA KVIDLTFDPD DEQLIILPLA 601 FGTRQGREFI WNDLLSLETG LIKLANGRVI EKTIYNKKIG RDEPALFVAL TFERREWDP 661 SNIKPVNLIG VDRGENIPAV IALTDPEGCP LPEFKDSSGG PTDILRIGEG YKEKQRAIQA 721 AKEVEQRRAG GYSRKFASKS RNLADDMVRN SARDLFYHAV THDAVLVFEN LSRGFGRQGK 781 RTFMTERQYT KMEDWLTAKL AYEGLTSKTY LSKTLAQYTS KTCSNCGFTI TTADYDGMLV 841 RLKKTSDGWA TTLNNKELKA EGQITYYNRY KRQTVEKELS AELDRLSEES GNNDISKWTK 901 GRRDEALFLL KKRFSHRPVQ EQFVCLDCGH EVHADEQAAL NIARSWLFLN SNSTEFKSYK 961 SGKQPFVGAW QAFYKRRLKE VWKPNA (SEQ ID NO: 1).

[0234] In some cases, a reference CasX protein is isolated or derived from Planctomycetes. In some embodiments, a reference CasX protein comprises a sequence identical to a sequence of: 1 MQEIKRINKI RRRLVKDSNT KKAGKTGPMK TLLVRVMTPD LRERLENLRK KPENIPQPIS 61 NTSRANLNKL LTDYTEMKKA ILHVYWEEFQ KDPVGLMSRV AQPAPKNIDQ RKLIPVKDGN 121 ERLTSSGFAC SQCCQPLYVY KLEQVNDKGK PHTNYFGRCN VSEHERLILL SPHKPEANDE 181 LVTYSLGKFG QRALDFYSIH VTRESNHPVK PLEQIGGNSC ASGPVGKALS DACMGAVASF 241 LTKYQDIILE HQKVIKKNEK RLANLKDIAS ANGLAFPKIT LPPQPHTKEG IEAYNNWAQ 301 IVIWVNLNLW QKLKIGRDEA KPLQRLKGFP SFPLVERQAN EVDWWDMVCN VKKLINEKKE 361 DGKVFWQNLA GYKRQEALLP YLSSEEDRKK GKKFARYQFG DLLLHLEKKH GEDWGKVYDE 421 AWERIDKKVE GLSKHIKLEE ERRSEDAQSK AALTDWLRAK ASFVIEGLKE ADKDEFCRCE 481 LKLQKWYGDL RGKPFAIEAE NSILDISGFS KQYNCAFIWQ KDGVKKLNLY LIINYFKGGK 541 LRFKKIKPEA FEANRFYTVI NKKSGEIVPM EVNFNFDDPN LIILPLAFGK RQGREFIWND 601 LLSLETGSLK LANGRVIEKT LYNRRTRQDE PALFVALTFE RREVLDSSNI KPMNLIGIDR 661 GENIPAVIAL TDPEGCPLSR FKDSLGNPTH ILRIGESYKE KQRTIQAAKE VEQRRAGGYS 721 RKYASKAKNL ADDMVRNTAR DLLYYAVTQD AMLIFENLSR GFGRQGKRTF MAERQYTRME 781 DWLTAKLAYE GLPSKTYLSK TLAQYTSKTC SNCGFTITSA DYDRVLEKLK KTATGWMTTI 841 NGKELKVEGQ ITYYNRYKRQ NWKDLSVEL DRLSEESVNN DISSWTKGRS GEALSLLKKR 901 FSHRPVQEKF VCLNCGFETH ADEQAALNIA RSWLFLRSQE YKKYQTNKTT GNTDKRAFVE 961 TWQSFYRKKL KEVWKPAV (SEQ ID NO: 2).

[0235] In some cases, a reference CasX protein is isolated or derived from Candidates Sungbacteria. In some embodiments, a reference CasX protein comprises a sequence identical to a sequence of 1 MDNANKPSTK SLVNTTRISD HFGVTPGQVT RVFSFGIIPT KRQYAIIERW FAAVEAARER 61 LYGMLYAHFQ ENPPAYLKEK FSYETFFKGR PVLNGLRDID PTIMTSAVFT ALRHKAEGAM 121 AAFHTNHRRL FEEARKKMRE YAECLKANEA LLRGAADIDW DKIVNALRTR LNTCLAPEYD 181 AVIADFGALC AFRALIAETN ALKGAYNHAL NQMLPALVKV DEPEEAEESP RLRFFNGRIN 241 DLPKFPVAER ETPPDTETII RQLEDMARVI PDTAEILGYI HRIRHKAARR KPGSAVPLPQ 301 RVALYCAIRM ERNPEEDPST VAGHFLGEID RVCEKRRQGL VRTPFDSQIR ARYMDIISFR 361 ATLAHPDRWT EIQFLRSNAA SRRVRAETIS APFEGFSWTS NRTNPAPQYG MALAKDANAP 421 ADAPELCICL SPSSAAFSVR EKGGDLIYMR PTGGRRGKDN PGKEITWVPG SFDEYPASGV 481 ALKLRLYFGR SQARRMLTNK TWGLLSDNPR VFAANAELVG KKRNPQDRWK LFFHMVISGP 541 PPVEYLDFSS DVRSRARTVI GINRGEVNPL AYAWSVEDG QVLEEGLLGK KEYIDQLIET 601 RRRISEYQSR EQTPPRDLRQ RVRHLQDTVL GSARAKIHSL IAFWKGILAI ERLDDQFHGR 661 EQKIIPKKTY LANKTGFMNA LSFSGAVRVD KKGNPWGGMI EIYPGGISRT CTQCGTVWLA 721 RRPKNPGHRD AMWIPDIVD DAAATGFDNV DCDAGTVDYG ELFTLSREWV RLTPRYSRVM 781 RGTLGDLERA IRQGDDRKSR QMLELALEPQ PQWGQFFCHR CGFNGQSDVL AATNLARRAI 841 SLIRRLPDTD TPPTP (SEQ ID NO: 3). b. Class 2, Type V CasX Variant Proteins

[0236] The present disclosure provides Class 2, Type V, CasX variants of a reference CasX protein or variants derived from other CasX variants (interchangeably referred to herein as “Class 2, Type V CasX variant”, “CasX variant” or “CasX variant protein”), wherein the Class 2, Type V CasX variants comprise at least one modification in at least one domain relative to the reference CasX protein, including but not limited to the sequences of SEQ ID NOS: 1-3, or at least one modification relative to another CasX variant. Any change in amino acid sequence of a reference CasX protein or to another CasX variant protein that leads to an improved characteristic of the CasX protein is considered a CasX variant protein of the disclosure. For example, CasX variants can comprise one or more amino acid substitutions, insertions, deletions, or swapped domains, or any combinations thereof, relative to a reference CasX protein sequence.

[0237] The CasX variants of the disclosure have one or more improved characteristics compared to a reference CasX protein of SEQ ID NO: 1, SEQ ID NO:2 or SEQ ID NO:3, or the variant from which it was derived; e.g. CasX 491 (SEQ ID NO: 138) or CasX 515 (SEQ ID NO: 145). Exemplary improved characteristics of the CasX variant embodiments include, but are not limited to improved folding of the variant, increased binding affinity to the gRNA, increased binding affinity to the target nucleic acid, improved ability to utilize a greater spectrum of PAM sequences in the editing and / or binding of target nucleic acid, improved unwinding of the target DNA, increased editing activity, improved editing efficiency, improved editing specificity for the target nucleic acid, decreased off-target editing or cleavage, increased percentage of a eukaryotic genome that can be efficiently edited, increased activity of the nuclease, increased target strand loading for double strand cleavage, decreased target strand loading for single strand nicking, increased binding of the non-target strand of DNA, improved protein stability, improved protein:gRNA (RNP) complex stability, and improved fusion characteristics. In the foregoing embodiments, the one or more of the improved characteristics of the CasX variant is at least about 1.1 to about 100,000-fold improved relative to the reference CasX protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or CasX 491 (SEQ ID NO: 138) or CasX 515 (SEQ ID NO: 145), when assayed in a comparable fashion. In other embodiments, the improvement is at least about 1.1-fold, at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 50-fold, at least about 100-fold, at least about 500-fold, at least about 1000-fold, at least about 5000-fold, at least about 10,000-fold, or at least about 100,000-fold compared to the reference CasX protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or CasX 491 (SEQ ID NO: 138) or CasX 515 (SEQ ID NO: 145). when assayed in a comparable fashion. In other cases, the one or more improved characteristics of an RNP of the CasX variant and the gRNA variant are at least about 1.1, at least about 10, at least about 100, at least about 1000, at least about 10,000, at least about 100,000-fold or more improved relative to an RNP of the reference CasX protein of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3 and the gRNA of Table 1 or CasX 491 or CasX 515 with gRNA 174. In other cases, the one or more of the improved characteristics of an RNP of the CasX variant and the gRNA variant are about 1.1 to 100,00fold, about 1.1 to 10,00-fold, about 1.1 to 1,000-fold, about 1.1 to 500-fold, about 1.1 to 100fold, about 1.1 to 50-fold, about 1.1 to 20-fold, about 10 to 100,00-fold, about 10 to 10,00-fold, about 10 to 1,000-fold, about 10 to 500-fold, about 10 to 100-fold, about 10 to 50-fold, about 10 to 20-fold, about 2 to 70-fold, about 2 to 50-fold, about 2 to 30-fold, about 2 to 20-fold, about 2 to 10-fold, about 5 to 50-fold, about 5 to 30-fold, about 5 to 10-fold, about 100 to 100,00-fold, about 100 to 10,00-fold, about 100 to 1,000-fold, about 100 to 500-fold, about 500 to 100,00fold, about 500 to 10,00-fold, about 500 to 1,000-fold, about 500 to 750-fold, about 1,000 to 100,00-fold, about 10,000 to 100,00-fold, about 20 to 500-fold, about 20 to 250-fold, about 20 to 200-fold, about 20 to 100-fold, about 20 to 50-fold, about 50 to 10,000-fold, about 50 to 1,000fold, about 50 to 500-fold, about 50 to 200-fold, or about 50 to 100-fold, improved relative to an RNP of the reference CasX protein of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3 and the gRNA of Table 1, or CasX 491 or CasX 515 with gRNA 174, when assayed in a comparable fashion. In other cases, the one or more improved characteristics of an RNP of the CasX variant and the gRNA variant are about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 25-fold, 30-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 110-fold, 120fold, 130-fold, 140-fold, 150-fold, 160-fold, 170-fold, 180-fold, 190-fold, 200-fold, 210-fold, 220-fold, 230-fold, 240-fold, 250-fold, 260-fold, 270-fold, 280-fold, 290-fold, 300-fold, 310fold, 320-fold, 330-fold, 340-fold, 350-fold, 360-fold, 370-fold, 380-fold, 390-fold, 400-fold, 425-fold, 450-fold, 475-fold, or 500-fold improved relative to an RNP of the reference CasX protein of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3 and the gRNA of Table 1, or CasX 491 or CasX 515 with gRNA 174, when assayed in a comparable fashion.

[0238] In some embodiments, the modification of the CasX variant is a mutation in one or more amino acids of the reference CasX. In other embodiments, the modification is an insertion or substitution of a part or all of a domain from a different CasX protein. In a particular embodiment, the CasX variants of SEQ ID NOS: 144-160, 40208-40369, 40828-40912 have a NTSB and helical IB domain of SEQ ID NO: 1, while the other domains are derived from SEQ ID NO: 2, in addition to individual modifications in select domains, described herein. Mutations can be introduced in any one or more domains of the reference CasX protein or in a CasX variant to result in a CasX variant, and may include, for example, deletion of part or all of one or more domains, or one or more amino acid substitutions, deletions, or insertions in any domain of the reference CasX protein or the CasX variant from which it was derived. The domains of CasX proteins include the non-target strand binding (NTSB) domain, the target strand loading (TSL) domain, the Helical I domain, the Helical II domain, the oligonucleotide binding domain (OBD), and the RuvC DNA cleavage domain. Without being bound to theory or mechanism, a NTSB domain in a CasX allows for binding to the non-target nucleic acid strand and may aid in unwinding of the non-target and target strands. The NTSB domain is presumed to be responsible for the unwinding, or the capture, of a non-target nucleic acid strand in the unwound state. An exemplary NTSB domain comprises amino acids 100-190 of SEQ ID NO: 1 or amino acids 102191 of SEQ ID NO: 2. In some embodiments, the NTSB domain of a reference CasX protein comprises a four-stranded beta sheet. In some embodiments, the TSL acts to place or capture the target-strand in a folded state that places the scissile phosphate of the target strand DNA backbone in the RuvC active site. An exemplary TSL comprises amino acids 824-933 of SEQ ID NO: 1 or amino acids 811-920 of SEQ ID NO: 2. Without wishing to be bound by theory, it is thought that in some cases the Helical I domain may contribute to binding of the protospacer adjacent motif (PAM). In some embodiments, the Helical I domain of a reference CasX protein comprises one or more alpha helices. Exemplary Helical I-I and I-II domains comprise amino acids 56-99 and 191-331 of SEQ ID NO: 1, respectively, or amino acids 58-101 and 192-332 of SEQ ID NO: 2, respectively. The Helical II domain is responsible for binding to the guide RNA scaffold stem loop as well as the bound DNA. An exemplary Helical II domain comprises amino acids 332-508 of SEQ ID NO: 1, or amino acids 333-500 of SEQ ID NO: 2. The OBD largely binds the RNA triplex of the guide RNA scaffold. The OBD may also be responsible for binding to the protospacer adjacent motif (PAM). Exemplary OBD I and II domains comprise amino acids 1-55 and 509-659 of SEQ ID NO: 1, respectively, or amino acids 1-57 and 501-646 of SEQ ID NO: 2, respectively. The RuvC has a DED motif active site that is responsible for cleaving both strands of DNA (one by one, most likely the non-target strand first at 11-14 nucleotides (nt) into the targeted sequence and then the target strand next at 2-4 nucleotides after the target sequence, resulting in a staggered cut). Specifically in CasX, the RuvC domain is unique in that it is also responsible for binding the guide RNA scaffold stem loop that is critical for CasX function. Exemplary RuvC I and II domains comprise amino acids 660-823 and 934986 of SEQ ID NO: 1, respectively, or amino acids 647-810 and 921-978of SEQ ID NO: 2, respectively, while CasX variants may comprise mutations at positions 1658 and A708 relative to SEQ ID NO: 2, or the mutations of CasX 515, described below.

[0239] In some embodiments, the CasX variant protein comprises at least one modification in at least 1 domain, in at least each of 2 domains, in at least each of 3 domains, in at least each of 4 domains or in at least each of 5 domains of the reference CasX protein, including the sequences of SEQ ID NOS: 1-3. In some embodiments, the CasX variant protein comprises two or more modifications in at least one domain of the reference CasX protein. In some embodiments, the CasX variant protein comprises at least two modifications in at least one domain of the reference CasX protein, at least three modifications in at least one domain of the reference CasX protein or at least four or more modifications in at least one domain of the reference CasX protein. In some embodiments, wherein the CasX variant comprises two or more modifications compared to a reference CasX protein, and each modification is made in a domain independently selected from the group consisting of a NTSB, TSL, Helical I domain, Helical II domain, OBD, and RuvC DNA cleavage domain. In some embodiments, wherein the CasX variant comprises two or more modifications compared to a reference CasX protein, a modification is made in two or more domains. In some embodiments, the at least one modification of the CasX variant protein comprises a deletion of at least a portion of one domain of the reference CasX protein of SEQ ID NOS: 1-3. In some embodiments, the deletion is in the NTSB domain, TSL domain, Helical I domain, Helical II domain, OBD, or RuvC DNA cleavage domain.

[0240] In some cases, the CasX variants of the disclosure comprise modifications in structural regions that may encompass one or more domains. In some embodiments, a CasX variant comprises at least one modification of a region of non-contiguous amino acid residues of the CasX variant that form a channel in which gRNA:target nucleic acid complexing with the CasX variant occurs. In other embodiments, a CasX variant comprises at least one modification of a region of non-contiguous amino acid residues of the CasX variant that form an interface which binds with the gRNA. In other embodiments, a CasX variant comprises at least one modification of a region of non-contiguous amino acid residues of the CasX variant that form a channel which binds with the non-target strand DNA. In other embodiments, a CasX variant comprises at least one modification of a region of non-contiguous amino acid residues of the CasX variant that form an interface which binds with the protospacer adjacent motif (PAM) of the target nucleic acid. In other embodiments, a CasX variant comprises at least one modification of a region of non-contiguous surface-exposed amino acid residues of the CasX variant. In other embodiments, a CasX variant comprises at least one modification of a region of non-contiguous amino acid residues that form a core through hydrophobic packing in a domain of the CasX variant. In the foregoing embodiments of the paragraph, the modifications of the region can comprise one or more of a deletion, an insertion, or a substitution of one or more amino acids of the region; or between 2 to 15 amino acid residues of the region of the CasX variant are substituted with charged amino acids; or between 2 to 15 amino acid residues of a region of the CasX variant are substituted with polar amino acids; or between 2 to 15 amino acid residues of a region of the CasX variant are substituted with amino acids that stack, or have affinity with DNA or RNA bases.

[0241] In other embodiments, the disclosure provides CasX variants wherein the CasX variants comprise at least one modification relative to another CasX variant; e.g., CasX variant 515 and 527 is a variant of CasX variant 491 and CasX variants 668 and 672 are variants of CasX 535. In some embodiments, the at least one modification is selected from the group consisting of an amino acid insertion, deletion, or substitution. All variants that improve one or more functions or characteristics of the CasX variant protein when compared to a reference CasX protein or the variant from which it was derived described herein are envisaged as being within the scope of the disclosure. As described in the Examples, a CasX variant can be mutagenized to create another CasX variant. In a particular embodiment, the disclosure provides, in Example 21, Table 30, variants of CasX 515 (SEQ ID NO: 145) created by introducing modifications to the encoding sequence resulting in amino acid substitutions, deletions, or insertions at one or more positions in one or more domains.

[0242] Suitable mutagenesis methods for generating CasX variant proteins of the disclosure may include, for example, Deep Mutational Evolution (DME), deep mutational scanning (DMS), error prone PCR, cassette mutagenesis, random mutagenesis, staggered extension PCR, gene shuffling, or domain swapping (described in PCT / US20 / 36506 and WO2020247883A2, incorporated by reference herein). In some embodiments, the CasX variants are designed, for example by selecting multiple desired mutations in a CasX variant identified, for example, using the assays described in the Examples. In certain embodiments, the activity of a reference CasX or the CasX variant protein prior to mutagenesis is used as a benchmark against which the activity of one or more resulting CasX variants are compared, thereby measuring improvements in function of the new CasX variants.

[0243] In some embodiments of the CasX variants described herein, the at least one modification comprises: (a) a substitution of 1 to 100 consecutive or non-consecutive amino acids in the CasX variant compared to a reference CasX of SEQ ID NO: 1, SEQ ID NO:2, SEQ IDNO:3, CasX variant 491 (SEQ ID NO: 138) or CasX variant 515 (SEQ ID NO: 145); (b) a deletion of 1 to 100 consecutive or non-consecutive amino acids in the CasX variant compared to a reference CasX or the variant from which it was derived; (c) an insertion of 1 to 100 consecutive or non-consecutive amino acids in the CasX compared to a reference CasX or the variant from which it was derived; or (d) any combination of (a)-(c). In some embodiments, the at least one modification comprises: (a) a substitution of 1-10 consecutive or non-consecutive amino acids in the CasX variant compared to a reference CasX of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, or the variant from which it was derived; (b) a deletion of 1-5 consecutive or non-consecutive amino acids in the CasX variant compared to a reference CasX or the variant from which it was derived; (c) an insertion of 1-5 consecutive or non-consecutive amino acids in the CasX compared to a reference CasX or the variant from which it was derived; or (d) any combination of (a)-(c).

[0244] In some embodiments, the CasX variant protein comprises or consists of a sequence that has at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at lease 80, at least 90, or at least 100 alterations relative to the sequence of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, CasX 491 or CasX 515. In some embodiments, the CasX variant protein comprises one more substitutions relative to CasX 491, or SEQ ID NO: 138. In some embodiments, the CasX variant protein comprises one more substitutions relative to CasX 515, or SEQ ID NO: 145. These alterations can be amino acid insertions, deletions, substitutions, or any combinations thereof. The alterations can be in one domain or in any domain or any combination of domains of the CasX variant. Any amino acid can be substituted for any other amino acid in the substitutions described herein. The substitution can be a conservative substitution (e.g., a basic amino acid is substituted for another basic amino acid). The substitution can be a nonconservative substitution (e.g., a basic amino acid is substituted for an acidic amino acid or vice versa). For example, a proline in a reference CasX protein can be substituted for any of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine or valine to generate a CasX variant protein of the disclosure.

[0245] Any permutation of the substitution, insertion and deletion embodiments described herein can be combined to generate a CasX variant protein of the disclosure. For example, a CasX variant protein can comprise at least one substitution and at least one deletion relative to a reference CasX protein sequence or a sequence of CasX 491 or CasX 515, at least one substitution and at least one insertion relative to a reference CasX protein sequence or a sequence of CasX 491 or CasX 515, at least one insertion and at least one deletion relative to a reference CasX protein sequence or a sequence of CasX 491 or CasX 515, or at least one substitution, one insertion and one deletion relative to a reference CasX protein sequence or a sequence of CasX 491 or CasX 515.

[0246] In some embodiments, the CasX variant protein comprises between 400 and 2000 amino acids, between 500 and 1500 amino acids, between 700 and 1200 amino acids, between 800 and 1100 amino acids, or between 900 and 1000 amino acids.

[0247] In some embodiments, a CasX variant protein comprises a sequence of SEQ ID NOS: 49-160, 40208-40369, or 40828-40912 as set forth in Table 3. In some embodiments, a CasX variant protein consists of a sequence of SEQ ID NOS: 49-160, 40208-40369, or 40828-40912 as set forth in Table 3. In other embodiments, a CasX variant protein comprises a sequence at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical to a sequence of SEQ ID NOS: 49-160, 40208-40369, or 40828-40912 as set forth in Table 3. In some embodiments, a CasX variant protein comprises or consists of a sequence of SEQ ID NOS: 49-160, 40208-40369, or 4082840912 as set forth in Table 3. In other embodiments, a CasX variant protein comprises a sequence at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical to a sequence of SEQ ID NOS: 85-160, 40208-40369, or 40828-40912. In some embodiments, a CasX variant protein comprises or consists of a sequence of SEQ ID NOS: 85-160, 40208-40369, or 4082840912. c. CasX Variant Proteins with Domains from Multiple Source Proteins

[0248] In certain embodiments, the disclosure provides a chimeric CasX protein for use in the AAV systems comprising protein domains from two or more different CasX proteins, such as two or more reference CasX proteins, or two or more CasX variant protein sequences as described herein. As used herein, a “chimeric CasX protein” refers to a CasX containing at least two domains isolated or derived from different sources, such as two naturally occurring proteins, which may, in some embodiments, be isolated from different species. For example, in some embodiments, a chimeric CasX protein comprises a first domain from a first CasX protein and a second domain from a second, different CasX protein. In some embodiments, the first domain can be selected from the group consisting of the NTSB, TSL, Helical I, Helical II, OBD and RuvC domains. In some embodiments, the second domain is selected from the group consisting of the NTSB, TSL, Helical I, Helical II, OBD and RuvC domains with the second domain being different from the foregoing first domain. For example, a chimeric CasX protein may comprise an NTSB, TSL, Helical I, Helical II, OBD domains from a CasX protein of SEQ ID NO: 2, and a RuvC domain from a CasX protein of SEQ ID NO: 1, or vice versa. As a further example, a chimeric CasX protein may comprise an NTSB, TSL, Helical II, OBD and RuvC domain from CasX protein of SEQ ID NO: 2, and a Helical I domain from a CasX protein of SEQ ID NO: 1, or vice versa. Thus, in certain embodiments, a chimeric CasX protein may comprise an NTSB, TSL, Helical II, OBD and RuvC domain from a first CasX protein, and a Helical I domain from a second CasX protein. In some embodiments of the chimeric CasX proteins, the domains of the first CasX protein are derived from the sequences of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, and the domains of the second CasX protein are derived from the sequences of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, and the first and second CasX proteins are not the same. In some embodiments, domains of the first CasX protein comprise sequences derived from SEQ ID NO: 1 and domains of the second CasX protein comprise sequences derived from SEQ ID NO: 2 In some embodiments, domains of the first CasX protein comprise sequences derived from SEQ ID NO: 1 and domains of the second CasX protein comprise sequences derived from SEQ ID NO: 3. In some embodiments, domains of the first CasX protein comprise sequences derived from SEQ ID NO: 2 and domains of the second CasX protein comprise sequences derived from SEQ ID NO: 3.

[0249] In some embodiments, a CasX variant protein comprises at least one chimeric domain comprising a first part from a first CasX protein and a second part from a second, different CasX protein. As used herein, a “chimeric domain” refers to a single domain containing at least two parts isolated or derived from different sources, such as two naturally occurring proteins or portions of domains from two reference CasX proteins. The at least one chimeric domain can be any of the NTSB, TSL, Helical I, Helical II, OBD or RuvC domains as described herein. In some embodiments, the first portion of a CasX domain comprises a sequence of SEQ ID NO: 1 and the second portion of a CasX domain comprises a sequence of SEQ ID NO: 2. In some embodiments, the first portion of the CasX domain comprises a sequence of SEQ ID NO: 1 and the second portion of the CasX domain comprises a sequence of SEQ ID NO: 3. In some embodiments, the first portion of the CasX domain comprises a sequence of SEQ ID NO: 2 and the second portion of the CasX domain comprises a sequence of SEQ ID NO: 3. In some embodiments, the at least one chimeric domain comprises a chimeric RuvC domain. As an example of the foregoing, the chimeric RuvC domain comprises amino acids 661 to 824 of SEQ ID NO: 1 and amino acids 922 to 978 of SEQ ID NO: 2. As an alternative example of the foregoing, a chimeric RuvC domain comprises amino acids 648 to 812 of SEQ ID NO: 2 and amino acids 935 to 986 of SEQ ID NO: 1. In some embodiments, a CasX protein comprises a first domain from a first CasX protein and a second domain from a second CasX protein, and at least one chimeric domain comprising at least two parts isolated from different CasX proteins using the approach of the embodiments described in this paragraph.

[0250] In some embodiments, a CasX variant protein for use in the AAV systems comprises a sequence set forth in Table 3. In other embodiments, a CasX variant protein comprises a sequence at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical to a sequence selected from the group consisting of the sequences as set forth in Table 3. Table 3: CasX Variant Sequences SEQ ID NO Variant Description of Variant 49 ND TSL, Helical I, Helical II, OBD and RuvC domains from SEQ ID NO:2 and an NTSB domain from SEQ ID NO:1 50 ND NTSB, Helical I, Helical II, OBD and RuvC domains from SEQ ID NO:2 and a TSL domain from SEQ ID NO:1. 51 ND TSL, Helical I, Helical II, OBD and RuvC domains from SEQ ID NO: 1 and an NTSB domain from SEQ ID NO:2 52 ND NTSB, Helical I, Helical II, OBD and RuvC domains from SEQ ID NO:1 and an TSL domain from SEQ ID NO:2. 53 ND NTSB, TSL, Helical I, Helical II and OBD domains SEQ ID NO:2 and an exogenous RuvC domain or a portion thereof from a second CasX protein. 54 ND No description 55 ND NTSB, TSL, Helical II, OBD and RuvC domains from SEQ ID NO:2 and a Helical I domain from SEQ ID NO:1 56 ND NTSB, TSL, Helical I, OBD and RuvC domains from SEQ ID NO:2 and a Helical II domain from SEQ ID NO:1 57 ND NTSB, TSL, Helical I, Helical II and RuvC domains from a first CasX protein and an exogenous OBD or a part thereof from a second CasX protein 58 ND No description SEQ ID NO Variant Description of Variant 59 ND No description 60 ND substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of P at position 793 and a substitution of T620P of SEQ ID NO:2 61 ND substitution of M771A of SEQ ID NO:2. 62 ND substitution of L379R, a substitution of A708K, a deletion of P at position 793 and a substitution of D732N of SEQ ID NO:2. 63 ND substitution of W782Q of SEQ ID NO:2. 64 ND substitution of M771Q of SEQ ID NO:2 65 ND substitution of R458I and a substitution of A739V of SEQ ID NO:2. 66 ND L379R, a substitution of A708K, a deletion of P at position 793 and a substitution of M77 IN of SEQ ID NO:2 67 ND substitution of L379R, a substitution of A708K, a deletion of P at position 793 and a substitution of A739T of SEQ ID NO :2 68 ND substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of P at position 793 and a substitution of D489S of SEQ ID NO:2. 69 ND substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of P at position 793 and a substitution of D732N of SEQ ID NO:2. 70 ND substitution of V71 IK of SEQ ID NO:2. 71 ND substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of P at position 793 and a substitution of Y797L of SEQ ID NO:2. 72 119 ND 73 ND substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of P at position 793 and a substitution of M77 IN of SEQ ID NO:2. 74 ND substitution of A708K, a deletion of P at position 793 and a substitution of E386S of SEQ ID NO:2. 75 ND substitution of L379R, a substitution of C477K, a substitution of A708K and a deletion of P at position 793 of SEQ ID NO:2. 76 ND substitution of L792D of SEQ ID NO:2. 77 ND substitution of G791F of SEQ ID NO:2. 78 ND substitution of A708K, a deletion of P at position 793 and a substitution of A739V of SEQ ID NO:2. 79 ND substitution of L379R, a substitution of A708K, a deletion of P at position 793 and a substitution of A739V of SEQ ID NO:2. 80 ND substitution of C477K, a substitution of A708K and a deletion of P at position 793 of SEQ ID SEQ ID NO Variant Description of Variant NO:2. 81 ND substitution of L249I and a substitution of M771N of SEQ ID NO:2. 82 ND substitution of V747K of SEQ ID NO:2. 83 ND substitution of L379R, a substitution of C477K, a substitution of A708K, a deletion of P at position 793 and a substitution of M779N of SEQ ID NO:2. 84 ND L379R, F755M 85 429 ND 86 430 ND 87 431 ND 88 432 ND 89 433 ND 90 434 ND 91 435 ND 92 436 ND 93 437 ND 94 438 ND 95 439 ND 96 440 ND 97 441 ND 98 442 ND 99 443 ND 100 444 ND 101 445 ND 102 446 ND 103 447 ND 104 448 ND 105 449 ND SEQ ID NO Variant Description of Variant 106 450 ND 107 451 ND 108 452 ND 109 453 ND 110 454 ND 111 455 ND 112 456 ND 113 457 ND 114 458 ND 115 459 ND 116 460 ND 117 278 ND 118 279 ND 119 280 ND 120 285 ND 121 286 ND 122 287 ND 123 288 ND 124 290 ND 125 291 ND 126 293 ND 127 300 ND 128 492 ND 129 493 ND 130 387 ND 131 395 ND SEQ ID NO Variant Description of Variant 132 485 ND 133 486 ND 134 487 ND 135 488 ND 136 489 ND 137 490 ND 138 491 ND 139 494 ND 140 328 ND 141 388 ND 142 389 ND 143 390 ND 144 514 ND 145 515 ND 146 516 ND 147 517 ND 148 518 ND 149 519 ND 150 520 ND 151 522 ND 152 523 ND 153 524 ND 154 525 ND 155 526 ND 156 527 ND 157 528 ND SEQ ID NO Variant Description of Variant 158 529 ND 159 530 ND 160 531 ND 40208 532 ND 40209 533 ND 40210 534 ND 40211 535 ND 40212 536 ND 40213 537 ND 40214 538 ND 40215 539 ND 40216 540 ND 40217 541 ND 40218 542 ND 40219 543 ND 40220 544 ND 40221 545 ND 40222 546 ND 40223 547 ND 40224 548 ND 40225 550 ND 40226 551 ND 40227 552 ND 40228 553 ND 40229 554 ND 40230 555 ND SEQ ID NO Variant Description of Variant 40231 556 ND 40232 557 ND 40233 558 ND 40234 559 ND 40235 560 ND 40236 561 ND 40237 562 ND 40238 563 ND 40239 564 ND 40240 565 ND 40241 566 ND 40242 567 ND 40243 568 ND 40244 569 ND 40246 570 ND 40247 571 ND 40248 572 ND 40249 573 ND 40250 574 ND 40251 575 ND 40252 576 ND 40253 577 ND 40254 578 ND 40255 579 ND 40256 580 ND 40257 581 ND SEQ ID NO Variant Description of Variant 40258 582 ND 40259 583 ND 40260 584 ND 40261 585 ND 40262 586 ND 40263 587 ND 40264 588 ND 40265 589 ND 40266 590 ND 40267 591 ND 40268 592 ND 40269 593 ND 40270 594 ND 40271 595 ND 40272 596 ND 40273 597 ND 40274 598 ND 40275 599 ND 40276 600 ND 40277 601 ND 40278 602 ND 40279 603 ND 40280 604 ND 40281 605 ND 40282 606 ND 40283 607 ND SEQ ID NO Variant Description of Variant 40284 608 ND 40285 609 ND 40286 610 ND 40287 611 ND 40288 612 ND 40289 613 ND 40290 614 ND 40291 615 ND 40292 616 ND 40293 617 ND 40294 618 ND 40295 619 ND 40296 620 ND 40297 621 ND 40298 622 ND 40299 623 ND 40300 624 ND 40301 625 ND 40302 626 ND 40303 627 ND 40304 628 ND 40305 629 ND 40306 630 ND 40307 631 ND 40308 632 ND 40309 633 ND SEQ ID NO Variant Description of Variant 40310 634 ND 40311 635 ND 40312 636 ND 40313 637 ND 40314 638 ND 40315 639 ND 40316 640 ND 40317 641 ND 40318 642 ND 40319 643 ND 40320 644 ND 40321 645 ND 40322 646 ND 40323 647 ND 40324 648 ND 40325 649 ND 40326 650 ND 40327 651 ND 40328 652 ND 40329 653 ND 40330 654 ND 40331 655 ND 40332 656 ND 40333 657 ND 40334 658 ND 40335 659 ND SEQ ID NO Variant Description of Variant 40336 660 ND 40337 661 ND 40338 662 ND 40339 663 ND 40340 664 ND 40341 665 ND 40342 666 ND 40343 667 ND 40344 668 ND 40345 669 ND 40346 671 ND 40347 672 ND 40348 673 ND 40349 674 ND 40350 675 ND 40351 676 ND 40352 677 ND 40353 678 ND 40354 679 ND 40355 680 ND 40356 681 ND 40357 682 ND 40358 683 ND 40359 684 ND 40360 685 ND 40361 686 ND SEQ ID NO Variant Description of Variant 40362 687 ND 40363 688 ND 40364 689 ND 40365 690 ND 40366 691 ND 40367 692 ND 40368 693 ND 40369 694 ND 40828 701 ND 40829 702 ND 40830 703 ND 40831 704 ND 40832 705 ND 40833 706 ND 40834 707 ND 40835 708 ND 40836 709 ND 40837 710 ND 40838 711 ND 40839 712 ND 40840 713 ND 40841 714 ND 40842 715 ND 40843 716 ND 40844 717 ND 40845 718 ND SEQ ID NO Variant Description of Variant 40846 719 ND 40847 720 ND 40848 721 ND 40849 722 ND 40850 723 ND 40851 724 ND 40852 725 ND 40853 726 ND 40854 727 ND 40855 728 ND 40856 729 ND 40857 730 ND 40858 731 ND 40859 732 ND 40860 733 ND 40861 734 ND 40862 735 ND 40863 736 ND 40864 737 ND 40865 738 ND 40866 739 ND 40867 740 ND 40868 741 ND 40869 742 ND 40870 743 ND 40871 744 ND SEQ ID NO Variant Description of Variant 40872 745 ND 40873 746 ND 40874 747 ND 40875 748 ND 40876 749 ND 40877 750 ND 40878 751 ND 40879 752 ND 40880 753 ND 40881 754 ND 40882 755 ND 40883 756 ND 40884 757 ND 40885 758 ND 40886 759 ND 40887 760 ND 40888 761 ND 40889 762 ND 40890 763 ND 40891 764 ND 40892 765 ND 40893 766 ND 40894 767 ND 40895 768 ND 40896 769 ND 40897 770 ND SEQ ID NO Variant Description of Variant 40898 777 ND 40899 778 ND 40900 779 ND 40901 780 ND 40902 781 ND 40903 782 ND 40904 783 ND 40905 784 ND 40906 785 ND 40907 786 ND 40908 787 ND 40909 788 ND 40910 789 ND 40911 790 ND 40912 791 ND d. Protein Affinity for the gRNA

[0251] In some embodiments, a CasX variant protein for use in the AAV systems of the disclosure has improved affinity for the gRNA relative to a reference CasX protein, leading to the formation of the ribonucleoprotein complex. Increased affinity of the CasX variant protein for the gRNA may, for example, result in a lower Ka for the generation of a RNP complex, which can, in some cases, result in a more stable ribonucleoprotein complex formation. In some embodiments, increased affinity of the CasX variant protein for the gRNA results in increased stability of the ribonucleoprotein complex when delivered to human cells. This increased stability can affect the function and utility of the complex in the cells of a subject, as well as result in improved pharmacokinetic properties in blood, when delivered to a subject. In some embodiments, increased affinity of the CasX variant protein, and the resulting increased stability of the ribonucleoprotein complex, allows for a lower dose of the CasX variant protein to be delivered to the subject or cells while still having the desired activity, for example in vivo or in vitro gene editing. In some embodiments, a higher affinity (tighter binding) of a CasX variant protein to a gRNA allows for a greater amount of editing events when both the CasX variant protein and the gRNA remain in an RNP complex. Increased editing events can be assessed using editing assays such as the EGFP disruption assay described herein.

[0252] In some embodiments, the Ka of a CasX variant protein for a gRNA is increased relative to a reference CasX protein by a factor of at least about 1.1, at least about 1.2, at least about 1.3, at least about 1.4, at least about 1.5, at least about 1.6, at least about 1.7, at least about 1.8, at least about 1.9, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, or at least about 100. In some embodiments, the CasX variant has about 1.1 to about 10-fold increased binding affinity to the gRNA compared to the reference CasX protein of SEQ ID NO: 2.

[0253] In some embodiments, increased affinity of the CasX variant protein for the gRNA results in increased stability of the ribonucleoprotein complex when delivered to mammalian cells, including in vivo delivery to a subject. This increased stability can affect the function and utility of the complex in the cells of a subject, as well as result in improved pharmacokinetic properties in blood, when delivered to a subject. In some embodiments, increased affinity of the CasX variant protein, and the resulting increased stability of the ribonucleoprotein complex, allows for a lower dose of the CasX variant protein to be delivered to the subject or cells while still having the desired activity; for example in vivo or in vitro gene editing. The increased ability to form RNP and keep them in stable form can be assessed using assays such as the in vitro cleavage assays described in the Examples herein. In some embodiments, RNP comprising the CasX variants of the disclosure are able to achieve a kcieave rate when complexed as an RNP that is at last 2-fold, at least 5-fold, or at least 10-fold higher compared to RNP comprising a reference CasX of SEQ ID NOS: 1-3.

[0254] In some embodiments, a higher affinity (tighter binding) of a CasX variant protein to a gRNA allows for a greater amount of editing events when both the CasX variant protein and the gRNA remain in an RNP complex. Increased editing events can be assessed using editing assays such as the assays described herein.

[0255] Without wishing to be bound by theory, in some embodiments amino acid changes in the Helical I domain can increase the binding affinity of the CasX variant protein with the gRNA targeting sequence, while changes in the Helical II domain can increase the binding affinity of the CasX variant protein with the gRNA scaffold stem loop, and changes in the oligonucleotide binding domain (OBD) increase the binding affinity of the CasX variant protein with the gRNA triplex.

[0256] Methods of measuring CasX protein binding affinity for a gRNA include in vitro methods using purified CasX protein and gRNA. The binding affinity for reference CasX and variant proteins can be measured by fluorescence polarization if the gRNA or CasX protein is tagged with a fluorophore. Alternatively, or in addition, binding affinity can be measured by biolayer interferometry, electrophoretic mobility shift assays (EMSAs), or filter binding. Additional standard techniques to quantify absolute affinities of RNA binding proteins such as the reference CasX and variant proteins of the disclosure for specific gRNAs such as reference gRNAs and variants thereof include, but are not limited to, isothermal calorimetry (ITC), and surface plasmon resonance (SPR), as well as the methods of the Examples. e. Affinity for Target Nucleic Acid

[0257] In some embodiments, a CasX variant protein for use in the AAV systems of the disclosure has improved binding affinity for a target nucleic acid sequence relative to the affinity of a reference CasX protein for a target nucleic acid sequence. CasX variants with higher affinity for their target nucleic acid may, in some embodiments, cleave the target nucleic acid sequence more rapidly than a reference CasX protein that does not have increased affinity for the target nucleic acid. In some embodiments, the improved affinity for the target nucleic acid sequence comprises improved affinity for the target nucleic acid sequence, improved binding affinity to a wider spectrum of PAM sequences, an improved ability to search DNA for the target nucleic acid sequence, or any combinations thereof. Without wishing to be bound by theory, it is thought that CRISPR / Cas system proteins such as CasX may find their target nucleic acid sequences by one-dimension diffusion along a DNA molecule. The process is thought to include (1) binding of the ribonucleoprotein to the DNA molecule followed by (2) stalling at the target nucleic acid sequence, either of which may be, in some embodiments, affected by improved affinity of CasX proteins for a target nucleic acid sequence, thereby improving function of the CasX variant protein compared to a reference CasX protein.

[0258] In some embodiments, a CasX variant protein for use in the AAV systems has improved binding affinity for the non-target strand of the target nucleic acid. As used herein, the term “non-target strand” refers to the strand of the DNA target nucleic acid sequence that does not form Watson and Crick base pairs with the targeting sequence in the gRNA, and is complementary to the target strand. In some embodiments, the CasX variant protein has about 1.1 to about 100-fold increased binding affinity to the non-target stand of the target nucleic acid compared to the reference protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or to the CasX variants 119 (SEQ ID NO: 72) and CasX 491 (SEQ ID NO: 138).

[0259] Methods of measuring CasX protein (such as reference or variant) affinity for a target nucleic acid molecule may include electrophoretic mobility shift assays (EMSAs), filter binding, isothermal calorimetry (ITC), and surface plasmon resonance (SPR), fluorescence polarization and biolayer interferometry (BLI). Further methods of measuring CasX protein affinity for a target include in vitro biochemical assays that measure DNA cleavage events over time.

[0260] In some embodiments, the CasX variant protein for use in the AAV systems is catalytically dead (dCasX). In some embodiments, the disclosure provides RNP comprising a catalytically-dead CasX protein that retains the ability to bind target DNA. An exemplary catalytically-dead CasX variant protein comprises one or more mutations in the active site of the RuvC domain of the CasX protein. In some embodiments, a catalytically-dead CasX variant protein comprises substitutions at residues 672, 769 and / or 935 of SEQ ID NO: 1. In some embodiments, a catalytically-dead CasX variant protein comprises substitutions of D672A, E769A and / or D935A in the reference CasX protein of SEQ ID NO: 1. In some embodiments, a catalytically-dead CasX protein comprises substitutions at amino acids 659, 765 and / or 922 of SEQ ID NO: 2. In some embodiments, a catalytically-dead CasX protein comprises D659A, E756A and / or D922A substitutions in a reference CasX protein of SEQ ID NO: 2. In further embodiments, a catalytically-dead reference CasX protein comprises deletions of all or part of the RuvC domain of the reference CasX protein. Exemplary dCasX sequences are provided as SEQ ID NOS: 40808-40827, 41006-41009 in Table 7.

[0261] In some embodiments, improved affinity for DNA of a CasX variant protein also improves the function of catalytically inactive versions of the CasX variant protein. In some embodiments, the catalytically inactive version of the CasX variant protein comprises one or mutations in the DED motif in the RuvC. Catalytically dead CasX variant proteins can, in some embodiments, be used for base editing or epigenetic modifications. With a higher affinity for DNA, in some embodiments, catalytically-dead CasX variant proteins can, relative to catalytically active CasX, find their target DNA faster, remain bound to target DNA for longer periods of time, bind target DNA in a more stable fashion, or a combination thereof, thereby improving the function of the catalytically-dead CasX variant protein. f. Improved Specificity for a Target Site

[0262] In some embodiments, a CasX variant protein for use in the AAV systems has improved specificity for a target nucleic acid sequence relative to a reference CasX protein. As used herein, “specificity,” interchangeably referred to as “target specificity,” refers to the degree to which a CRISPR / Cas system ribonucleoprotein complex cleaves off-target sequences that are similar, but not identical to the target nucleic acid sequence; e.g., a CasX variant RNP with a higher degree of specificity would exhibit reduced off-target cleavage of sequences relative to a reference CasX protein. The specificity, and the reduction of potentially deleterious off-target effects, of CRISPR / Cas system proteins can be vitally important in order to achieve an acceptable therapeutic index for use in mammalian subjects.

[0263] Without wishing to be bound by theory, it is possible that amino acid changes in the Helical I and II domains that increase the specificity of the CasX variant protein for the target nucleic acid strand can increase the specificity of the CasX variant protein for the target nucleic acid sequence overall. In some embodiments, amino acid changes that increase specificity of CasX variant proteins for target nucleic acid sequence may also result in decreased affinity of CasX variant proteins for DNA.

[0264] Methods of testing CasX protein (such as variant or reference) target specificity may include guide and Circularization for In vitro Reporting of Cleavage Effects by Sequencing (CIRCLE-seq), or similar methods. In brief, in CIRCLE-seq techniques, genomic DNA is sheared and circularized by ligation of stem-loop adapters, which are nicked in the stem-loop regions to expose 4 nucleotide palindromic overhangs. This is followed by intramolecular ligation and degradation of remaining linear DNA. Circular DNA molecules containing a CasX cleavage site are subsequently linearized with CasX, and adapter adapters are ligated to the exposed ends followed by high-throughput sequencing to generate paired end reads that contain information about the off-target site. Additional assays that can be used to detect off-target events, and therefore CasX protein specificity include assays used to detect and quantify indels (insertions and deletions) formed at those selected off-target sites such as mismatch-detection nuclease assays and next generation sequencing (NGS). Exemplary mismatch-detection assays include nuclease assays, in which genomic DNA from cells treated with CasX and sgRNA is PCR amplified, denatured and rehybridized to form hetero-duplex DNA, containing one wild type strand and one strand with an indel. Mismatches are recognized and cleaved by mismatch detection nucleases, such as Surveyor nuclease or T7 endonuclease I. g. Protospacer and PAM Sequences

[0265] Herein, the protospacer is defined as the DNA sequence complementary to the targeting sequence of the guide RNA and the DNA complementary to that sequence, referred to as the target strand and non-target strand, respectively. As used herein, the PAM is a nucleotide sequence proximal to the protospacer that, in conjunction with the targeting sequence of the gRNA, helps the orientation and positioning of the CasX for the potential cleavage of the protospacer strand(s).

[0266] PAM sequences may be degenerate, and specific RNP constructs may have different preferred and tolerated PAM sequences that support different efficiencies of cleavage. Following convention, unless stated otherwise, the disclosure refers to both the PAM and the protospacer sequence and their directionality according to the orientation of the non-target strand. This does not imply that the PAM sequence of the non-target strand, rather than the target strand, is determinative of cleavage or mechanistically involved in target recognition. For example, when reference is to a TTC PAM, it may in fact be the complementary GAA sequence that is required for target cleavage, or it may be some combination of nucleotides from both strands. In the case of the CasX proteins disclosed herein, the PAM is located 5’ of the protospacer with a single nucleotide separating the PAM from the first nucleotide of the protospacer. Thus, in the case of reference CasX, in which the canonical PAM is TTC, the PAM should be understood to mean a sequence following the formula 5’-.. ,NNTTCN(protospacer)NNNNNN.. .3’ where ‘N’ is any DNA nucleotide and ‘(protospacer)’ is a DNA sequence having identity with the targeting sequence of the guide RNA. In the case of a CasX variant with expanded PAM recognition, a TTC, CTC, GTC, or ATC PAM should be understood to mean a sequence following the formulae: 5’-.. ,NNTTCN(protospacer)NNNNNN... 3’; 5’-.. ,NNCTCN(protospacer)NNNNNN... 3’; 5’-..,NNGTCN(protospacer)NNNNNN...3’; or 5’-.. ,NNATCN(protospacer)NNNNNN.. .3’. Alternatively, a TC PAM should be understood to mean a sequence following the formula 5’-.. ,NNNTCN(protospacer)NNNNNN.. .3’.

[0267] Additionally, the CasX variant proteins of the disclosure have an enhanced ability to efficiently edit and / or bind target nucleic acid, when complexed with a gRNA as an RNP, utilizing a PAM TC motif, including PAM sequences selected from TTC, ATC, GTC, or CTC, (in a 5’ to 3’ orientation), compared to an RNP of a reference CasX protein and reference gRNA, or to an RNP of another CasX variant from which it was derived, such as CasX 491, and gRNA 174. In the foregoing, the PAM sequence is located at least 1 nucleotide 5’ to the non-target strand of the protospacer having identity with the targeting sequence of the gRNA in an assay system compared to the editing efficiency and / or binding of an RNP comprising a reference CasX protein and reference gRNA in a comparable assay system. In one embodiment, an RNP of a CasX variant and gRNA variant exhibits greater editing efficiency and / or binding of a target sequence in the target nucleic acid compared to an RNP comprising a reference CasX protein and a reference gRNA (or an RNP of another CasX variant from which it was derived, such as CasX 491, and gRNA 174) in a comparable assay system, wherein the PAM sequence of the target DNA is TTC. In another embodiment, an RNP of a CasX variant and gRNA variant exhibits greater editing efficiency and / or binding of a target sequence in the target nucleic acid compared to an RNP comprising a reference CasX protein and a reference gRNA (or an RNP of another CasX variant from which it was derived, such as CasX 491 and gRNA 174) in a comparable assay system, wherein the PAM sequence of the target DNA is ATC. In a particular embodiment of the foregoing, wherein the CasX variant exhibits enhanced editing with an ATC PAM, the CasX variant is 528 (SEQ ID NO: 157). In another embodiment, an RNP of a CasX variant and gRNA variant exhibits greater editing efficiency and / or binding of a target sequence in the target nucleic acid compared to an RNP comprising a reference CasX protein and a reference gRNA (or an RNP of another CasX variant from which it was derived, such as CasX 491, and gRNA 174) in a comparable assay system, wherein the PAM sequence of the target DNA is CTC. In another embodiment, an RNP of a CasX variant and gRNA variant exhibits greater editing efficiency and / or binding of a target sequence in the target nucleic acid compared to an RNP comprising a reference CasX protein and a reference gRNA (or an RNP of another CasX variant from which it was derived and gRNA 174) in a comparable assay system, wherein the PAM sequence of the target DNA is GTC. In the foregoing embodiments, the increased editing efficiency and / or binding affinity for the one or more PAM sequences is at least 1.5-fold, at least 2-fold, at least 4-fold, at least 10-fold, at least 20-fold, at least 30-fold, or at least 40-fold greater or more compared to the editing efficiency and / or binding affinity of an RNP of any one of the CasX proteins of SEQ ID NOS: 1-3 and the gRNA comprising a sequence of Table 1 for the PAM sequences. Exemplary assays demonstrating the improved editing are described herein, in the Examples. In some embodiments, a CasX protein can bind and / or modify (e.g., cleave, nick, methylate, demethylate, etc.) a target nucleic acid and / or a polypeptide associated with target nucleic acid (e.g., methylation or acetylation of a histone tail). In some embodiments, the CasX protein is catalytically-dead (dCasX) but retains the ability to bind a target nucleic acid. h. Affinity for Target RNA

[0268] In some embodiments, variants of a reference CasX protein for use in the AAV systems of the disclosure have increased specificity for a target RNA, and increased the activity with respect to a target RNA when compared to the reference CasX protein. For example, CasX variant proteins can display increased binding affinity for target RNAs, or increased cleavage of target RNAs, when compared to reference CasX proteins. In some embodiments, a ribonucleoprotein complex comprising a CasX variant protein binds to a target RNA and / or cleaves the target RNA. In some embodiments, a CasX variant has at least about two-fold to about 10-fold increased binding affinity to the target RNA compared to the reference protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. i. CasX Variant Proteins with Domains from Multiple Source Proteins

[0269] In some embodiments, the disclosure provides AAV encoding a chimeric CasX variant protein comprising protein domains from two or more different CasX proteins, such as two or more naturally occurring CasX proteins, or two or more CasX variant protein sequences as described herein. As used herein, a “chimeric CasX protein” refers to a CasX containing at least two domains isolated or derived from different sources, such as two naturally occurring proteins, which may, in some embodiments, be isolated from different species. For example, in some embodiments, a chimeric CasX protein comprises a first domain from a first CasX protein and a second domain from a second, different CasX protein. In some embodiments, the first domain can be selected from the group consisting of the NTSB, TSL, helical I, helical II, OBD and RuvC domains. In some embodiments, the second domain is selected from the group consisting of the NTSB, TSL, helical I, helical II, OBD and RuvC domains with the second domain being different from the foregoing first domain. For example, a chimeric CasX protein may comprise an NTSB, TSL, helical I, helical II, OBD domains from a CasX protein of SEQ ID NO: 2, and a RuvC domain from a CasX protein of SEQ ID NO: 1, or vice versa. As a further example, a chimeric CasX protein may comprise an NTSB, TSL, helical II, OBD and RuvC domain from CasX protein of SEQ ID NO: 2, and a helical I domain from a CasX protein of SEQ ID NO: 1, or vice versa. Thus, in certain embodiments, a chimeric CasX protein may comprise an NTSB, TSL, helical II, OBD and RuvC domain from a first CasX protein, and a helical I domain from a second CasX protein. In some embodiments of the chimeric CasX proteins, the domains of the first CasX protein are derived from the sequences of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, and the domains of the second CasX protein are derived from the sequences of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, and the first and second CasX proteins are not the same. In some embodiments, domains of the first CasX protein comprise sequences derived from SEQ ID NO: 1 and domains of the second CasX protein comprise sequences derived from SEQ ID NO: 2. In some embodiments, domains of the first CasX protein comprise sequences derived from SEQ ID NO: 1 and domains of the second CasX protein comprise sequences derived from SEQ ID NO: 3. In some embodiments, domains of the first CasX protein comprise sequences derived from SEQ ID NO: 2 and domains of the second CasX protein comprise sequences derived from SEQ ID NO: 3. As an example of the foregoing, the chimeric RuvC domain comprises amino acids 660 to 823 of SEQ ID NO: 1 and amino acids 921 to 978 of SEQ ID NO: 2. As an alternative example of the foregoing, a chimeric RuvC domain comprises amino acids 647 to 810 of SEQ ID NO: 2 and amino acids 934 to 986 of SEQ ID NO: 1. In some embodiments, the at least one chimeric domain comprises a chimeric helical I domain wherein the chimeric helical I domain comprises amino acids 56-99 of SEQ ID NO: 1 and amino acids 192-332 of SEQ ID NO: 2. In some embodiments, the chimeric CasX variant is further modified, including the CasX variants selected from the group consisting of the sequences of SEQ ID NO: 40959, SEQ ID NO: 40960, SEQ ID NO: 40968, SEQ ID NO: 40977, SEQ ID NO: 40969, SEQ ID NO: 40970, SEQ ID NO: 40971, SEQ ID NO: 40972, SEQ ID NO: 40973, SEQ ID NO: 40961, SEQ ID NO: 40978, SEQ ID NO: 40962, SEQ ID NO: 40979, SEQ ID NO: 40963, SEQ ID NO: 40980, SEQ ID NO: 40964, SEQ ID NO: 40981, SEQ ID NO: 40965, SEQ ID NO: 40982, SEQ ID NO: 40966, SEQ ID NO: 40983, SEQ ID NO: 40967, SEQ ID NO: 40974, SEQ ID NO: 40975, SEQ ID NO: 40976, SEQ ID NO: 40984, and SEQ ID NO: 40985. In some embodiments, the one or more additional modifications comprises an insertion, substitution or deletion as described herein.

[0270] In the case of split or non-contiguous domains such as helical I, RuvC and OBD, a portion of the non-contiguous domain can be replaced with the corresponding portion from any other source. For example, the helical I-I domain (sometimes referred to as helical I-a) in SEQ ID NO: 2 can be replaced with the corresponding helical I-I sequence from SEQ ID NO: 1, and the like. Domain sequences from reference CasX proteins, and their coordinates, are shown in Table 4. Representative examples of chimeric CasX proteins include the variants of CasX 472483, 485-491 and 515, the sequences of which are set forth in Table 3. Table 4. Domain coordinates in Reference CasX proteins Domain Name Coordinates in SEQ ID NO: 1 Coordinates in SEQ ID NO: 2 OBD-I 1-55 1-57 helical I-I 56-99 58-101 NTSB 100-190 102-191 helical I-II 191-331 192-332 helical II 332-508 333-500 OBD-II 509-659 501-646 RuvC-I 660-823 647-810 TSL 824-933 811-920 RuvC-II 934-986 921-978 *OBD I and II, helical I-I and I-II, and RuvC I and II are also referred to herein as OBD a and b, helical I a and b, and RuvC a and b.

[0271] Exemplary domain sequences are provided in Table 5 below. Table 5. Exemplary Domain Sequences Deltaproteobacter sp. (reference CasX of SEQ ID NO: 1) SEQ ID Domain Sequence 40986 OBD-I EKRINKIRKKLSADNATKPVSRSGPMKTLLVRVMTDDLKKRLEKRRKKPEVMPQ 40987 helical I-I VISNNAANNLRMLLDDYTKMKEAILQVYWQEFKDDHVGLMCKFA 40988 NTSB QPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNY FGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQ 40989 helical I II RALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKYQD IHEHQKWKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEVIARVR MWVNLNLWQ KLKLSRDDAKPLLRLKGFPSF 40990 helical II PVVERRENEVDWWNTINEVKKLIDAKRDMGRVFWSGVTAEKRNTILEGYNYLP NENDHKKREGSLENPKKPAKRQFGDLLLYLEKKYAGDWGKVFDEAWERIDKKI AGLTSHIEREEARNAEDAQSKAVLTDWLRAKASFVLERLKEMDEKEFYACEIQL QKWYGDLRG NPFAVEAE 40991 OBD-II NRWDISGFSIGSDGHSIQYRNLLAWKYLENGKREFYLLMNYGKKGRIRFTDGTD IKKSGKWQGLLYGGGKAKVIDLTFDPDDEQLIILPLAFGTRQGREFIWNDLLSLET GLIKLANGRVIEKTIYNKKIG RDEPALFVALTFERREWD 40992 RuvC-I PSNIKPVNLIGVDRGENIPAVIALTDPEGCPLPEFKDSSGGPTDILRIGEGYKEKQR AIQAAKEVEQRRAGGYSRKFASKSRNLADDMVRNSARDLFYHAVTHDAVLVFE NLSRGFGRQGKRTFMTERQYTKMEDWLTAKLAYEGLTSKTYLSKTLAQYTSKT C 40993 TSL SNCGFTITTADYDGMLVRLKKTSDGWATTLNNKELKAEGQITYYNRYKRQTVE KELSAELDRLSEESGNNDISKWTKGRRDEALFLLKKRFSHRPVQEQFVCLDCGHE VH 40994 RuvC-II ADEQAALNIARSWLFLN SNSTEFKSYKSGKQPFVGAWQAFYKRRLKEVWKPNA Planctomycetes sp. (Reference CasX of SEQ ID NO: 2) SEQ ID Domain Sequence 40995 OBD-I QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIP Q 40996 helical I II PISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVA 40997 NTSB QPAPKNIDQRKLIPVKDGNERLTSSGFACSQCCQPLYVYKLEQVNDKGKPHTNYF GRCNVSEHERLILLSPHKPEANDELVTYSLGKFGQ 40998 helical I II RALDFYSIHVTRESNHPVKPLEQIGGNSCASGPVGKALSDACMGAVASFLTKYQ DIILEHQKVIKKNEKRLANLKDIASANGLAFPKITLPPQPHTKEGIEAYNNWAQI VIWVNLNLWQKLKIGRDEAKPLQRLKGFPSF 40999 helical II PLVERQANEVDWWDMVCNVKKLINEKKEDGKVFWQNLAGYKRQEALLPYLSS EEDRKKGKKFARYQFGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLSKHIK LEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQKWYGDLR GKPFAIEAE 41000 OBD-II NSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRF YTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLK LANGRVIEKTLYNRRTRQDEPALFVALTFERREVLD 41001 RuvC-I SSNIKPMNLIGIDRGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEKQRT IQAAKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDAMLIFEN LSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLPSKTYLSKTLAQYTSKTC 41002 TSL SNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYNRYKRQNWK DLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFET H 41003 RuvC-II ADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEV WKPAV

[0272] A further exemplary helical II domain sequence is provided as SEQ ID NO: 41004, and a further exemplary RuvC a domain sequence is provided as SEQ ID NO: 41005.

[0273] In other embodiments, a CasX variant protein comprises a sequence of SEQ ID NOS: 49160, 40208-40286, or 40828-40912 as set forth in Table 3, and further comprises one or more NLS disclosed herein at or near either the N-terminus, the C-terminus, or both. In other embodiments, a CasX variant protein comprises a sequence of SEQ ID NOS: 72-160, 4020840286, or 40828-40912, and further comprises one or more NLS disclosed herein at or near either the N-terminus, the C-terminus, or both. In other embodiments, a CasX variant protein comprises a sequence of SEQ ID NOS: 144-160, 40208-40286, or 40828-40912, and further comprises one or more NLS disclosed herein at or near either the N-terminus, the C-terminus, or both. It will be understood that in some cases, the N-terminal methionine of the CasX variants of the Tables is removed from the expressed CasX variant during post-translational modification. The person of ordinary skill in the art will understand that an NLS near the N or C terminus of a protein can be within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20 or 20 amino acids of the N or C terminus. j. CasX variants derived from other CasX variants

[0274] In further iterations of the generation of variant proteins, a variant protein can be utilized to generate additional CasX variants of the disclosure. For example, CasX 119 (SEQ ID NO: 72), CasX 491 (SEQ ID NO: 138), and CasX 515 (SEQ ID NO: 145) are exemplary variant proteins that are modified to generate additional CasX variants of the disclosure having improvements or additional properties relative to a reference CasX or CasX variants from which they were derived. CasX 119 contains a substitution of L379R, a substitution of A708K and a deletion of P at position 793 of SEQ ID NO: 2. CasX 491 contains NTSB and Helical IB swap from SEQ ID NO: 1. CasX 515 was derived from CasX 491 by insertion of P at position 793 (relative to SEQ ID NO:2) and was used to create the CasX variants described in Example 21. For example, CasX 668 has an insertion of R at position 26 and a substitution of G223S relative to CasX 515. CasX 672 has substitutions of L169K and G223S relative to CasX 515. CasX 676 has substitutions of L169K and G223S and an insertion of R at position 26 relative to CasX 515.

[0275] Exemplary methods used to generate and evaluate CasX variants derived from other CasX variants are described in the Examples, which were created by introducing modifications to the encoding sequence resulting in amino acid substitutions, deletions, or insertions at one or more positions in one or more domains of the CasX variant. In particular, Example 21 describes the methods used to create variants of CasX 515 (SEQ ID NO: 145) that were then assayed to determine those positions in the sequence that, when modified by an amino acid insertion, deletion or substitution, resulted in an enrichment or improvement in the assays. For purposes of the disclosure, the sequences of the domains of CasX 515 are provided in Table 6 and include an OBD-I domain having the sequence of SEQ ID NO: 40995, an OBD-II domain having the sequence of SEQ ID NO: 41000, NTSB domain having the sequence of SEQ ID NO: 40988, a helical I-I domain having the sequence of SEQ ID NO: 40996, a helical I-II domain having the sequence of SEQ ID NO: 40989, a helical II domain having the sequence of SEQ ID NO: 41004, a RuvC-I domain having the sequence of SEQ ID NO: 41005, a RuvC-II domain having the sequence of SEQ ID NO: 41003, and a TSL domain having the sequence of SEQ ID NO: 41002. By the methods of the disclosure, individual positions in the domains of CasX 515 were modified, assayed, and the resulting positions and exemplary modifications leading to an enrichment or improvement that follow are provided, relative to their position in each domain or subdomain. In some cases, such positions are disclosed in Tables 30-33 of the Examples. In some embodiments, the disclosure provides CasX variants derived from CasX 515 comprising one or more modifications (i.e., an insertion, a deletion, or a substitution) at one or more amino acid positions in the NTSB domain relative to the NTSB domain sequence (SEQ ID NO: 40988) selected from the group consisting of P2, S4, Q9, E15, G20, G33, L41, Y51, F55, L68, A70, E75, K88, and G90, wherein the modification results in an improved characteristic relative to CasX 515. In a particular embodiment, the one or more modifications at one or more amino acid positions in the NTSB domain relative to the NTSB domain sequence (SEQ ID NO: 40988) are selected from the group consisting of AG2, AI4, AL4, Q9P, E15S, G20D, [S30], G33T, L41A, Y51T, F55V, L68D, L68E, L68K, A70Y, A70S, E75A, E75D, E75P, K88Q, and G90Q (where “A” represents and insertion and “[ ]” represents a deletion at that position). In some embodiments, the disclosure provides CasX variants derived from CasX 515 comprising one or more modifications at one or more amino acid positions in the helical I-II domain relative to the helical I-II domain sequence (SEQ ID NO: 40989)selected from the group consisting of 124, A25, Y29 G32, G44, S48, S51, Q54,156, V63, S73, L74, K97, V100, Ml 12, LI 16, G137, F138, and SI40, wherein the modification results in an improved characteristic relative to CasX 515. In a particular embodiment, the one or more modifications at one or more amino acid positions in the helical I-II domain are selected from the group consisting of AT24, AC25, Y29F,G32Y, G32N, G32H, G32S, G32T, G32A, G32V, [G32], G32S, G32T, G44L, G44H, S48H, S48T, S51T, Q54H, I56T, V63T, S73H, L74Y, K97G, K97S, K97D, K97E, V100L, M112T, M112W, M112R, M112K, L116K, G137R, G137K, G137N, AQ138, and S140Q. In some embodiments, the disclosure provides CasX variants derived from CasX 515 comprising one or more modifications at one or more amino acid positions in the helical II domain relative to the helical II domain sequence (SEQ ID NO: 41004) selected from the group consisting of L2, V3, E4, R5, Q6, A7, E9, V10, Dll, W12, W13, D14, M15, V16, C17, N18, V19, K20, L22,123, E25, K26, K31, Q35, L37, A38, K41,R 42, Q43, E44, L46, K57, Y65, G68, L70, L71, L72, E75, G79, D81, W82, K84, V85, Y86, D87,193, K95, K96, E98, L100, K102,1104, K105, E109, RI 10, D114, K118, A120, L121, W124, L125, R126, A127, A129,1133, E134, G135, L136, E138, D140, K141, D142, E143, F144, C145, C147, E148, L149, K150, L151, Q152, K153, L158, E166, and A167, wherein the modification results in an improved characteristic relative to CasX 515. In a particular embodiment, the one or more modifications at one or more amino acid positions in the helical II domain are selected from the group consisting of AA2, AH2, [L2]+[V3], V3E, V3Q, V3F, [V3], AD3, V3P, E4P, [E4], E4D, E4L, E4R, R5N, Q6V, AQ6, AG7, AH9, AA9, VD10, AT10, [V10], AF10, AD11, [Dll], DI IS, [W12], W12T, W12H, AP12, AQ13, AG12, AR13, W13P, W13D, AD13, W13L, AP14, AD14, [D14]+[M15], [M15], AT16, AP17, N18I, V19N, V19H, K20D, L22D, I23S, E25C, E25P, AG25, K26T, K27E, K31L, K31Y, Q35D, Q35P, AS37, [L37]+[A38], K41L, AR42, [Q43]+[E44], L46N, K57Q, Y65T, G68M, L70V, L71C, L72D, L72N, L72W, L72Y, E75F, E75L, E75Y,G79P, AE79, AT81, AR81, AW81, AY81, AW82, AY82, W82G, W82R, K84D, K84H, K84P, K84T, V85L, V85A, AL85, Y86C, D87G, D87M, D87P, I93C, K95T, K96R, E98G, L100A, K102H, I104T, I104S, I104Q, K105D, AK109, E109L, R110D, [RI 10], D114E, AD114, K118P, A120R, L121T, W124L, L125C, R126D, A127E, A127L, A129T, A129K, I133E, AC133, AS134, AG134, AR135, G135P, L136K, L136D, L136S, L136H, [E138], D140R, AD140, AP141, AD142, [E143]+[F144], AQ143, F144K, [F144], [F144]+[C145], C145R, AG145, C145K, C147D, AV148, E148D, AH149, L149R, K150R, L151H, Q152C, K153P, L158S, E166L, and AF167. In some embodiments, the disclosure provides CasX variants derived from CasX 515 comprising one or more modifications at one or more amino acid positions in the RuvC-I domain relative to the RuvC-I domain sequence (SEQ ID NO: 41005) selected from the group consisting of 14, K5, P6, M7, N8, L9, V12, G49, K63, K80, N83, R90, M125, and L146, wherein the modification results in an improved characteristic relative to CasX 515. In a particular embodiment, the one or more modifications at one or more amino acid positions in the RuvC-I domain are selected from the group consisting of AI4, AS5, AT6, AN6, AR7, AK7, AH8, AS8, V12L, G49W, G49R, S51R, S51K, K62S, K62T, K62E, V65A, K80E, N83G, R90H, R90G, M125S, M125A, L137Y, AP137, [L141], L141R, L141D, AQ142, AR143, AN143, E144N, AP146, L146F, P147A, K149Q, T150V, AR152, AH153, T155Q, AH155, AR155, AL156, [L156], AW156, AA157, AF157, A157S, Q158K, [Y159], T160Y, T160F, AI161, S161P, T163P, AN163, C164K, and C164M. In some embodiments, the disclosure provides CasX variants derived from CasX 515 comprising one or more modifications at one or more amino acid positions in the OBD-I domain relative to the OBD-I domain sequence (SEQ ID NO: 40995)selected from the group consisting of 14, K5, P6, M7, N8, L9, V12, G49, K63, K80, N83, R90, M125, and L146, wherein the modification results in an improved characteristic relative to CasX 515. In a particular embodiment, the one or more modifications at one or more amino acid positions in the OBD-I domain are selected from the group consisting of AG3,13G, I3E, AG4, K4G, K4P, K4S, K4W, K4W, R5P, AP5, AG5, R5S, AS5, R5A, R5P, R5G, R5L, I6A, I6L, AG6, N7Q, N7L, N7S, K8G, K15F, D16W, AF16, AF18, AP27, M28P, M28H, V33T, R34P, M36Y, R41P, L47P, AP48, E52P, AP55, [P55]+[Q56], Q56S, Q56P, AD56, AT56, and Q56P. In some embodiments, the disclosure provides CasX variants derived from CasX 515 comprising one or more modifications at one or more amino acid positions in the OBD-II domain relative to the OBD-II domain sequence (SEQ ID NO: 41000) selected from the group consisting of 14, K5, P6, M7, N8, L9, V12, G49, K63, K80, N83, R90, M125, and L146, wherein the modification results in an improved characteristic relative to CasX 515. In a particular embodiment, the one or more modifications at one or more amino acid positions in the OBD-I domain are selected from the group consisting of [S2], I3R, I3K, [I3]+[L4], [L4], KI IT, AP24, K37G, R42E, AS53, AR58, [K63], M70T, I82T, Q92I, Q92F, Q92V, Q92A, AA93, K110Q, R115Q, L121T, AA124, AR141, AD143, AA143, AW144, and AA145. In some embodiments, the disclosure provides CasX variants derived from CasX 515 comprising one or more modifications at one or more amino acid positions in the TSL domain relative to the TSL domain sequence (SEQ ID NO: 41002) selected from the group consisting of SI, N2, C3, G4, F5,17, K18, V58, S67, T76, G78, S80, G81, E82, S85, V96, and E98, wherein the modification results in an improved characteristic relative to CasX 515. In a particular embodiment, the one or more modifications at one or more amino acid positions in the OBD-I domain are selected from the group consisting of AM1, [N2], AV2, C3S, AG4, AW4, F5P, AW7, K18G, V58D, AA67, T76E, T76D, T76N, G78D, [S80], [G81], AE82, AN82, S85I, V96C, V96T, and E98D. It will be understood that combinations of any of the same foregoing modifications of the paragraph can similarly be introduced into the CasX variants of the disclosure, resulting in a CasX variant with improved characteristics. For example, in one embodiment, the disclosure provides CasX variant 535 (SEQ ID NO: 40211), which has a single mutation of G223S relative to CasX 515. In another embodiment, the disclosure provides CasX variant 668 (SEQ ID NO: 40344), which has an insertion of R at position 26 and a substitution of G223S relative to CasX 515. In another embodiment, the disclosure provides CasX 672 (SEQ ID NO: 40347), which has substitutions of L169K and G223S relative to CasX 515. In another embodiment, the disclosure provides CasX 676 (SEQ ID NO: 40351), which has substitutions of L169K and G223S and an insertion of R at position 26 relative to CasX 515. CasX variants with improved characteristics relative to CasX 515 include variants of Table 3.

[0276] Exemplary characteristics that can be improved in CasX variant proteins relative to the same characteristics in reference CasX proteins or relative to the CasX variant from which they were derived include, but are not limited to improved folding of the variant, increased binding affinity to the gRNA, increased binding affinity to the target nucleic acid, improved ability to utilize a greater spectrum of PAM sequences in the editing and / or binding of target nucleic acid, improved unwinding of the target DNA, increased editing activity, improved editing efficiency, improved editing specificity for the target nucleic acid, decreased off-target editing or cleavage, increased percentage of a eukaryotic genome that can be efficiently edited, increased activity of the nuclease, increased target strand loading for double strand cleavage, decreased target strand loading for single strand nicking, increased binding of the non-target strand of DNA, improved protein stability, improved protein:gRNA (RNP) complex stability, and improved fusion characteristics. In a particular embodiment, as described in the Examples, such improved characteristics can include, but are not limited to, improved cleavage activity in target nucleic acids having TTC, ATC, and CTC PAM sequences, increased specificity for cleavage of a target nucleic acid sequence, and decreased off-target cleavage of a target nucleic acid. Table 6: CasX 515 domain sequences Domain SEQ ID NO Amino Acid Sequence OBD-I 40995 48122 QEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLE NLRKKPENIPQ Helical I-I 40996 41824 PISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVA Domain SEQ ID NO Amino Acid Sequence NTSB 40988 41818 QPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQV SEKGKAYTNYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKF GQ Helical I-II 40989 40819 RALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGT IASFLSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPP QPHTKEGVDAYNEVIARVRMWVNLNLWQKLKLSRDDAKPLLRL KGFPSF Helical II 41004 41820 PLVERQANEVDWWDMVCNVKKLINEKKEDGKVFWQNLAGYKR QEALRPYLSSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKV YDEAWERIDKKVEGLSKHIKLEEERRSEDAQSKAALTDWLRAKA SFVIEGLKEADKDEFCRCELKLQKWYGDLRGKPFAIEAE OBD-II 41000 41823 NSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKI KPEAFEANRFYTVINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQ GREFIWNDLLSLETGSLKLANGRVIEKTLYNRRTRQDEPALFVAL TFERREVLD RuvC-I 41005 41812 S SNIKPMNLIGVDRGENIPAVIALTDPEGCPLSRFKD SLGNPTHILRI GESYKEKQRTIQAKKEVEQRRAGGYSRKYASKAKNLADDMVRN TARDLLYYAVTQDAMLIFENLSRGFGRQGKRTFMAERQYTRMED WLTAKLAYEGLPSKTYLSKTLAQYTSKTC TSL 41002 41825 SNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYN RYKRQNVVKDLSVELDRLSEESVNNDISSWTKGRSGEALSLLKKR FSHRPVQEKFVCLNCGFETH RuvC-II 41003 41826 ADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQS FYRKKLKEVWKPAV

[0277] The CasX variants of the embodiments described herein have the ability to form an RNP complex with the gRNA disclosed herein. In some embodiments, an RNP comprising the CasX variant protein and a gRNA of the disclosure, at a concentration of 20 pM or less, is capable of cleaving a double stranded DNA target with an efficiency of at least 80%. In some embodiments, the RNP at a concentration of 20 pM or less is capable of cleaving a double stranded DNA target with an efficiency of at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90% or at least 95%. In some embodiments, the RNP at a concentration of 50 pM or less, 40 pM or less, 30 pM or less, 20 pM or less, 10 pM or less, or 5 pM or less, is capable of cleaving a double stranded DNA target with an efficiency of at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90% or at least 95%. These improved characteristics are described in more detail, below. k. Catalytically-dead CasX variants

[0278] In some embodiments, for example those embodiments encompassing applications where cleavage of the target nucleic acid sequence is not a desired outcome, improving the catalytic activity of a CasX variant protein comprises altering, reducing, or abolishing the catalytic activity of the CasX variant protein. In some embodiments, the disclosure provides catalytically-dead CasX variant proteins that, while able to bind a target nucleic acid when complexed with a gRNA having a targeting sequence complementary to the target nucleic acid, are not able to cleave the target nucleic acid. Exemplary catalytically-dead CasX proteins comprise one or more mutations in the active site of the RuvC domain of the CasX protein. In some embodiments, a catalytically-dead CasX variant protein comprises substitutions at residues 672, 769 and / or 935 relative to SEQ ID NO: 1. In one embodiment, a catalytically-dead CasX variant protein comprises substitutions of D672A, E769A and / or D935A relative to a reference CasX protein of SEQ ID NO: 1. In other embodiments, a catalytically-dead CasX variant protein comprises substitutions at amino acids 659, 756 and / or 922 relative to a reference CasX protein of SEQ ID NO: 2. In some embodiments, a catalytically-dead CasX variant protein comprises D659A, E756A and / or D922A substitutions relative to a reference CasX protein of SEQ ID NO: 2. In some embodiments, a catalytically-dead CasX variant 527, 668 and 676 proteins comprise D660A, E757A, and D922A modifications to abolish the endonuclease activity. In further embodiments, a catalytically-dead CasX protein comprises deletions of all or part of the RuvC domain of the CasX protein. It will be understood that the same foregoing substitutions can similarly be introduced into the CasX variants of the disclosure, resulting in a catalytically-dead CasX (dCasX) variant. In one embodiment, all or a portion of the RuvC domain is deleted from the CasX variant, resulting in a dCasX variant. Catalytically inactive dCasX variant proteins can, in some embodiments, be used for base editing or epigenetic modifications. With a higher affinity for DNA, in some embodiments, catalytically inactive dCasX variant proteins can, relative to catalytically active CasX, find their target nucleic acid faster, remain bound to target nucleic acid for longer periods of time, bind target nucleic acid in a more stable fashion, or a combination thereof, thereby improving these functions of the catalytically-dead CasX variant protein compared to a CasX variant that retains its cleavage capability. Exemplary dCasX variant sequences are disclosed as SEQ ID NOS: 40808-40827 and 41006-41009 as set forth in Table 7. In some embodiments, a dCasX variant is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to a sequence of SEQ ID NOS: 40808-40827, 41006-41009 and retains the functional properties of a dCasX variant protein. In some embodiments, a dCasX variant comprises a sequence of SEQ ID NOS: 40808-40827, 4100641009. Table 7: Catalytically-dead CasX Variant Proteins SEQ ID NO: dCasX Amino Acid Sequence 40808 CAS 100 - 40809 CAS099 [d491] - 40810 CAS098 - 40811 CAS085 - 40812 CAS087 - 40813 CAS086 - 40814 CAS083 - 40815 CAS082 - 40816 CAS069 - 40817 CAS068 - 40818 CAS070 - 40819 CAS071 - 40820 CAS072 - 40821 CAS073 - 40822 CAS074 - 40823 CAS075 - 40824 CAS076 - 40825 CAS077 - 40826 CAS078 - 40827 CAS081 - 41006 CAS096 - 41007 CAS401 - 41008 CAS 142 - 41009 CAS402 - I. CasX Fusion Proteins

[0279] In some embodiments, the disclosure provides AAV encoding CasX proteins comprising a heterologous protein fused to the CasX. In some cases, the CasX is a CasX variant of any of the embodiments described herein. In some embodiments, a CasX variant comprises any one of the sequences as set forth in Table 3 fused to one or more proteins or domains thereof with an activity of interest.

[0280] In some embodiments, the CasX fusion protein comprises any one of the variants SEQ ID NOS: 49-160, 40208-40369, or 40828-40912 as set forth in Table 3, fused to one or more proteins or domains thereof that have a different activity of interest, resulting in a fusion protein. For example, in some embodiments, the CasX variant protein is fused to a protein (or domain thereof) that inhibits transcription, modifies a target nucleic acid, or modifies a polypeptide associated with a nucleic acid (e.g., histone modification).

[0281] In some embodiments, a heterologous polypeptide (or heterologous amino acid such as a cysteine residue or a non-natural amino acid) can be inserted at one or more positions within a CasX protein to generate a CasX fusion protein. In other embodiments, a cysteine residue can be inserted at one or more positions within a CasX protein followed by conjugation of a heterologous polypeptide described below. In some alternative embodiments, a heterologous polypeptide or heterologous amino acid can be added at the N- or C-terminus of the CasX variant protein. In other embodiments, a heterologous polypeptide or heterologous amino acid can be inserted internally within the sequence of the CasX protein.

[0282] In some embodiments, the CasX variant fusion protein retains RNA-guided sequence specific target nucleic acid binding and cleavage activity. In some cases, the CasX variant fusion protein has (retains) 50% or more of the activity (e.g., cleavage and / or binding activity) of the corresponding CasX variant protein that does not have the insertion of the heterologous protein. In some cases, the CasX variant fusion protein retains at least about 60%, or at least about 70% or more, at least about 80%, or at least about 90%, or at least about 92%, or at least about 95%, or at least about 98%, or at least about 100% of the activity (e.g., cleavage and / or binding activity) of the corresponding CasX protein that does not have the insertion of the heterologous protein.

[0283] In some cases, the CasX variant fusion protein retains (has) target nucleic acid binding activity relative to the activity of the CasX protein without the inserted heterologous amino acid or heterologous polypeptide. In some cases, the CasX variant fusion protein retains at least about 60%, or at least about 70% or more, at least about 80%, or at least about 90%, or at least about 92%, or at least about 95%, or at least about 98%, or at least about 100% of the binding activity of the corresponding CasX protein that does not have the insertion of the heterologous protein.

[0284] In some cases, the CasX variant fusion protein retains (has) target nucleic acid binding and / or cleavage activity relative to the activity of the parent CasX protein without the inserted heterologous amino acid or heterologous polypeptide. For example, in some cases, the CasX variant fusion protein has (retains) 50% or more of the binding and / or cleavage activity of the corresponding parent CasX protein (the CasX protein that does not have the insertion). For example, in some cases, the CasX variant fusion protein has (retains) 60% or more (70% or more, 80% or more, 90% or more, 92% or more, 95% or more, 98% or more, or 100%) of the binding and / or cleavage activity of the corresponding CasX parent protein (the CasX protein that does not have the insertion). Methods of measuring cleaving and / or binding activity of a CasX protein and / or a CasX fusion protein will be known to one of ordinary skill in the art and any convenient method can be used.

[0285] A variety of heterologous polypeptides are suitable for inclusion in a reference CasX or CasX variant fusion protein of the disclosure. In some cases, the fusion partner can modulate transcription (e.g., inhibit transcription, increase transcription) of a target DNA. For example, in some cases the fusion partner is a protein (or a domain from a protein) that inhibits transcription (e.g., a transcriptional repressor, a protein that functions via recruitment of transcription inhibitor proteins, modification of target DNA such as methylation, recruitment of a DNA modifier, modulation of histones associated with target DNA, recruitment of a histone modifier such as those that modify acetylation and / or methylation of histones, and the like). In some cases the fusion partner is a protein (or a domain from a protein) that increases transcription (e.g., a transcription activator, a protein that acts via recruitment of transcription activator proteins, modification of target DNA such as demethylation, recruitment of a DNA modifier, modulation of histones associated with target DNA, recruitment of a histone modifier such as those that modify acetylation and / or methylation of histones, and the like).

[0286] In some cases, a fusion partner has enzymatic activity that modifies a target nucleic acid sequence; e.g., nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity or glycosylase activity. In some embodiments, a CasX variant comprises any one of SEQ ID NOS: 49-160, 40208-40369, or 40828-40912 as set forth in Table 3 and a polypeptide with methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity or demyristoylation activity.

[0287] Examples of proteins (or fragments thereof) that can be used as a fusion partner to increase transcription include but are not limited to: transcriptional activators such as VP16, VP64, VP48, VP160, p65 subdomain (e.g., from NFkB), and activation domain of EDLL and / or TAL activation domain (e.g., for activity in plants); histone lysine methyltransferases such as SET1A, SET1B, MLL1 to 5, ASH1, SYMD2, NSD1, and the like; histone lysine demethylases such as JHDM2a / b, UTX, JMJD3, and the like; histone acetyltransferases such as GCN5, PCAF, CBP, p300, TAF1, TIP60 / PLIP, MOZ / MYST3, MORF / MYST4, SRC1, ACTR, Pl60, CLOCK, and the like; and DNA demethylases such as Ten-Eleven Translocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, ROS1, and the like.

[0288] Examples of proteins (or fragments thereof) that can be used as a fusion partner to decrease transcription include but are not limited to: transcriptional repressors such as the Kruppel associated box (KRAB or SKD); K0X1 repression domain; the Mad mSIN3 interaction domain (SID); the ERF repressor domain (ERD), the SRDX repression domain (e.g., for repression in plants), and the like; histone lysine methyltransferases such as Pr-SET7 / 8, SUV4-20H1, RIZ1, and the like; histone lysine demethylases such as JMJD2A / JHDM3A, JMJD2B, JMJD2C / GASC1, JMJD2D, JARID1A / RBP2, JARID1B / PLU-1, JARID 1C / SMCX, JARID1D / SMCY, and the like; histone lysine deacetylases such as HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, HDAC11, and the like; DNA methylases such as Hhal DNA m5c-methyltransferase (M.Hhal), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plants), ZMET2, CMT1, CMT2 (plants), and the like; and periphery recruitment elements such as Lamin A, Lamin B, and the like.

[0289] In some cases, the fusion partner has enzymatic activity that modifies the target nucleic acid sequence (e.g., ssRNA, dsRNA, ssDNA, dsDNA). Examples of enzymatic activity that can be provided by the fusion partner include but are not limited to: nuclease activity such as that provided by a restriction enzyme (e.g., FokI nuclease), methyltransferase activity such as that provided by a methyltransferase (e.g., Hhal DNA m5c-methyltransferase (M.Hhal), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plants), ZMET2, CMT1, CMT2 (plants), and the like); demethylase activity such as that provided by a demethylase (e.g., Ten-Eleven Translocation (TET) dioxygenase 1 (TET 1 CD), TET1, DME, DML1, DML2, ROS1, and the like) , DNA repair activity, DNA damage activity, deamination activity such as that provided by a deaminase (e.g., a cytosine deaminase enzyme, e.g., an APOBEC protein such as rat APOBEC1), dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity such as that provided by an integrase and / or resolvase (e.g., Gin invertase such as the hyperactive mutant of the Gin invertase, GinH106Y; human immunodeficiency virus type 1 integrase (IN); Tn3 resolvase; and the like), transposase activity, recombinase activity such as that provided by a recombinase (e.g., catalytic domain of Gin recombinase), polymerase activity, ligase activity, helicase activity, photolyase activity, and glycosylase activity).

[0290] In some cases, a CasX variant protein for use in the AAV systems of the present disclosure is fused to a polypeptide selected from a domain for increasing transcription (e.g., a VP16 domain, a VP64 domain), a domain for decreasing transcription (e.g., a KRAB domain, e.g., from the Koxl protein), a core catalytic domain of a histone acetyltransferase (e.g., histone acetyltransferase p300), a protein / domain that provides a detectable signal (e.g., a fluorescent protein such as GFP), a nuclease domain (e.g., a Fokl nuclease), or a base editor (e.g., cytidine deaminase such as APOBEC 1).

[0291] In some cases, the fusion partner has enzymatic activity that modifies a protein associated with the target nucleic acid (e.g., ssRNA, dsRNA, ssDNA, dsDNA) (e.g., a histone, an RNA binding protein, a DNA binding protein, and the like). Examples of enzymatic activity (that modifies a protein associated with a target nucleic acid) that can be provided by the fusion partner include but are not limited to: methyltransferase activity such as that provided by a histone methyltransferase (HMT) (e.g., suppressor of variegation 3-9 homolog 1 (SUV39H1, also known as KMT1A), euchromatic histone lysine methyltransferase 2 (G9A, also known as KMT1C and EHMT2), SUV39H2, ESET / SETDB 1, and the like, SET1A, SET1B, MLL1 to 5, ASH1, SYMD2, NSD1, DOT1L, Pr-SET7 / 8, SUV4-20H1, EZH2, RIZ1), demethylase activity such as that provided by a histone demethylase (e.g., Lysine Demethylase 1A (KDM1A also known as LSD1), JHDM2a / b, JMJD2A / JHDM3 A, JMJD2B, JMJD2C / GASC1, JMJD2D, JARID1A / RBP2, JARID1B / PLU-1, JARID1C / SMCX, JARID1D / SMCY, UTX, JMJD3, and the like), acetyltransferase activity such as that provided by a histone acetylase transferase (e.g., catalytic core / fragment of the human acetyltransferase p300, GCN5, PCAF, CBP, TAF1, TIP60 / PLIP, MOZ / MYST3, MORF / MYST4, HB01 / MYST2, HMOF / MYST1, SRC1, ACTR, P160, CLOCK, and the like), deacetylase activity such as that provided by a histone deacetylase (e.g, HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, HDAC11, and the like), kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity, and demyristoylation activity.

[0292] Additional examples of suitable fusion partners of the CasX variants are (i) a dihydrofolate reductase (DHFR) destabilization domain (e.g., to generate a chemically controllable subject RNA-guided polypeptide or a conditionally active RNA-guided polypeptide), and (ii) a chloroplast transit peptide.

[0293] In some embodiments, a CasX variant comprises any one of SEQ ID NOS: 49-160, 40208-40369, or 40828-40912 as set forth in Table 3, and a chloroplast transit peptide including, but are not limited to: MASMISSSAVTTVSRASRGQSAAMAPFGGLKSMTGFPVRKVNTDITSITSNGGR VKCMQVWPPIGKKKFETLSYLPPLTRDSRA (SEQ ID NO: 40790); MASMISSSAVTTVSRASRGQSAAMAPFGGLKSMTGFPVRKVNTDITSITSNGGRVKS (SEQ ID NO: 39980); MASSMLSSATMVASPAQATMVAPFNGLKSSAAFPATRKANNDITSITSNGGRVNCMQV WPPIEKKKFETLSYLPDLTDSGGRVNC (SEQ ID NO: 39968); MAQVSRICNGVQNPSLISNLSKSSQRKSPLSVSLKTQQHPRAYPISSSWGLKKSGMTLIG SELRPLKVMSSVSTAC (SEQ ID NO: 39969); MAQVSRICNGVWNPSLISNLSKSSQRKSPLSVSLKTQQHPRAYPISSSWGLKKSGMTLIG SELRPLKVMSSVSTAC (SEQ ID NO: 39970); MAQINNMAQGIQTLNPNSNFHKPQVPKSSSFLVFGSKKLKNSANSMLVLKKDSIFMQLF CSFRISASVATAC (SEQ ID NO: 39971); MAALVTSQLATSGTVLSVTDRFRRPGFQGLRPRNPADAALGMRTVGASAAPKQSRKPH RFDRRCLSMVV (SEQ ID NO: 39972); MAALTTSQLATSATGFGIADRSAPSSLLRHGFQGLKPRSPAGGDATSLSVTTSARATPKQ QRSVQRGSRRFPSVWC (SEQ ID NO: 39973); MASSVLSSAAVATRSNVAQANMVAPFTGLKSAASFPVSRKQNLDITSIASNGGRVQC (SEQ ID NO: 39974); MESLAATSVFAPSRVAVPAARALVRAGTWPTRRTSSTSGTSGVKCSAAVTPQASPVIS RSAAAA (SEQ ID NO: 39975); and MGAAATSMQSLKFSNRLVPPSRRLSPVPNNVTCNNLPKSAAPVRTVKCCASSWNSTING AAATTNGASAASS (SEQ ID NO: 39976).

[0294] In some cases, a CasX variant protein of the present disclosure for use in the AAV systems can include an endosomal escape peptide. In some cases, an endosomal escape polypeptide comprises the amino acid sequence GLFXALLXLLXSLWXLLLXA (SEQ ID NO: 39977), wherein each X is independently selected from lysine, histidine, and arginine. In some cases, an endosomal escape polypeptide comprises the amino acid sequence GLFHALLHLLHSLWHLLLHA (SEQ ID NO: 39978), or HHHHHHHHH (SEQ ID NO: 39979).

[0295] Non-limiting examples of fusion partners for use with a CasX variant when targeting ssRNA target nucleic acid sequences include (but are not limited to): splicing factors (e.g., RS domains); protein translation components (e.g., translation initiation, elongation, and / or release factors; e.g., eIF4G); RNA methylases; RNA editing enzymes (e.g., RNA deaminases, e.g., adenosine deaminase acting on RNA (ADAR), including A to I and / or C to U editing enzymes); helicases; RNA-binding proteins; and the like. It is understood that a heterologous polypeptide can include the entire protein or in some cases can include a fragment of the protein (e.g., a functional domain).

[0296] In some embodiments, a CasX variant of any one of SEQ ID NOS: 49-160, 4020840369, or 40828-40912 as set forth in Table 3, comprises a fusion partner of any domain capable of interacting with ssRNA (which, for the purposes of this disclosure, includes intramolecular and / or intermolecular secondary structures, e.g., double-stranded RNA duplexes such as hairpins, stem-loops, etc.), whether transiently or irreversibly, directly or indirectly, including but not limited to an effector domain selected from the group comprising; endonucleases (for example RNase III, the CRR22 DYW domain, Dicer, and PIN (PilT N-terminus) domains from proteins such as SMG5 and SMG6); proteins and protein domains responsible for stimulating RNA cleavage (for example CPSF, CstF, CFIm and CFIIm); exonucleases (for example XRN-1 or Exonuclease T); deadenylases (for example HNT3); proteins and protein domains responsible for nonsense mediated RNA decay (for example UPF1, UPF2, UPF3, UPF3b, RNP SI, Y14, DEK, REF2, and SRml60); proteins and protein domains responsible for stabilizing RNA (for example PABP); proteins and protein domains responsible for repressing translation (for example Ago2 and Ago4); proteins and protein domains responsible for stimulating translation (for example Staufen); proteins and protein domains responsible for (e.g., capable of) modulating translation (e.g., translation factors such as initiation factors, elongation factors, release factors, etc., e.g., eIF4G); proteins and protein domains responsible for polyadenylation of RNA (for example PAPI, GLD-2, and Star- PAP); proteins and protein domains responsible for polyuridinylation of RNA (for example CI DI and terminal uridylate transferase); proteins and protein domains responsible for RNA localization (for example from IMP1, ZBP1, She2p, She3p, and Bicaudal-D); proteins and protein domains responsible for nuclear retention of RNA (for example Rrp6); proteins and protein domains responsible for nuclear export of RNA (for example TAP, NXF1, THO, TREX, REF, and Aly); proteins and protein domains responsible for repression of RNA splicing (for example PTB, Sam68, and hnRNP Al); proteins and protein domains responsible for stimulation of RNA splicing (for example serine / arginine-rich (SR) domains); proteins and protein domains responsible for reducing the efficiency of transcription (for example FUS (TLS)); and proteins and protein domains responsible for stimulating transcription (for example CDK7 and HIV Tat). Alternatively, the effector domain may be selected from the group comprising endonucleases; proteins and protein domains capable of stimulating RNA cleavage; exonucleases; deadenylases; proteins and protein domains having nonsense mediated RNA decay activity; proteins and protein domains capable of stabilizing RNA; proteins and protein domains capable of repressing translation; proteins and protein domains capable of stimulating translation; proteins and protein domains capable of modulating translation (e.g., translation factors such as initiation factors, elongation factors, release factors, etc., e.g., eIF4G); proteins and protein domains capable of polyadenylation of RNA; proteins and protein domains capable of polyuridinylation of RNA; proteins and protein domains having RNA localization activity; proteins and protein domains capable of nuclear retention of RNA; proteins and protein domains having RNA nuclear export activity; proteins and protein domains capable of repression of RNA splicing; proteins and protein domains capable of stimulation of RNA splicing; proteins and protein domains capable of reducing the efficiency of transcription; and proteins and protein domains capable of stimulating transcription. Another suitable heterologous polypeptide is a PUF RNA-binding domain, which is described in more detail in WO2012068627, which is hereby incorporated by reference in its entirety.

[0297] Some RNA splicing factors that can be used (in whole or as fragments thereof) as a fusion partner for a CasX variant have modular organization, with separate sequence-specific RNA binding modules and splicing effector domains. For example, members of the serine / arginine-rich (SR) protein family contain N-terminal RNA recognition motifs (RRMs) that bind to exonic splicing enhancers (ESEs) in pre-mRNAs and C-terminal RS domains that promote exon inclusion. As another example, the hnRNP protein hnRNP Al binds to exonic splicing silencers (ESSs) through its RRM domains and inhibits exon inclusion through a C-terminal glycine-rich domain. Some splicing factors can regulate alternative use of splice site (ss) by binding to regulatory sequences between the two alternative sites. For example, ASF / SF2 can recognize ESEs and promote the use of intron proximal sites, whereas hnRNP Al can bind to ESSs and shift splicing towards the use of intron distal sites. One application for such factors is to generate ESFs that modulate alternative splicing of endogenous genes, particularly disease associated genes. For example, Bcl-x pre-mRNA produces two splicing isoforms with two alternative 5' splice sites to encode proteins of opposite functions. The long splicing isoform Bcl-xL is a potent apoptosis inhibitor expressed in long-lived post mitotic cells and is up-regulated in many cancer cells, protecting cells against apoptotic signals. The short isoform Bcl-xS is a pro-apoptotic isoform and expressed at high levels in cells with a high turnover rate (e.g., developing lymphocytes). The ratio of the two Bcl-x splicing isoforms is regulated by multiple cis-elements that are located in either the core exon region or the exon extension region (i.e., between the two alternative 5' splice sites). For more examples, see WO2010075303, which is hereby incorporated by reference in its entirety.

[0298] Further suitable fusion partners for use with a CasX variant include, but are not limited to, proteins (or fragments thereof) that are boundary elements (e.g., CTCF), proteins and fragments thereof that provide periphery recruitment (e.g., Lamin A, Lamin B, etc.), and protein docking elements (e.g., FKBP / FRB, Pill / Abyl, etc.).

[0299] In some cases, a heterologous polypeptide (a fusion partner) for use with a CasX variant provides for subcellular localization, i.e., the heterologous polypeptide contains a subcellular localization sequence (e.g., a nuclear localization signal (NLS) for targeting to the nucleus, a sequence to keep the fusion protein out of the nucleus, e.g., a nuclear export sequence (NES), a sequence to keep the fusion protein retained in the cytoplasm, a mitochondrial localization signal for targeting to the mitochondria, a chloroplast localization signal for targeting to a chloroplast, an ER retention signal, and the like). In some embodiments, a subject RNA-guided polypeptide or a conditionally active RNA-guided polypeptide and / or subject CasX fusion protein does not include a NLS so that the protein is not targeted to the nucleus (which can be advantageous, e.g., when the target nucleic acid sequence is an RNA that is present in the cytosol). In some embodiments, a fusion partner can provide a tag (i.e., the heterologous polypeptide is a detectable label) for ease of tracking and / or purification (e.g., a fluorescent protein, e.g., green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), mCherry, tdTomato, and the like; a histidine tag, e.g., a 6XHis tag; a hemagglutinin (HA) tag; a FLAG tag; a Myc tag; and the like).

[0300] In some cases, a CasX variant protein for use in the AAV systems includes (is fused to) a nuclear localization signal (NLS) for targeting the CasX / gRNA to the nucleus of the cell. In some cases, a CasX variant protein is fused to 2 or more, 3 or more, 4 or more, or 5 or more 6 or more, 7 or more, 8 or more NLSs. In some cases, one or more NLSs (2 or more, 3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 50 amino acids of) the N-terminus and / or the C-terminus. In some cases, one or more NLSs (2 or more, 3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 50 amino acids of) the N-terminus. In some cases, one or more NLSs (2 or more, 3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 50 amino acids of) the C-terminus. In some cases, an NLS is positioned at the N-terminus and an NLS is positioned at the C-terminus. In some cases, one or more NLSs (2 or more, 3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 50 amino acids of) both the N-terminus and the C-terminus. In some cases, a CasX variant protein includes (is fused to) between 1 and 10 NLSs (e.g., 1-9, 1-8, 1-7, 1-6, 1-5, 2-10, 2-9, 2-8, 2-7, 2- 6, or 2-5 NLSs). In some cases, a CasX variant protein includes (is fused to) between 2 and 5 NLSs (e.g., 2-4, or 2-3 NLSs). Non-limiting examples of NLSs suitable for use with a CasX variant include sequences having at least about 80%, at least about 90%, or at least about 95% identity or are identical to sequences derived from: the NLS of the SV40 virus large T-antigen, having the amino acid sequence PKKKRKV (SEQ ID NO: 196); the NLS from nucleoplasmin (e.g., the nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 197); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 248) or RQRRNELKRSP (SEQ ID NO: 161); the hRNPAl M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 162); the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 163) of the IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO: 164) and PPKKARED (SEQ ID NO: 165) of the myoma T protein; the sequence PQPKKKPL (SEQ ID NO: 166) of human p53; the sequence SALIKKKKKMAP (SEQ ID NO: 167) of mouse c-abl IV; the sequences DRLRR (SEQ ID NO: 168) and PKQKKRK (SEQ ID NO: 169) of the influenza virus NS1; the sequence RKLKKKIKKL (SEQ ID NO: 170) of the Hepatitis virus delta antigen; the sequence REKKKFLKRR (SEQ ID NO: 171) of the mouse Mxl protein; the sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 172) of the human poly(ADP-ribose) polymerase; the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 173) of the steroid hormone receptors (human) glucocorticoid; the sequence PRPRKIPR (SEQ ID NO: 174) of Borna disease virus P protein (BDV-P1); the sequence PPRKKRTVV (SEQ ID NO: 175) of hepatitis C virus nonstructural protein (HCV-NS5A); the sequence NLSKKKKRKREK (SEQ ID NO: 176) of LEF1; the sequence RRPSRPFRKP (SEQ ID NO: 177) of ORF57 simirae; the sequence KRPRSPSS (SEQ ID NO: 178) of EBV LANA; the sequence KRGINDRNFWRGENERKTR (SEQ ID NO: 179) of Influenza A protein; the sequence PRPPKMARYDN (SEQ ID NO: 180) of human RNA helicase A (RHA); the sequence KRSFSKAF (SEQ ID NO: 181) of nucleolar RNA helicase II; the sequence KLKIKRPVK (SEQ ID NO: 182) of TUS-protein; the sequence PKKKRKVPPPPAAKRVKLD (SEQ ID NO: 183) associated with importin-alpha; the sequence PKTRRRPRRSQRKRPPT (SEQ ID NO: 184) from the Rex protein in HTLV-1; the sequence MSRRRKANPTKLSENAKKLAKEVEN (SEQ ID NO: 185) from the EGL-13 protein of Caenorhabditis elegans; and the sequences KTRRRPRRSQRKRPPT (SEQ ID NO: 186), RRKKRRPRRKKRR (SEQ ID NO: 187), PKKKSRKPKKKSRK (SEQ ID NO: 188), HKKKHPDASVNFSEFSK (SEQ ID NO: 189), QRPGPYDRPQRPGPYDRP (SEQ ID NO: 190), LSPSLSPLLSPSLSPL (SEQ ID NO: 191), RGKGGKGLGKGGAKRHRK (SEQ ID NO: 192), PKRGRGRPKRGRGR (SEQ ID NO: 193), PKKKRKVPPPPAAKRVKLD (SEQ ID NO: 194) and PKKKRKVPPPPKKKRKV (SEQ ID NO: 195), PAKRARRGYKC (SEQ ID NO: 40188), KLGPRKATGRW (SEQ ID NO: 40189), PRRKREE (SEQ ID NO: 40190), PYRGRKE (SEQ ID NO: 40191), PLRKRPRR (SEQ ID NO: 40192), PLRKRPRRGSPLRKRPRR (SEQ ID NO: 40193), PAAKRVKLDGGKRTADGSEFESPKKKRKV (SEQ ID NO: 40194), PAAKRVKLDGGKRTADGSEFESPKKKRKVGIHGVPAA (SEQ ID NO: 40195), PAAKRVKLDGGKRTADGSEFESPKKKRKVAEAAAKEAAAKEAAAKA (SEQ ID NO: 40196), PAAKRVKLDGGKRTADGSEFESPKKKRKVPG (SEQ ID NO: 40197), KRKGSPERGERKRHW (SEQ ID NO: 40198), KRTADSQHSTPPKTKRKVEFEPKKKRKV (SEQ ID NO: 40199), and PKKKRKVGGSKRTADSQHSTPPKTKRKVEFEPKKKRKV (SEQ ID NO: 40200). Additional NLS for incorporation in the AAV systems of the disclosure are provided in Tables 15 and 16, indicating NLS for linking to the N- or C-terminus of the CasX. In some embodiments, the one or more NLS are linked to the CasX or to an adjacent NLS by a linker peptide wherein the linker peptide is selected from the group consisting of RS, (G)n (SEQ ID NO: 40201), (GS)n (SEQ ID NO: 40202), (GSGGS)n (SEQ ID NO: 208), (GGSGGS)n (SEQ ID NO: 209), (GGGS)n (SEQ ID NO: 210), GGSG (SEQ ID NO: 211), GGSGG (SEQ ID NO: 212), GSGSG (SEQ ID NO: 213), GSGGG (SEQ ID NO: 214), GGGSG (SEQ ID NO: 215), GSSSG (SEQ ID NO: 216), GPGP (SEQ ID NO: 217), GGP, PPP, PPAPPA (SEQ ID NO: 218), PPPG (SEQ ID NO: 40207), PPPGPPP (SEQ ID NO: 219), PPP(GGGS)n (SEQ ID NO: 40203), (GGGS)nPPP (SEQ ID NO: 40204), AEAAAKEAAAKEAAAKA (SEQ ID NO: 40205), and TPPKTKRKVEFE (SEQ ID NO: 40206), wherein n is 1 to 5. In some embodiments, the AAV constructs of the disclosure comprise polynucleic acids encoding the NLS and linker peptides of any of the foregoing embodiments of the paragraph, as well as the NLS of Tables 15 and 16, and can be, in some cases, configured in relation to the other components of the constructs as depicted in any one of FIGS. 24, 33-35 or 42.

[0301] In general, NLS (or multiple NLSs) are of sufficient strength to drive accumulation of a CasX variant fusion protein in the nucleus of a eukaryotic cell. Detection of accumulation in the nucleus may be performed by any suitable technique. For example, a detectable marker may be fused to a CasX variant fusion protein such that location within a cell may be visualized. Cell nuclei may also be isolated from cells, the contents of which may then be analyzed by any suitable process for detecting protein, such as immunohistochemistry, Western blot, or enzyme activity assay. Accumulation in the nucleus may also be determined indirectly.

[0302] In some cases, a CasX variant fusion protein for use in the AAV systems includes a "protein transduction domain" or PTD (also known as a CPP - cell penetrating peptide), which refers to a protein, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates traversing a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane. A PTD attached to another molecule, which can range from a small polar molecule to a large macromolecule and / or a nanoparticle, facilitates the molecule traversing a membrane, for example going from an extracellular space to an intracellular space, or from the cytosol to within an organelle. In some embodiments, a PTD is covalently linked to the amino terminus of a CasX variant fusion protein. In some embodiments, a PTD is covalently linked to the carboxyl terminus of a CasX variant fusion protein. In some cases, the PTD is inserted internally in the sequence of a CasX variant fusion protein at a suitable insertion site. In some cases, a CasX variant fusion protein includes (is conjugated to, is fused to) one or more PTDs (e.g., two or more, three or more, four or more PTDs). In some cases, a PTD includes one or more nuclear localization signals (NLS). Examples of PTDs include, but are not limited to, peptide transduction domain of HIV TAT comprising YGRKKRRQRRR (SEQ ID NO: 198), RKKRRQRR (SEQ ID NO: 199); YARAAARQARA (SEQ ID NO: 200); THRLPRRRRRR (SEQ ID NO: 201); and GGRRARRRRRR (SEQ ID NO: 202); a polyarginine sequence comprising a number of arginines sufficient to direct entry into a cell (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines) (SEQ ID NO: 203); a VP22 domain (Zender et al. (2002) Cancer Gene Ther. 9(6):489-96); an Drosophila Antennapedia protein transduction domain (Noguchi et al. (2003) Diabetes 52(7): 1732-1737); a truncated human calcitonin peptide (Trehin et al. (2004) Pharm. Research 21 :1248-1256); polylysine (Wender et al. (2000) Proc. Natl. Acad. Sci. USA 97: 13003-13008); RRQRRTSKLMKR (SEQ ID NO: 204); Transportan GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 205); I<ALAWEAI<LAI<ALAI<ALAI<HLAI<ALAI<ALI<CEA (SEQ ID NO: 206); and RQIKIWFQNRRMKWKK (SEQ ID NO: 207). In some embodiments, the PTD is an activatable CPP (ACPP) (Aguilera et al. (2009) IntegrBiol (Camb) June; 1(5-6): 371-381). ACPPs comprise a polycationic CPP (e.g., Arg9 or "R9") connected via a cleavable linker to a matching polyanion (e.g., Glu9 or "E9"), which reduces the net charge to nearly zero and thereby inhibits adhesion and uptake into cells. Upon cleavage of the linker, the polyanion is released, locally unmasking the polyarginine and its inherent adhesiveness, thus "activating" the ACPP to traverse the membrane.

[0303] In some embodiments, a CasX variant fusion protein can include a CasX protein that is linked to an internally inserted heterologous amino acid or heterologous polypeptide (a heterologous amino acid sequence) via a linker polypeptide (e.g., one or more linker polypeptides). In some embodiments, a CasX variant fusion protein can be linked at the C-terminal and / or N-terminal end to a heterologous polypeptide (fusion partner) via a linker polypeptide (e.g., one or more linker polypeptides). The linker polypeptide may have any of a variety of amino acid sequences. Proteins can be joined by a spacer peptide, generally of a flexible nature, although other chemical linkages are not excluded. Suitable linkers include polypeptides of between 4 amino acids and 40 amino acids in length, or between 4 amino acids and 25 amino acids in length. These linkers are generally produced by using synthetic, linker encoding oligonucleotides to couple the proteins. Peptide linkers with a degree of flexibility can be used. The linking peptides may have virtually any amino acid sequence, bearing in mind that the preferred linkers will have a sequence that results in a generally flexible peptide. The use of small amino acids, such as glycine and alanine, are of use in creating a flexible peptide. The creation of such sequences is routine to those of skill in the art. A variety of different linkers are commercially available and are considered suitable for use. Example linker polypeptides include glycine polymers (G)n, glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, glycine-proline polymers, proline polymers and proline-alanine polymers. Example linkers can comprise amino acid sequences including, but not limited to (G)n (SEQ ID NO: 40201), (GS)n (SEQ ID NO: 40202), (GSGGS)n (SEQ ID NO: 208), (GGSGGS)n (SEQ ID NO: 209), (GGGS)n (SEQ ID NO: 210), GGSG (SEQ ID NO: 211), GGSGG (SEQ ID NO: 212), GSGSG (SEQ ID NO: 213), GSGGG (SEQ ID NO: 214), GGGSG (SEQ ID NO: 215), GSSSG (SEQ ID NO: 216), GPGP (SEQ ID NO: 217), GGP, PPP, PPAPPA (SEQ ID NO: 218), PPPG (SEQ ID NO: 40207), PPPGPPP (SEQ ID NO: 219), PPP(GGGS)n (SEQ ID NO: 40203), (GGGS)nPPP (SEQ ID NO: 40204), AEAAAKEAAAKEAAAKA (SEQ ID NO: 40205), and TPPKTKRKVEFE (SEQ ID NO: 40206), where n is 1 to 5, where n is 1 to 5. The ordinarily skilled artisan will recognize that design of a peptide conjugated to any elements described above can include linkers that are all or partially flexible, such that the linker can include a flexible linker as well as one or more portions that confer less flexible structure. V. AAV Systems and Methods for Modification of Target Nucleic Acids

[0304] The AAV provided herein are useful for various applications, including as therapeutics, diagnost...

Claims

1. A polynucleotide comprising the following component sequences:a.     a first adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence;b. a second AAV ITR sequence;c. a first promoter sequence;d. a sequence encoding a CasX variant protein comprising at least 85% sequence identity to SEQ ID NO: 145, wherein the CasX variant protein is a chimeric CasX protein comprising protein domains from two different CasX proteins;e.     a sequence encoding a first guide RNA (gRNA); and,f.     optionally, at least one accessory element sequence selected from the groupconsisting of a polyadenylation signal sequence (poly(A) signal), an enhancer element, an intron, a posttranscriptional regulatory element (PTRE), a nuclear localization signal (NLS), a deaminase, a DNA glycosylase inhibitor, an additional promoter, a factor that stimulates CRISPR-mediated homology-directed repair, an activator or repressor of transcription, a self-cleaving sequence, and a fusion domain,wherein the polynucleotide is configured for incorporation into a single recombinant adeno-associated virus (rAAV).

2. The polynucleotide of claim 1, wherein the sequences encoding the CasX variant protein and the first gRNA are less than 3100, less than 3090, less than 3080, less than 3070, less than 3060, less than 3050, or less than 3040 nucleotides in combined length.

3. The polynucleotide of claim 1 or claim 2, wherein the first promoter sequence and the sequence encoding the CasX variant protein are operably linked,optionally wherein the first promoter is a pol II promoter,and / or optionally wherein the first promoter is selected from the group consisting of polyubiquitin C (UBC) promoter, cytomegalovirus (CMV) promoter, simian virus 40 (SV40) promoter, chicken beta-Actin promoter and rabbit beta-Globin splice acceptor site fusion (CAG), chicken p-actin promoter with cytomegalovirus enhancer (CB7), PGK promoter, Jens Tornoe (JeT) promoter, GUSB promoter, CBA hybrid (CBh) promoter, elongation factor-1 alpha (EF-1alpha) promoter, beta-actin promoter, Rous sarcoma virus (RSV)2021394878   03 Sep 2026promoter, silencing-prone spleen focus forming virus (SFFV) promoter, CMVd1 promoter, truncated human CMV (tCMVd2), minimal CMV promoter, hepB promoter, chicken p-actin promoter, HSV TK promoter, Mini-TK promoter, minimal IL-2 promoter, GRP94 promoter, Super Core Promoter 1, Super Core Promoter 2, Super Core Promoter 3, adenovirus major late (AdML) promoter, MLC promoter, MCK promoter, GRK1 protein promoter, Rho promoter, CAR protein promoter, hSyn Promoter, U1a promoter, Ribosomal Protein Large subunit 30 (Rpl30) promoter, Ribosomal Protein Small subunit 18 (Rps18) promoter, CMV53 promoter, minimal SV40 promoter, CMV53 promoter, SFCp promoter, Mecp2 promoter, pJB42CAT5 promoter, MLP promoter, EFS promoter, MeP426 promoter, MecP2 promoter, MHCK7 promoter, beta-glucuronidase (GUSB) promoter, CK7 promoter, and CK8e promoter.

4. The polynucleotide of any one of claims 1-3, further comprising a second promoter, optionally wherein the second promoter sequence and the sequence encoding the first gRNA are operably linked,and / or optionally wherein the second promoter is a pol III promoter,and / or optionally wherein the second promoter is selected from the group consisting of U6, mini U61, mini U62, mini U63, BiH1 (Bidrectional H1 promoter), BiU6 (Bidirectional U6 promoter), gorilla U6, rhesus U6, human 7sk, and human H1 promoters.

5. The polynucleotide of claim 4, wherein the sequences of the first promoter and the second promoter:(a) are greater than at least about 1300, at least about 1350, at least about 1360, at least about 1370, at least about 1380, at least about 1390, at least about 1400, at least about 1500, at least about 1600 nucleotides, at least 1650, at least about 1700, at least about 1750, at least about 1800, at least about 1850, or at least about 1900 nucleotides in combined length; or(b) and the at least one accessory element are greater than at least about 1300, at least about 1350, at least about 1360, at least about 1370, at least about 1380, at least about 1390, at least about 1400, at least about 1500, at least about 1600 nucleotides, at least 1650, at least about 1700, at least about 1750, at least about 1800, at least about 1850, or at least about 1900 nucleotides in combined length.2021394878   03 Sep 20266. The polynucleotide of any one of claims 1-5, wherein the encoded CasX variant protein comprises:a. a non-target strand binding (NTSB) domain comprising a sequence ofQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYT NYFGRCNVAEHEKLILLAQLKPEKDSDEAVTYSLGKFGQ (SEQ ID NO: 41818), or a sequence having at least 80% at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto;b. a helical I-II domain comprising a sequence ofRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKY QDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDAYNEV IARVRMWVNLNLWQKLKLSRDDAKPLLRLKGFPSF (SEQ ID NO: 41819), or a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto;c. a helical II domain comprising a sequence ofPLVERQANEVDWWDMVCNVKKLINEKKEDGKVFWQNLAGYKRQEALRPYL SSEEDRKKGKKFARYQLGDLLLHLEKKHGEDWGKVYDEAWERIDKKVEGLS KHIKLEEERRSEDAQSKAALTDWLRAKASFVIEGLKEADKDEFCRCELKLQK WYGDLRGKPFAIEAE (SEQ ID NO: 41820), or a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto; andd. a RuvC-I domain comprising a sequence ofSSNIKPMNLIGVDRGENIPAVIALTDPEGCPLSRFKDSLGNPTHILRIGESYKEK QRTIQAKKEVEQRRAGGYSRKYASKAKNLADDMVRNTARDLLYYAVTQDA MLIFENLSRGFGRQGKRTFMAERQYTRMEDWLTAKLAYEGLPSKTYLSKTLA QYTSKTC (SEQ ID NO: 41821), or a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity thereto, optionally wherein the encoded CasX variant protein comprises an oligonucleotide binding domain (OBD)-I domain comprising a sequence ofQEIKRINKIRRRLVKDSNTKKAGKTGPMKTLLVRVMTPDLRERLENLRKKPENIPQ (SEQ ID NO: 41822), or a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.2021394878   03 Sep 20267. The polynucleotide of claim 6 wherein the encoded CasX variant protein comprises:(a) an OBD-II domain comprising a sequence ofNSILDISGFSKQYNCAFIWQKDGVKKLNLYLIINYFKGGKLRFKKIKPEAFEANRFYT VINKKSGEIVPMEVNFNFDDPNLIILPLAFGKRQGREFIWNDLLSLETGSLKLANGRV IEKTLYNRRTRQDEPALFVALTFERREVLD (SEQ ID NO: 41823), or a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto;(b) a helical I-I domain comprising a sequence ofPISNTSRANLNKLLTDYTEMKKAILHVYWEEFQKDPVGLMSRVA (SEQ ID NO: 41824), or a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto;(c) a target strand loading (TSL) domain comprising a sequence ofSNCGFTITSADYDRVLEKLKKTATGWMTTINGKELKVEGQITYYNRYKRQNVVKD LSVELDRLSEESVNNDISSWTKGRSGEALSLLKKRFSHRPVQEKFVCLNCGFETH (SEQ ID NO: 41825), or a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto;(d) a RuvC-II domain comprising a sequence ofADEQAALNIARSWLFLRSQEYKKYQTNKTTGNTDKRAFVETWQSFYRKKLKEVWK PAV (SEQ ID NO: 41826), or a sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto; and / or(e) the sequence of SEQ ID NO: 145, or a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

8. The polynucleotide of any one of claims 1-7, wherein the sequence encoding the CasX variant protein comprises a sequence selected from the group consisting of SEQ ID NOS: 40577-40588, or a sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

9. The polynucleotide of any one of claims 1-8, wherein the polynucleotide encodes one or more NLS linked to the sequence encoding the CasX variant protein,optionally wherein the one or more encoded NLS are selected from the group of sequences consisting of PKKKRKV (SEQ ID NO: 196), KRPAATKKAGQAKKKK (SEQ2021394878   03 Sep 2026ID NO: 197), PAAKRVKLD (SEQ ID NO: 248), RQRRNELKRSP (SEQ ID NO: 161), NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 162), RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 163), VSRKRPRP (SEQ ID NO: 164), PPKKARED (SEQ ID NO: 165), PQPKKKPL (SEQ ID NO: 166), SALIKKKKKMAP (SEQ ID NO: 167), DRLRR (SEQ ID NO: 168), PKQKKRK (SEQ ID NO: 169), RKLKKKIKKL (SEQ ID NO: 170), REKKKFLKRR (SEQ ID NO: 171), KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 172), RKCLQAGMNLEARKTKK (SEQ ID NO: 173), PRPRKIPR (SEQ ID NO: 174), PPRKKRTVV (SEQ ID NO: 175), NLSKKKKRKREK (SEQ ID NO: 176), RRPSRPFRKP (SEQ ID NO: 177), KRPRSPSS (SEQ ID NO: 178), KRGINDRNFWRGENERKTR (SEQ ID NO: 179), PRPPKMARYDN (SEQ ID NO: 180), KRSFSKAF (SEQ ID NO: 181), KLKIKRPVK (SEQ ID NO: 182), PKKKRKVPPPPAAKRVKLD (SEQ ID NO: 183), PKTRRRPRRSQRKRPPT (SEQ ID NO: 184), SRRRKANPTKLSENAKKLAKEVEN (SEQ ID NO: 41827), KTRRRPRRSQRKRPPT (SEQ ID NO: 186), RRKKRRPRRKKRR (SEQ ID NO: 187), PKKKSRKPKKKSRK (SEQ ID NO: 188), HKKKHPDASVNFSEFSK (SEQ ID NO: 189), QRPGPYDRPQRPGPYDRP (SEQ ID NO: 190), LSPSLSPLLSPSLSPL (SEQ ID NO: 191), RGKGGKGLGKGGAKRHRK (SEQ ID NO: 192), PKRGRGRPKRGRGR (SEQ ID NO: 193), PKKKRKVPPPPKKKRKV (SEQ ID NO: 195), PAKRARRGYKC (SEQ ID NO: 40188), KLGPRKATGRW (SEQ ID NO: 40189), PRRKREE (SEQ ID NO: 40190), PYRGRKE (SEQ ID NO: 40191), PLRKRPRR (SEQ ID NO: 40192), PLRKRPRRGSPLRKRPRR (SEQ ID NO: 40193), PAAKRVKLDGGKRTADGSEFESPKKKRKV (SEQ ID NO: 40194), PAAKRVKLDGGKRTADGSEFESPKKKRKVGIHGVPAA (SEQ ID NO: 40195), PAAKRVKLDGGKRTADGSEFESPKKKRKVAEAAAKEAAAKEAAAKA (SEQ ID NO: 40196), PAAKRVKLDGGKRTADGSEFESPKKKRKVPG (SEQ ID NO: 40710), KRKGSPERGERKRHW (SEQ ID NO: 40198), KRTADSQHSTPPKTKRKVEFEPKKKRKV (SEQ ID NO: 41828), and PKKKRKVGGSKRTADSQHSTPPKTKRKVEFEPKKKRKV (SEQ ID NO: 40200) wherein the one or more NLS are linked to the CRISPR variant or to adjacent NLS with a linker peptide wherein the linker peptide is selected from the group consisting of RS, (G)n (SEQ ID NO: 40201), (GS)n (SEQ ID NO: 40202), (GSGGS)n (SEQ ID NO: 208), (GGSGGS)n (SEQ ID NO: 209), (GGGS)n (SEQ ID NO: 210), GGSG (SEQ ID NO: 211),2021394878   03 Sep 2026GGSGG (SEQ ID NO: 212), GSGSG (SEQ ID NO: 213), GSGGG (SEQ ID NO: 214), GGGSG (SEQ ID NO: 215), GSSSG (SEQ ID NO: 216), GPGP (SEQ ID NO: 217), GGP, PPP, PPAPPA (SEQ ID NO: 218), PPPG (SEQ ID NO: 40207), PPPGPPP (SEQ ID NO: 219), PPP(GGGS)n (SEQ ID NO: 40203), (GGGS)nPPP (SEQ ID NO: 40204), AEAAAKEAAAKEAAAKA (SEQ ID NO: 40205), and TPPKTKRKVEFE (SEQ ID NO: 40206), wherein n is 1 to 5.

10. The polynucleotide of any one of claims 1-9, wherein:(a) the encoded first gRNA comprises a sequence selected from the group consisting of SEQ ID NOS: 2101-2285, 39981-40026, 40913-40958, and 41817, or a sequence having at least 85%, at least 90%, at least 95%, at least 95%, at least 96%, at least 97%, at least 98% identity thereto;(b) the encoded first gRNA comprises a targeting sequence complementary to a target nucleic acid sequence;(c) the polynucleotide further comprises a sequence encoding a second gRNA and a third promoter operably linked to the second gRNA;(d) the encoded first and / or second gRNA comprises a scaffold sequence having one or more modifications relative to SEQ ID NO: 2238, wherein the one or more modifications result in an improved characteristic in the expressed first and second gRNA;(e) the 5’ and 3’ ITRs are derived from serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV 44.9, AAV-Rh74, or AAVRh10;(f) the polynucleotide comprises one or more sequences selected from the group consisting of the sequences of SEQ ID NOs: 40370-40442, 41010-41029, 40599-40643, 40502-40509, 40511-40559, 40561-40594, 41030-41055, 40645, 40647-40655, 40657, 40664, 40675-40677, 40679, 40680, 40682, 40683, 40685, 40686, 40446-40481, 40485, 40487-40494, 40576, 40702-40718, 40749, 40752, 40753, 40756, 40757, 40762, and 4105641816, or a sequence having at least 85%, at least 90%, at least 95%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto; and / or(g) the polynucleotide has a construct configuration of a construct depicted in any one of FIGS. 24, 33-35, or 42.2021394878   03 Sep 202611. The polynucleotide of claim 10:(a) the targeting sequence is selected from the group consisting of SEQ ID NOS: 4105641776, or a sequence having at least 80%, at least 90%, or at least 95% sequence identity thereto;(b) the encoded second gRNA comprises a sequence selected from the group consisting of SEQ ID NOS: 2101-2285, 39981-40026, 40913-40958, and 41817, or a sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% identity thereto;(c) the encoded second gRNA comprises a targeting sequence complementary to a target nucleic acid sequence different than the target nucleic acid of the first gRNA,(d) the encoded second gRNA comprises a targeting sequence selected from the group consisting of SEQ ID NOS: 41056-41776, or a sequence having at least 80%, at least 90%, or at least 95% sequence identity thereto;(e) the improved characteristic is one or more functional properties selected from the group consisting of increased editing activity, increased pseudoknot stem stability, increased triplex region stability, increased scaffold stem stability, extended stem stability, reduced off-target folding intermediates, and increased binding affinity to a CasX variant protein, optionally in an in vitro assay; and / or(f) the 5’ and 3’ ITRs are derived from serotype AAV2.

12. A recombinant adeno-associated virus vector (rAAV) comprising:a. an AAV capsid protein, andb. the polynucleotide of any one of claims 1-11.

13. A pharmaceutical composition, comprising the rAAV of claim 12 and a pharmaceutically acceptable carrier, diluent or excipient.

14. A method for modifying a target nucleic acid in a population of mammalian cells in vitro or ex vivo, comprising contacting a plurality of the cells with an effective amount of the rAAV of claim 12, wherein the target nucleic acid of a gene of the cells targeted by the expressed gRNA is modified by the expressed variant CasX protein,2021394878   03 Sep 2026optionally wherein the modifying comprises introducing an insertion, deletion, substitution, duplication, or inversion of one or more nucleotides in the target nucleic acid of the cells of the population.

15. An effective amount of the rAAV of claim 12, wherein the target nucleic acid of a gene of the cells targeted by the expressed gRNA is modified by the expressed variant CasX protein, for use in a method for modifying a target nucleic acid in a population of mammalian cells, optionally wherein the modifying comprises introducing an insertion, deletion, substitution, duplication, or inversion of one or more nucleotides in the target nucleic acid of the cells of the population.

16. A therapeutically effective dose of the rAAV of claim 12 for use in a method of treating a disease in a subject caused by one or more mutations in a gene of the subject.

17. A method of making an rAAV vector, comprising:a. providing a population of packaging cells; andb. transfecting the population of cells with:i) a vector comprising the polynucleotide of any one of claims 1-11;ii) a vector comprising an aap (assembly) gene; andiii) a vector comprising rep and cap genomes.

Images