Operated Class 2 V CRISPR system

Abstract

Provided herein are engineered class 2 and type V nucleases and guide RNAs useful for editing target nucleic acids. Methods for making and using such variants to modify nucleic acids are also provided.

Description

[Technical Field]

[0001] Cross-references to related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 121,196 filed on 3 December 2020, No. 63 / 162,346 filed on 17 March 2021, and No. 63 / 208,855 filed on 9 June 2021, the contents of each of these applications being incorporated herein by reference in whole.

[0002] Reference to sequence listings The contents of the text file submitted electronically with this specification are incorporated herein by reference in their entirety: a computer-readable copy of the sequence listing (filename: SCRB_031_03WO_SEQLIST_ST25, date recorded: December 1, 2021, file size: 5.61 megabytes). [Background technology]

[0003] The CRISPR-Cas systems in bacteria and archaea confer forms of adaptive immunity against phages and viruses. Intensive research over the past decade has revealed the biochemistry of these systems. A CRISPR-Cas system consists of Cas proteins involved in the acquisition, targeting, and cleavage of foreign DNA or RNA, and a CRISPR array containing serial repeats flanked by short spacer sequences that guide the Cas proteins to their targets. Class 2 CRISPR-Cas is a streamlined version in which a single Cas protein bound to RNA is involved in binding to and cleaving the target sequence. The programmable nature of these minimal systems has facilitated their use as versatile technologies that have revolutionized the field of genomics.

[0004] To date, only a small number of widely used Class 2 CRISPR / Cas systems have been discovered. Of these, type V is unique in that it recognizes a 5' PAM sequence distinct from the 3' PAM sequence recognized by Cas9, and utilizes a RuvC-like endonuclease (RuvC) domain to form alternating cleavages in target nucleic acids having a 5' overhang of 5, 7, or 10 nt (Yang et al., PAM-dependent target DNA recognition and cleavage by C2c1 CRISPR-Cas endonuclease. Cell 167:1814 (2016)). However, wild-type Cas and guide sequences of type V have low editing efficiency. Therefore, there is a need in this field for further Class 2, type V CRISPR / Cas systems (e.g., combinations of Cas protein and guide RNA) that are optimized and / or offer improvements over previous generations of systems for use in various therapeutic, diagnostic, and research applications. [Overview of the Initiative]

[0005] This disclosure relates to guide ribonucleic acid (gRNA), engineered class 2, V CRISPR proteins, and systems of engineered class 2, V CRISPR proteins and guide ribonucleic acid (gRNA) used to modify target nucleic acids of genes in eukaryotic cells. In some embodiments, this disclosure provides engineered class 2, V proteins that include one or more modifications to the domain of reference CasX and exhibit one or more improved features compared to the reference CasX protein of SEQ ID NO: 2. In other embodiments, this disclosure provides engineered sequence variants of CasX variant proteins such as CasX491 (SEQ ID NO: 336) or CasX515 (SEQ ID NO: 416), wherein the class 2, V proteins include at least one modification to the domain of the CasX variant and exhibit one or more improved features compared to the CasX variant protein. In some embodiments, a class 2, type V variant can form a complex with a guide ribonucleic acid (gRNA), the complex can bind to and cleave a target nucleic acid, the target nucleic acid comprising a non-target strand and a target strand.

[0006] In some embodiments, the disclosure provides a guide ribonucleic acid (gRNA) comprising a single guide composition capable of binding to a class 2, type V variant protein, wherein the gRNA comprises at least one modification in a certain region compared to the gRNA of SEQ ID NO: 2238 or SEQ ID NO: 2239. In some embodiments, the modified region of the gRNA scaffold comprises (a) an elongation stem loop, (b) a scaffold stem loop, (c) a triple helix, and (d) a pseudoknot. In some cases, the scaffold elongation stem of the variant gRNA further comprises a modification to a bubble. In other cases, the gRNA scaffold further comprises a modification to a triple helix loop region. In other cases, the variant gRNA scaffold further comprises heterologous RNA in the elongation stem comprising a hairpin sequence.

[0007] In some embodiments, the Disclosure provides gene editing pairs comprising an manipulated class 2, type V protein and gRNA variant from any of the embodiments described herein, wherein the gene editing pairs exhibit at least one improved feature compared to gene editing pairs comprising a reference CasX protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 and a gRNA of SEQ ID NO: 4 or SEQ ID NO: 5. In certain embodiments, the manipulated class 2, type V protein includes a sequence selected from the group consisting of sequences 247-592 and 1147-1231 shown in Table 3, or a sequence having at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto, and the gRNA is a sequence selected from the group consisting of sequences 2101-2332 and 2353-2398 shown in Table 2, or a sequence having at least about 85%, at least about 90%, at least about 95%, 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 certain embodiments, the manipulated class 2, type V protein comprises a sequence selected from the group consisting of sequences 270-592 and 1147-1231, or a sequence having at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto, and the gRNA comprises a sequence selected from the group consisting of sequences 2238-2332 and 2353-2398, or a sequence having at least about 85%, at least about 90%, at least about 95%, 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 certain embodiments, the manipulated class 2, type V protein comprises a sequence selected from the group consisting of sequences 415-592 and 1147-1231, or a sequence having at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto, and the gRNA comprises a sequence selected from the group consisting of sequences 2281-2332 and 2353-2398, or a sequence having at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto.

[0008] In some embodiments, this disclosure provides polynucleotides and vectors encoding the manipulated class 2, type V variant proteins, gRNA variants, and gene editing pairs described herein. In some embodiments, the vector is a viral vector, such as an adeno-associated virus (AAV) vector. In other embodiments, the vector is a CasX delivery particle called an XDP containing the gene editing pair RNP.

[0009] In some embodiments, the disclosure provides cells comprising polynucleotides, vectors, engineered class 2, type V proteins, and gRNAs as described herein. In other embodiments, the disclosure provides cells comprising target nucleic acids edited by the methods of the editing embodiments described herein.

[0010] In some embodiments, this disclosure provides kits comprising polynucleotides, vectors, engineered class 2, type V proteins, gRNAs, and gene editing pairs as described herein.

[0011] In some embodiments, the present disclosure provides a method for editing a target nucleic acid, comprising contacting the target nucleic acid with a class 2, type V protein, and gRNA variant described herein, wherein the contact results in editing or modification of the target nucleic acid.

[0012] In some embodiments, the Disclosure provides a method for editing a target nucleic acid in a population of cells, comprising contacting the cells with one or more of the gene editing pairs described herein, wherein the contact results in editing or modification of the target nucleic acid in the population of cells.

[0013] In other embodiments, the present disclosure provides a method for treating a subject requiring treatment, which includes administering a gene editing pair or a vector containing or encoding a gene editing pair, as described in any embodiment herein.

[0014] In another embodiment, gene editing pairs, compositions containing gene editing pairs, or vectors containing or encoding gene editing pairs are provided herein for use as pharmaceuticals.

[0015] In another embodiment, gene editing pairs, compositions comprising gene editing pairs, or vectors comprising or encoding gene editing pairs are provided herein for use in therapeutic methods, the methods comprising editing or modifying a target nucleic acid, optionally, the editing being performed in a subject having a mutation in a gene allele, the mutation causing a disease or impairment in the subject, preferably, the editing changing the mutation to a wild-type allele of the gene, or knocking down or knocking out the gene allele causing the disease or impairment in the subject.

[0016] Reference All publications, patents, and patent applications referenced herein are incorporated herein by reference as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference. The contents of International Publications 2020 / 247882, 2020 / 247883, and 2021 / 113772, which disclose CasX variants and gRNA variants and methods for delivering them, are incorporated herein by reference in their entirety. [Brief explanation of the drawing]

[0017] The novel features of the present invention are described in detail in the attached claims. The features and advantages of the present invention will be better understood by referring to the following detailed description of exemplary embodiments utilizing the principles of the present invention, as well as by the attached drawings. [Figure 1] This graph shows the results of an assay to quantify the active fraction of RNP formed by sgRNA 174 (SEQ ID NO: 2238) and CasX variants 119, 457, 488, and 491, as described in Example 8. The sequences corresponding to the sgRNA and CasX variants are provided in Tables 2 and 3, respectively. Equimolar amounts of RNP and target were co-incubated, and the amount of cleaved target was determined at the indicated time points. The mean and standard deviation of three independent replicates are shown for each time point. The two-phase fit of the combined replicas is shown. "2" refers to the reference CasX protein of SEQ ID NO: 2. [Figure 2] As described in Example 8, the quantification of the active fraction of RNP formed by CasX2 (reference CasX protein of SEQ ID NO: 2) and modified sgRNA is shown. Equimolar amounts of RNP and target were co-incubated, and the amount of cleaved target was determined at the indicated time points. The mean and standard deviation of three independent replicates are shown for each time point. The two-phase fit of the combined replicas is shown. [Figure 3]As described in Example 8, the quantification of the active fraction of RNP formed by CasX491 and modified sgRNA under guide restriction conditions is shown. Equimolar amounts of RNP and target were co-incubated, and the amount of cleaved target was determined at the indicated time. Two-phase fit of the data is shown. [Figure 4] The quantification of the cleavage rate of RNPs formed by sgRNA174 and CasX variants is shown, as described in Example 8. Target DNA was incubated with a 20-fold excess of the indicated RNPs, and the amount of target cleaved was determined at the indicated time points. The mean and standard deviation of three independent repeats are shown for each time point, except for 488 and 491, where single repeats are shown. Monophase fitting of combined repeats is shown. [Figure 5] As described in Example 8, the quantification of the cleavage rate of RNP formed by CasX2 and sgRNA variants is shown. Target DNA was incubated with a 20-fold excess of the indicated RNP, and the amount of target cleaved was determined at the indicated time points. The mean and standard deviation of three independent replicates are shown for each time point. A uniphase fit of the combined replicates is shown. [Figure 6] As described in Example 8, the initial velocity of RNPs formed by CasX2 and sgRNA variants is quantified. The initial cleavage velocity was determined by fitting the first two time points of the previous cleavage experiment to a linear model. [Figure 7] As described in Example 8, the cleavage rate of RNPs formed by CasX491 and sgRNA variants is quantified. Target DNA was incubated with a 20-fold excess of the indicated RNPs at 10°C, and the amount of target cleaved was determined at the indicated time points. Single-phase fit at the time points is shown. [Figure 8] As described in Example 8, the quantification of competent fractions of RNPs from CasX variants 515 and 526 complexed with gRNA variant 174 is shown, compared to the RNPs of reference CasX 2 complexed with gRNA 2, using equimolar amounts of indicated RNPs and complementary targets. Two-phase fit is shown for each time course or combined set of replications. [Figure 9]As described in Example 8, the cleavage rates of RNPs of CasX variants 515 and 526 complexed with gRNA variant 174 are quantified using a 20-fold excess of the indicated RNPs, compared to the RNPs of reference CasX 2 complexed with gRNA 2. [Figure 10A] As described in Example 5, the quantification of the cleavage rate of the CasX variant against TTC PAM is shown. Target DNA substrates having the same spacer and the indicated PAM sequence were incubated with a 20-fold excess of the indicated RNP at 37°C, and the amount of cleaved target was determined at the indicated time point. Single repeat monophase fitting is shown. [Figure 10B] As described in Example 5, the quantification of the cleavage rate of the CasX variant against CTC PAM is shown. Target DNA substrates having the same spacer and the indicated PAM sequence were incubated with a 20-fold excess of the indicated RNP at 37°C, and the amount of cleaved target was determined at the indicated time point. Single repeat monophase fitting is shown. [Figure 10C] As described in Example 5, the quantification of the cleavage rate of the CasX variant against GTC PAM is shown. Target DNA substrates having the same spacer and the indicated PAM sequence were incubated with a 20-fold excess of the indicated RNP at 37°C, and the amount of target cleaved was determined at the indicated time point. Single repeat monophase fitting is shown. [Figure 10D] As described in Example 5, the quantification of the cleavage rate of the CasX variant against ATC PAM is shown. Target DNA substrates having the same spacer and the indicated PAM sequence were incubated with a 20-fold excess of the indicated RNP at 37°C, and the amount of target cleaved was determined at the indicated time point. Single repeat monophase fitting is shown. [Figure 11A] As described in Example 5, the quantification of the cutting rate of RNP of CasX variant 491 and guide 174 against NTC PAM is shown. Time points were acquired over a 2-minute period, and the cut fractions were graphed for each target and time point, but for clarity, only the first 2 minutes of the time course are shown. [Figure 11B]As described in Example 5, the quantification of the RNP cutting rate of CasX variant 491 and guide 174 against NTT PAM is shown. Time points were acquired over a 10-minute period, and the cut fractions were graphed for each target and time point. [Figure 12A] As described in Example 9, the quantification of cleavage by RNP formed by sgRNA174 and CasX variant 515 is shown using 18, 19, or 20 nucleotide-length spacers. Target DNA was incubated with a 20-fold excess of the indicated RNP, and the amount of cleaved target was determined at the indicated time points. The mean and standard deviation of three independent replicates are shown for each time point. A uniphase fit of combined replicates is shown. [Figure 12B] As described in Example 9, the quantification of cleavage by RNP formed by sgRNA174 and CasX variant 526 is shown using 18, 19, or 20 nucleotide spacers. Target DNA was incubated with a 20-fold excess of the indicated RNP, and the amount of cleaved target was determined at the indicated time points. The mean and standard deviation of three independent replicates are shown for each time point. A uniphase fit of combined replicates is shown. [Figure 13] This is a schematic diagram showing an example of the CasX protein and scaffold DNA sequence for packaging in adeno-associated virus (AAV). The DNA segments between AAV inverted terminal repeats (ITRs), consisting of the CasX-coding DNA and its promoter, as well as the scaffold-coding DNA and its promoter, are packaged within the AAV capsid during AAV production. [Figure 14]This figure shows the results of an editing assay comparing gRNA scaffolds 229–237 with scaffold 174 in mouse neural progenitor cells (mNPCs) isolated from Ai9-tdtomato transgenic mice, as described in Example 21. Cells were nucleofected with CasX491, scaffolds, and p59 plasmids encoding spacer 11.30 (5'AAGGGGCUCCGCACCACGCC3', SEQ ID NO: 17) that targets mRHO. Editing at the mRHO locus was evaluated by NGS 5 days after transfection, showing that editing with constructs containing scaffolds 230, 231, 234, and 235 demonstrated greater editing compared to constructs containing scaffold 174 at both doses. [Figure 15] As described in Example 21, the results of an editing assay comparing gRNA scaffolds 229–237 and scaffold 174 in mNPC cells are shown. Cells were nucleofected with CasX491, scaffolds, and p59 plasmid encoding spacer 12.7 (5'CUGCAUUCUAGUUGUGGUUU 3', SEQ ID NO: 1146), targeting repeat elements that prevent the expression of tdTomato fluorescent protein, at the indicated doses. Five days after transfection, editing was evaluated by FACS to quantify the percentage of tdTomato-positive cells. Cells nucleofected with scaffolds 231–235 showed approximately 35% greater editing at high doses and approximately 25% greater editing at low doses compared to constructs with scaffold 174. [Figure 16] This figure shows an exemplary method for constructing CasX protein and guide RNA variants of this disclosure using Deep Mutational Evolution (DME). In some exemplary embodiments, DME constructs and tests virtually all possible mutations, insertions, and deletions in biomolecules and their combinations / combinations, providing a nearly comprehensive and unbiased assessment of the biomolecular compatibility landscape and pathways in sequence space toward desired outcomes. As described herein, DME can be applied to both CasX protein and guide RNA. [Figure 17A] As shown in Example 13, the CryoEM structure of the Deltaproteobacteria CasX protein:sgRNA RNP complex (PDB ID:6YN2), which includes two stem-loops, a pseudoknot, and a triple helix, is shown. [Figure 17B] The secondary structure of the sgRNA of Sequence ID No. 4, identified from the structure shown in (A), is shown using the tool RNAPDBee2.0 (rnapdbee.cs.put.poznan.pl / ), the tool 3DNAS / DSSR, and the VARNA visualization tool. The RNA region is shown. Residues that were not evident in the PDB crystal structure file are indicated by plain text characters (i.e., not circled) and are not included in the residue numbering. [Figure 18] This is a schematic diagram of the regions and domains of the guide RNA used to design the scaffold library, as described in Example 13. [Figure 19] As described in Example 13, this is a pie chart of the relative distribution and design of scaffold libraries having both unbiased mutations (double and single mutations) and targeted mutations (for triple strands, scaffold stem bubbles, pseudoknots, and elongated stems and loops). [Figure 20] This is a schematic diagram of triple-strand mutagenesis designed to specifically incorporate alternating triple-strand forming base pairs into the triple hemisphere, as described in Example 13. Solid lines represent Watson-Crick pairs in the triple hemisphere. Nucleotides in the third strand are shown as dotted lines representing non-canonical interactions with purines in the double hemisphere. In the library, each of the five indicated positions was replaced with all possible triple-strand motif (G: GC, T: AT, G: GC) = 243 sequences. Sequence ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCANNNAUCAAAG (Sequence ID 1022). [Figure 21] As described in Example 13, this is a bar graph showing the results of the concentrated values ​​of reference guide scaffolds 174 and 175 in each screening. [Figure 22]As described in Example 13, this is a scatter plot showing the log2 enrichment values ​​for each measured single nucleotide substitution, deletion, or insertion, measured in each of two independent screenings of the mutant library for guide scaffolds 174 and 175. [Figure 23] As described in Example 13, this is a heatmap for single mutants in guide scaffolds 174 and 175, showing specific mutable regions in the scaffold across sequences. Yellow shading reflects values ​​with similar enrichment to the reference scaffold. Red shading indicates increased enrichment (and therefore activity) compared to the reference scaffold. Blue shading indicates loss of activity compared to the wild-type scaffold. White indicates missing data (or substitutions resulting in a wild-type sequence). [Figure 24] This is a scatter plot comparing the log2 enrichment of single nucleotide mutations on reference guide scaffolds 174 and 175, as described in Example 13. Only mutations to similar positions are shown between 174 and 175. The results, overall, suggest that guide scaffold 174 is more resistant to change than 175. [Figure 25] This is a bar graph showing the mean (and 95% confidence interval) log2 enrichment values ​​for a set of shuffled pseudoknot pairs, as described in Example 13, where each new pseudoknot has the same base pair composition but a different order within the stem. Each bar represents a set of scaffolds having the indicated G:A (or A:G) pair position (see figure on the right). 291 pseudoknot stems were tested. The numbers above the bars indicate the number of stems having the G:A (or A:G) pair at each position. [Figure 26] Figures 55 and 56 are schematic diagrams of pseudoknot sequences (given from 5' to 3'), with the two strand sequences separated by an underline. [Figure 27]As described in Example 13, this is a bar graph showing the mean (and 95% confidence interval) log2 enrichment values ​​for scaffolds, separated by the predicted stability of the secondary structure in the pseudoknot stem region. Scaffolds with very stable stems (e.g., ΔG < -7 kcal / mol) had high enrichment values ​​on average, while scaffolds with destabilized stems (ΔG ≥ -5 kcal / mol) had low enrichment values ​​on average. [Figure 28] As described in Example 13, this is a heatmap of all double mutants at positions 7 and 29 on scaffold 175. The pseudoknot sequence is given on the right side, from 5' to 3'. [Figure 29A] We present the editing results in ARPE-19 nucleofect cells using the manipulated guide 235 compared to 174, which has an 11.1 spacer (containing CasX491) that targets the P23 site of the Rho locus, demonstrating the improved activity of the 235 variant, which increased on-target activity in WT exogenous RHO without off-target cleavage (by a non-targeting spacer) in the mutant RHO reporter gene, as described in Example 21. [Figure 29B] This is a bar graph showing the scaling factor change of the editing level of p59.491.235.11.1, normalized to the benchmark p59.491.174.11.1 level (set to a value of 1.0) in ARPE-1 cells nucleofected with 1000 ng of each plasmid, as described in Example 21. [Figure 30]As described in Example 17, we present the results of editing assays comparing Cas nucleases 2, 119, 491, 515, 527, 528, 529, 530, and 531 in the custom HEK293 cell line PASS_V1.01. Cells were lipofected with 2 μg of p67 plasmid encoding the indicated Cas proteins. After 5 days, genomic DNA was extracted from the cells. PCR amplification and next-generation sequencing were performed to isolate and quantify the fraction of cells edited at custom-designed on-target editing sites. For each sample, editing was evaluated at target sites (individual dots) consisting of the following PAM sequences: individual sites of 48 TTCs, 14 ATCs, 22 CTCs, and 11 GTCs, and the percentage of editing (%) was normalized to the vehicle control. Cells lipofected with any of the nucleases showed higher mean editing at TTC PAM target sites (horizontal bars) than those of wild-type nuclease Cas2, except for Cas528. Furthermore, the relative priority of any given nuclease against four different PAM sequences is represented in a violin plot. In particular, Cas nucleases 527, 528, and 529 exhibit substantially different PAM priorities than those of the wild-type nuclease Cas2. [Figure 31]As described in Example 18, the results of an editing assay comparing improved Cas nuclease 491 with improved nucleases 532 and 533 in the custom HEK293 cell line PASS_V1.01 are shown. Cells were lipofected in double pairs with 2 μg of p67 plasmid encoding the indicated Cas protein and puromycin resistance gene, and grown under puromycin selectivity. After 3 days, genomic DNA was extracted from the cells. PCR amplification and next-generation sequencing were performed to isolate and quantify the fraction of cells edited at custom-designed on-target editing sites. For each sample, editing was evaluated at target sites consisting of the following PAM sequences: individual sites of 48 TTCs, 14 ATCs, 22 CTCs, and 11 GTCs, and the percentage of editing was normalized to the vehicle control. Cells lipofected with CasX532 or 533 showed higher mean editing than Cas491 at each of the PAM sequences in the TTC PAM target sites, except for CasX533. The error bars represent the standard error of the mean for a biological sample of n=2. [Figure 32] This is a graph of a survival assay to determine the selective stringency of CcdB selection to different spacers when targeted by CasX protein 515 and scaffold 174, as described in Example 14. [Figure 33A] As described in Example 14, this is a heatmap of CasX515 variants demonstrating neutral or improved biochemical cleavage for each variant at the TTC PAM target site, as an average of three spacers. The figure shows the results over the full length of the CasX515 sequence. [Figure 33B] As described in Example 14, this is a heatmap of CasX515 variants demonstrating neutral or improved biochemical cleavage for each variant at the TTC PAM target site, as an average of three spacers. The figure shows the results over the full length of the CasX515 sequence. [Figure 33C]As described in Example 14, this is a heatmap of CasX515 variants demonstrating neutral or improved biochemical cleavage for each variant at the TTC PAM target site, as an average of three spacers. The figure shows the results over the full length of the CasX515 sequence. [Figure 33D] As described in Example 14, this is a heatmap of CasX515 variants demonstrating neutral or improved biochemical cleavage for each variant at the TTC PAM target site, as an average of three spacers. The figure shows the results over the full length of the CasX515 sequence. [Figure 33E] As described in Example 14, this is a heatmap of CasX515 variants demonstrating neutral or improved biochemical cleavage for each variant at the TTC PAM target site, as an average of three spacers. The figure shows the results over the full length of the CasX515 sequence. [Figure 34A] As described in Example 14, this is a heatmap of CasX515 variants demonstrating neutral or improved biochemical cleavage for each variant at the CTC PAM target site, as an average of three biochemical cleavages in a single spacer (biological replicates). The figure shows the results over the full length of the CasX515 sequence. [Figure 34B] As described in Example 14, this is a heatmap of CasX515 variants demonstrating neutral or improved biochemical cleavage for each variant at the CTC PAM target site, as an average of three biochemical cleavages in a single spacer (biological replicates). The figure shows the results over the full length of the CasX515 sequence. [Figure 34C]As described in Example 14, this is a heatmap of CasX515 variants demonstrating neutral or improved biochemical cleavage for each variant at the CTC PAM target site, as an average of three biochemical cleavages in a single spacer (biological replicates). The figure shows the results over the full length of the CasX515 sequence. [Figure 34D] As described in Example 14, this is a heatmap of CasX515 variants demonstrating neutral or improved biochemical cleavage for each variant at the CTC PAM target site, as an average of three biochemical cleavages in a single spacer (biological replicates). The figure shows the results over the full length of the CasX515 sequence. [Figure 34E] As described in Example 14, this is a heatmap of CasX515 variants demonstrating neutral or improved biochemical cleavage for each variant at the CTC PAM target site, as an average of three biochemical cleavages in a single spacer (biological replicates). The figure shows the results over the full length of the CasX515 sequence. [Figure 35A] As described in Example 14, this is a heatmap of CasX515 variants demonstrating neutral or improved biochemical cleavage for each variant at the CTC PAM target site, as an average of three biological replicates in a single spacer. The figure shows the results over the full length of the CasX515 sequence. [Figure 35B] As described in Example 14, this is a heatmap of CasX515 variants demonstrating neutral or improved biochemical cleavage for each variant at the CTC PAM target site, as an average of three biological replicates in a single spacer. The figure shows the results over the full length of the CasX515 sequence. [Figure 35C]As described in Example 14, this is a heatmap of CasX515 variants demonstrating neutral or improved biochemical cleavage for each variant at the CTC PAM target site, as an average of three biological replicates in a single spacer. The figure shows the results over the full length of the CasX515 sequence. [Figure 35D] As described in Example 14, this is a heatmap of CasX515 variants demonstrating neutral or improved biochemical cleavage for each variant at the CTC PAM target site, as an average of three biological replicates in a single spacer. The figure shows the results over the full length of the CasX515 sequence. [Figure 35E] As described in Example 14, this is a heatmap of CasX515 variants demonstrating neutral or improved biochemical cleavage for each variant at the CTC PAM target site, as an average of three biological replicates in a single spacer. The figure shows the results over the full length of the CasX515 sequence. [Figure 36A] As described in Example 14, this is a heatmap of CasX515 variants demonstrating neutral or improved biochemical cleavage for each variant at the ATC PAM target site, as an average of three biological replicates in a single spacer. The figure shows the results over the full length of the CasX515 sequence. [Figure 36B] As described in Example 14, this is a heatmap of CasX515 variants demonstrating neutral or improved biochemical cleavage for each variant at the ATC PAM target site, as an average of three biological replicates in a single spacer. The figure shows the results over the full length of the CasX515 sequence. [Figure 36C] As described in Example 14, this is a heatmap of CasX515 variants demonstrating neutral or improved biochemical cleavage for each variant at the ATC PAM target site, as an average of three biological replicates in a single spacer. The figure shows the results over the full length of the CasX515 sequence. [Figure 36D]As described in Example 14, this is a heatmap of CasX515 variants demonstrating neutral or improved biochemical cleavage for each variant at the ATC PAM target site, as an average of three biological replicates in a single spacer. The figure shows the results over the full length of the CasX515 sequence. [Figure 36E] As described in Example 14, this is a heatmap of CasX515 variants demonstrating neutral or improved biochemical cleavage for each variant at the ATC PAM target site, as an average of three biological replicates in a single spacer. The figure shows the results over the full length of the CasX515 sequence. [Figure 37A] Regarding spacer 15.3, as described in Example 15, this graph shows the effect of spacer length on the ability to edit target nucleic acids using RNP in Jurkat cells. [Figure 37B] Regarding spacer 15.5, as described in Example 15, this graph shows the effect of spacer length on the ability to edit target nucleic acids using RNP in Jurkat cells. [Figure 38] As described in Example 16, this is a bar graph of selected CasX variant proteins and their editing efficiencies for four different PAM sequences (TTC, ATC, CTC, and GTC) in replicated samples. The data are shown as editing percentage (%) + / - standard deviation. [Figure 39] As described in Example 19, this bar graph shows the mean editing efficiency of the selected CasX nuclease compared to CasX491 at 48 different TTC PAM target sites. The mean propagation standard errors of the two experiments are plotted as error bars. Asterisks indicate a significant difference between CasX527 and CasX491 (p=0.0000635, by Welch's two-tailed t-test). [Figure 40]As described in Example 19, this is a diagram based on the published CryoEM structure of homologous reference CasX1 (SEQ ID NO: 1: Protein Databank Identifier: 6NY2), showing the target DNA PAM sequence, PAM interaction loop, NTSB domain, and the physical location of amino acid position 26. [Figure 41] This is a violin plot of the selected CasX variant protein and its editing efficiency at 48 TTC PAM target sites, as described in Example 19. [Figure 42] As described in Example 19, this is a bar graph of the editing efficiencies of selected CasX variant proteins compared to CasX491 at 48 TTC PAM target sites. The data are presented as mean relative editing efficiencies, with CasX491 editing equal to 1.0. The gray dashed line indicates the editing efficiency of CasX119. For replicated samples, the error is + / - propagation SEM. [Figure 43] As described in Example 20, this is a bar graph showing the average editing efficiency compared to CasX491 and the average specificity ratio of the selected CasX nuclease. [Figure 44] As described in Example 21, this flowchart shows the qualitative relationship between the combinations of mutations tested and their effects on both the activity and specificity of the resulting CasX variants. [Figure 45A] As described in Example 21, the results of an AAV-mediated editing assay comparing gRNA scaffold 235 and scaffold 174, and guides 11.30 and 11.31, at the endogenous mouse Rho exon 1 locus in mNPC across the MOI range are shown. [Figure 45B] As described in Example 21, the editing results are shown as a magnification change in the editing level of scaffold 235 compared to guide 174 (set to 1.0) with spacer 11.30 in cells infected with 5.0e+5MOI. [Figure 46]This is a schematic diagram showing the modifications made to the elongation stem loop in gRNA variant 175 incorporated into gRNA variant 235. Elongation stem loop of sgRNA175: Sequence ID 1285, Elongation stem loop of sgRNA235: Sequence ID 1286. [Figure 47] This is a schematic diagram of gRNA variant 235, showing modifications in the triple helix, scaffolding stem bubble, and elongation stem loop compared to gRNA variants 174 and 175. Pseudoknot and triple helix loop: SEQ ID NO: 1287, scaffolding stem and elongation stem: SEQ ID NO: 1288. [Figure 48] This is a schematic diagram showing the positions of bases within the MS2 hairpin, as described in Example 23. MS2 sequence in the diagram: Sequence ID 1289. [Figure 49] This graph shows the percentage of editing at the tdTomato locus, as measured by tdTomato fluorescence, for XDP packaged with the indicated scaffold variants, where gRNA scaffolds 188 and 251 function as base variants, as described in Example 23. Two MS2 versions (MS2 353 and MS2 WT) were used. [Figure 50] As described in Example 23, we show the improvement in EC50 values ​​determined using NanoSight for editing at the tdTomato locus in NPC, compared to the titer of XDP packaged with the indicated gRNA scaffold variants (scaffolds 188 and 251 served as base controls). Two MS2 versions, MS2 353 and MS2 wild type (WT), were used. [Figure 51] As described in Example 23, the correlation between MS2 hairpin affinity (KD) and EC50 is shown for XDP packaged with the indicated gRNA scaffold variant. [Figure 52] As described in Example 23, the correlation between MS2 hairpin affinity (KD) and titer is shown for XDP packaged with the indicated gRNA scaffold variant. [Modes for carrying out the invention]

[0018] Preferred embodiments of the present invention have been shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided merely as examples. Those skilled in the art will be able to conceive of numerous variations, modifications, and substitutions without departing from the present invention. It should be understood that various substitutes for the embodiments of the present invention described herein may be used when carrying out the present invention. The following claims define the scope of the present invention, and the methods and structures within these claims, as well as their equivalents, are intended to be encompassed thereby.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in which the present invention pertains. Any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of these embodiments, but preferred methods and materials are described below. In case of any conflict, the present specification, including the definitions, shall prevail. In addition, the materials, methods, and examples are illustrative and not intended to limit the scope of the invention. Those skilled in the art will be able to conceive of numerous variations, modifications, and substitutions without departing from the present invention.

[0020] definition The terms “polynucleotide” and “nucleic acid” are used interchangeably herein and refer to polymeric forms of nucleotides of any length, which are either ribonucleotides or deoxyribonucleotides. Accordingly, the terms “polynucleotide” and “nucleic acid” encompass polymers containing single-stranded DNA, double-stranded DNA, multi-stranded DNA, single-stranded RNA, double-stranded RNA, multi-stranded RNA, genomic DNA, cDNA, DNA-RNA hybrids, and purines and pyrimidine bases or other natural, chemically or biochemically modified, unnatural, or derivatized nucleotide bases.

[0021] The terms "hybridizable" and "complementary" are used interchangeably and mean that a nucleic acid (e.g., RNA, DNA) contains a sequence of nucleotides that allows it to non-covalently bond to another nucleic acid in a sequence-specific antiparallel manner (i.e., form Watson-Crick base pairs and / or G / U base pairs) under appropriate in vitro and / or in vivo conditions of temperature and ionic strength of solution, "annealing" or "hybridizing" (i.e., the nucleic acid specifically binding to a complementary nucleic acid). It is understood that the sequence of a polynucleotide does not need to be 100% complementary to the sequence of its target nucleic acid in order to be specifically hybridizable. It may have at least about 70%, at least about 80%, at least about 90%, or at least about 95% sequence identity and still be able to hybridize to the target nucleic acid sequence. Furthermore, a polynucleotide can hybridize across one or more segments without intervening or adjacent segments being involved in the hybridization event (e.g., loop or hairpin structures, "bulges," "bubbles," etc.).

[0022] For the purposes of this disclosure, “gene” includes DNA regions that encode a gene product (e.g., protein, RNA), and all DNA regions that regulate the production of the gene product, whether such regulatory sequences are adjacent to the coding and / or transcription sequences. Thus, a gene may include, but is not limited to, accessory elements, which include promoter sequences, terminators, translation regulatory sequences (e.g., ribosome binding sites and internal ribosome entry sites), enhancers, silencers, insulators, boundary elements, origins of replication, matrix attachment sites, and locus regulatory regions. The coding sequence encodes the gene product during transcription or transcription and translation. The coding sequence in this disclosure may include fragments of an open reading frame, but does not need to include the full length. A gene may include both the transcribed strand and a complementary strand containing the anticodon.

[0023] The term "downstream" refers to a nucleotide sequence located 3' to the reference nucleotide sequence. In certain embodiments, the downstream nucleotide sequence relates to the sequence following the transcription start site. For example, the translation start codon of a gene is located downstream of the transcription start site.

[0024] The term "upstream" refers to a nucleotide sequence located 5' to the reference nucleotide sequence. In certain embodiments, the upstream nucleotide sequence refers to a sequence located 5' to the coding region or transcription start site. For example, most promoters are located upstream of the transcription start site.

[0025] With respect to a polynucleotide or amino acid sequence, the term “adjacent to” refers to sequences that are adjacent to or adjacent to each other in a polynucleotide or polypeptide. Those skilled in the art will understand that two sequences can be considered adjacent to each other and still contain a limited number of intervening sequences, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides or amino acids.

[0026] The term “accessory element” is used herein interchangeably with the term “accessory sequence,” and is intended to include, among other things, polyadenylation signals (poly(A) signals), enhancer elements, introns, posttranscriptional regulatory elements (PTREs), nuclear localization signals (NLSs), deaminases, DNA glycosylase inhibitors, further promoters, factors that stimulate CRISPR-mediated homologous recombination repair (e.g., cis or trans), transcription activators or repressors, autocleavage sequences, and fusion domains (e.g., fusion domains fused to CRISPR proteins). It is understood that the selection of appropriate one or more accessory elements depends on the encoded component being expressed (e.g., protein or RNA) or on whether the nucleic acid contains multiple components requiring different polymerases or is not intended to be expressed as a fusion protein.

[0027] The term “promoter” refers to a DNA sequence containing a transcription initiation site and further sequences to facilitate polymerase binding and transcription. Exemplary eukaryotic promoters include elements such as the TATA box and / or B recognition element (BRE), which assist or promote the transcription and expression of the associated transcriptable polynucleotide sequence and / or gene (or transgene). Promoters may be synthetically produced or derived from known or naturally occurring promoter sequences or other promoter sequences. Promoters may be proximal or distal to the gene being transcribed. Promoters may also include chimeric promoters, which include combinations of two or more heterologous sequences to confer specific characteristics. Promoters of this disclosure may include variants of promoter sequences that are similar in composition but not identical to known or other promoter sequences provided herein. Promoters may be classified according to criteria relating to the expression pattern of the associated coding or transcriptable sequence or gene operably linked to the promoter (e.g., constitutive, developmental, tissue-specific, inducible, etc.). Promoters may also be classified according to their strength. When used in the context of promoters, "strength" refers to the transcription rate of the gene controlled by the promoter. A "strong" promoter means a high transcription rate, while a "weak" promoter means a relatively low transcription rate.

[0028] The promoters of this disclosure may be polymerase II (Pol II) promoters. Polymerase II transcribes all protein-coding genes and many non-coding genes. Typical Pol II promoters include a core promoter, which is a sequence of approximately 100 base pairs surrounding a transcription start site, and function as a binding platform for Pol II polymerase and related common transcription factors. The promoter may include one or more core promoter elements (e.g., 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.

[0029] The promoters of this disclosure may be polymerase III (Pol III) promoters. Pol III transcribes DNA to synthesize small ribosomal RNAs such as 5S rRNA, tRNA, and other small RNAs. Typical Pol III promoters use internal regulatory sequences (sequences within the transcribed portion of a gene) to support transcription, but upstream elements such as TATA boxes may also be used. All Pol III promoters are assumed to be within the scope of this disclosure.

[0030] The term "enhancer" refers to a regulatory DNA sequence that modulates the expression of a related gene when bound to a specific protein called a transcription factor. Enhancers may be located in the intron of a gene, or at the 5' or 3' of the gene's coding sequence. Enhancers may be located proximal to the gene (i.e., within tens or hundreds of base pairs (bp) of the promoter) or distal to the gene (i.e., thousands, hundreds of thousands, or even millions of bp away from the promoter). A single gene may be regulated by two or more enhancers, all of which are assumed to be within the scope of this disclosure.

[0031] As used herein, a “post-transcriptional regulatory element (PRE)” (e.g., a hepatitis PRE) refers to a DNA sequence that, when transcribed, generates a tertiary structure that may exhibit post-transcriptional activity, thereby enhancing or promoting the expression of an associated gene operably ligated to it.

[0032] As used herein, “post-transcriptional regulatory element (PTRE)” (e.g., hepatitis PTRE) means a DNA sequence that, when transcribed, generates a tertiary structure that may exhibit post-transcriptional activity that enhances or promotes the expression of an associated gene operably ligated thereto.

[0033] As used herein, “recombinant” 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 structurally recognizable coding or non-coding sequence from endogenous nucleic acids found in natural systems. Generally, DNA sequences encoding structural coding sequences can be assembled from cDNA fragments and short oligonucleotide linkers, or from a series of synthetic oligonucleotides, providing synthetic nucleic acids that can be expressed from recombinant transcription units contained in cells or cell-free transcription and translation systems. Such sequences may typically be provided in the form of an open reading frame uninterrupted by internal non-coding sequences or introns present in eukaryotic genes. Genomic DNA containing the relevant sequences can also be used in the formation of recombinant genes or transcription units. The non-coding DNA sequence may be located at 5' or 3' of the open reading frame, where such sequence does not interfere with the manipulation or expression of the coding region, but can actually act to regulate the production of the desired product by various mechanisms (see “enhancer” and “promoter” above).

[0034] The terms “recombinant polynucleotides” or “recombinant nucleic acids” refer to those that do not exist naturally, for example, those created through human intervention by artificially combining two otherwise separated sequences. This artificial combination is often achieved by either chemical synthesis or artificial manipulation of isolated nucleic acid segments (e.g., genetic engineering techniques). This can typically be done by substituting codons with degenerate codons encoding the same or conserved amino acids, while introducing or removing sequence recognition sites. Alternatively, it can be done by ligating nucleic acid segments with desired functions together to produce a combination of desired functions. This artificial combination is often achieved by either chemical synthesis or artificial manipulation of isolated nucleic acid segments (e.g., genetic engineering techniques).

[0035] Similarly, the terms “recombinant polypeptide” or “recombinant protein” refer to polypeptides or proteins that do not exist in nature, for example, those created through human intervention by artificially combining two segments of an otherwise separated amino sequence. Therefore, for example, a protein containing a heterologous amino acid sequence is a recombinant.

[0036] As used herein, the term “in contact” means to establish a physical connection between two or more entities. For example, bringing a target nucleic acid into contact with a guide nucleic acid means that the target nucleic acid and the guide nucleic acid are made to share a physical connection (for example, they can hybridize if their sequences share sequence similarity).

[0037] "Dissociation constant" or "K" d The terms "A" and "B" are used interchangeably and represent the affinity between ligand "L" and protein "P" (i.e., how strongly the ligand binds to a particular protein). The dissociation constant is given by formula K d It can be calculated using the formula =[L][P] / [LP], where [P], [L], and [LP] represent the molar concentrations of the protein, ligand, and complex, respectively.

[0038] This disclosure provides systems and methods useful for editing target nucleic acid sequences. As used herein, “editing” is used interchangeably with “modification” and includes, but is not limited to, cleavage, nicking, deletion, knock-in, knockout, and the like.

[0039] "Cleavage" refers to the disruption of the covalent backbone of a target nucleic acid molecule (e.g., RNA, DNA). Cleavage can be initiated by various means, including, but not limited to, enzymatic or chemical hydrolysis of phosphodiester bonds. Both single-strand and double-strand breaks are possible, and a double-strand break can result from two separate single-strand break events.

[0040] The term "knockout" refers to the elimination of a gene or its expression. For example, a gene may be knocked out by either the deletion or addition of a nucleotide sequence that results in the disruption of the leading frame. Alternatively, a gene may be knocked out by replacing a portion of the gene with an unrelated sequence. As used herein, the term "knockdown" refers to the reduction of the expression of a gene or its gene product. As a result of gene knockdown, protein activity or function may be reduced, or protein levels may be reduced or eliminated.

[0041] As used herein, “homology-directed repair” (HDR) refers to a form of DNA repair that occurs during the repair of double-strand breaks in cells. This process requires homology of nucleotide sequences and uses a donor template to repair or knock out target DNA, resulting in the transfer of genetic information from the donor to the target. Homologous recombination repair can result in alteration of the target sequence by insertion, deletion, or mutation if the donor template differs from the target DNA sequence and some or all of the donor template sequence is incorporated into the target DNA.

[0042] As used herein, "non-homologous end joining" (NHEJ) refers to the repair of double-strand breaks in DNA by directly ligating the broken ends together without requiring a homologous template (as opposed to homologous recombination repair, which requires homologous sequences to induce repair). NHEJ often results in the loss (deletion) of nucleotide sequences near the double-strand break site.

[0043] As used herein, “microhomology-mediated end joining” (MMEJ) refers to a mutagenic DSB repair mechanism that does not require a homologous template (as opposed to homologous recombination repair, which requires a homologous sequence to induce repair) and is always associated with a deletion adjacent to the cleavage site. MMEJs often result in the loss (deletion) of nucleotide sequences near the double-strand break site. A polynucleotide or polypeptide has a certain percentage of “sequence similarity” or “sequence identity” with another polynucleotide or polypeptide, which means the percentage of the same bases or amino acids at the same relative positions when the two sequences are compared (aligned). Sequence similarity (also called percentage similarity, percentage identity, or homology) can be determined in several different ways. To determine sequence similarity, sequences can be aligned using methods and computer programs known in the art, including BLAST, which is available on the World Wide Web at ncbi.nlm.nih.gov / BLAST. Percent complementarity between specific stretches of nucleic acid sequences within a nucleic acid can be determined using any convenient method. Examples of methods include the BLAST program (a basic local sorting search tool) and the PowerBLAST program (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.), for example, using the default settings that employ the Smith and Waterman algorithm (Adv.Appl.Math., 1981, 2, 482-489)).

[0044] The terms “polypeptide” and “protein” are used interchangeably herein and refer to polymeric forms of amino acids of any length, which may include coding and non-coding amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having a modified peptide backbone. The term also includes fusion proteins (including, but not limited to, fusion proteins having heterologous amino acid sequences).

[0045] A "vector" or "expression vector" is a replicon such as a plasmid, phage, virus, or cosmid, to which another DNA segment (i.e., an "insert") can be attached, resulting in the replication or expression of the attached segment in a cell.

[0046] As used herein, the terms “naturally occurring,” “unmodified,” or “wild-type” refer to nucleic acids, polypeptides, cells, or organisms that are found in nature, when applied to nucleic acids, polypeptides, cells, or organisms.

[0047] As used herein, “mutation” means the insertion, deletion, substitution, duplication, or inversion of one or more amino acids or nucleotides compared to the wild-type or reference amino acid sequence, or compared to the wild-type or reference nucleotide sequence.

[0048] As used herein, the term “isolated” means a polynucleotide, polypeptide, or cell in an environment different from the environment in which the cell naturally exists. Isolated genetically modified host cells may be present in a mixed population of genetically modified host cells.

[0049] As used herein, “host cell” means a eukaryotic cell, prokaryotic cell, or cell derived from a multicellular organism (e.g., a cell line), and eukaryotic or prokaryotic cells include offspring of the original cell that has been genetically modified by the nucleic acid, used as the recipient of the nucleic acid (e.g., an expression vector). It is understood that single-cell offspring may not necessarily be completely identical to the original parent in morphology or in genomic or total DNA complementation, due to natural, accidental, or intentional mutations. A “recombinant host cell” (also referred to as a “genetically modified host cell”) is a host cell into which a different nucleic acid (e.g., an expression vector) has been introduced.

[0050] As used herein, the term “tropism” refers to the preferential entry of a virus-like particle (XDP, which may also be referred to herein as XDP) into a particular cell or tissue type, and / or preferential interaction with a cell surface that facilitates entry into a particular cell or tissue type, followed by the selective and preferably, expression of a sequence delivered to the cell by the XDP (e.g., transcription and selective translation).

[0051] As used herein, the terms “pseudotype” or “pseudotyping” refer to a viral envelope protein that has been replaced with the envelope protein of another virus having preferred characteristics. For example, HIV can be pseudotyped with the vesicular stomatitis virus G-protein (VSV-G) envelope protein (as described below herein, among other things), which allows HIV to infect a wider range of cells, as the HIV envelope protein primarily targets CD4+-presenting cells.

[0052] As used herein, the term “tropism factor” refers to a component incorporated into the surface of XDP that provides tropism to a particular cell or tissue type. Non-limiting examples of tropism factors include glycoproteins, antibody fragments (e.g., scFv, nanobodies, linear antibodies, etc.), receptors, and ligands for target cell markers.

[0053] "Target cell markers" refer to molecules expressed by target cells and include, but are not limited to, cell surface receptors, cytokine receptors, antigens, tumor-associated antigens, glycoproteins, oligonucleotides, enzyme substrates, antigenic determinants, or binding sites (which may be present on the surface of target tissues or cells that can function as ligands for antibody fragments or glycoprotein tropism factors).

[0054] The term "conservative amino acid substitution" refers to the interchangeability of amino acid residues with similar side chains in proteins. For example, amino acids with aliphatic side chains include glycine, alanine, valine, leucine, and isoleucine; amino acids with aliphatic hydroxyl side chains include serine and threonine; amino acids with amide-containing side chains include asparagine and glutamine; amino acids with aromatic side chains include phenylalanine, tyrosine, and tryptophan; amino acids with basic side chains include lysine, arginine, and histidine; and amino acids with sulfur-containing side chains include cysteine ​​and methionine. Exemplary conservative amino acid substitutions include valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.

[0055] As used herein, the term “antibody” encompasses a wide range of antibody structures, including, but is not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), nanobodies, single-domain antibodies (e.g., VHH antibodies), and antibody fragments (insofar as they exhibit the desired antigen-binding activity or immunological activity). Antibodies represent a large family of molecules, including several types of molecules such as IgD, IgG, IgA, IgM, and IgE.

[0056] An "antibody fragment" refers to a molecule other than the intact antibody that contains a portion of an intact antibody and binds to 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, single-domain antibodies, single-domain camel antibodies, single-chain variable fragment (scFv) antibody molecules, and multispecific antibodies (formed from antibody fragments).

[0057] As used herein, “treatment” or “to treat” is used interchangeably herein and refers to an approach to obtain beneficial or desired outcomes, including but not limited to therapeutic and / or preventive benefits. Therapeutic benefits mean the eradication or improvement of the underlying disorder or disease being treated. Therapeutic benefits can also be achieved by the eradication or improvement of one or more symptoms associated with the underlying disease, or by improvement of one or more clinical parameters, such that improvement is observed in the subject, even though the subject may still be suffering from the underlying disease.

[0058] The terms “therapeutic dose” and “therapeutic dosage,” as used herein, refer to the amount of a drug or bioagent, either alone or as part of a composition, that, when administered to a subject (e.g., a human or an experimental animal) in a single dose or repeated dose, can have any detectable beneficial effect on any symptom, aspect, measured parameter, or characteristic of a disease state or condition. Such effect does not need to be absolute to be beneficial.

[0059] As used herein, “administer” means a method of giving a subject a dosage of a compound (e.g., a composition of this disclosure) or a composition (e.g., a pharmaceutical composition).

[0060] The "subjects" are mammals. These include, but are not limited to, livestock, non-human primates, humans, dogs, rabbits, mice, rats, and other rodents.

[0061] All publications, patents, and patent applications referenced herein are incorporated herein by reference as if each individual publication, patent, or patent application were specifically and individually indicated to be invoked by reference.

[0062] I. General Methods The implementation of this invention will, unless otherwise indicated, utilize prior art in immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, including standard textbooks such 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 This can be found in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998) (their disclosures are incorporated herein by reference).

[0063] Where a range of values ​​is provided, it is understood that the endpoint is included, and that each intermediary value between the upper and lower limits of that range encompasses any other listed or intermediary values ​​within that range, up to one-tenth of the lower limit unit, unless the context otherwise explicitly indicates otherwise. The upper and lower limits of these smaller ranges may independently be included within smaller ranges and are also included according to any specifically excluded limits within the listed range. If a listed range includes one or both limits, it also includes ranges that exclude one or both of those included limits.

[0064] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art in which the present invention pertains. All publications referenced herein are incorporated herein by reference to disclose and describe the methods and / or materials by which the publications are cited.

[0065] Note that, as used herein and in the appended claims, the singular forms "a," "an," and "the" refer to multiple objects unless the context clearly indicates otherwise.

[0066] For clarity, it will be understood that certain features of the Disclosure described in the context of separate embodiments may be provided in combination in a single embodiment. In other cases, for brevity, various features of the Disclosure described in the context of a single embodiment may be provided separately or in any preferred partial combination. All combinations of embodiments relating to the Disclosure are specifically encompassed by the Disclosure and are intended to be disclosed herein as if every possible combination were disclosed individually and expressly. In addition, all partial combinations of various embodiments and their elements are also specifically encompassed by the Disclosure and are disclosed herein as if every possible partial combination were disclosed herein individually and expressly.

[0067] II. Systems for gene editing and gene editing pairs In a first aspect, the Disclosure provides a system comprising a class 2, type V CRISPR nuclease protein and one or more guide nucleic acids (e.g., gRNAs) for use in modifying or editing a target nucleic acid (including coding and non-coding regions) of a gene. Generally, any portion of a gene can be targeted using the programmable systems and methods provided herein. Where used herein, “systems” such as a CRISPR nuclease protein and one or more gRNAs of the Disclosure as a gene editing pair, a nucleic acid encoding the CRISPR nuclease protein and gRNAs, and a system comprising a vector containing the nucleic acid or CRISPR nuclease protein and one or more gRNAs of the Disclosure are used interchangeably with the term “composition.”

[0068] In some embodiments, this disclosure provides systems specifically designed to modify target nucleic acids of genes in eukaryotic cells, either in vitro, ex vivo, or in vivo. Generally, any portion of a gene can be targeted using the programmable systems and methods provided herein. In some embodiments, the CRISPR nuclease is a class 2, type V nuclease. While there are differences among the members of class 2 type V CRISPR-Cas nucleases, they share several common features that distinguish them from the Cas9 system. First, type V nucleases have a single RNA-inducible effector containing a RuvC domain but lacking an HNH domain, and recognize the PAM of a TC motif 5' upstream of the target region on the non-target strand. This differs from the Cas9 system, which relies on a G-rich PAM at the 3' side of the target sequence. Unlike Cas9, which produces a blunt end proximal to the PAM, type V nucleases produce a double-strand break distal to the PAM sequence. In addition, when activated by a cis-bound target dsDNA or ssDNA, the V-type nuclease degrades the ssDNA in trans. In some embodiments, the disclosure provides a class 2, V-type nuclease selected from the group consisting of Cas12a, Cas12b, Cas12c, Cas12d (CasY), Cas12j, Cas12k, C2c4, C2c8, C2c5, C2c10, C2c9, CasZ, and CasX. In some embodiments, the disclosure provides a system comprising one or more CasX variant proteins and one or more guide nucleic acid (gRNA) variants as a CasX:gRNA system.

[0069] A system comprising a class 2, type V protein and a gRNA variant, referred to herein as a gene editing pair, is provided herein. In some embodiments, the class 2, type V variant is a CasX variant (e.g., the sequence of SEQ ID NO: 416, but not limited to this). The terms CasX variant protein and CasX variant are used interchangeably herein. In some embodiments, the gRNA is a variant of another gRNA (e.g., the sequences of SEQ ID NOs: 2238 and 2239, but not limited to this). The gRNA and CasX protein can bind together via non-covalent interactions to form a gene editing pair complex (referred herein as a ribonucleoprotein (RNP) complex). In some embodiments, the use of a pre-complexed CasX:gRNA RNP provides advantages in the delivery of system components to cells or target nucleic acids for editing the target nucleic acid. In the RNP, the gRNA can provide target specificity to the RNP complex by including a targeting sequence (or "spacer") having a nucleotide sequence complementary to the sequence of the target nucleic acid sequence. In RNPs, the CasX protein of the pre-complexed CasX:gRNA provides specific activity and, upon association with the gRNA, is directed to a target site within the target nucleic acid sequence to be modified (e.g., further stabilized at the target site). The CasX variant protein of the RNP complex provides site-specific activity of the complex, such as binding, cleavage, or nicking of the target sequence by the CasX protein. This specification provides systems and cells comprising the CasX variant protein, gRNA variant, and CasX:gRNA gene editing pairs of any combination of CasX variant and gRNA variant as described herein, as well as a delivery mode containing CasX:gRNA. The use of each of these components and their use in editing target nucleic acids of genes is described herein below.

[0070] In some embodiments, the Disclosure provides a system of gene editing pairs comprising CasX variant proteins selected from any one of the CasX variant proteins (SEQ ID NOs. 247-592 and 1147-1231) listed in Table 3, or sequences having at least about 85%, at least about 90%, at least about 95%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereto, while the gRNA is as described herein. The gRNA variant is a gRNA variant (e.g., SEQ ID NOs. 2101-2332 and 2353-2398 shown in Table 2), or a sequence variant having at least 60%, at least 70%, at least about 80%, at least about 90%, or at least about 95% sequence identity thereto, wherein the gRNA contains a targeting sequence complementary to the target nucleic acid. In some embodiments, the disclosure includes a CasX variant protein selected from any one of the CasX variant proteins (SEQ ID NOs. 270-592 and 1147-1231) in Table 3. The present disclosure provides a gene editing pair system in which the gRNA is a gRNA variant described herein (e.g., SEQ ID NOs. 2238-2332 and 2353-2398), and the gRNA contains a targeting sequence complementary to the target nucleic acid. In some embodiments, the present disclosure provides a gene editing pair system comprising a CasX variant protein selected from any one of the CasX variant proteins in Table 3 (SEQ ID NOs. 415-592 and 1147-1231), and the gRNA is a gRNA variant described herein (e.g., sequence number (Sequence numbers 2281-2332 and 2353-2398), wherein the gRNA contains a targeting sequence complementary to the target nucleic acid. In other embodiments, the Disclosure provides a gene editing pair system comprising a CasX variant protein, a first gRNA variant described herein having a targeting sequence (e.g., SEQ ID NOs. 2101-2332 or 2353-2398 shown in Table 2), and a second gRNA variant, wherein the second gRNA variant has a targeting sequence complementary to a different or overlapping portion of the target nucleic acid compared to the targeting sequence of the first gRNA.In other embodiments, the Disclosure provides a system of gene editing pairs comprising a CasX variant protein, a first gRNA variant described herein having a targeting sequence (e.g., SEQ ID NOs. 2101-2332 or 2353-2398), and a second gRNA variant, wherein the second gRNA variant has a targeting sequence complementary to a different or overlapping portion of the target nucleic acid compared to the targeting sequence of the first gRNA. In some embodiments of the CasX:gRNA gene editing pairs of this disclosure, the CasX variant protein is selected from the group consisting of CasX variant proteins 515, 528, 529, 534-539, 668, 672, and 678 (SEQ ID NOs. 416, 428, 434-439, 567, 570, and 576) in Table 3, and the sgRNA variant is selected from the group consisting of gRNA variants 229-237 (SEQ ID NOs. 2286-2294) in Table 2. In certain embodiments, the gene editing pair comprises a CasX variant protein selected from any one of CasX variant proteins 668 (SEQ ID NOs. 567), 672 (SEQ ID NOs. 570), or 676 (SEQ ID NOs. 574), and a gRNA variant 235 (SEQ ID NOs. 2292).

[0071] In some embodiments, gene editing pairs can associate together to form a ribonucleoprotein complex (RNP). In other embodiments, gene editing pairs associate together within the ribonucleoprotein complex (RNP). In some embodiments, the RNP of a gene editing pair can bind to and cleave the double strand of a target nucleic acid containing coding sequences, complements to coding sequences, non-coding sequences, and regulatory elements. In some embodiments, the RNP of a gene editing pair can bind to the target nucleic acid and generate one or more single-stranded nicks in the target nucleic acid. In some embodiments, the RNP of a gene editing pair can bind to the target nucleic acid but cannot cleave it.

[0072] In some embodiments, the variant gene editing pair has one or more improved features compared to a reference gene editing pair comprising a reference CasX protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 and a reference gRNA of SEQ ID NO: 5 or SEQ ID NO: 4. In other embodiments, the variant gene editing pair of a CasX variant and a gRNA variant has one or more improved features compared to a gene editing pair comprising a CasX variant from which the variant is derived (e.g., CasX515, SEQ ID NO: 416) and a gRNA variant from which the variant is derived (e.g., gRNA scaffold 174 (SEQ ID NO: 2238) or 175 (SEQ ID NO: 2239)). In the embodiments described above, one or more improved features can be assayed in an in vitro assay under equivalent conditions for the gene editing pair as well as for the reference CasX and reference gRNA. As described herein, exemplary improved features include, in some embodiments, stability of the CasX:gRNA RNP complex, increased binding affinity between CasX and gRNA, improved dynamics of RNP complex formation, a higher percentage of cleavage-competent RNPs, increased RNP binding affinity to target nucleic acids, unwinding of target nucleic acids, increased editing activity, increased editing efficiency, increased editing specificity to target nucleic acids, reduced off-target editing or cleavage, increased nuclease activity, increased target strand loading for double-strand breaks, reduced target strand loading for single-strand nicking, increased binding of non-target strands of DNA, or increased resistance to nuclease activity. In the embodiments described above, the improvement is at least about 2 times, at least about 5 times, at least about 10 times, at least about 50 times, at least about 100 times, at least about 500 times, at least about 1000 times, at least about 5000 times, at least about 5000 times, at least about 1000 times, at least about 10,000 times, at least about 10,000 times, or at least about 100,000 times compared to the features of the reference CasX protein and reference gRNA pair, or the features of the CasX variant and gRNA variant from which the gene editing pair originated.In other cases, one or more of the improved features are approximately 1.1-100,000 times, 1.1-10,000 times, 1.1-1,000 times, 1.1-500 times, 1.1-100 times, 1.1-50 times, 1.1-20 times, 10-100,000 times, 10-10,000 times, 10-1,000 times, 10-500 times, 10-100 times, 10-50 times, 10-20 times, 2-70 times, 2-50 times, 2-30 times, 2-20 times, 2- 10 times, about 5 to 50 times, about 5 to 30 times, about 5 to 10 times, about 100 to 100,00 times, about 100 to 10,00 times, about 100 to 1,000 times, about 100~500x, approx. 500~100,00x, approx. 500~10,00x, approx. 500~1,000x, approx. 500~750x, approx. 1,000~ Improvements of 100,000 times, approximately 10,000 to 100,000 times, approximately 20 to 500 times, approximately 20 to 250 times, approximately 20 to 200 times, approximately 20 to 100 times, approximately 20 to 50 times, approximately 50 to 10,000 times, approximately 50 to 1,000 times, approximately 50 to 500 times, approximately 50 to 200 times, or approximately 50 to 100 times are possible. In other cases, one or more of the improved features were approximately 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, 25 times, 30 times, 40 times, 45 times, 50 times, compared to the features of the reference gene-edited pair, or the CasX variant and gRNA variant from which the gene-edited pair originated. It may be improved by 55 times, 60 times, 70 times, 80 times, 90 times, 100 times, 110 times, 120 times, 130 times, 140 times, 150 times, 160 times, 170 times, 180 times, 190 times, 200 times, 210 times, 220 times, 230 times, 240 times, 250 times, 260 times, 270 times, 280 times, 290 times, 300 times, 310 times, 320 times, 330 times, 340 times, 350 times, 360 times, 370 times, 380 times, 390 times, 400 times, 425 times, 450 times, 475 times, or 500 times or more.

[0073] In some embodiments, the gene editing pair comprises both the CasX variant protein and the gRNA variant described herein, and one or more features of the gene editing pair are improved beyond what can be achieved by altering the CasX protein or gRNA alone. In some embodiments, the CasX variant protein and the gRNA variant act additively to improve one or more features of the gene editing pair. In some embodiments, the CasX variant protein and the gRNA variant act synergistically to improve one or more features of the gene editing pair. In the embodiments described above, the improvement is at least about 2 times, at least about 5 times, at least about 10 times, at least about 50 times, at least about 100 times, at least about 500 times, at least about 1000 times, at least about 5000 times, at least about 1000 times, at least about 5000 times, at least about 10,000 times, or at least about 100,000 times compared to the features of the reference CasX protein and reference gRNA pair, or the features of the CasX variant and gRNA variant from which the gene editing pair is derived.

[0074] In some embodiments, the present disclosure provides a composition of any of the gene editing pairs disclosed herein for use as a pharmaceutical for the treatment of a subject having a disease.

[0075] In other embodiments, the system of the Disclosure comprises one or more CasX variant proteins, one or more guide nucleic acids (gRNAs), and one or more donor template nucleic acids comprising nucleic acids encoding a portion of a gene, wherein the donor template nucleic acid comprises a wild-type sequence for correcting a mutation, or comprises one or more nucleotide deletions, insertions, or mutations compared to a wild-type genomic nucleic acid sequence for knocking down or knocking out a gene.

[0076] In other embodiments, the disclosure provides CasX variants, gRNA variants, and vectors encoding or containing donor templates for the production and / or delivery of the CasX:gRNA system. Methods for producing CasX variant proteins and gRNA variants, including gene editing methods and therapeutic methods, as well as methods for using the CasX variants and gRNA variants, are also provided herein. The CasX variant protein and gRNA variant components of the CasX:gRNA system, their characteristics, and delivery modes and methods for using the system are described in more detail below.

[0077] The donor templates for the CasX:gRNA system are designed to be used to correct mutations in a target gene, to insert a transgene at a different locus in the genome ("knock-in"), or to disrupt the expression of an abnormal gene product (e.g., including one or more mutations that reduce gene product expression or cause protein dysfunction) ("knockdown" or "knockout"). In some embodiments, the donor template is a single-stranded DNA template or a single-stranded RNA template. In other embodiments, the donor template is a double-stranded DNA template. In some embodiments, the CasX:gRNA system used in editing a target nucleic acid includes a donor template having all or at least part of the open reading frame of the gene in the target nucleic acid for insertion of a modified wild-type sequence to correct a defective protein. In other cases, the donor template includes all or part of the wild-type gene for insertion at a different locus in the genome for gene product expression. In other cases, a portion of the gene may be inserted upstream of the mutation in the target nucleic acid ('5), and the donor template gene portion extends to the C-terminus of the gene or the 3' end of the mutated sequence, resulting in the expression of a functional gene product upon insertion into the target nucleic acid.

[0078] In some embodiments, the donor template sequence includes non-homologous sequences adjacent to two homologous regions (i.e., homologous arms) on the 5' and 3' sides of the cleavage site of the target nucleic acid, facilitating the insertion of non-homologous sequences in the target region, which can be mediated by homologous recombination repair (HDR) or homology-independent targeted integration (HITI). The exogenous donor template inserted by HITI may be of any length, e.g., a relatively short sequence of 10–50 nucleotides, or a longer sequence of about 50–1000 nucleotides. Lack of homology may, for example, have less than 20–50% sequence identity and / or lack specific hybridization with low strictness. In other cases, lack of homology may further include the criterion of having less than 5, 6, 7, 8, or 9 bp identity. In such cases, the use of homologous arms facilitates the insertion of non-homologous sequences at the cleavage site introduced by the nuclease. In some embodiments, the donor template polynucleotide comprises at least about 10, at least about 50, at least about 100, or at least about 200, or at least about 300, or at least about 400, or at least about 500, or at least about 600, or at least about 700, or at least about 800, or at least about 900, or at least about 1000, or at least about 10,000, or at least about 15,000 nucleotides. In other embodiments, the donor template comprises at least about 10 to about 15,000 nucleotides, or at least about 100 to about 10,000 nucleotides, or at least about 400 to about 8,000 nucleotides, or at least about 600 to about 5,000 nucleotides, or at least about 1,000 to about 2,000 nucleotides. The donor template sequence may contain differences from the genome sequence, such as specific sequences, restriction sites, nucleotide polymorphisms, and selection markers (e.g., drug resistance genes, fluorescent proteins, enzymes, etc.), which can be used to assess the success of donor nucleic acid insertion at cleavage sites, or, in some cases, for other purposes (e.g., to indicate expression at a target genomic locus).Alternatively, these sequence differences may include adjacent recombinant sequences (e.g., FLP sequences, loxP sequences, etc.) that can be activated later to remove marker sequences.

[0079] III. Guide to Systems for Gene Editing: Nucleic Acids In another aspect, the disclosure relates to a specially designed guide ribonucleic acid (gRNA) comprising a targeting sequence (also referred to herein as a spacer) that is complementary to (and therefore can hybridize with) a target nucleic acid sequence of a gene, which, when complexed with a CRISPR nuclease, is useful in genome editing of the target nucleic acid in cells. In some embodiments, it is envisioned that multiple gRNAs are delivered in the system for modification of the target nucleic acid. For example, a pair of gRNAs having targeting sequences for different or overlapping regions of the target nucleic acid sequence can be used, each complexed with a CRISPR nuclease to bind and cleave at two different or overlapping sites within a gene, and the gene is then edited by non-homologous end joining (NHEJ), homologous recombination repair (HDR), homology-independent targeted integration (HITI), microhomology-mediated end joining (MMEJ), single-strand annealing (SSA), or base excision repair (BER).

[0080] In some embodiments, this disclosure provides gRNAs that are utilized in a system, which have utility in genome editing of genes in eukaryotic cells. In certain embodiments, the gRNAs of the system can form complexes (ribonucleoprotein (RNP) complexes) with CRISPR nucleases, which are described in more detail below.

[0081] a. Reference gRNA and gRNA variants As used herein, “reference gRNA” refers to a CRISPR guide nucleic acid containing a wild-type sequence of a naturally occurring gRNA. In some embodiments, the reference gRNA of this disclosure may be subjected to one or more mutagenesis methods, for example, the mutagenesis methods described herein in the Examples (e.g., Example 13, and International Application PCT / US20 / 36506 and International Publication 2020247883(A2) incorporated herein by reference), to generate one or more guide nucleic acid variants (hereinafter referred to herein as “gRNA variants”) that are enhanced or altered compared to the reference gRNA, such mutagenesis methods include Deep Mutational Evolution (DME), Deep Mutational Scanning (DMS), Error Prone PCR, Cassette Mutagenesis, Random Mutagenesis, Staggered Extension PCR, Gene Shuffling, or Domain Swapping. gRNA variants also include variants containing one or more exogenous sequences, for example, fused to or inserted into either the 5' or 3' end. The activity of a reference gRNA or a variant derived therefrom may be used as a benchmark against which the activity of the gRNA variant is compared, thereby measuring improvements in the function or other characteristics of the gRNA variant. In other embodiments, the reference gRNA or gRNA variant may be subjected to one or more intentionally targeted mutations to produce a gRNA variant (e.g., a rationally designed variant).

[0082] The gRNAs of this disclosure comprise two segments: a targeting sequence and a protein-binding segment. The targeting segment of the gRNA comprises a nucleotide sequence (replaced, referred to interchangeably, as a guide sequence, spacer, targeter, or targeting sequence) that is complementary to (and therefore hybridizes with) a specific sequence (target site) within a target nucleic acid sequence (e.g., a target ssRNA, target ssDNA, or strand of a double-stranded target DNA) as described in more detail below. The targeting sequence of the gRNA can bind to the target nucleic acid sequence (including coding sequences, complements to coding sequences, and non-coding sequences) and regulatory elements. The protein-binding segment (or “activator” or “protein-binding sequence”) interacts with (e.g., binds to) the CasX protein as a complex forming an RNP (as described in more detail below). Alternatively, the protein-binding segment is referred to herein as a “scaffold,” which consists of several regions (as described in more detail below).

[0083] In the case of dual guide RNA (dgRNA), the targeter and activator portions each have a double-stranding segment, and the double-stranding segments of the targeter and activator are complementary to each other and hybridize to form a double-stranded double helix (dsRNA double helix for gRNA). When gRNA is gRNA, the terms “targeter” or “targeter RNA” are used herein to refer to the crRNA-like molecule (crRNA: “CRISPR RNA”) of CasX dual guide RNA (and therefore, of CasX single guide RNA when the “activator” and “targeter” are linked together, for example, by intervening nucleotides). crRNA has a 5' region that anneals with tracrRNA, followed by nucleotides of the targeting sequence. Thus, for example, guide RNA (dgRNA or sgRNA) includes a guide sequence and the double-stranding segment of crRNA (which may also be called a crRNA repeat). The corresponding tracrRNA-like molecule (activator) also includes a double-stranding stretch of nucleotides that forms the other half of the dsRNA double helix of the protein-binding segment of the guide RNA. Thus, the targeter and activator hybridize as a corresponding pair to form a dual guide RNA (hereinafter referred to as “dual-molecule gRNA”, “dgRNA”, “dual-molecule guide RNA”, or “two-molecule guide RNA”). Site-specific binding and / or cleavage of the target nucleic acid sequence (e.g., genomic DNA) by the CasX protein may occur at one or more locations (e.g., the sequence of the target nucleic acid) determined by the complementarity of base pairing between the targeting sequence of the gRNA and the target nucleic acid sequence. Therefore, for example, the gRNA of this disclosure has a sequence complementary to the target nucleic acid adjacent to a TC PAM motif or PAM sequence (such as ATC, CTC, GTC, or TTC), and can therefore hybridize with the target nucleic acid. Since the targeting sequence of the guide sequence hybridizes with the sequence of the target nucleic acid sequence, the targeter can be modified by the user to hybridize with a specific target nucleic acid sequence, insofar as the position of the PAM sequence is taken into consideration.Therefore, in some cases, the targeter sequence may be complementary to a sequence that does not exist in nature. In other cases, the targeter sequence may be a naturally occurring sequence derived from a complement to the gene being edited. In other embodiments, the activator and targeter of the gRNA are covalently linked to each other (rather than hybridizing with each other) and comprise a single molecule (hereinafter referred to herein as “single-molecule gRNA”, “single-guide RNA”, “single-molecule guide RNA”, “single-molecule guide RNA”, or “sgRNA”). In some embodiments, the sgRNA comprises an “activator” or a “targeter” and is therefore “activator RNA” and “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 encompasses naturally occurring molecules as well as sequence variants (e.g., modified nucleotides that do not exist in nature).

[0084] In summary, the constructed gRNAs of the present disclosure comprise four distinct regions or domains: an RNA triple, a scaffolding stem, an elongation stem, and a targeting sequence (specific to the target nucleic acid in the embodiments of the present disclosure and located at the 3' end of the gRNA). The RNA triple, scaffolding stem, and elongation stem together are referred to as the gRNA “scaffold” (gRNA scaffold). The gRNA scaffolds of the present disclosure may comprise RNA, or RNA and DNA. The gRNA scaffolds may comprise uracil (U), one or more uracils may be replaced by thymine (T).

[0085] b.RNA triplex In some embodiments of the guide RNA provided herein, the gRNA comprises an RNA triple helix, and the RNA triple helix optionally terminates with AAAG after two intervening stem-loops (a scaffolding stem-loop and an elongation stem-loop) in a UUU-N structure. XThe sequence contains a (approximately 4-15)-UUU stem-loop (SEQ ID NO: 241) and may form a pseudoknot that extends beyond the triple helix into a double-stranded pseudoknot. The triple-stranded UU-UUU-AAA sequence forms a nexus between the targeting sequence, the scaffolding stem, and the elongation stem. In the example gRNA, the UUU-loop-UUU region is encoded first, followed by the scaffolding stem-loop, then the elongation stem-loop linked by a tetraloop, and finally AAAG closes the triple helix before becoming the targeting sequence.

[0086] c. Scaffolding stem loop In some embodiments of the gRNAs of this disclosure, a scaffolding stem-loop follows the triple-stranded region. The scaffolding stem-loop is the region of the gRNA to which the CasX protein (e.g., reference or CasX variant protein) binds when an RNP is formed. In some embodiments, the scaffolding stem-loop is a fairly short and stable stem-loop, increasing the overall stability of the gRNA. In some cases, the scaffolding stem-loop does not tolerate much change and requires some form of RNA bubble. In some embodiments, the scaffolding stem is required for the function of the gRNA. This is perhaps analogous to the Cas9 guide nexus stem, which is a critical stem-loop, but the scaffolding stem of the gRNA has a required bulge (RNA bubble) in some embodiments, which differs from many other stem-loops found in the CRISPR / Cas system. In some embodiments, the presence of this bulge is conserved across gRNAs interacting with different CasX proteins. An exemplary sequence of a scaffolding stem-loop sequence of a gRNA includes the sequence CCAGCGACUAUGUCGUAUGG (SEQ ID NO: 242).

[0087] d. Elongated stem loop In some embodiments of the gRNAs of this disclosure, an elongation stem loop follows a scaffolding stem loop. In some embodiments, the elongation stem mainly comprises a synthetic tracrRNA and crRNA fusion that does not have a CasX protein bound to it. In some embodiments, the elongation stem loop may be highly malleable. In some embodiments, a single guide gRNA is constructed using a GAAA tetraloop linker or GAGAAA linker between the tracrRNA and crRNA in the elongation stem loop. In some cases, the targeter and activator of the sgRNA are linked to each other by intervening nucleotides, and the linker may have a length of 3 to 20 nucleotides. In some embodiments of the sgRNAs of this disclosure, the elongation stem is a large 32 bp loop located outside the CasX protein in the ribonucleoprotein complex. An exemplary sequence of the elongation stem loop sequence of the reference gRNA includes the sequence GCGCUUAUUUAUCGGAGAGAAAUCCGAUAAAUAAGAAGC (SEQ ID NO: 15).

[0088] e. Targeted sequences In some embodiments of the gRNAs of this disclosure, the elongated stem-loop is followed by a region that forms part of a triple helix, and then a targeting sequence (or "spacer") at the 3' end of the gRNA. The targeting sequence targets the CasX ribonucleoprotein holocomplex to a specific region of the target nucleic acid sequence of the gene to be modified. Thus, for example, the gRNA targeting sequence of this disclosure has, as a component of the RNP, a sequence complementary to (and therefore hybridizable to) a portion of a gene in a target nucleic acid of a eukaryotic cell (e.g., eukaryotic chromosomes, chromosome sequences, etc.) if one of the TC PAM motif or PAM sequences TTC, ATC, GTC, or CTC is located 1 nucleotide 5' to the non-target strand sequence complementary to the target sequence. The gRNA targeting sequence can be modified so that the gRNA can target a desired sequence of any desired target nucleic acid sequence, insofar as the position of the PAM sequence is taken into consideration. In some embodiments, the gRNA scaffold is located at the 5' end of the target sequence, and the targeting sequence is located at the 3' end of the gRNA. In some embodiments, the PAM motif sequence recognized by the RNP nuclease is TC. In other embodiments, the PAM sequence recognized by the RNP nuclease is NTC (i.e., ATC, CTC, GTC, or TTC).

[0089] In some embodiments, the Disclosure provides a gRNA whose targeting sequence is complementary to the target nucleic acid sequence of the gene to be modified. In some embodiments, the targeting sequence of the gRNA is complementary to the target nucleic acid sequence of the gene and contains one or more mutations compared to the wild-type gene sequence for the purpose of editing the mutation-containing sequence with the CasX: gRNA system of the Disclosure. In such cases, the modification brought about by the CasX: gRNA system can correct or compensate for the mutation, or knock down or knock out the expression of the mutation-containing gene product. In other embodiments, the targeting sequence of the gRNA is complementary to the target nucleic acid sequence of the wild-type gene for the purpose of editing the sequence into which the mutation is introduced with the CasX: gRNA system of the Disclosure in order to knock down or knock out the gene. In some embodiments, the targeting sequence of the gRNA is designed to be specific to the exon of the gene of the target nucleic acid. In other embodiments, the targeting sequence of the gRNA is designed to be specific to the intron of the gene of the target nucleic acid. In other embodiments, the targeting sequence of the gRNA is designed to be specific to the intron-exon junction of the gene of the target nucleic acid. In some embodiments, the gRNA targeting sequence is designed to be specific to a regulatory element of the gene. In some embodiments, the gRNA targeting sequence is designed to be complementary to a sequence containing one or more single nucleotide polymorphisms (SNPs) in the gene. SNPs in coding sequences or non-coding sequences are both within the scope of this disclosure. In other embodiments, the gRNA targeting sequence is designed to be complementary to a sequence in the intergenetic region of the target nucleic acid gene.

[0090] In some embodiments, the targeting sequence is designed to be specific to a regulatory element that modulates the expression of a gene product. Such regulatory elements include, but are not limited to, regions containing promoter regions, enhancer regions, intergenetic regions, 5' untranslated regions (5'UTR), 3' untranslated regions (3'UTR), conserved elements, and cis-regulatory elements. The promoter region is intended to contain nucleotides within 5 kb of the start of the coding sequence, or, in the case of gene enhancer elements or conserved elements, may be thousands of bp, hundreds of thousands of bp, or even millions of bp away from the coding sequence of the gene of the target nucleic acid. In the foregoing, the target is a target whose coding gene is intended to be knocked out or knocked down so that the gene product is not expressed or is expressed at a lower level in the cell.

[0091] In some embodiments, the gRNA targeting sequence has 14 to 35 consecutive nucleotides. In some embodiments, the gRNA targeting sequence has 10 to 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 other embodiments, the gRNA targeting sequence 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 contain 0-5, 0-4, 0-3, or 0-2 mismatches with respect to the target nucleic acid sequence, and can maintain sufficient binding specificity so that the RNP containing the gRNA containing the targeting sequence can form a complementary bond to the target nucleic acid.

[0092] 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, the second or additional gRNA having 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, resulting in the targeting of multiple points in the target nucleic acid, for example, by CasX introducing multiple cleavage in the target nucleic acid. In such cases, it will be understood that the second or additional gRNA is complexed with an additional copy of the CasX protein. By selecting the targeting sequence of the gRNA, a defined region of the target nucleic acid sequence surrounding a mutation can be modified or edited using the CasX:gRNA system described herein, for example, if mutation repetition occurs, or in some cases if the removal of the exon containing the mutation still results in the expression of a functional gene product, this includes facilitating the excision of DNA between insertion or cleavage sites of a donor template.

[0093] f.gRNA scaffold Apart from the targeted sequence region, the remaining region of the gRNA is referred to herein as the scaffold. In some embodiments, the gRNA scaffold is derived from a naturally occurring sequence described below as the reference gRNA. In other embodiments, the gRNA scaffold is a variant of another gRNA variant, introduced by mutation, insertion, deletion, or domain substitution to confer desired properties to the gRNA.

[0094] In some embodiments, the reference gRNA includes a sequence isolated or derived from Deltaproteobacteria. In some embodiments, the sequence is a CasX tracrRNA sequence. Exemplary reference tracrRNA sequences isolated or derived from Deltaproteobacteria may include ACAUCUGGCGCGUUUAUUCCAUUACUUUGGAGCCAGUCCCAGCGACUAUGUCGUAUGGACGAAGCGCUUAUUUAUCGGAGA (SEQ ID NO: 6) and ACAUCUGGCGCGUUUAUUCCAUUACUUUGGAGCCAGUCCCAGCGACUAUGUCGUAUGGACGAAGCGCUUAUUUAUCGG (SEQ ID NO: 7). Exemplary crRNA sequences isolated or derived from Deltaproteobacteria may include the sequence CCGAUAAGUAAAACGCAUCAAAG (SEQ ID NO: 243).

[0095] In some embodiments, the reference guide RNA includes a sequence isolated or derived from Planctomycetes. In some embodiments, the sequence is tracrRNA. Exemplary reference tracrRNA sequences isolated or derived from Planctomycetes include UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGGAGA (SEQ ID NO: 8) and The sequence UACUGGCGCUUUUAUCUCAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAUGGGUAAAGCGCUUAUUUAUCGG (Sequence ID 9) may be included. Exemplary crRNA sequences isolated or derived from Planctomycetes may include the sequence UCUCCGAUAAAUAAGAAGCAUCAAAG (Sequence ID 244).

[0096] In some embodiments, the reference gRNA includes sequences isolated or derived from Candidatus Sungbacteria. Exemplary CasX reference tracrRNA sequences isolated or derived from Candidatus Sungbacteria may include the sequences GUUUACACACUCCCUCUCAUAGGGU (SEQ ID NO: 10), GUUUACACACUCCCUCUCAUGAGGU (SEQ ID NO: 11), UUUUACAUACCCCCUCUCAUGGGAU (SEQ ID NO: 12), and GUUUACACACUCCCUCUCAUGGGGG (SEQ ID NO: 13).

[0097] Table 1 provides reference gRNA tracr, cr sequences and scaffold sequences. In some embodiments, the disclosure provides gRNA variant sequences, the gRNA having a scaffold comprising a sequence having at least one nucleotide modification compared to a reference gRNA sequence having one of the sequences of SEQ ID NOs: 4-16 in Table 1. In embodiments where the vector comprises DNA encoding the gRNA sequence or the gRNA is an RNA-DNA chimera, it will be understood that thymine (T) bases may be substituted with uracil (U) bases of any of the embodiments of the gRNA sequences described herein.

[0098] [Table 1]

[0099] g.gRNA variant In other embodiments, the disclosure relates to a gRNA variant that includes one or more modifications to a reference gRNA scaffold or is derived from another gRNA variant. As used herein, “scaffold” means all portions of the gRNA necessary for the function of the gRNA, excluding the targeting sequence.

[0100] In some embodiments, the gRNA variant includes one or more nucleotide substitutions, insertions, deletions, or swapped or substituted regions compared to the reference gRNA sequence of this disclosure. In some embodiments, mutations may occur in any region of the reference gRNA scaffold to generate the 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 with the sequence of SEQ ID NO: 4 or SEQ ID NO: 5. In other embodiments, the gRNA variant includes one or more nucleotide substitutions, insertions, deletions, or swapped or substituted regions compared to the gRNA variant sequence of this 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 with the sequence of SEQ ID NO: 2238 or SEQ ID NO: 2239.

[0101] In some embodiments, a gRNA variant includes one or more nucleotide changes within one or more regions of a reference gRNA scaffold that improve the characteristics of the reference gRNA. In other embodiments, a gRNA variant includes one or more nucleotide changes within one or more regions of the gRNA variant scaffold from which it originates that improve the characteristics compared to its gRNA. Exemplary regions include RNA triple helix, pseudoknots, scaffold stem-loops, and elongation stem-loops. In some cases, the variant scaffold stem further includes bubbles. In other cases, the variant scaffold further includes triple-helix loop regions. In yet other cases, the variant scaffold further includes a 5' unstructured region. In some embodiments, the gRNA variant scaffold includes a scaffold stem-loop having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 14. In other embodiments, the gRNA variant includes a scaffolding stem-loop having the sequence CCAGCGACUAUGUCGUAGUGG (SEQ ID NO: 245). In other embodiments, the Disclosure provides a modified elongated stem-loop with C18G substitution, G55 insertion, U1 deletion, and a modified elongated stem-loop with the original 6nt loop and the most-loop-proximal 13 base pairs (32 nucleotides total) replaced with a Uvsx hairpin (4nt loop and the most-loop-proximal 5 base pairs (14 nucleotides total)), and the loop-distal bases of the elongated stem are converted to a fully base-paired stem adjacent to the new Uvsx hairpin by A99 deletion and G65U substitution. In the embodiment described above, the gRNA scaffold is gRNA variant 174, and includes the sequence ACUGGCGCUUUUAUCUGAUUACUUUGAGAGCCAUCACCAGCGACUAUGUCGUAGUGGGUAAAGCUCCCUCUUCGGAGGGAGCAUCAAAG (Sequence ID 2238).

[0102] Any gRNA variant that, when compared to a reference gRNA or a mutagenerated gRNA variant to generate a novel gRNA variant described herein, has one or more improved features or adds one or more novel functions is assumed to be within the scope of this disclosure. A representative example of such a gRNA variant is Guide 235 (SEQ ID NO: 2292), whose design is described in the examples. In some embodiments, the gRNA variant adds a novel function to an RNP containing the gRNA variant. In some embodiments, the gRNA variant has improved features selected from: increased stability, increased gRNA transcription, increased resistance to nuclease activity, increased gRNA folding rate, decreased byproduct formation during folding, increased productive folding, increased binding affinity to the CasX protein, increased binding affinity to target nucleic acids when complexed with the CasX protein, increased gene editing when complexed with the CasX protein, increased specificity of target nucleic acid editing when complexed with the CasX protein, decreased off-target editing when complexed with the CasX protein, and increased ability to utilize one or more broadly categorized PAM sequences, including ATC, CTC, GTC, or TTC, in target nucleic acid editing when complexed with the CasX protein, or any combination thereof. In some cases, one or more of the improved features of the gRNA variant are increased by at least about 1.1 to about 100,000 times compared to the reference gRNA of SEQ ID NO: 4 or SEQ ID NO: 5 or gRNA variant 174 or 175. In other cases, one or more improved features of the gRNA variant are increased by at least approximately 1.1 times, at least approximately 10 times, at least approximately 100 times, at least approximately 1,000 times, at least approximately 10,000 times, at least approximately 100,000 times, or more compared to the reference gRNA of SEQ ID NO: 4 or SEQ ID NO: 5 or gRNA variant 174 or 175.In other cases, one or more of the improved features of the gRNA variant are approximately 1.1 to 100,000 times, approximately 1.1 to 10,000 times, approximately 1.1 to 1,000 times, approximately 1.1 to 500 times, approximately 1.1 to 100 times, approximately 1.1 to 50 times, approximately 1.1 to 20 times, approximately 10 to 100,000 times, approximately 10 to 10,000 times, approximately 10 to 1,000 times, approximately 10 to 500 times, approximately 10 to 100 times, approximately 10 to 50 times, approximately 10 to 20 times, approximately 2 to 70 times, approximately 2 to 50 times, approximately 2 to 30 times, approximately 2 to 20 times, approximately 2 ~10 times, about 5 to 50 times, about 5 to 30 times, about 5 to 10 times, about 100 to 100,00 times, about 100 to 10,00 times, about 100 to 1,000 times, Approximately 100 to 500 times, Approximately 500 to 100,00 times, Approximately 500 to 10,00 times, Approximately 500 to 1,000 times, Approximately 500 to 750 times, Approximately 1,000 It has increased by approximately 100,000 times, approximately 10,000 to 100,000 times, approximately 20 to 500 times, approximately 20 to 250 times, approximately 20 to 200 times, approximately 20 to 100 times, approximately 20 to 50 times, approximately 50 to 10,000 times, approximately 50 to 1,000 times, approximately 50 to 500 times, approximately 50 to 200 times, or approximately 50 to 100 times. In other cases, one or more improved features of the gRNA variant are approximately 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, 25 times, 30 times, 40 times, 45 times, compared to the reference gRNA of SEQ ID NO: 4 or SEQ ID NO: 5 or gRNA variant 174 or 175. It has increased by 50, 55, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 425, 450, 475, or 500 times.

[0103] In some embodiments, novel gRNA variants can be produced by subjecting a reference gRNA or gRNA variant to one or more mutagenesis methods, such as those described herein in the following examples, which may include comprehensive mutation evolution (DME), comprehensive mutation scanning (DMS), error-prone PCR, cassette mutagenesis, random mutagenesis, adhesion extension PCR, gene shuffling, or domain swapping. The activity of the reference gRNA or the activity of the mutagenesis-treated gRNA variant can be used as a benchmark for the activity of the gRNA variant, thereby allowing for the measurement of improvement in the function of the gRNA variant. In other embodiments, the reference gRNA or gRNA may be subjected to one or more intentionally targeted mutations, substitutions, or domain swaps to produce a gRNA variant (e.g., 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.

[0104] In some embodiments, the gRNA variant comprises one or more modifications compared to a reference gRNA or gRNA variant scaffold sequence, the one or more modifications being 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; substitution of all or part of a region of the gRNA; deletion of all or part of a region of the gRNA; or any combination thereof. In some cases, the modification is the substitution of 1 to 15 consecutive or non-consecutive nucleotides in one or more regions of the gRNA. In other cases, the modification is the deletion of 1 to 10 consecutive or non-consecutive nucleotides in one or more regions of the gRNA. In other cases, the modification is the insertion of 1 to 10 consecutive or non-consecutive nucleotides in one or more regions of the gRNA. In other cases, the modification is the substitution of the scaffold stem-loop or elongation stem-loop with an RNA stem-loop sequence from a heterologous RNA source having the proximal 5' and 3' ends. In some cases, the gRNA variants of this disclosure include two or more modifications in one region compared to the reference gRNA or gRNA variant. In other cases, the gRNA variants of this disclosure include modifications in two or more regions. In other cases, the gRNA variants include any combination of the modifications described in this paragraph.

[0105] In some embodiments, a 5'G is added to the gRNA variant sequence compared to the original gRNA for in vivo expression, because transcription from the U6 promoter is more efficient and consistent with respect to the start site when the +1 nucleotide is G. In other embodiments, two 5'Gs are added to the gRNA variant sequence to increase production efficiency in in vitro transcription, because T7 polymerase strongly prefers the G at position +1 and the purine at position +2. In some cases, the 5'G base is added to the reference scaffold in Table 1. In other cases, the 5'G base is added to the variant scaffold in Table 2.

[0106] Table 2 provides exemplary gRNA variant scaffold sequences. In some embodiments, the gRNA variant scaffold includes one of the sequences listed in Table 2, sequence numbers 2101-2332 or 2353-2398, 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%, or at least about 99% sequence identity thereto. In some embodiments, the gRNA variant scaffold includes one of the sequences of SEQ ID NOs. 2238-2332 or 2353-2398, 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%, or at least about 99% sequence identity with it. In some embodiments, the gRNA variant scaffold includes one of the sequences of SEQ ID NOs. 2281-2332 or 2353-2398, 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%, or at least about 99% sequence identity with it. In embodiments where the vector contains DNA encoding a gRNA sequence or the gRNA is a chimera of RNA and DNA, it will be understood that thymine (T) bases may be substituted with uracil (U) bases of any of the embodiments of the gRNA sequence described herein.

[0107] [Table 2-1]

[0108] [Table 2-2]

[0109] [Table 2-3]

[0110] Table 2-4

[0111] Table 2-5

[0112] Table 2-6

[0113] Table 2-7

[0114] Table 2-8

[0115] Table 2-9

[0116] Table 2-10

[0117] Table 2-11

[0118] In some embodiments, the sgRNA variant includes one or more additional modifications to the sequences of SEQ ID NOs: 2238, 2239, 2240, 2241, 2243, 2256, 2274, 2275, 2279, 2281, 2285, 2289, 2292, or 2308 in Table 2.

[0119] In some embodiments of the gRNA variants of this disclosure, the gRNA variant comprises at least one modification compared to the reference guide scaffold of Sequence ID No. 5, wherein at least one modification is selected from one or more of the following: (a) C18G substitution in a triple-stranded loop, (b) G55 insertion in a stem bubble, (c) U1 deletion, (d) modification of an elongated stem loop in which (i) a 6nt loop and 13 base pairs proximal to the loop are substituted by a Uvsx hairpin, and (ii) a deletion of A99 and substitution of G65U results in complete base pairing of the bases distal to the loop.

[0120] In some embodiments, the gRNA variant comprises an exogenous stem-loop having a long non-coding RNA (lncRNA). As used herein, lncRNA refers to non-coding RNA longer than approximately 200 bp. In some embodiments, the 5' and 3' ends of the exogenous stem-loop are base-paired (i.e., interact to form a region of double-stranded 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 and form a loop.

[0121] In some embodiments, the Disclosure provides gRNA variants having nucleotide modifications compared to a reference gRNA, including (a) substitution of 1 to 15 consecutive or non-consecutive nucleotides in one or more regions of the gRNA variant, (b) deletion of 1 to 10 consecutive or non-consecutive nucleotides in one or more regions of the gRNA variant, (c) insertion of 1 to 10 consecutive or non-consecutive nucleotides in one or more regions of the gRNA variant, (d) substitution of a scaffold stem-loop or elongation stem-loop with an RNA stem-loop sequence from a heterologous RNA source having the proximal 5' and 3' ends, or any combination of (a) to (d). Any combination of substitutions, insertions, and deletions described herein can be used to generate the gRNA variants of the Disclosure. For example, a gRNA variant may include, compared to the reference gRNA, at least one substitution and at least one deletion, at least one substitution and at least one insertion, at least one insertion and at least one deletion, or at least one substitution, one insertion, and one deletion compared to the reference gRNA.

[0122] In some embodiments, the sgRNA variants of this disclosure include one or more modifications to the sequence of a previously generated variant, where the previously generated variant itself functions as the sequence to be modified. In some cases, one or more modifications are introduced into the pseudoknot region of the scaffold. In other cases, one or more modifications are introduced into the triple-stranded region of the scaffold. In other cases, one or more modifications are introduced into the scaffold bubble. In other cases, one or more modifications are introduced into the elongation stem region of the scaffold. In yet other cases, one of the modifications is introduced into two or more of the aforementioned regions. Such modifications may include the insertion, deletion, or substitution of one or more nucleotides in the aforementioned regions, or any combination thereof. Exemplary methods for generating and evaluating modifications are described in Example 15.

[0123] In some embodiments, the sgRNA variant includes one or more modifications to the 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: 2289, SEQ ID NO: 2292, or SEQ ID NO: 2308.

[0124] In exemplary embodiments, a gRNA variant comprises one or more modifications to gRNA scaffold variant 174 (SEQ ID NO: 2238), and the resulting gRNA variant exhibits improved functional characteristics compared to parent 174 when evaluated in an in vitro or in vivo assay under equivalent conditions. In other exemplary embodiments, a gRNA variant comprises one or more modifications to gRNA scaffold variant 175 (SEQ ID NO: 2239), and the resulting gRNA variant exhibits improved functional characteristics compared to parent 175 when evaluated in an in vitro or in vivo assay under equivalent conditions. For example, variants with modifications to the triple loop of gRNA variant 175, particularly mutations to C15 or C17, show higher enrichment compared to the 175 scaffold. Additionally, changes to either member of the predicted pair in the pseudoknot stem between G7 and A29 are both expected to be highly concentrated compared to the 175 scaffold, converting A29 to C or T to form a canonical Watson-Crick pair (G7:C29), the second of which forms a GU fluctuation pair (G7:U29), both of which are expected to increase helix stability compared to the G:A pair. Moreover, the insertion of C at position 54 in guide scaffold 175 results in a concentrated modification.

[0125] In some embodiments, the disclosure provides a gRNA variant comprising one or more modifications to gRNA scaffold variant 174 (SEQ ID NO: 2238) selected from the group consisting of modifications in Table 19, wherein the resulting gRNA variant exhibits improved functional characteristics compared to parent 174 when evaluated in vitro or in vivo assays under equivalent conditions. In some embodiments, the improved functional characteristics are one or more functional properties selected from the group consisting of increased editing activity, increased pseudoknot stem stability, increased triple-stranded region stability, increased scaffold stem stability, elongation stem stability, reduced off-target folding intermediates, and increased binding affinity to class 2, type V CRISPR proteins. In the embodiments described above, gRNAs containing one or more modifications to gRNA scaffold variant 174 selected from the group of modifications in Table 16 (having a ligated targeting sequence and complexing with a class 2, type V CRISPR protein) exhibit improved enrichment scores (log2) in in vitro assays that are at least approximately 2.0, at least approximately 2.5, at least approximately 3, or at least approximately 3.5 higher than the score of the gRNA scaffold of SEQ ID NO: 2238.

[0126] In some embodiments, the disclosure provides a gRNA variant comprising one or more modifications to gRNA scaffold variant 175 (SEQ ID NO: 2239) selected from the group consisting of modifications in Table 20, wherein the resulting gRNA variant exhibits improved functional characteristics compared to parent 175 when evaluated in vitro or in vivo assays under equivalent conditions. In some embodiments, the improved functional characteristics are one or more functional properties selected from the group consisting of increased editing activity, increased pseudoknot stem stability, increased triple-stranded region stability, increased scaffold stem stability, elongation stem stability, reduced off-target folding intermediates, and increased binding affinity to class 2, type V CRISPR proteins. In the embodiments described above, gRNAs containing one or more modifications to gRNA scaffold variant 175 selected from the group of modifications in Table 16 (having a ligated targeting sequence and complexing with class 2, type V CRISPR protein) exhibit improved enrichment scores (log2) in in vitro assays that are at least approximately 1.2, at least approximately 1.5, at least approximately 2.0, at least approximately 2.5, at least approximately 3, or at least approximately 3.5 higher than the score of gRNA scaffold of SEQ ID NO: 2239.

[0127] In certain embodiments, one or more modifications of gRNA scaffold variant 174 are selected from the group consisting of nucleotide positions U11, U24, A29, U65, C66, C68, A69, U76, G77, A79, and A87. In certain embodiments, modifications of gRNA scaffold variant 174 are U11C, U24C, A29C, U65C, C66G, C68U, insertion of ACGGA at position 69, insertion of UCCGU at position 76, G77A, insertion of GA at position 79, and A87G. In another particular embodiment, modifications of gRNA scaffold variant 175 are selected from the group consisting of nucleotide positions C9, U11, C17, U24, A29, G54, C65, A89, and A96. In certain embodiments, modifications of gRNA scaffold variant 174 include C9U, U11C, C17G, U24C, A29C, a G insertion at position 54, a C insertion at position 65, A89G, and A96G.

[0128] In exemplary embodiments, the gRNA variant comprises one or more modifications to the gRNA scaffold variant 215 (SEQ ID NO: 2275), and the resulting gRNA variant exhibits improved functional characteristics compared to the parent 215 when evaluated in an in vitro or in vivo assay under equivalent conditions.

[0129] In exemplary embodiments, the gRNA variant comprises one or more modifications to the gRNA scaffold variant 221 (SEQ ID NO: 2281), and the resulting gRNA variant exhibits improved functional characteristics compared to the parent 221 when evaluated in an in vitro or in vivo assay under equivalent conditions.

[0130] In exemplary embodiments, the gRNA variant comprises one or more modifications to the gRNA scaffold variant 225 (SEQ ID NO: 2285), and the resulting gRNA variant exhibits improved functional characteristics compared to the parent 225 when evaluated in an in vitro or in vivo assay under equivalent conditions.

[0131] In exemplary embodiments, the gRNA variant comprises one or more modifications to the gRNA scaffold variant 235 (SEQ ID NO: 2292), and the resulting gRNA variant exhibits improved functional characteristics compared to the parent 225 when evaluated in an in vitro or in vivo assay under equivalent conditions.

[0132] In exemplary embodiments, the gRNA variant comprises one or more modifications to the gRNA scaffold variant 251 (SEQ ID NO: 2308), and the resulting gRNA variant exhibits improved functional characteristics compared to the parent 251 when evaluated in an in vitro or in vivo assay under equivalent conditions.

[0133] In the embodiments described above, the improved functional features include, but are not limited to, one or more of the following: increased stability, increased gRNA transcription, increased resistance to nuclease activity, increased gRNA folding rate, decreased byproduct formation during folding, increased productive folding, increased binding affinity to the CasX protein, increased binding affinity to target nucleic acids when complexed with the CasX protein, increased gene editing when complexed with the CasX protein, increased editing specificity when complexed with the CasX protein, decreased off-target editing when complexed with the CasX protein, and increased ability to utilize one or more broadly categorized PAM sequences, including ATC, CTC, GTC, or TTC, in the modification of target nucleic acids when complexed with the CasX protein. In some cases, one or more of the improved features of the gRNA variant are improved by at least about 1.1 to about 100,000 times compared to the gRNA from which it is derived. In other cases, one or more improved features of the gRNA variant are improved by at least approximately 1.1 times, at least approximately 10 times, at least approximately 100 times, at least approximately 1,000 times, at least approximately 10,000 times, at least approximately 100,000 times, or more compared to the gRNA from which it originates.In other cases, one or more of the improved features of the gRNA variant are approximately 1.1-100,000 times, 1.1-10,000 times, 1.1-1,000 times, 1.1-500 times, 1.1-100 times, 1.1-50 times, 1.1-20 times, 10-100,000 times, 10-10,000 times, 10-1,000 times, 10-500 times, 10-100 times, 10-50 times, 10-20 times, 2-70 times, 2-50 times, 2-30 times, 2-20 times, 2-10 times, 5-50 times, 5- Improvements of 30 times, approximately 5-10 times, approximately 100-100.00 times, approximately 100-10.00 times, approximately 100-1,000 times, approximately 100-500 times, approximately 500-100.00 times, approximately 500-10.00 times, approximately 500-750 times, approximately 1,000-100.00 times, approximately 10,000-100.00 times, approximately 20-500 times, approximately 20-250 times, approximately 20-200 times, approximately 20-100 times, approximately 20-50 times, approximately 50-10,000 times, approximately 50-1,000 times, approximately 50-500 times, approximately 50-200 times, or approximately 50-100 times. In other cases, one or more improved features of a gRNA variant are approximately 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, 25 times, 30 times, 40 times, 45 times, 50 times, 55 times, 60 times, 70 times compared to the gRNA from which it originates. It is improved by 80 times, 90 times, 100 times, 110 times, 120 times, 130 times, 140 times, 150 times, 160 times, 170 times, 180 times, 190 times, 200 times, 210 times, 220 times, 230 times, 240 times, 250 times, 260 times, 270 times, 280 times, 290 times, 300 times, 310 times, 320 times, 330 times, 340 times, 350 times, 360 times, 370 times, 380 times, 390 times, 400 times, 425 times, 450 times, 475 times, or 500 times.

[0134] In some embodiments, the gRNA variant includes an exogenous elongated stem-loop, the difference from the reference gRNA being as follows. In some embodiments, the exogenous elongated stem-loop has little to no identity with the reference stem-loop region disclosed herein (e.g., SEQ ID NO: 15). In some embodiments, the 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 includes an elongated stem-loop region containing 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 can bind to proteins, RNA structures, DNA sequences, or small molecules. In some embodiments, the exogenous stem-loop region substituting the stem-loop includes an RNA stem-loop or a hairpin, and the resulting gRNA has increased stability and, depending on the choice of loop, can interact with specific cellular proteins or RNAs.Such exogenous elongated stem-loops include, for example, thermostable RNA, MS2 hairpin (ACAUGAGGAUCACCCAUGU (SEQ ID NO: 1137)), Qβ hairpin (UGCAUGUCUAAGACAGCA (SEQ ID NO: 32)), U1 hairpin II (AAUCCAUUGCACUCCGGAUU (SEQ ID NO: 33)), Uvsx (CCUCUUCGGAGG (SEQ ID NO: 34)), PP7 hairpin (AGGAGUUUCUAUGGAAACCCU (SEQ ID NO: 35)), phage replication loop (AGGUGGGACGACCUCUCGGUCGUCCUAUCU (SEQ ID NO: 36)), kissing loop_a (UGCUCGCUCCGUUCGAGCA (SEQ ID NO: 37)), kissing loop It may include p_b1 (UGCUCGACGCGUCCUCGAGCA (SEQ ID NO: 38)), kissing loop_b2 (UGCUCGUUUGCGGCUACGAGCA (SEQ ID NO: 39)), G triple-stranded M3q (AGGGAGGGAGGGAGAGG (SEQ ID NO: 40)), G quadruple-stranded telomere basket (GGUUAGGGUUAGGGUUAGG (SEQ ID NO: 41)), salsin-lysine loop (CUGCUCAGUACGAGAGGAACCGCAG (SEQ ID NO: 42)), or pseudoknot (UACACUGGGAUCGCUGAAUUAGAGAUCGGCGUCCUUUCAUUCUAUAUACUUUGGAGUUUUAAAAUGUCUCUAAGUACA (SEQ ID NO: 43)). In some embodiments, one of the aforementioned hairpin sequences is incorporated into a stem-loop to help transport the incorporation of the gRNA (and associated CasX in the RNP complex) into the budding XDP when the corresponding ligand is incorporated into the Gag polyprotein of the XDP (as described in more detail below).

[0135] In some embodiments, the gRNA variant includes a terminal fusion partner. The term gRNA variant encompasses variants that include exogenous sequences such as terminal fusions or internal insertions. Exemplary terminal fusions may include the fusion of gRNA to a self-cleaved ribozyme or protein-binding motif. As used herein, “ribozyme” means RNA or a segment thereof having one or more catalytic activities similar to those of a protein enzyme. Exemplary ribozyme catalytic activities may include, for example, RNA cleavage and / or ligation, DNA cleavage and / or ligation, or peptide bond formation. In some embodiments, such fusions may either improve scaffold folding or mobilize DNA repair mechanisms. For example, in some embodiments, gRNA may be fused to the 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 ring virus (TRSV) ribozyme, WT virus hammerhead ribozyme (and reasonable variants), or the Twisted Sister 1 or RBMX mobilization motif. Hammerhead ribozymes are RNA motifs that catalyze reversible cleavage and ligation reactions at specific sites within an RNA molecule. Hammerhead ribozymes include type I, type II, and type III hammerhead ribozymes. HDV, pistol, and hatchet ribozymes have autocleavage activity. gRNA variants containing one or more ribozymes may enable extended gRNA function compared to the gRNA reference. For example, gRNAs containing self-cleaving ribozymes can, in some embodiments, be transcribed and processed into mature gRNA as part of a polycistronic transcript. Such fusions can occur at either the 5' or 3' end of the gRNA. In some embodiments, the gRNA variant includes fusions at both the 5' and 3' ends, each fusion independently as described herein.

[0136] In some embodiments of the gRNA variant, the gRNA variant further comprises a spacer (or targeting sequence) region located at the 3' end of the gRNA that can hybridize with the target nucleic acid, and the spacer is designed using a sequence that comprises at least 14 to about 35 nucleotides and is complementary to the target nucleic acid. In some embodiments, the encoded gRNA variant comprises a targeting sequence of at least 10 to 20 nucleotides that is complementary to the 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.

[0137] h. Class 2, complex formation with type V proteins. In some embodiments, at expression, the gRNA variant is complexed as an RNP with a class 2, type V protein containing a CasX variant protein that includes one of the sequences SEQ ID NOs. 247-592 or 1147-1231 in 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 with it. In some embodiments, during expression, the gRNA variant is complexed as an RNP with a CasX variant protein containing one of the sequences SEQ ID NOs. 270-592 or 1147-1231, 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 with it. In some embodiments, during expression, the gRNA variant is complexed as an RNP with a CasX variant protein containing one of the sequences SEQ ID NOs. 415-592 or 1147-1231, 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 with it.

[0138] In some embodiments, the gRNA variant has an improved ability to form a complex with the CasX variant protein compared to the reference gRNA, thereby improving its ability to form a cleavage-competent ribonucleoprotein (RNP) complex with the CasX protein, as described in the examples. Improving the formation of the ribonucleoprotein complex can improve the efficiency of constructing a functional RNP in some embodiments. In some embodiments, more than 90%, more than 93%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% of the RNP containing the gRNA variant and its targeting sequence are eligible for gene editing of the target nucleic acid.

[0139] Exemplary nucleotide alterations that can improve the ability of gRNA variants to complex with the CasX protein may, in some embodiments, involve replacing the scaffold stem with a thermostable stem-loop. While we do not wish to be bound by any theory, replacing the scaffold stem with a thermostable stem-loop may increase the overall binding stability between the gRNA variant and the CasX protein. Alternatively, or additionally, by removing a large portion of the stem-loop, the folding dynamics of the gRNA variant may be altered, for example, by reducing the degree to which the gRNA variant can "entangle" itself, thereby allowing functionally folded gRNAs to be structurally constructed more easily and rapidly. In some embodiments, the selection of the scaffold stem-loop sequence may vary depending on the different targeting sequences utilized for the gRNA. In some embodiments, the scaffold sequence may be tailored to the targeting sequence, and therefore the target sequence. Biochemical assays, including the assays of the examples, can be used to evaluate the binding affinity of the CasX protein to gRNA variants for RNP formation. For example, those skilled in the art can measure the change in the amount of fluorescently tagged gRNA bound to the immobilized CasX protein in response to an increase in the concentration of additional unlabeled "cold competitor" gRNA. Alternatively, or additionally, the changes in the fluorescence signal can be monitored or observed as different amounts of fluorescently labeled gRNA flow along the immobilized CasX protein. Alternatively, the ability to form RNPs can be evaluated using an in vitro cleavage assay against a defined target nucleic acid sequence, as described in the examples.

[0140] i. Chemically modified gRNA In some embodiments, the disclosure provides chemically modified gRNAs. In some embodiments, the disclosure provides chemically modified gRNAs that have guide NA functionality and reduced sensitivity to nuclease cleavage. A gRNA containing any nucleotide or deoxynucleotide other than the four canonical ribonucleotides (A, C, G, and U) is a chemically modified gRNA. In some cases, a chemically modified gRNA contains any backbone or nucleotide bond other than the native phosphodiester nucleotide bond. In certain embodiments, retained functionality includes the ability of the modified gRNA to bind to CasX in any of the embodiments described herein. In certain embodiments, retained functionality includes the ability of the modified gRNA to bind to a target nucleic acid sequence. In certain embodiments, retained functionality includes the ability to target the CasX protein or the ability of a pre-complexed RNP to bind to a target nucleic acid sequence. In certain embodiments, retained functionality includes the ability of the CasX-gRNA to introduce a nick into a target polynucleotide. In certain embodiments, retained functionality includes the ability of the CasX-gRNA to cleave a target nucleic acid sequence. In certain embodiments, the retained functionality is any other known functionality of the gRNA in the recombinant system having the CasX chimeric protein of the embodiments of this disclosure.

[0141] In some embodiments, the present disclosure provides chemically modified gRNAs, including 2'-OC 1~4 Alkyl compounds, for example, 2'-O-methyl (2'-OMe), 2'-deoxy (2'-H), 2'-OC 1-3 Alkyl-OC 1-3Modifications of the nucleotide sugar selected from the group consisting of alkyl, such as 2'-methoxyethyl ("2'-MOE"), 2'-fluoro ("2'-F"), 2'-amino ("2'-NH2"), 2'-arabinosyl ("2'-arabino") nucleotide, 2'-F-arabinosyl ("2'-F-arabino") nucleotide, 2'-locked nucleic acid ("LNA") nucleotide, 2'-unlocked nucleic acid ("ULNA") nucleotide, L-type sugar ("L-sugar"), and 4'-thioribosyl nucleotide are incorporated into the gRNA. In other embodiments, modifications of the internucleotide linkage selected from the group consisting of phosphorothioate "P(S)" (P(S)), phosphonocarboxylate (P(CH2) n COOR), such as phosphonoacetate "PACE" (P(CH2COO - )), thiophosphonocarboxylate ((S)P(CH2) n COOR), such as thiophosphonoacetate "thioPACE" ((S)P(CH2) n COO - )), alkylphosphonate (P(C 1-3 alkyl)), such as methylphosphonate - P(CH3), boranophosphonate (P(BH3)), and phosphorodithioate (P(S)₂) are incorporated into the guide RNA.

[0142] In certain embodiments, the Disclosure provides chemically modified gRNAs including 2-thiouracil ("2-thioU"), 2-thiocytosine ("2-thioC"), 4-thiouracil ("4-thioU"), 6-thioguanine ("6-thioG"), 2-aminoadenine ("2-aminoA"), 2-aminopurine, pseudouracil, hypoxanthine, 7-deazaguanine, 7-deaza-8-azaguanine, 7-deazaadenine, 7-deaza-8-azaadenine, 5-methylcytosine ("5-methylC"), 5-methyluracil ("5-methylU"), 5-hydroxymethylcytosine, 5-hydroxymethyluracil, and 5,6-dehydro Modifications to nucleic acid bases ("bases") selected from the group consisting of racil, 5-propynylcytosine, 5-propynyluracil, 5-ethinylcytosine, 5-ethinyluracil, 5-allyluracil ("5-allyl U"), 5-allylcytosine ("5-allyl C"), 5-aminoallyluracil ("5-aminoallyl U"), 5-aminoallyl-cytosine ("5-aminoallyl C"), debasalized nucleotides, Z bases, P bases, amorphous nucleic acids ("UNA"), isoguanines ("isoG"), isocytosine ("isoC"), 5-methyl-2-pyrimidine, x (A, G, C, T), and y (A, G, C, T) are incorporated into gRNA.

[0143] In other embodiments, the Disclosure provides one or more chemically modified gRNAs. 15 N, 13 C, 14 C, deuterium, 3 H, 32 P, 125 I, 131 One or more isotopic modifications are introduced into nucleotide sugars, nucleic acid bases, phosphodiester bonds, and / or nucleotide phosphates, including nucleotides containing an I atom or other atoms or elements used as tracers.

[0144] In some embodiments, the “terminus” modifications incorporated into the gRNA are selected from the group consisting of PEG (polyethylene glycol), hydrocarbon linkers (heteroatom (O,S,N) substituted hydrocarbon spacers, halo substituted hydrocarbon spacers, keto-, carboxyl-, amid-, thionyl-, carbamoyl-, thionocarbamaoil--containing hydrocarbon spacers), spermine linkers, fluorescent dyes conjugated to the linker (e.g., fluorescein, rhodamine, cyanine), such as 6-fluorescein-hexyl, quenchers (e.g., dabusil, BHQ), and other labels (e.g., biotin, digoxigenin, acridine, streptavidin, avidin, peptides, and / or proteins). In some embodiments, the “terminus” modifications include the conjugation (or ligation) of the gRNA to another molecule containing oligonucleotides of deoxynucleotides and / or ribonucleotides, peptides, proteins, sugars, oligosaccharides, steroids, lipids, folic acid, vitamins, and / or other molecules. In certain embodiments, the Disclosure provides a chemically modified gRNA in which the “terminal” modification (as described above) is located inside the gRNA sequence via a linker such as a 2-(4-butylamidefluorescein)propane-1,3-diolbis(phosphodiester)linker, and the linker is incorporated as a phosphodiester bond and can be incorporated anywhere between two nucleotides in the gRNA.

[0145] In some embodiments, the present disclosure provides chemically modified gRNA having terminal alterations, comprising terminal functional groups such as amines, thiols (or sulfhydryls), hydroxyls, carboxyls, carbonyls, thionyls, thiocarbonyls, carbamoyls, thiocarbamoyls, phosphoryls, alkenes, alkynes, halogens, or functional group-terminal linkers, which are subsequently labeled with fluorescent dyes, non-fluorescent labels, tags ( 14 For C, for example, biotin, avidin, streptavidin, or 15 N, 13 C, deuterium, 3 H, 32 P, 125A desired portion selected from the group consisting of oligonucleotides (including deoxynucleotides and / or ribonucleotides containing aptamers), amino acids, peptides, proteins, sugars, oligosaccharides, steroids, lipids, folic acid, and vitamins may be conjugated. Conjugation may be performed using, but are not limited to, standard chemistry well known in the art, such as coupling via N-hydroxysuccinimide, isothiocyanates, DCC (or DCI), and / or any other standard method (the contents of which are incorporated herein by reference in their entirety in "Bioconjugate Techniques" by Greg T. Hermanson, Publisher Eslsevier Science, 3). rd (As listed in ed. (2013))

[0146] IV. Class 2 and V CRISPR proteins for modifying target nucleic acids This disclosure provides a system comprising CRISPR nucleases useful in genome editing of eukaryotic cells. In some embodiments, the CRISPR nucleases used in the genome editing system are class 2, type V nucleases. While there are differences among the members of the class 2, type V CRISPR-Cas system, they share several common features that distinguish them from the Cas9 system. First, class 2, type V nucleases have a single RNA-inducible RuvC domain-containing effector but lack an HNH domain and recognize the PAM of a TC motif 5' upstream of the target region on the non-target strand, which differs from the Cas9 system, which relies on G-rich PAMs at the 3' end of the target sequence. Unlike Cas9, which produces blunt ends proximal to the PAM, type V nucleases produce double-strand breaks distal to the PAM sequence. In addition, when activated by target dsDNA or ssDNA bound in cis, type V nucleases degrade ssDNA in trans. In some embodiments, the V-type nuclease of this embodiment recognizes a 5'-TC PAM motif and generates a sticky end that is cleaved only by the RuvC domain. In some embodiments, the V-type nuclease is selected from the group consisting of Cas12a, Cas12b, Cas12c, Cas12d (CasY), Cas12j, Cas12k, C2c4, C2c8, C2c5, C2c10, C2c9, CasZ, and CasX. In some embodiments, the disclosure provides a system (CasX: gRNA system) comprising a CasX variant protein and one or more gRNA variants, which is specifically designed to modify a target nucleic acid sequence in eukaryotic cells.

[0147] As used herein, the term “CasX protein” refers to a family of proteins, and includes all naturally occurring CasX proteins, proteins that share at least 50% identity with naturally occurring CasX proteins, and CasX variants that have one or more improved characteristics compared to a naturally occurring reference CasX protein or another CasX variant from which it is derived.

[0148] The CasX protein of this disclosure comprises at least one of the following domains: a non-target strand binding (NTSB) domain, a target strand loading (TSL) domain, a helix I domain (further divided into helix II and I-II subdomains), a helix II domain, an oligonucleotide binding domain (further divided into OBD, OBD-I and OBD-II subdomains), and a RuvC DNA cleavage domain (further divided into RuvC-I and II subdomains). The RuvC domain may be modified or deleted in catalytically-dead CasX variants, which are described in more detail below.

[0149] In some embodiments, the CasX protein can bind to and / or modify (e.g., introduce nicks, catalyze double-strand breaks, methylate, demethylate, etc.) a specific sequence of target nucleic acid that is targeted by an associated gRNA that hybridizes to a sequence within the target nucleic acid sequence.

[0150] a. Reference CasX protein This disclosure provides a naturally occurring CasX protein (hereinafter referred to as the “reference CasX protein”) which has subsequently been modified to produce the CasX variants of this disclosure. For example, the reference CasX protein can be isolated from naturally occurring prokaryotes such as Deltaproteobacteria, Plantomycetes, or Candidatus Sungbacteria. The reference CasX protein is a type II CRISPR / Cas endonuclease belonging to the CasX (referred to interchangeably as Cas12e) family of proteins that interact with a guide RNA to form a ribonucleoprotein (RNP) complex.

[0151] In some cases, the reference CasX protein is isolated from or derived from Deltaproteobacter and has the following sequence: 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 QKVVKGNQKR LESLRELAGK ENLEYPSVTL PPQPHTKEGV DAYNEVIARV 301 RMWVNLNLWQ KLKLSRDDAK PLLRLKGFPS FPVVERRENE VDWWNTINEV KKLIDAKRDM 361 GRVFWSGVTA EKRNTILEGY NYLPNENDHK KREGSLENPK KPAKRQFGDL LLYLEKKYAG 421 DWGKVFDEAW ERIDKKIAGL TSHIEREEAR NAEDAQSKAV LTDWLRAKAS FVLERLKEMD 481 EKEFYACEIQ LQKWYGDLRG NPFAVEAENR VVDISGFSIG SDGHSIQYRN LLAWKYLENG 541 KREFYLLMNY GKKGRIRFTD GTDIKKSGKW QGLLYGGGKA KVIDLTFDPD DEQLIILPLA 601 FGTRQGREFI WNDLLSLETG LIKLANGRVI EKTIYNKKIG RDEPALFVAL TFERREVVDP 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). [[ID=IO]]

[0152] [[ID=II]] In some cases, the reference CasX protein is isolated from or derived from Planctomycetes and has the following sequence: 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 IEAYNNVVAQ 301 IVIWVNLNLW QKLKIGRDEA KPLQRLKGFP SFPLVERQAN EVDWWDMVCN VKKLINEKKE It should be noted that there seems to be a mistake in the original text where "配列番号1" is written as "配列番号1)" in the English translation. It should be "SEQ ID NO: 1". Also, "IO" in the original text might be a typo and should probably be "ID".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 NVVKDLSVEL DRLSEESVNN DISSWTKGRS GEALSLLKKR 901 FSHRPVQEKF VCLNCGFETH ADEQAALNIA RSWLFLRSQE YKKYQTNKTT GNTDKRAFVE 961 TWQSFYRKKL KEVWKPAV (Sequence ID 2).

[0153] In some cases, the reference CasX protein is isolated from or derived from Candidatus Sungbacteria and has the following sequence: 1 MDNANKPSTK SLVNTTRISD HFGVTPGQVT RVFSFGIIPT KRQYAIIERW FAAVEAARER 61 LYGMLYAHFQ ENPPAYLKEK FSYETFKGR PVNLGLRDID 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 AYAVVSVEDG QVLEEGLLGK KEYIDQLIET 601 RRRISEYQSR EQTPPRDLRQ RVRHLQDTVL GSARAKIHSL IAFWKGILAI ERLDDQFHGR 661 EQKIIPKKTY LANKTGFMNA LSFSGAVRVD KKGNPWGGMI EIYPGGISRT CTQCGTVWLA 721 RRPKNPGHRD AMVVIPDIVD DAAATGFDNV DCDAGTVDYG ELFTLSREWV RLTPRYSRVM 781 RGTLGDLERA IRQGDDRKSR QMLELALEPQ PQWGQFFCHR CGFNGQSDVL AATNLARRAI 841 SLIRRLPDTD TPPTP (Sequence ID 3).

[0154] b. Class 2, V-type CasX variant protein This disclosure provides variants derived from a Class 2, Type V, CasX variant or other CasX variant of a reference CasX protein (see, for example, Figure 44) (hereinafter interchangeably referred to as “Class 2, Type V CasX variant,” “CasX variant,” or “CasX variant protein”), where a Class 2, Type V CasX variant contains at least one modification in at least one domain compared to the reference CasX protein (including, but not limited to, the sequences of SEQ ID NOs: 1-3), or at least one modification compared to another CasX variant. Any changes in the amino acid sequence of the reference CasX protein or another CasX variant protein that result in improved characteristics of the CasX protein are considered CasX variant proteins of this disclosure. For example, a CasX variant may contain one or more amino acid substitutions, insertions, deletions, or swapped domains, or any combination thereof, compared to the reference CasX protein sequence.

[0155] The CasX variants of this disclosure have one or more improved features compared to the reference CasX protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or variants derived therefrom (e.g., CasX491 (SEQ ID NO: 336) or CasX515 (SEQ ID NO: 416)). Exemplary improved features of embodiments of the CasX variants include improved variant folding, increased binding affinity to gRNA, increased binding affinity to target nucleic acids, improved ability to utilize a wider range of PAM sequences in editing and / or binding of target nucleic acids, improved unwinding of target DNA, increased editing activity, improved editing efficiency, improved editing specificity for target nucleic acids, reduced off-target editing or cleavage, increased proportion of eukaryotic genomes that can be efficiently edited, increased nuclease activity, increased target strand loading for double-strand breaks, reduced target strand loading for single-strand nicking, increased binding of non-target strands of DNA, improved protein stability, and protein: Improved stability of the gRNA(RNP) complex and improved fusion characteristics are among the improvements, but are not limited to these. In the embodiments described above, one or more of the improved characteristics of the CasX variant are improved by at least about 1.1 to about 100,000 times compared to the reference CasX protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or CasX491 (SEQ ID NO: 336) or CasX515 (SEQ ID NO: 416), when assayed in an equivalent manner. In other embodiments, the improvements are at least about 1.1 times, at least about 2 times, at least about 5 times, at least about 10 times, at least about 50 times, at least about 100 times, at least about 500 times, at least about 1000 times, at least about 500 times, at least about 10,000 times, at least about 10,000 times, or at least about 100,000 times compared to the reference CasX protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or CasX491 or CasX515, when assayed in an equivalent manner.In other cases, one or more improved features of the RNPs of CasX variants and gRNA variants are improved by at least approximately 1.1, at least approximately 10, at least approximately 100, at least approximately 1,000, at least approximately 10,000, at least approximately 100,000, or more compared to the RNPs of the reference CasX protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 and the gRNAs in Table 1, or the RNPs of CasX491 or CasX515 and gRNA174. In other cases, one or more of the improved features of the RNPs of CasX variants and gRNA variants, when assayed in an equivalent manner, are approximately 1.1–100,000, 1.1–10,000, 1.1–1,000, 1.1–500, 1.1–100, 1.1–50, 1.1–20, 10–100,000, 10–10,000, 10–1,000, 10–500, 10–100, 10–50, 10–20 , about 2-70 times, about 2-50 times, about 2-30 times, about 2-20 times, about 2-10 times, about 5-50 times, about 5-30 times, about 5-10 times, about 100-100,00 times, about 100~10,00x, approx. 100~1,000x, approx. 100~500x, approx. 500~100,00x, approx. 500~10,00x, approx. 500~1,000x, approx. Improvements of 500-750 times, approximately 1,000-100,000 times, approximately 10,000-100,000 times, approximately 20-500 times, approximately 20-250 times, approximately 20-200 times, approximately 20-100 times, approximately 20-50 times, approximately 50-10,000 times, approximately 50-1,000 times, approximately 50-500 times, approximately 50-200 times, or approximately 50-100 times are observed.In other cases, one or more improved features of the RNPs of CasX variants and gRNA variants, when assayed in an equivalent manner, are approximately 1.1x, 1.2x, 1.3x, 1.4x, 1.5x, 1.6x, 1.7x, 1.8x, 1.9x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 11x, 12x, 13x, 14x, 15x, 16x, 11x, 16x, It is improved by 20 times, 17 times, 18 times, 19 times, 20 times, 25 times, 30 times, 40 times, 45 times, 50 times, 55 times, 60 times, 70 times, 80 times, 90 times, 100 times, 110 times, 120 times, 130 times, 140 times, 150 times, 160 times, 170 times, 180 times, 190 times, 200 times, 210 times, 220 times, 230 times, 240 times, 250 times, 260 times, 270 times, 280 times, 290 times, 300 times, 310 times, 320 times, 330 times, 340 times, 350 times, 360 times, 370 times, 380 times, 390 times, 400 times, 425 times, 450 times, 475 times, or 500 times.

[0156] In some embodiments, modifications to a CasX variant are mutations in one or more amino acids of the reference CasX. In other embodiments, modifications include insertions or substitutions of some or all of the domains derived from different CasX proteins. In certain embodiments, the CasX variants of SEQ ID NOs. 415-592 and 1147-1231 have the NTSB and helix 1B domains of SEQ ID NO. 1, in addition to the individual modifications in the selected domains described herein, while the other domains are derived from SEQ ID NO. 2. Mutations may be introduced into any one or more domains of the reference CasX protein or a CasX variant, and may include, for example, deletions of some or all of the domains in any domain of the reference CasX protein or a CasX variant from which it is derived, or substitutions, deletions, or insertions of one or more amino acids. The domains of the CasX protein include the non-target chain binding (NTSB) domain, the targeted chain loading (TSL) domain, the helix I domain, the helix II domain, the oligonucleotide binding domain (OBD), and the RuvC DNA cleavage domain. While not bound by theory or mechanism, the NTSB domain in CasX may enable binding to a non-target nucleic acid strand and assist in the unwinding of both the non-target and target strands. The NTSB domain is presumed to be involved in the unwinding or capture of the unwinded non-target nucleic acid strand. An exemplary NTSB domain includes amino acids 100-190 of SEQ ID NO: 1 or amino acids 102-191 of SEQ ID NO: 2. In some embodiments, the NTSB domain of the reference CasX protein includes a quadruple-stranded β-sheet. In some embodiments, the TSL acts to position or capture the folded target strand, placing cleavable phosphates of the target strand DNA backbone at the RuvC active site. An exemplary TSL includes amino acids 824-933 of SEQ ID NO: 1 or amino acids 811-920 of SEQ ID NO: 2. While not wishing to be bound by theory, in some cases, the helix I domain is thought to contribute to the binding of protospacer adjacent motifs (PAMs). In some embodiments, the helix I domain of the reference CasX protein contains one or more alpha helices.Exemplary helix II and I-II domains contain 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 helix II domain is involved in binding to the guide RNA scaffold stem loop and bound DNA. Exemplary helix II domains contain amino acids 332-508 of SEQ ID NO: 1, or amino acids 333-500 of SEQ ID NO: 2. The OBD primarily binds to the RNA triple helix of the guide RNA scaffold. The OBD may also be involved in binding to the protospacer adjacent motif (PAM). Exemplary OBD I and II domains contain 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. RuvC has a DED motif active site involved in cleaving both strands of DNA (most likely to be cleaved one by one, first the non-target strand at 11-14 nucleotides (nt) into the target sequence, and then the target strand cleaved 2-4 nucleotides after the target sequence, resulting in a shifted cleavage). CasX is particularly unique in that the RuvC domain is also involved in the binding of the guide RNA scaffold stem-loop, which is crucial for CasX function. Exemplary RuvC I and II domains include amino acids 660-823 and 934-986 of SEQ ID NO: 1, or amino acids 647-810 and 921-978 of SEQ ID NO: 2, respectively, while CasX variants may include mutations at positions I658 and A708 of SEQ ID NO: 2, or the CasX515 mutations described below.

[0157] In some embodiments, the CasX variant protein includes at least one modification in at least one domain of the reference CasX protein containing sequences 1-3, in each of at least two domains, in each of at least three domains, in each of at least four domains, or in each of at least five domains. In some embodiments, the CasX variant protein includes two or more modifications in at least one domain of the reference CasX protein. In some embodiments, the CasX variant protein includes 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, the CasX variant includes two or more modifications compared to reference CasX, and each modification is independently made in a domain selected from the group consisting of NTSB, TSL, helix I domain, helix II domain, OBD, and RuvC DNA cleavage domain. In some embodiments, the CasX variant comprises two or more modifications compared to the reference CasX protein, and the modifications are made in two or more domains. In some embodiments, 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 located in the NTSB domain, TSL domain, helix I domain, helix II domain, OBD, or RuvC DNA cleavage domain.

[0158] In some cases, the CasX variants of this disclosure include modifications in a structural region that may encompass one or more domains. In some embodiments, the CasX variant includes at least one modification of a non-contiguous amino acid residue region of the CasX variant that forms a channel in which target nucleic acid complexation with gRNA:CasX variant occurs. In other embodiments, the CasX variant includes at least one modification of a non-contiguous amino acid residue region of the CasX variant that forms an interface for binding to gRNA. In other embodiments, the CasX variant includes at least one modification of a non-contiguous amino acid residue region of the CasX variant that forms a channel for binding to non-target strand DNA. In other embodiments, the CasX variant includes at least one modification of a non-contiguous amino acid residue region of the CasX variant that forms an interface for binding to a protospacer adjacent motif (PAM) of the target nucleic acid. In other embodiments, the CasX variant includes at least one modification of a non-contiguous surface-exposed amino acid residue region of the CasX variant. In other embodiments, the CasX variant includes at least one modification of a region of non-adjacent amino acid residues that form a core via hydrophobic packing in the domain of the CasX variant. In the embodiments described above, the modification of the region may include one or more deletions, insertions, or substitutions of one or more amino acids in the region, or substitution of 2 to 15 amino acid residues in the region of the CasX variant with charged amino acids, or substitution of 2 to 15 amino acid residues in the region of the CasX variant with polar amino acids, or substitution of 2 to 15 amino acid residues in the region of the CasX variant with amino acids that stack with or have affinity for DNA or RNA bases.

[0159] In other embodiments, the Disclosure provides CasX variants comprising at least one modification compared to another CasX variant. For example, CasX variants 515 and 527 are variants of CasX variant 491, and CasX variants 668 and 672 are variants of CasX 535 (see Figure 44). In some embodiments, at least one modification is selected from the group consisting of amino acid insertions, deletions, or substitutions. All variants that improve one or more functions or characteristics of a CasX variant protein compared to a reference CasX protein described herein or a variant from which it is derived are assumed to be within the scope of the Disclosure. As described in the Examples, CasX variants may be mutagenic to produce another CasX variant. In certain embodiments, the Disclosure provides a variant of CasX 515 (SEQ ID NO: 416) produced in Example 14 by introducing a modification into the coding sequence that results in an amino acid substitution, deletion, or insertion at one or more positions in one or more domains.

[0160] Suitable mutagenesis methods for generating the CasX variant proteins of this disclosure may include, for example, comprehensive mutational evolution (DME), comprehensive mutational scanning (DMS), error-prone PCR, cassette mutagenesis, random mutagenesis, adhesion extension PCR, gene shuffling, or domain swapping (as described in International Application PCT / US20 / 36506 and International Publication 2020247883(A2), incorporated herein by reference). In some embodiments, CasX variants are designed by selecting several desired mutations in a CasX variant identified, for example, using the assay described in the Examples. In certain embodiments, the activity of a reference CasX or CasX variant protein is used as a benchmark against which the activity of one or more obtained CasX variants is compared before mutagenesis, thereby measuring the improvement in the function of the new CasX variant.

[0161] In some embodiments of the CasX variants described herein, at least one modification comprises: (a) 1 to 100 consecutive or non-consecutive amino acid substitutions in the CasX variant as compared to the reference CasX of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, CasX variant 491 (SEQ ID NO: 336), or CasX variant 515 (SEQ ID NO: 416); (b) 1 to 100 consecutive or non-consecutive amino acid deletions in the CasX variant as compared to the reference CasX or the variant from which it is derived; (c) 1 to 100 consecutive or non-consecutive amino acid insertions in the CasX as compared to the reference CasX or the variant from which it is derived; or (d) any combination of (a) to (c). In some embodiments, at least one modification comprises: (a) 1 to 10 consecutive or non-consecutive amino acid substitutions in the CasX variant as compared to the reference CasX of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or the variant from which it is derived; (b) 1 to 5 consecutive or non-consecutive amino acid deletions in the CasX variant as compared to the reference CasX or the variant from which it is derived; (c) 1 to 5 consecutive or non-consecutive amino acid insertions in the CasX as compared to the reference CasX or the variant from which it is derived; or (d) any combination of (a) to (c).

[0162] In some embodiments, a CasX variant protein comprises or consists of a sequence having 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 least 80, at least 90, or at least 100 modifications compared to the sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, CasX491, or CasX515. In some embodiments, a CasX variant protein comprises one or more substitutions compared to CasX491 or SEQ ID NO: 336. In some embodiments, a CasX variant protein comprises one or more substitutions compared to CasX515 or SEQ ID NO: 416. These modifications may be amino acid insertions, deletions, substitutions, or any combination thereof. The modifications may be in one domain of the CasX variant, or in any of the domains, or in any combination of domains. In the substitutions described herein, any amino acid may be substituted with any other amino acid. The substitution may be a conservative substitution (e.g., a basic amino acid is substituted for another basic amino acid). The substitution may also be a non-conservative substitution (e.g., a basic amino acid is substituted for an acidic amino acid, or vice versa). For example, the CasX variant protein of this disclosure can be generated by substituting proline in the reference CasX protein with any of arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, or valine.

[0163] Any permutation of substitutions, insertions, and deletions of the embodiments described herein can be combined to generate the CasX variant proteins of this disclosure. For example, a CasX variant protein may include at least one substitution and at least one deletion compared to a reference CasX protein sequence or the sequence of CasX491 or CasX515, may include at least one substitution and at least one insertion compared to a reference CasX protein sequence or the sequence of CasX491 or CasX515, may include at least one insertion and at least one deletion compared to a reference CasX protein sequence or the sequence of CasX491 or CasX515, or may include at least one substitution, at least one insertion, and at least one deletion compared to a reference CasX protein sequence or the sequence of CasX491 or CasX515.

[0164] In some embodiments, the CasX variant protein comprises 400-2000 amino acids, 500-1500 amino acids, 700-1200 amino acids, 800-1100 amino acids, or 900-1000 amino acids.

[0165] In some embodiments, the CasX variant protein comprises the sequences of SEQ ID NOs. 247-592 or 1147-1231 shown in Table 3. In other embodiments, the CasX variant protein contains sequences that are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 86%, at least 87%, at least 88%, at least 89%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and at least 99.5% identical to sequences of sequence numbers 270–592 or 1147–1231 shown in Table 3. In other embodiments, the CasX variant protein contains sequences that are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 86%, at least 87%, at least 88%, at least 89%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and at least 99.5% identical to sequences of sequence numbers 415–592 or 1147–1231.To the extent of the other implementation, the CasX variant protein contains sequences that are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 86%, at least 87%, at least 88%, at least 89%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and at least 99.5% identical to sequences of sequence numbers 416-592 or 1147-1231. (ND = Not described or otherwise not provided).

[0166] [Table 3-1]

[0167] [Table 3-2]

[0168] [Table 3-3]

[0169] [Table 3-4]

[0170] [Table 3-5]

[0171] [Table 3-6]

[0172] [Table 3-7]

[0173] c. CasX variant protein having domains derived from multiple source proteins In certain embodiments, the Disclosure provides a chimeric CasX protein comprising protein domains derived from two or more different CasX proteins (e.g., two or more naturally occurring CasX proteins or two or more CasX variant protein sequences described herein). As used herein, “chimeric CasX protein” means a CasX comprising at least two domains isolated or derived from different sources (e.g., two naturally occurring proteins), which in some embodiments may be isolated from different species. For example, in some embodiments, a chimeric CasX protein comprises a first domain derived from a first CasX protein and a second domain derived from a second different CasX protein. In some embodiments, the first domain may be selected from the group consisting of NTSB, TSL, helix I, helix II, OBD, and RuvC domains. In some embodiments, the second domain is selected from the group consisting of NTSB, TSL, helix I, helix II, OBD, and RuvC domains, and the second domain is different from the first domain described herein. In certain embodiments, the CasX variants 514-791 (SEQ ID NOs. 415-592 and 1147-1231) have an NTSB domain and a helix 1B domain derived from the sequence of SEQ ID NO: 1, while the other domains are derived from SEQ ID NO: 2, and it is understood that this variant has further amino acid changes at a selected position.

[0174] Protein affinity for d.gRNA In some embodiments, the CasX variant protein has improved affinity for gRNA compared to the reference CasX protein, leading to the formation of a ribonucleoprotein (RNP) complex. The increased affinity of the CasX variant protein for gRNA results in, for example, lower K for RNP complex formation. d This can result in the formation of a more stable ribonucleoprotein complex. In some embodiments, increased affinity of the CasX variant protein to 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 target cells and may result in improved pharmacokinetic properties in the blood when delivered to the target. In some embodiments, increased affinity of the CasX variant protein and the resulting increased stability of the ribonucleoprotein complex result in a lower dose of the CasX variant protein delivered to the target or cell while still having the desired activity, e.g., gene editing in vivo or in vitro. In some embodiments, higher affinity (stronger binding) of the CasX variant protein to gRNA allows for more editing events if both the CasX variant protein and gRNA remain in the RNP complex. The increase in editing events can be evaluated using an editing assay such as the tdTom editing assay described herein. In some embodiments, the K of the CasX variant protein to gRNA dThe binding affinity is increased by 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 times compared to the reference CasX protein. In some embodiments, the CasX variant has a binding affinity to gRNA that is increased by about 1.1 to about 10 times compared to the reference CasX protein of SEQ ID NO: 2.

[0175] In some embodiments, increased affinity of the CasX variant protein to gRNA results in increased stability of the ribonucleoprotein complex when delivered to mammalian cells, including in vivo delivery to the target. This increased stability may affect the function and utility of the complex in the target cells and may result in improved pharmacokinetic properties in the blood when delivered to the target. In some embodiments, increased affinity of the CasX variant protein and increased stability of the resulting ribonucleoprotein complex allow for lower doses of the CasX variant protein delivered to the target or cells while still having the desired activity (e.g., in vivo or in vitro gene editing). The increased ability to form RNPs and maintain them in a stable form can be evaluated using assays (e.g., in vitro cleavage assays described in the examples herein). In some embodiments, RNPs containing the CasX variant of this disclosure, when complexed as RNPs, exhibit at least 2-fold, at least 5-fold, or at least 10-fold higher kJ compared to RNPs containing the reference CasX of SEQ ID NOs: 1-3. 切断 Speed ​​can be achieved.

[0176] In some embodiments, a higher affinity (stronger binding) of the CasX variant protein to the gRNA allows for more editing events if both the CasX variant protein and gRNA remain in the RNP complex. The increase in editing events can be evaluated using editing assays such as those described herein.

[0177] While we do not wish to be bound by theory, in some embodiments, amino acid changes in the helix I domain can increase the binding affinity between the CasX variant protein and the gRNA targeting sequence, changes in the helix II domain can increase the binding affinity between the CasX variant protein and the gRNA scaffold stem-loop, and changes in the oligonucleotide-binding domain (OBD) can increase the binding affinity between the CasX variant protein and the gRNA triple helix.

[0178] Methods for measuring the binding affinity of CasX protein to gRNA include in vitro methods using purified CasX protein and gRNA. Binding affinity to reference CasX and variant proteins can be measured by fluorescence polarization if the gRNA or CasX protein is tagged with a fluorophore. Alternatively or additionally, binding affinity can be measured by biolayer interferometry, electrophoretic mobility shift assay (EMSA), or membrane binding. Further standard techniques for quantifying the absolute affinity of RNA-binding proteins, such as reference CasX and the variant proteins of this disclosure, to specific gRNAs, such as reference gRNA and its variants, include, but are not limited to, isothermal calorimetry (ITC) and surface plasmon resonance (SPR), as well as the methods described in the examples.

[0179] In some catalytically inactive embodiments, the CasX variant protein includes one or more modifications to a region of non-contiguous residues that forms a channel where gRNA:target nucleic acid complexation occurs. In some embodiments, the CasX variant protein includes one or more modifications to a region of non-contiguous residues that form an interface with gRNA. For example, in some embodiments of the reference CasX protein, the helix I, helix II, and OBD domains are all in contact with or adjacent to the gRNA:target nucleic acid complex, and one or more modifications to non-contiguous residues in any of these domains may improve the function of the CasX variant protein.

[0180] In some embodiments, the CasX variant protein includes one or more modifications to a region of non-contiguous residues that form a channel for binding to non-target strand DNA. For example, the CasX variant protein may include one or more modifications to non-contiguous residues in the NTSB domain. In some embodiments, the CasX variant protein includes one or more modifications to a region of non-contiguous residues that form an interface for binding to PAM. For example, the CasX variant protein may include one or more modifications to non-contiguous residues in the helix I domain or OBD. In some embodiments, the CasX variant protein includes one or more modifications that include a region of non-contiguous surface-exposed residues. As used herein, “surface-exposed residues” means amino acids on the surface of the CasX protein, or amino acids in which at least a portion (e.g., part of the backbone or side chain) is on the surface of the protein. Surface-exposed residues of cellular proteins (e.g., CasX) exposed to the aqueous intracellular environment are often selected from positively charged hydrophilic amino acids, such as arginine, asparagine, aspartic acid, glutamine, glutamic acid, histidine, lysine, serine, and threonine. Therefore, in some embodiments of the variants provided herein, for example, the surface-exposed residue region includes one or more insertions, deletions, or substitutions compared to a reference CasX protein. In some embodiments, one or more positively charged residues are replaced with one or more other positively charged residues, or negatively charged residues, or uncharged residues, or any combination thereof. In some embodiments, one or more amino acid residues for substitution are nearby bound nucleic acids (e.g., residues in the RuvC domain or helix I domain that contact the target nucleic acid, or residues in the OBD or helix II domain that bind to gRNA), and may be replaced with one or more positively charged amino acids or polar amino acids.

[0181] In some embodiments, the CasX variant protein includes one or more modifications to the region of non-contiguous residues that form a core via hydrophobic packing in the domain of the reference CasX protein. While we do not wish to be bound by any theory, the region that forms a core via hydrophobic packing is rich in hydrophobic amino acids (e.g., valine, isoleucine, leucine, methionine, phenylalanine, tryptophan, and cysteine). For example, in some reference CasX proteins, the RuvC domain includes a hydrophobic pocket adjacent to the active site. In some embodiments, 2 to 15 residues in the region are charged residues, polar residues, or base-stacking residues. Examples of charged amino acids (sometimes referred to herein as residues) include arginine, lysine, aspartic acid, and glutamic acid, the side chains of which can form salt bridges, provided that a cross-linking partner is also present (see Figure 14). Examples of polar amino acids include glutamine, asparagine, histidine, serine, threonine, tyrosine, and cysteine. Polar amino acids, in some embodiments, can form hydrogen bonds as proton donors or acceptors, depending on the identity of their side chains. As used herein, “base stacking” includes the interaction between the aromatic side chain of an amino acid residue (such as tryptophan, tyrosine, phenylalanine, or histidine) and the stacked nucleotide bases in a nucleic acid. Any modification to spatially adjacent, non-contiguous regions of amino acids to form a functional portion of a CasX variant protein is assumed to be within the scope of this disclosure.

[0182] e. CasX variant protein having domains derived from multiple source proteins In certain embodiments, the Disclosure provides chimeric CasX variant proteins comprising protein domains derived from two or more different CasX proteins (e.g., two or more naturally occurring CasX proteins or two or more CasX variant protein sequences described herein). As used herein, “chimeric CasX protein” means CasX comprising at least two domains isolated or derived from different sources (e.g., two naturally occurring proteins), which in some embodiments may be isolated from different species. For example, in some embodiments, the chimeric CasX protein comprises a first domain derived from a first CasX protein and a second domain derived from a second different CasX protein. In some embodiments, the first domain may be selected from the group consisting of NTSB, TSL, helix I, helix II, OBD, and RuvC domains. In some embodiments, the second domain is selected from the group consisting of NTSB, TSL, helix I, helix II, OBD, and RuvC domains, and the second domain is different from the first domain described herein. For example, a chimeric CasX protein may contain the NTSB, TSL, helix I, helix II, and OBD domains from the CasX protein of SEQ ID NO: 2, and the RuvC domain from the CasX protein of SEQ ID NO: 1, or vice versa. As a further example, a chimeric CasX protein may contain the NTSB, TSL, helix II, OBD, and RuvC domains from the CasX protein of SEQ ID NO: 2, and the helix I domain from the CasX protein of SEQ ID NO: 1, or vice versa. Therefore, in certain embodiments, a chimeric CasX protein may contain the NTSB, TSL, helix II, OBD, and RuvC domains from a first CasX protein, and the helix I domain from a second CasX protein. In some embodiments of the chimeric CasX protein, the domain of the first CasX protein is derived from the sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and the domain of the second CasX protein is derived from the sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and the first CasX protein and the second CasX protein are not the same.In some embodiments, the domain of the first CasX protein includes a sequence derived from SEQ ID NO: 1, and the domain of the second CasX protein includes a sequence derived from SEQ ID NO: 2. In some embodiments, the domain of the first CasX protein includes a sequence derived from SEQ ID NO: 1, and the domain of the third CasX protein includes a sequence derived from SEQ ID NO: 2. In some embodiments, the domain of the second CasX protein includes a sequence derived from SEQ ID NO: 1, and the domain of the third CasX protein includes a sequence derived from SEQ ID NO: 2. As an example of the foregoing, the chimeric RuvC domain includes amino acids 660-823 of SEQ ID NO: 1 and amino acids 921-978 of SEQ ID NO: 2. As an alternative example of the foregoing, the chimeric RuvC domain includes amino acids 647-810 of SEQ ID NO: 2 and amino acids 934-986 of SEQ ID NO: 1. In some embodiments, at least one chimeric domain includes a chimeric helix I domain, which includes 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 may be further modified to include a CasX variant selected from the group consisting of the sequences SEQ ID NOs: 270, 328, 336, 780, 412, 413, 414, 416, 435, 329, 781, 330, 782, 331, 783, 332, 784, 333, 785, 334, 786, 335, 567, 570, 574, 787, and 788. In some embodiments, one or more additional modifications may include insertions, substitutions, or deletions as described herein.

[0183] In the case of split or discontinuous domains such as helix I, RuvC, and OBD, a portion of the discontinuous domain can be replaced with a corresponding portion from any other source. For example, the helix II domain in SEQ ID NO: 2 (sometimes referred to as helix Ia) can be replaced with the corresponding helix II sequence from SEQ ID NO: 1. Table 4 shows the domain sequences and their coordinates from the reference CasX protein. Representative examples of chimeric CasX proteins include variants of CasX472-483, 485-491, and 515, whose sequences are shown in Table 3.

[0184] [Table 4] * OBDI and II, helix II and I-II, and RuvC I and II are also referred to herein as OBDa and b, helix Ia and b, and RuvCa and b.

[0185] Exemplary domain sequences are provided in Table 5 below.

[0186] [Table 5]

[0187] Further exemplary helix II domain sequences are provided as Sequence ID No. 2351, and further exemplary RuvCa domain sequences are provided as Sequence ID No. 2352.

[0188] In other embodiments, the CasX variant protein comprises the sequences of SEQ ID NOs. 247-592 or 1147-1231 shown in Table 3, and further comprises one or more NLSs disclosed herein at or near the N-terminus, C-terminus, or both. In other embodiments, the CasX variant protein comprises the sequences of SEQ ID NOs. 270-592 and 1147-1231, and further comprises one or more NLSs disclosed herein at or near the N-terminus, C-terminus, or both. In other embodiments, the CasX variant protein comprises the sequences of SEQ ID NOs. 415-592 and 1147-1231, and further comprises one or more NLSs disclosed herein at or near the N-terminus, C-terminus, or both. In some cases, it will be understood that the N-terminal methionine of the CasX variants in the table is removed from the expressed CasX variant during post-translational modification. Those skilled in the art will understand that the NLS near the N-terminus or C-terminus of a protein may 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-terminus or C-terminus.

[0189] f. CasX variants derived from other CasX variants In further iterations of variant protein generation, the variant proteins can be used to generate further CasX variants of the present disclosure. For example, as shown in Figure 44, CasX119 (SEQ ID NO: 270), CasX491 (SEQ ID NO: 336), and CasX515 (SEQ ID NO: 416) are exemplary variant proteins modified to generate additional CasX variants of the present disclosure that have improvements or additional properties compared to the reference CasX or CasX variant from which they are derived. CasX119 includes the L379R substitution, the A708K substitution, and the deletion of P at position 793 of SEQ ID NO: 2. CasX491 includes the NTSB and helix 1B swap derived from SEQ ID NO: 1. CasX515 was derived from CasX491 by inserting P at position 793 (relative to SEQ ID NO: 2) and was used to construct the CasX variants described in Examples 13 and 14. For example, CasX668 has an R insertion and a G223S substitution at position 26 compared to CasX515. CasX672 has L169K and G223S substitutions compared to CasX515. CasX676 has L169K and G223S substitutions and an R insertion at position 26 compared to CasX515.

[0190] Exemplary methods used to generate and evaluate CasX variants derived from other CasX variants are described in the Examples, which were generated by introducing modifications to the coding sequence that result in amino acid substitutions, deletions, or insertions at one or more positions in one or more domains of the CasX variant. In particular, Examples 14 and 15 describe methods used to generate variants of CasX515 (SEQ ID NO: 416), which were then assayed to determine the positions in the sequence that, when modified by amino acid insertions, deletions, or substitutions, result in enrichment or improvement in the assay. In some cases, the assay results were used to generate the heatmaps in Figures 34–36, which provide qualitative and quantitative data at given amino acid positions modified by the present method. For the purposes of this disclosure, the domain sequences of CasX515 are provided in Table 4, including the OBD-I domain having the sequence of SEQ ID NO: 2342, the OBD-II domain having the sequence of SEQ ID NO: 2347, the NTSB domain having the sequence of SEQ ID NO: 2335, the Helix II domain having the sequence of SEQ ID NO: 2343, the Helix I-II domain having the sequence of SEQ ID NO: 2336, the Helix II domain having the sequence of SEQ ID NO: 2351, the RuvC-I domain having the sequence of SEQ ID NO: 2352, the RuvC-II domain having the sequence of SEQ ID NO: 2350, and the TSL domain having the sequence of SEQ ID NO: 2349. The methods of this disclosure provide exemplary modifications that result in the obtained positions and subsequent enrichment or improvement by modifying and assaying individual positions in the domains of CasX515 and comparing them with those positions in each domain or subdomain. In some cases, such positions are disclosed in Tables 21-24 of the Examples. In some embodiments, the Disclosure provides CasX variants derived from CasX515 that, compared to SEQ ID NO: 2335, include one or more modifications (i.e., insertions, deletions, or substitutions) at one or more amino acid positions in the NTSB domain selected from the group consisting of P2, S4, Q9, E15, G20, G33, L41, Y51, F55, L68, A70, E75, K88, and G90, wherein the modifications result in improved characteristics compared to CasX515.In certain embodiments, compared to Sequence ID No. 2335, one or more modifications at one or more amino acid positions in the NTSB domain are selected from the group consisting of ^G2, ^I4, ^L4, Q9P, E15S, G20D, [S30], G33T, L41A, Y51T, F55V, L68D, L68E, L68K, A70Y, A70S, E75A, E75D, E75P, K88Q, and G90Q (where "^" represents an insertion at that position and "[]" represents a deletion at that position). In some embodiments, the disclosure provides CasX variants derived from CasX515, comprising one or more modifications at one or more amino acid positions in the helix I-II domain, selected from the group consisting of I24, A25, Y29, G32, G44, S48, S51, Q54, I56, V63, S73, L74, K97, V100, M112, L116, G137, F138, and S140, compared to SEQ ID NO: 2336, wherein the modifications result in improved characteristics compared to CasX515. In certain embodiments, one or more modifications at one or more amino acid positions in the helix I-II domain are selected from the group consisting of ^T24, ^C25, 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, ^Q138, and S140Q.In some embodiments, the present disclosure provides CasX variants derived from CasX515, compared to Sequence ID No. 2351, namely L2, V3, E4, R5, Q6, A7, E9, V10, D11, W12, W13, D14, M15, V16, C17, N18, V19, K20, L22, I23, E25, K26, K31, Q35, L37, A38, K41, R42, Q43, E44, L46, K57, Y65, G68, L70, L71, L72, E75, G79, D81, W82, K84, V85, Y86, D87, I93, K95, K96, E98, L100, K10 2, comprising one or more modifications at one or more amino acid positions in the helix II domain, selected from the group consisting of I104, K105, E109, R110, D114, K118, A120, L121, W124, L125, R126, A127, A129, I133, E134, G135, L136, E138, D140, K141, D142, E143, F144, C145, C147, E148, L149, K150, L151, Q152, K153, L158, E166, and A167, wherein the modifications result in improved characteristics compared to CasX515.In certain embodiments, one or more modifications at one or more amino acid positions in the helix II domain are ^A2, ^H2, [L2]+[V3], V3E, V3Q, V3F, [V3], ^D3, V3P, E4P, [E4], E4D, E4L, E4R, R5N, Q6V, ^Q6, ^G7, ^H9, ^A9, VD10, ^T10, [V10], ^F10, ^D11, [D11], D11S, [W12], W12T, W12H, ^P12, ^Q13, ^G12, ^R13, W13P, W13D, ^D13, W13L, ^P14, ^D1 4, [D14]+[M15], [M15], ^T16, ^P17, N18I, V19N, V19H, K20D, L22D, I23S , E25C, E25P, ^G25, K26T, K27E, K31L, K31Y, Q35D, Q35P, ^S37, [L37]+[A 38], K41L, ^R42, [Q43]+[E44], L46N, K57Q, Y65T, G68M, L70V, L71C, L72 D, L72N, L72W, L72Y, E75F, E75L, E75Y, G79P, ^E79, ^T81, ^R81, ^W81, ^Y 81, ^W82, ^Y82, W82G, W82R, K84D, K84H, K84P, K84T, V85L, V85A, ^L85, Y 86C, D87G, D87M, D87P, I93C, K95T, K96R, E98G, L100A, K102H, I104T, I1 04S, I104Q, K105D, ^K109, E109L, R110D, [R110], D114E, ^D114, K118P, A120R, L121T, W124L, L125C, R126D, A127E, A127L, A129T, A129K, I133E Selected from the group consisting of ^C133, ^S134, ^G134, ^R135, G135P, L136K, L136D, L136S, L136H, [E138], D140R, ^D140, ^P141, ^D142, [E143]+[F144], ^Q143, F144K, [F144], [F144]+[C145], C145R, ^G145, C145K, C147D, ^V148, E148D, ^H149, L149R, K150R, L151H, Q152C, K153P, L158S, E166L, and ^F167.In some embodiments, the disclosure provides CasX variants derived from CasX515, comprising one or more modifications at one or more amino acid positions in the RuvC-I domain, selected from the group consisting of I4, K5, P6, M7, N8, L9, V12, G49, K63, K80, N83, R90, M125, and L146, compared to SEQ ID NO: 2352, wherein the modifications result in improved characteristics compared to CasX515. In certain embodiments, one or more modifications at one or more amino acid positions in the RuvC-I domain are ^I4, ^S5, ^T6, ^N6, ^R7, ^K7, ^H8, ^S8, V12L, G49W, G49R, S51R, S51K, K62S, K62T, K62E, V65A, K80E, N83G, R90H, R90G, M125S, M125A, L137Y, ^P137, [L141], L141R, L141D, ^Q142 Selected from the group consisting of ^R143, ^N143, E144N, ^P146, L146F, P147A, K149Q, T150V, ^R152, ^H153, T155Q, ^H155, ^R155, ^L156, [L156], ^W156, ^A157, ^F157, A157S, Q158K, [Y159], T160Y, T160F, ^I161, S161P, T163P, ^N163, C164K, and C164M. In some embodiments, the disclosure provides CasX variants derived from CasX515, comprising one or more modifications at one or more amino acid positions in the OBD-I domain, selected from the group consisting of I4, K5, P6, M7, N8, L9, V12, G49, K63, K80, N83, R90, M125, and L146, compared to SEQ ID NO: 2342, wherein the modifications result in improved characteristics compared to CasX515.In certain embodiments, one or more modifications at one or more amino acid positions in the OBD-I domain are selected from the group consisting of ^G3, I3G, I3E, ^G4, K4G, K4P, K4S, K4W, K4W, R5P, ^P5, ^G5, R5S, ^S5, R5A, R5P, R5G, R5L, I6A, I6L, ^G6, N7Q, N7L, N7S, K8G, K15F, D16W, ^F16, ^F18, ^P27, M28P, M28H, V33T, R34P, M36Y, R41P, L47P, ^P48, E52P, ^P55, [P55]+[Q56], Q56S, Q56P, ^D56, ^T56, and Q56P. In some embodiments, the Disclosure provides CasX variants derived from CasX515, comprising one or more modifications at one or more amino acid positions in the OBD-II domain, selected from the group consisting of I4, K5, P6, M7, N8, L9, V12, G49, K63, K80, N83, R90, M125, and L146, compared to SEQ ID NO: 2347, wherein the modifications result in improved characteristics compared to CasX515. In certain embodiments, 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], K11T, ^P24, K37G, R42E, ^S53, ^R58, [K63], M70T, I82T, Q92I, Q92F, Q92V, Q92A, ^A93, K110Q, R115Q, L121T, ^A124, ^R141, ^D143, ^A143, ^W144, and ^A145. In some embodiments, the Disclosure provides CasX variants derived from CasX515, comprising one or more modifications at one or more amino acid positions in the TSL domain, selected from the group consisting of S1, N2, C3, G4, F5, I7, K18, V58, S67, T76, G78, S80, G81, E82, S85, V96, and E98, compared to SEQ ID NO: 2349, wherein the modifications result in improved characteristics compared to CasX515.In certain embodiments, one or more modifications at one or more amino acid positions in the OBD-I domain are selected from the group consisting of ^M1, [N2], ^V2, C3S, ^G4, ^W4, F5P, ^W7, K18G, V58D, ^A67, T76E, T76D, T76N, G78D, [S80], [G81], ^E82, ^N82, S85I, V96C, V96T, and E98D. Any combination of the same aforementioned modifications in the paragraph may similarly be introduced into the CasX variants of this disclosure, and it will be understood that this results in CasX variants having improved characteristics. For example, in one embodiment, this disclosure provides CasX variant 535 (SEQ ID NO: 435) having a single mutation of G223S compared to CasX515. Another embodiment. In a different embodiment, the disclosure provides CasX variant 668 (SEQ ID NO: 567) having an insertion of R at position 26 and a substitution of G223S compared to CasX515. In another embodiment, the disclosure provides CasX672 (SEQ ID NO: 570) having substitutions of L169K and G223S compared to CasX515. In yet another embodiment, the disclosure provides CasX676 (SEQ ID NO: 574) having substitutions of L169K and G223S at position 26 and an insertion of R compared to CasX515. CasX variants having improved features compared to CasX515 include the variants in Table 3.

[0191] Compared to the same features in a reference CasX protein and to CasX variants from which they are derived, exemplary features that may be improved in a CasX variant protein include, but are not limited to, improved variant folding, improved binding affinity to gRNA, improved binding affinity to target nucleic acids, improved ability to utilize a wider range of PAM sequences in editing and / or binding of target nucleic acids, improved unwinding of target DNA, increased editing activity, improved editing efficiency, improved editing specificity for target nucleic acids, reduced off-target editing or cleavage, increased proportion of eukaryotic genomes that can be efficiently edited, increased nuclease activity, increased target strand loading for double-strand breaks, reduced target strand loading for single-strand nicking, improved binding of the non-target strand of DNA, improved protein stability, improved stability of the protein:gRNA (RNP) complex, and improved fusion features. In certain embodiments, as described in the examples, such improved features may 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 target nucleic acid sequences, and reduced off-target cleavage of target nucleic acids.

[0192] [Table 6]

[0193] The CasX variants of the embodiments described herein have the ability to form an RNP complex with the gRNA disclosed herein. In some embodiments, the RNP comprising the CasX variant protein and gRNA of this disclosure can cleave double-stranded DNA targets with at least 80% efficiency at concentrations of 20 pM or less. In some embodiments, the RNP at concentrations of 20 pM or less can cleave double-stranded DNA targets with 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% efficiency. In some embodiments, the RNP at concentrations 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 can cleave double-stranded DNA targets with 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% efficiency. These improved features are described in more detail below.

[0194] g. Protein stability In some embodiments, the disclosure provides a CasX variant protein having improved stability compared to a reference CasX protein. In some embodiments, the improved stability of the CasX variant protein results in higher steady-state protein expression and improved editing efficiency. In some embodiments, the improved stability of the CasX variant protein results in a larger proportion of CasX protein remaining folded in a functional conformation, improving editing efficiency or improving purifiability for manufacturing purposes. As used herein, “functional conformation” refers to a CasX protein in a conformation that allows the protein to bind to gRNA and target nucleic acid. In embodiments where the CasX variant does not have one or more mutations that catalytically inactivate it, the CasX variant, when complexed with a gRNA having a targeting sequence that can hybridize with the target nucleic acid, can cleave, nick, or otherwise modify the target nucleic acid. A functional conformation of CasX refers to a conformation that is “cleavage competent”. In some embodiments, including those embodiments that result in a larger proportion of CasX protein remaining folded into a functional conformation, applications such as gene editing require lower concentrations of the CasX variant compared to the reference CasX protein. Therefore, in some embodiments, the CasX variant with improved stability has improved efficiency compared to the reference CasX in one or more gene editing situations.

[0195] In some embodiments, the disclosure provides CasX variant proteins having improved stability of the CasX variant protein:gRNA RNP complex compared to the reference CasX protein:gRNA complex, such that the RNPs remain in a functional form. Improved stability may include increased thermal stability, resistance to proteolysis, enhanced pharmacokinetic properties, stability across a range of pH conditions, salt conditions, and tonicity. Improved complex stability may result in improved editing efficiency in some embodiments. In some embodiments, the RNPs of the CasX variant and gRNA variant have at least 2-fold, at least 3-fold, or at least 4-fold higher proportions of cleavage-competent RNPs compared to the RNPs of the reference CasX (SEQ ID NOs. 1-3) and gRNA (SEQ ID NOs. 4 or 5) in Table 1. Exemplary data of increased cleavage-competent RNPs are provided in the examples.

[0196] In some embodiments, the improved stability of the CasX variant protein includes improved folding kinetics of the CasX variant protein compared to the reference CasX protein. In some embodiments, the folding kinetics of the CasX variant protein are improved by at least about 5 times, at least about 10 times, at least about 50 times, at least about 100 times, at least about 500 times, at least about 1,000 times, at least about 2,000 times, at least about 3,000 times, at least about 4,000 times, at least about 5,000 times, or at least about 10,000 times compared to the reference CasX protein. In some embodiments, the folding dynamics of the CasX variant protein are improved by at least about 1 kJ / mol, at least about 5 kJ / mol, at least about 10 kJ / mol, at least about 20 kJ / mol, at least about 30 kJ / mol, at least about 40 kJ / mol, at least about 50 kJ / mol, at least about 60 kJ / mol, at least about 70 kJ / mol, at least about 80 kJ / mol, at least about 90 kJ / mol, at least about 100 kJ / mol, at least about 150 kJ / mol, at least about 200 kJ / mol, at least about 250 kJ / mol, at least about 300 kJ / mol, at least about 350 kJ / mol, at least about 400 kJ / mol, at least about 450 kJ / mol, or at least about 500 kJ / mol compared to the reference CasX protein.

[0197] Exemplary amino acid changes that can increase the stability of a CasX variant protein compared to a reference CasX protein include, but are not limited to, amino acid changes that increase the number of hydrogen bonds in the CasX variant protein, increase the number of disulfide crosslinks in the CasX variant protein, increase the number of salt bridges in the CasX variant protein, strengthen the interactions between segments of the CasX variant protein, increase the buried hydrophobic surface of the CasX variant protein, or any combination thereof.

[0198] Protein affinity for h-gRNA In some embodiments, the CasX variant protein exhibits improved affinity for gRNA compared to the reference CasX protein or another CasX variant from which it is derived, leading to the formation of a ribonucleoprotein complex. The increased affinity of the CasX variant protein for gRNA results in, for example, lower K for RNP complex formation. d This can result in the formation of a more stable RNP complex, which in some cases leads to increased affinity of the CasX variant protein to the gRNA, resulting in increased stability of the RNP complex when delivered to human cells. This increased stability can affect the function and utility of the complex in the target cells and may result in improved pharmacokinetic properties in the blood when delivered to the target. In some embodiments, increased affinity of the CasX variant protein and the resulting increased stability of the RNP complex allow for lower doses of the CasX variant protein delivered to the target or cells while still having the desired activity, e.g., gene editing in vivo or in vitro.

[0199] In some embodiments, a higher affinity (stronger binding) of the CasX variant protein to the gRNA allows for more editing events if both the CasX variant protein and gRNA remain in the RNP complex. The increase in editing events can be evaluated using the editing assays described herein.

[0200] In some embodiments, the K of the CasX variant protein relative to gRNA dThe binding affinity is increased by 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 times compared to the reference CasX protein or another CasX variant from which it is derived. In some embodiments, the CasX variant has a binding affinity to gRNA that is increased by about 1.1 to about 10 times compared to the reference CasX protein of SEQ ID NO: 2.

[0201] While we do not wish to be bound by theory, in some embodiments, amino acid changes in the helix I domain can increase the binding affinity between the CasX variant protein and the gRNA targeting sequence, changes in the helix II domain can increase the binding affinity between the CasX variant protein and the gRNA scaffold stem-loop, and changes in the oligonucleotide-binding domain (OBD) can increase the binding affinity between the CasX variant protein and the gRNA triple helix.

[0202] Methods for measuring the binding affinity of CasX protein to gRNA include in vitro methods using purified CasX protein and gRNA. Binding affinity to reference CasX and variant proteins can be measured by fluorescence polarization if the gRNA or CasX protein is tagged with a fluorophore. Alternatively or additionally, binding affinity can be measured by biolayer interferometry, electrophoretic mobility shift assay (EMSA), or membrane binding. Further standard techniques for quantifying the absolute affinity of RNA-binding proteins, such as reference CasX and the variant proteins of this disclosure, to specific gRNAs, such as reference gRNA and its variants, include, but are not limited to, isothermal calorimetry (ITC) and surface plasmon resonance (SPR), as well as the methods of the examples.

[0203] i. Affinity for target nucleic acids In some embodiments, CasX variant proteins have improved binding affinity to the target nucleic acid compared to the affinity of the reference CasX protein or another CasX variant from which it is derived. CasX variants with higher affinity to the target nucleic acid can, in some embodiments, cleave the target nucleic acid sequence more rapidly than reference CasX proteins that do not have increased affinity to the target nucleic acid.

[0204] In some embodiments, improved affinity for a target nucleic acid includes improved affinity for the target sequence or protospacer sequence of the target nucleic acid, improved affinity for the PAM sequence, improved ability to search for DNA for the target sequence, or any combination thereof. While we do not wish to be bound by theory, it is thought that proteins in the CRISPR / Cas system, such as CasX, can find their target sequences by one-dimensional diffusion along the DNA molecule. This process is thought to include (1) binding of the ribonucleoprotein to the DNA molecule, followed by (2) termination at the target sequence, and in some embodiments, both of these may be influenced by improved affinity of the CasX protein for the target nucleic acid sequence, thereby improving the function of the CasX variant protein compared to the reference CasX protein.

[0205] In some embodiments, CasX variant proteins with improved target nucleic acid affinity have increased affinity for or ability to utilize specific PAM sequences other than the canonical TTC PAM recognized by the reference CasX protein of Sequence ID No. 2, including PAM sequences selected from the group consisting of TTC, ATC, GTC, and CTC, thereby increasing the amount of target nucleic acid that can be edited compared to the wild-type CasX nuclease or the CasX199 or 491 nuclease. While we do not wish to be bound by theory, these protein variants may interact more strongly with DNA overall and, due to their ability to utilize further PAM sequences beyond the PAM sequences of the wild-type reference CasX or the CasX199 or 491 nuclease, may have an increased ability to access and edit sequences within target nucleic acids, thereby enabling a more efficient search process for CasX proteins regarding target sequences. The higher overall affinity for DNA can also, in some embodiments, increase the frequency with which the CasX protein can effectively initiate and terminate the binding and unwinding steps, thereby facilitating target strand entry and R-loop formation, and ultimately cleavage of the target nucleic acid sequence.

[0206] While not theoretically bound, amino acid alterations in the NTSB domain may increase the efficiency of unwinding or capturing the unwinded non-target nucleic acid strand, thereby increasing the affinity of the CasX variant protein to the target nucleic acid. Alternatively, or additionally, amino acid alterations in the NTSB domain may increase the NTSB domain's ability to stabilize DNA during unwinding, thereby increasing the affinity of the CasX variant protein to the target nucleic acid. Alternatively, or additionally, amino acid alterations in the OBD may increase the affinity of the CasX variant protein to the protospacer adjacent motif (PAM), thereby increasing the affinity of the CasX variant protein to the target nucleic acid. Alternatively, or additionally, amino acid alterations in the helix I and / or helix II, RuvC, and TSL domains may increase the affinity of the CasX variant protein to the target nucleic acid strand, thereby increasing the affinity of the CasX variant protein to the target nucleic acid.

[0207] In some embodiments, the binding affinity of the CasX variant protein of this disclosure to a target nucleic acid molecule is increased by 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 times compared to a reference CasX protein or another CasX variant from which it is derived. In some embodiments, the CasX variant protein has a binding affinity to the target nucleic acid that is approximately 1.1 to 100 times higher compared to the reference protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or to the CasX 491 and 515 variants.

[0208] In some embodiments, the CasX variant protein has increased binding affinity to 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 nucleic acid strand. In some embodiments, the CasX variant protein has approximately 1.1 to approximately 100-fold increased binding affinity to the non-target strand 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 the CasX variant of SEQ ID NO: 270 or SEQ ID NO: 336.

[0209] Methods for measuring the affinity of a CasX protein (e.g., reference or variant) to target nucleic acid molecules and / or non-target nucleic acid molecules may include electrophoretic mobility shift assays (EMSA), membrane binding, isothermal calorimetry (ITC), and surface plasmon resonance (SPR), fluorescence polarization, and biolayer interferometry (BLI). Further methods for measuring the affinity of a CasX protein to a target include in vitro biochemical assays that measure DNA cleavage events over time.

[0210] j. Improved specificity for target sites In some embodiments, CasX variant proteins exhibit improved specificity to a target nucleic acid sequence compared to a reference CasX protein or another CasX variant from which it is derived. As used herein, “specificity” (sometimes interchangeably referred to as “target specificity”) refers to the degree to which the CRISPR / Cas system ribonucleoprotein complex cleaves off-target sequences that are similar to but not identical to the target nucleic acid sequence. For example, a CasX variant RNP with a higher degree of specificity exhibits reduced off-target cleavage of the sequence compared to a reference CasX protein. The specificity of CRISPR / Cas system proteins, and the reduction of potentially harmful off-target effects, can be critical to achieving an acceptable therapeutic index for use in mammalian subjects.

[0211] In some embodiments, the CasX variant protein exhibits improved specificity to target sites within target sequences that are complementary to the targeting sequence of the gRNA. Correlating with improved specificity, as described above, is a reduction in off-target editing. In some embodiments, the CasX variant protein shows reduced off-target editing or cleavage to target sites within target sequences that are not 100% complementary to the targeting sequence of the gRNA complexed with the CasX variant as an RNP. While we do not wish to be bound by theory, amino acid changes in the helix I and II domains that increase the specificity of the CasX variant protein to the target nucleic acid strand may also increase the specificity of the CasX variant protein to the entire target nucleic acid. In some embodiments, amino acid changes that increase the specificity of the CasX variant protein to the target nucleic acid may also result in a decrease in the affinity of the CasX variant protein to DNA.

[0212] Methods for testing the target specificity of CasX proteins (e.g., variants or reference) may include circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq), or similar methods. Briefly, the CIRCLE-seq technique involves shearing genomic DNA, circularizing it by ligating a stem-loop adapter, nicking the stem-loop region, and exposing a 4-nucleotide palindromic overhang. This is followed by intramolecular ligation and degradation of the remaining linear DNA. Subsequently, the circular DNA molecule containing the CasX cleavage site is linearized with CasX, the adapter adapter is ligated to the exposed ends, and then high-throughput sequencing is performed to generate paired-end reads containing information about off-target sites. Further assays that can be used to detect off-target events (and therefore the specificity of the CasX protein) include assays used to detect and quantify indels (insertions and deletions) formed at selected off-target sites (e.g., mismatch detection nuclease assays and next-generation sequencing, NGS). An exemplary mismatch detection assay is a nuclease assay, which involves PCR amplification, denaturation, and re-hybridization of genomic DNA from cells treated with CasX and sgRNA to form heterodouble-stranded DNA containing one wild-type strand and one indel-containing strand. The mismatch is recognized and cleaved by a mismatch detection nuclease such as Surveyor nuclease or T7 endonuclease I. Methods for evaluating the specificity of CasX variants are described in the examples, along with supporting data demonstrating the improved specificity of embodiments of the CasX variants.

[0213] k. Protospacer and PAM array In this specification, a protospacer is defined as a DNA sequence complementary to the targeting sequence of the guide RNA and DNA complementary to that sequence, referred to as the target strand and non-target strand, respectively. As used herein, PAM is a nucleotide sequence proximal to the protospacer, which, together with the targeting sequence of the gRNA, assists in the orientation and positioning of CasX for potential cleavage of the protospacer strand.

[0214] The PAM sequence may be degenerate, and a particular RNP construct may have different preferred acceptable PAM sequences supporting different cleavage efficiencies. By convention, unless otherwise stated, this disclosure refers to both the PAM and protospacer sequences, as well as their orientation according to the orientation of the non-target strand. This does not mean that the PAM sequence on the non-target strand rather than the target strand is a determinant of cleavage or is mechanistically involved in target recognition. For example, when referring to the TTC PAM, it may actually be a complementary GAA sequence required for target cleavage, or any combination of nucleotides from both strands. In the case of the CasX protein disclosed herein, the PAM is located at the 5' end of the protospacer, and a single nucleotide separates the PAM from the first nucleotide of the protospacer. Therefore, in the case of reference CasX, TTC PAM should be understood to mean a sequence that follows the formula 5'-...NNTTCN(protospacer)NNNNNN...3'(SEQ ID NO: 19) (wherein "N" is any DNA nucleotide and "(protospacer)" is a DNA sequence that is identical to the targeting sequence of the guide RNA). In the case of CasX variants with extended PAM recognition, TTC, CTC, GTC, or ATC PAM should be understood to mean a sequence that follows the following formulas: 5'-...NNTTCN(protospacer)NNNNNN...3'(SEQ ID NO: 19), 5'-...NNCTCN(protospacer)NNNNNN...3'(SEQ ID NO: 20); 5'-...NNGTCN(protospacer)NNNNNN...3'(SEQ ID NO: 21); or 5'-...NNATCN(protospacer)NNNNNN...3'(SEQ ID NO: 22). Alternatively, TC PAM should be understood to mean a sequence following formula 5'-...NNNTCN(protospacer)NNNNNN...3'(sequence number 23).

[0215] Additionally, when the CasX variant proteins of this disclosure are complexed with gRNA as RNPs using a PAM TC motif containing a PAM sequence selected from TTC, ATC, GTC, or CTC (in 5' to 3' orientation), their ability to efficiently edit and / or bind to target nucleic acids is enhanced compared to the RNPs of reference CasX proteins and reference gRNA, or the RNPs of other CasX variants from which they are derived (e.g., CasX491 and gRNA174). As stated above, the PAM sequence is located at least one nucleotide 5' to the non-target strand of a protospacer that is identical to the targeting sequence of gRNA in the assay system, compared to the editing efficiency and / or binding of RNPs containing reference CasX proteins and reference gRNA in an equivalent assay system. In one embodiment, the RNPs of the CasX variant and the gRNA variant exhibit higher editing efficiency and / or binding of the target sequence in the target nucleic acid compared to an RNP containing a reference CasX protein and reference gRNA in an equivalent assay system where the PAM sequence of the target DNA is TTC (or an RNP of another CasX variant from which it is derived, e.g., CasX491 and gRNA174). In another embodiment, the RNPs of the CasX variant and the gRNA variant exhibit higher editing efficiency and / or binding of the target sequence in the target nucleic acid compared to an RNP containing a reference CasX protein and reference gRNA in an equivalent assay system where the PAM sequence of the target DNA is ATC (or an RNP of another CasX variant from which it is derived, e.g., CasX491 and gRNA174). In the particular embodiment described above in which the CasX variant exhibits enhanced editing by ATC PAM, the CasX variant is 528 (SEQ ID NO: 428). In another embodiment, RNPs of CasX variants and gRNA variants exhibit higher editing efficiency and / or binding of target sequences to target nucleic acids compared to RNPs containing reference CasX protein and reference gRNA in an equivalent assay system where the PAM sequence of the target DNA is a CTC (or RNPs of other CasX variants from which they are derived, e.g., CasX491 and gRNA174).In another embodiment, RNPs of CasX variants and gRNA variants exhibit higher editing efficiency and / or binding of the target sequence to the target nucleic acid compared to RNPs containing a reference CasX protein and reference gRNA in an equivalent assay system where the PAM sequence of the target DNA is GTC (or RNPs of another CasX variant and gRNA174 from which it is derived). In the above embodiment, the increased editing efficiency and / or binding affinity for one or more PAM sequences is at least 1.5 times, at least 2 times, at least 4 times, at least 10 times, at least 20 times, at least 30 times, or at least 40 times greater than the editing efficiency and / or binding affinity of RNPs of any one of the CasX proteins of SEQ ID NOs. 1-3 and gRNAs of Table 1 for the PAM sequences. Exemplary assays demonstrating improved editing are described in the examples herein (see, for example, Figure 41). In some embodiments, the CasX protein can bind to and / or modify (e.g., cleavage, nicking, methylation, demethylation, etc.) a target nucleic acid and / or polypeptides that associate with the target nucleic acid (e.g., histone tail methylation or acetylation). In some embodiments, the CasX protein is catalytically inactive (dCasX) but retains the ability to bind to the target nucleic acid.

[0216] l. DNA rewinding In some embodiments, CasX variant proteins possess improved DNA unwinding ability compared to the reference CasX protein. Previous studies have shown that insufficient dsDNA unwinding impairs or hinders the DNA-cleaving ability of CRISPR / Cas system proteins AnaCas9 or Cas14. Therefore, while we do not wish to be bound by any theory, the increased DNA-cleaving activity by some of the CasX variant proteins in this disclosure is likely at least partially attributable to an increased ability to locate and unwind dsDNA at target sites. Methods for measuring the DNA-unwinding ability of a CasX protein (e.g., variant or reference) include, but are not limited to, in vitro assays observing increased on-rate of dsDNA targets in fluorescence polarization or biolayer interferometry.

[0217] While we do not wish to be bound by theory, it is thought that amino acid changes in the NTSB domain can produce CasX variant proteins with increased DNA unwinding characteristics. Alternatively, or additionally, amino acid changes in helix domain regions interacting with OBD or PAM can also generate CasX variant proteins with increased DNA unwinding characteristics.

[0218] Methods for measuring the ability of a CasX protein (e.g., variant or reference) to unwind DNA include, but are not limited to, in vitro assays that observe an increase in the on-rate of a dsDNA target in fluorescence polarization or biolayer interferometry.

[0219] m.Catalytic activity The CasX:gRNA system ribonucleoprotein complexes disclosed herein include a CasX variant complexed with a gRNA variant that binds to a target nucleic acid and, in some cases, cleaves the target nucleic acid. In some embodiments, the CasX variant protein has improved catalytic activity compared to a reference CasX protein or another CasX variant from which it is derived. While we do not wish to be bound by theory, in some cases, cleavage of the target strand may be a limiting factor for Cas12-like molecules in the generation of dsDNA cleavage. In some embodiments, the CasX variant protein improves the bending and cleavage of the target strand of DNA, resulting in an overall improvement in the efficiency of dsDNA cleavage by the CasX ribonucleoprotein complex.

[0220] In some embodiments, the CasX variant protein has increased nuclease activity compared to a reference CasX protein or to another CasX variant from which it is derived. Variants with increased nuclease activity can be generated, for example, through amino acid changes in the RuvC nuclease domain. In some embodiments, the CasX variant includes a RuvC nuclease domain having nickase activity. In the above, the CasX nickase in the CasX:gRNA system generates a single-strand break within 10-18 nucleotides on the 3' side of the PAM site on the non-target strand. In other embodiments, the CasX variant includes a RuvC nuclease domain having double-strand break activity. In the above, the CasX in the CasX:gRNA system generates double-strand breaks within 18-26 nucleotides on the 5' side of the PAM site on the target strand and within 10-18 nucleotides on the 3' side of the non-target strand. Nuclease activity can be assayed by various methods, including the method of the examples. In some embodiments, the CasX variant is at least 2 times, or at least 3 times, or at least 4 times, or at least 5 times, or at least 6 times, or at least 7 times, or at least 8 times, or at least 9 times, or at least 10 times larger than the reference CasX. 切断 It has a constant.

[0221] In some embodiments, the CasX variant protein has an improved characteristic of forming RNPs with gRNA, resulting in a higher percentage of cleavage-competent RNPs compared to the RNPs of the reference CasX protein and gRNA of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, as described in the Examples. Cleavage competence means that the formed RNPs have the ability to cleave target nucleic acids. In some embodiments, the RNPs of the CasX variant and gRNA exhibit at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, or at least 10-fold cleavage rates compared to the RNPs of the reference CasX protein and gRNA of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 and Table 2. In the embodiments described above, the improved competency rate can be demonstrated in in vitro assays as described in the Examples.

[0222] In some embodiments, CasX variant proteins have increased target strand loading for double-strand breaks compared to reference CasX. Variants with increased target strand loading activity may be generated, for example, through amino acid alterations in the TLS domain. While we do not wish to be bound by theory, amino acid alterations in the TSL domain may result in CasX variant proteins with improved catalytic activity. Alternatively, or additionally, amino acid alterations around the RNA:DNA double-strand binding channel can also improve the catalytic activity of CasX variant proteins.

[0223] In some embodiments, the CasX variant protein has increased incidental cleavage activity compared to the reference CasX protein. As used herein, “incidental cleavage activity” means further untargeted cleavage of the nucleic acid following recognition and cleavage of the target nucleic acid sequence. In some embodiments, the CasX variant protein has decreased incidental cleavage activity compared to the reference CasX protein.

[0224] Exemplary methods for characterizing the catalytic activity of the CasX protein include, but are not limited to, in vitro cleavage assays, including the methods described in the following examples. In some embodiments, electrophoresis of DNA products on an agarose gel can be used to investigate the dynamics of strand breaks.

[0225] n. Affinity for target RNA In some embodiments, a ribonucleoprotein complex containing a reference CasX protein or a variant thereof binds to a target RNA and cleaves the target nucleic acid. In some embodiments, a variant of the reference CasX protein increases the specificity of the CasX variant protein to the target RNA and increases the activity of the CasX variant protein to the target RNA compared to the reference CasX protein. For example, the CasX variant protein may show increased binding affinity to the target RNA or increased cleavage of the target RNA compared to the reference CasX protein. In some embodiments, a ribonucleoprotein complex containing the CasX variant protein binds to and / or cleaves the target RNA. In some embodiments, the CasX variant has at least about 2 to about 10 times increased binding affinity to the target nucleic acid compared to the reference protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, or the CasX variant of SEQ ID NO: 270 or SEQ ID NO: 336.

[0226] o. Catalytically inactive CasX variants In some embodiments, for example, embodiments encompassing applications where cleavage of the target nucleic acid sequence is not the desired result, improving the catalytic activity of the CasX variant protein includes altering, reducing, or eliminating the catalytic activity of the CasX variant protein. In some embodiments, the disclosure provides a catalytically inactive CasX variant protein that, when complexed with a gRNA having a targeting sequence complementary to the target nucleic acid, can bind to the target nucleic acid but cannot cleave it. An exemplary catalytically inactive CasX protein contains one or more mutations in the active site of the RuvC domain of the CasX protein. In some embodiments, the catalytically inactive CasX variant protein includes substitutions at residues 672, 769, and / or 935 compared to SEQ ID NO: 1. In one embodiment, the catalytically inactive CasX variant protein includes substitutions at D672A, E769A, and / or D935A compared to the reference CasX protein of SEQ ID NO: 1. In other embodiments, catalytically inactive CasX variant proteins include substitutions at amino acids 659, 756, and / or 922 compared to the reference CasX protein of SEQ ID NO: 2. In some embodiments, catalytically inactive CasX variant proteins include D659A, E756A, and / or D922A substitutions compared to the reference CasX protein of SEQ ID NO: 2. In some embodiments, catalytically inactive CasX variant proteins 527, 668, and 676 include D660A, E757A, and D922A modifications that eliminate endonuclease activity. In further embodiments, catalytically inactive CasX proteins include the deletion of all or part of the RuvC domain of the CasX protein. It will be understood that the same aforementioned substitutions can similarly be introduced into the CasX variants of this disclosure, resulting in catalytically inactive CasX (dCasX) variants. In one embodiment, all or part of the RuvC domain is deleted from the CasX variant, resulting in a dCasX variant. The catalytically inactive dCasX variant protein can be used for base editing or epigenetic modification in some embodiments.In some embodiments, catalytically inactive dCasX variant proteins having a higher affinity for DNA can find their target nucleic acids more quickly, remain bound to the target nucleic acids for longer periods, bind to the target nucleic acids in a more stable manner, or a combination thereof, compared to catalytically active CasX, thereby improving these functions of catalytically inactive CasX variant proteins compared to CasX variants that retain their cleavage ability. Exemplary dCasX variant sequences are disclosed as SEQ ID NOs. 44-62 and 1232-1235 shown in Table 7. In some embodiments, the dCasX variants are at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequences of SEQ ID NOs. 44-62 or 1232-1235, and retain the functional properties of the dCasX variant protein. In some embodiments, the dCasX variant includes the sequence of sequence numbers 44-62 or 1232-1235.

[0227] [Table 7]

[0228] p.CasX fusion protein In some embodiments, this disclosure provides CasX variant proteins comprising a heterologous protein fused to CasX, which includes a CasX variant of any of the embodiments described herein. This includes CasX variants comprising N-terminal, C-terminal, or internal fusion of CasX to a heterologous protein or its domain.

[0229] In some embodiments, the CasX fusion protein comprises one of the variants of SEQ ID NOs. 247-592 or 1147-1231, or any sequence from Table 3, fused to one or more proteins or their domains having different activities of interest that result in the fusion protein. In some embodiments, the CasX fusion protein comprises one of the variants of SEQ ID NOs. 270-592 or 1147-1231, fused to one or more proteins or their domains having different activities of interest. In some embodiments, the CasX fusion protein comprises one of the variants of SEQ ID NOs. 415-592 or 1147-1231, fused to one or more proteins or their domains having different activities of interest. For example, in some embodiments, the CasX variant protein is fused to a protein (or its domain) that inhibits transcription, modifies a target nucleic acid, or modifies a polypeptide that associates with a nucleic acid (e.g., histone modification).

[0230] 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 the CasX protein to generate a CasX fusion protein. In other embodiments, a cysteine ​​residue can be inserted at one or more positions within the CasX protein, followed by the conjugation of the heterologous polypeptide described below. In some alternative embodiments, the heterologous polypeptide or heterologous amino acid may be added to the N-terminus or C-terminus of a reference or CasX variant protein. In other embodiments, the heterologous polypeptide or heterologous amino acid may be internally inserted into the sequence of the CasX protein.

[0231] In some embodiments, the CasX variant fusion protein retains RNA-induced 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 activity and / or binding activity) of the corresponding CasX variant protein without heterologous protein insertion. In some cases, the CasX variant fusion protein retains at least about 60%, or at least about 70%, 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 activity and / or binding activity) of the corresponding CasX protein without heterologous protein insertion.

[0232] In some cases, a reference CasX or CasX variant fusion protein retains (has) target nucleic acid binding activity compared to the activity of a CasX protein without the inserted heterologous amino acid or heterologous polypeptide. In some cases, a reference CasX or CasX variant fusion protein retains at least about 60%, or at least about 70%, 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 about 100% of the binding activity of the corresponding CasX protein without the insertion of the heterologous protein.

[0233] In some cases, CasX variant fusion proteins retain (have) target nucleic acid binding and / or cleavage activity compared to the activity of the parental CasX protein without the inserted heterologous amino acid or heterologous polypeptide. For example, in some cases, CasX variant fusion proteins have (retain) 50% or more of the binding and / or cleavage activity of the corresponding parental CasX protein (CasX protein without the insertion). For example, in some cases, CasX variant fusion proteins have (retain) 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 parental CasX protein (CasX protein without the insertion). Methods for measuring the cleavage and / or binding activity of CasX proteins and / or CasX fusion proteins are known to those skilled in the art, and any convenient method can be used.

[0234] Various heterologous polypeptides are suitable for inclusion in the CasX variant fusion protein of this disclosure. In some cases, the fusion partner can regulate the transcription of a target nucleic acid (e.g., inhibit transcription, increase transcription). For example, in some cases, the fusion partner is a transcription-repressing protein (or protein-derived domain) (e.g., a protein that functions via transcriptional repressors, recruitment of transcription-repressing proteins, modification of target nucleic acids such as methylation, recruitment of DNA modifiers, regulation of histones associated with target nucleic acids, or recruitment of histone modifiers such as those that modify histone acetylation and / or methylation). In some cases, the fusion partner is a transcription-increasing protein (or protein-derived domain) (e.g., a protein that functions via transcriptional activators, recruitment of transcriptional activator proteins, modification of target nucleic acids such as demethylation, recruitment of DNA modifiers, regulation of histones associated with target nucleic acids, or recruitment of histone modifiers such as those that modify histone acetylation and / or methylation).

[0235] In some cases, the fusion partner may have enzymatic activity that modifies the target nucleic acid sequence, such as nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimerization activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, or glycosylase activity. In some embodiments, the CasX variant comprises one of SEQ ID NOs: 247-592 or 1147-1231 and a polypeptide having methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitination activity, adenylation activity, deadenylation activity, SUMOylation activity, deSUMOylation activity, ribosylation activity, deribosylation activity, myristoylation activity, or demyristoylation activity. In some embodiments, the CasX variant comprises one of SEQ ID NOs: 270-592 or 1147-1231 and the polypeptide described above. In some embodiments, the CasX variant comprises one of SEQ ID NOs: 415-592 or 1147-1231 and the polypeptide described above.

[0236] Examples of proteins (or fragments thereof) that can be used as fusion partners to increase transcription include transcription activators (e.g., VP16, VP64, VP48, VP160, p65 subdomain (e.g., derived from NFκB), and the activation domain and / or TAL activation domain of EDLL (e.g., active in plants); histone lysine methyltransferases (e.g., SET1A, SET1B, MLL1-5, ASH1, SYMD2, NSD1, etc.); histone lysine demethylases (e.g., JHDM2a / b, UTX, JMJD3, etc.); histone acetyltransferases (e.g., GCN5, PCAF, CBP, p300, TAF1, TIP60 / PLIP, MOZ / MYST3, MORF / MYST4, SRC1, ACTR, P160, CLOCK, etc.); and DNA demethylases (e.g., Ten-Eleven) Examples of translocation (TET) dioxygenases include, but are not limited to, translocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, ROS1, etc.

[0237] Examples of proteins (or fragments thereof) that can be used as fusion partners to reduce transcription include, but are not limited to, the following: transcriptional repressors (e.g., Kruppel-associated boxes (KRAB or SKD)); KOX1 repressor domains; Mad mSIN3 interaction domains (SID); ERF repressor domains (ERF repressor Histone lysine methyltransferases (e.g., ERD domain), SRDX repression domain (for repression in plants), etc.; histone lysine methyltransferases (e.g., Pr-SET7 / 8, SUV4-20H1, RIZ1, etc.); histone lysine demethylases (e.g., JMJD2A / JHDM3A, JMJD2B, JMJD2C / GASC1, JMJD2D, JARID1A / RBP2, JARID1B / PLU-1, JARID1C / SMCX, JARID1D / SMCY, etc.); histone lysine deacetylases (e.g., HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, HDAC11, etc.); DNA methylases (e.g., HhaI DNA m5c-methyltransferase (M.HhaI), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plant), ZMET2, CMT1, CMT2 (plant), etc.; and peripheral recruiting elements (e.g., Lamin A, Lamin B, etc.).

[0238] In some cases, the fusion partner for a CasX variant possesses enzymatic activity that modifies the target nucleic acid (e.g., ssRNA, dsRNA, ssDNA, dsDNA). Examples of enzymatic activity that may be provided by a fusion partner include nuclease activity (e.g., provided by restriction enzymes (e.g., FokI nuclease)), methyltransferase activity (e.g., provided by methyltransferases (e.g., HhaI DNA m5c-methyltransferase (M.Hhal), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plant), ZMET2, CMT1, CMT2 (plant), etc.)), demethylase activity (e.g., demethylase (e.g., Ten-Eleven Translocation (TET) dioxygenase 1 (TET1) (Provided by CD), TET1, DME, DML1, DML2, ROS1, etc.), DNA repair activity, DNA damage activity, deamination activity (e.g., provided by deaminase (e.g., cytosine deaminase enzyme, e.g., rat apolipoprotein B mRNA editing enzyme, APOBEC proteins such as catalytic peptide 1 {APOBEC1})), dismutase activity, alkylation activity, depurination activity, oxidative activity, pyrimidine dimerization activity, integrase activity (e.g., integrase and / or These include, but are not limited to, resolverase activity (e.g., provided by Gin invertase (e.g., a highly active variant of Gin invertase GinH106Y)), human immunodeficiency virus type 1 integrase (IN), Tn3 resolverase, etc.), transposase activity, recombinase activity (e.g., provided by recombinase (e.g., the catalytic domain of Gin recombinase)), polymerase activity, ligase activity, helicase activity, photolyase activity, and glycosylase activity.

[0239] In some cases, the CasX variant protein of this disclosure is fused to a polypeptide selected from domains for increasing transcription (e.g., VP16 domain, VP64 domain), domains for decreasing transcription (e.g., KRAB domain (e.g., derived from Kox1 protein)), core catalytic domains of histone acetyltransferases (e.g., histone acetyltransferase p300), proteins / domains that provide a detectable signal (e.g., fluorescent proteins such as GFP), nuclease domains (e.g., FokI nuclease), or base editors (e.g., cytidine deaminases such as APOBEC1).

[0240] In some embodiments, the CasX variant includes one of SEQ ID NOs: 247-592 or 1147-1231, or one of SEQ ID NOs: 270-592 or 1147-1231, or one of SEQ ID NOs: 415-592 or 1147-1231, or the sequences in Table 3, fused to a polypeptide selected from the group consisting of a domain for reducing transcription, a domain having enzymatic activity, a core catalytic domain for histone acetyltransferase, a protein / domain providing a detectable signal, a nuclease domain, and a base editor. In some embodiments, the CasX variant comprises one of SEQ ID NOs: 270-592 or 1147-1231 fused to the polypeptide described above. In some embodiments, the CasX variant comprises one of SEQ ID NOs: 415-592 or 1147-1231 fused to the polypeptide described above. In some embodiments, the CasX variant comprises one of SEQ ID NOs: 760-789 fused to a polypeptide selected from the group consisting of a domain for reducing transcription, a domain having enzymatic activity, a core catalytic domain for histone acetyltransferase, a protein / domain providing a detectable signal, a nuclease domain, and a base editor. In some embodiments, the CasX variant comprises one of SEQ ID NOs: 411-592 fused to a polypeptide selected from the group consisting of a domain for reducing transcription, a domain having enzymatic activity, a core catalytic domain for histone acetyltransferase, a protein / domain providing a detectable signal, a nuclease domain, and a base editor.

[0241] In some cases, the reference CasX protein or CasX variant of this disclosure is fused to a base editor. Base editors include those capable of modifying guanine, adenine, cytosine, thymine, or uracil bases on a nucleoside or nucleotide. Examples of base editors include, but are not limited to, adenosine deaminase, cytosine deaminase (e.g., APOBEC1), and guanine oxidase. Therefore, any of the CasX variants provided herein may include (i.e., be fused to) a base editor. For example, a CasX variant of this disclosure may be fused to an adenosine deaminase, cytosine deaminase, or guanine oxidase. In exemplary embodiments, a CasX variant of this disclosure containing any one of SEQ ID NOs. 247-592 or 1147-1231 is fused to an adenosine deaminase, cytosine deaminase, or guanine oxidase. In further exemplary embodiments, a CasX variant of the present disclosure comprising any one of SEQ ID NOs: 270-592 or 1147-1231 is fused to an adenosine deaminase, cytosine deaminase, or guanine oxidase. In further exemplary embodiments, a CasX variant of the present disclosure comprising any one of SEQ ID NOs: 415-592 or 1147-1231 is fused to an adenosine deaminase, cytosine deaminase, or guanine oxidase.

[0242] In some cases, a fusion partner for a CasX variant may possess enzymatic activity that modifies proteins (e.g., histones, RNA-binding proteins, DNA-binding proteins, etc.) that associate with the target nucleic acid (e.g., ssRNA, dsRNA, ssDNA, dsDNA). Examples of enzymatic activity that may be provided by a fusion partner for a CasX variant (modifying proteins that associate with the target nucleic acid) include methyltransferase activity (e.g., methyltransferase (histone)).Methyltransferase (HMT) (e.g., suppressor of variegated 3-9 homolog 1 (also known as SUV39H1, KMT1A), true chromatin histone lysine methyltransferase 2 (also known as G9A, KMT1C and EHMT2), SUV39H2, ESET / SETDB1, etc., SET1A, SET1B, MLL1~5, ASH1, SMYD2, NSD1, DOT1-like histone lysine methyltransferase (DOT1L), Pr-SET7 / 8, lysine methyltransferase Demethylase activity (e.g., histone demethylases (e.g., lysine demethylase 1A (KDM1A, also known as LSD1), JHDM2a / b, JMJD2A / JHDM3A, JMJD2B, JMJD2C / GASC1, JMJD2D, JARID1A / RBP2, JARID1B / PLU-1, JARID1C / SM)) (provided by CX, JARID1D / SMCY, UTX, JMJD3, etc.), acetyltransferase activity (e.g., histone acetyltransferase (e.g., catalytic core / fragment of human acetyltransferase p300, GCN5, PCAF, CBP, TAF1, TIP60 / PLIP, MOZ / MYST3, MORF / MYST4, HB01 / MYST2, HMOF / MYST1, SRC1, ACTR, P160, CLOCK, etc.)), deacetylase activity (e.g., Examples include, but are not limited to, histone deacetylases (e.g., those provided by HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, HDAC11, etc.), kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitination activity, adenylation activity, deadenylation activity, SUMOylation activity, deSUMOylation activity, ribosylation activity, deribosylation activity, myristoylation activity, and demyristoylation activity.

[0243] Further examples of suitable fusion partners for CasX variants include (i) dihydrofolate reductase (DHFR) destabilization domains (for example, to generate a chemically controllable target RNA-inducible polypeptide), and (ii) chloroplast-transfer peptides.

[0244] In some embodiments, the CasX variant is one of the sequence numbers 247-592 or 1147-1231, or one of the sequence numbers 270-592 or 1147-1231, or one of the sequence numbers 415-592 or 1147-1231, or the sequence in Table 3 and a chloroplast-transfer peptide (MASMISSSAVTTVSRASRGQSAAMAPFGGLKSMTGFPVRKVNTDITSITSNGGR VKCMQVWPPIGKKKFETLSYLPPLTRDSRA (sequence number 338); MASMISSSAVTTVSRASRGQSAAMAPFGGLKSMTGFPVRKVNTDITSITSNGGRVKS (sequence number 339); MASSMLSSATMVASPAQATMVAPFNGLKSSAAFPATRKANNDITSITSNGGRVNCMQV WPPIEKKKFETLSYLPDLTDSGGRVNC(Sequence ID 340);MAQVSRICNGVQNPSLISNLSKSSQRKSPLSVSLKTQQHPRAYPISSSWGLKKSGMTLIG SELRPLKVMSSVSTAC(Sequence ID 341);MAQVSRICNGVWNPSLISNLSKSSQRKSPLSVSLKTQQHPRAYPISSSWGLKKSGMTLIG SELRPLKVMSSVSTAC(Sequence ID 342);MAQINNMAQGIQTLNPNSNFHKPQVPKSSSFLVFGSKKLKNSANSMLVLKKDSIFMQLF CSFRISASVATAC(Sequence ID 343);MAALVTSQLATSGTVLSVTDRFRRPGFQGLRPRNPADAALGMRTVGASAAPKQSRKPH RFDRRCLSMVV(Sequence ID 344);MAALTTSQLATSATGFGIADRSAPSSLLRHGFQGLKPRSPAGGDATSLSVTTSARATPKQ QRSVQRGSRRFPSVVVC(Sequence ID 345);MASSVLSSAAVATRSNVAQANMVAPFTGLKSAASFPVSRKQNLDITSIASNGGRVQC(Sequence ID 346);MESLAATSVFAPSRVAVPAARALVRAGTVVPTRRTSSTSGTSGVKCSAAVTPQASPVIS RSAAAA(Sequence ID 347);This includes, but is not limited to, MGAAAATSMQSLKFSNRLVPPSRRLSPVPNNVTCNNLPKSAAPVRTVKCCASSWNSTINGAAATTNGASAASS (Sequence ID 348), and also includes;

[0245] In some cases, the CasX variant protein of this disclosure may include an endosomal escape peptide. In some cases, the endosomal escape polypeptide includes the amino acid sequence GLFXALLXLLXSLWXLLLXA (SEQ ID NO: 349), where each X is independently selected from lysine, histidine, and arginine. In some cases, the endosomal escape polypeptide includes the amino acid sequence GLFHALLHLLHSLWHLLLHA (SEQ ID NO: 350) or HHHHHHHHH (SEQ ID NO: 351). In some embodiments, the CasX variant includes one of SEQ ID NOs. 247-592 or 1147-1231, or one of SEQ ID NOs. 270-592 or 1147-1231, or one of SEQ ID NOs. 415-592 or 1147-1231, or a sequence from Table 3, and the endosomal escape polypeptide.

[0246] When targeting ssRNA target nucleic acid sequences, non-limiting examples of fusion partners for CasX variants for use include (but are not limited to) splicing factors (e.g., RS domains); protein translation components (e.g., translation initiation, elongation, and / or termination factors; e.g., eukaryotic translation initiation factor 4γ{eIF4G}); RNA methylases; RNA editing enzymes (e.g., RNA deaminases (e.g., adenosine deaminase acting on RNA, ADAR)), including editing enzymes A through I and / or C through U); helicases; RNA-binding proteins, etc. It is understood that heterologous polypeptides may include whole proteins or, in some cases, fragments of proteins (e.g., functional domains).

[0247] In some embodiments, any one of sequence numbers 247-592 or 1147-1231, or any one of sequence numbers 270-592 or 1147-1231, or any one of sequence numbers 415-592 or 1147-1231, or a CasX variant of a sequence in Table 3, includes a fusion partner of any domain capable of interacting with ssRNA (for the purposes of this disclosure, this includes intramolecular and / or intermolecular secondary structures, e.g., double-stranded RNA double helix (e.g., hairpin, stem-loop, etc.)). This includes, but is not limited to, effector domains selected from the group including, whether transient or irreversible, direct or indirect: endonucleases (e.g., RNase III, CRR22 DYW domain, Dicer, and protein-derived PIN (PilT N-terminal) domains such as SMG5 and SMG6); proteins and protein domains involved in stimulating RNA cleavage (e.g., cleavage and polyadenylation-specific factor {CPSF}, cleavage-stimulating factor {CstF}, CFIm, and CFIIm); exonucleases (e.g., chromatin-binding exonuclease XRN1 (XRN-1) or exonuclease T); deadenylases (e.g., DNA 5'-adenosine monophosphate hydrolase {HNT3}); factors and protein domains involved in nonsense-mediated RNA degradation (e.g., UPF1 RNA helicase and ATPase {UPF1}, UPF2, UPF3, UPF3b, RNP SI, RNA-binding motif protein 8A{Y14}, DEK proto-oncogene{DEK}, RNA processing protein REF2{REF2}, and serine-arginine repeat matrix 1{SRm160}); proteins and protein domains involved in RNA stabilization (e.g., cytoplasmic poly(A)-binding protein 1{PABP}); proteins and protein domains involved in translational repression (e.g., Argonaut RISC catalytic components 2{Ago2} and Ago4); proteins and protein domains involved in translational stimulation (e.g., Staufen);Proteins and protein domains involved in the regulation of translation (e.g., those capable of regulating translation) (e.g., translation factors (e.g., initiation factors, elongation factors, termination factors, etc., e.g., eIF4G)); proteins and protein domains involved in RNA polyadenylation (e.g., poly(A) polymerase (PAP1), PAP-related domain-containing proteins; poly(A)RNA polymerase gld-2 {GLD-2}, and Star-PAP); proteins and protein domains involved in RNA polyuridinylation (e.g., terminal uridylyltransferase {CID1} and terminal uridylate transferase); proteins and protein domains involved in RNA localization (e.g., insulin-like growth factor 2) mRNA-binding protein 1 {IMP1}, Z-DNA-binding protein 1 {ZBP1}, She2p, She3p, and Bicaudal-D-derived proteins); proteins and protein domains involved in the nuclear retention of RNA (e.g., Rrp6); proteins and protein domains involved in the nuclear export of RNA (e.g., nuclear RNA transport factor 1 {TAP}, nuclear RNA transport factor 1 {NXF1}, THO complex {THO}, TREX, REF, and Aly / REF transport factor {Aly}); proteins and protein domains involved in the repression of RNA splicing (e.g., polypyrimidine tract-binding protein 1 {PTB}, KH RNA-binding domain-containing, signal transduction-related 1 {Sam68}, and heteronuclear ribonucleoprotein A1 {hnRNP A1}); proteins and protein domains involved in the stimulation of RNA splicing (e.g., serine / arginine-rich (SR) domain); proteins and protein domains involved in the reduction of transcription efficiency (e.g., FUS RNA-binding proteins {FUS(TLS)}; and proteins and protein domains involved in transcriptional stimulation (e.g., cyclin-dependent kinase 7 {CDK7} and HIV Tat). Alternatively, effector domains can be selected from the group including: endonucleases; proteins and protein domains capable of stimulating RNA cleavage; exonucleases; deadenylases; proteins and protein domains with nonsense-mediated RNA degradation 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 regulating translation (e.g., translation factors (e.g., initiation factors, elongation factors, termination 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 retaining RNA in the nucleus; proteins and protein domains having RNA nuclear export activity; proteins and protein domains capable of repressing RNA splicing; proteins and protein domains capable of stimulating RNA splicing; proteins and protein domains capable of reducing transcription efficiency; and proteins and protein domains capable of stimulating transcription. Another preferred heterologous polypeptide is the PUF RNA-binding domain, which is described in detail in International Publication No. 2012 / 068627 (the entire publication of which is incorporated herein by reference).

[0248] Some RNA splicing factors that can be used (whole or in fragments) as fusion partners with CasX variants have a modular configuration with separate sequence-specific RNA-binding modules and splicing effector domains. For example, members of the serine / arginine-rich (SR) protein family contain an N-terminal RNA recognition motif (RRM) that binds to an exonic splicing enhancer (ESE) in premRNA and a C-terminal RS domain that promotes exon inclusion. As another example, the hnRNP protein hnRNP A1 binds to an exonic splicing silencer (ESS) via its RRM domain and inhibits exon inclusion via its C-terminal glycine-rich domain. Some splicing factors can modulate the selective use of splice sites (ss) by binding to regulatory sequences between two selective sites. For example, ASF / SF2 can recognize ESEs and promote the use of proximal intron sites, while hnRNP A1 can bind to ESSs and shift splicing to the use of distal intron sites. One application of such factors is the creation of ESFs that regulate alternative splicing of endogenous genes (particularly disease-associated genes). For example, BCL2-like 1 (Bcl-x) premRNA produces two splicing isoforms, each having two alternative 5' splice sites encoding proteins with opposite functions. The long splicing isoform, Bcl-xL, is a potent apoptosis inhibitor expressed in long-lived postmittal cells, upregulated in many cancer cells, and protects cells from apoptotic signaling. The short isoform, Bcl-xS, is a pro-apoptotic isoform and is expressed at high levels in cells with high metabolic rates (e.g., developing lymphocytes).The ratio of two Bcl-x splicing isoforms is regulated by multiple cc elements located either in the core exon region or the exon extension region (i.e., between two alternative 5' splice sites). For further examples, see International Publication 2010 / 075303, which is incorporated herein by reference in its entirety.

[0249] Further suitable fusion partners for use with CasX variants include, but are not limited to, boundary element proteins (e.g., CTCF) (or their fragments), peripheral recruitment-providing proteins and their fragments (e.g., lamin A, lamin B, etc.), and protein docking elements (e.g., FKBP / FRB, Pill / Abyl, etc.).

[0250] Additionally, or alternatively, the CasX variant proteins of this disclosure may be fused to polypeptide permeable domains to facilitate cellular uptake. Several permeable domains are known in the art and may be used in non-integrated polypeptides of this disclosure, including peptides, peptide mimes, and non-peptide carriers. For example, International Publication 2017 / 106569 and U.S. Patent Application Publication 2018 / 0363009(A1) (in whole, by reference) describe fusions of Cas proteins with one or more nuclear localization sequences (NLS) to facilitate cellular uptake. In other embodiments, the permeable peptide may be derived from the third α-helix of the Drosophila melanogaster transcription factor Antennapedia, called penetratin, containing the amino acid sequence RQIKIWFQNRRMKWKK (SEQ ID NO: 398). As another example, the permeable peptide may contain the HIV-1 tat basic region amino acid sequence, for example, amino acids 49-57 of the naturally occurring tat protein. Other permeable domains include polyarginine motifs, such as the amino acid region 34-56 of the HIV-1 rev protein, nonaarginine, and octaarginine. The fusion site may be selected to optimize the polypeptide's biological activity, secretion, or binding characteristics. The optimal site is determined through routine experiments.

[0251] In some cases, a heterologous polypeptide (fusion partner) for use with a CasX variant provides intracellular localization. That is, the heterologous polypeptide includes intracellular localization sequences (e.g., a nuclear localization signal (NLS) for targeting the nucleus, a sequence for retaining the fusion protein outside the nucleus (e.g., a nuclear export sequence (NES)), a sequence for retaining the fusion protein in the cytoplasm, a mitochondrial localization signal for targeting mitochondria, a chloroplast localization signal for targeting chloroplasts, an ER retention signal, etc.). In some embodiments, the target RNA guide polypeptide or conditionally active RNA guide polypeptide and / or the target CasX fusion protein does not contain an NLS so that the protein is not targeted to the nucleus (this may be advantageous, for example, when the target nucleic acid is RNA present in the cytosol). In some embodiments, the fusion partner may provide a tag for easier tracking and / or purification. In other words, heterogeneous polypeptides are detectable labels (e.g., fluorescent proteins, e.g., green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), mCherry, tdTomato, etc.; histidine tags, e.g., 6×His tag; hemagglutinin (HA) tag; FLAG tag; Myc tag, etc.). In some embodiments, the CasX variant comprises one of sequence numbers XX-XX and an intracellular localization sequence or tag.

[0252] In some cases, the reference protein or CasX variant protein contains (or is fused to) a nuclear localization signal (NLS). Non-limiting examples of NLSs suitable for use with CasX variants include sequences that have at least approximately 80%, at least approximately 90%, or at least approximately 95% identity with, or are identical to, sequences derived from: the SV40 virus large T antigen NLS having the amino acid sequence PKKKRKV (SEQ ID NO: 352); nucleoplasmin-derived NLS (e.g., the nucleoplasmin bifurcation NLS having the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 353)); c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 354) or RQRRNELKRSP (SEQ ID NO: 355); and hRNPAl M9 having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 356). NLS; Importin alpha-derived IBB domain sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 357); Myoma T protein sequences VSRKRPRP (SEQ ID NO: 358) and PPKKARED (SEQ ID NO: 359); Human p53 sequence PQPKKKPL (SEQ ID NO: 360); Mouse c-abl Sequence IV SALIKKKKKMAP (SEQ ID NO: 361); Sequences of influenza virus NS1 DRLRR (SEQ ID NO: 362) and PKQKKRK (SEQ ID NO: 363); Sequence of herpesvirus δ antigen RKLKKKIKKL (SEQ ID NO: 364); Sequence of mouse Mxl protein REKKKFLKRR (SEQ ID NO: 365); Sequence of human poly(ADP-ribose) polymerase KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 366); Sequence of steroid hormone receptor (human) glucocorticoid RKCLQAGMNLEARKTKK (SEQ ID NO: 367); Borna disease virus P protein,Sequence of BDV-P1) PRPRKIPR (SEQ ID NO: 368); Sequence of hepatitis C virus nonstructural protein (HCV-NS5A) PPRKKRTVV (SEQ ID NO: 369); Sequence of LEF1 NLSKKKKRKREK (SEQ ID NO: 370); Sequence of ORF57 simirae RRPSRPFRKP (SEQ ID NO: 371); Sequence of EBV LANA KRPRSPSS (SEQ ID NO: 372); Sequence of influenza A protein KRGINDRNFWRGENERKTR (SEQ ID NO: 373); Human RNA helicase A Sequence PRPPKMARYDN (SEQ ID NO: 374) of A, RHA; sequence KRSFSKAF (SEQ ID NO: 375) of nucleolar RNA helicase II; sequence KLKIKRPVK (SEQ ID NO: 376) of TUS-protein; sequence PKKKRKVPPPPAAKRVKLD (SEQ ID NO: 377) of importin α; sequence PKTRRRPRRSQRKRPPT (SEQ ID NO: 378) of HTLV-1 Rex protein; Caenorhabditis The sequences SRRRKANPTKLSENAKKLAKEVEN (SEQ ID NO: 379), KTRRRPRRSQRKRPPT (SEQ ID NO: 380), RRKKRRPRRKKRR (SEQ ID NO: 381), PKKKSRKPKKKSRK (SEQ ID NO: 382), HKKKHPDASVNFSEFSK (SEQ ID NO: 383), QRPGPYDRPQRPGPYDRP (SEQ ID NO: 384), LSPSLSPLLSPSLSPL (SEQ ID NO: 385), RGKGGKGLGKGGAKRHRK (SEQ ID NO: 386), PKRGRGRPKRGRGR (Sequence No. (Sequence ID 387), PKKKRKVPPPPAAKRVKLD (Sequence ID 388), PKKKRKVPPPPKKKRKV (Sequence ID 389), PAKRARRGYKC (Sequence ID 63), KLGPRKATGRW (Sequence ID 64), PRRKREE (Sequence ID 65), PYRGRKE (Sequence ID 66), PLRKRPRR (Sequence ID 67), PLRKRPRRGSPLRKRPRR (Sequence ID 68), PAAKRVKLDGGKRTADGSEFESPKKKRKV (Sequence ID 69), PAAKRVKLDGGKRTADGSEFESPKKKRKVGIHGVPAA (Sequence ID 70),PAAKRVKLDGGKRTADGSEFESPKKKRKVAEAAAKEAAAKEAAAKA (SEQ ID NO: 71), PAAKRVKLDGGKRTADGSEFESPKKKRKVPG (SEQ ID NO: 72), KRKGSPERGERKRHW (SEQ ID NO: 73), KRTADSQHSTPPKTKRKVEFEPKKKRKV (SEQ ID NO: 74), and PKKKRKVGGSKRTADSQHSTPPKTKRKVEFEPKKKRKV (SEQ ID NO: 75). In some embodiments, one or more NLSs are linked to a CRISPR protein or an adjacent NLS using a linker peptide, and the linker peptide is RS, (G)n (SEQ ID NO: 1023), (GS)n (SEQ ID NO: 1024), (GSGGS)n (SEQ ID NO: 399), (GGSGGS)n (SEQ ID NO: 400), (GGGS)n (SEQ ID NO: 401), GGSG (SEQ ID NO: 402), GGSGG (SEQ ID NO: 403), GSGSG (SEQ ID NO: 404), GSGGG (SEQ ID NO: 40 5) Selected from the group consisting of GGGSG (SEQ ID NO: 406), GSSSG (SEQ ID NO: 407), GPGP (SEQ ID NO: 408), GGP, PPP, PPAPPA (SEQ ID NO: 409), PPPG (SEQ ID NO: 24), PPPGPPP (SEQ ID NO: 410), PPP(GGGS)n (SEQ ID NO: 25), (GGGS)nPPP (SEQ ID NO: 26), AEAAAKEAAAKEAAAKA (SEQ ID NO: 1025), and TPPKTKRKVEFE (SEQ ID NO: 27) (wherein n is 1 to 5). Generally, NLS (or multiple NLS) are potent enough to drive the accumulation of CasX variant fusion proteins in the nucleus of eukaryotic cells. Detection of accumulation in the nucleus can be carried out by any suitable technique. For example, a detectable marker can be fused to the CasX variant fusion protein so that its intracellular location can be visualized. The cell nucleus can also be isolated from the cell, and its contents can then be analyzed by any suitable process for detecting proteins (e.g., immunohistochemistry, Western blotting, or enzyme activity assay). Accumulation in the nucleus can also be determined indirectly.

[0253] This disclosure envisions the assembly of multiple NLSs in various configurations for linking to a CRISPR protein. In some embodiments, one, two, three, four or more NLSs are linked to the N-terminus of a CRISPR protein by a linker peptide. In other embodiments, one, two, three, four or more NLSs are linked to the C-terminus of a CRISPR protein by a linker peptide. In some embodiments, the NLS linked to the N-terminus of a CRISPR protein are identical to the NLS linked to the C-terminus. In other embodiments, the NLS linked to the N-terminus of a CRISPR protein are different from the NLS linked to the C-terminus. In some embodiments, the NLS linked to the N-terminus of a CRISPR protein are selected from the group consisting of N-terminal sequences shown in Table 8. In some embodiments, the NLS linked to the C-terminus of a CRISPR protein are selected from the group consisting of C-terminal sequences shown in Table 8. Detection of accumulation in the nucleus can be carried out by any preferred technique. For example, a detectable marker can be fused to a reference or CasX variant fusion protein so that its intracellular location can be visualized. The cell nucleus can also be isolated from the cell, and its contents can then be analyzed by any suitable process for detecting proteins (e.g., immunohistochemistry, Western blotting, or enzyme activity assay). Accumulation in the nucleus can also be determined indirectly.

[0254] [Table 8-1]

[0255] [Table 8-2]

[0256] [Table 8-3]

[0257] [Table 8-4]

[0258] In some embodiments, the CasX variant includes one of sequence numbers 247-592 or 1147-1231, or one of sequence numbers 270-592 or 1147-1231, or one of sequence numbers 415-592 or 1147-1231, or one of sequence numbers 415-592 or 1147-1231, or one of sequence numbers 415-592 or 1147-1231, or one of the sequences in Table 3, fused to one or more NLSs of any of the sequences in Table 8. In some embodiments, one or more NLSs are fused to the N-terminus or vicinity of the CasX variant. In some embodiments, one or more NLSs are fused to the C-terminus or vicinity of the CasX variant. In some embodiments, one or more NLSs are fused to both the N-terminus and C-terminus of the CasX variant. In some embodiments, an NLS is linked to another NLS by a linker.

[0259] In some embodiments, the reference or CasX variant fusion protein includes a "protein transduction domain" or PTD (also known as a CPP cell permeable peptide), which refers to a protein, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates transposition across lipid bilayers, micelles, cell membranes, organelle membranes, or vesicle membranes. The PTD, bound to another molecule (which can range from small polar molecules to large macromolecules and / or nanoparticles), facilitates the molecule's transposition across membranes, e.g., from extracellular space to intracellular space, or from cytosol to organelle space. In some embodiments, the PTD is covalently bound to the amino terminus of the reference or CasX variant fusion protein. In some embodiments, the PTD is covalently bound to the carboxyl terminus of the reference or CasX variant fusion protein. In some embodiments, the PTD is inserted into the sequence of the reference or CasX variant fusion protein at a preferred insertion site. In some cases, the reference or CasX variant fusion protein is conjugated to or fused to one or more PTDs (e.g., two or more, three or more, or four or more PTDs). In some cases, the PTDs contain one or more nuclear localization signals (NLSs).Examples of PTDs include, but are not limited to, the following: peptide transduction domains of HIV TAT containing YGRKKRRQRRR (SEQ ID NO: 390) and RKKRRQRR (SEQ ID NO: 391); YARAAARQARA (SEQ ID NO: 392); THRLPRRRRRR (SEQ ID NO: 393); and GGRRARRRRRR (SEQ ID NO: 394); polyarginine sequences containing a sufficient number of arginines (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines) to direct cell entry (SEQ ID NO: 1026); VP22 domain (Zender et al. (2002) Cancer Gene Ther. 9(6): 489-96); Drosophila Antennapedia protein transduction domain (Noguchi et al. (2003) Diabetes 52(7): 1732-1737); truncated human calcitonin peptide (Trehin et al. al. (2004) Pharm. Research 21:1248-1256); polylysine (Wender et al. (2000) Proc. Natl. Acad. Sci. USA 97:13003-13008); RRQRRTSKLMKR (SEQ ID NO: 395); transportan GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 396); KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO: 397); and RQIKIWFQNRRMKWKK (SEQ ID NO: 398). In some embodiments, the PTD is activatable CPP (ACPP) (Aguilera et al. (2009) Integr Biol (Camb) June; 1(5-6):371-381). ACPP contains a polycationic CPP (e.g., Arg9 or "R9") linked to a matching polyanion (e.g., Glu9 or "E9") via a cleavage-competent linker, which reduces the net charge to nearly zero, thereby inhibiting adhesion to and uptake of cells. When the linker is cleaved, the polyanion is released, locally unmasking polyarginine and its inherent adhesiveness, and thus "activating" the ACPP to pass through the membrane.In some embodiments, the CasX variant includes any one of sequence numbers 247-592 or 1147-1231, or any one of sequence numbers 270-592 or 1147-1231, or any one of sequence numbers 415-592 or 1147-1231, or the sequences and PTDs in Table 3.

[0260] In some embodiments, the CasX variant fusion protein may include a CasX protein linked to heterologous amino acids or heterologous polypeptide (heterologous amino acid sequence) inserted internally via a linker polypeptide (e.g., one or more linker polypeptides). In some embodiments, the reference or CasX variant fusion protein may be linked to a heterologous polypeptide (fusion partner) via a linker polypeptide (e.g., one or more linker polypeptides) at its C-terminus and / or N-terminus. The linker polypeptide may have any of a variety of amino acid sequences. Proteins may be linked by spacer peptides of a generally flexible nature, but other chemical bonds are not excluded. Preferred linkers include polypeptides of 4 to 40 amino acids in length, or 4 to 25 amino acids in length. These linkers are generally produced by coupling proteins using oligonucleotides encoding synthetic linkers. Peptide linkers with some degree of flexibility can be used. The linked peptide may have substantially any amino acid sequence, considering that the preferred linker will generally have a sequence that results in a flexible peptide. The use of small amino acids such as glycine and alanine is useful in the production of flexible peptides. The production of such sequences is routine for those skilled in the art. Various different linkers are commercially available and are considered suitable for use. Exemplary linker polypeptides include glycine polymer (G)n, glycine-serine polymers (e.g., (GS)n (SEQ ID NO: 1024), (GSGGS)n (SEQ ID NO: 399), (GGSGGS)n (SEQ ID NO: 400), and (GGGS)n (SEQ ID NO: 401) (wherein n is at least an integer of 1), glycine-alanine polymer, alanine-serine polymer, glycine-proline polymer, proline polymer, and proline-alanine polymer.Exemplary linkers may include, but are not limited to, amino acid sequences such as RS, (G)n, (GS)n (SEQ ID NO: 1024), (GSGGS)n (SEQ ID NO: 399), (GGSGGS)n (SEQ ID NO: 400), (GGGS)n (SEQ ID NO: 401), GGSG (SEQ ID NO: 402), GGSGG (SEQ ID NO: 403), GSGSG (SEQ ID NO: 404), GSGGG (SEQ ID NO: 405), GGGSG (SEQ ID NO: 406), GSSSG (SEQ ID NO: 407), GPGP (SEQ ID NO: 408), GGP, PPP, PPAPPA (SEQ ID NO: 409), PPPG (SEQ ID NO: 24), PPPGPPP (SEQ ID NO: 410), PPP(GGGS)n (SEQ ID NO: 25), (GGGS)nPPP (SEQ ID NO: 26), AEAAAKEAAAKEAAAKA (SEQ ID NO: 1025), and TPPKTKRKVEFE (SEQ ID NO: 27) (wherein n is 1 to 5). Those skilled in the art will recognize that the design of a peptide conjugated to any of the above elements may include a linker that is all or partially flexible, so that the linker may include one or more parts that confer a flexible linker as well as a less flexible structure.

[0261] Method for producing V.CasX variant proteins and gRNA variants The CasX variant proteins and gRNA variants described herein can be constructed by a variety of methods. Such methods include, for example, the following and examples, as well as the comprehensive mutational evolution (DME) described in international application PCT / US20 / 36506 and international publication 2020247883(A2) (incorporated herein by reference).

[0262] a. Comprehensive Variation Evolution (DME) In some embodiments, DME is used to identify CasX protein and sgRNA scaffold variants with improved functionality. In some embodiments, the DME method involves constructing and testing a comprehensive set of mutations against an initiating biomolecule to generate a library of biomolecular variants (e.g., a library of CasX variant proteins or sgRNA scaffold variants). DME may encompass constructing all possible substitutions of amino acids (for proteins) or nucleotides (for RNA or DNA), as well as all possible minor insertions and all possible deletions, against the initiating biomolecule. Figure 16 shows a schematic diagram illustrating the DME method. In some embodiments, DME includes a subset of all such possible substitutions, insertions, and deletions. In certain embodiments of DME, one or more libraries of variants are constructed and evaluated for functional changes, and this information is used to construct one or more additional libraries. Such iterative construction and evaluation of variants may result in the identification of mutation themes that lead to specific functional outcomes, such as regions of proteins or RNA that, when mutated in a particular way, result in one or more improved functions. Subsequently, such stratification of identified mutations can further improve function, for example, through additive or synergistic interactions. DME includes library design, library construction, and library screening. In some embodiments, multiple rounds of design, construction, and screening are performed.

[0263] b. Library Design The DME method generates variants of biomolecules, which are polymers of many monomers. In some embodiments, the biomolecules include protein or ribonucleic acid (RNA) molecules, and the monomer units are amino acids or ribonucleotides, respectively. The basic units of biomolecular mutation include (1) replacing one monomer with another monomer of different identity (substitution), (2) inserting one or more additional monomers into a biomolecule (insertion), or (3) removing one or more monomers from a biomolecule (deletion). A DME library containing substitutions, insertions, and deletions of any one or more monomers in any biomolecule described herein, either individually or in combination, is considered to be within the scope of the present invention.

[0264] In some embodiments, DME is used to construct and test a comprehensive set of mutations in a biomolecule, encompassing all possible substitutions, as well as small insertions and deletions of amino acids (in the case of proteins) or nucleotides (in the case of RNA). The construction and functional readout of these mutations can be achieved using a variety of established molecular biology methods. In some embodiments, the library contains a subset of all possible modifications to a monomer. For example, in some embodiments, the library collectively represents single modifications of a single monomer for at least 10% of the total monomer positions in the biomolecule, with each single modification selected from the group consisting of substitutions, single insertions, and single deletions. In some embodiments, the library collectively represents single modifications of a single monomer for at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or up to 100% of the total monomer positions in the starting biomolecule. In certain embodiments, for a specific proportion of the total monomer positions in the starting biomolecule, the library collectively represents each possible single modification of a single monomer, e.g., all possible substitutions by 19 other naturally occurring amino acids (in the case of proteins) or 3 other naturally occurring ribonucleotides (in the case of RNA), each of 20 naturally occurring amino acids (in the case of proteins) or 4 naturally occurring ribonucleotides (in the case of RNA), or monomer deletion. In further embodiments, each insertion at each position is independently the insertion of more than one monomer, e.g., two or more, three or more, or four or more monomers, or one to four, two to four, or one to three monomers. In some embodiments, a deletion at a position is independently the deletion of more than one monomer, e.g., two or more, three or more, or four or more monomers, or one to four, two to four, or one to three monomers. Examples of such libraries of CasX variants and gRNA variants are described in Examples 14 and 15, respectively.

[0265] In some embodiments, the biomolecule is a protein, and the individual monomers are amino acids. In embodiments where the biomolecule is a protein, the number of possible DME mutations at each monomer (amino acid) position in the protein includes 19 amino acid substitutions, 20 amino acid insertions, and 1 amino acid deletion, resulting in a total of 40 possible mutations per amino acid in the protein.

[0266] In some embodiments, the DME library of CasX variant proteins containing insertions is a 1-amino acid insertion library, a 2-amino acid insertion library, a 3-amino acid insertion library, a 4-amino acid insertion library, a 5-amino acid insertion library, a 6-amino acid insertion library, a 7-amino acid insertion library, an 8-amino acid insertion library, a 9-amino acid insertion library, or a 10-amino acid insertion library. In some embodiments, the DME library of CasX variant proteins containing insertions contains 1 to 4 amino acid insertions.

[0267] In some embodiments, the biomolecule is RNA. In embodiments where the biomolecule is RNA, the number of possible DME mutations at each monomer (ribonucleotide) position in the RNA includes 3 nucleotide substitutions, 4 nucleotide insertions, and 1 nucleotide deletion, resulting in a total of 8 possible mutations per nucleotide.

[0268] In some embodiments, the design of a DME library involves enumerating all possible mutations for each of one or more target monomers in a biomolecule. As used herein, “target monomer” means a monomer in a biomolecular polymer that targets DME having the substitutions, insertions, and deletions described herein. For example, a target monomer may be an amino acid at a specific position in a protein, or a nucleotide at a specific position in RNA. A biomolecule may have at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, or more target monomers, which are systematically mutated to produce a DME library of biomolecular variants. In some embodiments, all monomers in a biomolecule are target monomers. For example, in a DME of a protein with two target amino acids, the design of the DME library involves enumerating 40 possible DME mutations for each of the two target amino acids. In a further example, in a DME of RNA with four target nucleotides, the design of the DME library involves enumerating 8 possible DME mutations for each of the four target nucleotides. In some embodiments, each target monomer of a biomolecule is independently and randomly selected or selected by deliberate design. Thus, in some embodiments, the DME library includes random variants, or designed variants, or variants containing both random and designed mutations within a single biomolecule, or any combination thereof.

[0269] In some embodiments of the DME method, the DME mutation is incorporated into double-stranded DNA encoding a biomolecule. This DNA can be maintained and replicated in a standard cloning vector (e.g., a bacterial plasmid referred to herein as a target plasmid). An exemplary target plasmid includes a DNA sequence encoding the starting biomolecule to be subjected to DME, a bacterial origin for replication, and a suitable antibiotic resistance expression cassette. In some embodiments, the antibiotic resistance cassette confers resistance to kanamycin, ampicillin, spectinomycin, bleomycin, streptomycin, erythromycin, tetracycline, or chloramphenicol. In some embodiments, the antibiotic resistance cassette confers resistance to kanamycin.

[0270] Libraries containing the variant can be constructed in various ways. In certain embodiments, the library is constructed using plasmid recombination. Such methods can incorporate the mutation into a plasmid encoding a reference biomolecule using DNA oligonucleotides encoding one or more mutations. In the case of biomolecular variants having multiple mutations, two or more oligonucleotides are used in some embodiments. In some embodiments, the DNA oligonucleotide encodes one or more mutations, and the mutation region is adjacent to 10 to 100 nucleotides homologous to the target plasmid at both the 5' and 3' ends relative to the mutation. Such oligonucleotides may be commercially synthesized in some embodiments and used in PCR amplification. Exemplary templates for mutation-encoding oligonucleotides are provided below. 5'-(N) 10-100 -Mutation-(N') 10-100 -3'

[0271] In this exemplary oligonucleotide design, N represents a sequence identical to that of the target plasmid (referred to herein as a homology arm). When a particular monomer in a biomolecule targets a mutation, these homology arms are directly adjacent to the DNA encoding the monomer in the target plasmid. In some exemplary embodiments where the biomolecule receiving DME is a protein, 40 different oligonucleotides using the same set of homology arms are used to encode 40 different amino acid mutations, listed for each amino acid residue in the protein targeting DME. If the mutation is a single amino acid mutation, the region encoding one or more desired mutations contains three nucleotides (in the case of substitution or a single insertion) or zero nucleotides (in the case of deletion) encoding the amino acids. In some embodiments, the oligonucleotide encodes more than one amino acid insertion. For example, if the oligonucleotide encodes an insertion of X amino acids, the region encoding the desired mutation contains 3 × X nucleotides encoding the X amino acids. In some embodiments, the mutation region encodes two or more mutations, for example, mutations in two or more monomers of a biomolecule that are in close proximity (e.g., adjacent to each other or within one, two, three, four, five, six, seven, eight, nine, or ten or more monomers of each other).

[0272] In some exemplary embodiments where the biomolecule receiving DME is RNA, eight different oligonucleotides, using the same set of homology arms, encode eight different single-nucleotide mutations for each nucleotide in the RNA targeting DME. If the mutation is a single ribonucleotide mutation, the region of the oligo encoding the mutation may consist of the following nucleotide sequences: one nucleotide (in the case of substitution or insertion) or zero nucleotides (in the case of deletion). In some embodiments, the oligonucleotides are synthesized as single-stranded DNA oligonucleotides. In some embodiments, all oligonucleotides targeting specific amino acids or nucleotides of the biomolecule subjected to DME are pooled. In some embodiments, all oligonucleotides targeting the biomolecule subjected to DME are pooled. There is no limit to the types or number of mutations that can be generated simultaneously in a DME library.

[0273] c. Library screening Any suitable method for screening or selecting a DME library is within the scope of the present invention and is assumed to be as follows: High-throughput methods can be used to evaluate large libraries with thousands of individual mutations. In some embodiments, the throughput of the library screening or selection assay is such that it is the throughput of millions of individual cells. In some embodiments, phenotypic and genotypic assays utilizing live cells are preferred because they are physically related in live cells by properties contained within the same lipid bilayer. Live cells can also be used to directly amplify subpopulations of the entire library. In other embodiments, smaller assays are used in the DME method, for example, to screen focused libraries developed through multiple rounds of mutation and evaluation. Exemplary methods for screening libraries are described in Examples 14 and 15.

[0274] In some embodiments, DME libraries screened or selected for highly functional variants are further characterized. In some embodiments, further characterization of the DME library involves analyzing DME variants individually by sequencing (e.g., Sanger sequencing) to identify one or more specific mutations that resulted in highly functional variants. Individual variants of biomolecules can be isolated by standard molecular biology techniques for subsequent functional analysis. In some embodiments, further characterization of the DME library involves high-throughput sequencing of both the I library and one or more libraries of highly functional variants. This approach may, in some embodiments, allow for the rapid identification of overexpressed mutations in one or more libraries of highly functional variants compared to a naive DME library. While we do not wish to be bound by any theory, overexpressed mutations in one or more libraries of highly functional variants are likely to be associated with the activity of the highly functional variants. In some embodiments, further characterizing the DME library includes both sequencing of individual variants and high-throughput sequencing of both naive libraries and libraries of one or more highly functional variants.

[0275] High-throughput sequencing can generate high-throughput data demonstrating the functional effects of library members. In embodiments where one or more libraries represent all possible mutations at all monomer positions, such high-throughput sequencing can evaluate the functional effects of all possible DME mutations. Such sequencing can also be used to evaluate one or more highly functional subpopulations of a given library, which in some embodiments may lead to the identification of mutations that result in improved function. Extensive Mutation Scanning

[0276] In some embodiments, comprehensive mutation scanning (DMS) is used to identify CasX variant proteins with improved function. Comprehensive mutation scanning assesses the plasticity of a protein in relation to its function. In the DMS method, all amino acids in a protein are replaced with all other amino acids to assay absolute protein function. For example, all amino acids in a CasX protein can be replaced with all other amino acids, and the ability of the mutated CasX protein to bind to or cleave DNA can be assayed. Exemplary assays, such as CRISPRi assays or bacterial-based cleavage assays, that can be used to characterize collections of DMS CasX variant proteins are described in Oakes et al. (2016) “Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch” Nat Biotechnol 34(6): 646-51 and Liu et al. (2019) “CasX enzymes comprise a distinct family of RNA-guided genome editors” Nature doi.org / 10.1038 / s41586-019-0908 (these contents are incorporated herein by reference).

[0277] In some embodiments, DMS is used to identify CasX proteins with improved DNA-binding activity. In some embodiments, DNA-binding activity is assayed using a CRISPRi assay. In a non-limiting exemplary embodiment of the CRISPRi assay, cells expressing a fluorescent protein, such as green fluorescent protein (GFP) or red fluorescent protein (RFP), are assayed using FACS to identify CasX variants that can repress the expression of the fluorescent protein in an sgRNA-dependent manner. In this example, catalytically inactive CasX (dCasX) is used to generate a collection of DMS variants to be assayed. The wild-type CasX protein binds to its homologous sgRNA, forming a protein-RNA complex. This complex binds to a specific DNA target (in this case, the DNA sequence encoding the fluorescent protein) by Watson-Crick base pairing between the sgRNA and the DNA target. In the case of wild-type CasX, the DNA is cleaved by the nuclease activity of the CasX protein. However, while not wanting to be bound by theory, it is likely that dCasX can still complex with sgRNA and bind to specific DNA targets. When dCasX targeting occurs in the protein-coding region, it blocks RNA polymerase II and transcription initiation and / or elongation, resulting in a decrease in the expression of fluorescent proteins that can be detected by FAC.

[0278] In some embodiments, DMS is used to identify CasX proteins with improved DNA cleavage activity. Methods for assaying the DNA cleavage efficiency of CasX variant proteins will be apparent to those skilled in the art. For example, a CasX protein complexed with an sgRNA having a spacer complementary to a specific target nucleic acid sequence can be used to cleave the DNA target sequence in vitro or in vivo in a suitable cell type, and the frequency of insertions and deletions at the cleavage site can be assayed. While not wishing to be bound by theory, cleavage or nicking by CasX generates a double-strand break in the DNA, and subsequent repair by the non-homologous end joining pathway (NHEJ) results in small insertions or deletions (indels) at the double-strand break site. The frequency of indels at CasX cleavage sites can be measured using high-throughput sequencing or Sanger sequencing of the target sequence. Alternatively, or additionally, the frequency of indel generation by CasX cleavage of the target sequence can be measured using a mismatch assay (e.g., T7 Endonuclease I (T7EI) or Surveyor mismatch assay).

[0279] In some embodiments, following DMS, a map (e.g., a heatmap) of genotypes of DMS variants associated with their resulting phenotypes is generated and used to characterize the fundamental principles of the protein. All possible mutations are characterized as those that result in functional or non-functional protein products in order to establish the functional landscape of the protein.

[0280] d. Error-prone PCR In some embodiments, error-prone PCR is used to generate CasX proteins or sgRNA scaffold variants with improved functionality. Polymerases that replicate DNA have different levels of fidelity. One method of introducing random mutations into a gene is by using an error-prone polymerase that will incorporate incorrect nucleotides at a certain frequency. This frequency can be adjusted depending on the desired outcome. In some embodiments, conditions of polymerase and polymerase activity are selected that result in a frequency of nucleotide changes that produce an average of n 1 to 4 amino acid changes in the protein sequence. An exemplary error-prone polymerase is Agilent's GeneMorphII kit. The GeneMorphII kit can be used to amplify the DNA sequence encoding the wild-type CasX protein (e.g., the protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3) according to the manufacturer's protocol, thereby subjecting the protein to unbiased random mutagenesis and generating a diverse population of CasX variant proteins. Next, this diverse population of CasX variant proteins can be assayed using the same assay described above for DMS to observe how genotype changes relate to phenotypic changes.

[0281] e. Cassette mutagenesis In some embodiments, cassette mutagenesis is used to generate CasX variant proteins or sgRNA scaffold variants with improved function. Cassette mutagenesis utilizes specific restriction enzyme sites substituted with degenerate nucleotides to create highly diverse subregions in the selective region of the gene of interest, such as the CasX protein or sgRNA scaffold. In an exemplary cassette mutagenesis protocol, restriction enzymes are used to cleave near the mutagenesis-targeted sequence on the DNA molecule encoding the CasX protein or sgRNA scaffold contained in a suitable vector. This step removes everything between the mutagenesis-targeted sequence and the restriction site. A synthetic double-stranded DNA molecule containing the desired mutation and ends complementary to the restriction digestion ends is then ligated in place of the sequence removed by restriction digestion, and suitable cells (e.g., E. coli) are transformed with the ligated vector. In some embodiments, cassette mutagenesis can be used to generate one or more specific mutations in the CasX protein or sgRNA scaffold. In some embodiments, cassette mutagenesis can be used to generate a library of CasX variant proteins or sgRNA scaffold variants that can be screened or selected for improved functionality using the methods described herein. For example, when generating CasX variants using cassette mutagenesis, a portion of the non-target strand-binding (NTSB) domain can be replaced with a sequence of degenerate nucleotides. The sequence of degenerate nucleotides may be highly localized to a region of the CasX protein (e.g., a region of the NTSB of interest because they are highly mobile elements or because they are in direct contact with DNA). The library of CasX variant proteins generated via cassette mutagenesis can then be screened using the assays described herein for DME, DMS, and error-prone PCR, and variants can be sele...

Claims

1. A chimeric class 2, type V CRISPR protein, a. A non-target chain binding (NTSB) domain obtained from Deltaproteobacteria containing the sequence of Sequence ID No. 2335, 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, b. A helix I-II domain obtained from Deltaproteobacteria containing the sequence of Sequence ID No. 2336, 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, c. A helix II domain obtained from Plantomycetes containing the sequence of Sequence ID No. 2351, 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, d. An OBD-I domain obtained from Plantomycetes containing the sequence of Sequence ID No. 2342, 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, e. An OBD-II domain obtained from Plantomycetes containing the sequence of Sequence ID No. 2347, 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, f. A helix I-I domain obtained from Plantomycetes containing the sequence of Sequence ID No. 2343, 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, g. A TSL domain obtained from Plantomycetes containing the sequence of Sequence ID No. 2349, 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, h. A RuvC-II domain obtained from Plantomycetes containing the sequence of Sequence ID No. 2350, 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, i. A RuvC-I domain obtained from Plantomycetes containing the sequence of Sequence ID No. 2352, 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, Includes, The RuvC-I domain contains a proline amino acid residue at position 793 compared to SEQ ID NO: 336, indicating that the chimeric class 2, type V CRISPR protein has a lower ratio of off-target editing to on-target editing compared to SEQ ID NO:

336. Chimeric class 2, type V CRISPR protein.

2. A chimeric class 2, type V CRISPR protein according to claim 1, comprising one or more nuclear localization signals (NLS), wherein the one or more NLS are linked to the chimeric class 2, type V CRISPR protein or adjacent NLS using a linker peptide.

3. A chimeric class 2, type V CRISPR protein according to claim 1 or 2, comprising the sequence of sequence number 416, or a sequence having at least 90% identity therewith.

4. A chimeric class 2, type V CRISPR protein containing a sequence selected from the group consisting of sequence numbers 415-592 and 1147-1231 shown in Table 3.

5. A gene editing pair comprising gRNA and a chimeric class 2, type V CRISPR protein, wherein the pair is a. A gRNA comprising a gRNA scaffold and a targeting sequence complementary to the target nucleic acid sequence at the 3' end of the gRNA scaffold, b. A gRNA comprising a chimeric class 2, type V CRISPR protein according to any one of claims 1 to 4, wherein the gRNA and the class 2, type V CRISPR protein can form a ribonucleoprotein complex (RNP), and the RNP of the class 2, type V CRISPR protein and the gRNA exhibits at least one improved feature compared to the RNP of the gRNA containing the reference protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and the sequence of SEQ ID NO: 4, or SEQ ID NO: 5, and the PAM sequence TTC, ATC, GTC, or If any one of the CTCs is located one nucleotide 5' to the non-target strand of a protospacer having identity with the targeting sequence of the gRNA in a cell assay system, then the RNP comprising the class 2, type V CRISPR protein and the gRNA exhibits higher editing efficiency and / or binding of the target nucleic acid sequence in the target nucleic acid compared to the editing efficiency and / or binding of the RNP comprising the reference protein of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 and the reference gRNA in an equivalent assay system. Gene-editing pair.

6. A catalytically inactive class 2, type V CRISPR protein containing a sequence selected from the group consisting of sequence numbers 44-62 and 1232-1235 shown in Table 7.

7. A nucleic acid comprising a sequence encoding a chimeric class 2, type V CRISPR protein according to any one of claims 1 to 4.

8. A vector comprising a chimeric class 2, type V CRISPR protein according to any one of claims 1 to 4, or the nucleic acid according to claim 7.

9. The aforementioned vector, (a) including a promoter; and / or (b) Selected from the group consisting of retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus (AAV) vectors, herpes simplex virus (HSV) vectors, plasmids, minicircles, nanoplasmides, DNA vectors, RNA vectors, and lipid nanoparticles (LNPs), The vector according to claim 8.

10. A host cell comprising the vector according to claim 8 or 9.

11. A method for modifying a target nucleic acid in a cell in vitro or ex vivo, comprising contacting the target nucleic acid of the cell with i) the gene editing pair described in claim 5, ii) the gene editing pair described in claim 5 and a donor template, iii) one or more nucleic acids encoding or containing the gene editing pair of (i) or (ii), iv) a vector containing the nucleic acid of (i) or (ii), or v) two or more combinations of (i) to (iv), wherein the contact of the target nucleic acid modifies the target nucleic acid.

12. (a) Including contact of the target with a first and second gRNA or a plurality of gene editing pairs comprising a plurality of gRNAs, which include targeting sequences complementary to different or overlapping regions of the target nucleic acid; or (b) The method comprises contacting the target with a plurality of nucleic acids encoding a gene editing pair, which includes first and second gRNAs or a plurality of gRNAs, that contain targeting sequences complementary to different or overlapping regions of the target nucleic acid. The method according to claim 11, The contact includes binding the target nucleic acid and introducing one or more double-strand breaks into the target nucleic acid, and the modification includes introducing mutations, insertions, or deletions into the target nucleic acid. method.

13. The method according to claim 11 or 12, wherein the modification occurs in the target cell having a mutation in a gene allele, and the mutation causes a disease or disorder in the target.

14. The aforementioned cells, (a) self-derived with respect to the subject; or (b) The same type of self-derived object as the object The method according to claim 13.

15. The aforementioned vector, (a) is an adeno-associated virus (AAV) vector; or (b) A lentiviral vector, The method according to any one of claims 11 to 14.

16. (a) comprising the chimeric class 2, type V CRISPR protein according to any one of claims 1 to 4; (b) a gRNA comprising a gRNA scaffold and a targeting sequence complementary to a target nucleic acid sequence at the 3' end of the gRNA scaffold, and a chimeric class 2, type V CRISPR protein according to any one of claims 1 to 4, wherein the chimeric class 2, type V CRISPR protein and the gRNA are associated together in a ribonucleoprotein complex (RNP); or (c) comprising the gene editing pair described in claim 5, composition.

17. (a) comprising the chimeric class 2, type V CRISPR protein and container according to any one of claims 1 to 4; (b) a gRNA comprising a gRNA scaffold and a targeting sequence complementary to a target nucleic acid sequence at the 3' end of the gRNA scaffold, a chimeric class 2, type V CRISPR protein and a container according to any one of claims 1 to 4; or (c) comprising the gene editing pair and container described in claim 5, kit.

18. A composition for use as a pharmaceutical for the treatment of a subject having a disease, comprising (a) a chimeric class 2, type V CRISPR protein according to any one of claims 1 to 4, and (b) a gRNA comprising a gRNA scaffold and a targeting sequence complementary to a target nucleic acid sequence at the 3' end of the gRNA scaffold.

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