Type v cas proteins and applications thereof

Abstract

Type V Cas proteins, for example Type V Cas proteins referred to as ZWGD, ZJHK, ZIKV, ZZFT, YYAN, ZZGY, ZKBG, ZZKD, ZXPB, ZPPX, ZXHQ, ZQKH, ZRGM, ZTAE, ZSQQ, ZSYN, ZRBH, ZWPU, ZZQE, and ZRXE Type V Cas proteins; gRNAs for Type V Cas proteins; systems comprising Type V Cas proteins and gRNAs; nucleic acids encoding the Type V Cas proteins, gRNAs and systems; particles comprising the foregoing; pharmaceutical compositions of the foregoing; and uses of the foregoing, for example to alter the genomic DNA of a cell.

Description

1. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of U.S. provisional application no. 63 / 574,354, filed April 4, 2024, the contents of which are incorporated herein in their entireties by reference thereto. 2. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML Sequence Listing, created on March 25, 2025, is named ALA-013WO_SL.xml and is 679,601 bytes in size. 3. BACKGROUND

[0003] CRISPR-Cas systems (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated proteins) are powerful tools with the potential to treat a variety of genetic diseases. The CRISPR-Cas systems are classified into two classes (Class 1 and 2) that are subdivided into six types (Type 1 through VI). Class 1 (Type I, III and IV) systems use multiple Cas proteins in their CRISPR ribonucleoprotein effector nucleases, and Class 2 systems (Type II, V and VI) use a single Cas protein. Cas9, belonging to Class 2 Type II CRISPR-Cas system, is the most extensively used tool for genome editing.

[0004] However, there are some challenges in using CRISPR-Cas9 systems. For example, packaging a large Cas protein such as SpCas9 together with a guide RNA into a single AAV vector (Adeno-associated viral vectors) can be challenging due to the limited packaging capacity of AAVs. Type V Cas proteins such as Cas12a target T-rich sequences, which in principle allow Type V Cas proteins to access different genomic regions as compared to Cas9. Type V Cas proteins typically produce staggered ends when it creates a double stranded DNA cut (while Cas9 creates a blund end), which may be an advantage in certain situations such as during gene insersions and substitutions. Type V Cas proteins also typically produce mid sized deletions at the target site (generally tens of nucleotides) allowing for the removal of target sequences locally (e.g. binding sites fortranscription factors, splice sites, etc). In comparason, Cas9 produces relatively small indels (generally insertion or deletion of a few nucleotides). Type V Cas proteins such as Cas12a are typically capable of processing their own crRNA from larger transcripts, which can make multiplexing easier.

[0005] Thus, there is a need for new Cas nucleases, especially Type V Cas nucleases. 4. SUMMARY

[0006] This disclosure is based, in part, on the discovery of a Type V Cas protein from an unclassified bacterium from the Candidatus Saccharibacteria phylum (referred to herein as “wildtype ZWGD type V Cas”); a Type V Cas protein from an unclassified bacterium from the Clostridiaceae family (referred to herein as “wildtype ZJHK type V Cas”); a Type V Cas protein from an unclassified bacterium from the Firmucutes phylum (referred to herein as “wildtype ZIKV type V Cas”); a Type V Cas protein from an unclassified bacterium from the Bacteroidota phylum (referred to herein as “wildtype ZZFT type V Cas”); a Type V Cas protein from an unclassified bacterium from the Firmicutes phylum (referred to herein as “wildtype YYAN type V Cas”); a Type V Cas protein from an unclassified bacterium from the Succinivibrionaceae family (referred to herein as “wildtype ZZGY type V Cas”); a Type V Cas protein from an unclassified bacterium from the Muribaculaceae family (referred to herein as “wildtype ZKBG type V Cas”); a Type V Cas protein from Mogibacterium kristiansenii (referred to herein as “wildtype ZZKD type V Cas”); a Type V Cas protein from an unclassified bacterium from the Bacteroidales order (referred to herein as “wildtype ZXPB type V Cas”); a Type V Cas protein from an unclassified bacterium from the Prevotellaceae family (referred to herein as “wildtype ZPPX type V Cas”); a Type V Cas protein from an unclassified bacterium from the phylum Candidatus Roizmanbacteria (referred to herein as “wildtype ZXHQ type V Cas”); a Type V Cas protein from an unclassified bacterium from the phylum Bacteroidota (referred to herein as “wildtype ZQKH type V Cas”); a Type V Cas protein from an unclassified bacterium from the phylum Firmicutes (referred to herein as “wildtype ZRGM type V Cas”); a Type V Cas protein from an unclassified bacterium from the phylum Kiritimatiellaeota (referred to herein as “wildtype ZTAE type V Cas”); a Type V Cas protein from an unclassified bacterium from the phylum Fibrobacteres (referred to herein as “wildtype ZSQQ type V Cas”); a Type V Cas protein from an unclassified bacterium from the phylum Firmicutes (referred to herein as “wildtype ZSYN type V Cas”); a Type V Cas protein from an unclassified bacterium from the phylum Firmicutes (referred to herein as “wildtype ZRBH type V Cas”); a Type V Cas protein from an unclassified bacterium from the phylum Bacteroidota (referred to herein as “wildtype ZWPU type V Cas”); a Type V Cas protein from an unclassified bacterium from the Prevotellaceae family (referred to herein as “wildtype ZZQE type V Cas”); and a Type V Cas protein from an unclassified bacterium from the phylum Bacteroidota (referred to herein as “wildtype ZRXE type V Cas”).

[0007] In one aspect, the disclosure provides Type V Cas proteins whose amino acid sequence comprises an amino acid sequence that is at least 50% identical (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identical, or 100% identical) to SEQ ID NO:1 (such proteins referred to herein as “ZWGD Type V Cas proteins”). Exemplary ZWGD Type V Cas protein sequences are set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.

[0008] In one aspect, the disclosure provides Type V Cas proteins whose amino acid sequence comprises an amino acid sequence that is at least 50% identical (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identical, or 100% identical) to SEQ ID NO:7 (such proteins referred to herein as “ZJHK Type V Cas proteins”). Exemplary ZJHK Type V Cas protein sequences are set forth in SEQ ID NOT, SEQ ID NO:8, and SEQ ID NO:9.

[0009] In one aspect, the disclosure provides Type V Cas proteins whose amino acid sequence comprises an amino acid sequence that is at least 50% identical (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identical, or 100% identical) to SEQ ID NO:13 (such proteins referred to herein as “ZIKV Type V Cas proteins”). Exemplary ZIKV Type V Cas protein sequences are set forth in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15.

[0010] In one aspect, the disclosure provides Type V Cas proteins whose amino acid sequence comprises an amino acid sequence that is at least 50% identical (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identical, or 100% identical) to SEQ ID NO:19 (such proteins referred to herein as “ZZFT Type V Cas proteins”). Exemplary ZZFT Type V Cas protein sequences are set forth in SEQ ID NO:19, SEQ ID NQ:20, and SEQ ID NO:21.

[0011] In one aspect, the disclosure provides Type V Cas proteins whose amino acid sequence comprises an amino acid sequence that is at least 50% identical (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identical, or 100% identical) to SEQ ID NO:25 (such proteins referred to herein as “YYAN Type V Cas proteins”). Exemplary YYAN Type V Cas protein sequences are set forth in SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27.

[0012] In one aspect, the disclosure provides Type V Cas proteins whose amino acid sequence comprises an amino acid sequence that is at least 50% identical (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identical, or 100% identical) to SEQ ID NO:31 (such proteins referred to herein as “ZZGY Type V Cas proteins”). Exemplary ZZGY Type V Cas protein sequences are set forth in SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33.

[0013] In one aspect, the disclosure provides Type V Cas proteins whose amino acid sequence comprises an amino acid sequence that is at least 50% identical (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identical, or 100% identical) to SEQ ID NO:37 (such proteins referred to herein as “ZKBG Type V Cas proteins”). Exemplary ZKBG Type V Cas protein sequences are set forth in SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39.

[0014] In one aspect, the disclosure provides Type V Cas proteins whose amino acid sequence comprises an amino acid sequence that is at least 50% identical (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identical, or 100% identical) to SEQ ID NO:43 (such proteins referred to herein as “ZZKD Type V Cas proteins”). Exemplary ZZKD Type V Cas protein sequences are set forth in SEQ ID NO:43, SEQ ID NO:44, and SEQ ID NO:45.

[0015] In one aspect, the disclosure provides Type V Cas proteins whose amino acid sequence comprises an amino acid sequence that is at least 50% identical (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identical, or 100% identical) to SEQ ID NO:49 (such proteins referred to herein as “ZXPB Type V Cas proteins”). Exemplary ZXPB Type V Cas protein sequences are set forth in SEQ ID NO:49, SEQ ID NQ:50, and SEQ ID NO:51.

[0016] In one aspect, the disclosure provides Type V Cas proteins whose amino acid sequence comprises an amino acid sequence that is at least 50% identical (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identical, or 100% identical) to SEQ ID NO:55 (such proteins referred to herein as “ZPPX Type V Cas proteins”). Exemplary ZPPX Type V Cas protein sequences are set forth in SEQ ID NO:55, SEQ ID NO:56, and SEQ ID NO:57.

[0017] In one aspect, the disclosure provides Type V Cas proteins whose amino acid sequence comprises an amino acid sequence that is at least 50% identical (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identical, or 100% identical) to SEQ ID NO:61 (such proteins referred to herein as “ZXHQ Type V Cas proteins”). Exemplary ZXHQ Type V Cas protein sequences are set forth in SEQ ID NO:61, SEQ ID NO:62, and SEQ ID NO:63.

[0018] In one aspect, the disclosure provides Type V Cas proteins whose amino acid sequence comprises an amino acid sequence that is at least 50% identical (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identical, or 100% identical) to SEQ ID NO:67 (such proteins referred to herein as “ZQKH Type V Cas proteins”). Exemplary ZQKH Type V Cas protein sequences are set forth in SEQ ID NO:67, SEQ ID NO:68, and SEQ ID NO:69.

[0019] In one aspect, the disclosure provides Type V Cas proteins whose amino acid sequence comprises an amino acid sequence that is at least 50% identical (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identical, or 100% identical) to SEQ ID NO:73 (such proteins referred to herein as “ZRGM Type V Cas proteins”). Exemplary ZRGM Type V Cas protein sequences are set forth in SEQ ID NO:73, SEQ ID NO:74, and SEQ ID NO:75.

[0020] In one aspect, the disclosure provides Type V Cas proteins whose amino acid sequence comprises an amino acid sequence that is at least 50% identical (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identical, or 100% identical) to SEQ ID NO:79 (such proteins referred to herein as “ZTAE Type V Cas proteins”). Exemplary ZTAE Type V Cas protein sequences are set forth in SEQ ID NO:79, SEQ ID NQ:80, and SEQ ID NO:81.

[0021] In one aspect, the disclosure provides Type V Cas proteins whose amino acid sequence comprises an amino acid sequence that is at least 50% identical (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identical, or 100% identical) to SEQ ID NO:85 (such proteins referred to herein as “ZSQQ Type V Cas proteins”). Exemplary ZSQQ Type V Cas protein sequences are set forth in SEQ ID NO:85, SEQ ID NO:86, and SEQ ID NO:87.

[0022] In one aspect, the disclosure provides Type V Cas proteins whose amino acid sequence comprises an amino acid sequence that is at least 50% identical (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identical, or 100% identical) to SEQ ID NO:91 (such proteins referred to herein as “ZSYN Type V Cas proteins”). Exemplary ZSYN Type V Cas protein sequences are set forth in SEQ ID NO:91, SEQ ID NO:92, and SEQ ID NO:93.

[0023] In one aspect, the disclosure provides Type V Cas proteins whose amino acid sequence comprises an amino acid sequence that is at least 50% identical (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identical, or 100% identical) to SEQ ID NO:97 (such proteins referred to herein as “ZRBH Type V Cas proteins”). Exemplary ZRBH Type V Cas protein sequences are set forth in SEQ ID NO:97, SEQ ID NO:98, and SEQ ID NO:99.

[0024] In one aspect, the disclosure provides Type V Cas proteins whose amino acid sequence comprises an amino acid sequence that is at least 50% identical (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identical, or 100% identical) to SEQ ID NQ:103 (such proteins referred to herein as “ZWPU Type V Cas proteins”). Exemplary ZWPU Type V Cas protein sequences are set forth in SEQ ID NQ:103, SEQ ID NQ:104, and SEQ ID NQ:105.

[0025] In one aspect, the disclosure provides Type V Cas proteins whose amino acid sequence comprises an amino acid sequence that is at least 50% identical (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identical, or 100% identical) to SEQ ID NQ:109 (such proteins referred to herein as “ZZQE Type V Cas proteins”). Exemplary ZZQE Type V Cas protein sequences are set forth in SEQ ID NQ:109, SEQ ID NQ:110, and SEQ ID NO:111.

[0026] In one aspect, the disclosure provides Type V Cas proteins whose amino acid sequence comprises an amino acid sequence that is at least 50% identical (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identical, or 100% identical) to SEQ ID NO:115 (such proteins referred to herein as “ZRXE Type V Cas proteins”). Exemplary ZRXE Type V Cas protein sequences are set forth in SEQ ID NO:115, SEQ ID NO:116, and SEQ ID NO:117.

[0027] In another aspect, the disclosure provides Type V Cas proteins comprising an amino acid sequence having at least 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%) sequence identity to a WED-1 domain, REC1 domain, REC2 domain, WED-II domain, PI domain, WED-HI domain, RuvC-l domain, BH domain, RuvC-ll domain, NUC domain, or RuvC-lll domain of a ZWGD Type V Cas protein, a ZJHK Type V Cas protein, a ZIKV Type V Cas protein, a ZZFT Type V Cas protein, a YYAN Type V Cas protein, a ZZGY Type V Cas protein, a ZKBG Type V Cas protein, a ZZKD Type V Cas protein, a ZXPB Type V Cas protein, a ZPPX Type V Cas protein, a ZXHQ Type V Cas protein, a ZQKH Type V Cas protein, a ZRGM Type V Cas protein, a ZTAE Type V Cas protein, a ZSQQ Type V Cas protein, a ZSYN Type V Cas protein, a ZRBH Type V Cas protein, a ZWPU Type V Cas protein, a ZZQE Type V Cas protein, or a ZRXE Type V Cas protein.

[0028] In some embodiments, a Type V Cas protein of the disclosure is a chimeric Type V Cas protein, for example, comprising one or more domains from a ZWGD, ZJHK, ZIKV, ZZFT, YYAN, ZZGY, ZKBG, ZZKD, ZXPB, ZPPX, ZXHQ, ZQKH, ZRGM, ZTAE, ZSQQ, ZSYN, ZRBH, ZWPU, ZZQE, and / or ZRXE Type V Cas protein(s) and one or more domains from a different Type V Cas protein such as AsCas12a.

[0029] In some embodiments, the Type V Cas proteins of the disclosure are in the form of a fusion protein, for example, comprising a ZWGD Type V Cas protein, a ZJHK Type V Cas protein, a ZIKV Type V Cas protein, a ZZFT Type V Cas protein, a YYAN Type V Cas protein, a ZZGY Type V Cas protein, a ZKBG Type V Cas protein, a ZZKD Type V Cas protein, a ZXPB Type V Cas protein, a ZPPX Type V Cas protein, a ZXHQ Type V Cas protein, a ZQKH Type V Cas protein, a ZRGM Type V Cas protein, a ZTAE Type V Cas protein, a ZSQQ Type V Cas protein, a ZSYN Type V Cas protein, a ZRBH Type V Cas protein, a ZWPU Type V Cas protein, a ZZQE Type V Cas protein, or a ZRXE Type V Cas protein sequence fused to one or more additional amino acid sequences, for example, one or more nuclear localization signals and / or one or more tags. Other exemplary fusion partners can enable base editing (e.g., where the fusion partner is nucleoside deaminase) or prime editing (e.g., where the fusion partner is a reverse transcriptase).

[0030] Exemplary features of Type V Cas proteins of the disclosure are described in Section 6.2 and specific embodiments 1 to 329 and 660 to 671, infra.

[0031] In further aspects, the disclosure provides guide (gRNA) molecules and combinations of two or more gRNA molecules. In various embodiments, the disclosure provides gRNAs that can be used with a ZWGD, ZJHK, ZIKV, ZZFT, YYAN, ZZGY, ZKBG, ZZKD, ZXPB, ZPPX, ZXHQ, ZQKH, ZRGM, ZTAE, ZSQQ, ZSYN, ZRBH, ZWPU, ZZQE, or ZRXE Type V Cas protein of the disclosure. Exemplary features of the gRNAs and combinations of gRNAs of the disclosure of the disclosure are described in Section 6.3 and specific embodiments 330 to 578, infra.

[0032] In further aspects, the disclosure provides systems comprising a Type V Cas protein of the disclosure and one or more gRNAs. For example, a system can comprise a ribonucleoprotein (RNP) comprising a Type V Cas protein complexed with a gRNA. Exemplary features of systems are described in Section 6.4 and specific embodiments 579 to 594, infra.

[0033] In another aspect, the disclosure provides nucleic acids and pluralities of nucleic acids encoding a Type V Cas protein of the disclosure and, optionally, a gRNA. In some embodiments, the nucleic acids comprise a Type V Cas protein of the disclosure operably linked to a heterologous promoter, e.g., a mammalian promoter, for example a human promoter.

[0034] In another aspect, the disclosure provides nucleic acids encoding a gRNA, and, optionally, a Type V Cas protein, for example a ZWGD Type V Cas protein, a ZJHK Type V Cas protein, a ZIKV Type V Cas protein, a ZZFT Type V Cas protein, a YYAN Type V Cas protein, a ZZGY Type V Cas protein, a ZKBG Type V Cas protein, a ZZKD Type V Cas protein, a ZXPB Type V Cas protein, a ZPPX Type V Cas protein, a ZXHQ Type V Cas protein, a ZQKH Type V Cas protein, a ZRGM Type V Cas protein, a ZTAE Type V Cas protein, a ZSQQ Type V Cas protein, a ZSYN Type V Cas protein, a ZRBH Type V Cas protein, a ZWPU Type V Cas protein, a ZZQE Type V Cas protein, or a ZRXE Type V Cas protein. Exemplary features of nucleic acids and pluralities of nucleic acids are described in Section 6.5 and specific embodiments 595 to 659, infra.

[0035] In further aspects, the disclosure provides particles comprising the Type V Cas proteins, gRNAs, nucleic acids, and systems of the disclosure. Exemplary features of particles of the disclosure are described in Section 6.6 and specific embodiments 672 to 687, infra.

[0036] In another aspect, the disclosure provides cells and populations of cells containing or contacted with a Type V Cas protein, gRNA, nucleic acid, plurality of nucleic acids, system, or particle of the disclosure. Exemplary features of such cells and cell populations are described in Section 6.6 and specific embodiments 689 to 699 and 737, infra.

[0037] In another aspect, the disclosure provides pharmaceutical compositions comprising a Type V Cas protein, gRNA, nucleic acid, plurality of nucleic acids, system, particle, cell, or population of cells together with one or more excipients. Exemplary features of pharmaceutical compositions are described in Section 6.7 and specific embodiment 688, infra.

[0038] In another aspect, the disclosure provides methods of altering cells {e.g., editing the genome of a cell) using the Type V Cas proteins, gRNAs, nucleic acids, systems, particles, and pharmaceutical compositions of the disclosure. Cells altered according to the methods of the disclosure can be used, for example, to treat subjects having a disease or disorder, e.g., genetic disease or disorder. Features of exemplary methods of altering cells are described in Section 6.8 and specific embodiments 700 to 736, infra.

[0039] In another aspect, the disclosure provides methods of detecting a target nucleic acid using the Type V Cas proteins, gRNAs, and systems of the disclosure, and use of the foregoing in such methods. Features of exemplary methods of detecting target nucleic acids, and Type V Cas proteins, gRNAs, and systems for use in methods of detecting a target nucleic acid are described in Section 6.9 and specific embodiments 738 to 740, infra. 5. BRIEF DESCRIPTION OF THE FIGURES

[0040] FIGS. 1A-1E illustrate exemplary Type V-A Cas protein crRNAs (corresponding DNA sequences shown). Schematic representation of the hairpin structure generated for visualization using RNAplot after in silico folding using RNAalifold v2.4.17 of the crRNA scaffolds (not including the spacer sequence) for ZWGD Type V-A Cas protein (FIG. 1 A), ZJHK Type V-A Cas protein (FIG. 1B), ZIKV Type V-A Cas protein (FIG. 1C), ZZFT Type V-A Cas protein (FIG. 1D) and YYAN Type V-A Cas protein (FIG. 1E) are shown. Figures disclose SEQ ID NOS 390-394, respectively, in order of appearance.

[0041] FIGS. 2A-2E illustrate exemplary Type V-A Cas protein crRNAs (corresponding DNA sequences shown). Schematic representation of the hairpin structure generated for visualization using RNAplot after in silico folding using RNAalifold v2.4.17 of the crRNA scaffolds (not including the spacer sequence) for ZZGY Type V-A Cas protein (FIG. 2A), ZKBG Type V-A Cas protein (FIG. 2B), ZZKD Type V-A Cas protein (FIG. 2C), ZXPB Type V-A Cas protein (FIG. 2D) or ZPPX Type V-A Cas protein (FIG. 2E). Figures disclose SEQ ID NOS 395-399, respectively, in order of appearance.

[0042] FIGS. 3A-3E illustrate in silico predicted PAM specificities for ZWGD, ZJHK, ZIKV, ZZFT and YYAN Type V-A Cas proteins. PAM sequence logos for ZWGD (FIG. 3A), ZJHK (FIG. 3B), ZIKV (FIG. 3C), ZZFT (FIG. 3D) and YYAN (FIG. 3E) Type V-A Cas proteins are shown.

[0043] FIGS. 4A-4E illustrate in silico predicted PAM specificities for ZZGY, ZKBG, ZZKD, ZXPB and ZPPX Type V-A Cas proteins. PAM sequence logos for ZZGY (FIG. 4A), ZKGB (FIG. 4B), ZZKD (FIG. 4C), ZXPB (FIG. 4D) and ZPPX (FIG. 4E) Type V-A Cas proteins are shown.

[0044] FIG. 5 illustrates activity of Type V-A Cas proteins against an EGFP reporter in mammalian cells. The activity of the selected Type V-A Cas proteins was evaluated after transient electroporation of plasmids encoding each nuclease together with the indicated guide RNAs in U2OS cells stably expressing EGFP. For each Cas protein, 2 different gRNAs targeting the same two positions of the EGFP coding sequence were evaluated. Loss of EGFP fluorescence, expressed as % of EGFP-negative cells, was measured by cytofluorimetry. Data presented as mean ± SEM of n>2 biologically independent runs. Untreated U2OS cells (U2OS sample) are included as a measurement of the background loss of fluorescence.

[0045] FIGS. 6A-6C illustrate activity of ZZKD Type V-A Cas protein against benchmark endogenous genomic loci in mammalian cells. The activity of ZZKD Type V-A Cas protein was evaluated after transient electroporation of plasmids encoding each nuclease together with the indicated guide RNAs in U2OS cells. Several gRNAs targeting the TRAC (FIG. 6A), B2M (FIG. 6B) and PD1 (FIG. 6C) benchmark loci were evaluated. Editing activity was measured by Sanger chromatogram deconvolution 3 days after transfection. Data presented as mean ± SEM of n>2 biologically independent runs.

[0046] FIGS. 7A-7E illustrate exemplary Type V-A Cas protein crRNAs (corresponding DNA sequences shown). Schematic representation of the hairpin structure generated for visualization using RNAplot after in silico folding using RNAalifold v2.4.17 of the crRNA scaffolds (not including the spacer sequence) for ZXHQ Type V-A Cas protein (Fig. 7A), ZQKH Type V-A Cas protein (Fig. 7B), ZRGM Type V-A Cas protein (Fig. 7C), ZTAE Type V-A Cas protein (Fig. 7D) and ZSQQ Type V-A Cas protein (FIG. 7E) are shown. Figures disclose SEQ ID NOS 400-404, respectively, in order of appearance.

[0047] FIGS. 8A-8E illustrate exemplary Type V-A Cas protein crRNAs (corresponding DNA sequences shown). Schematic representation of the hairpin structure generated for visualization using RNAplot after in silico folding using RNAalifold v2.4.17 of the crRNA scaffolds (not including the spacer sequence) for ZSYN Type V-A Cas protein (FIG. 8A), ZRBH Type V-A Cas protein (FIG. 8B), ZWPU Type V-A Cas protein (FIG. 8C), ZZQE Type V-A Cas protein (FIG. 8D) and ZRXE Type V-A Cas protein (FIG. 8E) are shown. Figures disclose SEQ ID NOS 405-409, respectively, in order of appearance.

[0048] FIG. 9 illustrates in silico prediction of ZZQE Type V-A Cas protein PAM specificity. PAM sequence logo for ZZQE Type V-A Cas protein is shown.

[0049] FIG. 10 shows activity of novel Type V-A Cas proteins in human cells. Evaluation of the activity of novel Type V-A Cas protein after transient electroporation in U2OS-EGFP cells. Two different guide RNAs were evaluated (target sequences are common for all proteins) and EGFP downregulation was measured by flow cytometry 5 days post-electroporation. A non-transfected control sample has been included to measure the assay background (NT Ctrl). 23nt spacers were used. Data represented as mean ± SD of n=2 independent biological replicates.

[0050] FIG. 11 shows activity of selected Type V-A Cas proteins towards endogenous genomic loci in human cells. The editing activity of ZZKD, ZRGM and ZZQE Type V-A Cas proteins was evaluated for the benchmark TRAC-g3, B2M-g2 and PD1-g2 genomic loci after transient transfection in HEK293T cells. Given the PAM compatibility among the different proteins the same spacers were used (23nt in length). For ZZKD activity on the TRAC locus, data represented as mean ± SD of n=3 independent biological replicates.

[0051] FIGS. 12A-12C show in vitro analysis of PAM preferences of ZZKD Type V-A Cas protein. A PAM sequence logo is shown in FIG. 12A and PAM heatmap is shown in FIG. 12B for ZZKD Type V-A Cas protein FIG. 12C shows validation of the PAM preferences by measurement of indel formation after transient transfection of HEK293T cells using crRNAs associated with PAMs shown to be preferentially cut by the PAM assay. The PAM associated with each guide is reported on the graph. Data represented as mean ± SD of n>2 independent biological replicates.

[0052] FIGS. 13A-13D show analysis of PAM preferences of ZRGM and ZZQE Type V-A Cas proteins. A PAM sequence logo is shown in FIG. 13A and a PAM heatmap is shown in FIG. 13B for ZRGM Type V-A Cas protein. A PAM sequence logo is shown in FIG. 13C and a PAM heatmap is shown in FIG. 13D for ZZQE Type V-A Cas protein.

[0053] FIGS. 14A-14B illustrate in vitro determination of the double strand break profile of ZZKD Type V-A Cas protein. In vitro cleavage reactions using a PCR-generated target (TRAC-g3) and recombinant ZZKD Type V-A Cas protein were run on an agarose gel and the separated fragments were independently Sanger sequenced using a forward and a reverse primer to sequence both DNA strands. Based on the drop in the chromatographic signal in the two sequencing reactions (FIG. 14A) it was possible to determine that ZZKD type V-A Cas protein produces a 6 nucleotide staggered cut, as indicated by the solid lines in the scheme shown in FIG. 14B. Figure discloses SEQ ID NOS 410-411, 410, and 412, respectively, in order of appearance.

[0054] FIG. 15 shows an evaluation of alternative nuclear localization signal (NLS) designs to improve the activity of ZZKD Type V-A Cas protein. FIG. 15 plots indel formation at the TRAC locus (g3) after transient transfection of HEK293T cells with alternative versions of ZZKD Type V-A Cas proteins characterized by different nuclear localization signal sequences positioned either at the N- or the C-terminus of the protein, as indicated on the graph. The amino acid sequence of each evaluated NLS is reported in the figure. Data represented as mean ± SD of n>2 independent biological replicates. Figure discloses SEQ ID NOS 179, 122, 180, and 125, respectively, in order of appearance.

[0055] FIGS. 16A-16C show alternative crRNA scaffolds for selected Type V-A Cas proteins. Schematic representation of the hairpin structure generated for visualization using the RNAfold webserver (www.unafold.org) of the crRNA trimmed scaffolds (not including the spacer sequence) for ZZKD Type VACas protein (FIG. 16A) (SEQ ID NO:211), ZZQE Type V-A Cas protein (FIG. 16B) (SEQ ID NO:212) and ZRGM Type V-A Cas protein (FIG. 16C) (SEQ ID NO:213).

[0056] FIGS. 17A-17B show the activity of alternative crRNA scaffolds for selected Type V-A Cas proteins. FIG. 17A shows indel formation measured after transient transfection ofHEK293T cells with alternative versions (full-length or trimmed) of the crRNAs targeting the TRAC-g3 locus for ZZKD, ZZQE and ZRGM Type V-A Cas proteins. FIG. 17B shows indel formation measured after transient transfection of HEK293T cells with alternative versions (full-length or trimmed) of ZZKD Type V-A Cas protein crRNAs targeting the BCL11A, TRAC, AAVS1 and B2M loci, as indicated on the graph. Data represented as mean ± SD of n=2 independent biological replicates.

[0057] FIGS. 18A-18B illustrate the effect of alternative spacer lengths on ZZKD Type V-A Cas protein editing activity. Indel formation in HEK293T cells after transient transfection of ZZKD Type V-A Cas protein in combination with families of crRNAs characterized by different spacer lengths (from 20nt to 24nt) targeting either the Match6 (FIG. 18A) or the TRAC locus (g3, FIG. 18B). Data represented as mean ± SD of n=2 independent biological replicates.

[0058] FIG. 19 shows a side-by-side comparison of ZZKD Type V-A Cas protein activity with AsCs12a Ultra. The figure shows a violin plot summarizing the editing activity of ZZKD Type V-A Cas protein and AsCas12a Ultra on a panel of endogenous genomic loci (TRAC, PD1, B2M, EMX1, AAVS1, BCL11a, PCSK9, Match6, VEGFA) after transient transfection of HEK292T cells, using crRNAs for the two nucleases that overlap on each locus. Each point on the graph represents the mean of n=2 independent runs except for B2M-g1_21 nt for AsCasI 2a Ultra (n=1).

[0059] FIGS. 20A-20D show activity of ZZKD Type V-A Cas protein in subsaturating conditions. Titration curves obtained by measuring indel formation at the BCL11A-g4 (FIG. 20A), VEGFA-g1 (FIG. 20B), B2M-g1_21nt (FIG. 20C) and B2M-g2_21nt (FIG. 20D) target sites after a 2-fold serial dilution of the amount of ZZKD and crRNA plasmids transiently transfected in HEK293T cells. The activity of AsCasI 2a Ultra was measured in the same study conditions as a benchmark and is reported on each graph. Data represented as mean ± SD of n=2 independent biological replicates.

[0060] FIGS. 21A-21C show activity of ZZKD Type V-A Cas after direct ribonucleoprotein delivery in human cell lines. Indel formation after ZZKD Type V-A Cas RNP electroporation in U2OS cells to target either the TRAC-g3 locus (FIG. 21 A) or the B2M-g2 locus (FIG. 21B). Cells were also transfected with plasmids expressing ZZKD and its crRNA as a positive control. IVT, in vitro transcribed crRNA; syn, unmodified chemically synthesized crRNA; AltR, chemically synthesized crRNA including commercially available AltR modifications from IDT. FIG. 21C shows the results of a titration study in U2OS cells delivering different amounts of recombinant ZZKD and cognate crRNA targeting the B2M-g2 locus by electroporation. The amount (pmol) of recombinant protein and crRNA used in each condition is indicated below each bar. Data represented as mean ± SD of n>2 independent biological replicates, except for B2M-g2 IVT and panel (FIG. 21C) where only one replicate is available.

[0061] FIG. 22 shows activity of ZZKD Type V-A Cas after direct ribonucleoprotein delivery in primary human T cells. The figure shows percentage of TRAC-negative cells measured by flow cytometry after ZZKD Type V-A Cas RNP electroporation in commercial human primary T cells to target the TRAC-g3 locus. 6. DETAILED DESCRIPTION

[0062] In one aspect, the disclosure provides Type V Cas proteins, e.g., a ZWGD Type V Cas protein, a ZJHK Type V Cas protein, a ZIKV Type V Cas protein, a ZZFT Type V Cas protein, a YYAN Type V Cas protein, a ZZGY Type V Cas protein, a ZKBG Type V Cas protein, a ZZKD Type V Cas protein, a ZXPB Type V Cas protein, a ZPPX Type V Cas protein, a ZXHQ Type V Cas protein, a ZQKH Type V Cas protein, a ZRGM Type V Cas protein, a ZTAE Type V Cas protein, a ZSQQ Type V Cas protein, a ZSYN Type V Cas protein, a ZRBH Type V Cas protein, a ZWPU Type V Cas protein, a ZZQE Type V Cas protein, and a ZRXE Type V Cas protein. Type V Cas proteins of the disclosure can be in the form of fusion proteins. Unless required otherwise by context, disclosures relating to Type V Cas proteins encompass Type V Cas proteins which are not fusion proteins and Type V Cas proteins which are in the form of fusion proteins (e.g., Type V Cas protein comprising one or more nuclear localization signals and / or one or more tags).

[0063] In some embodiments, a Type V Cas protein of the disclosure comprises an amino acid sequence having at least 50% {e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%) sequence identity to a WED-1 domain, REC1 domain, REC2 domain, WED-II domain, PI domain, WED-HI domain, RuvC-l domain, BH domain, RuvC-ll domain, NUC domain, or RuvC-lll domain of a ZWGD Type V Cas protein, a ZJHK Type V Cas protein, a ZIKV Type V Cas protein, a ZZFT Type V Cas protein, a YYAN Type V Cas protein, a ZZGY Type V Cas protein, a ZKBG Type V Cas protein, a ZZKD Type V Cas protein, a ZXPB Type V Cas protein, a ZPPX Type V Cas protein, a ZXHQ Type V Cas protein, a ZQKH Type V Cas protein, a ZRGM Type V Cas protein, a ZTAE Type V Cas protein, a ZSQQ Type V Cas protein, a ZSYN Type V Cas protein, a ZRBH Type V Cas protein, a ZWPU Type V Cas protein, a ZZQE Type V Cas protein, or a ZRXE Type V Cas protein.

[0064] In some embodiments, a Type V Cas protein of the disclosure is a chimeric Type V Cas protein, for example, comprising one or more domains from a ZWGD Type V Cas protein and / or a ZJHK Type V Cas protein and / or a ZIKV Type V Cas protein and / or a ZZFT Type V Cas protein and / or a YYAN Type V Cas protein and / or a ZZGY Type V Cas protein and / or a ZKBG Type V Cas protein and / or a ZZKD Type V Cas protein and / or a ZXPB Type V Cas protein and / or a ZPPX Type V Cas protein and / or a ZXHQ Type V Cas protein and / or a ZQKH Type V Cas protein and / or a ZRGM Type V Cas protein and / or a ZTAE Type V Cas protein and / or a ZSQQ Type V Cas protein and / or a ZSYN Type V Cas protein and / or a ZRBH Type V Cas protein and / or a ZWPU Type V Cas protein and / or a ZZQE Type V Cas protein and / or a ZRXE Type V Cas protein, and one or more domains from a different Type V Cas protein such as AsCas12a.

[0065] Exemplary features of Type V Cas proteins of the disclosure are described in Section 6.2.

[0066] In further aspects, the disclosure provides guide (gRNA) molecules and combinations of guide RNA molecules, for example combinations of two or more gRNAs. Exemplary features of the gRNAs and combinations of gRNAs of the disclosure are further described in Section 6.3.

[0067] In further aspects, the disclosure provides systems comprising a Type V Cas protein of the disclosure and one or more gRNAs. Exemplary features of systems are described in Section 6.4.

[0068] In further aspects, the disclosure provides nucleic acids and pluralities of nucleic acids encoding a Type V Cas protein of the disclosure and, optionally, a gRNA , and provides nucleic acids encoding a gRNA, of the disclosure and, optionally, a Type V Cas protein. Exemplary features of nucleic and pluralities of nucleic acids of the disclosure are described in Section 6.5.

[0069] In further aspects, the disclosure provides particles comprising the Type V Cas proteins, gRNAs, nucleic acids, and systems of the disclosure. Exemplary features of particles of the disclosure are described in Section 6.6.

[0070] In another aspect, the disclosure provides cells and populations of cells containing or contacted with a Type V Cas protein, gRNA, nucleic acid, plurality of nucleic acids, system, or particle of the disclosure. Exemplary features of such cells and cell populations are described in Section 6.6.

[0071] In another aspect, the disclosure provides pharmaceutical compositions comprising a Type V Cas protein, gRNA, nucleic acid, plurality of nucleic acids, system, particle, cell, or population of cells together with one or more excipients. Exemplary features of pharmaceutical compositions are described in Section 6.7.

[0072] In another aspect, the disclosure provides methods of altering cells (e.g., editing the genome of a cell) using the Type V Cas proteins, gRNAs, nucleic acids, systems, particles, and pharmaceutical compositions of the disclosure. Features of exemplary methods of altering cells are described in Section 6.8. 6.1. Definitions

[0073] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs. The following definitions are provided for the full understanding of terms used in this specification.

[0074] As used in the specification and claims, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “an agent” includes a plurality of agents, including mixtures thereof.

[0075] Unless indicated otherwise, an “or” conjunction is intended to be used in its correct sense as a Boolean logical operator, encompassing both the selection of features in the alternative (A or B, where the selection of A is mutually exclusive from B) and the selection of features in conjunction (A or B, where both A and B are selected). In some places in the text, the term “and / or” is used for the same purpose, which shall not be construed to imply that “or” is used with reference to mutually exclusive alternatives.

[0076] AsCas12a refers to a Cas12a protein havin the following amino acid sequence: MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYADQCLQLVQLD WENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLK QLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVP SLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKND ETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHI FISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQ KTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEME PSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKA LSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPE KEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLY HISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYR PKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKD RRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTI QQFDYQKKLDNREKERVAARQAWSWGTIKDLKQGYLSQVIHEIVDLMIHYQAVWLENLNFGFKSKRTG IAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPL TGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNET QFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVAL IRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDL KLQNGISNQDWLAYIQELRN (SEQ ID N0:121)

[0077] A Type V Cas protein refers to a wild-type or engineered Type V Cas protein. Engineered Type V Cas proteins can also be referred to as Type V Cas variants. For the avoidance of doubt, any disclosure pertaining to a “Type V Cas” or “Type V Cas protein” pertains to wild-type Type V Cas proteins and Type V Cas variants, unless the context dictates otherwise. A Type V Cas protein can have nuclease activity or be catalytically inactive (e.g., as in a dCas).

[0078] As used herein, the percentage identity between two nucleotide sequences or between two amino acid sequences is calculated by multiplying the number of matches between a pair of aligned sequences by 100, and dividing by the length of the aligned region. Identity scoring only counts perfect matches and does not consider the degree of similarity of amino acids to one another, nor does it consider substitutions or deletions as matches. For calculation of the percent sequence identity (% sequence identity), two sequences are aligned using the EMBOSS Needle Pairwise Sequence Alignment software tool based on the Needleman and Wunsch algorithm (available at www.ebi.ac.uk / jdispatcher / psa / emboss_needle) with the following parameters: Matrix: BLOSUM62 (for protein sequences) or DNAfull (for DNA sequences); Gap Open: 10; Gap Extend: 0.5; End Gap Penalty: false; End Gap Open: 10; and End Gap Extend: 0.5.

[0079] Guide RNA molecule (gRNA) refers to an RNA capable of forming a complex with a Type V Cas protein and which can direct the Type V Cas protein to a target DNA. gRNAs typically comprise a spacer of 15 to 30 nucleotides in length. gRNAs of the disclosure typically comprise a crRNA scaffold region at the 5’ end of the molecule and a spacer at the 3’ end of the molecule. Various non-limiting examples of crRNA scaffolds are described in Section 6.3.

[0080] An gRNA can in some embodiments comprise no uracil base at the 3’ end of the gRNA sequence. Alternatively, a gRNA can comprise one or more uracil bases at the 3’ end of the sgRNA sequence. For example, a gRNA can comprise 1 uracil (U) at the 3’ end of the gRNA sequence, 2 uracil (UU) at the 3’ end of the gRNA sequence, 3 uracil (UUU) at the 3’ end of the gRNA sequence, 4 uracil (UUUU) at the 3’ end of the gRNA sequence, 5 uracil (UUUUU) at the 3’ end of the gRNA sequence, 6 uracil (UUUUUU) at the 3’ end of the gRNA sequence, 7 uracil (UUUUUUU) at the 3’ end of the gRNA sequence, or 8 uracil (UUUUUUUU) at the 3’ end of the gRNA sequence. Different length stretches of uracil can be appended at the 3’ end of a gRNA as terminators.

[0081] A gRNA can in some embodiments comprise a 5’ guanine (G) at it’s 5’ end. A 5’-G can promote efficient transcription from a U6 promoter.

[0082] Peptide, protein, and polypeptide are used interchangeably to refer to a natural or synthetic molecule comprising two or more amino acids linked by the carboxyl group of one amino acid to the alpha amino group of another. The amino acids may be natural or synthetic, and can contain chemical modifications such as disulfide bridges, substitution of radioisotopes, phosphorylation, substrate chelation (e.g., chelation of iron or copper atoms), glycosylation, acetylation, formylation, amidation, biotinylation, -13- and a wide range of other modifications. A polypeptide may be attached to other molecules, for instance molecules required for function. Examples of molecules which may be attached to a polypeptide include, without limitation, cofactors, polynucleotides, lipids, metal ions, phosphate, etc. Non-limiting examples of polypeptides include peptide fragments, denatured / unstructured polypeptides, polypeptides having quaternary or aggregated structures, etc. There is expressly no requirement that a polypeptide must contain an intended function; a polypeptide can be functional, non-functional, function for unexpected / unintended purposes, or have unknown function. A polypeptide is comprised of approximately twenty, standard naturally occurring amino acids, although natural and synthetic amino acids which are not members of the standard twenty amino acids may also be used. The standard twenty amino acids include alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamine (Gin, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine, (His, H), isoleucine (lie, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Vai, V). The terms “polypeptide sequence” or “amino acid sequence” are an alphabetical representation of a polypeptide molecule.

[0083] Polynucleotide and oligonucleotide are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: a gene or gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, primers and gRNAs. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine (T) when the polynucleotide is RNA. Thus, the term “nucleotide sequence” is the alphabetical representation of a polynucleotide molecule. The letters used in polynucleotide sequences described herein correspond to IUPAC notation. For example, the letter “N” in a nucleotide sequence represents a nucleotide which can be A, T, C, or G in a DNA sequence, or A, U, C, or G in a RNA sequence; the letter “R” in a nucleotide sequence represents a nucleotide which can be A or G; the letter “V” in a nucleotide sequence represents a nucleotide which can be A, C, or G; and the letter “Y” in a nucleotide sequence represents a nucleotide which can be C or T.

[0084] Protospacer adjacent motif (PAM) refers to a DNA sequence upstream (e.g., immediately upstream) of a target sequence on the non-target strand recognized by a Type V Cas protein. A PAM sequence is located 5’ of the target sequence on the non-target strand.

[0085] Spacer refers to a region of a gRNA molecule which is partially or fully complementary to a target sequence found in the + or - strand of genomic DNA. When complexed with a Type V Cas protein, the gRNA directs the Type V Cas to the target sequence in the genomic DNA. A spacer of a Type V Cas -14- gRNA is typically 15 to 30 nucleotides in length (e.g., 20-25 nucleotides). The nucleotide sequence of a spacer can be, but is not necessarily, fully complementary to the target sequence. For example, a spacer can contain one or more mismatches with a target sequence, e.g., the spacer can comprise one, two, or three mismatches with the target sequence. 6.2. Type V Cas Proteins 6.2.1. ZWGD Type V Cas Proteins

[0086] In one aspect, the disclosure provides ZWGD Type V Cas proteins. ZWGD Type V Cas proteins can be further classified as Type V-A Cas proteins. The ZWGD Type V Cas proteins typically comprise an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:1. In some embodiments, the ZWGD Type V Cas proteins comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:1. In some embodiments, a ZWGD Type V Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:1.

[0087] Exemplary ZWGD Type V Cas protein sequences and nucleotide sequences encoding exemplary ZWGD Type V Cas proteins are set forth in Table 1 A. TABLE1A ZWGD Type V Cas Sequences Name Sequence SEQ ID NO. Wildtype amino acid sequence (without N-terminal methionine) VSEKENTPTFNSLTNLYSVSKTLRFELRPQYSTLDHIKDDQIVDKGEELKNHYKTFKKILD QVFSRIINDSLDKTYLDQKYISTYQDLVFKHRDRLTDKDRAELKALKETLKKQIDKSLDHK DKKAIFSDPVNFLIDNESDFADLIGDNRPSIEAFNRQKGYLSGYLQNRANIFDHTTNETSV AFRIVEENLAIFLNNRLTLQHFFEKVADKDGLLKFLQETLSQLGFKLKLEDLLSLDYFNRT LSQPGIDQYNLLISGKALEDGKKMQGINEVLNQYLQQHQEEKLHKIKLKQLYKQILSESK TESFTLDFVEDNKGLAAMLLQFIDFVNKLIEEKMLLLDMIQGLKDSSVSSEFLSRLYLERK NIKRLSNFIYKDYGYIEQSLEENFLSTIEGKITKKALEEHRKQDAFTIHEILVALQKQQYEK DGALESADHLLLPGVVDFLYQNLDCKHSTLLEKVGSEKQPLLDLFNEKQLLEGQDAESH ASKYSDRPFNDHEIKVVKTALDFYKNLQSNFAIFQIPDENLKLDSEFYSEFDEFYQGLKNI IPVYNKSRNFLTKKPFSTEKTKLIFNNPQLLDGWSKSKESDCLGTIFIKDGKYYVGIINSAT NAKNTLFEPNNFANFDQKQYFEKMNLFFLSDLKRDFPKKYFSEKWHNQHPVPADLREK YDYYRIDEHKDERKNDLKYHHQLIAYYQDCLKKDTEWQIYQFKYKAPEEYSDVNEFLSE LTPNTYKMEFNKIPAEYIKKLVDDGKLYFFQIYSKDFSEFAKGKPNLHTLYLKAVFDQKNA EEFNYNYKISGSAEIFYRPASIETRVTHPKNQPIKNKNKNNPKAESVFQYDLCKDRRYMS DKFFLHLPIELNRIPLLANDSSVNSMVNQVVSSRNQNYFLGIDRGERHLIYLVLIDQNGRII KQQTLNQITSSYQEKANNQTVEVITDYHDLLNDKEKLRKKNLQEWQSVENIKELKAGYL SNVVNEIGKIIVEYQPVIMLENLNTGFKNSRIKIEKQVYQKFEKALIDKFNYFMRKDLDSSA IGGLYHALQLTKEYSKQYNGKQNGIIYYIPASYTSNIDPTTGFISAFIQTRYENVEKTKSLIE KFNDITYDAEESLFCFSADYKKFSPEAKLWQQTIWQIYTNGDRIYTFKNKEEWQSKNYIL VEEFKDLFAKYHIDYCRDLKAQILSQTDASFFKQFLFLLRLTLQMRNSRTTELNGTDADT KKRENDYIISPVKNQYGKFYDSRKDYVDWPENADANGAYNIARKGLIMLKHLKEGLPEK RICDISTEEWVQFVEELNK 1 Wildtype amino acid sequence (with N-terminal methionine) MVSEKENTPTFNSLTNLYSVSKTLRFELRPQYSTLDHIKDDQIVDKGEELKNHYKTFKKIL DQVFSRIINDSLDKTYLDQKYISTYQDLVFKHRDRLTDKDRAELKALKETLKKQIDKSLDH KDKKAIFSDPVNFLIDNESDFADLIGDNRPSIEAFNRQKGYLSGYLQNRANIFDHTTNETS VAFRIVEENLAIFLNNRLTLQHFFEKVADKDGLLKFLQETLSQLGFKLKLEDLLSLDYFNR TLSQPGIDQYNLLISGKALEDGKKMQGINEVLNQYLQQHQEEKLHKIKLKQLYKQILSES KTESFTLDFVEDNKGLAAMLLQFIDFVNKLIEEKMLLLDMIQGLKDSSVSSEFLSRLYLER KNIKRLSNFIYKDYGYIEQSLEENFLSTIEGKITKKALEEHRKQDAFTIHEILVALQKQQYE KDGALESADHLLLPGVVDFLYQNLDCKHSTLLEKVGSEKQPLLDLFNEKQLLEGQDAES HASKYSDRPFNDHEIKVVKTALDFYKNLQSNFAIFQIPDENLKLDSEFYSEFDEFYQGLK NIIPVYNKSRNFLTKKPFSTEKTKLIFNNPQLLDGWSKSKESDCLGTIFIKDGKYYVGIINS ATNAKNTLFEPNNFANFDQKQYFEKMNLFFLSDLKRDFPKKYFSEKWHNQHPVPADLR EKYDYYRIDEHKDERKNDLKYHHQLIAYYQDCLKKDTEWQIYQFKYKAPEEYSDVNEFL SELTPNTYKMEFNKIPAEYIKKLVDDGKLYFFQIYSKDFSEFAKGKPNLHTLYLKAVFDQK NAEEFNYNYKISGSAEIFYRPASIETRVTHPKNQPIKNKNKNNPKAESVFQYDLCKDRRY MSDKFFLHLPIELNRIPLLANDSSVNSMVNQVVSSRNQNYFLGIDRGERHLIYLVLIDQNG 2 TABLE1A ZWGD Type V Cas Sequences Name Sequence SEQID NO. RIIKQQTLNQITSSYQEKANNQTVEVITDYHDLLNDKEKLRKKNLQEWQSVENIKELKAG YLSNVVNEIGKIIVEYQPVIMLENLNTGFKNSRIKIEKQVYQKFEKALIDKFNYFMRKDLDS SAIGGLYHALQLTKEYSKQYNGKQNGIIYYIPASYTSNIDPTTGFISAFIQTRYENVEKTKS LIEKFNDITYDAEESLFCFSADYKKFSPEAKLWQQTIWQIYTNGDRIYTFKNKEEWQSKN YILVEEFKDLFAKYHIDYCRDLKAQILSQTDASFFKQFLFLLRLTLQMRNSRTTELNGTDA DTKKRENDYIISPVKNQYGKFYDSRKDYVDWPENADANGAYNIARKGLIMLKHLKEGLP EKRICDISTEEWVQFVEELNK Expression construct (with N-terminal methionine, V5-tag and C-terminal NLS) aa sequence MGVSEKENTPTFNSLTNLYSVSKTLRFELRPQYSTLDHIKDDQIVDKGEELKNHYKTFK KILDQVFSRIINDSLDKTYLDQKYISTYQDLVFKHRDRLTDKDRAELKALKETLKKQIDKS LDHKDKKAIFSDPVNFLIDNESDFADLIGDNRPSIEAFNRQKGYLSGYLQNRANIFDHTT NETSVAFRIVEENLAIFLNNRLTLQHFFEKVADKDGLLKFLQETLSQLGFKLKLEDLLSLD YFNRTLSQPGIDQYNLLISGKALEDGKKMQGINEVLNQYLQQHQEEKLHKIKLKQLYKQI LSESKTESFTLDFVEDNKGLAAMLLQFIDFVNKLIEEKMLLLDMIQGLKDSSVSSEFLSRL YLERKNIKRLSNFIYKDYGYIEQSLEENFLSTIEGKITKKALEEHRKQDAFTIHEILVALQK QQYEKDGALESADHLLLPGVVDFLYQNLDCKHSTLLEKVGSEKQPLLDLFNEKQLLEG QDAESHASKYSDRPFNDHEIKVVKTALDFYKNLQSNFAIFQIPDENLKLDSEFYSEFDEF YQGLKNIIPVYNKSRNFLTKKPFSTEKTKLIFNNPQLLDGWSKSKESDCLGTIFIKDGKYY VGIINSATNAKNTLFEPNNFANFDQKQYFEKMNLFFLSDLKRDFPKKYFSEKWHNQHPV PADLREKYDYYRIDEHKDERKNDLKYHHQLIAYYQDCLKKDTEWQIYQFKYKAPEEYSD VNEFLSELTPNTYKMEFNKIPAEYIKKLVDDGKLYFFQIYSKDFSEFAKGKPNLHTLYLKA VFDQKNAEEFNYNYKISGSAEIFYRPASIETRVTHPKNQPIKNKNKNNPKAESVFQYDLC KDRRYMSDKFFLHLPIELNRIPLLANDSSVNSMVNQVVSSRNQNYFLGIDRGERHLIYLV LIDQNGRIIKQQTLNQITSSYQEKANNQTVEVITDYHDLLNDKEKLRKKNLQEWQSVENI KELKAGYLSNVVNEIGKIIVEYQPVIMLENLNTGFKNSRIKIEKQVYQKFEKALIDKFNYFM RKDLDSSAIGGLYHALQLTKEYSKQYNGKQNGIIYYIPASYTSNIDPTTGFISAFIQTRYE NVEKTKSLIEKFNDITYDAEESLFCFSADYKKFSPEAKLWQQTIWQIYTNGDRIYTFKNK EEWQSKNYILVEEFKDLFAKYHIDYCRDLKAQILSQTDASFFKQFLFLLRLTLQMRNSRT TELNGTDADTKKRENDYIISPVKNQYGKFYDSRKDYVDWPENADANGAYNIARKGLIML KHLKEGLPEKRICDISTEEWVQFVEELNKSRKRTADGSEFESPKKKRKVGSGKPIPNPL LGLDST 3 Wildtype coding sequence (with N-terminal methionine and stop codon) ATGGTGTCCGAAAAAGAAAATACACCAACTTTTAATAGTCTAACCAATCTCTATAGTG TTTCAAAGACTCTTAGATTTGAACTTAGGCCACAATATTCAACTCTAGATCACATTAA AGATGACCAAATTGTTGACAAAGGTGAAGAACTAAAAAACCACTACAAAACTTTCAA GAAAATTCTTGATCAGGTC Illi CAAGGATCATCAACGATAGCCTAGATAAAACCTA TCTTGATCAAAAATATATTTCCACCTACCAAGATCTTGTATTCAAGCATCGAGACCGA CTAACAGACAAAGACCGTGCAGAACTAAAGGCCTTAAAAGAAACACTCAAAAAGCA GATCGACAAAAGCCTCGATCATAAAGATAAAAAAGCTATCTTCAGTGATCCCGTAAA I I I I C I CAI CGACAAI GAAI CGGA Illi GCTGACTTAATTGGTGATAATCGTCCTAGT ATTGAAGCTTTCAACCGTCAAAAAGGTTATCTTTCCGGATATCTCCAAAATCGCGCA AATATCTTCGATCACACCACAAATGAAACTTCAGTCGCGTTTCGTATTGTCGAGGAA AACCTCGCTATC I I I I I AAA I AA I OGOU I CACAI IACAGCAI I I I I I CGAGAAAGTTG CAGATAAAGATGGGCTATTAAAATTTTTACAAGAGACACTTTCTCAGTTAGGTTTTAA GTTGAAACTCGAAGACCTTCTTTCCCTTGATTA Illi AATCGTACCCTATCTCAACCC GGCATCGATCAGTATAACCTCCTAATCTCTGGCAAGGCGCTAGAAGATGGAAAGAA AATGCAGGGAATTAATGAGGTCCTCAATCAATATCTCCAACAACATCAAGAAGAGAA GCTACATAAAATCAAACTCAAGCAACTCTATAAGCAGATCCTCTCAGAGTCAAAAAC TGAATCATTTACCCTTGA Illi GTGGAAGATAATAAAGGGCTTGCTGCCATGCTCCT ACAGTTTATCGA Illi GTAAACAAGCTGATTGAAGAGAAAATGCTTCTCCTTGATATG ATTCAGGGGCTAAAAGATAGCTCAGTTTCATCAGAATTTCTTTCACGACTCTATCTT GAACGCAAAAACATCAAGCGTCTTTCGAATTTTATCTATAAAGATTATGGCTATATTG AGCAATCCTTGGAAGAGAACTTTCTCTCGACAATTGAAGGCAAGATTACCAAGAAG GCACTCGAGGAACATCGCAAACAGGATGCTTTCACAATCCATGAAATCTTAGTTGC CCTACAAAAGCAACAATATGAAAAGGATGGAGCTCTAGAGTCCGCAGATCATC Illi ACTTCCTGGTGTTGTTGACTTCCTCTACCAGAATTTGGATTGCAAACACTCCACTCT ACTTGAAAAAGTCGGGTCAGAAAAACAGCCACTACTCGACCTCTTCAACGAAAAAC AATTATTGGAAGGTCAAGACGCAGAATCTCATGCTTCCAAATATTCTGATCGTCCAT TCAACGACCACGAAATAAAGGTTGTTAAAACTGCTTTGGATTTTTATAAAAATCTACA GAGTAATTTTGCGATCTTTCAAATCCCGGATGAAAACCTTAAACTAGATTCCGAATTT TATTCCGAGTTTGATGAA Illi ATCAAGGTCTCAAGAATATTATTCCAGTCTATAACA AGTCCAGAAATTTCCTCACTAAAAAACCATTCTCAACCGAAAAGACCAAGCTCATTT TTAACAACCCGCAACTACTTGACGGATGGAGTAAATCAAAAGAGTCAGATTGTTTAG GCACGATTTTTATTAAAGACGGCAAATATTATGTTGGCATTATTAATAGTGCTACGAA TGCTAAAAATACTTTATTTGAGCCTAACAA Illi GCAAACTTCGACCAAAAACAATAT TTTGAAAAGATGAACC I I I I C I I CO I I I CGGAC I I GAAGCGAGA Illi CCTAAGAAAT 4 TABLE1A ZWGD Type V Cas Sequences Name Sequence SEQID NO. AT I I I I CTGAAAAGTGGCATAATCAACACCCAGTTCCAGCCGATCTTCGTGAAAAGT ATGATTATTATCGAATCGACGAACATAAGGATGAGCGCAAAAATGATCTAAAATATC ATCATCAACTTATCGCCTATTATCAAGACTGTCTTAAAAAAGACACGGAATGGCAGA TTTATCAATTCAAATATAAGGCCCCTGAAGAATATTCAGATGTCAATGAATTCTTATC CGAGCTTACTCCAAATACCTACAAAATGGAGTTCAATAAAATCCCAGCTGAATATAT CAAAAAGCTTGTTGATGATGGAAAATTATATTTCTTCCAAATTTATTCCAAAGATTTTT CTGAGTTTGCAAAAGGTAAACCAAATCTCCATACTCTCTATCTAAAAGCGGTCTTTG ATCAGAAAAATGCGGAAGAGTTCAACTATAATTATAAAATTTCTGGTAGTGCCGAAA TCTTCTATCGTCCAGCCAGCATTGAAACTCGTGTCACTCATCCAAAAAATCAACCAA TCAAGAATAAGAATAAAAATAATCCAAAGGCTGAATCTGTCTTCCAGTATGATCTTTG TAAAGATCGTCGCTATATGTCAGATAAATTCTTTTTGCATCTTCCGATCGAATTAAAT CGTATTCCGTTACTCGCTAACGACTCCTCGGTAAATAGTATGGTCAATCAAGTCGTT AGTTCTCGTAATCAGAATTATTTCCTTGGTATTGACCGTGGCGAGAGGCATCTAATT TATCTAGTCCTGATCGATCAAAACGGTAGAATCATTAAACAGCAAACCTTAAATCAG ATCACTAGTTCATACCAAGAAAAAGCCAATAACCAAACGGTTGAAGTTATTACGGAT TATCATGATCTCTTGAATGACAAAGAAAAACTGCGAAAGAAGAATCTCCAAGAGTGG CAATCCGTCGAAAATATCAAGGAGTTAAAGGCTGGGTACCTAAGTAATGTGGTGAA TGAAATCGGTAAGATTATCGTTGAATATCAGCCAGTTATTATGCTGGAAAATCTTAAT ACTGGATTTAAAAACTCACGAATTAAAATTGAGAAACAGGTGTACCAGAAATTTGAG AAGGCGCTCATTGATAAGTTTAACTACTTTATGAGAAAAGATCTCGACTCTTCAGCT ATTGGTGGTCTCTATCACGCTTTGCAGTTGACTAAGGAATACTCTAAGCAGTACAAC GGCAAGCAGAATGGTATCATCTACTATATTCCTGCAAGCTACACTAGTAATATTGAT CCAACTACTGGTTTCATCTCGGCCTTTATACAGACTAGATACGAAAACGTCGAGAAA ACAAAATCCTTAATCGAAAAGTTTAATGATATCACTTATGATGCAGAAGAATCTCTCT TCTGCTTCTCCGCAGATTACAAGAAATTTAGTCCAGAGGCCAAGCTTTGGCAGCAG ACGATTTGGCAGATTTATACTAATGGCGATCGTATTTATACATTTAAGAACAAAGAAG AGTGGCAGAGCAAAAACTACATCCTCGTTGAGGAGTTCAAAGATCTCTTTGCTAAAT ATCACATCGATTATTGCAGGGACCTTAAGGCGCAGATTCTGTCACAAACTGACGCG AGCTTCTTCAAGCAGTTCCTCTTCTTGTTGCGACTAACCTTGCAGATGCGAAATAGT CGCACTACCGAATTAAATGGAACTGATGCTGATACTAAAAAACGTGAGAATGATTAT ATTATTTCTCCAGTTAAGAATCAGTATGGCAAGTTCTATGATTCCCGCAAGGATTAT GTGGACTGGCCAGAAAATGCAGATGCAAATGGCGCATACAATATTGCCAGAAAAGG TCTCATCATGCTAAAACACCTAAAAGAAGGTCTTCCCGAAAAACGTATCTGTGATAT ATCGACTGAAGAATGGGTACAGTTTGTCGAAGAACTAAATAAATAG Codon optimized coding sequence (no N-terminal methionine, no stop codon) GTGTCTGAAAAGGAAAACACCCCTACCTTCAACTCTCTGACCAACCTGTACAGCGTT TCTAAAACCCTGCGGTTCGAGCTGCGGCCTCAGTACAGCACCCTGGACCACATCAA GGACGATCAGATCGTGGACAAGGGAGAGGAGCTAAAGAACCACTACAAGACATTC AAAAAAATCCTGGACCAGGTGTTCTCTCGGATCATCAACGACTCTCTGGATAAAACT TACCTGGATCAGAAGTACATCTCCACCTACCAGGATCTGGTGTTCAAGCACAGAGA TAGACTGACAGATAAGGACAGAGCCGAACTGAAGGCCCTGAAGGAGACACTGAAG AAGCAGATCGACAAAAGCCTGGATCACAAAGACAAGAAGGCTATCTTCTCCGACCC TGTGAACTTCCTGATCGACAATGAGAGCGACTTCGCCGACCTGATTGGAGACAACC GGCCCAGCATCGAGGCCTTTAACCGCCAGAAGGGATATCTGTCCGGCTACCTGCA GAATAGAGCCAACATCTTCGATCATACAACCAACGAAACCAGCGTTGCTTTCAGAAT CGTGGAAGAGAACCTCGCCATCTTCCTCAACAACCGCCTGACCCTGCAGCATTTCT TCGAGAAAGTGGCCGACAAAGACGGACTGCTGAAGTTCCTGCAGGAGACACTGAG CCAGCTGGGCTTCAAGCTGAAGCTGGAGGATCTGCTGAGCCTGGATTACTTTAACC GGACACTGAGCCAGCCTGGCATCGACCAATACAACCTGCTGATCAGCGGAAAGGC CCTGGAAGATGGCAAGAAGATGCAGGGCATCAATGAAGTGCTGAACCAGTACCTG CAGCAGCACCAGGAGGAAAAGCTGCACAAAATCAAGCTGAAGCAGCTGTATAAGCA AATCCTGAGCGAAAGCAAGACAGAGAGCTTCACGCTGGACTTCGTGGAGGACAAC AAGGGCCTGGCCGCCATGCTGCTGCAGTTTATCGATTTCGTGAACAAGTTAATAGA AGAGAAGATGCTGCTGCTGGATATGATCCAGGGACTGAAAGACAGCAGTGTGTCCA GCGAGTTCTTGAGCCGGCTTTACCTGGAAAGAAAGAACATCAAGCGGCTGAGCAAC TTCATCTACAAGGACTATGGCTATATCGAGCAGTCCCTGGAAGAAAACTTCCTGAG CACCATCGAGGGCAAGATCACTAAGAAGGCCCTGGAAGAGCATAGAAAACAGGAC GCCTTTACCATTCACGAGATCCTGGTCGCACTGCAGAAACAACAGTACGAAAAGGA CGGCGCCCTAGAGAGCGCCGACCACCTGCTGCTTCCAGGCGTGGTGGATTTCCTC TACCAAAACCTGGACTGTAAGCACAGCACGCTGCTGGAAAAGGTGGGCAGCGAGA AGCAGCCCCTGCTGGATC Illi CAACGAAAAGCAGCTGCTTGAGGGCCAGGACGC CGAGTCCCACGCCTCTAAGTACAGCGATCGGCCTTTCAACGACCACGAGATCAAG GTGGTGAAAACCGCCCTGGACTTCTACAAGAACCTGCAATCTAACTTTGCTATCTTC CAGATCCCCGACGAAAACCTGAAGCTGGATAGCGAG Illi ACAGCGAGTTTGATGA GTTCTACCAGGGCCTGAAAAATATTATTCCTGTGTACAACAAAAGCCGGAACTTCCT 5 TABLE1A ZWGD Type V Cas Sequences Name Sequence SEQID NO. GACAAAAAAGCCGTTCAGCACCGAAAAGACCAAACTGATCTTCAACAACCCCCAGC TGCTCGATGGCTGGAGCAAGAGCAAGGAAAGCGACTGTCTGGGGACCATCTTCAT CAAAGACGGCAAGTACTATGTGGGAATCATCAACAGCGCCACCAACGCTAAGAATA CACTGTTCGAGCCTAACAACTTCGCCAATTTCGACCAAAAACAATACTTCGAGAAGA TGAACCTGTTCTTCCTGAGCGATCTGAAGCGAGACTTCCCCAAGAAGTATTTCTCC GAGAAGTGGCACAACCAGCACCCCGTGCCCGCTGACCTTAGAGAAAAGTACGACT ACTACCGGATCGACGAGCATAAGGATGAGAGAAAGAATGACCTGAAATACCACCAC CAGTTAATCGCCTACTACCAAGACTGCCTGAAAAAGGATACAGAGTGGCAGATCTA CCAGTTCAAGTACAAGGCCCCTGAGGAGTACAGCGACGTGAACGAGTTCCTGAGT GAACTGACCCCTAATACCTACAAGATGGAGTTCAACAAGATTCCTGCCGAGTACATT AAGAAGCTGGTGGATGACGGCAAGCTGTACTTTTTTCAGATATACTCCAAAGACTTT AGCGAATTTGCCAAGGGCAAGCCAAACCTGCACACCCTCTACCTGAAGGCCGTGTT CGACCAGAAGAACGCCGAGGAGTTCAACTACAACTATAAAATATCTGGATCTGCTG AAATCI I I IACAGACCTGCTTCTATCGAGACAAGAGTGACCCACCCTAAGAATCAGC CTATCAAGAACAAGAACAAGAACAATCCTAAGGCTGAAAGCGTGTTCCAGTACGAC CTGTGCAAGGACCGGCGGTACATGTCCGACAAGTTCTTCCTGCACCTTCCCATCGA ACTTAACAGAATCCCTCTGCTGGCTAACGATTCCTCCGTGAATAGCATGGTCAACCA GGTGGTGAGCAGCAGAAACCAGAACTACTTCCTGGGCATCGATAGAGGCGAGAGA CACCTGATCTACCTGGTGCTGATCGACCAGAACGGTAGAATCATCAAGCAACAGAC CCTGAATCAGATTACAAGCAGCTACCAAGAAAAGGCCAACAACCAGACAGTGGAGG TGATCACAGACTACCACGACCTGCTGAACGACAAGGAAAAGCTCAGAAAGAAGAAT CTTCAGGAGTGGCAGTCCGTGGAGAATATCAAAGAGCTGAAGGCCGGCTACCTGA GCAACGTGGTCAACGAGATCGGCAAGATCATCGTGGAGTACCAGCCTGTGATCAT GCTGGAAAACCTCAACACCGGATTTAAAAACTCAAGAATCAAGATTGAGAAGCAGG TGTACCAGAAGTTCGAGAAGGCCTTAATCGATAAGTTCAATTACTTCATGCGGAAGG ATCTGGACTCTAGCGCCATCGGCGGCCTGTACCACGCCCTGCAGCTGACCAAAGA GTATAGCAAGCAGTACAACGGCAAGCAGAACGGCATCATCTACTACATCCCAGCTT CTTACACCTCTAATATCGACCCCACCACCGGCTTTATTAGCGCCTTCATCCAGACCA GATACGAGAACGTGGAAAAGACCAAGTCTCTGATCGAGAAATTTAATGACATCACCT ACGACGCCGAAGAGTCGCTGTTCTGCTTCAGCGCCGATTACAAGAAA Illi CACCT GAAGCTAAGCTGTGGCAGCAAACCATCTGGCAGATCTATACCAACGGCGACAGAAT CTACACCTTCAAGAACAAGGAAGAGTGGCAAAGCAAGAACTACATTCTGGTGGAGG AGTTTAAGGACCTGTTCGCCAAATACCACATCGACTATTGCAGGGACCTGAAAGCC CAGATCCTGAGCCAGACCGACGCATCTTTTTTCAAGCAGTTTCTCTTCCTGCTGAGA CTGACACTGCAAATGAGAAATAGTCGTACCACAGAGCTGAACGGCACCGACGCCG ACACCAAGAAAAGAGAGAATGACTACATCATCTCTCCAGTGAAAAATCAGTACGGC AAATTCTATGATTCCCGCAAGGACTACGTGGACTGGCCTGAGAACGCCGACGCCAA TGGCGCCTACAACATCGCCAGAAAGGGCCTGATCATGCTGAAGCACCTGAAGGAA GGACTGCCTGAGAAGAGGATCTGCGACATCAGCACAGAAGAATGGGTTCAGTTTGT GGAAGAACTGAACAAG Expression construct (with N-terminal methionine and stop codon, includes V5-tag and C-terminal NLS) ATGggcGTGTCTGAAAAGGAAAACACCCCTACCTTCAACTCTCTGACCAACCTGTAC AGCGTTTCTAAAACCCTGCGGTTCGAGCTGCGGCCTCAGTACAGCACCCTGGACC ACATCAAGGACGATCAGATCGTGGACAAGGGAGAGGAGCTAAAGAACCACTACAA GACATTCAAAAAAATCCTGGACCAGGTGTTCTCTCGGATCATCAACGACTCTCTGGA TAAAACTTACCTGGATCAGAAGTACATCTCCACCTACCAGGATCTGGTGTTCAAGCA CAGAGATAGACTGACAGATAAGGACAGAGCCGAACTGAAGGCCCTGAAGGAGACA CTGAAGAAGCAGATCGACAAAAGCCTGGATCACAAAGACAAGAAGGCTATCTTCTC CGACCCTGTGAACTTCCTGATCGACAATGAGAGCGACTTCGCCGACCTGATTGGAG ACAACCGGCCCAGCATCGAGGCCTTTAACCGCCAGAAGGGATATCTGTCCGGCTA CCTGCAGAATAGAGCCAACATCTTCGATCATACAACCAACGAAACCAGCGTTGCTTT CAGAATCGTGGAAGAGAACCTCGCCATCTTCCTCAACAACCGCCTGACCCTGCAGC ATTTCTTCGAGAAAGTGGCCGACAAAGACGGACTGCTGAAGTTCCTGCAGGAGACA CTGAGCCAGCTGGGCTTCAAGCTGAAGCTGGAGGATCTGCTGAGCCTGGATTACTT TAACCGGACACTGAGCCAGCCTGGCATCGACCAATACAACCTGCTGATCAGCGGA AAGGCCCTGGAAGATGGCAAGAAGATGCAGGGCATCAATGAAGTGCTGAACCAGT ACCTGCAGCAGCACCAGGAGGAAAAGCTGCACAAAATCAAGCTGAAGCAGCTGTAT AAGCAAATCCTGAGCGAAAGCAAGACAGAGAGCTTCACGCTGGACTTCGTGGAGG ACAACAAGGGCCTGGCCGCCATGCTGCTGCAGTTTATCGATTTCGTGAACAAGTTA ATAGAAGAGAAGATGCTGCTGCTGGATATGATCCAGGGACTGAAAGACAGCAGTGT GTCCAGCGAGTTCTTGAGCCGGCTTTACCTGGAAAGAAAGAACATCAAGCGGCTGA GCAACTTCATCTACAAGGACTATGGCTATATCGAGCAGTCCCTGGAAGAAAACTTC CTGAGCACCATCGAGGGCAAGATCACTAAGAAGGCCCTGGAAGAGCATAGAAAAC AGGACGCCTTTACCATTCACGAGATCCTGGTCGCACTGCAGAAACAACAGTACGAA AAGGACGGCGCCCTAGAGAGCGCCGACCACCTGCTGCTTCCAGGCGTGGTGGATT 6 TABLE1A ZWGD Type V Cas Sequences Name Sequence SEQ ID NO. TCCTCTACCAAAACCTGGACTGTAAGCACAGCACGCTGCTGGAAAAGGTGGGCAG CGAGAAGCAGCCCCTGCTGGATC Illi CAACGAAAAGCAGCTGCTTGAGGGCCAG GACGCCGAGTCCCACGCCTCTAAGTACAGCGATCGGCCTTTCAACGACCACGAGA TCAAGGTGGTGAAAACCGCCCTGGACTTCTACAAGAACCTGCAATCTAACTTTGCTA TCTTCCAGATCCCCGACGAAAACCTGAAGCTGGATAGCGAG Illi ACAGCGAGTTT GATGAGTTCTACCAGGGCCTGAAAAATATTATTCCTGTGTACAACAAAAGCCGGAAC TTCCTGACAAAAAAGCCGTTCAGCACCGAAAAGACCAAACTGATCTTCAACAACCC CCAGCTGCTCGATGGCTGGAGCAAGAGCAAGGAAAGCGACTGTCTGGGGACCATC TTCATCAAAGACGGCAAGTACTATGTGGGAATCATCAACAGCGCCACCAACGCTAA GAATACACTGTTCGAGCCTAACAACTTCGCCAATTTCGACCAAAAACAATACTTCGA GAAGATGAACCTGTTCTTCCTGAGCGATCTGAAGCGAGACTTCCCCAAGAAGTATT TCTCCGAGAAGTGGCACAACCAGCACCCCGTGCCCGCTGACCTTAGAGAAAAGTA CGACTACTACCGGATCGACGAGCATAAGGATGAGAGAAAGAATGACCTGAAATACC ACCACCAGTTAATCGCCTACTACCAAGACTGCCTGAAAAAGGATACAGAGTGGCAG ATCTACCAGTTCAAGTACAAGGCCCCTGAGGAGTACAGCGACGTGAACGAGTTCCT GAGTGAACTGACCCCTAATACCTACAAGATGGAGTTCAACAAGATTCCTGCCGAGT ACATTAAGAAGCTGGTGGATGACGGCAAGCTGTACTTTTTTCAGATATACTCCAAAG ACTTTAGCGAATTTGCCAAGGGCAAGCCAAACCTGCACACCCTCTACCTGAAGGCC GTGTTCGACCAGAAGAACGCCGAGGAGTTCAACTACAACTATAAAATATCTGGATCT GCTGAAATCI I I IACAGACCTGCTTCTATCGAGACAAGAGTGACCCACCCTAAGAAT CAGCCTATCAAGAACAAGAACAAGAACAATCCTAAGGCTGAAAGCGTGTTCCAGTA CGACCTGTGCAAGGACCGGCGGTACATGTCCGACAAGTTCTTCCTGCACCTTCCCA TCGAACTTAACAGAATCCCTCTGCTGGCTAACGATTCCTCCGTGAATAGCATGGTCA ACCAGGTGGTGAGCAGCAGAAACCAGAACTACTTCCTGGGCATCGATAGAGGCGA GAGACACCTGATCTACCTGGTGCTGATCGACCAGAACGGTAGAATCATCAAGCAAC AGACCCTGAATCAGATTACAAGCAGCTACCAAGAAAAGGCCAACAACCAGACAGTG GAGGTGATCACAGACTACCACGACCTGCTGAACGACAAGGAAAAGCTCAGAAAGAA GAATCTTCAGGAGTGGCAGTCCGTGGAGAATATCAAAGAGCTGAAGGCCGGCTAC CTGAGCAACGTGGTCAACGAGATCGGCAAGATCATCGTGGAGTACCAGCCTGTGA TCATGCTGGAAAACCTCAACACCGGATTTAAAAACTCAAGAATCAAGATTGAGAAGC AGGTGTACCAGAAGTTCGAGAAGGCCTTAATCGATAAGTTCAATTACTTCATGCGGA AGGATCTGGACTCTAGCGCCATCGGCGGCCTGTACCACGCCCTGCAGCTGACCAA AGAGTATAGCAAGCAGTACAACGGCAAGCAGAACGGCATCATCTACTACATCCCAG CTTCTTACACCTCTAATATCGACCCCACCACCGGCTTTATTAGCGCCTTCATCCAGA CCAGATACGAGAACGTGGAAAAGACCAAGTCTCTGATCGAGAAATTTAATGACATC ACCTACGACGCCGAAGAGTCGCTGTTCTGCTTCAGCGCCGATTACAAGAAAI I I IC ACCTGAAGCTAAGCTGTGGCAGCAAACCATCTGGCAGATCTATACCAACGGCGACA GAATCTACACCTTCAAGAACAAGGAAGAGTGGCAAAGCAAGAACTACATTCTGGTG GAGGAGTTTAAGGACCTGTTCGCCAAATACCACATCGACTATTGCAGGGACCTGAA AGCCCAGATCCTGAGCCAGACCGACGCATCTTTTTTCAAGCAGTTTCTCTTCCTGCT GAGACTGACACTGCAAATGAGAAATAGTCGTACCACAGAGCTGAACGGCACCGAC GCCGACACCAAGAAAAGAGAGAATGACTACATCATCTCTCCAGTGAAAAATCAGTA CGGCAAATTCTATGATTCCCGCAAGGACTACGTGGACTGGCCTGAGAACGCCGAC GCCAATGGCGCCTACAACATCGCCAGAAAGGGCCTGATCATGCTGAAGCACCTGA AGGAAGGACTGCCTGAGAAGAGGATCTGCGACATCAGCACAGAAGAATGGGTTCA GTTTGTGGAAGAACTGAACAAGtctagaAAGCGGACAGCAGACGGCTCCGAATTTGAA AGCCCTAAGAAAAAGAGAAAGGTGggatccGGCAAACCTATCCCCAATCCCCTGCTG GGCCTGGACAGCACCTGA

[0088] In some embodiments a ZWGD Type V Cas protein comprises an amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. In some embodiments, a ZWGD Type V Cas protein has nickase activity, for example resulting from one or more amino acid substitutions relative to the sequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D891 substitution, wherein the position of the D891 substitution is defined with respect to the amino acid numbering of SEQ ID NO:2 (corresponding to amino acid 908 of SEQ ID NO:121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise an E990 substitution, wherein the position of the E990 substitution is defined -19- with respect to the amino acid numbering of SEQ ID NO:2 (corresponding to amino acid 993 of SEQ ID NO:121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a R1200 substitution, wherein the position of the R1200 substitution is defined with respect to the amino acid numbering of SEQ ID NO:2 (corresponding to amino acid 1226 of SEQ ID NO:121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D1248 substitution, wherein the position of the D1248 substitution is defined with respect to the amino acid numbering of SEQ ID NO:2 (corresponding to amino acid 1263 of SEQ ID NO:121). In some embodiments, a ZWGD Type V Cas protein is catalytically inactive, for example due to a R1200 substitution in combination with a D891 substitution, a E990 substitution, and / or D1248 substitution. 6.2.2. ZJHK Type V Cas Proteins

[0089] In one aspect, the disclosure provides ZJHK Type V Cas proteins. ZJHK Type V Cas proteins can be further classified as Type V-A Cas proteins. The ZJHK Type V Cas proteins typically comprise an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:7. In some embodiments, the ZJHK Type V Cas proteins comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:7. In some embodiments, a ZJHK Type V Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:7.

[0090] Exemplary ZJHK Type V Cas protein sequences and nucleotide sequences encoding exemplary ZJHK Type V Cas proteins are set forth in Table 1B. TABLE1B ZJHK Type V Cas Sequences Name Sequence SEQ ID NO. Wildtype amino acid sequence (without N-terminal methionine) KSIYENFIGLESKNLTLRFALNPEAKTQENLKLYWDKLRDEERDRAYPIVKKILDKE YQQLISEGLKLLENQNVLDWTELAEYIRTSDLSKKKKEDKRLRKLIAQNLKAHPLV DKLKVKNAFGKNGYLETLPLGKEEKEAVKVFAGFGGFFNNYNKNRENYFSTEEK STAIANRIVNENFSKHFSNVEIVTKIQKEVPELIQIVEAQFKGYDTIFTVNGYNTALS QAGIDTYNEMVAIWNKEANLYAQKAGKLPDGHPLKKKRNYLLSALFKQIGSEKEH LIQIDRFDGDEEVIEALTGVKKMLQEADVFEKLNMLVEDMENWDYSKIYLSAQSLS NVSVFLNNLYEDERENSWNYLDNVLREKWQIELQGKKKGTDLEEAIRKKKQSFY SIEELQEAVNAIEETDKCYNVSKWLLGAMKSERVIEEKKKDVEDFCTQWKNERNS LKETDITALKEYLEQWIFLARYCKSFYANGIEKKEKDEAFYHILEDVLYVLDEVIYFY NKVRNYVTKKPYSLEKMHLKFGHNELANGWSVNKEENYGTAILRRNGKYYLAITN SLNKKMSIPTQLESTGNNYEKMVLNVFPNVFRMIPKCTTGRNDVKSCFERKEPNE YFFIDTPEFVNPFKVTREEYELNKITYDGVKKWQSDYSKNTQDEKGYKEAVTKWI QFCMRFLQSYKSTAIYDYSTLQQPEKYETVDSFYHDVEKILYECHFEYVPANKIEQ LEEEGRIFLFQIYNKDFSENRRPDSKKNLHTLYWEALFSEENRKAKVIQLNGKAEI FRREKSIEHPIVHKAGEVLVNKRTKDGEPIPDDIYKDLSNYFNGRNVTSEKEEYKE CLDKVYTSTKKYDITKDKRFTETKYEFHVPITLNYQADGVKYLNQKILHVLRDNPD VNIIGLDRGERNLISYVVLNREGKIVNNQQGSFNIVGKMDYQKKLYQKEKNRDKE RKTWKNIETIKDLKEGYISQVVHELTDMAIRNNAIIVMEDLNFGFKRGRTKVERQV YQKFELALLKKLHYLVTDKTEGEAMLKPGGVLQGYQLAREVKTLKEIGKQCGCVF YVPPGYTSKIDPTTGFVDVFNMSGVTNREKKKAFFEKFDNMFYDEKRDMFGFSF NYEKFTTYQSSYRNDWTVYSNGSKYVWNSLKRTDELIDVTKELKLLFEKYAIDYR NEALFEQIMSQDTDKNNADFWNKLFWYFRVLLRLRNSSDELDQIVSPVLNQNGE FFETPKKITEKSYLSDYPMDADTNGAYHIALKGLYLIQEKIADESVDLDNKLPKDFY KISNAEWFMFRQKEK 7 Wildtype amino acid sequence (with N-terminal methionine) MKSIYENFIGLESKNLTLRFALNPEAKTQENLKLYWDKLRDEERDRAYPIVKKILDK EYQQLISEGLKLLENQNVLDWTELAEYIRTSDLSKKKKEDKRLRKLIAQNLKAHPL VDKLKVKNAFGKNGYLETLPLGKEEKEAVKVFAGFGGFFNNYNKNRENYFSTEE KSTAIANRIVNENFSKHFSNVEIVTKIQKEVPELIQIVEAQFKGYDTIFTVNGYNTAL SQAGIDTYNEMVAIWNKEANLYAQKAGKLPDGHPLKKKRNYLLSALFKQIGSEKE HLIQIDRFDGDEEVIEALTGVKKMLQEADVFEKLNMLVEDMENWDYSKIYLSAQSL 8 TABLE1B ZJHK Type V Cas Sequences Name Sequence SEQ ID NO. SNVSVFLNNLYEDERENSWNYLDNVLREKWQIELQGKKKGTDLEEAIRKKKQSF YSIEELQEAVNAIEETDKCYNVSKWLLGAMKSERVIEEKKKDVEDFCTQWKNERN SLKETDITALKEYLEQWIFLARYCKSFYANGIEKKEKDEAFYHILEDVLYVLDEVIYF YNKVRNYVTKKPYSLEKMHLKFGHNELANGWSVNKEENYGTAILRRNGKYYLAIT NSLNKKMSIPTQLESTGNNYEKMVLNVFPNVFRMIPKCTTGRNDVKSCFERKEPN EYFFIDTPEFVNPFKVTREEYELNKITYDGVKKWQSDYSKNTQDEKGYKEAVTKW IQFCMRFLQSYKSTAIYDYSTLQQPEKYETVDSFYHDVEKILYECHFEYVPANKIE QLEEEGRIFLFQIYNKDFSENRRPDSKKNLHTLYWEALFSEENRKAKVIQLNGKAE IFRREKSIEHPIVHKAGEVLVNKRTKDGEPIPDDIYKDLSNYFNGRNVTSEKEEYKE CLDKVYTSTKKYDITKDKRFTETKYEFHVPITLNYQADGVKYLNQKILHVLRDNPD VNIIGLDRGERNLISYVVLNREGKIVNNQQGSFNIVGKMDYQKKLYQKEKNRDKE RKTWKNIETIKDLKEGYISQVVHELTDMAIRNNAIIVMEDLNFGFKRGRTKVERQV YQKFELALLKKLHYLVTDKTEGEAMLKPGGVLQGYQLAREVKTLKEIGKQCGCVF YVPPGYTSKIDPTTGFVDVFNMSGVTNREKKKAFFEKFDNMFYDEKRDMFGFSF NYEKFTTYQSSYRNDWTVYSNGSKYVWNSLKRTDELIDVTKELKLLFEKYAIDYR NEALFEQIMSQDTDKNNADFWNKLFWYFRVLLRLRNSSDELDQIVSPVLNQNGE FFETPKKITEKSYLSDYPMDADTNGAYHIALKGLYLIQEKIADESVDLDNKLPKDFY KISNAEWFMFRQKEK Expression construct (with N-terminal methionine, V5-tag and C-terminal NLS) aa sequence MGKSIYENFIGLESKNLTLRFALNPEAKTQENLKLYWDKLRDEERDRAYPIVKKILD KEYQQLISEGLKLLENQNVLDWTELAEYIRTSDLSKKKKEDKRLRKLIAQNLKAHP LVDKLKVKNAFGKNGYLETLPLGKEEKEAVKVFAGFGGFFNNYNKNRENYFSTE EKSTAIANRIVNENFSKHFSNVEIVTKIQKEVPELIQIVEAQFKGYDTIFTVNGYNTA LSQAGIDTYNEMVAIWNKEANLYAQKAGKLPDGHPLKKKRNYLLSALFKQIGSEK EHLIQIDRFDGDEEVIEALTGVKKMLQEADVFEKLNMLVEDMENWDYSKIYLSAQ SLSNVSVFLNNLYEDERENSWNYLDNVLREKWQIELQGKKKGTDLEEAIRKKKQS FYSIEELQEAVNAIEETDKCYNVSKWLLGAMKSERVIEEKKKDVEDFCTQWKNER NSLKETDITALKEYLEQWIFLARYCKSFYANGIEKKEKDEAFYHILEDVLYVLDEVIY FYNKVRNYVTKKPYSLEKMHLKFGHNELANGWSVNKEENYGTAILRRNGKYYLAI TNSLNKKMSIPTQLESTGNNYEKMVLNVFPNVFRMIPKCTTGRNDVKSCFERKEP NEYFFIDTPEFVNPFKVTREEYELNKITYDGVKKWQSDYSKNTQDEKGYKEAVTK WIQFCMRFLQSYKSTAIYDYSTLQQPEKYETVDSFYHDVEKILYECHFEYVPANKI EQLEEEGRIFLFQIYNKDFSENRRPDSKKNLHTLYWEALFSEENRKAKVIQLNGKA EIFRREKSIEHPIVHKAGEVLVNKRTKDGEPIPDDIYKDLSNYFNGRNVTSEKEEYK ECLDKVYTSTKKYDITKDKRFTETKYEFHVPITLNYQADGVKYLNQKILHVLRDNP DVNIIGLDRGERNLISYVVLNREGKIVNNQQGSFNIVGKMDYQKKLYQKEKNRDK ERKTWKNIETIKDLKEGYISQVVHELTDMAIRNNAIIVMEDLNFGFKRGRTKVERQ VYQKFELALLKKLHYLVTDKTEGEAMLKPGGVLQGYQLAREVKTLKEIGKQCGCV FYVPPGYTSKIDPTTGFVDVFNMSGVTNREKKKAFFEKFDNMFYDEKRDMFGFS FNYEKFTTYQSSYRNDWTVYSNGSKYVWNSLKRTDELIDVTKELKLLFEKYAIDY RNEALFEQIMSQDTDKNNADFWNKLFWYFRVLLRLRNSSDELDQIVSPVLNQNG EFFETPKKITEKSYLSDYPMDADTNGAYHIALKGLYLIQEKIADESVDLDNKLPKDF YKISNAEWFMFRQKEKSRKRTADGSEFESPKKKRKVGSGKPIPNPLLGLDST 9 Wildtype coding sequence (with N-terminal methionine and stop codon) ATGAAAAGTATTTATGAAAAI I I IATTGGATTGGAGTCAAAAAATTTGACGCTG CGCTTTGCGTTGAATCCAGAAGCTAAGACACAAGAAAATTTGAAGTTGTACTG GGACAAATTGCGTGATGAGGAGAGAGATAGGGCGTATCCAATTGTAAAAAAG ATATTGGATAAGGAATATCAGCAGCTGATTTCGGAAGGACTGAAATTATTAGA GAATCAGAATGTGTTGGATTGGACAGAATTAGCAGAGTATATACGGACAAGTG ATTTAAGTAAGAAGAAAAAAGAAGATAAACGCTTAAGAAAATTAATAGCACAAA ATTTAAAAGCGCATCCGTTAGTTGACAAACTGAAAGTAAAAAATGCATTTGGTA AAAATGGCTATCTTGAAACTTTACCGTTGGGAAAAGAAGAGAAAGAGGCAGTA AAAG I I I I IGCCGG Illi GGCGGC I I I I I CAATAACTACAATAAAAACAGGGAA AATTATTTTTCAACCGAGGAAAAAAGCACTGCAATCGCAAACCGAATTGTAAAT GAAAAI I I I ICAAAACAI I I I ICAAATGTAGAAATAGTTACCAAAATTCAAAAGG AAGTGCCAGAATTAATTCAAATCGTGGAAGCACAATTCAAGGGATATGATACT ATCTTTACAGTAAATGGTTATAATACGGCATTGTCACAGGCAGGGATTGATAC ATATAATGAGATGGTTGCAATCTGGAATAAAGAAGCAAATTTGTATGCGCAAA AGGCAGGAAAACTTCCAGATGGACATCCGTTAAAGAAAAAGAGAAATTACTTA TTGTCGGCATTGTTTAAACAGATTGGGAGTGAAAAGGAGCATTTGATTCAAAT TGATAGATTTGATGGAGATGAAGAGGTGATTGAGGCATTGACGGGTGTGAAA AAAATGCTTCAAGAGGCAGATGTATTTGAAAAATTGAATATGCTTGTGGAGGA TATGGAGAATTGGGATTATAGTAAAATATATTTGTCAGCACAGAGTTTATCCAA TGTTTCTGTGTTCCTAAATAATTTATATGAGGATGAACGGGAGAACTCATGGAA TTATCTTGATAATGTCCTAAGAGAAAAATGGCAAATAGAATTACAGGGAAAGAA AAAGGGGACAGATCTGGAAGAAGCGATTCGGAAGAAAAAACAAAGTTTCTATT 10 TABLE1B ZJHK Type V Cas Sequences Name Sequence SEQ ID NO. CAATAGAAGAACTTCAAGAGGCAGTGAATGCCATAGAAGAAACAGATAAATGT TATAATGTATCTAAATGGCTTCTAGGAGCAATGAAAAGCGAAAGGGTAATAGA AGAAAAAAAGAAGGATGTGGAAGATTTTTGCACACAGTGGAAAAATGAAAGAA ACTCGCTGAAAGAGACAGATATAACTGCACTGAAAGAATATCTGGAGCAATGG AT I I I I I I GGCAAGAIAI I GCAAAI C I I I I IATGCAAATGGAATTGAAAAAAAAG AAAAAGATGAAGCAI I I IAI CAIAI I I IAGAAGAIGIGI I G IAI G I I I I GGATG AAGTAATATATTTTTATAATAAAGTTCGAAATTATGTAACGAAGAAGCCATATTC TCTTGAAAAAATGCATTTAAAATTTGGTCATAATGAACTGGCAAATGGATGGTC TGTTAACAAAGAAGAGAACTATGGTACGGCAATATTGAGGCGAAATGGCAAAT ACTATTTGGCAATTACAAATTCATTGAATAAAAAGATGAGTATTCCCACTCAAT TAGAAAGTACAGGAAATAATTATGAAAAGATGGTATTGAATGTATTCCCAAATG TATTTCGGATGATACCAAAATGTACTACAGGAAGAAATGATGTGAAAAGTTGTT TTGAAAGAAAAGAGCCAAATGAGTATTTCTTTATTGATACACCGGAATTTGTTA ACCCATTTAAAGTTACGCGCGAGGAATATGAGTTAAATAAGATAACTTATGATG GTGTTAAAAAGTGGCAATCTGATTATTCAAAAAATACGCAGGATGAAAAAGGA TACAAAGAGGCAGTGACAAAATGGATTCAG I I I I G IAI GCGC I I I I IACAATCT TATAAGAGTACAGCAATATATGATTATTCAACTTTACAGCAACCGGAGAAATAT GAGACGGTGGATTCTTTTTATCATGACGTTGAAAAAATATTATATGAATGTCAT TTTGAGTACGTTCCGGCTAATAAAATAGAGCAGTTGGAAGAAGAAGGAAGAAT I I I I C I G I I I CAGAI I IACAACAAAGAI I I I I CGGAAAACAGACGCCCGGACA GCAAAAAGAATTTGCATACACTTTATTGGGAGGCATTG Illi CAGAAGAAAATC GGAAAGCAAAAGTGATACAATTAAATGGCAAAGCTGAAATATTTCGGAGAGAA AAAAGCATTGAACATCCGATTGTTCATAAAGCTGGGGAAGTGTTAGTGAATAA ACGAACGAAAGACGGGGAACCAATACCAGATGATATTTATAAAGATTTGAGCA ACTA I I I IAACGGAAGAAATGTAACATCTGAAAAGGAAGAGTATAAGGAATGT CTGGATAAAGTGTATACTTCGACCAAAAAATATGATATTACAAAGGATAAACGT TTTACTGAAACCAAATATGAATTTCATGTTCCGATTACCTTGAACTATCAGGCG GACGGTGTTAAATATTTGAATCAGAAAATACTTCATGTGCTGAGGGATAATCC AGATGTGAATATTATAGGTCTAGATAGAGGCGAGCGTAATCTGATTTCCTACG TAGTATTGAACCGAGAAGGCAAGATTGTTAACAATCAGCAGGGGAGTTTCAAT ATTGTGGGTAAGATGGACTATCAGAAGAAACTGTATCAAAAAGAAAAGAATCG TGACAAAGAACGAAAAACTTGGAAAAATATCGAAACAATAAAGGATTTGAAGG AAGGATATATTTCACAAGTCGTTCATGAATTGACCGATATGGCGATTCGCAAT AATGCAATTATTGTGATGGAAGATCTGAA Illi GGATTTAAAAGGGGACGCAC CAAAGTGGAACGGCAGGTATATCAGAAGTTTGAGCTGGCGCTTCTGAAGAAA TTGCATTATCTGGTTACGGATAAAACAGAAGGTGAGGCTATGCTTAAGCCTGG CGGTGTCCTTCAAGGTTATCAGCTTGCAAGAGAAGTAAAAACCCTAAAAGAAA TCGGAAAGCAATGCGGATGTGTA Illi ATGTTCCACCGGGATATACTTCTAAA ATCGATCCAACAACCGGATTTGTTGATGTGTTTAACATGTCAGGTGTTACGAA TCGTGAAAAGAAAAAAGCATTTTTTGAAAAGTTCGATAATATGTTCTATGATGA AAAGCGGGATATGTTTGGA Illi CATTTAACTATGAGAAGTTTACAACATATCA AAGTTCTTATAGAAATGATTGGACTGTATATTCGAATGGAAGCAAATATGTGTG GAACTCTTTAAAAAGGACAGACGAGCTTATTGATGTTACAAAAGAATTGAAACT GCTCTTTGAAAAGTATGCAATTGATTACAGAAACGAAGCATTGTTTGAACAAAT CATGTCCCAAGATACGGATAAAAACAATGCTGAC Illi GGAATAAATTGTTCTG GTAI I I I CG IG I I I I GCTCCGTCTGAGAAACAGTTCAGATGAATTAGATCAGAT TGTTTCACCGGTACTTAATCAAAACGGAGAATTTTTTGAAACACCGAAAAAAAT CACGGAGAAAAGTTATTTGTCTGATTATCCGATGGATGCGGATACCAATGGTG CGTATCACATCGCTTTAAAAGGGTTGTATCTCATACAGGAAAAAATTGCAGAT GAGAGCGTAGATTTGGATAACAAATTACCAAAAGATTTTTACAAGATCTCTAAT GCAGAGTGGTTTATGTTTAGGCAGAAGGAGAAGTAA Codon optimized coding sequence (no N-terminal methionine, no stop codon) AAGAGCATCTACGAGAACTTCATCGGTCTTGAGAGCAAGAACCTGACACTGA GATTCGCCCTGAACCCTGAGGCTAAAACCCAGGAGAACCTGAAGCTGTACTG GGACAAACTGAGGGACGAAGAAAGAGATAGAGCCTACCCTATCGTGAAAAAA ATCCTCGACAAGGAGTATCAGCAGCTCATCAGCGAGGGCCTGAAACTGCTGG AAAATCAAAACGTGCTGGACTGGACCGAACTGGCCGAGTACATCAGAACCAG CGATCTGTCTAAGAAGAAGAAGGAGGACAAGAGACTGCGCAAGCTGATCGCC CAGAACCTGAAAGCCCACCCCCTGGTCGACAAGCTGAAGGTGAAGAATGCCT TCGGCAAGAACGGCTACCTGGAAACCCTGCCATTAGGAAAGGAAGAAAAAGA GGCCGTGAAGGTGTTTGCCGGATTCGGAGGCTTTTTCAACAACTACAACAAG AATCGGGAGAACTACTTCAGTACCGAGGAGAAGTCCACCGCCATCGCCAACA GAATCGTGAACGAGAACTTCAGCAAGCACTTCAGCAACGTGGAAATCGTTACA AAGATCCAAAAAGAAGTGCCAGAGCTGATTCAAATCGTGGAAGCTCAGTTCAA GGGTTACGACACCATCTTTACCGTGAACGGCTACAACACCGCCCTGAGCCAG 11 TABLE1B ZJHK Type V Cas Sequences Name Sequence SEQ ID NO. GCTGGCATCGACACATACAACGAAATGGTGGCCATCTGGAACAAGGAGGCAA ACCTGTACGCTCAAAAAGCCGGCAAGCTGCCAGACGGCCACCCGCTGAAGA AGAAGCGTAACTACCTGCTGAGCGCCCTCTTCAAACAGATCGGCAGCGAAAA AGAACACCTGATCCAGATCGACAGATTCGACGGCGACGAGGAAGTGATCGAA GCCCTGACTGGCGTGAAAAAGATGCTGCAGGAGGCCGACGTGTTCGAGAAG CTGAACATGCTGGTCGAGGACATGGAAAATTGGGATTACTCCAAGATCTACCT GTCTGCCCAGAGCCTGAGTAACGTGTCCGTGTTCCTGAACAACCTGTATGAA GATGAACGGGAGAACAGCTGGAACTACCTGGATAACGTGCTGAGAGAGAAGT GGCAGATTGAACTGCAGGGCAAAAAAAAGGGAACAGATCTGGAAGAGGCCAT TAGAAAGAAGAAGCAGAGC I I I IACTCTATCGAGGAACTTCAGGAGGCAGTG AACGCCATCGAGGAAACCGACAAGTGCTACAATGTGTCTAAATGGCTGCTGG GAGCCATGAAGAGCGAGAGAGTGATCGAGGAGAAGAAGAAAGACGTGGAGG ATTTCTGCACACAGTGGAAGAACGAGAGAAACAGCCTCAAGGAAACCGACAT CACCGCCCTGAAGGAGTACCTGGAGCAGTGGATCTTCCTGGCTAGGTACTGC AAGAGCTTCTACGCCAATGGCATCGAAAAGAAAGAGAAGGATGAGGCTTTTTA CCACATCCTGGAGGATGTGCTGTACGTGCTGGACGAAGTGATCTACTTCTAC AACAAGGTGCGGAACTACGTGACCAAAAAGCCTTACAGTCTGGAGAAGATGC ACCTGAAGTTCGGCCACAACGAGCTGGCCAACGGCTGGAGCGTGAACAAGG AAGAAAATTACGGCACCGCCATCCTGAGAAGAAACGGCAAGTACTACCTGGC CATCACCAACAGCCTGAACAAGAAAATGAGCATCCCTACCCAGCTGGAGAGC ACAGGAAATAATTATGAGAAGATGGTCCTGAACGTTTTTCCCAACGTGTTCCG GATGATCCCAAAGTGCACCACAGGCAGGAACGACGTGAAGTCATGCTTCGAG AGAAAGGAACCCAACGAGTACTTCTTCATCGACACCCCTGAGTTCGTGAACC CCTTTAAGGTCACACGGGAGGAGTACGAACTGAATAAGATCACCTACGACGG AGTTAAGAAGTGGCAGAGCGACTACAGCAAGAACACACAGGACGAAAAGGGC TATAAGGAAGCCGTGACCAAGTGGATTCAG Illi GTATGCGGTTCCTGCAGTC TTATAAGAGCACCGCCATATATGACTACAGCACCCTGCAGCAACCTGAAAAAT ACGAAACAGTGGACAGCTTCTATCATGATGTGGAAAAGATCCTGTACGAGTGC CACTTCGAGTACGTGCCCGCTAACAAGATCGAGCAGCTTGAAGAAGAGGGAA GAATCTTCCTGTTCCAGATCTACAACAAGGATTTTTCTGAGAACAGACGGCCT GATAGCAAGAAAAACCTCCACACCCTGTACTGGGAGGCGCTGTTCTCCGAAG AGAATAGAAAGGCCAAGGTGATTCAGCTGAATGGCAAGGCCGAGATCTTCAG ACGGGAGAAATCAATCGAGCACCCTATCGTGCATAAGGCTGGCGAGGTGCTG GTGAACAAGCGGACCAAAGATGGCGAACCTATTCCTGACGACATCTACAAGG ACCTGAGCAACTATTTCAACGGCAGAAACGTTACCTCTGAGAAGGAAGAGTAC AAGGAGTGTCTGGACAAGGTGTACACCAGCACCAAAAAGTACGATATCACCA AGGACAAAAGATTCACCGAGACAAAGTACGAGTTCCACGTGCCTATCACCCT GAACTACCAGGCCGACGGCGTGAAGTACCTGAATCAGAAGATCCTGCACGTG CTGCGGGACAACCCTGATGTTAACATCATCGGCCTGGATAGAGGCGAAAGAA ACCTGATCTCTTATGTTGTGCTGAACAGAGAGGGCAAGATCGTGAACAATCAG CAGGGTTCTTTCAACATCGTGGGCAAAATGGACTACCAGAAAAAGCTGTACCA GAAGGAGAAAAACCGGGATAAAGAACGGAAAACGTGGAAAAACATCGAAACC ATCAAGGACCTGAAGGAGGGCTATATCAGCCAGGTGGTACACGAGCTGACCG ATATGGCCATCCGGAATAACGCGATCATCGTGATGGAAGATCTGAATTTCGGA TTCAAGCGGGGCCGGACCAAGGTGGAACGGCAGGTGTACCAGAAGTTTGAG CTGGCCCTGCTGAAGAAGCTGCACTACCTCGTGACCGACAAGACCGAGGGA GAAGCTATGCTGAAACCCGGCGGCGTGCTGCAAGGCTACCAGCTGGCTAGA GAAGTCAAGACCCTGAAAGAGATCGGCAAGCAGTGCGGCTGTGTGTTCTACG TGCCCCCTGGCTACACAAGCAAGATCGACCCTACAACCGGCTTCGTCGACGT GTTCAACATGTCTGGAGTTACAAACCGCGAGAAAAAGAAAGCCTTTTTCGAAA AATTTGATAACATGTTCTACGACGAGAAGAGAGACATGTTCGGCTTCAGCTTC AATTACGAAAAGTTTACTACCTACCAGAGCAGCTACAGAAACGACTGGACCGT GTACAGCAACGGCAGCAAGTATGTGTGGAACTCCCTTAAGAGAACAGACGAG TTAATTGACGTGACAAAGGAGCTCAAGCTGCTGTTCGAGAAGTACGCCATCG ATTACCGGAACGAAGCTCTGTTTGAGCAGATCATGAGCCAGGATACAGATAA GAACAACGCCGACTTCTGGAACAAACTGTTCTGGTACTTCCGGGTGCTGCTG CGGCTGAGAAATAGCAGCGACGAACTGGACCAAATCGTCAGCCCTGTGCTGA ATCAGAACGGAGAGTTCTTCGAAACCCCTAAGAAAATCACAGAGAAGTCCTAC CTGTCTGATTACCCTATGGACGCCGATACAAACGGCGCCTACCACATCGCCC TGAAGGGCCTGTACCTGATCCAGGAGAAGATCGCTGACGAATCTGTGGACCT GGACAACAAGCTGCCTAAGGACTTCTACAAGATCAGCAACGCCGAGTGGTTC ATGTTTAGACAGAAAGAAAAA Expression construct (with ATGggcAAGAGCATCTACGAGAACTTCATCGGTCTTGAGAGCAAGAACCTGAC ACTGAGATTCGCCCTGAACCCTGAGGCTAAAACCCAGGAGAACCTGAAGCTG 12 TABLE1B ZJHK Type V Cas Sequences Name Sequence SEQ ID NO. N-terminal methionine and stop codon, includes V5-tag and C-terminal NLS) TACTGGGACAAACTGAGGGACGAAGAAAGAGATAGAGCCTACCCTATCGTGA AAAAAATCCTCGACAAGGAGTATCAGCAGCTCATCAGCGAGGGCCTGAAACT GCTGGAAAATCAAAACGTGCTGGACTGGACCGAACTGGCCGAGTACATCAGA ACCAGCGATCTGTCTAAGAAGAAGAAGGAGGACAAGAGACTGCGCAAGCTGA TCGCCCAGAACCTGAAAGCCCACCCCCTGGTCGACAAGCTGAAGGTGAAGAA TGCCTTCGGCAAGAACGGCTACCTGGAAACCCTGCCATTAGGAAAGGAAGAA AAAGAGGCCGTGAAGGTGTTTGCCGGATTCGGAGGCTTTTTCAACAACTACA ACAAGAATCGGGAGAACTACTTCAGTACCGAGGAGAAGTCCACCGCCATCGC CAACAGAATCGTGAACGAGAACTTCAGCAAGCACTTCAGCAACGTGGAAATC GTTACAAAGATCCAAAAAGAAGTGCCAGAGCTGATTCAAATCGTGGAAGCTCA GTTCAAGGGTTACGACACCATCTTTACCGTGAACGGCTACAACACCGCCCTG AGCCAGGCTGGCATCGACACATACAACGAAATGGTGGCCATCTGGAACAAGG AGGCAAACCTGTACGCTCAAAAAGCCGGCAAGCTGCCAGACGGCCACCCGC TGAAGAAGAAGCGTAACTACCTGCTGAGCGCCCTCTTCAAACAGATCGGCAG CGAAAAAGAACACCTGATCCAGATCGACAGATTCGACGGCGACGAGGAAGTG ATCGAAGCCCTGACTGGCGTGAAAAAGATGCTGCAGGAGGCCGACGTGTTC GAGAAGCTGAACATGCTGGTCGAGGACATGGAAAATTGGGATTACTCCAAGA TCTACCTGTCTGCCCAGAGCCTGAGTAACGTGTCCGTGTTCCTGAACAACCT GTATGAAGATGAACGGGAGAACAGCTGGAACTACCTGGATAACGTGCTGAGA GAGAAGTGGCAGATTGAACTGCAGGGCAAAAAAAAGGGAACAGATCTGGAAG AGGCCATTAGAAAGAAGAAGCAGAGC I I I IACTCTATCGAGGAACTTCAGGAG GCAGTGAACGCCATCGAGGAAACCGACAAGTGCTACAATGTGTCTAAATGGC TGCTGGGAGCCATGAAGAGCGAGAGAGTGATCGAGGAGAAGAAGAAAGACG TGGAGGATTTCTGCACACAGTGGAAGAACGAGAGAAACAGCCTCAAGGAAAC CGACATCACCGCCCTGAAGGAGTACCTGGAGCAGTGGATCTTCCTGGCTAGG TACTGCAAGAGCTTCTACGCCAATGGCATCGAAAAGAAAGAGAAGGATGAGG CTTTTTACCACATCCTGGAGGATGTGCTGTACGTGCTGGACGAAGTGATCTAC TTCTACAACAAGGTGCGGAACTACGTGACCAAAAAGCCTTACAGTCTGGAGAA GATGCACCTGAAGTTCGGCCACAACGAGCTGGCCAACGGCTGGAGCGTGAA CAAGGAAGAAAATTACGGCACCGCCATCCTGAGAAGAAACGGCAAGTACTAC CTGGCCATCACCAACAGCCTGAACAAGAAAATGAGCATCCCTACCCAGCTGG AGAGCACAGGAAATAATTATGAGAAGATGGTCCTGAACGTTTTTCCCAACGTG TTCCGGATGATCCCAAAGTGCACCACAGGCAGGAACGACGTGAAGTCATGCT TCGAGAGAAAGGAACCCAACGAGTACTTCTTCATCGACACCCCTGAGTTCGT GAACCCCTTTAAGGTCACACGGGAGGAGTACGAACTGAATAAGATCACCTAC GACGGAGTTAAGAAGTGGCAGAGCGACTACAGCAAGAACACACAGGACGAAA AGGGCTATAAGGAAGCCGTGACCAAGTGGATTCAG Illi GTATGCGGTTCCT GCAGTCTTATAAGAGCACCGCCATATATGACTACAGCACCCTGCAGCAACCT GAAAAATACGAAACAGTGGACAGCTTCTATCATGATGTGGAAAAGATCCTGTA CGAGTGCCACTTCGAGTACGTGCCCGCTAACAAGATCGAGCAGCTTGAAGAA GAGGGAAGAATCTTCCTGTTCCAGATCTACAACAAGGATTTTTCTGAGAACAG ACGGCCTGATAGCAAGAAAAACCTCCACACCCTGTACTGGGAGGCGCTGTTC TCCGAAGAGAATAGAAAGGCCAAGGTGATTCAGCTGAATGGCAAGGCCGAGA TCTTCAGACGGGAGAAATCAATCGAGCACCCTATCGTGCATAAGGCTGGCGA GGTGCTGGTGAACAAGCGGACCAAAGATGGCGAACCTATTCCTGACGACATC TACAAGGACCTGAGCAACTATTTCAACGGCAGAAACGTTACCTCTGAGAAGGA AGAGTACAAGGAGTGTCTGGACAAGGTGTACACCAGCACCAAAAAGTACGAT ATCACCAAGGACAAAAGATTCACCGAGACAAAGTACGAGTTCCACGTGCCTAT CACCCTGAACTACCAGGCCGACGGCGTGAAGTACCTGAATCAGAAGATCCTG CACGTGCTGCGGGACAACCCTGATGTTAACATCATCGGCCTGGATAGAGGCG AAAGAAACCTGATCTCTTATGTTGTGCTGAACAGAGAGGGCAAGATCGTGAAC AATCAGCAGGGTTCTTTCAACATCGTGGGCAAAATGGACTACCAGAAAAAGCT GTACCAGAAGGAGAAAAACCGGGATAAAGAACGGAAAACGTGGAAAAACATC GAAACCATCAAGGACCTGAAGGAGGGCTATATCAGCCAGGTGGTACACGAGC TGACCGATATGGCCATCCGGAATAACGCGATCATCGTGATGGAAGATCTGAA TTTCGGATTCAAGCGGGGCCGGACCAAGGTGGAACGGCAGGTGTACCAGAA GTTTGAGCTGGCCCTGCTGAAGAAGCTGCACTACCTCGTGACCGACAAGACC GAGGGAGAAGCTATGCTGAAACCCGGCGGCGTGCTGCAAGGCTACCAGCTG GCTAGAGAAGTCAAGACCCTGAAAGAGATCGGCAAGCAGTGCGGCTGTGTGT TCTACGTGCCCCCTGGCTACACAAGCAAGATCGACCCTACAACCGGCTTCGT CGACGTGTTCAACATGTCTGGAGTTACAAACCGCGAGAAAAAGAAAGCC Illi TCGAAAAATTTGATAACATGTTCTACGACGAGAAGAGAGACATGTTCGGCTTC AGCTTCAATTACGAAAAGTTTACTACCTACCAGAGCAGCTACAGAAACGACTG GACCGTGTACAGCAACGGCAGCAAGTATGTGTGGAACTCCCTTAAGAGAACA TABLE1B ZJHK Type V Cas Sequences Name Sequence SEQ ID NO. GACGAGTTAATTGACGTGACAAAGGAGCTCAAGCTGCTGTTCGAGAAGTACG CCATCGATTACCGGAACGAAGCTCTGTTTGAGCAGATCATGAGCCAGGATAC AGATAAGAACAACGCCGACTTCTGGAACAAACTGTTCTGGTACTTCCGGGTG CTGCTGCGGCTGAGAAATAGCAGCGACGAACTGGACCAAATCGTCAGCCCTG TGCTGAATCAGAACGGAGAGTTCTTCGAAACCCCTAAGAAAATCACAGAGAAG TCCTACCTGTCTGATTACCCTATGGACGCCGATACAAACGGCGCCTACCACAT CGCCCTGAAGGGCCTGTACCTGATCCAGGAGAAGATCGCTGACGAATCTGTG GACCTGGACAACAAGCTGCCTAAGGACTTCTACAAGATCAGCAACGCCGAGT GGTTCATGTTTAGACAGAAAGAAAAAtctagaAAGCGGACAGCAGACGGCTCCG AATTTGAAAGCCCTAAGAAAAAGAGAAAGGTGggatccGGCAAACCTATCCCCA ATCCCCTGCTGGGCCTGGACAGCACCTGA

[0091] In some embodiments a ZJHK Type V Cas protein comprises an amino acid sequence of SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9. In some embodiments, a ZJHK Type V Cas protein has nickase activity, for example resulting from one or more amino acid substitutions relative to the sequence of SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9. In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D900 substitution, wherein the position of the D900 substitution is defined with respect to the amino acid numbering of SEQ ID NO:8 (corresponding to amino acid 908 of SEQ ID NO:121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise an E987 substitution, wherein the position of the E987 substitution is defined with respect to the amino acid numbering of SEQ ID NO:8 (corresponding to amino acid 993 of SEQ ID NO:121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a R1203 substitution, wherein the position of the R1203 substitution is defined with respect to the amino acid numbering of SEQ ID NO:8 (corresponding to amino acid 1226 of SEQ ID NO:121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D1244 substitution, wherein the position of the D1244 substitution is defined with respect to the amino acid numbering of SEQ ID NO:121 (corresponding to amino acid 1263 of SEQ ID NO:121). In some embodiments, a ZJHK Type V Cas protein is catalytically inactive, for example due to a R1203 substitution in combination with a D900 substitution, a E987 substitution, and / or D1244 substitution. 6.2.3. ZIKV Type V Cas Proteins

[0092] In one aspect, the disclosure provides ZIKV Type V Cas proteins. ZIKV Type V Cas proteins can be further classified as Type V-A Cas proteins. The ZIKV Type V Cas proteins typically comprise an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:13. In some embodiments, the ZIKV Type V Cas proteins comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:13. In some embodiments, a ZIKV Type V Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:13.

[0093] Exemplary ZIKV Type V Cas protein sequences and nucleotide sequences encoding exemplary ACEE Type V Cas proteins are set forth in Table 1C. TABLE1C ZIKV Type V Cas Sequences Name Sequence SEQ ID NO. Wildtype amino acid sequence (without N-terminal methionine) NIYENFTNMYQVNKTIRMGLKPICKTGENIAKFLEEDKETSDKYKIAKEVIDKENRA FIEDRLKDFSISGLDEYLELLKQKKNLTKNQNKMKKEISTQLTKIQNKMRDEISTQL KGFPQFDNKYKFKYITDKEDIEILKYFKDKKFITFFEEFNTNRKNVYSKENISTSIGH RIVHENLPKFISNFRILNKAIEAFGISKINEDFKNNGINVTVEELNKIDYFNKVLTQSG IDLYNNLIGILNQNINLYNQQQKVKKNKIGKLEILYKQILSKTDKVSFIEEFTEDNQLL ECIDEYFKEKYSLITVDLNNLLENIDTYNLNGIFIKSDKSLGNISNYLYKDWWYISNLI NEEYDYKHKNKVRDDKYYETRKKAIDKIKYFSIGHIDELLKDKNVPMVENYFKEKIN LVVKEFNAYLNKFNEYKFINELKTDEIAVEIIKNLCDSIKNVQGIVKPLIITGNDKDDD FYVEINYIWDELNKFDKIYNMVRNYLTKKDYIEEKIRMMFSKSSFMDGWGKDYGT KKAHIVYHDKNYYLVIVDKKLKLEDIDKLYKPGGDTVHYVYNYQSTENGNIPRKFIY SKGKRFAPSVEKYNLPIEDVIEVYNNEYHTTDYEKKNPEIYKKSLTSLIDYFKIGVN RDMDFEKFDFRLKDSNEYKNIKEFYDNLETCCYKLQEEKVNFNVLEELSYSGKIYL FKIYNKDFSENSKGIPNLHTLYFKMLFDKENLENPIYKLSGKAKMFFRKGSLNLDK KTVDYDKKPIDKKENDKKIKNRRYKVDSFTLHMSIITNFQSYENKNVNETVNRALK YCDDVYAIGIDRGIRNLLYACVVNSKGEIVKQVPLNIINNKDYHNLLAEREEKKKNS RKNWKIIDNIRNLKEGYLSQAIHIITDLMVEYNAVLVLENLNFRFKEKQMKFESNVY QKFEKMLIDKLNFLVDKKLDKNANGGLFNAYQLTEKFTNFKDMKNQNGIIFYIPAW MTSKIDPVTGFTNLFYIKYESIEKAKEFFGKFKSIKFNKVDNYFEFEFDYNDFTDRA QGTRSKWTVCSFGPRIEGFRNPEKNNSWDGREIDITEKIKKLLDDYNVSLDKDIKA QIMDINTKDFFEKFIKYFKLVLQMRNSKTGTDIDYIISPVKNKQNEFFDSRKQNEKM PMDADANGAYNIARKGLMFIDIIKETEDKDLKMPKLFIKNKDWLNYVQKSDL 13 Wildtype amino acid sequence (with N-terminal methionine) MNIYENFTNMYQVNKTIRMGLKPICKTGENIAKFLEEDKETSDKYKIAKEVIDKENR AFIEDRLKDFSISGLDEYLELLKQKKNLTKNQNKMKKEISTQLTKIQNKMRDEISTQ LKGFPQFDNKYKFKYITDKEDIEILKYFKDKKFITFFEEFNTNRKNVYSKENISTSIG HRIVHENLPKFISNFRILNKAIEAFGISKINEDFKNNGINVTVEELNKIDYFNKVLTQS GIDLYNNLIGILNQNINLYNQQQKVKKNKIGKLEILYKQILSKTDKVSFIEEFTEDNQL LECIDEYFKEKYSLITVDLNNLLENIDTYNLNGIFIKSDKSLGNISNYLYKDWWYISN LINEEYDYKHKNKVRDDKYYETRKKAIDKIKYFSIGHIDELLKDKNVPMVENYFKEK INLVVKEFNAYLNKFNEYKFINELKTDEIAVEIIKNLCDSIKNVQGIVKPLIITGNDKDD DFYVEINYIWDELNKFDKIYNMVRNYLTKKDYIEEKIRMMFSKSSFMDGWGKDYG TKKAHIVYHDKNYYLVIVDKKLKLEDIDKLYKPGGDTVHYVYNYQSTENGNIPRKFI YSKGKRFAPSVEKYNLPIEDVIEVYNNEYHTTDYEKKNPEIYKKSLTSLIDYFKIGV NRDMDFEKFDFRLKDSNEYKNIKEFYDNLETCCYKLQEEKVNFNVLEELSYSGKI YLFKIYNKDFSENSKGIPNLHTLYFKMLFDKENLENPIYKLSGKAKMFFRKGSLNL DKKTVDYDKKPIDKKENDKKIKNRRYKVDSFTLHMSIITNFQSYENKNVNETVNRA LKYCDDVYAIGIDRGIRNLLYACVVNSKGEIVKQVPLNIINNKDYHNLLAEREEKKK NSRKNWKIIDNIRNLKEGYLSQAIHIITDLMVEYNAVLVLENLNFRFKEKQMKFESN VYQKFEKMLIDKLNFLVDKKLDKNANGGLFNAYQLTEKFTNFKDMKNQNGIIFYIP AWMTSKIDPVTGFTNLFYIKYESIEKAKEFFGKFKSIKFNKVDNYFEFEFDYNDFTD RAQGTRSKWTVCSFGPRIEGFRNPEKNNSWDGREIDITEKIKKLLDDYNVSLDKDI KAQIMDINTKDFFEKFIKYFKLVLQMRNSKTGTDIDYIISPVKNKQNEFFDSRKQNE KMPMDADANGAYNIARKGLMFIDIIKETEDKDLKMPKLFIKNKDWLNYVQKSDL 14 Expression construct (with N-terminal methionine, V5-tag and C-terminal NLS) aa sequence MGNIYENFTNMYQVNKTIRMGLKPICKTGENIAKFLEEDKETSDKYKIAKEVIDKEN RAFIEDRLKDFSISGLDEYLELLKQKKNLTKNQNKMKKEISTQLTKIQNKMRDEIST QLKGFPQFDNKYKFKYITDKEDIEILKYFKDKKFITFFEEFNTNRKNVYSKENISTSI GHRIVHENLPKFISNFRILNKAIEAFGISKINEDFKNNGINVTVEELNKIDYFNKVLTQ SGIDLYNNLIGILNQNINLYNQQQKVKKNKIGKLEILYKQILSKTDKVSFIEEFTEDN QLLECIDEYFKEKYSLITVDLNNLLENIDTYNLNGIFIKSDKSLGNISNYLYKDWWYI SNLINEEYDYKHKNKVRDDKYYETRKKAIDKIKYFSIGHIDELLKDKNVPMVENYFK EKINLVVKEFNAYLNKFNEYKFINELKTDEIAVEIIKNLCDSIKNVQGIVKPLIITGNDK DDDFYVEINYIWDELNKFDKIYNMVRNYLTKKDYIEEKIRMMFSKSSFMDGWGKD YGTKKAHIVYHDKNYYLVIVDKKLKLEDIDKLYKPGGDTVHYVYNYQSTENGNIPR KFIYSKGKRFAPSVEKYNLPIEDVIEVYNNEYHTTDYEKKNPEIYKKSLTSLIDYFKI GVNRDMDFEKFDFRLKDSNEYKNIKEFYDNLETCCYKLQEEKVNFNVLEELSYSG KIYLFKIYNKDFSENSKGIPNLHTLYFKMLFDKENLENPIYKLSGKAKMFFRKGSLN LDKKTVDYDKKPIDKKENDKKIKNRRYKVDSFTLHMSIITNFQSYENKNVNETVNR ALKYCDDVYAIGIDRGIRNLLYACVVNSKGEIVKQVPLNIINNKDYHNLLAEREEKK KNSRKNWKIIDNIRNLKEGYLSQAIHIITDLMVEYNAVLVLENLNFRFKEKQMKFES NVYQKFEKMLIDKLNFLVDKKLDKNANGGLFNAYQLTEKFTNFKDMKNQNGIIFYI PAWMTSKIDPVTGFTNLFYIKYESIEKAKEFFGKFKSIKFNKVDNYFEFEFDYNDFT DRAQGTRSKWTVCSFGPRIEGFRNPEKNNSWDGREIDITEKIKKLLDDYNVSLDK DIKAQIMDINTKDFFEKFIKYFKLVLQMRNSKTGTDIDYIISPVKNKQNEFFDSRKQ 15 TABLE1C ZIKV Type V Cas Sequences Name Sequence SEQ ID NO. NEKMPMDADANGAYNIARKGLMFIDIIKETEDKDLKMPKLFIKNKDWLNYVQKSDL SRKRTADGSEFESPKKKRKVGSGKPIPNPLLGLDST Wildtype coding sequence (with N-terminal methionine and stop codon) ATGAACATTTACGAAAAI I I I AC rAATATGTATCAGGTAAATAAGACTATAAGAA TGGGGTTAAAGCCAATATGTAAAACTGGTGAAAATATTGCTAAATTTCTTGAGG AAGATAAGGAAACAAGTGATAAATACAAGATAGCTAAAGAAGTAATTGATAAG GAAAATAGAGC I I I IAIAGAGGAI AGA I IAAAGGAI I I I I CAATTTCAGGGTTG GATGAATATTTGGAATTGCTTAAACAAAAAAAGAATTTAACCAAAAATCAAAAT AAAATGAAAAAGGAAATTTCAACACAGTTAACAAAAATACAAAATAAAATGAGA GATGAAATTTCAACACAGTTAAAAGGCTTCCCTCAATTTGATAATAAATATAAA TTCAAATATATTACAGATAAAGAAGATATAGAAAI I I IAAAAIAI I I IAAAGATA AGAAATTTATTACTTTCTTTGAAGAATTTAATACTAATAGAAAAAATGTCTACTC TAAAGAAAATATTTCAACTTCTATTGGACACAGAATTGTTCACGAAAATCTTCC AAAATTTATTTCAAA Illi AGGA Illi AAATAAAGCAATAGAGGCGTTTGGAATA AGTAAAATAAATGAAGA Illi AAGAATAATGGAATTAATGTTACAGTTGAAGAA CTTAATAAAATAGATTAI I I IAACAAGGI I I IAACTCAATCAGGAATAGATTTGT ATAATAATTTGATAGGTA Illi AAATCAAAATATAAATCTATATAATCAACAACA GAAAGTAAAAAAGAATAAAATTGGAAAGTTAGAAATATTATATAAGCAAAI I I IA AGTAAAACAGATAAAGTATCGTTTATTGAAGAATTTACTGAAGATAACCAACTT TTGGAATGTATTGATGAATATTTTAAAGAAAAATATAGTTTGATAACTGTAGATT TAAATAATTTACTTGAAAATATTGATACTTATAATTTGAATGGTATCTTTATTAAA AGTGATAAGTCCTTGGGAAATATATCTAATTATTTATATAAAGATTGGTGGTAT ATATCAAATCTTATAAACGAAGAATACGATTATAAACATAAGAATAAGGTAAGA GATGATAAGTATTATGAAACAAGAAAAAAAGCTATAGATAAGATTAAATATTTTT CCATAGGACATATTGATGAATTGTTAAAAGATAAAAATGTTCCTATGGTAGAAA ACTATTTCAAAGAAAAGATAAATTTAGTAGTAAAAGAATTTAATGCTTATTTAAA CAAATTTAATGAATATAAGTTTATAAATGAGCTAAAAACTGATGAAATTGCTGT CGAAATAATAAAAAATTTATGTGATTCAATAAAGAATGTACAGGGGATAGTAAA GCCTTTAATAATTACTGGAAATGATAAAGACGATGATTTTTATGTGGAAATCAA TTATATATGGGACGAGCTTAATAAGTTTGATAAAATATATAATATGGTTAGAAAT TATCTTACAAAAAAGGATTACATAGAGGAAAAAATTAGAATGATG Illi CAAAG AGCAG Illi ATGGATGGTTGGGGAAAAGATTATGGAACAAAAAAAGCACATAT AGTTTATCATGATAAAAATTATTATTTAGTAATAGTAGACAAGAAATTAAAATTA GAGGATATAGATAAATTATATAAACCAGGTGGAGATACTGTACATTATGTATAT AATTACCAATCAACAGAAAATGGAAATATTCCTAGAAAATTCATATATTCTAAG GGTAAAAGATTTGCACCATCTGTAGAAAAATATAATTTACCAATAGAAGATGTT ATCGAAGTGTATAACAATGAATATCATACAACAGATTACGAAAAGAAAAATCCT GAAATTTACAAGAAATCATTAACATCCTTAATTGATTAI I I IAAAATAGGGGTAA ATAGGGATATGGA Illi GAAAAAI I I GA I I I IAGATTAAAAGATTCAAACGAAT ACAAAAATATAAAAGAA Illi ATGATAATTTGGAAACTTGTTGCTATAAGTTACA AGAAGAAAAAGTTAA Illi AATGTACTTGAAGAGCTTTCATATAGTGGAAAAAT TTATTTATTTAAAATATACAATAAGGATTTTTCTGAAAATAGCAAAGGAATACCT AATCTTCATACTTTATA Illi AAAATGCTATTTGACAAAGAAAACCTTGAAAATC CGATTTATAAACTTAGTGGAAAGGCTAAAATGTTTTTTAGAAAGGGTAGTCTTA ATTTAGACAAAAAAACTGTTGATTATGATAAAAAGCCAATAGATAAGAAAGAAA ATGACAAAAAAATTAAAAATAGAAGATATAAAGTTGATAG Illi ACATTACATAT GTCAATTATTACGAACTTTCAGTCATATGAAAATAAAAATGTAAATGAAACTGT AAATAGGGCTTTAAAATATTGTGATGATGTTTATGCCATAGGTATAGACAGAG GAATAAGAAATTTATTATATGCTTGTGTAGTAAATTCAAAGGGAGAAATAGTAA AACAAGTTCCTTTAAATATTATAAATAATAAAGATTATCACAATTTACTTGCAGA AAGAGAAGAGAAGAAAAAGAATAGTAGGAAAAATTGGAAAATCATTGATAATA TAAGGAATTTAAAGGAAGGCTATTTAAGTCAGGCCATACATATAATAACTGACC TTATGGTTGAATATAATGCTGTACTTG I I I IAGAGAAI I IGAAI I I IAGATTTAA AGAAAAACAAATGAAATTTGAAAGTAATGTTTATCAAAAATTTGAAAAGATGCT TATTGATAAATTGAATTTCTTAGTTGATAAAAAGCTTGATAAGAACGCCAATGG TGGATTGTTTAATGCGTATCAATTAACAGAAAAATTTACAAACTTTAAAGATATG AAAAATCAAAATGGTATAATAI I I IATATTCCTGCTTGGATGACAAGCAAAATT GACCCAGTTACAGGATTTACAAATTTATTCTATATTAAATATGAGAGTATTGAA AAGGCTAAAGAGTTTTTTGGTAAGTTTAAATCAATAAAATTTAATAAGGTAGAC AACTAI I I IGAAI I IGAAI I IGAI IAIAAIGAI I I IACTGACAGAGCTCAAGGTA CAAGGTCTAAATGGACAGTTTGTAG Illi GGCCCTAGAATTGAAGGTTTTAGA AATCCTGAAAAAAATAATAGTTGGGATGGTAGAGAAATAGATATAACAGAGAA AATTAAAAAATTACTTGATGATTATAATGTATCTTTAGATAAAGATATTAAAGCT CAAATTATGGATATAAATACTAAGGATTTCTTTGAAAAATTTATTAAATAI I I IAA 16 TABLE1C ZIKV Type V Cas Sequences Name Sequence SEQ ID NO. ACTTGTATTGCAAATGAGAAACAGTAAAACAGGTACAGATATTGATTATATCAT TTCTCCGGTTAAAAATAAGCAAAATGAATTTTTTGACAGTAGAAAGCAAAATGA AAAAATGCCTATGGATGCAGATGCAAATGGTGCTTATAATATTGCTAGAAAAG GCTTAATGTTTATTGATATAATAAAAGAAACTGAAGATAAAGATTTAAAGATGC CTAAATTGTTCATTAAAAATAAAGATTGGTTAAATTATGTACAAAAGAGTGATTT GTAA Codon optimized coding sequence (no N-terminal methionine, no stop codon) AATATCTATGAGAACTTCACCAACATGTACCAGGTGAACAAGACAATCCGCAT GGGCCTGAAGCCTATCTGTAAAACCGGAGAAAACATCGCCAAGTTCCTGGAG GAGGACAAGGAAACCAGCGACAAGTACAAGATCGCCAAGGAGGTCATCGACA AGGAGAACAGAGCCTTTATCGAGGACAGACTGAAGGACTTCAGCATCAGCGG CCTGGACGAGTACCTGGAACTGCTGAAGCAGAAGAAAAACCTGACAAAGAAC CAGAACAAGATGAAAAAGGAAATCTCCACCCAGCTGACAAAGATCCAGAACAA GATGCGGGACGAGATATCGACACAGCTGAAGGGCTTCCCTCAGTTCGATAAC AAATACAAGTTCAAATATATCACAGACAAGGAGGACATCGAAATCCTCAAGTA CTTCAAGGATAAGAAGTTCATTACATTCTTTGAGGAATTTAATACCAATCGGAA AAACGTGTACAGCAAGGAAAACATCAGCACCTCTATCGGCCATAGAATCGTG CACGAGAACCTGCCAAAGTTCATCAGCAACTTCAGAATCCTGAATAAGGCCAT CGAGGCCTTCGGCATCTCTAAAATCAATGAGGACTTCAAGAACAATGGCATCA ACGTGACCGTAGAAGAACTGAACAAGATCGACTACTTCAACAAGGTCCTGACA CAGAGCGGCATTGACCTGTACAACAACCTGATTGGCATCCTGAACCAGAACA TCAACCTGTACAATCAGCAGCAGAAGGTGAAGAAGAACAAAATCGGAAAGCT GGAAATCCTGTACAAGCAAATCTTGTCCAAAACCGACAAGGTGTCTTTCATTG AGGAGTTCACCGAGGACAACCAGCTGCTGGAGTGCATCGACGAGTACTTTAA AGAGAAATACAGCCTGATCACCGTGGACCTGAACAACCTGCTTGAAAATATCG ACACCTACAATCTCAACGGCATCTTCATCAAATCTGATAAAAGCCTGGGCAAC ATCAGCAACTACCTGTACAAGGATTGGTGGTACATCAGCAACCTGATCAACGA AGAATACGACTACAAGCACAAGAACAAGGTCAGAGATGATAAGTACTACGAGA CAAGAAAGAAGGCCATCGACAAGATCAAGTACTTCTCTATCGGACACATCGAT GAGCTGCTGAAGGACAAGAACGTTCCAATGGTGGAAAACTACTTCAAGGAGA AGATCAACCTGGTCGTGAAGGAGTTCAATGCTTATCTGAACAAGTTCAATGAA TATAAATTCATCAACGAGCTGAAAACAGACGAGATCGCCGTGGAAATCATCAA GAACCTGTGCGACAGCATCAAGAACGTGCAGGGCATCGTGAAGCCCCTGATC ATCACCGGCAACGACAAGGATGATGATTTTTACGTGGAGATCAACTACATCTG GGATGAGCTTAACAAGTTCGACAAAATCTACAACATGGTCAGGAATTACCTAA CCAAGAAGGACTACATCGAGGAAAAGATCAGAATGATG Illi CCAAGAGCAG CTTTATGGACGGCTGGGGCAAGGACTACGGCACCAAGAAGGCCCACATCGT GTACCACGACAAGAACTACTACCTGGTGATCGTGGACAAGAAGCTGAAACTG GAAGATATCGACAAACTATACAAGCCAGGCGGCGACACAGTTCACTACGTGT ACAACTACCAGTCTACCGAGAACGGAAACATCCCTCGGAAGTTCATCTACTCT AAGGGCAAGCGGTTCGCCCCTAGCGTGGAAAAATATAACCTGCCTATTGAAG ATGTGATTGAGGTGTACAACAACGAGTACCACACCACCGACTATGAGAAAAAG AACCCTGAGATATACAAAAAGTCCCTGACCAGCCTGATCGACTATTTCAAGAT CGGCGTGAACAGAGATATGGACTTCGAGAAGTTTGA Illi CGGCTAAAGGACT CCAACGAATACAAGAACATCAAGGAGTTCTACGATAACCTGGAGACATGCTGC TACAAGCTGCAGGAGGAAAAGGTGAACTTCAACGTGCTGGAGGAACTGAGCT ACAGCGGAAAGATCTACCTGTTCAAGATCTACAACAAAGATTTCAGCGAGAAT AGCAAAGGCATCCCTAACCTGCATACCCTGTACTTCAAAATGCTGTTCGACAA AGAGAACCTGGAGAACCCCATCTACAAGCTGTCTGGAAAAGCTAAGATG Illi TCAGAAAGGGCAGCCTGAACCTGGACAAAAAAACCGTTGACTATGACAAAAAA CCTATCGATAAGAAGGAAAACGACAAAAAAATCAAGAATAGGCGGTACAAGGT GGACAGCTTCACCCTGCACATGAGCATCATCACCAACTTCCAGAGCTACGAG AACAAGAACGTTAATGAGACTGTGAACCGGGCCCTGAAGTACTGCGACGACG TGTACGCCATCGGCATCGACCGCGGAATCCGGAACCTGCTGTACGCTTGTGT GGTGAACAGCAAGGGCGAGATCGTGAAGCAAGTGCCCCTCAACATCATTAAC AATAAGGATTACCACAACCTGCTGGCCGAGAGAGAAGAAAAGAAGAAAAACA GCAGAAAGAATTGGAAGATCATAGACAACATCAGAAACCTGAAGGAAGGCTA CCTGAGCCAGGCCATCCACATCATCACCGACCTGATGGTGGAATACAACGCC GTGCTGGTGCTGGAGAACCTGAATTTCAGATTCAAGGAGAAGCAGATGAAGT TTGAAAGCAATGTGTACCAAAAATTCGAAAAAATGCTGATCGACAAGCTGAAT TTCCTGGTCGATAAAAAACTGGACAAGAATGCCAATGGCGGACTGTTTAACGC CTATCAGCTGACAGAGAAGTTCACCAACTTTAAGGATATGAAGAATCAGAACG GCATCATCTTCTACATCCCCGCCTGGATGACAAGCAAGATCGATCCCGTGAC CGGCTTCACAAACCTG Illi ATATCAAATACGAGAGCATCGAGAAGGCAAAGG 17 TABLE1C ZIKV Type V Cas Sequences Name Sequence SEQ ID NO. AGTTCTTCGGCAAGTTTAAGTCTATCAAGTTCAATAAGGTGGACAATTATTTCG AGTTCGAGTTCGACTACAACGACTTTACCGACAGAGCTCAAGGCACCAGAAG CAAGTGGACCGTGTGTAGCTTCGGTCCTCGGATCGAGGGCTTCAGAAACCCC GAGAAAAACAATTCCTGGGACGGCAGAGAAATCGACATCACAGAGAAGATCA AGAAGCTGCTGGATGACTACAATGTGAGCCTGGACAAAGACATCAAAGCCCA GATCATGGACATCAACACCAAGGATTTCTTCGAGAAGTTCATCAAGTACTTCA AGCTGGTGCTGCAGATGAGAAACAGCAAGACCGGCACCGACATCGATTACAT TATCTCCCCTGTGAAGAACAAGCAGAACGAGTTTTTCGACTCCAGAAAGCAGA ACGAGAAGATGCCTATGGACGCTGATGCCAACGGCGCCTACAACATCGCTAG AAAGGGGCTGATGTTCATCGATATCATCAAGGAAACAGAGGACAAGGACCTG AAAATGCCTAAGCTGTTCATAAAGAACAAGGATTGGCTGAACTATGTGCAGAA ATCAGATCTG Expression construct (with N-terminal methionine and stop codon, includes V5-tag and C-terminal NLS) ATGggcAATATCTATGAGAACTTCACCAACATGTACCAGGTGAACAAGACAATC CGCATGGGCCTGAAGCCTATCTGTAAAACCGGAGAAAACATCGCCAAGTTCC TGGAGGAGGACAAGGAAACCAGCGACAAGTACAAGATCGCCAAGGAGGTCA TCGACAAGGAGAACAGAGCCTTTATCGAGGACAGACTGAAGGACTTCAGCAT CAGCGGCCTGGACGAGTACCTGGAACTGCTGAAGCAGAAGAAAAACCTGACA AAGAACCAGAACAAGATGAAAAAGGAAATCTCCACCCAGCTGACAAAGATCCA GAACAAGATGCGGGACGAGATATCGACACAGCTGAAGGGCTTCCCTCAGTTC GATAACAAATACAAGTTCAAATATATCACAGACAAGGAGGACATCGAAATCCT CAAGTACTTCAAGGATAAGAAGTTCATTACATTCTTTGAGGAATTTAATACCAA TCGGAAAAACGTGTACAGCAAGGAAAACATCAGCACCTCTATCGGCCATAGA ATCGTGCACGAGAACCTGCCAAAGTTCATCAGCAACTTCAGAATCCTGAATAA GGCCATCGAGGCCTTCGGCATCTCTAAAATCAATGAGGACTTCAAGAACAATG GCATCAACGTGACCGTAGAAGAACTGAACAAGATCGACTACTTCAACAAGGTC CTGACACAGAGCGGCATTGACCTGTACAACAACCTGATTGGCATCCTGAACC AGAACATCAACCTGTACAATCAGCAGCAGAAGGTGAAGAAGAACAAAATCGG AAAGCTGGAAATCCTGTACAAGCAAATCTTGTCCAAAACCGACAAGGTGTCTT TCATTGAGGAGTTCACCGAGGACAACCAGCTGCTGGAGTGCATCGACGAGTA CTTTAAAGAGAAATACAGCCTGATCACCGTGGACCTGAACAACCTGCTTGAAA ATATCGACACCTACAATCTCAACGGCATCTTCATCAAATCTGATAAAAGCCTG GGCAACATCAGCAACTACCTGTACAAGGATTGGTGGTACATCAGCAACCTGAT CAACGAAGAATACGACTACAAGCACAAGAACAAGGTCAGAGATGATAAGTACT ACGAGACAAGAAAGAAGGCCATCGACAAGATCAAGTACTTCTCTATCGGACA CATCGATGAGCTGCTGAAGGACAAGAACGTTCCAATGGTGGAAAACTACTTCA AGGAGAAGATCAACCTGGTCGTGAAGGAGTTCAATGCTTATCTGAACAAGTTC AATGAATATAAATTCATCAACGAGCTGAAAACAGACGAGATCGCCGTGGAAAT CATCAAGAACCTGTGCGACAGCATCAAGAACGTGCAGGGCATCGTGAAGCCC CTGATCATCACCGGCAACGACAAGGATGATGATTTTTACGTGGAGATCAACTA CATCTGGGATGAGCTTAACAAGTTCGACAAAATCTACAACATGGTCAGGAATT ACCTAACCAAGAAGGACTACATCGAGGAAAAGATCAGAATGATG Illi CCAAG AGCAGCTTTATGGACGGCTGGGGCAAGGACTACGGCACCAAGAAGGCCCAC ATCGTGTACCACGACAAGAACTACTACCTGGTGATCGTGGACAAGAAGCTGA AACTGGAAGATATCGACAAACTATACAAGCCAGGCGGCGACACAGTTCACTA CGTGTACAACTACCAGTCTACCGAGAACGGAAACATCCCTCGGAAGTTCATCT ACTCTAAGGGCAAGCGGTTCGCCCCTAGCGTGGAAAAATATAACCTGCCTATT GAAGATGTGATTGAGGTGTACAACAACGAGTACCACACCACCGACTATGAGA AAAAGAACCCTGAGATATACAAAAAGTCCCTGACCAGCCTGATCGACTATTTC AAGATCGGCGTGAACAGAGATATGGACTTCGAGAAGTTTGA Illi CGGCTAAA GGACTCCAACGAATACAAGAACATCAAGGAGTTCTACGATAACCTGGAGACAT GCTGCTACAAGCTGCAGGAGGAAAAGGTGAACTTCAACGTGCTGGAGGAACT GAGCTACAGCGGAAAGATCTACCTGTTCAAGATCTACAACAAAGATTTCAGCG AGAATAGCAAAGGCATCCCTAACCTGCATACCCTGTACTTCAAAATGCTGTTC GACAAAGAGAACCTGGAGAACCCCATCTACAAGCTGTCTGGAAAAGCTAAGA TGTTTTTCAGAAAGGGCAGCCTGAACCTGGACAAAAAAACCGTTGACTATGAC AAAAAACCTATCGATAAGAAGGAAAACGACAAAAAAATCAAGAATAGGCGGTA CAAGGTGGACAGCTTCACCCTGCACATGAGCATCATCACCAACTTCCAGAGC TACGAGAACAAGAACGTTAATGAGACTGTGAACCGGGCCCTGAAGTACTGCG ACGACGTGTACGCCATCGGCATCGACCGCGGAATCCGGAACCTGCTGTACG CTTGTGTGGTGAACAGCAAGGGCGAGATCGTGAAGCAAGTGCCCCTCAACAT CATTAACAATAAGGATTACCACAACCTGCTGGCCGAGAGAGAAGAAAAGAAG AAAAACAGCAGAAAGAATTGGAAGATCATAGACAACATCAGAAACCTGAAGGA AGGCTACCTGAGCCAGGCCATCCACATCATCACCGACCTGATGGTGGAATAC 18 TABLE1C ZIKV Type V Cas Sequences Name Sequence SEQ ID NO. AACGCCGTGCTGGTGCTGGAGAACCTGAATTTCAGATTCAAGGAGAAGCAGA TGAAGTTTGAAAGCAATGTGTACCAAAAATTCGAAAAAATGCTGATCGACAAG CTGAATTTCCTGGTCGATAAAAAACTGGACAAGAATGCCAATGGCGGACTGTT TAACGCCTATCAGCTGACAGAGAAGTTCACCAACTTTAAGGATATGAAGAATC AGAACGGCATCATCTTCTACATCCCCGCCTGGATGACAAGCAAGATCGATCC CGTGACCGGCTTCACAAACCTG Illi ATATCAAATACGAGAGCATCGAGAAGG CAAAGGAGTTCTTCGGCAAGTTTAAGTCTATCAAGTTCAATAAGGTGGACAAT TATTTCGAGTTCGAGTTCGACTACAACGACTTTACCGACAGAGCTCAAGGCAC CAGAAGCAAGTGGACCGTGTGTAGCTTCGGTCCTCGGATCGAGGGCTTCAGA AACCCCGAGAAAAACAATTCCTGGGACGGCAGAGAAATCGACATCACAGAGA AGATCAAGAAGCTGCTGGATGACTACAATGTGAGCCTGGACAAAGACATCAA AGCCCAGATCATGGACATCAACACCAAGGATTTCTTCGAGAAGTTCATCAAGT ACTTCAAGCTGGTGCTGCAGATGAGAAACAGCAAGACCGGCACCGACATCGA TTACATTATCTCCCCTGTGAAGAACAAGCAGAACGAGTTTTTCGACTCCAGAA AGCAGAACGAGAAGATGCCTATGGACGCTGATGCCAACGGCGCCTACAACAT CGCTAGAAAGGGGCTGATGTTCATCGATATCATCAAGGAAACAGAGGACAAG GACCTGAAAATGCCTAAGCTGTTCATAAAGAACAAGGATTGGCTGAACTATGT GCAGAAATCAGATCTGtctagaAAGCGGACAGCAGACGGCTCCGAATTTGAAAG CCCTAAGAAAAAGAGAAAGGTGggatccGGCAAACCTATCCCCAATCCCCTGCT GGGCCTGGACAGCACCTGA

[0094] In some embodiments a ZIKV Type V Cas protein comprises an amino acid sequence of SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15. In some embodiments, a ZIKV Type V Cas protein has nickase activity, for example resulting from one or more amino acid substitutions relative to the sequence of SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15. In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D814 substitution, wherein the position of the D814 substitution is defined with respect to the amino acid numbering of SEQ ID NO:14 (corresponding to amino acid 908 of SEQ ID NO:121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise an E899 substitution, wherein the position of the E899 substitution is defined with respect to the amino acid numbering of SEQ ID NO:14 (corresponding to amino acid 993 of SEQ ID NO:121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a R1111 substitution, wherein the position of the R1111 substitution is defined with respect to the amino acid numbering of SEQ ID NO:14 (corresponding to amino acid 1226 of SEQ ID NO:121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D1148 substitution, wherein the position of the D1148 substitution is defined with respect to the amino acid numbering of SEQ ID NO:14 (corresponding to amino acid 1263 of SEQ ID NO:121). In some embodiments, a ZIKV Type V Cas protein is catalytically inactive, for example due to a R1111 substitution in combination with a D814 substitution, a E899 substitution, and / or D1148 substitution. 6.2.4. ZZFT Type V Cas Proteins

[0095] In one aspect, the disclosure provides ZZFT Type V Cas proteins. ZZFT Type V Cas proteins can be further classified as Type V-A Cas proteins. The ZZFT Type V Cas proteins typically comprise an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:19. In some embodiments, the ZZFT Type V Cas proteins comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:19. In some embodiments, a ZZFT Type V Cas protein comprises an amino acid sequence that is identical to SEQ ID N0:19.

[0096] Exemplary ZZFT Type V Cas protein sequences and nucleotide sequences encoding exemplary ZZFT Type V Cas proteins are set forth in Table 1D. TABLE1D ZZFT Type V Cas Sequences Name Sequence SEQ ID NO. Wildtype amino acid sequence (without N-terminal methionine) EISNRFTNKYQVSKTLRFRLEPTGGTDDLLCQAQIIEGDERRNKEAITMKQILDNC HKQIIERVLSDFNFKEHSLEEFFKVYTRNDDDREKDIENLQAKMRKEIAAAFTKQD VTKLFSSKFKDFVERGLIKYASNEKERNIVSRFKGFATYFTGFNTNRLNMYSEEAK STAISFRLINQNLIKFIDNILVYKKVSQTLPSDVLSNIYIDFKAIINTSSLEEFFSINNYN NILTQKQIEIFNAVIGGKKDKDEKIITKGFNQYINEYNQTNKNIRLPKMMRLFNQILS DREGVSARPEPFNNANETISSVRDCFTNEISKQITILSETTSKIESFDIDRIYIKGGE DLRALSNSIYGYFNYIHDRIADKWKHNNPQGKKSPESYQKNLNAYLKGIKSVSLHS IANICGDNKVIEYFRNLGAENTVDFQRENVVSLIDNKYNCASNLLSDAQITDEELRT NSRSIKDLLDAVKSAQRFFRLLCGSGNEPDKDHSFYDEYTPAFEALENSINPLYNK VRSFVTKKDFSTDKFKLNFDSSSFLSGWAKKSEYEKSSAFIFIRDNQYYLGINKCL SKEDIAYLEDSTSSSDTKRVVYMFQKVDATNIPRIFIRSKGSNLAPAVNEFQLPIETI LDIYDNKFFTTSYQKKDRTKWKESLTKLIDYYKLGFSQHKSYADFDLKWKASSEY NDINDFLADVQRFCYRIEFININWDKLIEFTEDGKFYLFRIANKDLSGNSTGLPNLH TIYWKMLFDESNLKDIVYKLSGNAEVFMRYNSLKNPIVHKAGVEIKNKCPFTEKKT SIFDYDIIKDRRYTKDQLELHVPILMNFKSPSAAKGKAFNKECLEYIRNNGIKHIIGID RGERNLLYMVITDLDGNIVEQKSLNQIASNPKLPLFRQDYNKLLKTKADANAQARR DWETIDTVKEIKFGFLSQIVHEIAMAIIKYDAIVVLENLNRGFMQKRGLENNVYQKF EQMLLDKLSYYVDKTKHPEEAGGALHAYQLSDTYANFNSLSKNAMVRQSGFVFY IPAWLTSKIDPVTGFASFLKFHRDDSMATIKSTISKFDCFKYDKECDMFHIRIDYNK FSTSCSGGQRKWDLFTFGDRILAERNTMQNSRYVYQTVNLTSEFKNLFATKDIDI SGNLKDSICKIEDVGFFRKLSQLLSLTLQLRNSNAETGEDFLISPVADKDGNFFDS RNCPDSLPKDADANGAYNIARKGLMLVEQLKRCKDVSKFKPAIKNEDWLDYVQR 19 Wildtype amino acid sequence (with N-terminal methionine) MEISNRFTNKYQVSKTLRFRLEPTGGTDDLLCQAQIIEGDERRNKEAITMKQILDN CHKQIIERVLSDFNFKEHSLEEFFKVYTRNDDDREKDIENLQAKMRKEIAAAFTKQ DVTKLFSSKFKDFVERGLIKYASNEKERNIVSRFKGFATYFTGFNTNRLNMYSEEA KSTAISFRLINQNLIKFIDNILVYKKVSQTLPSDVLSNIYIDFKAIINTSSLEEFFSINNY NNILTQKQIEIFNAVIGGKKDKDEKIITKGFNQYINEYNQTNKNIRLPKMMRLFNQIL SDREGVSARPEPFNNANETISSVRDCFTNEISKQITILSETTSKIESFDIDRIYIKGG EDLRALSNSIYGYFNYIHDRIADKWKHNNPQGKKSPESYQKNLNAYLKGIKSVSLH SIANICGDNKVIEYFRNLGAENTVDFQRENVVSLIDNKYNCASNLLSDAQITDEELR TNSRSIKDLLDAVKSAQRFFRLLCGSGNEPDKDHSFYDEYTPAFEALENSINPLYN KVRSFVTKKDFSTDKFKLNFDSSSFLSGWAKKSEYEKSSAFIFIRDNQYYLGINKC LSKEDIAYLEDSTSSSDTKRVVYMFQKVDATNIPRIFIRSKGSNLAPAVNEFQLPIE TILDIYDNKFFTTSYQKKDRTKWKESLTKLIDYYKLGFSQHKSYADFDLKWKASSE YNDINDFLADVQRFCYRIEFININWDKLIEFTEDGKFYLFRIANKDLSGNSTGLPNL HTIYWKMLFDESNLKDIVYKLSGNAEVFMRYNSLKNPIVHKAGVEIKNKCPFTEKK TSIFDYDIIKDRRYTKDQLELHVPILMNFKSPSAAKGKAFNKECLEYIRNNGIKHIIGI DRGERNLLYMVITDLDGNIVEQKSLNQIASNPKLPLFRQDYNKLLKTKADANAQAR RDWETIDTVKEIKFGFLSQIVHEIAMAIIKYDAIVVLENLNRGFMQKRGLENNVYQK FEQMLLDKLSYYVDKTKHPEEAGGALHAYQLSDTYANFNSLSKNAMVRQSGFVF YIPAWLTSKIDPVTGFASFLKFHRDDSMATIKSTISKFDCFKYDKECDMFHIRIDYN KFSTSCSGGQRKWDLFTFGDRILAERNTMQNSRYVYQTVNLTSEFKNLFATKDID ISGNLKDSICKIEDVGFFRKLSQLLSLTLQLRNSNAETGEDFLISPVADKDGNFFDS RNCPDSLPKDADANGAYNIARKGLMLVEQLKRCKDVSKFKPAIKNEDWLDYVQR 20 Expression construct (with N-terminal methionine, V5-tag and C-terminal NLS) aa sequence MGEISNRFTNKYQVSKTLRFRLEPTGGTDDLLCQAQIIEGDERRNKEAITMKQILD NCHKQIIERVLSDFNFKEHSLEEFFKVYTRNDDDREKDIENLQAKMRKEIAAAFTK QDVTKLFSSKFKDFVERGLIKYASNEKERNIVSRFKGFATYFTGFNTNRLNMYSE EAKSTAISFRLINQNLIKFIDNILVYKKVSQTLPSDVLSNIYIDFKAIINTSSLEEFFSIN NYNNILTQKQIEIFNAVIGGKKDKDEKIITKGFNQYINEYNQTNKNIRLPKMMRLFN QILSDREGVSARPEPFNNANETISSVRDCFTNEISKQITILSETTSKIESFDIDRIYIK GGEDLRALSNSIYGYFNYIHDRIADKWKHNNPQGKKSPESYQKNLNAYLKGIKSV SLHSIANICGDNKVIEYFRNLGAENTVDFQRENVVSLIDNKYNCASNLLSDAQITDE ELRTNSRSIKDLLDAVKSAQRFFRLLCGSGNEPDKDHSFYDEYTPAFEALENSINP LYNKVRSFVTKKDFSTDKFKLNFDSSSFLSGWAKKSEYEKSSAFIFIRDNQYYLGI NKCLSKEDIAYLEDSTSSSDTKRVVYMFQKVDATNIPRIFIRSKGSNLAPAVNEFQ LPIETILDIYDNKFFTTSYQKKDRTKWKESLTKLIDYYKLGFSQHKSYADFDLKWKA 21 TABLE1D ZZFT Type V Cas Sequences Name Sequence SEQ ID NO. SSEYNDINDFLADVQRFCYRIEFININWDKLIEFTEDGKFYLFRIANKDLSGNSTGL PNLHTIYWKMLFDESNLKDIVYKLSGNAEVFMRYNSLKNPIVHKAGVEIKNKCPFT EKKTSIFDYDIIKDRRYTKDQLELHVPILMNFKSPSAAKGKAFNKECLEYIRNNGIK HIIGIDRGERNLLYMVITDLDGNIVEQKSLNQIASNPKLPLFRQDYNKLLKTKADAN AQARRDWETIDTVKEIKFGFLSQIVHEIAMAIIKYDAIVVLENLNRGFMQKRGLENN VYQKFEQMLLDKLSYYVDKTKHPEEAGGALHAYQLSDTYANFNSLSKNAMVRQS GFVFYIPAWLTSKIDPVTGFASFLKFHRDDSMATIKSTISKFDCFKYDKECDMFHIR IDYNKFSTSCSGGQRKWDLFTFGDRILAERNTMQNSRYVYQTVNLTSEFKNLFAT KDIDISGNLKDSICKIEDVGFFRKLSQLLSLTLQLRNSNAETGEDFLISPVADKDGN FFDSRNCPDSLPKDADANGAYNIARKGLMLVEQLKRCKDVSKFKPAIKNEDWLD YVQRSRKRTADGSEFESPKKKRKVGSGKPIPNPLLGLDST Wildtype coding sequence (with N-terminal methionine and stop codon) ATGGAAATTTCGAACCGATTCACAAACAAGTATCAAGTAAGCAAGACCCTCCG CTTTCGCCTTGAGCCAACCGGAGGTACTGATGATTTACTTTGCCAAGCACAAA TCATCGAGGGAGACGAGCGCCGCAATAAAGAGGCTATAACAATGAAACAGAT TTTGGACAATTGTCACAAACAGATAATTGAGCGCGTATTGTCCGACTTTAATTT TAAAGAGCATTCTCTTGAAGAGTTTTTCAAAGTGTATACCAGAAACGATGATGA CCGCGAAAAGGACATTGAAAATCTCCAAGCAAAAATGCGCAAAGAAATAGCC GCCGCCTTCACCAAACAGGATGTTACGAAAC Illi CTCAAGCAAATTCAAGGA Illi GTTGAAAGAGGCTTGATTAAATATGCATCAAACGAGAAGGAACGCAACA TCGTTTCCCGCTTCAAAGG Illi GCCACTTACTTTACAGGGTTCAATACCAATA GACTGAATATGTACTCAGAAGAAGCAAAATCCACAGCTATATCATTCAGATTAA TTAATCAAAACTTGATAAAGTTCATAGACAACATCCTTGTATATAAAAAAGTGT CTCAAACGTTGCCTTCAGATGTGCTATCAAACATTTATATAGACTTTAAGGCAA TCATCAACACATCAAGTCTTGAAGAATTCTTCTCCATAAACAACTACAATAACA TACTCACCCAGAAACAGATTGAGA Illi CAATGCAGTTATCGGAGGTAAAAAA GACAAGGATGAAAAAATAATAACCAAAGGATTCAACCAATATATAAACGAATAC AACCAGACCAATAAAAACATCCGTCTGCCTAAGATGATGCGGTTATTCAATCA AATCCTAAGCGACAGAGAAGGTGTTTCTGCAAGACCAGAGCCATTCAATAACG CGAACGAGACAATCAGTTCCGTCCGTGATTG Illi ACAAACGAAATATCAAAA CAAATAACGATATTGTCTGAAACAACATCCAAAATTGAATCATTCGACATTGAT AGAATTTACATTAAGGGCGGAGAAGATCTGAGAGCATTATCCAACAGTATATA TGGATATTTCAATTATATCCATGACCGTATCGCAGACAAATGGAAACACAACAA TCCTCAGGGCAAAAAGAGCCCCGAAAGCTACCAAAAAAACCTCAACGCATAT CTGAAAGGCATAAAAAGCGTCTCTTTACACAGTATTGCAAACATCTGTGGTGA CAACAAAGTTATTGAGTATTTCAGGAATCTTGGTGCAGAAAACACTGTTGATTT CCAAAGAGAGAACGTTGTATCATTAATCGACAACAAATACAACTGCGCTTCAA ATCI I I IATCCGACGCCCAAATTACGGATGAAGAACTTCGCACAAACAGTCGC TCAATTAAAGACTTGCTTGACGCCGTCAAGAGTGCCCAACGATTTTTCCGTCT ACTGTGCGGTTCTGGCAACGAACCAGACAAAGACCACTCTTTTTATGACGAGT ATACACCAGCATTTGAAGCACTTGAGAATTCAATAAATCCCCTATATAACAAAG TCAGGAG Illi GTAACCAAAAAAGATTTCTCCACCGATAAATTCAAATTGAATT TCGACAGCAGCAGCTTTCTATCCGGTTGGGCAAAGAAATCAGAATATGAGAA GAGTTCTGCATTTATATTTATTCGCGACAATCAATATTACTTAGGAATAAACAA ATGCCTTAGCAAAGAAGACATTGCCTACCTTGAGGACTCAACAAGCTCATCAG ATACAAAAAGAGTGGTATATATGTTCCAAAAAGTGGACGCCACGAATATTCCC AGAATATTCATCCGTTCCAAAGGTTCCAATTTAGCTCCTGCTGTCAACGAATTC CAACTGCCGATAGAAACCATTCTTGACATTTATGACAATAAGTTCTTCACTACC AGTTATCAGAAAAAAGACCGGACTAAATGGAAAGAATCATTGACCAAACTCAT TGACTATTACAAGCTTGGATTCAGCCAGCACAAGTCATACGCAGATTTCGACT TAAAATGGAAAGCATCCAGTGAATATAACGACATAAATGACTTTCTTGCAGAC GTACAGAGATTCTGCTACAGAATCGAATTTATAAATATCAATTGGGACAAGCT GATAGAATTCACAGAAGATGGCAAAI I I IACCTATTCCGCATTGCAAATAAAGA TTTATCAGGCAATAGCACAGGTCTGCCCAATTTGCACACGATTTATTGGAAAA TGCT Illi GACGAAAGCAACCTCAAAGATATTGTCTATAAATTGTCGGGCAATG CGGAAGTCTTTATGCGCTATAATTCATTAAAAAATCCAATTGTGCATAAAGCGG GAGTGGAGATTAAAAACAAATGCCC Illi ACTGAAAAAAAGACAAGCATATTTG ACTACGACATTATAAAAGACCGTCGCTATACAAAAGATCAGCTTGAACTGCAT GTTCCAATCCTAATGAACTTCAAAAGCCCATCGGCAGCAAAAGGCAAAGCTTT CAACAAAGAATGCTTGGAATACATAAGAAATAATGGTATAAAGCATATTATAGG AATAGACCGAGGTGAACGGAATCTACTTTATATGGTTATAACAGACCTTGACG GCAACATCGTTGAGCAAAAGTCTTTGAACCAAATTGCGAGCAATCCGAAATTG CCTC Illi CAGACAAGACTACAACAAGCTGCTGAAGACAAAGGCTGATGCAAA CGCACAAGCACGTCGTGATTGGGAAACAATAGACACCGTAAAGGAGATAAAA TTCGGCTTCTTGAGTCAGATTGTACATGAGATAGCAATGGCTATCATAAAATAC 22 TABLE1D ZZFT Type V Cas Sequences Name Sequence SEQ ID NO. GATGCAATTGTTG Illi GGAGAATCTGAACAGAGGGTTTATGCAGAAACGAGG TCTTGAAAACAACGTCTATCAGAAATTCGAACAAATGCTGCTTGACAAGTTGA GCTACTATGTCGACAAAACGAAACATCCGGAAGAGGCCGGAGGAGCTTTGCA CGCATATCAGCTCTCTGACACTTACGCGAACTTCAATTCTCTGTCGAAGAATG CGATGGTGCGACAGTCGGG I I I IG I I I I CTATATTCCTGCATGGCTTACAAGC AAAATAGACCCCGTCACAGGATTCGCCTCCTTTTTGAAATTTCACAGAGATGA CAGTATGGCAACAATCAAATCTACAATTTCAAAGTTTGATTGTTTCAAATACGA CAAGGAATGCGACATGTTCCACATCCGCATTGACTATAACAAGTTTAGCACAA GCTGCAGCGGAGGTCAACGCAAATGGGACTTGTTCAC Illi GGCGATCGAAT CTTGGCAGAACGCAATACAATGCAAAACAGCAGATATGTTTACCAAACAGTCA ATTTAACTTCTGAATTCAAAAACTTATTTGCCACAAAGGATATCGACATTTCAG GCAACCTGAAGGACTCTATATGCAAAATTGAGGATGTTGGCTTTTTCAGAAAA CTAAGCCAACTCTTGTCACTCACGCTTCAATTACGCAACAGCAATGCTGAAAC AGGAGAAGACTTCTTGATTTCCCCAGTAGCTGACAAAGATGGCAATTTCTTCG ATTCAAGAAACTGTCCCGACTCTCTCCCAAAAGACGCAGATGCCAATGGCGC ATACAACATTGCTAGGAAGGGATTAATGCTTGTCGAGCAATTGAAGAGATGCA AAGATGTATCAAAATTCAAGCCCGCGATAAAAAACGAGGACTGGTTAGACTAT GTTCAACGCTGA Codon optimized coding sequence (no N-terminal methionine, no stop codon) GAAATCAGTAATCGGTTTACAAACAAGTACCAGGTGTCTAAGACCCTGCGGTT CAGACTGGAGCCTACAGGCGGGACCGATGACCTGCTGTGCCAGGCCCAGAT CATCGAGGGCGATGAGCGGCGCAACAAAGAAGCCATCACCATGAAACAGATC CTCGACAACTGTCACAAGCAGATCATCGAAAGAGTGCTGTCCGACTTCAACTT CAAAGAGCACTCCCTGGAAGAGTTCTTTAAGGTGTACACACGGAACGACGAT GACAGAGAGAAGGATATCGAGAACCTGCAGGCAAAGATGCGCAAGGAAATCG CCGCCGCCTTTACTAAGCAAGACGTGACAAAACTG Illi CTTCCAAGTTTAAA GACTTTGTCGAAAGGGGTCTGATCAAGTACGCCAGCAACGAGAAGGAGCGGA ATATCGTGTCCCGGTTCAAGGGCTTTGCCACATACTTCACCGGCTTCAACACA AACCGCCTGAACATGTACAGCGAGGAAGCCAAATCTACGGCCATTAGCTTCC GGCTGATCAACCAGAACCTCATCAAATTCATCGACAATATCCTGGTGTACAAG AAGGTGTCTCAGACCCTCCCTTCTGATGTCCTGAGCAACATCTACATCGACTT CAAGGCCATCATCAATACCAGCAGCCTGGAGGAGTTCTTCTCCATCAACAACT ACAACAACATCCTGACCCAGAAGCAGATCGAGATCTTCAACGCTGTGATCGG CGGAAAGAAGGATAAGGATGAGAAAATTATCACAAAGGGCTTCAACCAGTACA TCAATGAATATAATCAGACCAACAAGAATATCAGACTGCCAAAGATGATGAGA CTGTTCAATCAGATACTGAGCGACCGGGAAGGCGTGTCAGCTAGACCTGAGC CCTTCAACAACGCCAACGAGACAATCAGCTCCGTGAGAGACTG Illi ACAAAC GAAATCAGCAAGCAGATCACCATCCTGTCTGAAACCACCAGTAAGATCGAGA GCTTCGACATCGATAGAATCTACATCAAGGGCGGAGAGGACCTGCGGGCCCT GAGCAACAGCATCTACGGCTACTTCAACTACATCCACGATAGAATCGCTGATA AGTGGAAGCACAACAATCCTCAGGGCAAGAAGAGCCCCGAGAGCTACCAAAA GAATCTGAACGCCTACCTGAAGGGCATAAAGAGCGTGAGCCTGCATTCTATC GCCAACATCTGTGGCGACAACAAGGTGATCGAATA Illi AGAAATCTCGGCGC CGAGAACACAGTGGA Illi CAGAGAGAAAACGTGGTGTCCCTAATTGACAACA AATACAACTGTGCCTCAAACCTGCTGTCCGACGCCCAAATCACCGACGAGGA GCTGAGGACCAACAGCAGAAGCATCAAGGATCTGCTCGACGCCGTGAAGAGT GCCCAGAGATTCTTCAGACTGCTGTGCGGTTCTGGCAATGAGCCTGATAAAG ACCACAGC Illi ATGACGAGTACACCCCTGCTTTCGAGGCCCTGGAAAACAG CATCAACCCCCTGTACAACAAGGTCCGCAGCTTCGTGACCAAAAAGGACTTC AGCACAGACAAGTTCAAACTGAACTTCGACAGCAGCAGCTTCCTGAGCGGAT GGGCCAAGAAAAGCGAGTACGAGAAGAGCAGCGCTTTCATCTTCATCAGGGA TAATCAGTACTACCTGGGAATTAATAAGTGCCTGAGTAAAGAGGACATCGCCT ACCTGGAGGACAGCACCTCTAGCAGCGACACAAAGAGAGTGGTGTACATGTT TCAGAAGGTGGATGCCACCAATATCCCAAGAATCTTCATCAGATCCAAGGGCA GCAACCTGGCCCCTGCTGTGAACGAGTTCCAGCTGCCTATCGAAACCATCCT GGATATCTACGACAACAAGTTCTTCACCACCAGTTACCAGAAGAAGGATAGAA CCAAATGGAAGGAAAGCCTGACCAAGCTGATCGACTACTACAAGCTGGGCTT TAGCCAGCACAAGTCCTATGCCGATTTCGATTTAAAGTGGAAAGCCAGCTCAG AATACAATGACATCAATGATTTCCTGGCCGACGTGCAGAGATTCTGCTACAGA ATTGAGTTCATCAATATCAATTGGGACAAGCTCATCGAGTTCACAGAGGACGG CAAGTTCTACCTGTTTAGAATCGCCAACAAAGACCTGTCTGGCAACAGCACTG GCCTGCCCAATCTGCACACCATCTACTGGAAGATGCTGTTCGACGAGAGCAA CCTGAAGGACATCGTGTACAAGCTGAGCGGCAACGCTGAGGTGTTTATGCGC TACAACAGCCTGAAGAACCCCATTGTGCACAAGGCCGGAGTGGAAATCAAGA 23 TABLE1D ZZFT Type V Cas Sequences Name Sequence SEQ ID NO. ATAAGTGTCCTTTCACCGAGAAGAAAACCAGCATCTTTGACTACGACATTATC AAGGACCGCAGATACACCAAGGACCAGCTGGAACTGCATGTGCCTATCCTGA TGAACTTCAAGTCTCCATCTGCCGCTAAAGGCAAAGCCTTTAACAAGGAGTGC CTGGAATACATCAGAAACAACGGCATCAAGCACATCATCGGCATCGACAGAG GAGAGCGGAATCTGCTTTACATGGTGATCACAGACCTGGACGGCAACATCGT GGAACAGAAGTCTCTGAACCAGATCGCCTCCAATCCAAAGCTGCCTCTGTTCA GACAGGACTACAACAAGCTGCTGAAAACCAAAGCTGACGCCAACGCACAAGC CAGAAGAGACTGGGAGACAATAGACACCGTGAAGGAGATTAAGTTCGGCTTC CTGAGCCAGATCGTGCACGAGATCGCTATGGCCATCATCAAGTACGACGCCA TTGTGGTCCTGGAAAACCTGAACAGAGGCTTCATGCAAAAACGGGGCCTGGA AAACAACGTGTATCAGAAGTTCGAGCAAATGCTCCTCGATAAACTGAGCTACT ATGTCGACAAGACCAAACACCCTGAGGAAGCTGGCGGAGCCCTGCACGCCT ATCAGTTAAGCGATACCTACGCCAACTTCAATTCCTTGAGCAAGAACGCTATG GTGAGACAGTCTGGCTTCGTGTTCTACATCCCCGCCTGGCTGACCAGCAAGA TCGATCCTGTGACCGGCTTCGCCTCTTTCCTGAAGTTCCACAGAGATGATAGC ATGGCCACCATCAAGAGCACCATCTCCAAATTCGACTGCTTCAAGTACGACAA GGAATGCGACATGTTCCACATCAGAATAGATTACAACAAATTTAGCACTTCAT GCAGCGGTGGCCAGCGGAAGTGGGATCTGTTCACATTCGGAGACAGAATCCT GGCCGAGAGAAACACCATGCAGAACAGTAGATACGTTTACCAGACAGTTAAC CTGACCTCTGAGTTCAAGAACCTGTTCGCCACAAAGGATATCGATATAAGCGG GAACCTGAAGGATAGCATCTGCAAGATCGAGGACGTGGGCTTCTTCCGGAAG CTGAGCCAGCTGCTGAGCCTGACACTACAGCTTCGGAACAGCAACGCTGAAA CCGGAGAAGATTTCCTGATCAGCCCTGTGGCCGACAAGGACGGCAACTTCTT TGACAGCAGAAACTGCCCCGACAGCCTGCCAAAGGATGCAGACGCGAATGG CGCTTATAACATTGCCAGGAAGGGCCTGATGCTGGTGGAGCAACTGAAGCGG TGCAAGGACGTGAGCAAGTTCAAGCCTGCTATCAAGAACGAGGACTGGCTGG ACTACGTGCAGCGG Expression construct (with N-terminal methionine and stop codon, includes V5-tag and C-terminal NLS) ATGggcGAAATCAGTAATCGGTTTACAAACAAGTACCAGGTGTCTAAGACCCTG CGGTTCAGACTGGAGCCTACAGGCGGGACCGATGACCTGCTGTGCCAGGCC CAGATCATCGAGGGCGATGAGCGGCGCAACAAAGAAGCCATCACCATGAAAC AGATCCTCGACAACTGTCACAAGCAGATCATCGAAAGAGTGCTGTCCGACTTC AACTTCAAAGAGCACTCCCTGGAAGAGTTCTTTAAGGTGTACACACGGAACGA CGATGACAGAGAGAAGGATATCGAGAACCTGCAGGCAAAGATGCGCAAGGAA ATCGCCGCCGCCTTTACTAAGCAAGACGTGACAAAACTG Illi CTTCCAAGTT TAAAGACTTTGTCGAAAGGGGTCTGATCAAGTACGCCAGCAACGAGAAGGAG CGGAATATCGTGTCCCGGTTCAAGGGCTTTGCCACATACTTCACCGGCTTCAA CACAAACCGCCTGAACATGTACAGCGAGGAAGCCAAATCTACGGCCATTAGC TTCCGGCTGATCAACCAGAACCTCATCAAATTCATCGACAATATCCTGGTGTA CAAGAAGGTGTCTCAGACCCTCCCTTCTGATGTCCTGAGCAACATCTACATCG ACTTCAAGGCCATCATCAATACCAGCAGCCTGGAGGAGTTCTTCTCCATCAAC AACTACAACAACATCCTGACCCAGAAGCAGATCGAGATCTTCAACGCTGTGAT CGGCGGAAAGAAGGATAAGGATGAGAAAATTATCACAAAGGGCTTCAACCAG TACATCAATGAATATAATCAGACCAACAAGAATATCAGACTGCCAAAGATGAT GAGACTGTTCAATCAGATACTGAGCGACCGGGAAGGCGTGTCAGCTAGACCT GAGCCCTTCAACAACGCCAACGAGACAATCAGCTCCGTGAGAGACTGI IHA CAAACGAAATCAGCAAGCAGATCACCATCCTGTCTGAAACCACCAGTAAGATC GAGAGCTTCGACATCGATAGAATCTACATCAAGGGCGGAGAGGACCTGCGG GCCCTGAGCAACAGCATCTACGGCTACTTCAACTACATCCACGATAGAATCGC TGATAAGTGGAAGCACAACAATCCTCAGGGCAAGAAGAGCCCCGAGAGCTAC CAAAAGAATCTGAACGCCTACCTGAAGGGCATAAAGAGCGTGAGCCTGCATT CTATCGCCAACATCTGTGGCGACAACAAGGTGATCGAATAI I I IAGAAATCTC GGCGCCGAGAACACAGTGGA Illi CAGAGAGAAAACGTGGTGTCCCTAATTG ACAACAAATACAACTGTGCCTCAAACCTGCTGTCCGACGCCCAAATCACCGAC GAGGAGCTGAGGACCAACAGCAGAAGCATCAAGGATCTGCTCGACGCCGTG AAGAGTGCCCAGAGATTCTTCAGACTGCTGTGCGGTTCTGGCAATGAGCCTG ATAAAGACCACAGC Illi ATGACGAGTACACCCCTGCTTTCGAGGCCCTGGAA AACAGCATCAACCCCCTGTACAACAAGGTCCGCAGCTTCGTGACCAAAAAGG ACTTCAGCACAGACAAGTTCAAACTGAACTTCGACAGCAGCAGCTTCCTGAGC GGATGGGCCAAGAAAAGCGAGTACGAGAAGAGCAGCGCTTTCATCTTCATCA GGGATAATCAGTACTACCTGGGAATTAATAAGTGCCTGAGTAAAGAGGACATC GCCTACCTGGAGGACAGCACCTCTAGCAGCGACACAAAGAGAGTGGTGTACA TGTTTCAGAAGGTGGATGCCACCAATATCCCAAGAATCTTCATCAGATCCAAG GGCAGCAACCTGGCCCCTGCTGTGAACGAGTTCCAGCTGCCTATCGAAACCA TCCTGGATATCTACGACAACAAGTTCTTCACCACCAGTTACCAGAAGAAGGAT 24 TABLE1D ZZFT Type V Cas Sequences Name Sequence SEQ ID NO. AGAACCAAATGGAAGGAAAGCCTGACCAAGCTGATCGACTACTACAAGCTGG GCTTTAGCCAGCACAAGTCCTATGCCGATTTCGATTTAAAGTGGAAAGCCAGC TCAGAATACAATGACATCAATGATTTCCTGGCCGACGTGCAGAGATTCTGCTA CAGAATTGAGTTCATCAATATCAATTGGGACAAGCTCATCGAGTTCACAGAGG ACGGCAAGTTCTACCTGTTTAGAATCGCCAACAAAGACCTGTCTGGCAACAGC ACTGGCCTGCCCAATCTGCACACCATCTACTGGAAGATGCTGTTCGACGAGA GCAACCTGAAGGACATCGTGTACAAGCTGAGCGGCAACGCTGAGGTGTTTAT GCGCTACAACAGCCTGAAGAACCCCATTGTGCACAAGGCCGGAGTGGAAATC AAGAATAAGTGTCCTTTCACCGAGAAGAAAACCAGCATCTTTGACTACGACAT TATCAAGGACCGCAGATACACCAAGGACCAGCTGGAACTGCATGTGCCTATC CTGATGAACTTCAAGTCTCCATCTGCCGCTAAAGGCAAAGCCTTTAACAAGGA GTGCCTGGAATACATCAGAAACAACGGCATCAAGCACATCATCGGCATCGAC AGAGGAGAGCGGAATCTGCTTTACATGGTGATCACAGACCTGGACGGCAACA TCGTGGAACAGAAGTCTCTGAACCAGATCGCCTCCAATCCAAAGCTGCCTCT GTTCAGACAGGACTACAACAAGCTGCTGAAAACCAAAGCTGACGCCAACGCA CAAGCCAGAAGAGACTGGGAGACAATAGACACCGTGAAGGAGATTAAGTTCG GCTTCCTGAGCCAGATCGTGCACGAGATCGCTATGGCCATCATCAAGTACGA CGCCATTGTGGTCCTGGAAAACCTGAACAGAGGCTTCATGCAAAAACGGGGC CTGGAAAACAACGTGTATCAGAAGTTCGAGCAAATGCTCCTCGATAAACTGAG CTACTATGTCGACAAGACCAAACACCCTGAGGAAGCTGGCGGAGCCCTGCAC GCCTATCAGTTAAGCGATACCTACGCCAACTTCAATTCCTTGAGCAAGAACGC TATGGTGAGACAGTCTGGCTTCGTGTTCTACATCCCCGCCTGGCTGACCAGC AAGATCGATCCTGTGACCGGCTTCGCCTCTTTCCTGAAGTTCCACAGAGATGA TAGCATGGCCACCATCAAGAGCACCATCTCCAAATTCGACTGCTTCAAGTACG ACAAGGAATGCGACATGTTCCACATCAGAATAGATTACAACAAATTTAGCACTT CATGCAGCGGTGGCCAGCGGAAGTGGGATCTGTTCACATTCGGAGACAGAAT CCTGGCCGAGAGAAACACCATGCAGAACAGTAGATACGTTTACCAGACAGTT AACCTGACCTCTGAGTTCAAGAACCTGTTCGCCACAAAGGATATCGATATAAG CGGGAACCTGAAGGATAGCATCTGCAAGATCGAGGACGTGGGCTTCTTCCGG AAGCTGAGCCAGCTGCTGAGCCTGACACTACAGCTTCGGAACAGCAACGCTG AAACCGGAGAAGATTTCCTGATCAGCCCTGTGGCCGACAAGGACGGCAACTT CTTTGACAGCAGAAACTGCCCCGACAGCCTGCCAAAGGATGCAGACGCGAAT GGCGCTTATAACATTGCCAGGAAGGGCCTGATGCTGGTGGAGCAACTGAAGC GGTGCAAGGACGTGAGCAAGTTCAAGCCTGCTATCAAGAACGAGGACTGGCT GGACTACGTGCAGCGGtctagaAAGCGGACAGCAGACGGCTCCGAATTTGAAA GCCCTAAGAAAAAGAGAAAGGTGggatccGGCAAACCTATCCCCAATCCCCTGC TGGGCCTGGACAGCACCTGA

[0097] In some embodiments a ZZFT Type V Cas protein comprises an amino acid sequence of SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21. In some embodiments, a ZZFT Type V Cas protein has nickase activity, for example resulting from one or more amino acid substitutions relative to the sequence of SEQ ID NO:19, SEQ ID NQ:20, or SEQ ID NO:21. In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D856 substitution, wherein the position of the D856 substitution is defined with respect to the amino acid numbering of SEQ ID NQ:20 (corresponding to amino acid 908 of SEQ ID NO:121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise an E949 substitution, wherein the position of the E949 substitution is defined with respect to the amino acid numbering of SEQ ID NQ:20 (corresponding to amino acid 993 of SEQ ID NO:121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a R1166 substitution, wherein the position of the R1166 substitution is defined with respect to the amino acid numbering of SEQ ID NQ:20 (corresponding to amino acid 1226 of SEQ ID NO:121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D1203 substitution, wherein the position of the D1203 substitution is defined with respect to the amino acid numbering of SEQ ID NQ:20 (corresponding to amino acid 1263 of SEQ ID NO:121). In some embodiments, a ZZFT Type V Cas protein is catalytically inactive, for example due to a R1166 substitution in combination with a D856 substitution, a E949 substitution, and / or D1203 substitution. 6.2.5. YYAN Type V Cas Proteins

[0098] In one aspect, the disclosure provides YYAN Type V Cas proteins. YYAN Type V Cas proteins can be further classified as Type V-A Cas proteins. The YYAN Type V Cas proteins typically comprise an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:25. In some embodiments, the YYAN Type V Cas proteins comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:25. In some embodiments, a YYAN Type V Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:25.

[0099] Exemplary YYAN Type V Cas protein sequences and nucleotide sequences encoding exemplary YYAN Type V Cas proteins are set forth in Table 1E. TABLE 1E YYAN Type V Cas Sequences Name Sequence SEQ ID NO. Wildtype amino acid sequence (without N-terminal methionine) KINAFINCYSMSKTLRFKLAPEYETEKNLLEKGFLDRDKLRADDYDLMKKVIDKYHKH FIDKALEGFKFDLLQEYAEAFYSQSADDDGKKLEEIKKKMCKELATCFSKQDEFKLLD KKELVEKLIPAAEFIEDEEKDIAKRFKGFTTYFTGFNENRQNLYAAELKHGTIAFRLIEE NLPAFLYNCKKGVKIFEGLDAVDAETLNNELGEILSIENVKDVLSVEYYNKTLTQNGID VYNRIIGGYTQEDGTKIKGVNEYVNLYNQTHDKKLPSLAKLKKQILSDSYSLSFLPAKF NDDSELLLSLKKFYSTVNEETGLSVEKAIQEMRDVFSHIDDCDLHNVFIDAKFINKVSN DVFGNWSVLIDGINAEYEKLNPFNGKNLDNYEEKRKAFLNKIESYSVDALQAYSGKEE KIADYVQKRAVELYDSVACAYENMSNKVINAREGKVKLYQDDEKTEIIKTFLDAVQEF KKFAEMFCYDGTDGDTTFYGEFANYYGQIAEIIPLYNKCRNYLTKKPYSEDKIKINFDN AELLHGWDANKEKNYLTVLLFKNGSYYLGILDKKHKNVLIKDVPEKTQEEPCFKKMIY KLLPDPKRNMPRIILHAKSNKKLFEPSDEIYRIYETESFKTDIDDCHRLIDFYKESISKYE DWKTFGFKFKETSEYKNIGQFYNEVKEQGYKISFTDIPESYVKDLVNDGKLYLFRLAN KDFSPYSKGKKNLHTMYFEGIFDPENIKEKVYALNGGGELFFRCASLNYDKPTHPKN VPIKNKTYDFRTDNAKKETSTFEYDLIKDKRYTKDQYTLHCPVTLNFKERGIERINDLV RQSLRESDDNYVIGIDRGERNLIYISVIDGKGKIVEQFSMNNLLSGNDVSIDFHKMLET REHERDASRKNWNTIDNIKDLKQGYLSYVVKKICDLVVKYDAIVAMEDLNVGFKHGR EKFERQVYQKFEKALVDKMSYIVNKNASPHSDGGLFRAYQLTNKKYNENEKQNGFIF YVRAWNTSKIDPTTGFVNMLPLKYQSKEKSKEFFDKFEDIFYDENKDMFGFTFRYDD FGINIDHKNEWTAYSNGERIITVRNSFGKWDKAKIVLTPAFKKLFDDYNVDCRGDVKR QIMNVDDKDFFVRLYKLLSYTMQLRNSDDVDDYILSPVVNAEGKFFDSRNSDGSLPC DADANGAYHIAKKAMWAIGKIKEADEESFKKTSLAIDNKTWLEFVQKA 25 Wildtype amino acid sequence (with N-terminal methionine) MKINAFINCYSMSKTLRFKLAPEYETEKNLLEKGFLDRDKLRADDYDLMKKVIDKYHK HFIDKALEGFKFDLLQEYAEAFYSQSADDDGKKLEEIKKKMCKELATCFSKQDEFKLL DKKELVEKLIPAAEFIEDEEKDIAKRFKGFTTYFTGFNENRQNLYAAELKHGTIAFRLIE ENLPAFLYNCKKGVKIFEGLDAVDAETLNNELGEILSIENVKDVLSVEYYNKTLTQNGI DVYNRIIGGYTQEDGTKIKGVNEYVNLYNQTHDKKLPSLAKLKKQILSDSYSLSFLPAK FNDDSELLLSLKKFYSTVNEETGLSVEKAIQEMRDVFSHIDDCDLHNVFIDAKFINKVS NDVFGNWSVLIDGINAEYEKLNPFNGKNLDNYEEKRKAFLNKIESYSVDALQAYSGK EEKIADYVQKRAVELYDSVACAYENMSNKVINAREGKVKLYQDDEKTEIIKTFLDAVQ EFKKFAEMFCYDGTDGDTTFYGEFANYYGQIAEIIPLYNKCRNYLTKKPYSEDKIKINF DNAELLHGWDANKEKNYLTVLLFKNGSYYLGILDKKHKNVLIKDVPEKTQEEPCFKK MIYKLLPDPKRNMPRIILHAKSNKKLFEPSDEIYRIYETESFKTDIDDCHRLIDFYKESIS KYEDWKTFGFKFKETSEYKNIGQFYNEVKEQGYKISFTDIPESYVKDLVNDGKLYLFR LANKDFSPYSKGKKNLHTMYFEGIFDPENIKEKVYALNGGGELFFRCASLNYDKPTH PKNVPIKNKTYDFRTDNAKKETSTFEYDLIKDKRYTKDQYTLHCPVTLNFKERGIERIN DLVRQSLRESDDNYVIGIDRGERNLIYISVIDGKGKIVEQFSMNNLLSGNDVSIDFHKM LETREHERDASRKNWNTIDNIKDLKQGYLSYVVKKICDLVVKYDAIVAMEDLNVGFKH GREKFERQVYQKFEKALVDKMSYIVNKNASPHSDGGLFRAYQLTNKKYNENEKQNG FIFYVRAWNTSKIDPTTGFVNMLPLKYQSKEKSKEFFDKFEDIFYDENKDMFGFTFRY DDFGINIDHKNEWTAYSNGERIITVRNSFGKWDKAKIVLTPAFKKLFDDYNVDCRGDV KRQIMNVDDKDFFVRLYKLLSYTMQLRNSDDVDDYILSPVVNAEGKFFDSRNSDGSL PCDADANGAYHIAKKAMWAIGKIKEADEESFKKTSLAIDNKTWLEFVQKA 26 TABLE 1E YYAN Type V Cas Sequences Name Sequence SEQ ID NO. Expression construct (with N-terminal methionine, V5-tag and C-terminal NLS) aa sequence MGKINAFINCYSMSKTLRFKLAPEYETEKNLLEKGFLDRDKLRADDYDLMKKVIDKYH KHFIDKALEGFKFDLLQEYAEAFYSQSADDDGKKLEEIKKKMCKELATCFSKQDEFKL LDKKELVEKLIPAAEFIEDEEKDIAKRFKGFTTYFTGFNENRQNLYAAELKHGTIAFRLI EENLPAFLYNCKKGVKIFEGLDAVDAETLNNELGEILSIENVKDVLSVEYYNKTLTQNG IDVYNRIIGGYTQEDGTKIKGVNEYVNLYNQTHDKKLPSLAKLKKQILSDSYSLSFLPA KFNDDSELLLSLKKFYSTVNEETGLSVEKAIQEMRDVFSHIDDCDLHNVFIDAKFINKV SNDVFGNWSVLIDGINAEYEKLNPFNGKNLDNYEEKRKAFLNKIESYSVDALQAYSG KEEKIADYVQKRAVELYDSVACAYENMSNKVINAREGKVKLYQDDEKTEIIKTFLDAV QEFKKFAEMFCYDGTDGDTTFYGEFANYYGQIAEIIPLYNKCRNYLTKKPYSEDKIKIN FDNAELLHGWDANKEKNYLTVLLFKNGSYYLGILDKKHKNVLIKDVPEKTQEEPCFKK MIYKLLPDPKRNMPRIILHAKSNKKLFEPSDEIYRIYETESFKTDIDDCHRLIDFYKESIS KYEDWKTFGFKFKETSEYKNIGQFYNEVKEQGYKISFTDIPESYVKDLVNDGKLYLFR LANKDFSPYSKGKKNLHTMYFEGIFDPENIKEKVYALNGGGELFFRCASLNYDKPTH PKNVPIKNKTYDFRTDNAKKETSTFEYDLIKDKRYTKDQYTLHCPVTLNFKERGIERIN DLVRQSLRESDDNYVIGIDRGERNLIYISVIDGKGKIVEQFSMNNLLSGNDVSIDFHKM LETREHERDASRKNWNTIDNIKDLKQGYLSYVVKKICDLVVKYDAIVAMEDLNVGFKH GREKFERQVYQKFEKALVDKMSYIVNKNASPHSDGGLFRAYQLTNKKYNENEKQNG FIFYVRAWNTSKIDPTTGFVNMLPLKYQSKEKSKEFFDKFEDIFYDENKDMFGFTFRY DDFGINIDHKNEWTAYSNGERIITVRNSFGKWDKAKIVLTPAFKKLFDDYNVDCRGDV KRQIMNVDDKDFFVRLYKLLSYTMQLRNSDDVDDYILSPVVNAEGKFFDSRNSDGSL PCDADANGAYHIAKKAMWAIGKIKEADEESFKKTSLAIDNKTWLEFVQKASRKRTAD GSEFESPKKKRKVGSGKPIPNPLLGLDST 27 Wildtype coding sequence (with N-terminal methionine and stop codon) ATGAAAATTAACGC Illi ATCAACTGTTATTCGATGTCCAAGACGTTGCGATTCAA GCTTGCGCCCGAATACGAGACGGAAAAGAACC Illi GGAAAAGGGATTTCTTGAT CGCGACAAATTGCGCGCGGACGATTATGATTTAATGAAAAAAGTTATCGATAAAT ATCACAAACA Illi ATCGATAAAGCGTTGGAAGGTTTCAAATTCGATTTATTGCAA GAGTATGCCGAAGCGTTTTATTCGCAATCGGCCGATGACGACGGCAAAAAACTT GAAGAAATCAAAAAGAAAATGTGCAAGGAGTTGGCGACTTGTTTTTCGAAACAAG ACGAGTTTAAATTACTCGATAAAAAAGAACTGGTCGAAAAACTAATCCCTGCTGCC GAATTTATTGAAGACGAAGAAAAAGATATTGCGAAGAGATTCAAGGGGTTTACGA CCTATTTCACGGGATTCAACGAAAACAGGCAAAACTTATACGCCGCAGAACTGAA ACACGGGACGATTGCGTTCAGATTGATTGAAGAAAATTTGCCTGCATTTTTGTACA ACTGCAAAAAGGGAGTAAAAATATTCGAGGGACTCGACGCAGTCGATGCAGAAA CGCTTAATAATGAACTTGGAGAGATTCTTTCAATCGAAAACGTAAAAGATGTATTA AGCGTAGAGTATTACAATAAAACGCTCACGCAAAACGGCATAGACGTTTACAACC GGATTATAGGCGGCTATACACAGGAAGACGGGACGAAAATCAAAGGTGTCAACG AGTACGTCAATTTGTATAACCAGACGCACGACAAAAAACTTCCGTCGCTCGCAAA ACTCAAAAAACAGA Illi AAGCGACAG I I AI ICG I IG ICG I I I I I GCCCGCAAAAT TCAACGACGATTCCGAATTGC I I I IAICGCI IAAAAAGI I I IATTCGACGGTAAAC GAAGAGACCGGTTTAAGCGTAGAAAAGGCGATACAGGAAATGCGCGACGTTTTT TCACACATCGATGACTGTGATTTGCATAACGTTTTTATCGACGCAAAATTTATAAA CAAGGTTTCAAACGACG I I I ICGGGAAI I GGAGCG I I I IGATTGACGGCATAAAT GCGGAATATGAGAAACTCAATCCGTTCAACGGGAAAAACCTCGACAATTATGAGG AAAAACGCAAAGCGTTTTTAAACAAGATCGAAAGCTATTCTGTTGACGCGTTGCA GGCATATTCGGGTAAAGAAGAAAAAATCGCCGACTACGTTCAAAAACGTGCGGTC GAACTTTACGATAGTGTCGCATGCGCATATGAGAATATGAGTAATAAGGTAATAAA TGCGCGAGAAGGGAAGGTTAAACTTTATCAGGACGATGAAAAAACCGAAATAATC AAAACG I I I I I GGACGCGG IACAGGAA I I CAAAAAG I I I GCCGAGAI G I I I I GCT ATGACGGCACCGACGGCGATACGACG I I I IACGGCGAATTTGCGAATTATTACG GACAAATTGCCGAAATTATACCGCTTTACAATAAATGCAGGAACTATTTGACGAAA AAGCCGTATTCCGAAGACAAAATCAAAATAAACTTTGACAACGCTGAGC Illi GCA TGGATGGGACGCAAACAAAGAAAAGAATTATCTGACTGTATTATTATTTAAAAACG GCAGTTATTATCTCGGTATTCTGGATAAAAAGCATAAGAACG Illi GATCAAAGAC GTGCCCGAAAAGACGCAGGAGGAGCCGTGTTTCAAGAAAATGATTTACAAATTAC TCCCTGATCCGAAACGAAATATGCCTAGAATAATATTACATGCAAAAAGTAACAAG AAGTTGTTTGAGCCTAGTGATGAGATATATAGGATATATGAAACAGAATCGTTTAA AACTGACATTGACGACTGCCATAGGTTGATTGATTTTTATAAAGAAAGTATAAGCA AGTACGAGGACTGGAAGACGTTCGGGTTCAAGTTCAAAGAAACGAGCGAGTATA AAAACATAGGGCAA Illi ATAACGAAGTTAAAGAGCAGGGATATAAGATTTCATTC ACGGATATACCCGAAAGTTACGTCAAAGACTTGGTAAACGACGGGAAACTGTATT TATTCAGGCTTGCTAATAAAGATTTTTCTCCGTACAGCAAGGGCAAAAAGAATTTG CATACGATGTATTTCGAGGGAATATTTGATCCTGAAAACATAAAAGAAAAGGTTTA TGCGCTTAACGGCGGCGGCGAGTTG I I I I I CAGATGCGCGAGCTTGAATTACGA 28 TABLE 1E YYAN Type V Cas Sequences Name Sequence SEQ ID NO. CAAACCGACGCATCCGAAAAACGTACCGATTAAAAACAAAACGTATGATTTCCGC ACCGATAATGCGAAAAAAGAAACAAGCACGTTTGAATACGACCTCATAAAAGATA AGCGATATACGAAAGATCAATACACGTTGCATTGTCCGGTGACGCTTAAI I I IAA GGAAAGAGGAATCGAAAGAATAAACGATCTCGTAAGGCAATCGTTGCGTGAAAGT GACGACAACTACGTAATCGGCATTGATCGGGGCGAAAGAAACTTAATTTACATCA GTGTTATCGACGGAAAAGGAAAGATTGTCGAGCAATTCTCGATGAACAATTTGTT AAGCGGTAACGACGTGTCGATAGATTTCCACAAAATGCTCGAAACGCGGGAGCA CGAGCGCGACGCGTCCAGAAAAAACTGGAATACAATCGACAATATCAAAGACTTG AAGCAAGGATATTTAAGTTATGTCGTAAAGAAAATTTGCGACCTTGTCGTAAAATA CGACGCGATTGTCGCAATGGAAGACTTAAACGTCGGGTTCAAGCACGGACGAGA AAAGTTCGAGCGACAGGTATATCAGAAATTTGAAAAAGCACTTGTCGACAAAATG AGTTATATCGTAAACAAGAACGCGTCGCCGCATTCCGACGGAGGTTTGTTCAGG GCATACCAGCTGACCAATAAAAAGTATAATGAAAACGAAAAACAAAACGG I I I I AT TTTCTATGTCAGAGCGTGGAATACCAGTAAGATCGATCCGACGACCGGGTTTGTA AACATGCTTCCGTTAAAATATCAGAGCAAAGAAAAATCAAAAGAATTTTTCGATAA ATTTGAAGATAI I I I I IACGAI GAAAACAAGGAIAIG I I UGG Illi ACATTCAGATA TGACGATTTCGGTATAAATATCGATCATAAAAACGAATGGACGGCTTATTCAAACG GCGAACGAATAATCACCGTACGAAATTCGTTCGGCAAGTGGGATAAAGCGAAGA TCGTATTGACGCCGGCATTTAAGAAACTGTTTGACGACTATAACGTGGATTGTCG CGGCGACGTCAAACGACAGATTATGAACGTTGACGACAAAGACTTTTTCGTTAGG TTATATAAGC Illi GTCGTATACGATGCAGTTGAGAAACTCCGACGATGTTGACGA CTATA Illi GTCGCCCGTCGTTAATGCGGAAGGGAAGTTCTTTGACAGTCGCAAT TCGGACGGCAGTTTGCCTTGCGACGCGGACGCAAACGGAGCGTATCATATTGCC AAAAAGGCAATGTGGGCAATCGGGAAGATAAAAGAAGCGGACGAAGAAAG Illi AAAAAGACAAGTCTTGCAATCGACAACAAGACGTGGCTTGAATTCGTTCAAAAGG CATAA Codon optimized coding sequence (no N-terminal methionine, no stop codon) AAGATCAACGCI I I IATCAACTGTTACAGCATGAGCAAGACCCTGAGATTCAAGC TGGCCCCTGAGTACGAAACCGAGAAGAACCTGCTGGAAAAGGGCTTTCTGGACC GGGACAAGCTGAGAGCCGACGACTACGACCTGATGAAGAAGGTGATAGACAAGT ACCACAAGCACTTCATCGACAAGGCCCTGGAAGGCTTCAAGTTTGACCTGCTGC AAGAATACGCTGAGGCC Illi ACAGCCAGAGCGCCGACGACGACGGCAAGAAGC TCGAAGAGATCAAGAAGAAGATGTGCAAGGAGCTGGCCACATGCTTCAGCAAGC AAGACGAGTTCAAGCTACTGGATAAGAAAGAGCTGGTGGAAAAGCTGATCCCAG CCGCTGAGTTCATCGAGGACGAGGAAAAAGACATTGCCAAGAGATTCAAAGGCT TTACAACCTACTTTACCGGCTTCAATGAAAACAGACAGAATCTGTACGCCGCCGA GCTGAAGCACGGAACAATCGCCTTCAGACTGATCGAGGAGAACTTGCCTGCCTT CCTGTACAATTGCAAGAAGGGTGTTAAGATCTTCGAGGGCCTGGACGCTGTGGA TGCTGAGACTCTCAACAACGAGCTGGGCGAGATCCTGAGCATCGAAAACGTGAA GGACGTGCTGTCCGTGGAGTACTACAACAAAACCCTGACCCAAAACGGCATCGA TGTGTACAATAGAATCATCGGCGGCTACACCCAGGAGGATGGCACCAAGATCAA GGGAGTGAACGAGTACGTGAACCTGTATAACCAGACACACGACAAGAAACTGCC TTCTCTGGCTAAGCTGAAGAAGCAAATCCTGTCTGACTCCTATTCTCTGTCATTCC TGCCCGCCAAGTTTAACGACGACTCTGAGCTCCTGCTCAGCCTGAAGAAGI I I IA CAGCACCGTGAACGAGGAAACAGGACTGAGCGTGGAGAAAGCTATCCAGGAGAT GAGAGATGTGTTCAGCCACATTGACGACTGCGACCTTCACAACGTCTTTATCGAT GCCAAGTTCATCAACAAGGTGAGCAACGACGTGTTCGGCAACTGGTCGGTCCTG ATCGATGGCATCAATGCCGAGTACGAGAAGCTGAACCCCTTCAACGGCAAGAAC CTGGACAACTACGAGGAAAAAAGAAAGGCCTTTCTGAACAAAATCGAGAGCTATA GCGTGGACGCCCTGCAGGCCTACAGCGGCAAGGAAGAGAAGATCGCCGATTAT GTGCAGAAACGGGCCGTTGAACTGTACGACAGCGTGGCTTGTGCTTACGAAAAC ATGAGCAACAAAGTGATCAACGCCCGGGAAGGCAAGGTGAAGCTGTACCAGGAC GACGAAAAGACCGAGATTATCAAGACCTTCCTGGATGCTGTTCAGGAGTTCAAGA AGTTCGCCGAAATGTTCTGCTACGATGGAACAGATGGAGATACCACCTTCTACGG CGAGTTCGCCAATTATTACGGCCAGATCGCCGAGATAATCCCCCTGTACAACAAG TGCAGAAACTATCTGACAAAGAAACCTTACAGCGAGGACAAGATTAAGATCAACT TCGATAACGCGGAACTGCTGCATGGATGGGACGCCAACAAGGAAAAGAACTACC TGACAGTCCTGCTGTTCAAAAATGGATCATATTACCTGGGCATCCTGGATAAAAA GCATAAGAACGTGCTGATTAAGGACGTTCCTGAAAAGACACAGGAAGAGCCCTG TTTCAAAAAAATGATCTACAAGCTGCTGCCTGATCCCAAGCGGAATATGCCTAGG ATCATCTTGCACGCCAAAAGCAATAAAAAACTGTTCGAGCCTAGCGATGAGATCT ACAGAATCTATGAGACAGAGAGCTTCAAGACCGACATCGACGATTGCCACAGACT GATCGATTTCTACAAGGAATCCATCAGCAAGTACGAGGACTGGAAAACCTTTGGA TTTAAATTCAAAGAAACCAGCGAGTACAAGAACATCGGACAGTTCTACAACGAGG 29 TABLE 1E YYAN Type V Cas Sequences Name Sequence SEQ ID NO. TGAAGGAACAGGGCTACAAGATTAGCTTCACCGACATCCCTGAGAGCTACGTGA AGGATCTGGTGAATGATGGCAAGCTGTATCTGTTTAGACTCGCCAACAAGGATTT CTCTCCATACTCCAAGGGCAAAAAGAACCTGCACACCATGTACTTCGAGGGAATC TTCGACCCCGAAAACATCAAGGAGAAAGTGTACGCCCTGAACGGCGGCGGCGA GCTGTTCTTCCGCTGTGCCTCTCTGAACTACGACAAGCCTACCCACCCCAAGAAC GTGCCTATCAAGAACAAGACCTACGA Illi AGAACCGATAACGCTAAGAAAGAAA CCAGTACATTCGAGTACGACCTGATCAAAGATAAACGGTACACAAAGGACCAGTA CACACTGCACTGCCCTGTGACACTGAATTTCAAGGAGCGTGGAATCGAACGCAT CAACGACCTGGTGCGGCAGAGCCTGCGGGAAAGCGACGACAACTACGTCATCG GCATCGACAGAGGGGAGAGAAATCTGATCTACATCTCTGTGATCGACGGCAAGG GCAAGATCGTCGAGCAGTTCAGCATGAACAACCTGCTGTCCGGCAACGACGTCA GCATCGACTTCCACAAGATGCTGGAAACCAGAGAGCACGAGCGGGACGCCTCCA GAAAGAACTGGAACACCATCGACAACATCAAGGACCTGAAGCAGGGCTACCTGA GTTACGTGGTGAAAAAGATCTGCGACCTGGTCGTGAAGTATGATGCCATCGTGG CTATGGAGGATCTGAACGTGGGCTTTAAACACGGCAGAGAGAAGTTCGAGAGAC AGGTGTACCAGAAGTTTGAGAAAGCCCTGGTGGACAAGATGAGCTACATCGTGA ATAAAAATGCTAGTCCTCACAGCGATGGCGGCCTGTTCAGAGCTTATCAGCTGAC CAACAAGAAATACAACGAGAATGAAAAGCAGAACGGATTCATCI I I IACGTGAGA GCCTGGAATACCAGCAAGATCGACCCAACAACAGGCTTCGTGAACATGTTGCCA CTGAAATACCAATCTAAGGAAAAGTCCAAGGAGTTCTTCGACAAGTTCGAGGATA TCTTCTATGATGAAAACAAAGACATGTTCGGCTTCACCTTCCGGTACGACGACTT CGGCATCAACATCGACCACAAGAATGAATGGACCGCCTACAGCAATGGTGAGCG GATCATCACCGTGCGGAACAGCTTCGGCAAATGGGATAAAGCGAAGATCGTGCT GACCCCTGC Illi AAGAAGCTGTTCGATGATTACAACGTGGACTGCAGAGGCGA CGTGAAGCGACAGATTATGAACGTGGACGACAAAGATTTCTTCGTGCGGCTGTA CAAGCTGCTGAGCTACACCATGCAGCTGAGAAACAGCGACGACGTGGACGATTA CATCCTGAGCCCCGTGGTGAATGCCGAAGGCAAGTTCTTCGACAGCAGAAACTC TGACGGCTCTCTGCCTTGTGACGCCGATGCCAACGGCGCCTACCACATCGCCAA GAAGGCCATGTGGGCCATCGGCAAGATCAAGGAAGCCGATGAGGAATC I I I IAA GAAAACCTCCCTCGCCATCGACAACAAAACCTGGCTGGAGTTCGTGCAGAAAGC C Expression construct (with N-terminal methionine and stop codon, includes V5-tag and C-terminal NLS) ATGggcAAGATCAACGC I I I IAI CAACTGTTACAGCATGAGCAAGACCCTGAGATT CAAGCTGGCCCCTGAGTACGAAACCGAGAAGAACCTGCTGGAAAAGGGCTTTCT GGACCGGGACAAGCTGAGAGCCGACGACTACGACCTGATGAAGAAGGTGATAG ACAAGTACCACAAGCACTTCATCGACAAGGCCCTGGAAGGCTTCAAGTTTGACCT GCTGCAAGAATACGCTGAGGCC Illi ACAGCCAGAGCGCCGACGACGACGGCAA GAAGCTCGAAGAGATCAAGAAGAAGATGTGCAAGGAGCTGGCCACATGCTTCAG CAAGCAAGACGAGTTCAAGCTACTGGATAAGAAAGAGCTGGTGGAAAAGCTGAT CCCAGCCGCTGAGTTCATCGAGGACGAGGAAAAAGACATTGCCAAGAGATTCAA AGGCTTTACAACCTACTTTACCGGCTTCAATGAAAACAGACAGAATCTGTACGCC GCCGAGCTGAAGCACGGAACAATCGCCTTCAGACTGATCGAGGAGAACTTGCCT GCCTTCCTGTACAATTGCAAGAAGGGTGTTAAGATCTTCGAGGGCCTGGACGCT GTGGATGCTGAGACTCTCAACAACGAGCTGGGCGAGATCCTGAGCATCGAAAAC GTGAAGGACGTGCTGTCCGTGGAGTACTACAACAAAACCCTGACCCAAAACGGC ATCGATGTGTACAATAGAATCATCGGCGGCTACACCCAGGAGGATGGCACCAAG ATCAAGGGAGTGAACGAGTACGTGAACCTGTATAACCAGACACACGACAAGAAA CTGCCTTCTCTGGCTAAGCTGAAGAAGCAAATCCTGTCTGACTCCTATTCTCTGT CATTCCTGCCCGCCAAGTTTAACGACGACTCTGAGCTCCTGCTCAGCCTGAAGAA Gill IACAGCACCGTGAACGAGGAAACAGGACTGAGCGTGGAGAAAGCTATCCA GGAGATGAGAGATGTGTTCAGCCACATTGACGACTGCGACCTTCACAACGTCTTT ATCGATGCCAAGTTCATCAACAAGGTGAGCAACGACGTGTTCGGCAACTGGTCG GTCCTGATCGATGGCATCAATGCCGAGTACGAGAAGCTGAACCCCTTCAACGGC AAGAACCTGGACAACTACGAGGAAAAAAGAAAGGCCTTTCTGAACAAAATCGAGA GCTATAGCGTGGACGCCCTGCAGGCCTACAGCGGCAAGGAAGAGAAGATCGCC GATTATGTGCAGAAACGGGCCGTTGAACTGTACGACAGCGTGGCTTGTGCTTAC GAAAACATGAGCAACAAAGTGATCAACGCCCGGGAAGGCAAGGTGAAGCTGTAC CAGGACGACGAAAAGACCGAGATTATCAAGACCTTCCTGGATGCTGTTCAGGAG TTCAAGAAGTTCGCCGAAATGTTCTGCTACGATGGAACAGATGGAGATACCACCT TCTACGGCGAGTTCGCCAATTATTACGGCCAGATCGCCGAGATAATCCCCCTGTA CAACAAGTGCAGAAACTATCTGACAAAGAAACCTTACAGCGAGGACAAGATTAAG ATCAACTTCGATAACGCGGAACTGCTGCATGGATGGGACGCCAACAAGGAAAAG AACTACCTGACAGTCCTGCTGTTCAAAAATGGATCATATTACCTGGGCATCCTGG ATAAAAAGCATAAGAACGTGCTGATTAAGGACGTTCCTGAAAAGACACAGGAAGA 30 TABLE 1E YYAN Type V Cas Sequences Name Sequence SEQ ID NO. GCCCTGTTTCAAAAAAATGATCTACAAGCTGCTGCCTGATCCCAAGCGGAATATG CCTAGGATCATCTTGCACGCCAAAAGCAATAAAAAACTGTTCGAGCCTAGCGATG AGATCTACAGAATCTATGAGACAGAGAGCTTCAAGACCGACATCGACGATTGCCA CAGACTGATCGATTTCTACAAGGAATCCATCAGCAAGTACGAGGACTGGAAAACC TTTGGATTTAAATTCAAAGAAACCAGCGAGTACAAGAACATCGGACAGTTCTACAA CGAGGTGAAGGAACAGGGCTACAAGATTAGCTTCACCGACATCCCTGAGAGCTA CGTGAAGGATCTGGTGAATGATGGCAAGCTGTATCTGTTTAGACTCGCCAACAAG GATTTCTCTCCATACTCCAAGGGCAAAAAGAACCTGCACACCATGTACTTCGAGG GAATCTTCGACCCCGAAAACATCAAGGAGAAAGTGTACGCCCTGAACGGCGGCG GCGAGCTGTTCTTCCGCTGTGCCTCTCTGAACTACGACAAGCCTACCCACCCCA AGAACGTGCCTATCAAGAACAAGACCTACGAI I I IAGAACCGATAACGCTAAGAA AGAAACCAGTACATTCGAGTACGACCTGATCAAAGATAAACGGTACACAAAGGAC CAGTACACACTGCACTGCCCTGTGACACTGAATTTCAAGGAGCGTGGAATCGAA CGCATCAACGACCTGGTGCGGCAGAGCCTGCGGGAAAGCGACGACAACTACGT CATCGGCATCGACAGAGGGGAGAGAAATCTGATCTACATCTCTGTGATCGACGG CAAGGGCAAGATCGTCGAGCAGTTCAGCATGAACAACCTGCTGTCCGGCAACGA CGTCAGCATCGACTTCCACAAGATGCTGGAAACCAGAGAGCACGAGCGGGACG CCTCCAGAAAGAACTGGAACACCATCGACAACATCAAGGACCTGAAGCAGGGCT ACCTGAGTTACGTGGTGAAAAAGATCTGCGACCTGGTCGTGAAGTATGATGCCAT CGTGGCTATGGAGGATCTGAACGTGGGCTTTAAACACGGCAGAGAGAAGTTCGA GAGACAGGTGTACCAGAAGTTTGAGAAAGCCCTGGTGGACAAGATGAGCTACAT CGTGAATAAAAATGCTAGTCCTCACAGCGATGGCGGCCTGTTCAGAGCTTATCAG CTGACCAACAAGAAATACAACGAGAATGAAAAGCAGAACGGATTCATCI I I IACG TGAGAGCCTGGAATACCAGCAAGATCGACCCAACAACAGGCTTCGTGAACATGT TGCCACTGAAATACCAATCTAAGGAAAAGTCCAAGGAGTTCTTCGACAAGTTCGA GGATATCTTCTATGATGAAAACAAAGACATGTTCGGCTTCACCTTCCGGTACGAC GACTTCGGCATCAACATCGACCACAAGAATGAATGGACCGCCTACAGCAATGGT GAGCGGATCATCACCGTGCGGAACAGCTTCGGCAAATGGGATAAAGCGAAGATC GTGCTGACCCCTGC Illi AAGAAGCTGTTCGATGATTACAACGTGGACTGCAGAG GCGACGTGAAGCGACAGATTATGAACGTGGACGACAAAGATTTCTTCGTGCGGC TGTACAAGCTGCTGAGCTACACCATGCAGCTGAGAAACAGCGACGACGTGGACG ATTACATCCTGAGCCCCGTGGTGAATGCCGAAGGCAAGTTCTTCGACAGCAGAA ACTCTGACGGCTCTCTGCCTTGTGACGCCGATGCCAACGGCGCCTACCACATCG CCAAGAAGGCCATGTGGGCCATCGGCAAGATCAAGGAAGCCGATGAGGAATCTT TTAAGAAAACCTCCCTCGCCATCGACAACAAAACCTGGCTGGAGTTCGTGCAGAA AGCCtctagaAAGCGGACAGCAGACGGCTCCGAATTTGAAAGCCCTAAGAAAAAGA GAAAGGTGggatccGGCAAACCTATCCCCAATCCCCTGCTGGGCCTGGACAGCAC CTGA

[0100] In some embodiments a YYAN Type V Cas protein comprises an amino acid sequence of SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:27. In some embodiments, a YYAN Type V Cas protein has nickase activity, for example resulting from one or more amino acid substitutions relative to the sequence of SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:27. In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D838 substitution, wherein the position of the D838 substitution is defined with respect to the amino acid numbering of SEQ ID NO:26 (corresponding to amino acid 908 of SEQ ID NO:121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise an E928 substitution, wherein the position of the E928 substitution is defined with respect to the amino acid numbering of SEQ ID NO:26 (corresponding to amino acid 993 of SEQ ID NO:121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a R1135 substitution, wherein the position of the R1135 substitution is defined with respect to the amino acid numbering of SEQ ID NO:26 (corresponding to amino acid 1226 of SEQ ID NO:121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D1170 substitution, wherein the position of the D1170 substitution is defined with respect to the amino acid numbering of SEQ ID NO:26 (corresponding to amino acid 1263 of SEQ ID NO:121). In some embodiments, a YYAN Type V Cas protein is catalytically inactive, for example due to a R1135 substitution in combination with a D838 substitution, a E928 substitution, and / or D1170 substitution. 6.2.6. ZZGY Type V Cas Proteins

[0101] In one aspect, the disclosure provides ZZGY Type V Cas proteins. ZZGY Type V Cas proteins can be further classified as Type V-A Cas proteins. The ZZGY Type V Cas proteins typically comprise an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:31. In some embodiments, the ZZGY Type V Cas proteins comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:31. In some embodiments, a ZZGY Type V Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:31.

[0102] Exemplary ZZGY Type V Cas protein sequences and nucleotide sequences encoding exemplary ZZGY Type V Cas proteins are set forth in Table 1F. TABLE 1F ZZGY Type V Cas Sequences Name Sequence SEQ ID NO. Wildtype amino acid sequence (without N-terminal methionine) SKLSTFNEHFQKTLTLRNELVPVGKTLENIISSNVLINDEKRSEDYKKAKEIIDSYHREFI EKSLSSVNVDWNDLYSYLSKKEPEDYAQKQKFLEELENILLEKRKIIVKQFEQYVFGS YTDSKGKKTKDLKFENLFKSELFDYLLPNFLKNDEDKKVIGSFNKFTSYFTGFYENRK NLYKSEPLPTAVAYRIVNENFPKFISNKNIFRVWKDNVPQFIEIAKTKLREEGISDLNIEL KFDLTNFNSCLNQTGIDTYNDLIGQLNFAINLECQKDKNLCDLLRKKRSLKMVPLYKQI LSDNDSSFSIDEFDNDESAIKDVISFYKKMIGENCPQRTLSELLHGLSSHDLEKIFVQG KNLNSVSKNLFGGKNWSLLRDAVIEEKSKEKVFKKVIKSNSTADELDKVLSKEEFSISF LSKVSGKDLSVEIDKFVKKQDELLVENNIQNWPSSLKNSEEKNLIKAPLDFLLNFYRFA QSFSSNNIDKDMSFYADFDESLSSLENVIGLYNKVRNYATKKPYTLEKIKLNFENPNLA SGWSESKENDCLSIILLKEKKYFLGIFNKNNKPNFSEGISHSLSSNGCYRKMRYLLFK GFNKMLPKCAFTGEVKDHFKESSDDFSLFNKDTFISPLVITKEIFDLACSKEKVKKYQK EYEKINRAEYRQSLVKWITFGLKFLSSYKTTTQFDLSNLKRPEEYCDLKEFYEDVDNL TYKIEFLNIKEEDVDALVEKGQLYLFEIRNKDFAKNASGTPNLHTLYFKSIFDSKNLEN GIVKLNGEAEIFYRKKSLKKDDITVHREGSYLVNKVCVDPNSGKTEQIPDKIYENIYAF VNGKSRDLSKEDEVYYAKATIKKATHEIVKDRRFTVDKFFFHCPITINYKSKDKPSKFN DKVLDFLRNNKDINIIGIDRGERNLIYVTVINQNGEIIDCKSFNTIKHQSSTVNYDVDYH NKLQEREKNRKEEKRSWNSITKIADLKEGYLSAVIHEVSLMMVKYNAIVVMENLNQGF KRIRGGIAERSVYQKFEKMLIDKLNYFVIKNENWTNPGGVLNGYQLTNKVSTIKDIGN QCGFLFYVPATYTSKIDPSTGFVNLINFNKYKNSEDRRKLICSFDKICFVQNENLFKFSI DYGKLCPDSKIAIKKWDVFSYGTRIIKENLTTGHIEENPEYDPTEELKSLLSSRGIEYQK GQNLLETIPTSDMTREFWNSLFKIFKAILQMRNSLTNSPIDRLLSPVKGKDGTFFDTDK VEGTKFEKLKDADANGAYNIALKGLLVLEKNDSVESNKDLKNVKKISLEDWLKFVQITL RD 31 Wildtype amino acid sequence (with N-terminal methionine) MSKLSTFNEHFQKTLTLRNELVPVGKTLENIISSNVLINDEKRSEDYKKAKEIIDSYHRE FIEKSLSSVNVDWNDLYSYLSKKEPEDYAQKQKFLEELENILLEKRKIIVKQFEQYVFG SYTDSKGKKTKDLKFENLFKSELFDYLLPNFLKNDEDKKVIGSFNKFTSYFTGFYENR KNLYKSEPLPTAVAYRIVNENFPKFISNKNIFRVWKDNVPQFIEIAKTKLREEGISDLNI ELKFDLTNFNSCLNQTGIDTYNDLIGQLNFAINLECQKDKNLCDLLRKKRSLKMVPLYK QILSDNDSSFSIDEFDNDESAIKDVISFYKKMIGENCPQRTLSELLHGLSSHDLEKIFVQ GKNLNSVSKNLFGGKNWSLLRDAVIEEKSKEKVFKKVIKSNSTADELDKVLSKEEFSI SFLSKVSGKDLSVEIDKFVKKQDELLVENNIQNWPSSLKNSEEKNLIKAPLDFLLNFYR FAQSFSSNNIDKDMSFYADFDESLSSLENVIGLYNKVRNYATKKPYTLEKIKLNFENP NLASGWSESKENDCLSIILLKEKKYFLGIFNKNNKPNFSEGISHSLSSNGCYRKMRYL LFKGFNKMLPKCAFTGEVKDHFKESSDDFSLFNKDTFISPLVITKEIFDLACSKEKVKK YQKEYEKINRAEYRQSLVKWITFGLKFLSSYKTTTQFDLSNLKRPEEYCDLKEFYEDV DNLTYKIEFLNIKEEDVDALVEKGQLYLFEIRNKDFAKNASGTPNLHTLYFKSIFDSKNL ENGIVKLNGEAEIFYRKKSLKKDDITVHREGSYLVNKVCVDPNSGKTEQIPDKIYENIY AFVNGKSRDLSKEDEVYYAKATIKKATHEIVKDRRFTVDKFFFHCPITINYKSKDKPSK FNDKVLDFLRNNKDINIIGIDRGERNLIYVTVINQNGEIIDCKSFNTIKHQSSTVNYDVDY 32 TABLE 1F ZZGY Type V Cas Sequences Name Sequence SEQ ID NO. HNKLQEREKNRKEEKRSWNSITKIADLKEGYLSAVIHEVSLMMVKYNAIVVMENLNQ GFKRIRGGIAERSVYQKFEKMLIDKLNYFVIKNENWTNPGGVLNGYQLTNKVSTIKDIG NQCGFLFYVPATYTSKIDPSTGFVNLINFNKYKNSEDRRKLICSFDKICFVQNENLFKF SIDYGKLCPDSKIAIKKWDVFSYGTRIIKENLTTGHIEENPEYDPTEELKSLLSSRGIEY QKGQNLLETIPTSDMTREFWNSLFKIFKAILQMRNSLTNSPIDRLLSPVKGKDGTFFDT DKVEGTKFEKLKDADANGAYNIALKGLLVLEKNDSVESNKDLKNVKKISLEDWLKFVQ ITLRD Expression construct (with N-terminal methionine, V5-tag and C-terminal NLS) aa sequence MGSKLSTFNEHFQKTLTLRNELVPVGKTLENIISSNVLINDEKRSEDYKKAKEIIDSYHR EFIEKSLSSVNVDWNDLYSYLSKKEPEDYAQKQKFLEELENILLEKRKIIVKQFEQYVF GSYTDSKGKKTKDLKFENLFKSELFDYLLPNFLKNDEDKKVIGSFNKFTSYFTGFYEN RKNLYKSEPLPTAVAYRIVNENFPKFISNKNIFRVWKDNVPQFIEIAKTKLREEGISDLN IELKFDLTNFNSCLNQTGIDTYNDLIGQLNFAINLECQKDKNLCDLLRKKRSLKMVPLY KQILSDNDSSFSIDEFDNDESAIKDVISFYKKMIGENCPQRTLSELLHGLSSHDLEKIFV QGKNLNSVSKNLFGGKNWSLLRDAVIEEKSKEKVFKKVIKSNSTADELDKVLSKEEFS ISFLSKVSGKDLSVEIDKFVKKQDELLVENNIQNWPSSLKNSEEKNLIKAPLDFLLNFY RFAQSFSSNNIDKDMSFYADFDESLSSLENVIGLYNKVRNYATKKPYTLEKIKLNFEN PNLASGWSESKENDCLSIILLKEKKYFLGIFNKNNKPNFSEGISHSLSSNGCYRKMRY LLFKGFNKMLPKCAFTGEVKDHFKESSDDFSLFNKDTFISPLVITKEIFDLACSKEKVK KYQKEYEKINRAEYRQSLVKWITFGLKFLSSYKTTTQFDLSNLKRPEEYCDLKEFYED VDNLTYKIEFLNIKEEDVDALVEKGQLYLFEIRNKDFAKNASGTPNLHTLYFKSIFDSKN LENGIVKLNGEAEIFYRKKSLKKDDITVHREGSYLVNKVCVDPNSGKTEQIPDKIYENI YAFVNGKSRDLSKEDEVYYAKATIKKATHEIVKDRRFTVDKFFFHCPITINYKSKDKPS KFNDKVLDFLRNNKDINIIGIDRGERNLIYVTVINQNGEIIDCKSFNTIKHQSSTVNYDVD YHNKLQEREKNRKEEKRSWNSITKIADLKEGYLSAVIHEVSLMMVKYNAIWMENLNQ GFKRIRGGIAERSVYQKFEKMLIDKLNYFVIKNENWTNPGGVLNGYQLTNKVSTIKDIG NQCGFLFYVPATYTSKIDPSTGFVNLINFNKYKNSEDRRKLICSFDKICFVQNENLFKF SIDYGKLCPDSKIAIKKWDVFSYGTRIIKENLTTGHIEENPEYDPTEELKSLLSSRGIEY QKGQNLLETIPTSDMTREFWNSLFKIFKAILQMRNSLTNSPIDRLLSPVKGKDGTFFDT DKVEGTKFEKLKDADANGAYNIALKGLLVLEKNDSVESNKDLKNVKKISLEDWLKFVQ ITLRDSRKRTADGSEFESPKKKRKVGSGKPIPNPLLGLDST 33 Wildtype coding sequence (with N-terminal methionine and stop codon) ATGTCTAAATTATCAAC Illi AATGAACA Illi CAAAAAACGTTAACTTTAAGAAAC GAACTAGTTCCTGTAGGAAAAACTCTTGAAAATATCATATCTTCAAATGTATTGATA AATGATGAGAAAAGAAGTGAAGATTATAAAAAGGCTAAAGAGATCATAGATTCTTA TCATCGAGAGTTTATAGAGAAATCACTTTCATCAGTAAATGTTGATTGGAATGATC TGTACTCGTATTTATCCAAAAAAGAACCAGAAGACTATGCTCAAAAGCAGAAGTTC CTCGAAGAGTTAGAAAATATTCTCCTTGAAAAGAGAAAAATTATTGTTAAACAGTT TGAGCAATACG Illi CGGATCATATACAGATTCAAAAGGTAAAAAAACAAAAGATC TAAAATTTGAGAATC I I I I I AAA ICAGAG I I G I I I GA I IAIC I I I I GCCAAATTTCC TAAAAAATGATGAAGATAAAAAAGTAATAGGTAGI I I IAATAAATTTACATCGTATT TTACAGGTI I I IACGAAAATCGAAAGAATTTATATAAATCAGAGCCATTGCCAACA GCTGTGGCTTATAGAATAGTTAACGAAAACTTTCCTAAATTCATTTCTAATAAAAAT ATCTTTCGCGTGTGGAAAGATAATGTTCCTCAGTTTATAGAAATAGCGAAAACTAA ACTAAGAGAAGAAGGCATTTCTGATTTAAATATAGAATTAAAATTTGATTTAACTAA TTTCAATTCATGCTTAAATCAAACTGGAATTGATACTTACAATGACTTGATAGGTCA ACTCAACTTTGCAATTAACCTTGAATGTCAGAAAGACAAGAATTTATGTGACCTTT TAAGGAAGAAAAGAAGCCTTAAAATGGTACCTCTGTATAAACAGA Illi ATCTGAT AATGATTCTTCATTCAGTATTGATGAATTTGATAATGATGAATCGGCAATAAAAGAT GTAATTTCTTTTTATAAGAAAATGATTGGTGAAAATTGTCCTCAACGAACACTATCT GAATTGCTACATGGTTTGTCATCTCACGATCTTGAAAAGATATTTGTTCAAGGTAA AAACTTAAATTCGGTTTCTAAAAATTTATTTGGAGGGAAGAACTGGTCTTTACTAA GGGATGCAGTTATAGAAGAAAAGTCAAAAGAAAAAGTCTTCAAAAAGGTTATAAA GTCAAATTCTACCGCAGATGAATTAGACAAAGTTCTTTCCAAGGAAGAA Illi CAA TTTCATTCTTATCAAAAGTGAGCGGTAAAGATTTATCAGTAGAAATTGATAAATTTG TAAAAAAACAAGACGAACTACTTGTTGAAAATAATATACAAAATTGGCCAAGTTCT CTTAAGAACAGCGAAGAGAAAAATCTCATAAAAGCTCCTTTAGATTTCTTACTTAA TTTTTATAGATTTGCACAATCATTCTCTTCAAATAATATTGATAAGGATATGTCATTT TATGCTGACTTTGATGAATCTCTATCGTCTTTAGAAAATGTAATAGGTCTTTATAAC AAAGTCAGAAACTATGCAACTAAGAAACCTTATACACTCGAAAAGATCAAATTGAA Illi GAAAATCCAAATTTAGCTTCTGGATGGAGTGAAAGCAAAGAAAATGATTGTT TATCAATTATCTTATTAAAAGAGAAAAAATAI Illi IAGGAAI I I I CAACAAAAATAA TAAACCTAATTTTTCTGAAGGCATTTCTCATTCACTTTCTTCAAATGGTTGCTACAG AAAAATGAGGTATTTATTATTCAAGGGATTCAATAAAATGCTTCCTAAATGTGCTTT TACAGGAGAAGTTAAAGATCAI I I IAAAGAAICAICGGAI GA I I I I IC I C I I I I I AA 34 TABLE 1F ZZGY Type V Cas Sequences Name Sequence SEQ ID NO. CAAGGATAC I I I IA I'CTCTCCTCTTGTAATTACCAAAGAGATCTTTGATTTAGCATG TAGTAAAGAAAAGGTAAAAAAATATCAAAAAGAATATGAAAAGATCAATCGTGCTG AATATAGACAATCATTGGTTAAGTGGATTAC I I I I GGI C l I AAA I Illi GTCATCAT ATAAAACTACAACTCAATTTGATTTATCAAATTTAAAAAGACCTGAAGAATACTGCG ATCTAAAGGAAI I I IAI GAAGAI G I AGA IAAI C I IACAIACAAGAIAGAAI I I I IAA ATATAAAAGAAGAAGATGTAGATGCATTGGTTGAAAAAGGTCAACTGTATTTATTT GAAATTCGAAATAAAGA Illi GCAAAAAATGCAAGTGGCACTCCTAATCTACATAC TCTCTAI I I IAAAAGIAI I I I CGATTCGAAAAATTTAGAGAATGGCATTGTCAAGCT TAATGGTGAAGCAGAGATAI I I IATAGAAAGAAAAGCTTGAAGAAAGATGACATAA CTGTTCATCGAGAAGGCAGTTATCTTGTAAATAAGGTGTGTGTCGATCCTAATTCT GGAAAAACAGAACAGATTCCTGACAAAATTTATGAAAATATTTATGCTTTCGTAAA TGGTAAATCAAGAGATTTATCTAAGGAGGATGAAGTATATTATGCAAAAGCCACAA TAAAAAAAGCTACCCATGAGATCGTAAAAGATAGACGCTTTACTGTAGATAAATTC TTTTTCCACTGCCCTATTACTATTAACTATAAATCTAAAGATAAACCTTCAAAATTC AATGACAAGG Illi AGATTTCTTAAGAAATAATAAAGACATCAACATTATAGGCATA GATCGAGGAGAGAGAAATCTTATTTATGTAACTGTAATTAATCAAAATGGCGAAAT TATTGATTGCAAATCATTTAATACTATCAAACATCAGTCTTCAACAGTGAATTACGA TGTTGATTATCACAACAAATTACAAGAAAGAGAAAAAAATAGAAAAGAAGAAAAGA GATCTTGGAATAGTATTACTAAAATTGCAGATCTCAAAGAAGGCTATCTTTCTGCT GTAATTCATGAAGTTTCATTAATGATGGTTAAGTACAATGCCATTGTCGTTATGGA AAATTTGAATCAAGG Illi AAGAGAATTAGAGGAGGAATTGCTGAAAGATCCGTAT ACCAAAAATTTGAAAAGATGCTGATAGATAAACTGAATTA Illi GTTATAAAAAATG AAAATTGGACAAATCCTGGTGGGGTCCTCAATGGATATCAGTTAACTAACAAAGT GTCTACAATCAAAGATATCGGTAATCAGTGTGGAI I I I IAI I I IACGTTCCTGCAA CTTATACCTCAAAGATTGATCCTTCTACAGGCTTTGTTAATTTAATTAATTTCAATA AATATAAAAATTCAGAAGATCGAAGAAAACTCATTTGTAGCTTTGACAAGATATGC TTTGTACAGAATGAGAATTTATTTAAA Illi CTATAGATTATGGAAAATTATGCCCA GATAGCAAAATTGCTATAAAAAAATGGGATG Illi CTCCTACGGAACAAGAATTAT TAAGGAAAATCTAACAACTGGTCATATAGAAGAAAATCCTGAATACGATCCGACA GAAGAGCTTAAATCTCTGCTTTCCTCAAGAGGAATTGAGTATCAAAAAGGTCAAAA TTTACTAGAAACAATACCTACTAGTGATATGACTAGAGAA Illi GGAAI IC IC I I I I CAAGAI I I I IAAAGCAA Illi ACAAATGAGAAACAGTCTAACTAATTCACCAATAGA CAGGC Illi ATCTCCAGTTAAAGGAAAAGATGGAACCTTCTTTGATACAGATAAAG TAGAAGGTACTAAGTTTGAAAAGTTAAAAGACGCTGATGCAAACGGAGCATATAA CATTGCGTTAAAAGGATTGTTAGTCCTCGAGAAAAATGATTCTGTAGAGTCCAATA AGGATCTAAAAAATGTTAAGAAAATTAGTCTTGAGGATTGGTTAAAGTTTGTCCAA ATCACATTAAGAGATTAA Codon optimized coding sequence (no N-terminal methionine, no stop codon) AGCAAATTGTCGACCTTCAATGAGCACTTTCAGAAAACCCTGACCCTGCGGAATG AGCTGGTGCCCGTGGGCAAGACACTGGAGAACATCATCAGCTCTAACGTGCTGA TCAACGACGAGAAGCGGTCCGAGGACTACAAAAAGGCCAAGGAAATCATTGACA GCTATCACCGGGAGTTCATCGAGAAAAGCCTGAGCTCTGTGAATGTGGACTGGA ATGATCTGTACAGCTACCTGAGCAAGAAAGAACCCGAGGACTATGCCCAGAAAC AGAAGTTCCTGGAGGAGTTAGAGAACATCCTGCTGGAAAAGAGAAAGATCATCGT GAAGCAGTTCGAGCAGTACGTGTTCGGTTCCTATACCGACAGCAAGGGAAAAAA GACCAAGGACCTGAAATTCGAAAACCTGTTTAAGTCCGAACTCTTTGACTACCTG CTGCCTAACTTCTTGAAAAACGACGAGGATAAGAAGGTGATTGGCTCCTTCAATA AGTTCACCAGCTATTTCACCGGC I I I IACGAGAACAGAAAAAACCTGTACAAGAG CGAGCCTCTGCCTACCGCCGTCGCCTACAGAATCGTGAACGAGAACTTCCCCAA GTTTATCTCTAACAAGAACATCTTTAGAGTGTGGAAGGACAACGTCCCTCAATTCA TCGAGATCGCAAAGACCAAACTGAGAGAAGAAGGCATCTCTGATCTGAACATCGA GCTGAAGTTTGATTTGACAAATTTCAACTCCTGCCTGAATCAGACCGGCATCGAT ACCTACAACGACCTGATCGGCCAGCTGAACTTTGCTATCAACCTCGAATGTCAGA AGGACAAGAACCTTTGTGACCTGCTGCGCAAGAAGCGGAGCCTTAAGATGGTGC CACTGTACAAGCAAATCCTGTCCGACAACGATAGCAGCTTCAGCATCGACGAGTT CGACAATGATGAAAGCGCCATCAAGGACGTTATCAGCTTCTACAAGAAGATGATC GGCGAGAACTGCCCTCAGCGGACCCTGTCTGAGCTGCTGCACGGCCTGTCTAG CCACGATCTGGAGAAAA Illi CGTGCAAGGGAAGAACCTGAACAGCGTGTCCAA GAACCTGTTCGGCGGCAAGAACTGGTCCCTGCTGCGGGACGCCGTGATCGAGG AAAAAAGCAAAGAGAAGGTGTTCAAGAAGGTGATCAAGAGCAACAGCACCGCTG ATGAGCTGGATAAGGTGCTGTCTAAGGAGGAGTTCAGCATCTCTTTCCTATCCAA GGTGTCCGGCAAGGATCTGAGCGTGGAAATCGACAAGTTCGTCAAAAAACAGGA CGAGCTTCTGGTGGAGAACAATATCCAGAACTGGCCTTCTTCTCTCAAGAATAGC GAAGAAAAGAACCTGATCAAGGCCCCTCTGGAC I I I I I GTTGAATTTCTACAGGT 35 TABLE 1F ZZGY Type V Cas Sequences Name Sequence SEQ ID NO. TCGCCCAGAGCTTCAGCAGCAACAACATCGATAAAGATATGTCCTTCTACGCTGA Illi GACGAGTCTCTGTCAAGCCTGGAAAATGTGATAGGCCTGTACAACAAAGTG CGGAACTACGCCACCAAGAAACCTTACACACTGGAAAAGATCAAGCTAAACTTCG AGAACCCTAACCTGGCCTCTGGATGGAGTGAGAGCAAGGAAAACGATTGCCTGA GTATCATCCTGCTGAAGGAGAAGAAATACTTCCTGGGCATCTTCAACAAGAACAA CAAGCCCAAC Illi CAGAGGGCATCAGCCACAGCCTGTCAAGCAACGGCTGTTA CCGGAAGATGAGATACCTGCTGTTCAAGGGATTCAACAAGATGCTGCCTAAGTG CGCCTTCACAGGAGAGGTGAAGGACCACTTCAAGGAAAGCTCCGATGACTTCAG CCTGTTCAACAAGGACACCTTCATCAGCCCCCTGGTGATCACCAAGGAAAI I I I C GATCTGGCTTGCAGCAAGGAAAAAGTGAAGAAGTACCAAAAAGAATACGAGAAAA TCAACAGAGCCGAGTACCGGCAGTCTCTGGTGAAGTGGATCACCTTTGGCCTGA AGTTTCTGTCTAGCTACAAAACCACCACCCAGTTCGACCTGAGCAATTTGAAGCG CCCCGAGGAATACTGCGACCTGAAAGAAI I I IACGAGGACGTGGATAACTTAACC TACAAGATTGAGTTCCTGAACATTAAAGAGGAGGACGTGGACGCTCTGGTCGAG AAAGGCCAGCTGTACCTGTTTGAGATTAGAAACAAGGACTTCGCCAAGAATGCCA GCGGCACGCCCAACCTGCATACACTGTATTTCAAGAGCATCTTCGATAGCAAGAA CCTGGAAAATGGCATCGTGAAACTGAACGGCGAGGCCGAAA Illi CTACAGAAA GAAGAGCCTGAAGAAGGATGATATCACCGTGCACAGAGAGGGAAGCTACCTCGT CAACAAAGTCTGCGTGGACCCTAATTCCGGCAAGACAGAGCAGATCCCAGATAA GATCTACGAGAACATCTACGCCTTCGTCAACGGCAAGTCACGGGACCTGAGCAA GGAGGACGAGGTGTACTACGCCAAAGCCACCATCAAGAAGGCTACCCACGAGAT CGTGAAGGATCGAAGATTCACCGTCGACAAGTTCTTCTTCCACTGCCCCATCACT ATCAACTACAAGAGCAAAGACAAGCCAAGCAAGTTTAACGACAAAGTGCTGGACT TCCTGAGAAATAACAAGGACATCAATATCATCGGCATCGACAGAGGCGAAAGAAA CTTGATCTACGTGACCGTGATCAACCAGAACGGAGAGATCATCGACTGTAAGAG CTTCAATACCATTAAGCACCAGAGCAGCACAGTGAACTACGACGTGGACTACCAC AACAAGCTGCAGGAGCGGGAAAAGAACAGAAAGGAAGAAAAGAGATCTTGGAAC AGCATCACCAAGATCGCCGATCTGAAAGAGGGCTACCTGTCTGCCGTGATTCAC GAGGTTAGCCTGATGATGGTGAAGTACAACGCCATAGTTGTGATGGAAAACCTGA ACCAGGGCTTCAAGAGAATCCGGGGCGGCATCGCCGAACGGAGCGTGTACCAA AAGTTTGAAAAGATGCTCATCGACAAGCTGAACTACTTCGTGATCAAGAACGAGA ACTGGACCAATCCTGGCGGAGTGCTGAATGGATACCAGCTGACAAACAAGGTGT CCACAATCAAGGATATTGGAAATCAGTGCGGCTTCCTGTTCTACGTGCCCGCCAC TTATACATCTAAAATCGATCCTAGCACTGGATTTGTGAACCTGATCAACTTCAACA AGTACAAGAACAGCGAGGACAGAAGGAAGCTGATCTGTAGCTTCGACAAGATCT GCTTTGTGCAGAATGAGAACCTGTTCAAGTTCTCTATCGATTACGGCAAACTGTG CCCTGACAGCAAGATCGCCATCAAAAAGTGGGACGTATTCTCCTATGGCACCAG GATCATCAAGGAAAACCTGACAACAGGCCACATCGAAGAAAATCCAGAGTACGA CCCTACAGAGGAACTGAAATCCCTGCTTTCCAGCAGAGGCATCGAGTACCAGAA GGGCCAAAACCTGCTAGAAACCATCCCTACCAGCGACATGACCAGAGAGTTCTG GAATAGCCTGTTCAAGATCTTCAAGGCCATCCTGCAGATGAGAAACTCTCTGACA AACTCTCCTATCGACCGGCTGCTAAGCCCTGTGAAGGGGAAAGATGGAACCTTC TTCGACACCGACAAGGTGGAAGGCACAAAATTTGAGAAACTGAAGGACGCTGAC GCTAACGGCGCCTACAACATCGCCCTGAAGGGCCTGCTGGTGCTGGAAAAAAAC GACTCTGTCGAGAGCAACAAGGACCTCAAGAACGTGAAGAAAATCTCACTGGAG GACTGGCTGAAATTCGTGCAGATCACACTTAGAGAC Expression construct (with N-terminal methionine and stop codon, includes V5-tag and C-terminal NLS) ATGggcAGCAAATTGTCGACCTTCAATGAGCACTTTCAGAAAACCCTGACCCTGCG GAATGAGCTGGTGCCCGTGGGCAAGACACTGGAGAACATCATCAGCTCTAACGT GCTGATCAACGACGAGAAGCGGTCCGAGGACTACAAAAAGGCCAAGGAAATCAT TGACAGCTATCACCGGGAGTTCATCGAGAAAAGCCTGAGCTCTGTGAATGTGGA CTGGAATGATCTGTACAGCTACCTGAGCAAGAAAGAACCCGAGGACTATGCCCA GAAACAGAAGTTCCTGGAGGAGTTAGAGAACATCCTGCTGGAAAAGAGAAAGAT CATCGTGAAGCAGTTCGAGCAGTACGTGTTCGGTTCCTATACCGACAGCAAGGG AAAAAAGACCAAGGACCTGAAATTCGAAAACCTGTTTAAGTCCGAACTCTTTGACT ACCTGCTGCCTAACTTCTTGAAAAACGACGAGGATAAGAAGGTGATTGGCTCCTT CAATAAGTTCACCAGCTATTTCACCGGC I I I IACGAGAACAGAAAAAACCTGTACA AGAGCGAGCCTCTGCCTACCGCCGTCGCCTACAGAATCGTGAACGAGAACTTCC CCAAGTTTATCTCTAACAAGAACATCTTTAGAGTGTGGAAGGACAACGTCCCTCA ATTCATCGAGATCGCAAAGACCAAACTGAGAGAAGAAGGCATCTCTGATCTGAAC ATCGAGCTGAAGTTTGATTTGACAAATTTCAACTCCTGCCTGAATCAGACCGGCA TCGATACCTACAACGACCTGATCGGCCAGCTGAACTTTGCTATCAACCTCGAATG TCAGAAGGACAAGAACCTTTGTGACCTGCTGCGCAAGAAGCGGAGCCTTAAGAT GGTGCCACTGTACAAGCAAATCCTGTCCGACAACGATAGCAGCTTCAGCATCGA 36 TABLE 1F ZZGY Type V Cas Sequences Name Sequence SEQ ID NO. CGAGTTCGACAATGATGAAAGCGCCATCAAGGACGTTATCAGCTTCTACAAGAAG ATGATCGGCGAGAACTGCCCTCAGCGGACCCTGTCTGAGCTGCTGCACGGCCT GTCTAGCCACGATCTGGAGAAAA Illi CGTGCAAGGGAAGAACCTGAACAGCGT GTCCAAGAACCTGTTCGGCGGCAAGAACTGGTCCCTGCTGCGGGACGCCGTGA TCGAGGAAAAAAGCAAAGAGAAGGTGTTCAAGAAGGTGATCAAGAGCAACAGCA CCGCTGATGAGCTGGATAAGGTGCTGTCTAAGGAGGAGTTCAGCATCTCTTTCCT ATCCAAGGTGTCCGGCAAGGATCTGAGCGTGGAAATCGACAAGTTCGTCAAAAA ACAGGACGAGCTTCTGGTGGAGAACAATATCCAGAACTGGCCTTCTTCTCTCAAG AATAGCGAAGAAAAGAACCTGATCAAGGCCCCTCTGGACTTTTTGTTGAATTTCTA CAGGTTCGCCCAGAGCTTCAGCAGCAACAACATCGATAAAGATATGTCCTTCTAC GCTGATTTTGACGAGTCTCTGTCAAGCCTGGAAAATGTGATAGGCCTGTACAACA AAGTGCGGAACTACGCCACCAAGAAACCTTACACACTGGAAAAGATCAAGCTAAA CTTCGAGAACCCTAACCTGGCCTCTGGATGGAGTGAGAGCAAGGAAAACGATTG CCTGAGTATCATCCTGCTGAAGGAGAAGAAATACTTCCTGGGCATCTTCAACAAG AACAACAAGCCCAAC Illi CAGAGGGCATCAGCCACAGCCTGTCAAGCAACGGC TGTTACCGGAAGATGAGATACCTGCTGTTCAAGGGATTCAACAAGATGCTGCCTA AGTGCGCCTTCACAGGAGAGGTGAAGGACCACTTCAAGGAAAGCTCCGATGACT TCAGCCTGTTCAACAAGGACACCTTCATCAGCCCCCTGGTGATCACCAAGGAAAT TTTCGATCTGGCTTGCAGCAAGGAAAAAGTGAAGAAGTACCAAAAAGAATACGAG AAAATCAACAGAGCCGAGTACCGGCAGTCTCTGGTGAAGTGGATCACCTTTGGC CTGAAGTTTCTGTCTAGCTACAAAACCACCACCCAGTTCGACCTGAGCAATTTGA AGCGCCCCGAGGAATACTGCGACCTGAAAGAA Illi ACGAGGACGTGGATAACT TAACCTACAAGATTGAGTTCCTGAACATTAAAGAGGAGGACGTGGACGCTCTGGT CGAGAAAGGCCAGCTGTACCTGTTTGAGATTAGAAACAAGGACTTCGCCAAGAAT GCCAGCGGCACGCCCAACCTGCATACACTGTATTTCAAGAGCATCTTCGATAGCA AGAACCTGGAAAATGGCATCGTGAAACTGAACGGCGAGGCCGAAA Illi CTACA GAAAGAAGAGCCTGAAGAAGGATGATATCACCGTGCACAGAGAGGGAAGCTACC TCGTCAACAAAGTCTGCGTGGACCCTAATTCCGGCAAGACAGAGCAGATCCCAG ATAAGATCTACGAGAACATCTACGCCTTCGTCAACGGCAAGTCACGGGACCTGA GCAAGGAGGACGAGGTGTACTACGCCAAAGCCACCATCAAGAAGGCTACCCACG AGATCGTGAAGGATCGAAGATTCACCGTCGACAAGTTCTTCTTCCACTGCCCCAT CACTATCAACTACAAGAGCAAAGACAAGCCAAGCAAGTTTAACGACAAAGTGCTG GACTTCCTGAGAAATAACAAGGACATCAATATCATCGGCATCGACAGAGGCGAAA GAAACTTGATCTACGTGACCGTGATCAACCAGAACGGAGAGATCATCGACTGTAA GAGCTTCAATACCATTAAGCACCAGAGCAGCACAGTGAACTACGACGTGGACTA CCACAACAAGCTGCAGGAGCGGGAAAAGAACAGAAAGGAAGAAAAGAGATCTTG GAACAGCATCACCAAGATCGCCGATCTGAAAGAGGGCTACCTGTCTGCCGTGAT TCACGAGGTTAGCCTGATGATGGTGAAGTACAACGCCATAGTTGTGATGGAAAAC CTGAACCAGGGCTTCAAGAGAATCCGGGGCGGCATCGCCGAACGGAGCGTGTA CCAAAAGTTTGAAAAGATGCTCATCGACAAGCTGAACTACTTCGTGATCAAGAAC GAGAACTGGACCAATCCTGGCGGAGTGCTGAATGGATACCAGCTGACAAACAAG GTGTCCACAATCAAGGATATTGGAAATCAGTGCGGCTTCCTGTTCTACGTGCCCG CCACTTATACATCTAAAATCGATCCTAGCACTGGATTTGTGAACCTGATCAACTTC AACAAGTACAAGAACAGCGAGGACAGAAGGAAGCTGATCTGTAGCTTCGACAAG ATCTGCTTTGTGCAGAATGAGAACCTGTTCAAGTTCTCTATCGATTACGGCAAACT GTGCCCTGACAGCAAGATCGCCATCAAAAAGTGGGACGTATTCTCCTATGGCAC CAGGATCATCAAGGAAAACCTGACAACAGGCCACATCGAAGAAAATCCAGAGTA CGACCCTACAGAGGAACTGAAATCCCTGCTTTCCAGCAGAGGCATCGAGTACCA GAAGGGCCAAAACCTGCTAGAAACCATCCCTACCAGCGACATGACCAGAGAGTT CTGGAATAGCCTGTTCAAGATCTTCAAGGCCATCCTGCAGATGAGAAACTCTCTG ACAAACTCTCCTATCGACCGGCTGCTAAGCCCTGTGAAGGGGAAAGATGGAACC TTCTTCGACACCGACAAGGTGGAAGGCACAAAATTTGAGAAACTGAAGGACGCT GACGCTAACGGCGCCTACAACATCGCCCTGAAGGGCCTGCTGGTGCTGGAAAAA AACGACTCTGTCGAGAGCAACAAGGACCTCAAGAACGTGAAGAAAATCTCACTG GAGGACTGGCTGAAATTCGTGCAGATCACACTTAGAGACtctagaAAGCGGACAGC AGACGGCTCCGAATTTGAAAGCCCTAAGAAAAAGAGAAAGGTGggatccGGCAAAC CTATCCCCAATCCCCTGCTGGGCCTGGACAGCACCTGA

[0103] In some embodiments a ZZGY Type V Cas protein comprises an amino acid sequence of SEQ ID NO:31, SEQ ID NO:32, or SEQ ID NO:33. In some embodiments, a ZZGY Type V Cas protein has nickase activity, for example resulting from one or more amino acid substitutions relative to the sequence of SEQ ID NO:31, SEQ ID NO:32, or SEQ ID NO:33. In some embodiments, the one or more amino acid -45- substitutions providing nickase activity comprise a D905 substitution, wherein the position of the D905 substitution is defined with respect to the amino acid numbering of SEQ ID NO:32 (corresponding to amino acid 908 of SEQ ID NO:121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise an E998 substitution, wherein the position of the E998 substitution is defined with respect to the amino acid numbering of SEQ ID NO:32 (corresponding to amino acid 993 of SEQ ID NO:121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a R1214 substitution, wherein the position of the R1214 substitution is defined with respect to the amino acid numbering of SEQ ID NO:32 (corresponding to amino acid 1226 of SEQ ID NO:121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D1254 substitution, wherein the position of the D1254 substitution is defined with respect to the amino acid numbering of SEQ ID NO:32 (corresponding to amino acid 1263 of SEQ ID NO:121). In some embodiments, a ZZGY Type V Cas protein is catalytically inactive, for example due to a R1214 substitution in combination with a D905 substitution, a E998 substitution, and / or D1254 substitution. 6.2.7. ZKBG Type V Cas Proteins

[0104] In one aspect, the disclosure provides ZKBG Type V Cas proteins. ZKBG Type V Cas proteins can be further classified as Type V-A Cas proteins. The ZKBG Type V Cas proteins typically comprise an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:37. In some embodiments, the ZKBG Type V Cas proteins comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:37. In some embodiments, a ZKBG Type V Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:37.

[0105] Exemplary ZKBG Type V Cas protein sequences and nucleotide sequences encoding exemplary ZKBG Type V Cas proteins are set forth in Table 1G. TABLE1G ZKBG Type V Cas Sequences Name Sequence SEQ ID NO. Wildtype amino acid sequence (without N-terminal methionine) KRLIDFTNIYQRSKTLRFRLEPIGKTADYIKNSQSLETDARLAKESKKVKELADEYHKE FIGDVLSSLELPLSKINELWDIYIYIYMSNDTDREIKFKKLQENLRKVIAEAFSKDKRFG NLFKKEIITDILPEFLQDKDDDIKIVNRFKGFTTYFYAFHKNRENMYVSEEKSTAIPYRIV NQNLVKYFDNYKTFKEKVMPLLKDKNIVESIERDFKDILNEKSIEDVFGLANFTHTLCQ ADIEKYNTLIGGLVVKNEKKEIKGINQYINEHNQTSKKGNGIPKLKPLFNQILSDRKSLS FTLDDIKKTSEAIRTIKDEYENLRDKLATIERLIKSIKEYDLAGIYIKMGEDTSTISQHWF GAYYKIIEAIADAWERRNPKKNRESKAYSKYVSSLKSISLQEIDDLKIGEPIENYFATFG TTCSDRTSGVSSLNRIKAAYTEFVNKFPEGFEDGDDCNDAYFKANVEVVKNLLDSIK DFQRFVKPLLGNEDERDKDEAFYGEFVPTYTDMDNIITPLYNRVRNFATKKPYSTDKI KINFENVVLLKGWDKNKESDYASIILMKDGQYFLGVLRNGSKSTLKTILPNTGDCYQK MVYKYFKDIKSNLPRCTTQRKDVKAHFAESSDDYTLLDTKAFVSALTISREVFELYNA PDKEKKFKKEYLKNTNDSIGYANAVSVCKRFCLEFLKKYRSTAIYDLSDVETSVDSFD DLSSFYQEIDKRLYSISFENVSVDSVNELVDNGNMLLFRIANKDFSPNSKGRPNLHTIY WRMLFDPANLKDVVYQLNGNAEIFFRKASVTRTEPTHPANVAIKNKSEYNKQNKPYS TFKYGLIKDRRYTTDQFEFHVPITMNFKQPESSKLQDKLNKQVLDFLKQDGVRHIIGID RGERNLLYLVMVDMEGKIKKQISLNEIAGNPKNPEFKQDFLALLHEREGDRLESRRS WNTIQSIKELKEGYMSLVVHEIANMMLENDAIVVLENLNRSFMQKRGGIEKSVYQKFE KMLIDKLGYIVDKTKDVSDNGGALHAVQLADTFENFNKTQKGAIRQCGFIFYIPAWRT SKIDPVTGFVPMLRCQYESIVESKKFFGKFDSIYYDATGKYFVFQTDFTKFNTESKGGI QKWDICTYGDRIYAPRTKDRNNNPVSERVNLTEEMKSLFVSHNINIQGDIKAGIMQQT DKEFFESLHRLLRLTLQIRNSKKSTGKDYEDYIISPVMGKDGRFFDSRNADATQPKDA DANGAYNIARKGLMLLRQIQAQEKQDLSNGKWLEFAQR 37 Wildtype amino acid sequence (with N-terminal methionine) MKRLIDFTNIYQRSKTLRFRLEPIGKTADYIKNSQSLETDARLAKESKKVKELADEYHK EFIGDVLSSLELPLSKINELWDIYIYIYMSNDTDREIKFKKLQENLRKVIAEAFSKDKRFG NLFKKEIITDILPEFLQDKDDDIKIVNRFKGFTTYFYAFHKNRENMYVSEEKSTAIPYRIV NQNLVKYFDNYKTFKEKVMPLLKDKNIVESIERDFKDILNEKSIEDVFGLANFTHTLCQ ADIEKYNTLIGGLVVKNEKKEIKGINQYINEHNQTSKKGNGIPKLKPLFNQILSDRKSLS FTLDDIKKTSEAIRTIKDEYENLRDKLATIERLIKSIKEYDLAGIYIKMGEDTSTISQHWF GAYYKIIEAIADAWERRNPKKNRESKAYSKYVSSLKSISLQEIDDLKIGEPIENYFATFG TTCSDRTSGVSSLNRIKAAYTEFVNKFPEGFEDGDDCNDAYFKANVEVVKNLLDSIK DFQRFVKPLLGNEDERDKDEAFYGEFVPTYTDMDNIITPLYNRVRNFATKKPYSTDKI KINFENVVLLKGWDKNKESDYASIILMKDGQYFLGVLRNGSKSTLKTILPNTGDCYQK MVYKYFKDIKSNLPRCTTQRKDVKAHFAESSDDYTLLDTKAFVSALTISREVFELYNA PDKEKKFKKEYLKNTNDSIGYANAVSVCKRFCLEFLKKYRSTAIYDLSDVETSVDSFD DLSSFYQEIDKRLYSISFENVSVDSVNELVDNGNMLLFRIANKDFSPNSKGRPNLHTIY WRMLFDPANLKDVVYQLNGNAEIFFRKASVTRTEPTHPANVAIKNKSEYNKQNKPYS TFKYGLIKDRRYTTDQFEFHVPITMNFKQPESSKLQDKLNKQVLDFLKQDGVRHIIGID RGERNLLYLVMVDMEGKIKKQISLNEIAGNPKNPEFKQDFLALLHEREGDRLESRRS WNTIQSIKELKEGYMSLVVHEIANMMLENDAIVVLENLNRSFMQKRGGIEKSVYQKFE KMLIDKLGYIVDKTKDVSDNGGALHAVQLADTFENFNKTQKGAIRQCGFIFYIPAWRT SKIDPVTGFVPMLRCQYESIVESKKFFGKFDSIYYDATGKYFVFQTDFTKFNTESKGGI QKWDICTYGDRIYAPRTKDRNNNPVSERVNLTEEMKSLFVSHNINIQGDIKAGIMQQT DKEFFESLHRLLRLTLQIRNSKKSTGKDYEDYIISPVMGKDGRFFDSRNADATQPKDA DANGAYNIARKGLMLLRQIQAQEKQDLSNGKWLEFAQR 38 Expression construct (with N-terminal methionine, V5-tag and C-terminal NLS) aa sequence MGKRLIDFTNIYQRSKTLRFRLEPIGKTADYIKNSQSLETDARLAKESKKVKELADEYH KEFIGDVLSSLELPLSKINELWDIYIYIYMSNDTDREIKFKKLQENLRKVIAEAFSKDKRF GNLFKKEIITDILPEFLQDKDDDIKIVNRFKGFTTYFYAFHKNRENMYVSEEKSTAIPYRI VNQNLVKYFDNYKTFKEKVMPLLKDKNIVESIERDFKDILNEKSIEDVFGLANFTHTLC QADIEKYNTLIGGLVVKNEKKEIKGINQYINEHNQTSKKGNGIPKLKPLFNQILSDRKSL SFTLDDIKKTSEAIRTIKDEYENLRDKLATIERLIKSIKEYDLAGIYIKMGEDTSTISQHWF GAYYKIIEAIADAWERRNPKKNRESKAYSKYVSSLKSISLQEIDDLKIGEPIENYFATFG TTCSDRTSGVSSLNRIKAAYTEFVNKFPEGFEDGDDCNDAYFKANVEVVKNLLDSIK DFQRFVKPLLGNEDERDKDEAFYGEFVPTYTDMDNIITPLYNRVRNFATKKPYSTDKI KINFENVVLLKGWDKNKESDYASIILMKDGQYFLGVLRNGSKSTLKTILPNTGDCYQK MVYKYFKDIKSNLPRCTTQRKDVKAHFAESSDDYTLLDTKAFVSALTISREVFELYNA PDKEKKFKKEYLKNTNDSIGYANAVSVCKRFCLEFLKKYRSTAIYDLSDVETSVDSFD DLSSFYQEIDKRLYSISFENVSVDSVNELVDNGNMLLFRIANKDFSPNSKGRPNLHTIY WRMLFDPANLKDVVYQLNGNAEIFFRKASVTRTEPTHPANVAIKNKSEYNKQNKPYS TFKYGLIKDRRYTTDQFEFHVPITMNFKQPESSKLQDKLNKQVLDFLKQDGVRHIIGID RGERNLLYLVMVDMEGKIKKQISLNEIAGNPKNPEFKQDFLALLHEREGDRLESRRS WNTIQSIKELKEGYMSLVVHEIANMMLENDAIVVLENLNRSFMQKRGGIEKSVYQKFE KMLIDKLGYIVDKTKDVSDNGGALHAVQLADTFENFNKTQKGAIRQCGFIFYIPAWRT SKIDPVTGFVPMLRCQYESIVESKKFFGKFDSIYYDATGKYFVFQTDFTKFNTESKGGI QKWDICTYGDRIYAPRTKDRNNNPVSERVNLTEEMKSLFVSHNINIQGDIKAGIMQQT DKEFFESLHRLLRLTLQIRNSKKSTGKDYEDYIISPVMGKDGRFFDSRNADATQPKDA DANGAYNIARKGLMLLRQIQAQEKQDLSNGKWLEFAQRSRKRTADGSEFESPKKKR KVGSGKPIPNPLLGLDST 39 Wildtype coding sequence (with N-terminal methionine and stop codon) ATGAAACGCCTAATTGACTTTACAAACATCTATCAGCGATCAAAGACTTTGAGGTT TCGATTGGAGCCTATCGGTAAAACGGCCGACTATATTAAGAATTCTCAGTCCCTC GAAACTGATGCGCGTTTGGCAAAAGAGAGCAAGAAGGTAAAAGAGCTTGCTGAT GAATATCACAAAGAGTTTATTGGAGATGTCCTGTCTTCGTTGGAATTGCCTTTAAG CAAAATCAACGAGTTATGGGATATATATATATATATATATATGTCCAATGATACAGA CCGCGAGATAAAATTCAAAAAACTGCAAGAGAACCTGCGAAAGGTGATTGCAGA GGCTTTTAGTAAGGACAAACGGTTTGGTAATTTATTCAAAAAGGAGATAATCACAG ACATTCTGCCGGAATTCTTGCAAGATAAGGATGATGATATTAAGATCGTAAATAGA TTCAAAGGATTTACCACATATTTTTACGCCTTTCATAAGAATAGGGAAAATATGTAT GTCTCGGAAGAGAAATCGACTGCAATACCATATCGAATTGTGAATCAAAATCTCG TCAAGTA Illi GACAACTACAAGACGTTCAAAGAGAAGGTAATGCCTCTTCTGAAA GACAAGAATATAGTCGAAAGCATAGAGAGAGACTTCAAAGACATCTTGAACGAAA AATCAATAGAGGATGTTTTTGGCCTTGCCAACTTCACTCATACTTTATGTCAGGCT GACATCGAGAAATACAATACGTTGATAGGTGGCCTTGTCGTCAAAAACGAAAAAA AAGAGATTAAAGGTATTAATCAGTACATTAACGAACATAACCAAACGAGTAAAAAA GGGAATGGAATTCCGAAACTAAAGCCGTTGTTCAATCAGA Illi GAGCGATAGAA AATCGTTATCGTTTACCTTAGACGATATCAAAAAAACGTCGGAGGCTATTCGCAC CATTAAGGATGAGTATGAAAATCTCCGAGACAAGTTGGCGACCATCGAAAGGCTT ATTAAGTCTATCAAGGAGTATGATCTTGCAGGTATTTACATCAAGATGGGAGAGG ATACTTCGACAATATCGCAGCATTGGTTTGGTGCGTATTATAAAATCATCGAAGCG ATAGCAGATGCATGGGAACGACGAAATCCGAAGAAAAACAGAGAATCCAAGGCA TATAGCAAGTATGTATCGTCCCTAAAAAGCATCAGTCTCCAAGAAATAGATGATCT CAAAATCGGAGAGCCTATAGAGAACTACTTCGCAAC Illi GGCACGACTTGTTCA 40 GACCGAACAAGTGGAGTTTCTTCGCTCAATAGGATAAAAGCTGCTTATACCGAGT TCGTGAACAAATTTCCTGAAGGATTTGAAGATGGCGATGACTGTAACGATGCCTA CTTTAAGGCTAATGTGGAAGTCGTCAAAAATCTGCTGGATTCAATTAAAGA1 1 1 1 C AGCGTTTTGTGAAGCCTTTGCTTGGCAATGAGGACGAAAGAGACAAAGACGAGG CATTCTATGGAGAGTTTGTCCCGACATACACAGATATGGATAACATCATAACCCCT CTATACAACCGTGTACGCAA Illi GCCACCAAGAAACCATACTCTACAGACAAGA TAAAAATCAACTTTGAAAACGTAGTATTGCTAAAAGGATGGGACAAAAACAAGGA GTCAGACTACGCATCCATCATATTGATGAAAGACGGACAATACTTTTTAGGGGTA CTCCGTAATGGTTCAAAAAGTACTCTTAAAACCATATTGCCTAACACAGGTGATTG CTATCAAAAAATGGTTTATAAGTA Illi AAGGATATAAAATCAAATCTTCCCCGGTG TACGACCCAGAGGAAAGACGTGAAAGCGCACTTTGCCGAATCGAGCGACGATTA CACTC Illi AGATACAAAGGCCTTTGTTTCGGCACTGACTATCAGCAGAGAAGTG TTCGAACTATACAATGCCCCCGATAAGGAGAAAAAATTCAAAAAGGAATATTTGAA GAACACAAACGATAGTATAGGCTACGCCAATGCTGTATCCGTATGTAAACGCTTC TGTTTGGAGTTCCTAAAAAAATATCGCAGCACTGCCATATATGATCTTTCGGATGT TGAAACTTCAGTCGATTCGTTTGACGATTTGTCCTCATTCTATCAAGAGATAGACA AAAGGCTGTACAGCATCTCATTCGAAAATGTATCTGTCGATTCCGTCAATGAGCTT GTAGACAATGGCAATATGCTTCTATTCCGTATCGCGAATAAAGATTTTTCGCCTAA CAGCAAGGGCCGTCCCAATCTTCATACTATATATTGGCGAATGC Illi CGACCCG GCCAACCTGAAGGATGTTGTATATCAGCTCAATGGTAATGCCGAAATATTCTTCC GTAAGGCAAGCGTTACGAGGACGGAGCCTACACATCCGGCTAACGTTGCCATCA AAAACAAGAGCGAATATAACAAACAGAATAAGCCGTATAGTACATTCAAGTACGG TTTAATCAAGGATAGGCGCTACACTACCGACCAGTTCGAGTTTCATGTACCCATC ACAATGAACTTCAAGCAACCAGAGTCGTCTAAACTACAGGACAAGCTCAACAAGC AAGTGCTTGACTTCTTGAAACAGGACGGCGTACGCCATATTATAGGCATTGATCG GGGCGAACGTAATCTGCTATACTTGGTGATGGTAGATATGGAGGGCAAAATCAAA AAACAAATATCACTCAACGAGATAGCCGGTAATCCGAAGAATCCCGAGTTCAAAC AAGACTTCCTTGCACTACTGCACGAGCGCGAAGGTGACCGTTTGGAGTCACGTC GCAGTTGGAACACCATTCAGAGCATTAAAGAACTCAAAGAAGGTTACATGAGCTT GGTGGTTCATGAAATAGCGAATATGATGCTTGAGAATGATGCTATAGTAGTGCTC GAAAATCTGAATCGCTCGTTTATGCAAAAGCGCGGCGGCATAGAAAAGTCTGTAT ACCAAAAGTTCGAAAAGATGCTTATCGACAAGTTGGGATACATCGTGGATAAGAC TAAAGATGTGTCCGACAACGGAGGCGCACTACATGCTGTACAGCTTGCTGATAC GTTTGAAAACTTCAATAAGACCCAAAAAGGAGCTATTCGTCAATGTGGATTCATAT TCTATATTCCTGCATGGCGTACCAGCAAGATTGACCCCGTTACCGGCTTTGTGCC AATGCTTAGGTGTCAATATGAAAGCATCGTAGAATCCAAAAAATTCTTCGGAAAGT TCGACAGTATATACTACGATGCGACAGGAAAGTA Illi GTCTTCCAAACTGACTTT ACCAAATTCAATACCGAGAGCAAAGGAGGAATCCAAAAATGGGATATATGCACCT ATGGAGACAGAATATATGCTCCTCGCACCAAAGACCGGAATAATAACCCTGTTTC GGAACGTGTAAACCTTACTGAGGAGATGAAATCACTGTTTGTATCGCATAATATCA ATATTCAAGGCGATATCAAAGCCGGAATTATGCAGCAGACAGACAAGGAGTTCTT CGAGTCACTGCATCGATTGCTTCGACTTACGTTGCAAATACGCAATAGCAAAAAA TCTACAGGCAAAGACTATGAAGACTATATCATATCGCCGGTGATGGGCAAGGAC GGTCGTTTCTTTGATTCGCGTAACGCGGATGCTACGCAACCTAAGGATGCAGATG CCAATGGCGCGTACAATATTGCACGCAAAGGCTTGATGCTGCTTCGCCAGATTCA AGCCCAAGAGAAGCAAGACCTATCCAACGGAAAATGGCTTGAATTTGCCCAAAG GTGA Codon optimized coding sequence (no N-terminal methionine, no stop codon) AAGCGGCTCATCGACTTCACCAACATCTACCAGCGTTCTAAGACCCTGAGATTCA GACTGGAACCTATCGGCAAGACCGCGGACTACATCAAAAACAGCCAGTCCCTGG AAACAGACGCCAGACTGGCCAAGGAATCCAAGAAAGTGAAGGAACTGGCCGATG AGTACCACAAAGAGTTTATCGGCGACGTGCTGAGCAGCCTGGAGCTGCCCCTGA GCAAAATCAACGAGCTGTGGGACATCTATATCTACATCTACATGAGCAACGACAC CGATCGGGAAATCAAATTTAAGAAGCTCCAGGAGAACCTGCGGAAGGTGATCGC CGAGGCCTTTAGCAAGGATAAGAGATTCGGCAACCTGTTCAAGAAAGAAATCATC ACAGATATCCTGCCCGAGTTCCTGCAAGATAAAGATGACGATATCAAAATCGTGA ACCGGTTCAAGGG 1 1 1 1ACAACCTACTTCTACGCCTTCCACAAGAATCGGGAAAA CATGTACGTGTCTGAAGAGAAGAGCACAGCCATCCCCTACAGAATCGTGAATCAA AACCTGGTGAAATACTTCGATAACTACAAGACI 1 1 1AAGGAGAAGGTGATGCCTC TGCTGAAGGACAAGAACATCGTCGAAAGCATCGAGCGCGACTTCAAGGACATCC TGAACGAGAAAAGCATCGAGGACGTGTTCGGCCTGGCCAATTTCACCCACACCC TGTGCCAGGCTGACATCGAGAAGTACAACACCTTGATAGGCGGACTGGTGGTGA AGAACGAAAAGAAGGAGATCAAGGGCATCAACCAGTATATTAACGAGCACAACCA GACCTCTAAGAAGGGCAACGGCATCCCAAAGCTGAAGCCTCTGTTTAACCAGAT CCTGAGCGACAGAAAATCTCTCAGCTTCACCCTGGATGATATCAAGAAAACCAGC GAGGCCATCAGAACAATTAAGGACGAGTATGAGAACCTGAGAGATAAGCTGGCC ACAATCGAACGGCTGATCAAGAGCATCAAGGAATACGACCTGGCCGGCATCTAC ATCAAGATGGGCGAGGACACCTCTACCATCTCCCAGCACTGGTTCGGTGCCTAT TACAAGATTATCGAAGCCATCGCCGACGCCTGGGAGAGAAGAAACCCAAAGAAA 41 Expression construct (with N-terminal methionine and stop codon, includes V5-tag and C-terminal NLS) AACAGAGAGAGCAAGGCCTACAGCAAGTACGTGAGCAGCCTTAAGAGCATCAGC CTGCAGGAGATCGACGACCTGAAGATCGGCGAGCCTATCGAGAATTACTTCGCC ACCTTTGGAACAACATGTAGCGACCGGACATCTGGCGTGAGCTCTCTGAACCGG ATCAAAGCCGCCTACACCGAGTTCGTGAACAAGTTCCCCGAGGGCTTTGAGGAT GGCGATGATTGCAACGACGCTTACTTCAAAGCCAATGTGGAGGTGGTGAAGAAC TTGCTGGATAGCATAAAAGACTTCCAGAGATTTGTGAAGCCTCTACTGGGCAATG AGGACGAGCGGGACAAAGATGAGGCCTTCTACGGCGAGTTCGTTCCTACCTACA CAGATATGGACAACATCATCACGCCTCTGTATAATAGAGTCAGAAACTTCGCTAC CAAGAAGCCTTACAGTACAGACAAGATCAAAATAAACTTCGAAAACGTGGTACTG CTGAAGGGCTGGGATAAGAACAAGGAGAGCGACTATGCCAGCATCATCCTGATG AAGGACGGCCAGTACTTTCTGGGAGTGCTGAGAAACGGATCTAAGAGCACTCTG AAAACCATCCTGCCTAACACCGGTGACTGCTACCAGAAAATGGTGTACAAGTATT TCAAGGATATCAAGTCTAACCTGCCCAGATGCACCACCCAGAGAAAGGACGTGA AGGCACATTTCGCTGAAAGCAGCGATGATTACACCCTGCTTGATACAAAAGCCTT CGTGAGCGCTCTGACGATCTCCAGAGAGGTGTTCGAACTGTACAACGCTCCTGA TAAGGAAAAGAAATTCAAGAAGGAATACCTGAAGAACACCAACGACTCCATCGGC TACGCCAATGCAGTGAGCGTGTGCAAGAGATTCTGCCTGGAGTTCCTGAAAAAG TACCGGAGCACCGCCATCTACGACCTGAGCGATGTTGAAACCTCTGTGGACAGT TTCGACGACCTGAGCAGCTTCTACCAGGAGATCGATAAGAGACTGTACAGCATCA GCTTCGAAAACGTGAGCGTGGACAGCGTGAACGAGCTGGTGGATAACGGCAATA TGCTGCTGTTCAGAATCGCCAACAAGGATTTCTCTCCTAATAGCAAGGGCAGACC TAATCTGCACACAATTTACTGGAGAATGCTGTTCGACCCTGCTAATCTCAAGGAC GTCGTGTACCAACTGAACGGCAATGCCGAAATCTTCTTCCGGAAGGCCAGCGTT ACAAGGACAGAACCAACACACCCCGCCAATGTGGCCATCAAGAACAAGAGCGAG TACAACAAGCAGAACAAACCTTACAGCACCTTCAAGTACGGCCTCATCAAGGACC GGCGATACACCACCGATCAGTTCGAGTTCCACGTGCCTATCACCATGAACTTCAA GCAACCTGAGTCATCTAAGCTGCAGGACAAACTGAATAAGCAAGTGCTGGACTTC CTGAAGCAAGACGGCGTGCGGCACATCATCGGCATCGACCGGGGAGAAAGAAA CCTGCTGTACCTGGTGATGGTCGACATGGAAGGAAAAATCAAGAAGCAGATCAG CCTGAATGAAATCGCCGGAAACCCAAAGAACCCTGAGTTTAAGCAGGACTTCTTA GCTCTGCTGCATGAGAGAGAGGGCGATAGACTGGAGTCCAGAAGAAGTTGGAAC ACCATCCAGAGCATCAAGGAGCTGAAAGAAGGCTACATGTCCCTGGTGGTGCAC GAGATCGCTAACATGATGCTGGAGAATGATGCCATCGTGGTCTTGGAAAACCTTA ACAGATCCTTTATGCAGAAGAGAGGCGGCATTGAGAAAAGCGTGTACCAGAAGT TTGAGAAAATGCTGATCGACAAGCTGGGCTACATCGTGGACAAAACAAAAGATGT GTCAGATAATGGCGGAGCCCTGCACGCCGTGCAGCTGGCTGACACCTTCGAGAA CTTTAACAAGACCCAGAAAGGCGCCATCCGGCAGTGCGGCTTCATCTTTTATATC CCCGCCTGGCGGACAAGCAAAATTGACCCGGTAACCGGCTTTGTGCCCATGCTG AGATGTCAGTACGAATCTATCGTGGAATCCAAGAAGTTCTTTGGCAAATTCGACT CTATCTACTACGACGCCACCGGAAAGTACTTCGTGTTCCAGACCGACTTTACCAA GTTCAACACCGAGTCTAAGGGGGGCATCCAGAAGTGGGACATCTGTACCTACGG AGACAGAATCTACGCCCCTAGAACCAAAGACAGAAATAACAACCCTGTGTCCGAA AGAGTGAACCTGACAGAAGAAATGAAGAGCCTGTTCGTAAGCCACAATATCAACA TCCAGGGCGACATCAAGGCCGGCATTATGCAGCAGACAGACAAGGAGTTCTTCG AGTCGCTGCACAGACTGCTGAGACTGACCCTGCAGATCCGGAACAGCAAGAAAA GCACCGGCAAGGACTACGAGGACTACATTATCAGTCCTGTGATGGGCAAGGACG GAAGATTCTTCGACAGCCGGAACGCCGACGCCACCCAGCCCAAGGACGCCGAC GCAAACGGCGCCTACAACATTGCCAGAAAAGGCCTGATGCTGCTGCGCCAGATC CAGGCCCAGGAGAAGCAGGACCTGTCTAATGGGAAGTGGCTGGAGTTCGCCCA GCGG_______________________________________________ ATGggcAAGCGGCTCATCGACTTCACCAACATCTACCAGCGTTCTAAGACCCTGA GATTCAGACTGGAACCTATCGGCAAGACCGCGGACTACATCAAAAACAGCCAGT CCCTGGAAACAGACGCCAGACTGGCCAAGGAATCCAAGAAAGTGAAGGAACTGG CCGATGAGTACCACAAAGAGTTTATCGGCGACGTGCTGAGCAGCCTGGAGCTGC CCCTGAGCAAAATCAACGAGCTGTGGGACATCTATATCTACATCTACATGAGCAA CGACACCGATCGGGAAATCAAATTTAAGAAGCTCCAGGAGAACCTGCGGAAGGT GATCGCCGAGGCCTTTAGCAAGGATAAGAGATTCGGCAACCTGTTCAAGAAAGA AATCATCACAGATATCCTGCCCGAGTTCCTGCAAGATAAAGATGACGATATCAAA ATCGTGAACCGGTTCAAGGGTTTTACAACCTACTTCTACGCCTTCCACAAGAATC GGGAAAACATGTACGTGTCTGAAGAGAAGAGCACAGCCATCCCCTACAGAATCG TGAATCAAAACCTGGTGAAATACTTCGATAACTACAAGACTTTTAAGGAGAAGGT GATGCCTCTGCTGAAGGACAAGAACATCGTCGAAAGCATCGAGCGCGACTTCAA GGACATCCTGAACGAGAAAAGCATCGAGGACGTGTTCGGCCTGGCCAATTTCAC CCACACCCTGTGCCAGGCTGACATCGAGAAGTACAACACCTTGATAGGCGGACT GGTGGTGAAGAACGAAAAGAAGGAGATCAAGGGCATCAACCAGTATATTAACGA GCACAACCAGACCTCTAAGAAGGGCAACGGCATCCCAAAGCTGAAGCCTCTGTT TAACCAGATCCTGAGCGACAGAAAATCTCTCAGCTTCACCCTGGATGATATCAAG AAAACCAGCGAGGCCATCAGAACAATTAAGGACGAGTATGAGAACCTGAGAGAT 42 AAGCTGGCCACAATCGAACGGCTGATCAAGAGCATCAAGGAATACGACCTGGCC GGCATCTACATCAAGATGGGCGAGGACACCTCTACCATCTCCCAGCACTGGTTC GGTGCCTATTACAAGATTATCGAAGCCATCGCCGACGCCTGGGAGAGAAGAAAC CCAAAGAAAAACAGAGAGAGCAAGGCCTACAGCAAGTACGTGAGCAGCCTTAAG AGCATCAGCCTGCAGGAGATCGACGACCTGAAGATCGGCGAGCCTATCGAGAAT TACTTCGCCACCTTTGGAACAACATGTAGCGACCGGACATCTGGCGTGAGCTCT CTGAACCGGATCAAAGCCGCCTACACCGAGTTCGTGAACAAGTTCCCCGAGGGC TTTGAGGATGGCGATGATTGCAACGACGCTTACTTCAAAGCCAATGTGGAGGTG GTGAAGAACTTGCTGGATAGCATAAAAGACTTCCAGAGATTTGTGAAGCCTCTAC TGGGCAATGAGGACGAGCGGGACAAAGATGAGGCCTTCTACGGCGAGTTCGTTC CTACCTACACAGATATGGACAACATCATCACGCCTCTGTATAATAGAGTCAGAAA CTTCGCTACCAAGAAGCCTTACAGTACAGACAAGATCAAAATAAACTTCGAAAAC GTGGTACTGCTGAAGGGCTGGGATAAGAACAAGGAGAGCGACTATGCCAGCATC ATCCTGATGAAGGACGGCCAGTACTTTCTGGGAGTGCTGAGAAACGGATCTAAG AGCACTCTGAAAACCATCCTGCCTAACACCGGTGACTGCTACCAGAAAATGGTGT ACAAGTATTTCAAGGATATCAAGTCTAACCTGCCCAGATGCACCACCCAGAGAAA GGACGTGAAGGCACATTTCGCTGAAAGCAGCGATGATTACACCCTGCTTGATACA AAAGCCTTCGTGAGCGCTCTGACGATCTCCAGAGAGGTGTTCGAACTGTACAAC GCTCCTGATAAGGAAAAGAAATTCAAGAAGGAATACCTGAAGAACACCAACGACT CCATCGGCTACGCCAATGCAGTGAGCGTGTGCAAGAGATTCTGCCTGGAGTTCC TGAAAAAGTACCGGAGCACCGCCATCTACGACCTGAGCGATGTTGAAACCTCTG TGGACAGTTTCGACGACCTGAGCAGCTTCTACCAGGAGATCGATAAGAGACTGT ACAGCATCAGCTTCGAAAACGTGAGCGTGGACAGCGTGAACGAGCTGGTGGATA ACGGCAATATGCTGCTGTTCAGAATCGCCAACAAGGATTTCTCTCCTAATAGCAA GGGCAGACCTAATCTGCACACAATTTACTGGAGAATGCTGTTCGACCCTGCTAAT CTCAAGGACGTCGTGTACCAACTGAACGGCAATGCCGAAATCTTCTTCCGGAAG GCCAGCGTTACAAGGACAGAACCAACACACCCCGCCAATGTGGCCATCAAGAAC AAGAGCGAGTACAACAAGCAGAACAAACCTTACAGCACCTTCAAGTACGGCCTCA TCAAGGACCGGCGATACACCACCGATCAGTTCGAGTTCCACGTGCCTATCACCA TGAACTTCAAGCAACCTGAGTCATCTAAGCTGCAGGACAAACTGAATAAGCAAGT GCTGGACTTCCTGAAGCAAGACGGCGTGCGGCACATCATCGGCATCGACCGGG GAGAAAGAAACCTGCTGTACCTGGTGATGGTCGACATGGAAGGAAAAATCAAGA AGCAGATCAGCCTGAATGAAATCGCCGGAAACCCAAAGAACCCTGAGTTTAAGC AGGACTTCTTAGCTCTGCTGCATGAGAGAGAGGGCGATAGACTGGAGTCCAGAA GAAGTTGGAACACCATCCAGAGCATCAAGGAGCTGAAAGAAGGCTACATGTCCC TGGTGGTGCACGAGATCGCTAACATGATGCTGGAGAATGATGCCATCGTGGTCT TGGAAAACCTTAACAGATCCTTTATGCAGAAGAGAGGCGGCATTGAGAAAAGCGT GTACCAGAAGTTTGAGAAAATGCTGATCGACAAGCTGGGCTACATCGTGGACAAA ACAAAAGATGTGTCAGATAATGGCGGAGCCCTGCACGCCGTGCAGCTGGCTGAC ACCTTCGAGAACTTTAACAAGACCCAGAAAGGCGCCATCCGGCAGTGCGGCTTC ATCI I I IATATCCCCGCCTGGCGGACAAGCAAAATTGACCCGGTAACCGGCTTTG TGCCCATGCTGAGATGTCAGTACGAATCTATCGTGGAATCCAAGAAGTTCTTTGG CAAATTCGACTCTATCTACTACGACGCCACCGGAAAGTACTTCGTGTTCCAGACC GACTTTACCAAGTTCAACACCGAGTCTAAGGGGGGCATCCAGAAGTGGGACATC TGTACCTACGGAGACAGAATCTACGCCCCTAGAACCAAAGACAGAAATAACAACC CTGTGTCCGAAAGAGTGAACCTGACAGAAGAAATGAAGAGCCTGTTCGTAAGCC ACAATATCAACATCCAGGGCGACATCAAGGCCGGCATTATGCAGCAGACAGACA AGGAGTTCTTCGAGTCGCTGCACAGACTGCTGAGACTGACCCTGCAGATCCGGA ACAGCAAGAAAAGCACCGGCAAGGACTACGAGGACTACATTATCAGTCCTGTGA TGGGCAAGGACGGAAGATTCTTCGACAGCCGGAACGCCGACGCCACCCAGCCC AAGGACGCCGACGCAAACGGCGCCTACAACATTGCCAGAAAAGGCCTGATGCTG CTGCGCCAGATCCAGGCCCAGGAGAAGCAGGACCTGTCTAATGGGAAGTGGCT GGAGTTCGCCCAGCGGtctagaAAGCGGACAGCAGACGGCTCCGAATTTGAAAGC CCTAAGAAAAAGAGAAAGGTGggatccGGCAAACCTATCCCCAATCCCCTGCTGGG CCTGGACAGCACCTGA

[0106] In some embodiments a ZKBG Type V Cas protein comprises an amino acid sequence of SEQ ID NO:37, SEQ ID NO:38, or SEQ ID NO:39. In some embodiments, a ZKBG Type V Cas protein has nickase activity, for example resulting from one or more amino acid substitutions relative to the sequence of SEQ ID NO:37, SEQ ID NO:38, or SEQ ID NO:39. In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D885 substitution, wherein the position of the D885 substitution is defined with respect to the amino acid numbering of SEQ ID NO:38 (corresponding to amino acid 908 of SEQ ID NO:121). In some embodiments, the one or more amino acid substitutions -50- providing nickase activity comprise an E978 substitution, wherein the position of the E978 substitution is defined with respect to the amino acid numbering of SEQ ID NO:38 (corresponding to amino acid 993 of SEQ ID NO:121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a R1194 substitution, wherein the position of the R1194 substitution is defined with respect to the amino acid numbering of SEQ ID NO:38 (corresponding to amino acid 1226 of SEQ ID NO:121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D1234 substitution, wherein the position of the D1234 substitution is defined with respect to the amino acid numbering of SEQ ID NO:38 (corresponding to amino acid 1263 of SEQ ID NO:121). In some embodiments, a ZKBG Type V Cas protein is catalytically inactive, for example due to a R1194 substitution in combination with a D885 substitution, a E978 substitution, and / or D1234 substitution. 6.2.8. ZZKD Type V Cas Proteins

[0107] In one aspect, the disclosure provides ZZKD Type V Cas proteins. ZZKD Type V Cas proteins can be further classified as Type V-A Cas proteins. The ZZKD Type V Cas proteins typically comprise an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:43. In some embodiments, the ZZKD Type V Cas proteins comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:43. In some embodiments, a ZZKD Type V Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:43.

[0108] Exemplary ZZKD Type V Cas protein sequences and nucleotide sequences encoding exemplary ZZKD Type V Cas proteins are set forth in Table 1H. TABLE1H ZZKD Type V Cas Sequences Name Sequence SEQ ID NO. Wildtype amino acid sequence (without N-terminal methionine) AEMFKDFTNLYPVSKTLRFELIPEGETLHYLEKNGVLENDEKRNEDYKKLKKLMDEY YRAYIDEALSNVHLSDLDRYAELYSIQNKSDEENVEFENVQLRLRTQIVGFLESRETY SSLFKKELIEKELPKFFIRREEELNLIKSFKGFTTMCTGFWENRKNMFSAEEKSTAIA YRVVHENLPKFMNNIRIFRLFIDEKLDCSEKLLEKAGVNSLSEVFELDYFNNTLSQRG IELYNCILGGFTEDEKHKIQGVNELINLYNQQTKEKKIPQLQPLYKQILSDTKSLSFLA DAFENDGGVLATVKALYDEFHEEILSERGLISTTLQNIEKYDSKGIFVKNDLTITGLSN SLFGDWKAINGSLNSWYEENVPRKERTEEKHVEVRKAYFKKLKSISLEFIEEAGLSE LRCKYKALLLEKAEAVCDAYKNAEELFSEAYNENTNLIADGKSVEKIKALLDSMKELE AVILMLSGTGEEAERDELFYGEFEKHRFVLNLLDNVFNKTRNYVTKKPYKTEKIKLTF DSPTLLDGWDRNKETSNKSVILMKDGYYYLGIMNKANNKAFENLKDTGGKCYSKM DYKLLPGPNKMLPKVFFAKKNIDYYAPSEDLLQKYKEGTHKKGKKFNLEDCHALIDF FKDSIAKHPEWNEFGFDFSDTKSYRDISDFYKEVSEQGYKISYRNVSVNYIDSLVRE GKLYLFKIYNKDFSPYSKGRPNLHTMYWKALFANKNFENRIYKLNGQAEMFYRKKSI PEDKRVIHSAKEPIDQRRNTDEKSLFDYDIIKDRRYTVDKFQFNVPITMNYTAPGSGR INRKMREAIKNCENMHIIGIDRGERHLLYVTVIDMQGNIKEQFSLNRILSEYKANNVAK SVETDYKTLLTKKEIERQDARKQWKSIENIKELKDGYMSQVVHVIAELMIKYNAIVVM EDLNFGFKRGRQKVERQVYQKFEKALIDKLNYLVDKTASEMENTGLYAALQLTEKF ESFKKMGKQNGGLFYVNAWNTSKMDPTTGFVNLLYPKYESIEKSKAYIEKFKDIQFC DDDEYGKYLAISFDYNDFTEKAKGAKTEWTICSYGKRLYNHRNKDGYWEEQELDLT EEYFNLFEEFGINAASNIKEQVIAQNSADFFRRFMWLLKMTLQIRNSETNGETDYML SPVKNEDGKFFNSDEVKDDTLPENADANGAYNIARKGLLLVERIKDCPDEELDKVDL KVTNLDWMKFAQR 43 Wildtype amino acid sequence (with N-terminal methionine) MAEMFKDFTNLYPVSKTLRFELIPEGETLHYLEKNGVLENDEKRNEDYKKLKKLMDE YYRAYIDEALSNVHLSDLDRYAELYSIQNKSDEENVEFENVQLRLRTQIVGFLESRET YSSLFKKELIEKELPKFFIRREEELNLIKSFKGFTTMCTGFWENRKNMFSAEEKSTAI AYRVVHENLPKFMNNIRIFRLFIDEKLDCSEKLLEKAGVNSLSEVFELDYFNNTLSQR GIELYNCILGGFTEDEKHKIQGVNELINLYNQQTKEKKIPQLQPLYKQILSDTKSLSFL ADAFENDGGVLATVKALYDEFHEEILSERGLISTTLQNIEKYDSKGIFVKNDLTITGLS NSLFGDWKAINGSLNSWYEENVPRKERTEEKHVEVRKAYFKKLKSISLEFIEEAGLS 44 ELRCKYKALLLEKAEAVCDAYKNAEELFSEAYNENTNLIADGKSVEKIKALLDSMKEL EAVILMLSGTGEEAERDELFYGEFEKHRFVLNLLDNVFNKTRNYVTKKPYKTEKIKLT FDSPTLLDGWDRNKETSNKSVILMKDGYYYLGIMNKANNKAFENLKDTGGKCYSKM DYKLLPGPNKMLPKVFFAKKNIDYYAPSEDLLQKYKEGTHKKGKKFNLEDCHALIDF FKDSIAKHPEWNEFGFDFSDTKSYRDISDFYKEVSEQGYKISYRNVSVNYIDSLVRE GKLYLFKIYNKDFSPYSKGRPNLHTMYWKALFANKNFENRIYKLNGQAEMFYRKKSI PEDKRVIHSAKEPIDQRRNTDEKSLFDYDIIKDRRYTVDKFQFNVPITMNYTAPGSGR INRKMREAIKNCENMHIIGIDRGERHLLYVTVIDMQGNIKEQFSLNRILSEYKANNVAK SVETDYKTLLTKKEIERQDARKQWKSIENIKELKDGYMSQVVHVIAELMIKYNAIVVM EDLNFGFKRGRQKVERQVYQKFEKALIDKLNYLVDKTASEMENTGLYAALQLTEKF ESFKKMGKQNGGLFYVNAWNTSKMDPTTGFVNLLYPKYESIEKSKAYIEKFKDIQFC DDDEYGKYLAISFDYNDFTEKAKGAKTEWTICSYGKRLYNHRNKDGYWEEQELDLT EEYFNLFEEFGINAASNIKEQVIAQNSADFFRRFMWLLKMTLQIRNSETNGETDYML SPVKNEDGKFFNSDEVKDDTLPENADANGAYNIARKGLLLVERIKDCPDEELDKVDL KVTNLDWMKFAQR Expression construct (with N-terminal methionine, V5-tag and C-terminal NLS) aa sequence MGAEMFKDFTNLYPVSKTLRFELIPEGETLHYLEKNGVLENDEKRNEDYKKLKKLM DEYYRAYIDEALSNVHLSDLDRYAELYSIQNKSDEENVEFENVQLRLRTQIVGFLES RETYSSLFKKELIEKELPKFFIRREEELNLIKSFKGFTTMCTGFWENRKNMFSAEEKS TAIAYRVVHENLPKFMNNIRIFRLFIDEKLDCSEKLLEKAGVNSLSEVFELDYFNNTLS QRGIELYNCILGGFTEDEKHKIQGVNELINLYNQQTKEKKIPQLQPLYKQILSDTKSLS FLADAFENDGGVLATVKALYDEFHEEILSERGLISTTLQNIEKYDSKGIFVKNDLTITG LSNSLFGDWKAINGSLNSWYEENVPRKERTEEKHVEVRKAYFKKLKSISLEFIEEAG LSELRCKYKALLLEKAEAVCDAYKNAEELFSEAYNENTNLIADGKSVEKIKALLDSMK ELEAVILMLSGTGEEAERDELFYGEFEKHRFVLNLLDNVFNKTRNYVTKKPYKTEKIK LTFDSPTLLDGWDRNKETSNKSVILMKDGYYYLGIMNKANNKAFENLKDTGGKCYS KMDYKLLPGPNKMLPKVFFAKKNIDYYAPSEDLLQKYKEGTHKKGKKFNLEDCHALI DFFKDSIAKHPEWNEFGFDFSDTKSYRDISDFYKEVSEQGYKISYRNVSVNYIDSLV REGKLYLFKIYNKDFSPYSKGRPNLHTMYWKALFANKNFENRIYKLNGQAEMFYRK KSIPEDKRVIHSAKEPIDQRRNTDEKSLFDYDIIKDRRYTVDKFQFNVPITMNYTAPG SGRINRKMREAIKNCENMHIIGIDRGERHLLYVTVIDMQGNIKEQFSLNRILSEYKAN NVAKSVETDYKTLLTKKEIERQDARKQWKSIENIKELKDGYMSQVVHVIAELMIKYNA IVVMEDLNFGFKRGRQKVERQVYQKFEKALIDKLNYLVDKTASEMENTGLYAALQLT EKFESFKKMGKQNGGLFYVNAWNTSKMDPTTGFVNLLYPKYESIEKSKAYIEKFKDI QFCDDDEYGKYLAISFDYNDFTEKAKGAKTEWTICSYGKRLYNHRNKDGYWEEQE LDLTEEYFNLFEEFGINAASNIKEQVIAQNSADFFRRFMWLLKMTLQIRNSETNGETD YMLSPVKNEDGKFFNSDEVKDDTLPENADANGAYNIARKGLLLVERIKDCPDEELDK VDLKVTNLDWMKFAQRSRKRTADGSEFESPKKKRKVGSGKPIPNPLLGLDST 45 Wildtype coding sequence (with N-terminal methionine and stop codon) ATGGCTGAGATGTTTAAAGA Illi ACGAATTTGTATCCTGTTTCAAAAACCTTGC G 1 1 1 1GAATTAATTCCTGAAGGGGAAACATTGCATTATCTTGAAAAAAATGGCGT TCTGGAAAACGATGAGAAGCGAAACGAAGATTATAAGAAGTTGAAAAAACTGAT GGATGAATATTACCGTGCATACATCGATGAAGCTTTATCTAATGTTCATCTTTCA GATTTGGATAGATATGCAGAATTATATTCAATTCAGAATAAATCGGATGAAGAAA ATGTAGAATTCGAAAATGTTCAACTGAGATTGAGAACACAAATTGTTGGATTCTT AGAATCCAGAGAAACCTATTCTTCAC Illi CAAAAAAGAACTGATTGAGAAGGAA CTTCCTAAATTCTTTATTCGGAGAGAAGAGGAGCTTAATTTAATCAAATCATTTAA AGGI 1 1 1ACAACGA 1 G 1 GCACCGGC 1 1 C 1 GGGAAAA 1 CGGAAAAA1A1 G 1 1 1 1 CT GCCGAAGAAAAATCTACAGCAATAGCATATCGTGTAGTCCATGAAAACCTACCTA AGTTTATGAATAATATAAGAATTTTTCGTTTGTTCATTGATGAAAAGTTGGACTGT TCTGAAAAATTGCTGGAAAAAGCCGGAGTGAATTCTCTGAGTGAAGTGTTTGAA CTTGATTA Illi AACAATACATTATCCCAACGTGGCATTGAATTGTATAACTGTAT ATTGGGCGGATTTACCGAGGATGAAAAGCATAAGATTCAAGGCGTAAACGAATT GATTAATTTGTACAATCAGCAGACAAAAGAGAAGAAGATTCCACAGTTGCAGCC GCTGTACAAGCAGATTCTCAGCGATACCAAGAGCCTTTCATTTCTTGCAGATGC ATTTGAAAACGACGGGGGGGTCTTAGCGACTGTAAAAGCATTATATGATGAATTT CATGAAGAGA Illi GAGCGAAAGGGGATTAATCTCTACGACATTACAGAATATTG AAAAGTATGATTCAAAAGGCATCTTCGTAAAAAACGATTTAACGATTACCGGTTT ATCAAATAGTTTGTTCGGCGACTGGAAGGCTATTAATGGTAGTTTAAATTCGTGG TATGAGGAGAACGTGCCTCGAAAAGAAAGAACTGAAGAGAAACATGTAGAGGTA AGAAAAGCCTA Illi AAAAAGTTAAAATCAATAAGCCTGGAATTTATCGAGGAGG CCGGATTGTCGGAACTCCGTTGCAAATATAAAGCCCTTC Illi AGAAAAAGCAGA GGCTGTTTGCGATGCGTACAAAAATGCAGAAGAGCTTTTTAGTGAAGCTTATAAT GAAAATACTAACCTTATTGCCGATGGAAAGTCTGTGGAAAAAATAAAAGCGCTAT TGGATTCTATGAAAGAGCTTGAAGCGGTGATTCTTATGCTTTCCGGAACCGGAG AGGAAGCAGAACGGGATGAATTG Illi ACGGCGAATTTGAAAAACATAGGTTCG TATTGAATCTCTTAGACAACGTATTTAATAAAACGAGAAATTACGTAACAAAGAAA CCATATAAGACTGAGAAGATTAAATTAACATTTGATTCCCCAACGCTGCTAGACG GGTGGGATCGTAATAAAGAAACATCAAACAAGTCCGTGATACTTATGAAAGATG GCTATTATTACCTTGGAATTATGAACAAGGCAAATAACAAAGCCTTTGAGAATTT 46 GAAAGACACAGGCGGGAAATGCTATAGCAAGATGGATTACAAAC Illi GCCTGG ACCAAACAAGATGTTGCCGAAGGTGTTTTTTGCAAAGAAAAACATCGACTATTAT GCACCAAGCGAAGACTTGCTACAGAAATATAAAGAGGGAACACATAAAAAAGGA AAGAAATTTAATCTAGAGGATTGTCACGCGTTAATAGACTTTTTTAAAGACTCAAT TGCAAAGCATCCAGAATGGAACGAGTTTGGATTTGATTTTTCAGATACGAAATCA TATCGAGATATTAGTGATTTCTATAAGGAGGTTTCAGAGCAGGGATACAAAATCA GTTATCGAAATGTATCTGTTAATTACATAGATTCTCTAGTAAGAGAAGGGAAATT GTATTTGTTCAAAATTTATAATAAAGATTTTTCACCGTACAGCAAAGGCAGACCAA ATCTTCATACGATGTATTGGAAAGCGTTATTCGCTAATAAGAA Illi GAAAATCG CATATATAAGTTAAATGGCCAGGCAGAAATGTTCTATCGAAAAAAGAGCATTCCG GAAGACAAGAGGGTGATTCACTCGGCAAAAGAACCAATCGATCAGAGAAGAAAT ACGGATGAAAAGAGCCTCTTTGATTATGACATTATTAAAGATCGGCGATATACTG TGGACAAATTCCAATTTAATGTTCCGATTACGATGAATTACACTGCACCGGGTTC CGGCCGAATTAACAGAAAAATGCGGGAAGCGATTAAGAACTGTGAAAATATGCA TATTATCGGAATAGATAGAGGCGAACGTCATTTGCTGTATGTGACGGTTATCGAT ATGCAGGGAAACATTAAAGAACAG Illi CATTAAATCGAATCCTGAGTGAGTACA AGGCAAACAATGTGGCTAAAAGTGTCGAAACGGACTACAAAACACTCCTGACAA AAAAAGAAATTGAACGACAGGATGCAAGAAAGCAGTGGAAGAGCATTGAAAATA TTAAGGAATTAAAAGACGGCTACATGAGCCAGGTTGTGCATGTGATTGCCGAAC TCATGATAAAGTACAATGCGATTGTGGTTATGGAGGATTTGAATTTCGGATTCAA GCGAGGAAGACAGAAGGTTGAGAGACAGGTTTACCAGAAGTTTGAGAAGGCAT TAATTGATAAATTGAACTATTTGGTTGATAAAACAGCCTCTGAAATGGAGAACAC CGGTCTGTATGCGGCATTGCAGCTTACAGAAAAATTTGAGAGCTTTAAGAAAAT GGGCAAACAAAATGGTGGATTA Illi ATGTAAACGCATGGAATACCAGTAAAATG GATCCAACAACCGG Illi GTGAACCTTCTCTATCCTAAATATGAGAGCATTGAAA AAAGCAAAGCGTATATTGAGAAATTCAAGGATATTCAG Illi GTGATGATGACGA ATATGGAAAGTACCTTGCAATATC Illi GATTATAACGATTTCACGGAGAAGGCA AAGGGCGCAAAAACGGAATGGACCATTTGCTCTTATGGAAAGAGATTGTATAAT CACAGAAATAAAGATGGGTATTGGGAAGAGCAGGAATTGGATCTTACAGAAGAG TATTTCAATCTGTTTGAAGAATTTGGAATTAATGCAGCGTCTAATATTAAAGAACA AGTCATCGCACAGAATTCTGCAGACTTTTTTAGACGGTTTATGTGGCTTTTGAAA ATGACCTTACAGATTAGAAACAGTGAAACAAATGGGGAGACGGATTATATGCTTT CTCCGGTAAAAAATGAAGACGGAAAATTCTTTAATTCAGATGAAGTCAAGGATGA CACGCTTCCGGAAAATGCGGATGCGAATGGTGCATACAACATCGCTAGAAAAG GATTACTGCTTGTGGAAAGAATTAAAGACTGTCCGGACGAAGAACTTGATAAGG TTGATTTGAAGGTAACAAATTTAGATTGGATGAAATTTGCACAGAGGTAA Codon optimized coding sequence (no N-terminal methionine, no stop codon) GCCGAAATGTTCAAGGACTTCACCAACCTGTACCCAGTGTCCAAAACCCTCCGG TTCGAATTGATCCCCGAGGGCGAAACACTGCACTACCTAGAAAAGAACGGAGTG CTGGAAAACGACGAGAAGAGAAATGAGGATTACAAGAAGCTGAAGAAACTCATG GATGAATACTACCGGGCCTACATCGACGAGGCCTTATCTAATGTCCACCTGTCC GATCTGGACCGGTACGCCGAACTGTATTCTATCCAGAACAAGAGCGATGAGGA GAACGTGGAGTTCGAGAATGTGCAGCTGCGCCTGAGAACCCAGATCGTGGGCT TCCTGGAAAGCAGAGAAACCTACAGCAGCCTGTTCAAGAAGGAGCTGATCGAAA AAGAACTGCCTAAGTTTTTCATCAGAAGAGAGGAAGAGCTGAACCTGATAAAGA GCTTTAAGGGCTTTACCACTATGTGCACCGGCTTCTGGGAAAATCGGAAGAACA TGTTCAGCGCCGAGGAAAAGTCCACAGCCATCGCCTATAGAGTGGTCCATGAAA ACCTGCCCAAGTTCATGAACAACATTAGAATCTTCCGGCTGTTTATCGACGAGAA GCTGGATTGTAGCGAGAAGCTGCTGGAGAAGGCCGGCGTGAACAGCCTGAGC GAGGTGTTCGAGCTTGACTATTTCAATAACACCCTGAGCCAGAGAGGCATCGAG CTGTACAACTGCATCCTGGGCGGATTCACCGAGGATGAAAAACACAAGATCCAG GGAGTGAACGAGTTGATCAACCTGTACAACCAGCAGACAAAGGAGAAGAAAATT CCTCAGCTGCAACCTCTGTACAAACAGATCCTGTCTGACACGAAGTCGCTGTCC TTTCTGGCTGATGCCTTTGAAAACGACGGAGGAGTGCTGGCTACAGTGAAGGCT TTATATGATGAGTTTCACGAGGAAATCCTGAGCGAGAGAGGCCTGATCAGCACA ACCCTGCAGAACATTGAGAAGTACGATAGTAAGGGCATCTTTGTTAAGAACGAT CTCACCATTACAGGCCTGTCCAACAGCCTGTTTGGAGATTGGAAGGCCATCAAT GGAAGCCTGAACAGCTGGTACGAGGAGAACGTGCCCCGGAAGGAGCGAACAG AAGAGAAACACGTGGAAGTGAGAAAGGCTTAI 1 1 1AAGAAGCTGAAGTCTATCA GCCTGGAGTTCATCGAGGAGGCCGGACTGAGCGAGCTGCGGTGCAAGTACAA GGCCCTGCTGCTGGAGAAAGCCGAGGCTGTGTGCGACGCGTACAAGAACGCC GAGGAGCTGTTTAGCGAGGCCTATAATGAGAACACCAATCTGATCGCCGATGG CAAATCTGTGGAAAAAATCAAAGCCCTGCTGGACAGCATGAAGGAGCTGGAGG CCGTGATCCTGATGCTGAGCGGCACAGGCGAGGAGGCCGAGCGGGACGAACT Gill IATGGCGAGTTCGAAAAACATAGATTCGTGCTGAATCTGCTGGACAACGT GTTCAACAAGACCAGAAACTACGTGACCAAGAAGCCTTACAAGACCGAGAAGAT CAAGCTCACCTTCGACAGCCCTACCCTTCTGGATGGCTGGGACCGTAACAAGG AGACAAGCAACAAGAGCGTGATCCTGATGAAGGATGGCTACTACTACCTGGGC ATCATGAACAAAGCCAACAACAAGGCCTTCGAGAACCTGAAGGACACAGGAGG 47 CAAATGCTACAGCAAGATGGACTACAAGCTGCTGCCTGGCCCTAACAAGATGCT GCCTAAGGTGTTCTTTGCCAAAAAGAACATCGACTACTACGCCCCTAGCGAGGA CCTGCTGCAGAAGTACAAGGAGGGCACCCACAAGAAAGGGAAGAAGTTCAATC TTGAGGACTGTCACGCCCTGATCGACTTCTTCAAGGACAGCATCGCTAAACACC CCGAGTGGAACGAGTTCGGCTTCGAC Illi CTGACACCAAGTCTTATAGAGACA TCTCGGATTTCTACAAGGAGGTCAGCGAACAGGGCTACAAGATTAGCTACCGGA ACGTGAGTGTTAACTACATCGACAGTCTGGTGCGGGAAGGTAAGCTGTACCTGT TCAAGATCTACAACAAGGACTTCAGCCCATACTCCAAAGGACGTCCCAACCTGC ACACCATGTACTGGAAAGCCCTGTTCGCCAATAAAAACTTCGAAAACCGGATCT ACAAGCTGAACGGCCAGGCCGAAATGTTCTACAGAAAGAAATCTATCCCTGAAG ATAAGCGGGTGATCCACAGCGCCAAAGAACCTATCGATCAGAGAAGAAACACC GACGAAAAGTCTCTGTTTGACTACGACATCATCAAGGACAGACGGTACACCGTG GACAAGTTCCAGTTCAACGTGCCAATCACAATGAACTACACCGCCCCTGGCAGC GGCAGAATCAACAGAAAGATGCGGGAAGCTATCAAGAATTGCGAGAATATGCAC ATCATCGGCATCGACCGGGGAGAGCGGCACCTGCTGTACGTGACCGTGATCGA CATGCAGGGCAACATCAAAGAACAGTTCTCTCTCAACCGCATCCTGTCTGAGTA CAAGGCCAATAACGTCGCCAAGAGCGTGGAGACAGACTACAAAACACTGCTGA CGAAAAAAGAGATCGAGAGACAGGACGCTAGAAAGCAATGGAAGAGCATCGAA AACATCAAAGAGCTGAAAGACGGCTATATGAGCCAGGTGGTGCACGTGATAGC AGAGCTGATGATCAAGTACAACGCCATAGTTGTGATGGAGGACCTGAATTTCGG CTTCAAGAGAGGCCGGCAAAAGGTGGAGAGACAGGTGTACCAGAAATTCGAGA AGGCCCTGATCGATAAGCTGAATTACCTGGTGGATAAGACAGCTTCCGAGATGG AAAACACCGGCCTGTACGCCGCCCTGCAGCTGACAGAGAAGTTCGAATCCTTC AAGAAGATGGGCAAACAGAACGGCGGCTTGTTCTACGTGAACGCCTGGAACAC CAGCAAGATGGACCCTACCACCGGATTCGTGAACCTGCTGTACCCTAAGTACGA ATCTATCGAAAAGAGCAAGGCCTATATCGAGAAATTCAAGGATATCCAG 1 1 1 1 GT GACGACGATGAATACGGCAAATACCTGGCAATTTCTTTCGACTACAACGACTTC ACAGAAAAGGCCAAGGGCGCCAAGACCGAGTGGACCATCTGCAGCTACGGCAA AAGACTGTACAACCACAGAAATAAGGACGGCTACTGGGAGGAGCAGGAGCTGG ATCTGACCGAGGAGTACTTCAACCTGTTCGAAGAGTTCGGCATCAACGCTGCCA GCAACATCAAGGAACAAGTGATCGCTCAGAACAGCGCCGATTTCTTCAGAAGAT TCATGTGGCTGCTGAAGATGACCCTGCAGATCAGGAACTCTGAAACTAACGGCG AAACCGATTACATGCTGAGCCCTGTGAAGAACGAGGACGGCAAATTCTTCAACT CTGACGAGGTGAAGGACGACACCCTGCCCGAGAATGCCGACGCCAACGGCGC CTACAACATCGCAAGAAAGGGCCTGCTGCTGGTCGAACGTATCAAGGATTGCC CCGACGAAGAACTAGACAAGGTGGACCTGAAGGTCACCAACCTGGACTGGATG AAATTCGCCCAAAGA Expression construct (with N-terminal methionine and stop codon, includes V5-tag and C-terminal NLS) ATGggcGCCGAAATGTTCAAGGACTTCACCAACCTGTACCCAGTGTCCAAAACCC TCCGGTTCGAATTGATCCCCGAGGGCGAAACACTGCACTACCTAGAAAAGAACG GAGTGCTGGAAAACGACGAGAAGAGAAATGAGGATTACAAGAAGCTGAAGAAA CTCATGGATGAATACTACCGGGCCTACATCGACGAGGCCTTATCTAATGTCCAC CTGTCCGATCTGGACCGGTACGCCGAACTGTATTCTATCCAGAACAAGAGCGAT GAGGAGAACGTGGAGTTCGAGAATGTGCAGCTGCGCCTGAGAACCCAGATCGT GGGCTTCCTGGAAAGCAGAGAAACCTACAGCAGCCTGTTCAAGAAGGAGCTGA TCGAAAAAGAACTGCCTAAGTTTTTCATCAGAAGAGAGGAAGAGCTGAACCTGA TAAAGAGCTTTAAGGGCTTTACCACTATGTGCACCGGCTTCTGGGAAAATCGGA AGAACATGTTCAGCGCCGAGGAAAAGTCCACAGCCATCGCCTATAGAGTGGTC CATGAAAACCTGCCCAAGTTCATGAACAACATTAGAATCTTCCGGCTGTTTATCG ACGAGAAGCTGGATTGTAGCGAGAAGCTGCTGGAGAAGGCCGGCGTGAACAG CCTGAGCGAGGTGTTCGAGCTTGACTATTTCAATAACACCCTGAGCCAGAGAGG CATCGAGCTGTACAACTGCATCCTGGGCGGATTCACCGAGGATGAAAAACACAA GATCCAGGGAGTGAACGAGTTGATCAACCTGTACAACCAGCAGACAAAGGAGA AGAAAATTCCTCAGCTGCAACCTCTGTACAAACAGATCCTGTCTGACACGAAGT CGCTGTCCTTTCTGGCTGATGCCTTTGAAAACGACGGAGGAGTGCTGGCTACA GTGAAGGCTTTATATGATGAGTTTCACGAGGAAATCCTGAGCGAGAGAGGCCTG ATCAGCACAACCCTGCAGAACATTGAGAAGTACGATAGTAAGGGCATCTTTGTT AAGAACGATCTCACCATTACAGGCCTGTCCAACAGCCTGTTTGGAGATTGGAAG GCCATCAATGGAAGCCTGAACAGCTGGTACGAGGAGAACGTGCCCCGGAAGGA GCGAACAGAAGAGAAACACGTGGAAGTGAGAAAGGCTTA Illi AAGAAGCTGAA GTCTATCAGCCTGGAGTTCATCGAGGAGGCCGGACTGAGCGAGCTGCGGTGCA AGTACAAGGCCCTGCTGCTGGAGAAAGCCGAGGCTGTGTGCGACGCGTACAAG AACGCCGAGGAGCTGTTTAGCGAGGCCTATAATGAGAACACCAATCTGATCGCC GATGGCAAATCTGTGGAAAAAATCAAAGCCCTGCTGGACAGCATGAAGGAGCT GGAGGCCGTGATCCTGATGCTGAGCGGCACAGGCGAGGAGGCCGAGCGGGAC GAACTG Illi ATGGCGAGTTCGAAAAACATAGATTCGTGCTGAATCTGCTGGAC AACGTGTTCAACAAGACCAGAAACTACGTGACCAAGAAGCCTTACAAGACCGAG AAGATCAAGCTCACCTTCGACAGCCCTACCCTTCTGGATGGCTGGGACCGTAAC AAGGAGACAAGCAACAAGAGCGTGATCCTGATGAAGGATGGCTACTACTACCTG 48 GGCATCATGAACAAAGCCAACAACAAGGCCTTCGAGAACCTGAAGGACACAGG AGGCAAATGCTACAGCAAGATGGACTACAAGCTGCTGCCTGGCCCTAACAAGAT GCTGCCTAAGGTGTTCTTTGCCAAAAAGAACATCGACTACTACGCCCCTAGCGA GGACCTGCTGCAGAAGTACAAGGAGGGCACCCACAAGAAAGGGAAGAAGTTCA ATCTTGAGGACTGTCACGCCCTGATCGACTTCTTCAAGGACAGCATCGCTAAAC ACCCCGAGTGGAACGAGTTCGGCTTCGAC Illi CTGACACCAAGTCTTATAGAG ACATCTCGGATTTCTACAAGGAGGTCAGCGAACAGGGCTACAAGATTAGCTACC GGAACGTGAGTGTTAACTACATCGACAGTCTGGTGCGGGAAGGTAAGCTGTAC CTGTTCAAGATCTACAACAAGGACTTCAGCCCATACTCCAAAGGACGTCCCAAC CTGCACACCATGTACTGGAAAGCCCTGTTCGCCAATAAAAACTTCGAAAACCGG ATCTACAAGCTGAACGGCCAGGCCGAAATGTTCTACAGAAAGAAATCTATCCCT GAAGATAAGCGGGTGATCCACAGCGCCAAAGAACCTATCGATCAGAGAAGAAA CACCGACGAAAAGTCTCTGTTTGACTACGACATCATCAAGGACAGACGGTACAC CGTGGACAAGTTCCAGTTCAACGTGCCAATCACAATGAACTACACCGCCCCTGG CAGCGGCAGAATCAACAGAAAGATGCGGGAAGCTATCAAGAATTGCGAGAATAT GCACATCATCGGCATCGACCGGGGAGAGCGGCACCTGCTGTACGTGACCGTGA TCGACATGCAGGGCAACATCAAAGAACAGTTCTCTCTCAACCGCATCCTGTCTG AGTACAAGGCCAATAACGTCGCCAAGAGCGTGGAGACAGACTACAAAACACTG CTGACGAAAAAAGAGATCGAGAGACAGGACGCTAGAAAGCAATGGAAGAGCAT CGAAAACATCAAAGAGCTGAAAGACGGCTATATGAGCCAGGTGGTGCACGTGA TAGCAGAGCTGATGATCAAGTACAACGCCATAGTTGTGATGGAGGACCTGAATT TCGGCTTCAAGAGAGGCCGGCAAAAGGTGGAGAGACAGGTGTACCAGAAATTC GAGAAGGCCCTGATCGATAAGCTGAATTACCTGGTGGATAAGACAGCTTCCGAG ATGGAAAACACCGGCCTGTACGCCGCCCTGCAGCTGACAGAGAAGTTCGAATC CTTCAAGAAGATGGGCAAACAGAACGGCGGCTTGTTCTACGTGAACGCCTGGA ACACCAGCAAGATGGACCCTACCACCGGATTCGTGAACCTGCTGTACCCTAAGT ACGAATCTATCGAAAAGAGCAAGGCCTATATCGAGAAATTCAAGGATATCCAGTT TTGTGACGACGATGAATACGGCAAATACCTGGCAATTTCTTTCGACTACAACGA CTTCACAGAAAAGGCCAAGGGCGCCAAGACCGAGTGGACCATCTGCAGCTACG GCAAAAGACTGTACAACCACAGAAATAAGGACGGCTACTGGGAGGAGCAGGAG CTGGATCTGACCGAGGAGTACTTCAACCTGTTCGAAGAGTTCGGCATCAACGCT GCCAGCAACATCAAGGAACAAGTGATCGCTCAGAACAGCGCCGATTTCTTCAGA AGATTCATGTGGCTGCTGAAGATGACCCTGCAGATCAGGAACTCTGAAACTAAC GGCGAAACCGATTACATGCTGAGCCCTGTGAAGAACGAGGACGGCAAATTCTT CAACTCTGACGAGGTGAAGGACGACACCCTGCCCGAGAATGCCGACGCCAACG GCGCCTACAACATCGCAAGAAAGGGCCTGCTGCTGGTCGAACGTATCAAGGAT TGCCCCGACGAAGAACTAGACAAGGTGGACCTGAAGGTCACCAACCTGGACTG GATGAAATTCGCCCAAAGGtctagaAAGCGGACAGCAGACGGCTCCGAATTTGAA AGCCCTAAGAAAAAGAGAAAGGTGggatccGGCAAACCTATCCCCAATCCCCTGC TGGGCCTGGACAGCACCTGA

[0109] In some embodiments a ZZKD Type V Cas protein comprises an amino acid sequence of SEQ ID NO:43, SEQ ID NO:44, or SEQ ID NO:45. In some embodiments, a ZZKD Type V Cas protein has nickase activity, for example resulting from one or more amino acid substitutions relative to the sequence of SEQ ID NO:43, SEQ ID NO:44, or SEQ ID NO:45. In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D828 substitution, wherein the position of the D828 substitution is defined with respect to the amino acid numbering of SEQ ID NO:44 (corresponding to amino acid 908 of SEQ ID NO:121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise an E925 substitution, wherein the position of the E925 substitution is defined with respect to the amino acid numbering of SEQ ID NO:44 (corresponding to amino acid 993 of SEQ ID NO:121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a R1138 substitution, wherein the position of the R1138 substitution is defined with respect to the amino acid numbering of SEQ ID NO:44 (corresponding to amino acid 1226 of SEQ ID NO:121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D1176 substitution, wherein the position of the D1176 substitution is defined with respect to the amino acid numbering of SEQ ID NO:44 (corresponding to amino acid 1263 of SEQ ID NO:121). In some embodiments, a ZZKD Type V Cas protein is catalytically inactive, for example due to a R1138 substitution in combination with a D828 substitution, a E925 substitution, and / or D1176 substitution. 6.2.9. ZXPB Type V Cas Proteins

[0110] In one aspect, the disclosure provides ZXPB Type V Cas proteins. ZXPB Type V Cas proteins can be further classified as Type V-A Cas proteins. The ZXPB Type V Cas proteins typically comprise an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:49. In some embodiments, the ZXPB Type V Cas proteins comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:49. In some embodiments, a ZXPB Type V Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:49.

[0111] Exemplary ZXPB Type V Cas protein sequences and nucleotide sequences encoding exemplary ZXPB Type V Cas proteins are set forth in Table 11. TABLE 11 ZXPB Type V Cas Sequences Name Sequence SEQ ID NO. Wildtype amino acid sequence (without N-terminal methionine) KLEDFTNLYSLSKTLRFELRPIGKTRENIENGGLLRQDEDRAEKYVHIKKLIDEYHKAYI DKQLSGLVLQYADIGKANSLEEYYHSTRKSKDSDKDKIVKIQDNLRKQIVKRLKDSDE FKRIDKKELIQSDLAEFIKPAEDRALIAEFKNFTTYFTGFNENRQNMYSDKAISTAIAYR LIHENLPKFIDNIETFDRIAGITELYDQTSSDAEIFRLEHFSETLSQKQIDAYNSVMGRY NMLINEYNQTHKQSRLPKFKMLYKQILSDREHPSWLPEQFESDTAVLTAIRECYDDLR IPMANLKTLLEGLGNYDPSGIFLRNDQHLSQISKRLTGDRSSIERSVTEDLLTSRRLNK RKSRTTDEEESRKLFKQKGSLSIGYIADTAKIDVERYFAKLGAINTVTEQSENLFAKAE NARTTADELLANDYPAGKRLVQSNDDIALLKNLLDALMELQWFVKPLLGTGDEAGKD ERFYGEFAQIWEQLDRITPLYNMVRNYVTRKPYSTDKFKLNFESAALLGGWDKNKEP DCLSVILRKDEQYYLGIINKNHKKIFENDILPCEGECYDKMVYKLLPGANKMLPKVFFS ASRIAEFAPSDEVKRIYNDKTFQKGEKFDLNDCRTLIDFYKASIDKHEEWNKFGFEFS DTNNYEDISGFFREVDRQGYKMSFRPVAASYIETLVEEGKLYLFQIYNKDFSAYSKGT PNMHTLYWRMLFDERNLSDVVYQLNGGAELFFRRKSLQNGRPTHPANIPIKNKNSR NDKKESLFDYDLIKDRRYTVDKFQFHVPITLNFKSDGAGRINERVREYLRSADDVHVI GIDRGERNLLYLVVTDMDGNICEQFSLNEICNTDYHSLLDEREHKRMQERQSWQAIE GIKELKEGYLSQVVHRIATLMVKYRAIVVLEDLNFGFMRSRQKVEKSVYQKFEHMLID KLNYLVDKKANPTTPGGLLKAYQLTDKFESFQKLGKQSGFLFYVPAWNTSKIDPATG FVNMLDLGYESIDKAKTLLCKFDSIRYNACKDWFEFALDYDKFGSKATGTRTKWTVC TYGQRIDTYRNKDSQWVSRDVDLTNELKSLFSEHGIDIYSNLKDAIVAQNDKEFFANM QRILKLTMQMRNSKTGTDTDYIVSPVADANGRFFDSRQADATMPKDADANGAYNIAR KGIMLVQQIKQSDDLRTMKFDISNKSWLRFAQHTNQADE 49 Wildtype amino acid sequence (with N-terminal methionine) MKLEDFTNLYSLSKTLRFELRPIGKTRENIENGGLLRQDEDRAEKYVHIKKLIDEYHKA YIDKQLSGLVLQYADIGKANSLEEYYHSTRKSKDSDKDKIVKIQDNLRKQIVKRLKDSD EFKRIDKKELIQSDLAEFIKPAEDRALIAEFKNFTTYFTGFNENRQNMYSDKAISTAIAY RLIHENLPKFIDNIETFDRIAGITELYDQTSSDAEIFRLEHFSETLSQKQIDAYNSVMGR YNMLINEYNQTHKQSRLPKFKMLYKQILSDREHPSWLPEQFESDTAVLTAIRECYDDL RIPMANLKTLLEGLGNYDPSGIFLRNDQHLSQISKRLTGDRSSIERSVTEDLLTSRRLN KRKSRTTDEEESRKLFKQKGSLSIGYIADTAKIDVERYFAKLGAINTVTEQSENLFAKA ENARTTADELLANDYPAGKRLVQSNDDIALLKNLLDALMELQWFVKPLLGTGDEAGK DERFYGEFAQIWEQLDRITPLYNMVRNYVTRKPYSTDKFKLNFESAALLGGWDKNKE PDCLSVILRKDEQYYLGIINKNHKKIFENDILPCEGECYDKMVYKLLPGANKMLPKVFF SASRIAEFAPSDEVKRIYNDKTFQKGEKFDLNDCRTLIDFYKASIDKHEEWNKFGFEF SDTNNYEDISGFFREVDRQGYKMSFRPVAASYIETLVEEGKLYLFQIYNKDFSAYSKG TPNMHTLYWRMLFDERNLSDVVYQLNGGAELFFRRKSLQNGRPTHPANIPIKNKNS RNDKKESLFDYDLIKDRRYTVDKFQFHVPITLNFKSDGAGRINERVREYLRSADDVHV IGIDRGERNLLYLVVTDMDGNICEQFSLNEICNTDYHSLLDEREHKRMQERQSWQAIE GIKELKEGYLSQVVHRIATLMVKYRAIVVLEDLNFGFMRSRQKVEKSVYQKFEHMLID KLNYLVDKKANPTTPGGLLKAYQLTDKFESFQKLGKQSGFLFYVPAWNTSKIDPATG FVNMLDLGYESIDKAKTLLCKFDSIRYNACKDWFEFALDYDKFGSKATGTRTKWTVC TYGQRIDTYRNKDSQWVSRDVDLTNELKSLFSEHGIDIYSNLKDAIVAQNDKEFFANM 50 TABLE 11 ZXPB Type V Cas Sequences Name Sequence SEQ ID NO. QRILKLTMQMRNSKTGTDTDYIVSPVADANGRFFDSRQADATMPKDADANGAYNIAR KGIMLVQQIKQSDDLRTMKFDISNKSWLRFAQHTNQADE Expression construct (with N-terminal methionine, V5-tag and C-terminal NLS) aa sequence MGKLEDFTNLYSLSKTLRFELRPIGKTRENIENGGLLRQDEDRAEKYVHIKKLIDEYHK AYIDKQLSGLVLQYADIGKANSLEEYYHSTRKSKDSDKDKIVKIQDNLRKQIVKRLKDS DEFKRIDKKELIQSDLAEFIKPAEDRALIAEFKNFTTYFTGFNENRQNMYSDKAISTAIA YRLIHENLPKFIDNIETFDRIAGITELYDQTSSDAEIFRLEHFSETLSQKQIDAYNSVMG RYNMLINEYNQTHKQSRLPKFKMLYKQILSDREHPSWLPEQFESDTAVLTAIRECYD DLRIPMANLKTLLEGLGNYDPSGIFLRNDQHLSQISKRLTGDRSSIERSVTEDLLTSRR LNKRKSRTTDEEESRKLFKQKGSLSIGYIADTAKIDVERYFAKLGAINTVTEQSENLFA KAENARTTADELLANDYPAGKRLVQSNDDIALLKNLLDALMELQWFVKPLLGTGDEA GKDERFYGEFAQIWEQLDRITPLYNMVRNYVTRKPYSTDKFKLNFESAALLGGWDK NKEPDCLSVILRKDEQYYLGIINKNHKKIFENDILPCEGECYDKMVYKLLPGANKMLPK VFFSASRIAEFAPSDEVKRIYNDKTFQKGEKFDLNDCRTLIDFYKASIDKHEEWNKFG FEFSDTNNYEDISGFFREVDRQGYKMSFRPVAASYIETLVEEGKLYLFQIYNKDFSAY SKGTPNMHTLYWRMLFDERNLSDVVYQLNGGAELFFRRKSLQNGRPTHPANIPIKN KNSRNDKKESLFDYDLIKDRRYTVDKFQFHVPITLNFKSDGAGRINERVREYLRSADD VHVIGIDRGERNLLYLVVTDMDGNICEQFSLNEICNTDYHSLLDEREHKRMQERQSW QAIEGIKELKEGYLSQVVHRIATLMVKYRAIVVLEDLNFGFMRSRQKVEKSVYQKFEH MLIDKLNYLVDKKANPTTPGGLLKAYQLTDKFESFQKLGKQSGFLFYVPAWNTSKIDP ATGFVNMLDLGYESIDKAKTLLCKFDSIRYNACKDWFEFALDYDKFGSKATGTRTKW TVCTYGQRIDTYRNKDSQWVSRDVDLTNELKSLFSEHGIDIYSNLKDAIVAQNDKEFF ANMQRILKLTMQMRNSKTGTDTDYIVSPVADANGRFFDSRQADATMPKDADANGAY NIARKGIMLVQQIKQSDDLRTMKFDISNKSWLRFAQHTNQADESRKRTADGSEFESP KKKRKVGSGKPIPNPLLGLDST 51 Wildtype coding sequence (with N-terminal methionine and stop codon) ATGAAATTAGAAGA Illi ACCAACCTGTATTCGTTATCCAAGACTCTGCGTTTCGA ACTGCGGCCGATCGGCAAGACACGTGAAAATATCGAAAACGGAGGCC Illi GAG GCAGGACGAGGATCGTGCTGAAAAATATGTACACATAAAAAAACTAATCGATGAA TATCATAAAGCATATATCGATAAACAATTGTCGGGTTTAGTGCTGCAATACGCCGA TATCGGTAAAGCCAATTCATTGGAGGAGTATTATCACTCCACAAGAAAGAGCAAA GATTCGGACAAGGATAAGATTGTCAAAATCCAGGATAATCTGCGTAAACAAATTG TCAAACGGTTGAAAGACTCAGACGAATTCAAGCGTATCGATAAAAAAGAGTTGAT TCAATCGGATCTGGCAGAGTTCATAAAACCAGCCGAAGACAGAGCTTTGATTGCC GAATTCAAAAACTTCACAACATA Illi ACCGGATTCAATGAAAACAGACAGAACAT GTATTCGGACAAAGCTATATCTACGGCAATAGCTTATCGTCTGATACATGAGAATC TTCCGAAGTTCATAGACAACATAGAGACTTTCGATCGCATCGCCGGTATAACGGA ATTGTACGACCAAACCTCCTCCGATGCCGAAATTTTCCGTCTGGAACATTTTTCG GAAACACTGAGCCAAAAGCAGATCGATGCCTATAACTCCGTTATGGGCAGATATA ACATGCTTATCAATGAGTACAATCAGACGCATAAACAGTCGCGCCTACCTAAATT CAAAATGCTGTACAAACAGATTCTTAGCGACCGCGAACACCCCTCGTGGCTGCC CGAGCAGTTCGAGTCGGACACGGCTGTATTGACAGCCATTCGCGAATGTTACGA TGATCTGCGCATACCTATGGCCAATTTGAAAACGC Illi AGAGGGGTTGGGCAAC TATGACCCGAGTGGAATATTTTTGCGTAATGACCAACATCTCTCTCAGATATCCAA ACGATTGACAGGTGATCGGAGTAGCATTGAACGTAGCGTAACAGAAGACCTTCT GACATCGAGGAGACTCAACAAGCGAAAAAGCCGCACAACCGACGAGGAGGAATC GAGAAAACTGTTCAAGCAAAAGGGTAGTCTGAGTATAGGCTATATAGCTGACACG GCCAAAATCGATGTCGAAAGATACTTTGCCAAACTCGGTGCAATAAATACGGTAA CGGAGCAGAGCGAGAATCTATTCGCCAAGGCTGAGAATGCCCGCACGACAGCG GATGAGCTGCTCGCAAATGATTACCCGGCAGGCAAGAGGCTCGTTCAGTCCAAC GACGACATAGCATTGCTGAAAAATCTGCTCGATGCTTTAATGGAGCTGCAATGGT TCGTCAAGCCGCTGCTTGGCACGGGGGACGAAGCCGGCAAAGACGAACGTTTC TATGGAGAATTTGCACAGATATGGGAGCAGCTGGATCGTATAACGCCTCTCTATA ACATGGTGCGCAACTATGTTACCCGCAAGCCGTATTCGACCGACAAATTCAAGCT CAACTTTGAGAGCGCAGCGCTTCTCGGCGGCTGGGACAAGAACAAGGAGCCGG ACTGTCTGTCGGTAATCTTACGCAAGGATGAGCAATATTATCTCGGCATAATCAAT AAGAATCACAAAAAGATATTCGAGAACGATATCTTGCCGTGCGAAGGGGAGTGTT ACGACAAAATGGTATATAAACTCCTGCCCGGCGCAAACAAGATGCTGCCGAAAGT ATTCTTCTCGGCTTCGCGTATCGCCGAATTTGCACCGAGCGACGAAGTAAAACG GATATACAATGATAAGACTTTCCAAAAAGGCGAAAAGTTCGACTTGAACGATTGTC GCACACTGATCGACTTCTACAAGGCTTCTATCGACAAACATGAGGAGTGGAACAA GTTTGGATTCGAATTCTCGGATACGAACAATTATGAAGACATAAGCGGATTCTTTC GCGAGGTCGACAGGCAAGGCTATAAAATGTCATTCCGCCCGGTCGCAGCATCGT ATATCGAAACCCTTGTTGAAGAGGGCAAACTCTATC Illi CCAAATATATAATAAG GA 1 1 1 1 1 CGGCATATAGCAAAGGTACTCCCAATATGCACACGCTGTATTGGAGGA 52 TABLE 11 ZXPB Type V Cas Sequences Name Sequence SEQ ID NO. TGCTCTTCGACGAGCGCAATCTATCGGATGTCGTATATCAGCTCAACGGCGGAG CAGAGTTGTTCTTCCGAAGAAAGAGTCTTCAAAACGGCCGTCCGACGCATCCGG CAAATATTCCTATCAAAAACAAAAACAGTCGGAATGACAAAAAAGAGAGCCTGTTC GACTACGATTTGATCAAAGACAGACGCTATACTGTGGACAAATTTCAGTTCCATGT CCCGATAACCCTCAATTTCAAGAGCGACGGGGCGGGCAGGATCAACGAGCGTGT AAGGGAATATCTCCGCTCGGCGGACGACGTTCACGTCATAGGCATCGACCGCGG AGAACGCAATCTGCTGTATCTGGTCGTGACGGATATGGACGGCAATATCTGCGA ACAATTCTCGCTCAACGAAATTTGTAATACTGATTATCATTCTTTGTTGGATGAAC GCGAACACAAACGTATGCAGGAGAGACAGAGCTGGCAGGCGATAGAGGGCATC AAGGAGTTGAAAGAAGGTTATCTGTCTCAGGTCGTACACCGAATCGCGACACTCA TGGTTAAATATCGCGCCATTGTCGTACTGGAAGATCTCAACTTCGGCTTCATGCG TAGCCGCCAGAAGGTAGAGAAGTCTGTATACCAGAAATTCGAACACATGCTCATA GATAAGCTCAATTATCTGGTCGACAAGAAAGCCAATCCGACAACGCCGGGCGGT CTGCTAAAAGCCTATCAGTTGACAGACAAATTCGAGAGCTTCCAGAAGCTCGGCA AACAGAGCGGATTTCTATTCTACGTTCCGGCATGGAATACATCGAAGATCGATCC AGCAACCGGATTCGTCAACATGCTCGATCTCGGATACGAGAGCATCGACAAAGC CAAAACACTGCTCTGCAAGTTCGACTCTATACGCTACAATGCGTGCAAAGACTGG TTCGAGTTCGCTCTCGATTACGACAAGTTCGGCAGCAAGGCCACCGGTACCCGC ACGAAATGGACTGTTTGCACCTACGGACAACGTATCGATACTTATCGCAACAAAG ATTCGCAGTGGGTCAGCCGCGACGTCGATTTGACAAATGAGCTGAAATCACTCTT CTCCGAACACGGCATAGACATTTACAGCAATCTGAAAGATGCAATAGTCGCACAA AACGACAAAGAATTTTTCGCGAACATGCAGCGGATATTGAAACTGACCATGCAAA TGCGAAACAGCAAAACGGGTACCGACACAGACTATATCGTCTCGCCCGTCGCCG ATGCCAACGGCAGATTCTTCGACAGCAGGCAGGCCGATGCGACCATGCCCAAAG ATGCGGATGCGAACGGAGCGTATAATATCGCACGTAAGGGCATTATGCTCGTAC AGCAGATCAAGCAGTCCGACGATCTGCGTACAATGAAGTTCGACATAAGCAACAA GAGCTGGCTGCGCTTCGCCCAACATACGAACCAGGCGGACGAGTAA Codon optimized coding sequence (no N-terminal methionine, no stop codon) AAGCTGGAGGACTTCACCAATCTGTACTCTCTGAGCAAGACCCTGCGGTTTGAG CTGCGGCCTATCGGCAAGACAAGAGAAAACATCGAAAACGGCGGACTGCTGCGT CAAGACGAGGACAGAGCCGAAAAGTACGTGCATATTAAAAAGCTGATCGATGAAT ACCACAAGGCTTATATCGACAAGCAACTGAGTGGCCTGGTCCTGCAATACGCCG ATATCGGCAAGGCCAATTCTCTGGAGGAGTACTACCACAGCACTAGAAAAAGCAA GGACTCTGACAAGGATAAGATAGTCAAGATCCAGGACAACCTGCGCAAGCAGAT CGTCAAGAGATTGAAGGACAGCGATGAGTTTAAGAGGATCGATAAGAAGGAACT GATCCAGTCTGACCTGGCAGAGTTCATCAAGCCAGCCGAGGACAGGGCCCTGAT AGCCGAGTTCAAGAACTTCACCACCTACTTCACAGGATTCAACGAAAATAGGCAG AACATGTACAGCGATAAGGCTATCAGCACCGCCATCGCCTACCGGCTGATCCAC GAGAACCTGCCTAAGTTCATCGACAACATCGAAACCTTCGACCGGATCGCGGGC ATCACAGAGCTGTATGACCAGACATCCAGCGACGCAGAGATCTTTAGACTGGAG CACTTCAGTGAGACACTGAGCCAGAAGCAGATCGATGCCTATAACAGCGTGATG GGCCGGTACAACATGCTGATCAACGAATATAACCAGACCCACAAGCAATCTCGG CTGCCTAAATTCAAAATGCTGTACAAGCAGATCCTGAGCGACCGGGAGCACCCC AGCTGGCTGCCGGAACAGTTCGAGAGCGACACCGCCGTGCTGACCGCCATCAG AGAGTGTTACGACGACCTGAGAATCCCTATGGCCAACTTAAAAACCCTGCTTGAG GGCCTGGGAAATTACGATCCCTCTGGCATCTTCCTGCGGAACGATCAGCACCTG TCTCAGATCAGCAAAAGACTCACCGGAGACAGATCCAGCATCGAACGGAGCGTG ACCGAGGACTTATTAACGAGCCGGAGACTGAACAAAAGAAAGAGCAGAACCACC GATGAAGAGGAAAGCAGAAAGCTGTTCAAGCAAAAAGGCAGCCTGAGCATCGGC TACATCGCCGACACAGCCAAGATCGACGTGGAGAGATACTTCGCCAAGCTGGGA GCCATTAATACCGTGACCGAGCAGTCTGAGAACCTCTTCGCTAAGGCCGAGAAC GCCAGAACCACTGCTGACGAGCTGCTGGCCAACGACTACCCTGCCGGCAAAAGA CTGGTGCAGAGCAACGACGACATCGCTCTGCTGAAGAACCTATTGGACGCCCTG ATGGAACTGCAATGGTTCGTGAAGCCCCTGCTGGGCACCGGCGACGAGGCCGG CAAAGACGAACGGTTCTATGGCGAGTTCGCTCAGATCTGGGAGCAGCTGGATAG AATCACCCCTCTGTACAACATGGTGCGGAATTACGTGACAAGAAAGCCCTACTCC ACAGACAAGTTCAAGCTGAACTTCGAATCTGCCGCCCTGCTGGGCGGATGGGAC AAGAACAAAGAACCTGACTGCCTGTCCGTGATTCTGAGAAAGGACGAGCAGTAC TACCTGGGCATCATCAACAAGAACCACAAGAAGATCTTCGAGAATGACATTCTGC CTTGCGAGGGCGAGTGCTACGACAAGATGGTCTACAAGCTGCTGCCTGGCGCTA ACAAAATGCTGCCTAAGGTGTTCTTTAGCGCCTCCAGAATCGCTGAGTTCGCCCC TTCTGATGAGGTGAAAAGAATTTACAACGATAAGACCTTCCAGAAGGGCGAGAAG TTCGATCTGAACGACTGCAGAACCCTCATCGATTTCTACAAGGCTTCTATCGATAA GCACGAGGAGTGGAATAAATTTGGCTTCGAGTTTAGCGACACCAACAACTACGA 53 TABLE 11 ZXPB Type V Cas Sequences Name Sequence SEQ ID NO. GGACATCAGCGGCTTCTTCCGGGAGGTGGACAGACAGGGCTACAAGATGAGCTT TAGACCCGTGGCCGCCAGCTACATCGAAACGTTGGTGGAAGAGGGCAAACTGTA CCTGTTCCAGATCTACAACAAAGATTTCAGCGCCTACAGCAAGGGCACCCCTAAT ATGCACACCCTGTACTGGAGAATGCTGTTTGACGAGCGGAACCTGAGCGACGTG GTGTACCAGCTGAACGGCGGAGCTGAACTGTTCTTTAGACGCAAGTCCCTCCAG AACGGCCGGCCTACACACCCTGCCAACATCCCTATCAAGAACAAGAACAGCAGA AACGATAAAAAGGAATCACTGTTCGACTACGATCTCATCAAGGATCGTAGATACA CAGTGGATAAGTTCCAGTTCCACGTGCCAATCACACTGAATTTCAAGAGCGATGG CGCTGGCAGAATTAACGAGAGAGTGCGGGAGTACCTGAGATCTGCCGATGACGT GCACGTGATCGGCATCGACAGAGGCGAGCGGAACCTGCTGTACCTCGTGGTGA CCGATATGGACGGCAACATCTGCGAACAGTTTAGCCTGAACGAAATCTGTAATAC CGACTACCACAGCCTGTTGGATGAGAGAGAGCACAAAAGAATGCAGGAAAGACA GAGCTGGCAGGCCATCGAGGGAATCAAGGAGCTGAAGGAAGGCTACCTGTCCC AAGTGGTCCACAGAATCGCCACCCTGATGGTGAAGTACAGAGCGATCGTGGTGC TGGAGGACCTGAACTTCGGCTTCATGCGGAGCAGACAGAAAGTGGAAAAAAGCG TGTACCAGAAGTTCGAGCACATGCTGATCGACAAACTGAACTACCTGGTGGACAA GAAAGCCAACCCTACCACACCCGGCGGCCTGCTGAAGGCCTACCAGCTGACAG ACAAGTTCGAGAGCTTCCAGAAGCTGGGCAAGCAGTCTGGATTCCTGI 1 1 IATGT GCCCGCCTGGAACACAAGCAAGATCGACCCTGCTACCGGATTCGTGAACATGCT GGATCTGGGCTATGAGAGCATCGACAAGGCCAAAACCCTGCTGTGCAAGTTTGA CTCCATCAGATACAACGCCTGCAAGGACTGGTTCGAGTTTGCCCTGGACTACGA CAAGTTCGGCAGCAAGGCCACAGGCACACGGACCAAGTGGACAGTGTGCACCT ACGGCCAGCGGATCGATACTTATAGAAACAAGGACAGCCAGTGGGTGTCTCGGG ACGTGGATCTGACCAATGAGCTGAAGAGCCTG Illi CTGAACATGGCATCGACAT CTACAGCAACCTGAAAGACGCCATCGTGGCCCAAAATGACAAAGAGTTCTTCGC CAACATGCAGAGAATCCTGAAGCTGACCATGCAGATGAGAAATTCTAAAACTGGA ACAGATACAGACTACATTGTGTCCCCTGTTGCCGATGCTAACGGAAGATTCTTCG ACAGCAGACAAGCCGACGCCACCATGCCAAAGGACGCCGACGCCAACGGCGCC TACAACATCGCTAGAAAGGGCATCATGCTGGTTCAGCAGATCAAGCAGAGCGAT GACCTCCGCACCATGAAATTCGACATCAGCAACAAGAGCTGGCTGAGATTCGCC CAGCATACCAACCAGGCCGATGAG Expression construct (with N-terminal methionine and stop codon, includes V5-tag and C-terminal NLS) ATGggcAAGCTGGAGGACTTCACCAATCTGTACTCTCTGAGCAAGACCCTGCGGT TTGAGCTGCGGCCTATCGGCAAGACAAGAGAAAACATCGAAAACGGCGGACTGC TGCGTCAAGACGAGGACAGAGCCGAAAAGTACGTGCATATTAAAAAGCTGATCG ATGAATACCACAAGGCTTATATCGACAAGCAACTGAGTGGCCTGGTCCTGCAATA CGCCGATATCGGCAAGGCCAATTCTCTGGAGGAGTACTACCACAGCACTAGAAA AAGCAAGGACTCTGACAAGGATAAGATAGTCAAGATCCAGGACAACCTGCGCAA GCAGATCGTCAAGAGATTGAAGGACAGCGATGAGTTTAAGAGGATCGATAAGAA GGAACTGATCCAGTCTGACCTGGCAGAGTTCATCAAGCCAGCCGAGGACAGGGC CCTGATAGCCGAGTTCAAGAACTTCACCACCTACTTCACAGGATTCAACGAAAAT AGGCAGAACATGTACAGCGATAAGGCTATCAGCACCGCCATCGCCTACCGGCTG ATCCACGAGAACCTGCCTAAGTTCATCGACAACATCGAAACCTTCGACCGGATCG CGGGCATCACAGAGCTGTATGACCAGACATCCAGCGACGCAGAGATCTTTAGAC TGGAGCACTTCAGTGAGACACTGAGCCAGAAGCAGATCGATGCCTATAACAGCG TGATGGGCCGGTACAACATGCTGATCAACGAATATAACCAGACCCACAAGCAATC TCGGCTGCCTAAATTCAAAATGCTGTACAAGCAGATCCTGAGCGACCGGGAGCA CCCCAGCTGGCTGCCGGAACAGTTCGAGAGCGACACCGCCGTGCTGACCGCCA TCAGAGAGTGTTACGACGACCTGAGAATCCCTATGGCCAACTTAAAAACCCTGCT TGAGGGCCTGGGAAATTACGATCCCTCTGGCATCTTCCTGCGGAACGATCAGCA CCTGTCTCAGATCAGCAAAAGACTCACCGGAGACAGATCCAGCATCGAACGGAG CGTGACCGAGGACTTATTAACGAGCCGGAGACTGAACAAAAGAAAGAGCAGAAC CACCGATGAAGAGGAAAGCAGAAAGCTGTTCAAGCAAAAAGGCAGCCTGAGCAT CGGCTACATCGCCGACACAGCCAAGATCGACGTGGAGAGATACTTCGCCAAGCT GGGAGCCATTAATACCGTGACCGAGCAGTCTGAGAACCTCTTCGCTAAGGCCGA GAACGCCAGAACCACTGCTGACGAGCTGCTGGCCAACGACTACCCTGCCGGCA AAAGACTGGTGCAGAGCAACGACGACATCGCTCTGCTGAAGAACCTATTGGACG CCCTGATGGAACTGCAATGGTTCGTGAAGCCCCTGCTGGGCACCGGCGACGAG GCCGGCAAAGACGAACGGTTCTATGGCGAGTTCGCTCAGATCTGGGAGCAGCTG GATAGAATCACCCCTCTGTACAACATGGTGCGGAATTACGTGACAAGAAAGCCCT ACTCCACAGACAAGTTCAAGCTGAACTTCGAATCTGCCGCCCTGCTGGGCGGAT GGGACAAGAACAAAGAACCTGACTGCCTGTCCGTGATTCTGAGAAAGGACGAGC AGTACTACCTGGGCATCATCAACAAGAACCACAAGAAGATCTTCGAGAATGACAT TCTGCCTTGCGAGGGCGAGTGCTACGACAAGATGGTCTACAAGCTGCTGCCTGG 54 TABLE 11 ZXPB Type V Cas Sequences Name Sequence SEQ ID NO. CGCTAACAAAATGCTGCCTAAGGTGTTCTTTAGCGCCTCCAGAATCGCTGAGTTC GCCCCTTCTGATGAGGTGAAAAGAATTTACAACGATAAGACCTTCCAGAAGGGCG AGAAGTTCGATCTGAACGACTGCAGAACCCTCATCGATTTCTACAAGGCTTCTAT CGATAAGCACGAGGAGTGGAATAAATTTGGCTTCGAGTTTAGCGACACCAACAAC TACGAGGACATCAGCGGCTTCTTCCGGGAGGTGGACAGACAGGGCTACAAGATG AGCTTTAGACCCGTGGCCGCCAGCTACATCGAAACGTTGGTGGAAGAGGGCAAA CTGTACCTGTTCCAGATCTACAACAAAGATTTCAGCGCCTACAGCAAGGGCACCC CTAATATGCACACCCTGTACTGGAGAATGCTGTTTGACGAGCGGAACCTGAGCG ACGTGGTGTACCAGCTGAACGGCGGAGCTGAACTGTTCTTTAGACGCAAGTCCC TCCAGAACGGCCGGCCTACACACCCTGCCAACATCCCTATCAAGAACAAGAACA GCAGAAACGATAAAAAGGAATCACTGTTCGACTACGATCTCATCAAGGATCGTAG ATACACAGTGGATAAGTTCCAGTTCCACGTGCCAATCACACTGAATTTCAAGAGC GATGGCGCTGGCAGAATTAACGAGAGAGTGCGGGAGTACCTGAGATCTGCCGAT GACGTGCACGTGATCGGCATCGACAGAGGCGAGCGGAACCTGCTGTACCTCGT GGTGACCGATATGGACGGCAACATCTGCGAACAGTTTAGCCTGAACGAAATCTG TAATACCGACTACCACAGCCTGTTGGATGAGAGAGAGCACAAAAGAATGCAGGA AAGACAGAGCTGGCAGGCCATCGAGGGAATCAAGGAGCTGAAGGAAGGCTACC TGTCCCAAGTGGTCCACAGAATCGCCACCCTGATGGTGAAGTACAGAGCGATCG TGGTGCTGGAGGACCTGAACTTCGGCTTCATGCGGAGCAGACAGAAAGTGGAAA AAAGCGTGTACCAGAAGTTCGAGCACATGCTGATCGACAAACTGAACTACCTGGT GGACAAGAAAGCCAACCCTACCACACCCGGCGGCCTGCTGAAGGCCTACCAGC TGACAGACAAGTTCGAGAGCTTCCAGAAGCTGGGCAAGCAGTCTGGATTCCTGT TTTATGTGCCCGCCTGGAACACAAGCAAGATCGACCCTGCTACCGGATTCGTGA ACATGCTGGATCTGGGCTATGAGAGCATCGACAAGGCCAAAACCCTGCTGTGCA AGTTTGACTCCATCAGATACAACGCCTGCAAGGACTGGTTCGAGTTTGCCCTGGA CTACGACAAGTTCGGCAGCAAGGCCACAGGCACACGGACCAAGTGGACAGTGT GCACCTACGGCCAGCGGATCGATACTTATAGAAACAAGGACAGCCAGTGGGTGT CTCGGGACGTGGATCTGACCAATGAGCTGAAGAGCCTG Illi CTGAACATGGCA TCGACATCTACAGCAACCTGAAAGACGCCATCGTGGCCCAAAATGACAAAGAGTT CTTCGCCAACATGCAGAGAATCCTGAAGCTGACCATGCAGATGAGAAATTCTAAA ACTGGAACAGATACAGACTACATTGTGTCCCCTGTTGCCGATGCTAACGGAAGAT TCTTCGACAGCAGACAAGCCGACGCCACCATGCCAAAGGACGCCGACGCCAAC GGCGCCTACAACATCGCTAGAAAGGGCATCATGCTGGTTCAGCAGATCAAGCAG AGCGATGACCTCCGCACCATGAAATTCGACATCAGCAACAAGAGCTGGCTGAGA TTCGCCCAGCATACCAACCAGGCCGATGAGtctagaAAGCGGACAGCAGACGGCTC CGAATTTGAAAGCCCTAAGAAAAAGAGAAAGGTGggatccGGCAAACCTATCCCCA ATCCCCTGCTGGGCCTGGACAGCACCTGA

[0112] In some embodiments a ZXPB Type V Cas protein comprises an amino acid sequence of SEQ ID NO:49, SEQ ID NO:50, or SEQ ID NO:51. In some embodiments, a ZXPB Type V Cas protein has nickase activity, for example resulting from one or more amino acid substitutions relative to the sequence of SEQ ID NO:49, SEQ ID NQ:50, or SEQ ID NO:51. In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D821 substitution, wherein the position of the D821 substitution is defined with respect to the amino acid numbering of SEQ ID NQ:50 (corresponding to amino acid 908 of SEQ ID NO:121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise an E906 substitution, wherein the position of the E906 substitution is defined with respect to the amino acid numbering of SEQ ID NQ:50 (corresponding to amino acid 993 of SEQ ID NO:121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a R1116 substitution, wherein the position of the R1116 substitution is defined with respect to the amino acid numbering of SEQ ID NQ:50 (corresponding to amino acid 1226 of SEQ ID NO:121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D1153 substitution, wherein the position of the D1153 substitution is defined with respect to the amino acid numbering of SEQ ID NO:50 (corresponding to amino acid 1263 of SEQ ID NO:121). In some embodiments, a ZXPB Type V Cas protein is catalytically inactive, for example due to a R1116 substitution in combination with a D821 substitution, a E906 substitution, and / or D1153 substitution. 6.2.10. ZPPX Type V Cas Proteins

[0113] In one aspect, the disclosure provides ZPPX Type V Cas proteins. ZPPX Type V Cas proteins can be further classified as Type V-A Cas proteins. The ZPPX Type V Cas proteins typically comprise an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:55. In some embodiments, the ZPPX Type V Cas proteins comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:55. In some embodiments, a ZPPX Type V Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:55.

[0114] Exemplary ZPPX Type V Cas protein sequences and nucleotide sequences encoding exemplary ZPPX Type V Cas proteins are set forth in Table 1 J. TABLE 1J ZPPX Type V Cas Sequences Name Sequence SEQ ID NO. Wildtype amino acid sequence (without N-terminal methionine) MKDLTGQYSLSKTLRFELKPIGKTLEHIEQKGLLTQDEQRAEEYEQMKGIIDRYHKAFI TMCLRNCKIKVNNTDDELDSLEEYSSLLSKSKRDADDENKLEKIKENLRKQIVNAFKS GNTYGDLFTKELIKNHLPDFVTDEEEKQVVEHFCNFTTYFTGFHDNRKNMYSDKAKS TAIAYRLIHENFPRFFDNLRSFAKISESEVANRFPEIESAFSLYLNVEHIADMFHVDYFP VVLTQEQIDVYNNIIGGKTEEDGTKIQGINEYINLYNQHHPDVKLPFLKPLYKMILSDKV ALSWLPEEFENDEEMLTAINDFYKSVQPVVFGDDENCIRHLLTNIAEYNTDHIYISNDL GLTGISQQLFDQYSIFEDVIKDELRRNVKQTPKEKRNPELLEERIKNLFKKEKSFSISYL DSLIKDKGEDTIESYYAKLGAFDRDGKQTVNLLTQIEMAYIAAKEVLDGKYDNINQSEE ATKYIKDLLDAFKSLQHYIKPLLGSGEEAEKDNVFSSQLLNVWEALDVVTPLYNKVRN WLTRKPYSTKKIKLNFENVQLLGGWPNIEAYSCAIFMKDDNTYYLGILDNAYKTLLRD FPEPAEEKDTIGLMHYLQGGDMGKNIQNLMVVDGKVRKVNGRKEKSGINVGQNIRLE EAKKRYLPTEINRIRKLGTYSVSNPNYNKQDLITIIDYYKPLACEYYASYTFHFKDSSEY NSFAEFTDDINQQAYQLGFVPFSQQYLNKLVDEGKLYLFQIWNKDFSDYSKGTPNMH TLYWKALFDKANLADVVYKLNGRQAEVFYRKRSLQKENTTVHKALQPIKNKNTQNEK STSTFDYDIVKDRRYTVDKFHFHVPITINFKSSGKPNINEHVLDIIRHHGIEHVIGIDRGE RHLLYLSLIDLKGRIIKQMTLNEIKQQTGGNYGTNYKELLAAREGDRAEARRNWKKIE NIKDLKAGYLSQVVHVIAQMMVEYNAIVVLEDLNMGFMRGRQKIERSVYEQFEHMLID KLNFYVDKKKEACAPGGLLHGLQLANKFESFNKLGKQSGCLFYVPAWNTSKIDPVTG FVNMLDARYESVESSRRFFSRFDVIRYNEEKNWFEFTFDYNNFHAKLDGTKTQWTL CTYGSRIKTFRNPAKLNQWDNEEVVLTDEFKKVFANAGINIHGNLKEAICSLAKREHL EPLMHLMKLLLQLRNSKTNSEVDYMLSPVADNGVFYDSRSCNGNLPIDADANGAYNI ARKGLWVLRQIQDSKPGDKLNLALSNKEWLRFVQEKSNFE 55 Wildtype amino acid sequence (with N-terminal methionine) MKDLTGQYSLSKTLRFELKPIGKTLEHIEQKGLLTQDEQRAEEYEQMKGIIDRYHKAFI TMCLRNCKIKVNNTDDELDSLEEYSSLLSKSKRDADDENKLEKIKENLRKQIVNAFKS GNTYGDLFTKELIKNHLPDFVTDEEEKQVVEHFCNFTTYFTGFHDNRKNMYSDKAKS TAIAYRLIHENFPRFFDNLRSFAKISESEVANRFPEIESAFSLYLNVEHIADMFHVDYFP VVLTQEQIDVYNNIIGGKTEEDGTKIQGINEYINLYNQHHPDVKLPFLKPLYKMILSDKV ALSWLPEEFENDEEMLTAINDFYKSVQPVVFGDDENCIRHLLTNIAEYNTDHIYISNDL GLTGISQQLFDQYSIFEDVIKDELRRNVKQTPKEKRNPELLEERIKNLFKKEKSFSISYL DSLIKDKGEDTIESYYAKLGAFDRDGKQTVNLLTQIEMAYIAAKEVLDGKYDNINQSEE ATKYIKDLLDAFKSLQHYIKPLLGSGEEAEKDNVFSSQLLNVWEALDVVTPLYNKVRN WLTRKPYSTKKIKLNFENVQLLGGWPNIEAYSCAIFMKDDNTYYLGILDNAYKTLLRD FPEPAEEKDTIGLMHYLQGGDMGKNIQNLMVVDGKVRKVNGRKEKSGINVGQNIRLE EAKKRYLPTEINRIRKLGTYSVSNPNYNKQDLITIIDYYKPLACEYYASYTFHFKDSSEY NSFAEFTDDINQQAYQLGFVPFSQQYLNKLVDEGKLYLFQIWNKDFSDYSKGTPNMH TLYWKALFDKANLADVVYKLNGRQAEVFYRKRSLQKENTTVHKALQPIKNKNTQNEK STSTFDYDIVKDRRYTVDKFHFHVPITINFKSSGKPNINEHVLDIIRHHGIEHVIGIDRGE RHLLYLSLIDLKGRIIKQMTLNEIKQQTGGNYGTNYKELLAAREGDRAEARRNWKKIE NIKDLKAGYLSQVVHVIAQMMVEYNAIVVLEDLNMGFMRGRQKIERSVYEQFEHMLID 56 TABLE 1J ZPPX Type V Cas Sequences Name Sequence SEQ ID NO. KLNFYVDKKKEACAPGGLLHGLQLANKFESFNKLGKQSGCLFYVPAWNTSKIDPVTG FVNMLDARYESVESSRRFFSRFDVIRYNEEKNWFEFTFDYNNFHAKLDGTKTQWTL CTYGSRIKTFRNPAKLNQWDNEEVVLTDEFKKVFANAGINIHGNLKEAICSLAKREHL EPLMHLMKLLLQLRNSKTNSEVDYMLSPVADNGVFYDSRSCNGNLPIDADANGAYNI ARKGLWVLRQIQDSKPGDKLNLALSNKEWLRFVQEKSNFE Expression construct (with N-terminal methionine, V5-tag and C-terminal NLS) aa sequence MGKDLTGQYSLSKTLRFELKPIGKTLEHIEQKGLLTQDEQRAEEYEQMKGIIDRYHKA FITMCLRNCKIKVNNTDDELDSLEEYSSLLSKSKRDADDENKLEKIKENLRKQIVNAFK SGNTYGDLFTKELIKNHLPDFVTDEEEKQVVEHFCNFTTYFTGFHDNRKNMYSDKAK STAIAYRLIHENFPRFFDNLRSFAKISESEVANRFPEIESAFSLYLNVEHIADMFHVDYF PVVLTQEQIDVYNNIIGGKTEEDGTKIQGINEYINLYNQHHPDVKLPFLKPLYKMILSDK VALSWLPEEFENDEEMLTAINDFYKSVQPVVFGDDENCIRHLLTNIAEYNTDHIYISND LGLTGISQQLFDQYSIFEDVIKDELRRNVKQTPKEKRNPELLEERIKNLFKKEKSFSISY LDSLIKDKGEDTIESYYAKLGAFDRDGKQTVNLLTQIEMAYIAAKEVLDGKYDNINQSE EATKYIKDLLDAFKSLQHYIKPLLGSGEEAEKDNVFSSQLLNVWEALDVVTPLYNKVR NWLTRKPYSTKKIKLNFENVQLLGGWPNIEAYSCAIFMKDDNTYYLGILDNAYKTLLR DFPEPAEEKDTIGLMHYLQGGDMGKNIQNLMVVDGKVRKVNGRKEKSGINVGQNIR LEEAKKRYLPTEINRIRKLGTYSVSNPNYNKQDLITIIDYYKPLACEYYASYTFHFKDSS EYNSFAEFTDDINQQAYQLGFVPFSQQYLNKLVDEGKLYLFQIWNKDFSDYSKGTPN MHTLYWKALFDKANLADVVYKLNGRQAEVFYRKRSLQKENTTVHKALQPIKNKNTQ NEKSTSTFDYDIVKDRRYTVDKFHFHVPITINFKSSGKPNINEHVLDIIRHHGIEHVIGID RGERHLLYLSLIDLKGRIIKQMTLNEIKQQTGGNYGTNYKELLAAREGDRAEARRNWK KIENIKDLKAGYLSQVVHVIAQMMVEYNAIVVLEDLNMGFMRGRQKIERSVYEQFEH MLIDKLNFYVDKKKEACAPGGLLHGLQLANKFESFNKLGKQSGCLFYVPAWNTSKID PVTGFVNMLDARYESVESSRRFFSRFDVIRYNEEKNWFEFTFDYNNFHAKLDGTKT QWTLCTYGSRIKTFRNPAKLNQWDNEEVVLTDEFKKVFANAGINIHGNLKEAICSLAK REHLEPLMHLMKLLLQLRNSKTNSEVDYMLSPVADNGVFYDSRSCNGNLPIDADAN GAYNIARKGLWVLRQIQDSKPGDKLNLALSNKEWLRFVQEKSNFESRKRTADGSEF ESPKKKRKVGSGKPIPNPLLGLDST 57 Wildtype coding sequence (with N-terminal methionine and stop codon) ATGAAAGACCTGACAGGGCAATATAGCCTGTCGAAAACTTTACGATTTGAGTTAA AACCTATCGGTAAAACTCTTGAGCACATTGAGCAAAAAGGACTCTTGACACAGGA CGAACAAAGAGCAGAAGAGTACGAGCAAATGAAAGGTATCATCGACCGATATCA CAAGGCATTTATTACCATGTGTTTGAGAAACTGCAAAATCAAGGTAAATAATACAG ACGACGAATTAGACTCATTAGAAGAATACTCCTCATTACTTTCCAAAAGTAAAAGA GATGCTGATGATGAGAACAAATTGGAAAAGATTAAGGAAAATCTTCGCAAGCAAA TCGTCAATGCTTTCAAAAGCGGCAACACTTATGGCGACTTGTTCACAAAGGAACT GATTAAGAATCATCTGCCCGACTTCGTCACAGACGAGGAAGAAAAGCAAGTGGT GGAGCATTTCTGCAAI I I IACCACAIAI I I IACGGGTTTCCACGACAACCGCAAAA ACATGTACTCAGATAAGGCTAAATCCACGGCAATAGCCTATCGCCTGATACATGA GAATTTCCCTCGG I I I I I IGACAAI C I I CGC ICHII GCAAAGATTTCAGAAAGCG AGGTGGCAAATCGGTTCCCTGAGATAGAATCTGCTTTCTCTCTGTATCTCAACGT GGAACACATCGCCGACATGTTCCACGTTGACTATTTCCCAGTTGTTCTTACCCAA GAACAAATTGATGTGTATAATAATATTATTGGAGGCAAGACGGAAGAAGATGGGA CAAAAATACAGGGCATCAATGAATACATCAACCTTTATAACCAACATCACCCAGAT GTAAAGTTGCCGTTCTTGAAACCTCTATACAAGATGATTCTTAGCGACAAGGTTG CGCTTTCATGGTTGCCGGAGGAGTTTGAGAATGATGAAGAGATGTTGACGGCCA TAAATGAI I I I IACAAG ICAG I ICAGCC I G I CG I I I I CGGGGATGACGAGAATTGT ATCCGTCATCTTCTGACGAATATTGCCGAATACAATACGGATCACATATACATTTC AAACGATTTAGGATTGACTGGAATATCCCAGCAATTGTTCGACCAATACAGCATCT TTGAAGACGTCATTAAAGATGAGTTGAGGCGTAATGTCAAACAGACGCCCAAAGA GAAACGCAATCCTGAATTGTTGGAAGAAAGAATAAAGAACTTGTTCAAGAAAGAG AAGAGTTTCTCCATCTCTTACCTGGACTCTCTCATTAAGGATAAGGGTGAGGATA CGATCGAGTCTTATTATGCCAAACTTGGTGCGTTTGACAGAGACGGTAAGCAAAC AGTGAATTTGCTCACGCAAATTGAAATGGCATACATAGCGGCAAAGGAGGTGCTT GATGGTAAGTATGACAACATTAACCAGTCTGAAGAAGCAACGAAATATATTAAAGA TCTTCTTGATGCGTTCAAGTCTTTGCAACACTACATCAAACCGCTGTTAGGTAGTG GCGAAGAAGCAGAAAAGGATAATGTGTTTAGTTCGCAACTGCTCAATGTTTGGGA GGCGTTAGACGTTGTGACTCCTCTTTATAACAAAGTTCGCAACTGGCTCACACGC AAGCCTTACTCAACAAAAAAGATAAAGCTGAACTTTGAGAATGTCCAACTGCTTG GCGGCTGGCCAAATATAGAAGCGTATTCATGTGCTATTTTTATGAAGGATGATAAT ACTTACTATCTTGGAATACTGGACAATGCATATAAAACTTTATTAAGAGA Illi CCA GAGCCTGCCGAAGAGAAGGATACTATTGGGCTAATGCATTACCTCCAAGGAGGC GATATGGGAAAAAATATTCAGAATTTGATGGTGGTAGATGGAAAGGTTCGGAAAG TTAATGGGCGCAAAGAGAAGTCAGGAATTAATGTTGGGCAGAATATTCGATTAGA 58 TABLE 1J ZPPX Type V Cas Sequences Name Sequence SEQ ID NO. AGAAGCAAAAAAGAGATACCTGCCAACAGAAATCAATAGAATAAGGAAGTTGGGA ACGTATTCTGTTTCAAATCCAAATTATAACAAACAAGATTTGATAACCATAATCGAT TATTACAAGCCACTGGCTTGTGAATACTATGCTTCCTATACATTCCATTTCAAGGA TTCTTCCGAGTATAATTCGTTCGCGGAGTTTACAGACGATATCAATCAGCAAGCG TATCAACTTGGGTTTGTACCTTTTTCTCAACAATACTTAAACAAACTTGTAGACGAA GGCAAACTCTACC Illi CCAAATATGGAATAAAGATTTCTCTGATTATAGTAAAGG CACTCCCAATATGCATACCCTTTATTGGAAGGCGCTCTTTGATAAAGCAAATCTTG CCGATGTTGTCTACAAACTTAATGGTCGTCAGGCAGAGGTGTTCTATCGGAAAAG AAGCCTCCAAAAAGAGAATACGACTGTGCACAAAGCATTGCAGCCTATAAAGAAT AAAAACACGCAGAATGAGAAAAGCACCAGTACGTTTGACTATGACATCGTAAAAG ATCGTCGTTATACAGTTGATAAATTCCATTTCCATGTGCCCATTACTATTAACTTTA AGTCATCTGGAAAACCTAATATCAATGAACACGI I I IAGATATTATCCGTCACCAT GGCATTGAGCATGTCATCGGAATCGACCGTGGCGAGCGCCATCTATTATATCTTT CTCTTATAGATCTCAAGGGAAGAATAATCAAGCAAATGACGCTTAATGAGATAAAG CAGCAAACAGGCGGTAACTATGGCACAAATTATAAAGAACTCTTGGCCGCAAGAG AAGGCGATCGTGCGGAAGCGCGTCGTAACTGGAAAAAGATAGAGAATATTAAAG ACCTTAAAGCTGGCTATCTCAGTCAGGTTGTACATGTGATAGCCCAAATGATGGT GGAATACAATGCCATCGTTGTGCTCGAAGACCTCAATATGGGCTTTATGCGTGGG CGGCAGAAAATCGAGCGGAGCGTATACGAGCAGTTCGAACACATGCTGATAGAT AAGTTGAACTTCTATGTTGATAAGAAAAAGGAAGCATGTGCCCCCGGAGGTCTGC TTCATGGTCTCCAATTAGCCAATAAATTTGAGAGCTTCAATAAGCTTGGGAAACAG AGCGGTTGCCTTTTTTATGTACCGGCATGGAATACCAGCAAAATAGATCCTGTCA CAGGGTTTGTCAATATGCTTGATGCACGCTATGAAAGTGTAGAAAGTTCGCGCCG CTTCTTCTCTCGTTTCGATGTTATTCGTTACAATGAGGAAAAGAATTGGTTTGAAT TTAC Illi GATTATAATAACTTCCATGCAAAGTTGGACGGGACAAAAACCCAATGG ACGCTTTGCACATACGGCAGTCGCATCAAAACATTCCGCAACCCCGCAAAACTCA ATCAATGGGATAATGAAGAGGTGGTTCTTACCGATGAATTTAAGAAGGTATTTGC CAATGCTGGTATCAATATTCATGGGAATTTGAAAGAGGCCATTTGCTCTCTTGCTA AACGGGAGCATTTAGAACCGTTGATGCATTTGATGAAACTGCI I I IACAGTTGCG CAACAGCAAGACCAACTCAGAGGTCGACTATATGCTTTCTCCTGTGGCAGATAAT GGCGTG Illi ACGACAGCCGTTCTTGCAATGGCAATTTGCCTATAGATGCCGATG CCAATGGGGCATACAACATTGCCCGGAAAGGATTATGGG Illi GCGCCAAATTCA GGACTCTAAGCCTGGCGACAAACTGAATTTGGCTTTGTCGAACAAGGAATGGTTG CGATTTGTTCAAGAAAAGAGCAACTTTGAATAA Codon optimized coding sequence (no N-terminal methionine, no stop codon) AAGGATCTGACAGGCCAGTACAGCCTCTCTAAGACCCTCAGATTTGAACTGAAGC CTATCGGCAAGACCCTGGAGCACATCGAGCAAAAGGGCCTGCTGACCCAGGAC GAGCAGAGAGCCGAGGAATACGAGCAGATGAAGGGAATTATTGACAGATACCAC AAGGCCTTCATCACTATGTGCCTGAGAAATTGCAAGATCAAGGTGAACAACACCG ACGATGAGCTGGACAGCCTGGAAGAGTACAGCAGCCTGCTGTCAAAGTCTAAGC GGGACGCCGACGACGAGAACAAACTGGAGAAGATCAAGGAAAACCTGAGAAAG CAGATCGTCAATGCCTTCAAGAGCGGAAACACCTACGGCGATCTGTTCACCAAG GAGCTGATCAAGAACCACCTCCCCGA Illi GTGACCGACGAGGAAGAAAAGCAG GTGGTGGAACACTTCTGCAACTTCACCACCTACTTCACCGGCTTTCACGACAACC GCAAGAACATGTACAGCGACAAGGCCAAGAGCACAGCCATCGCCTACAGACTGA TCCACGAGAACTTTCCAAGATTTTTCGATAATCTGCGGAGCTTTGCCAAGATCTC CGAATCTGAAGTGGCCAACAGATTCCCAGAAATCGAGAGCGCCTTTAGCCTGTA CCTGAATGTGGAACATATCGCCGATATGTTCCACGTGGACTACTTCCCAGTGGTG CTGACCCAGGAGCAGATTGACGTGTACAACAACATCATCGGAGGCAAGACCGAG GAAGATGGCACAAAGATTCAGGGCATCAACGAGTATATCAACCTGTACAACCAAC ACCATCCTGACGTCAAACTGCCCTTCCTGAAGCCTCTGTATAAGATGATCCTGAG CGACAAGGTGGCCCTGAGCTGGCTGCCTGAAGAGTTCGAGAACGACGAGGAAA TGCTGACCGCCATCAATGATTTCTACAAGTCTGTGCAGCCTGTGGTGTTCGGCGA TGACGAGAACTGTATCAGACACCTGCTGACAAACATCGCCGAGTACAACACCGAT CACATTTACATCAGCAATGACCTGGGACTGACTGGCATCTCTCAGCAGCTGTTCG ACCAGTACTCTATCTTCGAAGATGTGATCAAGGACGAGCTACGGCGGAACGTGA AGCAAACACCTAAGGAGAAGCGGAACCCCGAACTGCTGGAAGAGAGAATCAAGA ACCTGTTCAAGAAAGAAAAGAGCTTCTCCATCAGCTACCTGGATAGCCTGATCAA GGACAAAGGAGAAGATACCATCGAGAGCTACTACGCCAAGCTGGGCGCCTTCGA CAGAGATGGCAAGCAGACAGTGAACCTGCTCACCCAGATCGAGATGGCCTACAT CGCCGCTAAGGAAGTGCTGGATGGCAAGTACGACAACATCAACCAGAGCGAGGA AGCTACAAAGTACATCAAGGATCTGCTTGACGCCTTCAAGAGCCTGCAGCACTAC ATCAAGCCCCTGCTGGGCAGCGGCGAGGAGGCCGAAAAAGACAACGTGTTCAG CAGCCAGCTCCTGAACGTGTGGGAGGCTCTGGACGTGGTGACGCCTCTGTACAA 59 TABLE 1J ZPPX Type V Cas Sequences Name Sequence SEQ ID NO. CAAGGTCAGAAATTGGCTGACAAGAAAGCCCTACAGTACCAAGAAAATCAAACTG AACTTCGAGAATGTTCAACTGCTGGGCGGATGGCCTAACATCGAGGCCTATAGC TGCGCCATTTTTATGAAAGACGACAACACCTACTACTTAGGCATCCTGGACAACG CCTATAAAACACTACTTCGGGACTTTCCTGAACCTGCTGAAGAAAAGGACACAAT CGGCCTGATGCACTACCTGCAAGGAGGCGACATGGGCAAGAACATCCAGAACCT GATGGTCGTCGACGGGAAGGTGCGGAAGGTGAACGGCCGTAAGGAAAAGTCCG GCATCAACGTGGGCCAGAATATCCGGCTGGAGGAGGCCAAGAAGAGATACCTG CCTACAGAGATCAACAGAATCAGAAAGCTGGGCACCTACTCTGTGAGCAACCCTA ATTATAACAAGCAGGATCTGATTACAATCATCGACTACTACAAGCCACTGGCCTG CGAGTACTACGCCTCTTATACATTCCACTTCAAGGACAGCAGCGAGTACAACAGC TTCGCCGAGTTCACCGATGATATCAACCAGCAGGCCTACCAGTTGGGCTTCGTG CCTTTCTCCCAGCAATACCTCAACAAACTGGTGGACGAGGGCAAGCTGTACCTGT TCCAGATCTGGAATAAGGACTTCTCTGACTACTCTAAGGGCACCCCCAACATGCA CACCCTGTACTGGAAGGCCCTGTTTGACAAGGCCAATCTGGCTGATGTGGTTTAC AAGCTGAACGGCAGACAGGCCGAGGTG Illi ACAGAAAGAGAAGCCTGCAGAAA GAGAACACAACCGTGCACAAGGCTCTGCAGCCCATCAAGAATAAGAACACACAG AACGAGAAATCTACCAGCACATTCGATTACGATATCGTGAAGGACAGAAGATACA CCGTGGACAAGTTCCATTTCCACGTTCCTATCACCATCAACTTCAAGTCCAGCGG CAAGCCTAACATCAACGAGCATGTGCTGGATATCATCAGACACCACGGCATCGA GCACGTGATCGGCATCGACCGCGGCGAAAGGCACCTGCTGTACCTGTCCCTGAT CGACCTGAAAGGACGGATCATAAAGCAGATGACCCTTAACGAGATCAAACAACA GACCGGCGGCAACTACGGCACAAACTACAAAGAGCTGCTGGCCGCCAGAGAAG GCGACAGAGCCGAGGCTAGAAGAAACTGGAAGAAAATCGAGAACATCAAGGACC TGAAGGCCGGCTACCTGAGCCAGGTGGTGCACGTGATTGCTCAGATGATGGTGG AATACAACGCCATTGTAGTGCTGGAGGACCTGAACATGGGCTTCATGAGAGGCA GACAGAAGATCGAGAGAAGCGTGTACGAGCAGTTCGAGCACATGCTGATTGACA AGCTGAACTTCTACGTGGACAAAAAGAAGGAAGCATGCGCCCCTGGCGGACTTC TGCACGGCCTGCAGCTGGCCAACAAATTCGAGTCTTTCAACAAACTGGGCAAGC AATCCGGCTGTCTGTTCTACGTGCCCGCCTGGAACACCAGCAAGATCGATCCTG TGACCGGATTCGTGAACATGCTGGACGCCCGGTACGAGAGCGTGGAGAGCTCC CGGCGGTTCTTCTCCAGATTTGACGTGATCAGATACAACGAGGAGAAGAACTGG TTCGAGTTCACCTTTGATTATAACAACTTCCACGCCAAACTGGATGGCACCAAGA CCCAGTGGACACTGTGCACCTACGGCAGCAGAATCAAGACCTTTAGAAATCCTG CTAAGCTGAATCAGTGGGACAATGAAGAGGTGGTTCTGACCGACGAATTTAAGAA GGTGTTCGCCAACGCCGGAATCAATATCCACGGCAACCTGAAGGAAGCTATCTG CAGCCTGGCCAAAAGAGAGCACCTGGAACCTCTGATGCACCTGATGAAACTGCT GCTGCAACTTCGGAATAGCAAAACCAACAGCGAGGTCGACTACATGCTGTCTCC AGTGGCCGATAATGGAGTGTTCTACGACAGCAGAAGCTGTAACGGTAACCTGCC TATCGACGCCGACGCCAACGGAGCCTACAATATCGCTAGAAAAGGTCTGTGGGT CCTCAGGCAAATCCAGGATAGCAAGCCCGGCGACAAGCTGAACCTGGCTCTGAG CAACAAGGAATGGCTGCGATTTGTACAGGAGAAAAGCAATTTCGAG Expression construct (with N-terminal methionine and stop codon, includes V5-tag and C-terminal NLS) ATGggcAAGGATCTGACAGGCCAGTACAGCCTCTCTAAGACCCTCAGATTTGAACT GAAGCCTATCGGCAAGACCCTGGAGCACATCGAGCAAAAGGGCCTGCTGACCCA GGACGAGCAGAGAGCCGAGGAATACGAGCAGATGAAGGGAATTATTGACAGATA CCACAAGGCCTTCATCACTATGTGCCTGAGAAATTGCAAGATCAAGGTGAACAAC ACCGACGATGAGCTGGACAGCCTGGAAGAGTACAGCAGCCTGCTGTCAAAGTCT AAGCGGGACGCCGACGACGAGAACAAACTGGAGAAGATCAAGGAAAACCTGAG AAAGCAGATCGTCAATGCCTTCAAGAGCGGAAACACCTACGGCGATCTGTTCAC CAAGGAGCTGATCAAGAACCACCTCCCCGA Illi GTGACCGACGAGGAAGAAAA GCAGGTGGTGGAACACTTCTGCAACTTCACCACCTACTTCACCGGCTTTCACGAC AACCGCAAGAACATGTACAGCGACAAGGCCAAGAGCACAGCCATCGCCTACAGA CTGATCCACGAGAACTTTCCAAGATTTTTCGATAATCTGCGGAGCTTTGCCAAGA TCTCCGAATCTGAAGTGGCCAACAGATTCCCAGAAATCGAGAGCGCCTTTAGCCT GTACCTGAATGTGGAACATATCGCCGATATGTTCCACGTGGACTACTTCCCAGTG GTGCTGACCCAGGAGCAGATTGACGTGTACAACAACATCATCGGAGGCAAGACC GAGGAAGATGGCACAAAGATTCAGGGCATCAACGAGTATATCAACCTGTACAACC AACACCATCCTGACGTCAAACTGCCCTTCCTGAAGCCTCTGTATAAGATGATCCT GAGCGACAAGGTGGCCCTGAGCTGGCTGCCTGAAGAGTTCGAGAACGACGAGG AAATGCTGACCGCCATCAATGATTTCTACAAGTCTGTGCAGCCTGTGGTGTTCGG CGATGACGAGAACTGTATCAGACACCTGCTGACAAACATCGCCGAGTACAACAC CGATCACATTTACATCAGCAATGACCTGGGACTGACTGGCATCTCTCAGCAGCTG TTCGACCAGTACTCTATCTTCGAAGATGTGATCAAGGACGAGCTACGGCGGAAC GTGAAGCAAACACCTAAGGAGAAGCGGAACCCCGAACTGCTGGAAGAGAGAATC 60 TABLE 1J ZPPX Type V Cas Sequences Name Sequence SEQ ID NO. AAGAACCTGTTCAAGAAAGAAAAGAGCTTCTCCATCAGCTACCTGGATAGCCTGA TCAAGGACAAAGGAGAAGATACCATCGAGAGCTACTACGCCAAGCTGGGCGCCT TCGACAGAGATGGCAAGCAGACAGTGAACCTGCTCACCCAGATCGAGATGGCCT ACATCGCCGCTAAGGAAGTGCTGGATGGCAAGTACGACAACATCAACCAGAGCG AGGAAGCTACAAAGTACATCAAGGATCTGCTTGACGCCTTCAAGAGCCTGCAGC ACTACATCAAGCCCCTGCTGGGCAGCGGCGAGGAGGCCGAAAAAGACAACGTG TTCAGCAGCCAGCTCCTGAACGTGTGGGAGGCTCTGGACGTGGTGACGCCTCTG TACAACAAGGTCAGAAATTGGCTGACAAGAAAGCCCTACAGTACCAAGAAAATCA AACTGAACTTCGAGAATGTTCAACTGCTGGGCGGATGGCCTAACATCGAGGCCT ATAGCTGCGCCATTTTTATGAAAGACGACAACACCTACTACTTAGGCATCCTGGA CAACGCCTATAAAACACTACTTCGGGACTTTCCTGAACCTGCTGAAGAAAAGGAC ACAATCGGCCTGATGCACTACCTGCAAGGAGGCGACATGGGCAAGAACATCCAG AACCTGATGGTCGTCGACGGGAAGGTGCGGAAGGTGAACGGCCGTAAGGAAAA GTCCGGCATCAACGTGGGCCAGAATATCCGGCTGGAGGAGGCCAAGAAGAGAT ACCTGCCTACAGAGATCAACAGAATCAGAAAGCTGGGCACCTACTCTGTGAGCA ACCCTAATTATAACAAGCAGGATCTGATTACAATCATCGACTACTACAAGCCACTG GCCTGCGAGTACTACGCCTCTTATACATTCCACTTCAAGGACAGCAGCGAGTACA ACAGCTTCGCCGAGTTCACCGATGATATCAACCAGCAGGCCTACCAGTTGGGCT TCGTGCCTTTCTCCCAGCAATACCTCAACAAACTGGTGGACGAGGGCAAGCTGT ACCTGTTCCAGATCTGGAATAAGGACTTCTCTGACTACTCTAAGGGCACCCCCAA CATGCACACCCTGTACTGGAAGGCCCTGTTTGACAAGGCCAATCTGGCTGATGT GGTTTACAAGCTGAACGGCAGACAGGCCGAGGTG Illi ACAGAAAGAGAAGCCT GCAGAAAGAGAACACAACCGTGCACAAGGCTCTGCAGCCCATCAAGAATAAGAA CACACAGAACGAGAAATCTACCAGCACATTCGATTACGATATCGTGAAGGACAGA AGATACACCGTGGACAAGTTCCATTTCCACGTTCCTATCACCATCAACTTCAAGTC CAGCGGCAAGCCTAACATCAACGAGCATGTGCTGGATATCATCAGACACCACGG CATCGAGCACGTGATCGGCATCGACCGCGGCGAAAGGCACCTGCTGTACCTGTC CCTGATCGACCTGAAAGGACGGATCATAAAGCAGATGACCCTTAACGAGATCAAA CAACAGACCGGCGGCAACTACGGCACAAACTACAAAGAGCTGCTGGCCGCCAG AGAAGGCGACAGAGCCGAGGCTAGAAGAAACTGGAAGAAAATCGAGAACATCAA GGACCTGAAGGCCGGCTACCTGAGCCAGGTGGTGCACGTGATTGCTCAGATGAT GGTGGAATACAACGCCATTGTAGTGCTGGAGGACCTGAACATGGGCTTCATGAG AGGCAGACAGAAGATCGAGAGAAGCGTGTACGAGCAGTTCGAGCACATGCTGAT TGACAAGCTGAACTTCTACGTGGACAAAAAGAAGGAAGCATGCGCCCCTGGCGG ACTTCTGCACGGCCTGCAGCTGGCCAACAAATTCGAGTCTTTCAACAAACTGGGC AAGCAATCCGGCTGTCTGTTCTACGTGCCCGCCTGGAACACCAGCAAGATCGAT CCTGTGACCGGATTCGTGAACATGCTGGACGCCCGGTACGAGAGCGTGGAGAG CTCCCGGCGGTTCTTCTCCAGATTTGACGTGATCAGATACAACGAGGAGAAGAA CTGGTTCGAGTTCACCTTTGATTATAACAACTTCCACGCCAAACTGGATGGCACC AAGACCCAGTGGACACTGTGCACCTACGGCAGCAGAATCAAGACCTTTAGAAAT CCTGCTAAGCTGAATCAGTGGGACAATGAAGAGGTGGTTCTGACCGACGAATTTA AGAAGGTGTTCGCCAACGCCGGAATCAATATCCACGGCAACCTGAAGGAAGCTA TCTGCAGCCTGGCCAAAAGAGAGCACCTGGAACCTCTGATGCACCTGATGAAAC TGCTGCTGCAACTTCGGAATAGCAAAACCAACAGCGAGGTCGACTACATGCTGT CTCCAGTGGCCGATAATGGAGTGTTCTACGACAGCAGAAGCTGTAACGGTAACC TGCCTATCGACGCCGACGCCAACGGAGCCTACAATATCGCTAGAAAAGGTCTGT GGGTCCTCAGGCAAATCCAGGATAGCAAGCCCGGCGACAAGCTGAACCTGGCT CTGAGCAACAAGGAATGGCTGCGATTTGTACAGGAGAAAAGCAATTTCGAGtctaga AAGCGGACAGCAGACGGCTCCGAATTTGAAAGCCCTAAGAAAAAGAGAAAGGTG ggatccGGCAAACCTATCCCCAATCCCCTGCTGGGCCTGGACAGCACCTGA

[0115] In some embodiments a ZPPX Type V Cas protein comprises an amino acid sequence of SEQ ID NO:55, SEQ ID NO:56, or SEQ ID NO:57. In some embodiments, a ZPPX Type V Cas protein has nickase activity, for example resulting from one or more amino acid substitutions relative to the sequence of SEQ ID NO:55, SEQ ID NO:56, or SEQ ID NO:57. In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D877 substitution, wherein the position of the D877 substitution is defined with respect to the amino acid numbering of SEQ ID NO:56 (corresponding to amino acid 908 of SEQ ID NO:121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise an E969 substitution, wherein the position of the E969 substitution is defined with respect to the amino acid numbering of SEQ ID NO:56 (corresponding to amino acid 993 of SEQ ID NO:121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a R1181 substitution, wherein the position of the R1181 substitution is defined with respect to the amino acid numbering of SEQ ID NO:56 (corresponding to amino acid 1226 of SEQ ID NO:121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D1217 substitution, wherein the position of the D1217 substitution is defined with respect to the amino acid numbering of SEQ ID NO:56 (corresponding to amino acid 1263 of SEQ ID NO:121). In some embodiments, a ZPPX Type V Cas protein is catalytically inactive, for example due to a R1181 substitution in combination with a D877 substitution, a E969 substitution, and / or D1217 substitution. 6.2.11. ZXHQ Type V Cas Proteins

[0116] In one aspect, the disclosure provides ZXHQ Type V Cas proteins. ZXHQ Type V Cas proteins can be further classified as Type V-A Cas proteins. The ZXHQ Type V Cas proteins typically comprise an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:61. In some embodiments, the ZXHQ Type V Cas proteins comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:61. In some embodiments, a ZXHQ Type V Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:61.

[0117] Exemplary ZXHQ Type V Cas protein sequences and nucleotide sequences encoding exemplary ZXHQ Type V Cas proteins are set forth in Table 1K. TABLE1K ZXHQ Type V Cas Sequences Name Sequence SEQ ID NO. Wildtype amino acid sequence (without N-terminal methionine) TNTSIFKTFTNQYSLSKTLRFELRPHPMTSGLDDIISLDTGIKKLYENEMKPLFDEL HFEFISQSLVQVSFPSEKLEVLLNKYRSLKDQKAKNIEKELEGPLQELRTIITDTFES TGNNWKKEWLQQGFKIKSSGYKVLTEEGILEVLSVRKKDKADAINKFKGFFTYFS GFNMNRENYYSSEDKKTAVAYRVINENLIRYMDNILLLQNVLAKAPEFKKFEDILSL TTFGKYINQEGITTYNNNVVATINLELNTYHQHNPKIFSRLPKLKLLYKQIGSPKED KRIFTIEKRTEWQSLEDLIQKQNKVVEHEKKNVEILSNLKAAYISFFTNTDETILSEV YFNKRSLNTISSFWFTGGWQTLLLKLKEFKLANQNKDGDIVVPKALSLAELKQVLD SLEEQDPAVNHLFKEMYSDCYKENLWQTFIAIWQCEITSKFNLLEGYIQECNAVKE DTFDKKKHKNIIKNICDTYLDIEQISKYIIVHESLPKYDALYDAVILYLQESSLRSYYD AFRNLISKRPVNEEKVKLNFQNSTLLDGWDMNKESANLCVLLKNNIGEYFLAVMN KKSNMVFDQKKNSALYSAGNESSFQKLEYKLLPGPNKMLPKVIFAKSNEKYFIIPE El VQIREEESFKKGKKFDKHALKTWIRFMQESIEKYPGWKTFDFTFKKPEEYEDVS KFYKDVEEQGYKLNWKDINEEELLSLVEQKKVYLFQIKSKDIGETKEHGNKNLHTL LFLELLKPENTSRLKLLGGGEMFYRAPSMEKVYKTVNEKQVLDSKGNPILEAKRY YEPKFFLHFPIQVKGSENGYKTEMNPKILRAISTSKEVNIIGIDRGEKHLLYYYVIKP DGTPITQGSLNTISLGLDKNQNPRLVDERTFKILERDSKGKPSKISDFESTGKKVD YIDYHNILTYYETKRNIARRSWDTIGAIKNFKEGYLSQAIHQIYQLMLKYNAVVVLE DLNTEFKAKRTAKVEKSVYEKFEIALAKKLNHLIIKGTDPAEAGSVINPYQLTPAITA DTLSDFKKSKQWGPLFYIRANYTSTTDPITGWRKHIYIPSGASDKEIKTYFCKQGE KEPLIQISYDTALTAFAFTYTHEGKEWTLHATKDTQRMRYDSKKRKMEPVEIFDRL RELFIDFSFEESLTDQLEATLSFDWKTLAFLWTMLNQIRNTDREAEGNDGDFIQSP VAPFYDSRDPENKTNGLPVNGDANGAFNIARKGAILIKRIQEYAKKDPTFEKMREK DGLNLYISDAEWDTEIS 61 Wildtype amino acid sequence (with MTNTSIFKTFTNQYSLSKTLRFELRPHPMTSGLDDIISLDTGIKKLYENEMKPLFDE LHFEFISQSLVQVSFPSEKLEVLLNKYRSLKDQKAKNIEKELEGPLQELRTIITDTFE STGNNWKKEWLQQGFKIKSSGYKVLTEEGILEVLSVRKKDKADAINKFKGFFTYF SGFNMNRENYYSSEDKKTAVAYRVINENLIRYMDNILLLQNVLAKAPEFKKFEDIL 62 TABLE1K ZXHQ Type V Cas Sequences Name Sequence SEQ ID NO. N-terminal methionine) SLTTFGKYINQEGITTYNNNVVATINLELNTYHQHNPKIFSRLPKLKLLYKQIGSPKE DKRIFTIEKRTEWQSLEDLIQKQNKVVEHEKKNVEILSNLKAAYISFFTNTDETILSE VYFNKRSLNTISSFWFTGGWQTLLLKLKEFKLANQNKDGDIVVPKALSLAELKQVL DSLEEQDPAVNHLFKEMYSDCYKENLWQTFIAIWQCEITSKFNLLEGYIQECNAV KEDTFDKKKHKNIIKNICDTYLDIEQISKYIIVHESLPKYDALYDAVILYLQESSLRSY YDAFRNLISKRPVNEEKVKLNFQNSTLLDGWDMNKESANLCVLLKNNIGEYFLAV MNKKSNMVFDQKKNSALYSAGNESSFQKLEYKLLPGPNKMLPKVIFAKSNEKYFII PEEIVQIREEESFKKGKKFDKHALKTWIRFMQESIEKYPGWKTFDFTFKKPEEYED VSKFYKDVEEQGYKLNWKDINEEELLSLVEQKKVYLFQIKSKDIGETKEHGNKNLH TLLFLELLKPENTSRLKLLGGGEMFYRAPSMEKVYKTVNEKQVLDSKGNPILEAK RYYEPKFFLHFPIQVKGSENGYKTEMNPKILRAISTSKEVNIIGIDRGEKHLLYYYVI KPDGTPITQGSLNTISLGLDKNQNPRLVDERTFKILERDSKGKPSKISDFESTGKK VDYIDYHNILTYYETKRNIARRSWDTIGAIKNFKEGYLSQAIHQIYQLMLKYNAVVV LEDLNTEFKAKRTAKVEKSVYEKFEIALAKKLNHLIIKGTDPAEAGSVINPYQLTPAI TADTLSDFKKSKQWGPLFYIRANYTSTTDPITGWRKHIYIPSGASDKEIKTYFCKQ GEKEPLIQISYDTALTAFAFTYTHEGKEWTLHATKDTQRMRYDSKKRKMEPVEIF DRLRELFIDFSFEESLTDQLEATLSFDWKTLAFLWTMLNQIRNTDREAEGNDGDFI QSPVAPFYDSRDPENKTNGLPVNGDANGAFNIARKGAILIKRIQEYAKKDPTFEKM REKDGLNLYISDAEWDTEIS Expression construct (with N-terminal methionine, V5-tag and C-terminal NLS) aa sequence MGSGTNTSIFKTFTNQYSLSKTLRFELRPHPMTSGLDDIISLDTGIKKLYENEMKPL FDELHFEFISQSLVQVSFPSEKLEVLLNKYRSLKDQKAKNIEKELEGPLQELRTIITD TFESTGNNWKKEWLQQGFKIKSSGYKVLTEEGILEVLSVRKKDKADAINKFKGFF TYFSGFNMNRENYYSSEDKKTAVAYRVINENLIRYMDNILLLQNVLAKAPEFKKFE DILSLTTFGKYINQEGITTYNNNVVATINLELNTYHQHNPKIFSRLPKLKLLYKQIGS PKEDKRIFTIEKRTEWQSLEDLIQKQNKVVEHEKKNVEILSNLKAAYISFFTNTDETI LSEVYFNKRSLNTISSFWFTGGWQTLLLKLKEFKLANQNKDGDIVVPKALSLAELK QVLDSLEEQDPAVNHLFKEMYSDCYKENLWQTFIAIWQCEITSKFNLLEGYIQEC NAVKEDTFDKKKHKNIIKNICDTYLDIEQISKYIIVHESLPKYDALYDAVILYLQESSL RSYYDAFRNLISKRPVNEEKVKLNFQNSTLLDGWDMNKESANLCVLLKNNIGEYF LAVMNKKSNMVFDQKKNSALYSAGNESSFQKLEYKLLPGPNKMLPKVIFAKSNEK YFIIPEEIVQIREEESFKKGKKFDKHALKTWIRFMQESIEKYPGWKTFDFTFKKPEE YEDVSKFYKDVEEQGYKLNWKDINEEELLSLVEQKKVYLFQIKSKDIGETKEHGN KNLHTLLFLELLKPENTSRLKLLGGGEMFYRAPSMEKVYKTVNEKQVLDSKGNPIL EAKRYYEPKFFLHFPIQVKGSENGYKTEMNPKILRAISTSKEVNIIGIDRGEKHLLY YYVIKPDGTPITQGSLNTISLGLDKNQNPRLVDERTFKILERDSKGKPSKISDFEST GKKVDYIDYHNILTYYETKRNIARRSWDTIGAIKNFKEGYLSQAIHQIYQLMLKYNA VVVLEDLNTEFKAKRTAKVEKSVYEKFEIALAKKLNHLIIKGTDPAEAGSVINPYQL TPAITADTLSDFKKSKQWGPLFYIRANYTSTTDPITGWRKHIYIPSGASDKEIKTYF CKQGEKEPLIQISYDTALTAFAFTYTHEGKEWTLHATKDTQRMRYDSKKRKMEPV EIFDRLRELFIDFSFEESLTDQLEATLSFDWKTLAFLWTMLNQIRNTDREAEGNDG DFIQSPVAPFYDSRDPENKTNGLPVNGDANGAFNIARKGAILIKRIQEYAKKDPTF EKMREKDGLNLYISDAEWDTEISSRKRTADGSEFESPKKKRKVGSGKPIPNPLLG LDST 63 Wildtype coding sequence (with N-terminal methionine and stop codon) ATGACTAACACATCTA Illi CAAAACCTTCACTAATCAATATTCACTTTCAAAAA CGTTGCGGTTTGAGTTGAGACCTCATCCGATGACTAGTGGTCTAGATGATATC ATTTCATTAGATACTGGCATAAAAAAATTGTATGAAAACGAGATGAAGCCGCTA TTTGATGAACTTCA Illi GAATTTATCTCTCAGTCGCTAGTTCAAGTATCATTCC CTTCAGAAAAACTGGAAG Illi GCTAAACAAGTATAGGTCTCTTAAGGATCAG AAAGCTAAAAATATAGAAAAAGAACTGGAAGGCCCATTACAGGAACTAAGAAC AATTATTACTGACACCTTTGAATCCACTGGTAACAACTGGAAAAAAGAATGGCT ACAACAAGGGTTTAAAATCAAAAGCTCGGGATACAAAGTACTAACAGAAGAGG GAATATTAGAAGTATTGTCTGTTCGTAAAAAAGATAAAGCGGATGCAATCAATA AATTTAAAGGATTCTTCACGTAC Illi CAGGGTTTAACATGAACCGTGAAAATT ATTATTCATCGGAAGATAAAAAAACAGCTGTAGCGTATAGGGTAATTAATGAAA ACCTTATCCGGTATATGGATAACATTCTCCTCCTTCAGAATGI I I IAGCAAAAG CTCCTGAGTTTAAAAAGTTTGAAGATA Illi AAGTCTTACTACATTTGGAAAATA CATAAATCAGGAAGGAATAACTACATATAATAATAACGTAGTTGCAACAATTAA TCTTGAACTTAATACGTACCATCAGCATAATCCAAAAATC Illi CTCGCCTGCC AAAGTTAAAATTGCTTTATAAACAAATTGGTTCACCAAAAGAGGACAAACGCAT Illi ACTATTGAAAAAAGAACGGAATGGCAGAGTTTGGAAGACTTAATACAAAA ACAGAATAAAGTTGTTGAACACGAAAAAAAGAATGTTGAAATCCTGTCAAATTT GAAAGCAGCATACATTTCTTTTTTCACGAACACAGATGAAACAATCTTAAGCGA GGTATATTTCAATAAGCGTTCTCTTAATACAATTTCTTCTTTCTGGTTTACGGGT GGCTGGCAAACACTGCTTCTTAAACTAAAAGAGTTTAAATTGGCCAATCAAAA 64 TABLE1K ZXHQ Type V Cas Sequences Name Sequence SEQ ID NO. CAAAGATGGTGATATAGTAGTCCCTAAAGCATTATCCCTTGCTGAACTAAAAC AGGTGCTTGATTCGTTAGAAGAGCAAGACCCTGCTGTTAATCATTTATTTAAG GAAATGTACTCAGATTGTTACAAAGAAAACCTATGGCAGACCTTTATAGCTATC TGGCAATGTGAAATTACATCAAAATTTAACCTGCTCGAAGGGTATATTCAAGAA TGTAATGCTGTTAAAGAAGACACCTTTGATAAAAAAAAGCATAAAAATATTATC AAAAACATCTGCGATACATACCTGGATATTGAGCAGATATCAAAATACATAATA GTACATGAAAGTCTTCCTAAATATGATGCGCTATATGATGCGGTAATACTTTAT TTGCAGGAATCTTCTTTACGCAGTTATTACGATGCCTTCCGCAACCTTATTAGC AAGCGACCTGTTAACGAAGAAAAAGTTAAGCTCAACTTTCAGAACTCTACCCT GCTTGATGGCTGGGATATGAATAAAGAAAGCGCTAACTTATGCGTATTACTGA AAAACAATATAGGTGAATACTTCCTTGCTGTAATGAATAAAAAGAGCAACATGG TTTTTGATCAGAAGAAAAACTCTGCCCTTTACTCTGCTGGGAATGAAAGTAGTT TTCAGAAGCTGGAGTATAAACTGTTGCCTGGGCCTAACAAAATGCTGCCAAAA GTAATTTTTGCAAAATCGAACGAAAAATATTTCATCATACCGGAAGAAATTGTG CAGATTAGAGAAGAAGAATCGTTTAAAAAAGGAAAAAAATTTGATAAGCATGC ATTGAAAACGTGGATCAGGTTTATGCAGGAATCAATTGAAAAATACCCAGGTT GGAAGACATTCGACTTTACCTTTAAAAAACCGGAAGAGTACGAAGATGTCAGC AAGTTCTATAAAGATGTAGAAGAACAGGGGTATAAACTAAACTGGAAAGATAT TAACGAGGAAGAGCTCCTGTCACTTGTAGAACAAAAAAAAGTATATCTGTTTC AGATAAAAAGCAAAGATATCGGAGAAACAAAGGAGCACGGCAACAAGAACCT TCACACATTGTTATTTTTAGAACTCCTCAAACCGGAAAATACCAGCAGGTTAAA GCTACTGGGCGGTGGCGAAATG Illi ATCGTGCGCCAAGTATGGAAAAGGTA TACAAAACCGTAAATGAAAAACAGGTTCTGGATTCAAAAGGTAACCCCAI I I IA GAAGCAAAACGGTACTATGAACCAAAGTTTTTCCTTCACTTCCCTATTCAGGTC AAAGGGAGCGAAAATGGTTATAAAACAGAAATGAATCCGAAAATATTGCGGGC AATTAGCACTTCAAAAGAAGTAAATATAATAGGAATAGACCGTGGAGAAAAGC ATTTACTCTATTATTACGTTATAAAGCCAGACGGAACTCCAATTACTCAAGGAA GCCTGAATACAATTAGTTTAGGTTTAGATAAAAATCAAAATCCCAGACTTGTTG ACGAGCGTACCTTCAAGA Illi GGAGAGAGATTCCAAGGGAAAACCATCAAAA ATATCAGA Illi GAATCTACAGGGAAAAAAGTTGATTACATAGATTATCACAAT ATACTTACCTATTACGAAACAAAACGCAATATAGCACGCCGTTCGTGGGATAC TATTGGGGCAATAAAAAACTTTAAAGAGGGGTACTTGTCTCAGGCGATTCACC AGATTTATCAGCTTATGTTGAAGTATAACGCTGTGGTAG Illi GGAAGATCTTA ATACGGAGTTTAAGGCAAAACGAACCGCAAAAGTTGAAAAATCCGTGTACGAA AAGTTTGAAATTGCCCTTGCTAAAAAACTGAACCACTTAATTATTAAAGGAACT GACCCTGCAGAAGCAGGAAGCGTAATAAATCCGTATCAGCTTACTCCAGCAAT TACAGCTGATACATTAAGCGACTTTAAGAAATCAAAACAATGGGGTCCGCTTT TCTATATTAGAGCAAACTATACCTCTACGACTGACCCTATAACCGGCTGGCGT AAACACATATATATCCCGTCCGGAGCTTCAGATAAAGAAATTAAAACATATTTC TGTAAACAGGGCGAAAAAGAACCTTTGATTCAGATTTCATATGATACAGCGCT TACCGCGTTTGCATTTACCTATACCCATGAAGGCAAAGAATGGACATTACACG CAACGAAAGATACTCAGCGTATGCGTTATGACAGTAAGAAGCGGAAGATGGA ACCCGTAGAAATATTTGATAGACTACGAGAGCI I I I I Al AGA I I I IAGTTTCGA AGAATCGTTAACAGATCAACTAGAAGCAACACTTTCCTTTGACTGGAAAACAC TGGCCTTTTTGTGGACAATGTTAAACCAGATACGTAATACCGACAGAGAAGCA GAAGGGAATGACGGTGACTTTATTCAGTCTCCGGTTGCTCCGI I I IATGATAG TCGAGATCCGGAAAATAAAACAAATGGACTTCCTGTTAACGGAGATGCTAATG GGGCTTTCAATATAGCCAGAAAAGGTGCAATCCTGATAAAACGTATTCAAGAA TATGCAAAAAAAGACCCCACCTTTGAAAAGATGAGAGAAAAAGATGGTCTCAA TTTGTATATATCTGATGCAGAGTGGGATACAGAAATAAGCTAA Codon optimized coding sequence (no N-terminal methionine, no stop codon) ACAAACACTAGCATCTTCAAGACATTCACCAACCAATACAGCCTCTCCAAGAC CCTGCGGTTTGAGCTCAGACCCCACCCTATGACCTCCGGCCTGGACGACATC ATCAGCCTGGACACCGGAATCAAAAAGCTGTACGAGAACGAAATGAAGCCTC TGTTCGACGAGCTGCACTTCGAGTTCATCAGCCAGAGCCTGGTCCAGGTCAG CTTCCCTAGCGAGAAGCTCGAAGTGCTGCTGAACAAGTACCGGAGCCTGAAG GACCAGAAAGCTAAGAACATCGAGAAGGAACTGGAGGGCCCCCTGCAGGAG CTGAGAACCATCATCACCGACACCTTCGAGAGCACCGGCAACAACTGGAAGA AAGAGTGGCTGCAGCAGGGGTTCAAGATCAAAAGCAGTGGATACAAGGTGCT GACAGAGGAGGGCATCCTGGAAGTGCTTTCCGTGCGGAAGAAGGATAAGGC CGATGCTATAAACAAGTTCAAAGGATTCTTCACCTACTTCAGCGGCTTCAACA TGAACAGAGAGAACTACTACAGCAGCGAAGATAAAAAAACAGCCGTGGCCTA CAGAGTGATCAACGAGAACCTGATCCGGTACATGGATAACATCCTGCTCCTG CAGAACGTGCTGGCCAAAGCCCCTGAGTTCAAGAAATTTGAAGATATCCTGA GTCTGACCACCTTCGGCAAGTACATCAACCAGGAGGGCATCACAACCTACAA 65 TABLE1K ZXHQ Type V Cas Sequences Name Sequence SEQ ID NO. CAACAACGTTGTGGCCACCATCAACCTGGAGCTGAACACCTACCACCAGCAC AACCCAAAAATCTTCAGCAGACTGCCCAAACTGAAGCTGCTGTACAAGCAGAT CGGTTCTCCAAAGGAGGACAAGCGCATCTTCACCATCGAGAAGAGAACAGAA TGGCAGAGCCTGGAGGACCTGATCCAGAAGCAGAACAAGGTCGTGGAACAC GAAAAGAAGAACGTGGAGATCCTGTCTAATCTGAAGGCCGCCTATATCAGCTT CTTCACAAACACCGACGAAACCATCCTGTCTGAGGTGTACTTCAACAAGAGAA GCCTGAATACGATCAGCAGCTTCTGGTTCACCGGCGGATGGCAAACCCTGCT GCTGAAACTGAAGGAATTTAAGCTGGCTAATCAGAACAAAGACGGCGATATC GTGGTTCCCAAGGCCCTGAGCCTGGCCGAGCTGAAGCAGGTGCTGGACTCC CTGGAAGAGCAGGACCCCGCCGTGAATCACCTGTTCAAGGAAATGTACAGCG ACTGCTACAAGGAAAACCTGTGGCAAACATTTATCGCCATCTGGCAATGTGAA ATCACAAGCAAGTTCAACCTGCTGGAGGGCTATATCCAAGAGTGCAACGCCG TGAAAGAGGACACCTTTGACAAGAAAAAGCACAAAAACATCATCAAGAACATC TGCGACACGTACCTGGACATTGAGCAGATCAGTAAGTACATCATCGTGCACG AAAGCCTGCCTAAATACGACGCCCTCTATGATGCCGTCATCCTGTACCTGCAG GAGTCTAGTCTGCGGTCCTACTACGACGCCTTTAGAAACCTGATTTCTAAGCG GCCAGTGAACGAGGAAAAGGTGAAGCTGAATTTCCAGAATAGCACCCTGCTG GATGGCTGGGACATGAATAAAGAAAGCGCCAATCTTTGTGTGCTGCTGAAGA ACAACATCGGAGAGTACTTTCTGGCCGTGATGAACAAAAAAAGCAACATGGTT TTTGACCAGAAAAAAAACAGCGCCCTGTATAGCGCTGGCAATGAATCTAGCTT CCAGAAGCTGGAGTACAAGCTGTTGCCCGGCCCTAACAAGATGCTGCCTAAG GTGATCTTTGCCAAGTCCAATGAGAAGTACTTCATCATCCCTGAGGAGATCGT GCAGATCAGGGAGGAAGAGAGCTTCAAGAAAGGCAAAAAATTCGATAAGCAC GCGCTGAAAACCTGGATCAGATTCATGCAGGAGTCTATCGAGAAGTATCCTG GCTGGAAAACCTTTGACTTCACATTCAAAAAGCCTGAGGAATACGAGGATGTG TCCAAGTTCTACAAAGACGTGGAAGAGCAGGGCTACAAACTGAACTGGAAGG ATATCAACGAGGAAGAACTGCTGAGCCTGGTGGAACAGAAGAAGGTGTACCT Illi CAGATCAAGTCCAAAGACATAGGCGAGACAAAGGAACACGGAAATAAGA ACCTGCACACCCTGCTCTTCCTAGAATTGCTGAAGCCTGAGAACACAAGTCG GCTGAAGCTGTTGGGCGGCGGAGAAATGTTCTACCGGGCCCCTTCTATGGAA AAAGTCTACAAAACAGTGAACGAGAAGCAGGTGCTGGATTCTAAAGGCAACC CTATCCTGGAGGCCAAGCGCTACTACGAGCCTAAGTTTTTTCTGCATTTCCCC ATCCAGGTGAAGGGCTCTGAGAACGGCTATAAGACCGAGATGAACCCCAAAA TCCTCAGAGCCATCAGCACCAGCAAGGAAGTGAACATCATTGGCATCGACAG AGGCGAGAAGCACCTGCTGTACTATTACGTGATCAAGCCCGACGGAACACCT ATCACCCAGGGCAGCCTGAACACCATCTCCCTGGGCCTTGATAAGAATCAAA ATCCTAGACTGGTGGACGAGAGAACCTTCAAGATCCTGGAAAGAGATAGCAA GGGCAAGCCAAGCAAGATCTCAGA Illi GAAAGCACAGGCAAGAAGGTCGAC TACATCGACTACCACAACATCCTGACATACTATGAAACCAAGAGAAATATCGC CAGAAGAAGCTGGGACACAATTGGCGCCATCAAGAATTTCAAGGAGGGATAC CTCTCTCAGGCCATCCACCAGATCTACCAGCTGATGCTGAAATATAACGCCGT GGTGGTGCTAGAGGACCTGAACACCGAGTTCAAGGCAAAGAGAACCGCCAA GGTGGAAAAAAGCGTGTACGAAAAGTTTGAGATAGCTCTGGCCAAGAAGCTG AATCACCTGATCATCAAGGGCACCGACCCAGCCGAGGCCGGATCTGTGATCA ACCCTTACCAGCTGACCCCTGCTATTACAGCCGACACACTGAGCGATTTCAAG AAGAGCAAACAATGGGGCCCTCTGTTCTACATCCGGGCCAACTACACCAGCA CAACCGACCCTATCACAGGCTGGAGAAAGCACATCTACATCCCCAGCGGAGC CAGTGACAAGGAAATCAAGACCTACTTCTGCAAGCAGGGCGAGAAGGAGCCT CTGATCCAGATTAGCTACGACACCGCCCTGACCGCCTTCGCCTTCACATACA CCCACGAAGGCAAGGAGTGGACCCTACATGCCACAAAGGATACCCAAAGAAT GCGGTACGACAGCAAGAAGAGAAAGATGGAACCCGTGGAAATCTTCGACAGA CTGAGAGAGCTGTTCATCGACTTCTCTTTCGAGGAAAGCCTGACCGACCAGC TGGAGGCAACCCTGTCCTTCGACTGGAAAACCCTGGC Illi CTGTGGACAAT GCTGAATCAGATCAGAAACACCGATAGAGAGGCTGAAGGCAACGACGGCGA CTTCATCCAGTCTCCTGTGGCCCCTTTCTATGATAGCCGGGACCCAGAGAAC AAGACCAATGGCCTGCCCGTTAACGGCGACGCCAACGGCGCCTTCAACATCG CTAGAAAGGGGGCTATCCTGATCAAGAGAATCCAGGAATACGCCAAGAAGGA CCCTACATTCGAGAAGATGCGGGAAAAGGACGGTTTAAACCTGTACATCAGC GATGCTGAGTGGGATACCGAGATCAGC Expression construct (with N-terminal methionine and stop ATGggctccggaACAAACACTAGCATCTTCAAGACATTCACCAACCAATACAGCCT CTCCAAGACCCTGCGGTTTGAGCTCAGACCCCACCCTATGACCTCCGGCCTG GACGACATCATCAGCCTGGACACCGGAATCAAAAAGCTGTACGAGAACGAAA TGAAGCCTCTGTTCGACGAGCTGCACTTCGAGTTCATCAGCCAGAGCCTGGT CCAGGTCAGCTTCCCTAGCGAGAAGCTCGAAGTGCTGCTGAACAAGTACCGG 66 TABLE1K ZXHQ Type V Cas Sequences Name Sequence SEQ ID NO. codon, includes V5-tag and C-terminal NLS) AGCCTGAAGGACCAGAAAGCTAAGAACATCGAGAAGGAACTGGAGGGCCCC CTGCAGGAGCTGAGAACCATCATCACCGACACCTTCGAGAGCACCGGCAACA ACTGGAAGAAAGAGTGGCTGCAGCAGGGGTTCAAGATCAAAAGCAGTGGATA CAAGGTGCTGACAGAGGAGGGCATCCTGGAAGTGCTTTCCGTGCGGAAGAA GGATAAGGCCGATGCTATAAACAAGTTCAAAGGATTCTTCACCTACTTCAGCG GCTTCAACATGAACAGAGAGAACTACTACAGCAGCGAAGATAAAAAAACAGCC GTGGCCTACAGAGTGATCAACGAGAACCTGATCCGGTACATGGATAACATCC TGCTCCTGCAGAACGTGCTGGCCAAAGCCCCTGAGTTCAAGAAATTTGAAGA TATCCTGAGTCTGACCACCTTCGGCAAGTACATCAACCAGGAGGGCATCACA ACCTACAACAACAACGTTGTGGCCACCATCAACCTGGAGCTGAACACCTACC ACCAGCACAACCCAAAAATCTTCAGCAGACTGCCCAAACTGAAGCTGCTGTAC AAGCAGATCGGTTCTCCAAAGGAGGACAAGCGCATCTTCACCATCGAGAAGA GAACAGAATGGCAGAGCCTGGAGGACCTGATCCAGAAGCAGAACAAGGTCG TGGAACACGAAAAGAAGAACGTGGAGATCCTGTCTAATCTGAAGGCCGCCTA TATCAGCTTCTTCACAAACACCGACGAAACCATCCTGTCTGAGGTGTACTTCA ACAAGAGAAGCCTGAATACGATCAGCAGCTTCTGGTTCACCGGCGGATGGCA AACCCTGCTGCTGAAACTGAAGGAATTTAAGCTGGCTAATCAGAACAAAGACG GCGATATCGTGGTTCCCAAGGCCCTGAGCCTGGCCGAGCTGAAGCAGGTGC TGGACTCCCTGGAAGAGCAGGACCCCGCCGTGAATCACCTGTTCAAGGAAAT GTACAGCGACTGCTACAAGGAAAACCTGTGGCAAACATTTATCGCCATCTGG CAATGTGAAATCACAAGCAAGTTCAACCTGCTGGAGGGCTATATCCAAGAGTG CAACGCCGTGAAAGAGGACACCTTTGACAAGAAAAAGCACAAAAACATCATCA AGAACATCTGCGACACGTACCTGGACATTGAGCAGATCAGTAAGTACATCATC GTGCACGAAAGCCTGCCTAAATACGACGCCCTCTATGATGCCGTCATCCTGT ACCTGCAGGAGTCTAGTCTGCGGTCCTACTACGACGCCTTTAGAAACCTGATT TCTAAGCGGCCAGTGAACGAGGAAAAGGTGAAGCTGAATTTCCAGAATAGCA CCCTGCTGGATGGCTGGGACATGAATAAAGAAAGCGCCAATCTTTGTGTGCT GCTGAAGAACAACATCGGAGAGTACTTTCTGGCCGTGATGAACAAAAAAAGC AACATGGTTTTTGACCAGAAAAAAAACAGCGCCCTGTATAGCGCTGGCAATGA ATCTAGCTTCCAGAAGCTGGAGTACAAGCTGTTGCCCGGCCCTAACAAGATG CTGCCTAAGGTGATCTTTGCCAAGTCCAATGAGAAGTACTTCATCATCCCTGA GGAGATCGTGCAGATCAGGGAGGAAGAGAGCTTCAAGAAAGGCAAAAAATTC GATAAGCACGCGCTGAAAACCTGGATCAGATTCATGCAGGAGTCTATCGAGA AGTATCCTGGCTGGAAAACCTTTGACTTCACATTCAAAAAGCCTGAGGAATAC GAGGATGTGTCCAAGTTCTACAAAGACGTGGAAGAGCAGGGCTACAAACTGA ACTGGAAGGATATCAACGAGGAAGAACTGCTGAGCCTGGTGGAACAGAAGAA GGTGTACCTTTTTCAGATCAAGTCCAAAGACATAGGCGAGACAAAGGAACAC GGAAATAAGAACCTGCACACCCTGCTCTTCCTAGAATTGCTGAAGCCTGAGAA CACAAGTCGGCTGAAGCTGTTGGGCGGCGGAGAAATGTTCTACCGGGCCCC TTCTATGGAAAAAGTCTACAAAACAGTGAACGAGAAGCAGGTGCTGGATTCTA AAGGCAACCCTATCCTGGAGGCCAAGCGCTACTACGAGCCTAAGTTTTTTCTG CATTTCCCCATCCAGGTGAAGGGCTCTGAGAACGGCTATAAGACCGAGATGA ACCCCAAAATCCTCAGAGCCATCAGCACCAGCAAGGAAGTGAACATCATTGG CATCGACAGAGGCGAGAAGCACCTGCTGTACTATTACGTGATCAAGCCCGAC GGAACACCTATCACCCAGGGCAGCCTGAACACCATCTCCCTGGGCCTTGATA AGAATCAAAATCCTAGACTGGTGGACGAGAGAACCTTCAAGATCCTGGAAAG AGATAGCAAGGGCAAGCCAAGCAAGATCTCAGA Illi GAAAGCACAGGCAAG AAGGTCGACTACATCGACTACCACAACATCCTGACATACTATGAAACCAAGAG AAATATCGCCAGAAGAAGCTGGGACACAATTGGCGCCATCAAGAATTTCAAG GAGGGATACCTCTCTCAGGCCATCCACCAGATCTACCAGCTGATGCTGAAAT ATAACGCCGTGGTGGTGCTAGAGGACCTGAACACCGAGTTCAAGGCAAAGAG AACCGCCAAGGTGGAAAAAAGCGTGTACGAAAAGTTTGAGATAGCTCTGGCC AAGAAGCTGAATCACCTGATCATCAAGGGCACCGACCCAGCCGAGGCCGGAT CTGTGATCAACCCTTACCAGCTGACCCCTGCTATTACAGCCGACACACTGAG CGATTTCAAGAAGAGCAAACAATGGGGCCCTCTGTTCTACATCCGGGCCAAC TACACCAGCACAACCGACCCTATCACAGGCTGGAGAAAGCACATCTACATCC CCAGCGGAGCCAGTGACAAGGAAATCAAGACCTACTTCTGCAAGCAGGGCGA GAAGGAGCCTCTGATCCAGATTAGCTACGACACCGCCCTGACCGCCTTCGCC TTCACATACACCCACGAAGGCAAGGAGTGGACCCTACATGCCACAAAGGATA CCCAAAGAATGCGGTACGACAGCAAGAAGAGAAAGATGGAACCCGTGGAAAT CTTCGACAGACTGAGAGAGCTGTTCATCGACTTCTCTTTCGAGGAAAGCCTGA CCGACCAGCTGGAGGCAACCCTGTCCTTCGACTGGAAAACCCTGGC I I I I CT GTGGACAATGCTGAATCAGATCAGAAACACCGATAGAGAGGCTGAAGGCAAC GACGGCGACTTCATCCAGTCTCCTGTGGCCCCTTTCTATGATAGCCGGGACC TABLE1K ZXHQ Type V Cas Sequences Name Sequence SEQ ID NO. CAGAGAACAAGACCAATGGCCTGCCCGTTAACGGCGACGCCAACGGCGCCT TCAACATCGCTAGAAAGGGGGCTATCCTGATCAAGAGAATCCAGGAATACGC CAAGAAGGACCCTACATTCGAGAAGATGCGGGAAAAGGACGGTTTAAACCTG T ACATCAGCGATGCTGAGTGGGATACCGAGATCAGCtctagaAAGCGGACAGCAG ACGGCTCCGAATTTGAAAGCCCTAAGAAAAAGAGAAAGGTGggatccGGCAAAC CTATCCCCAATCCCCTGCTGGGCCTGGACAGCACCTGA

[0118] In some embodiments a ZXHQ Type V Cas protein comprises an amino acid sequence of SEQ ID NO:61, SEQ ID NO:62, or SEQ ID NO:63. In some embodiments, a ZXHQ Type V Cas protein has nickase activity, for example resulting from one or more amino acid substitutions relative to the sequence of SEQ ID NO:61, SEQ ID NO:62, or SEQ ID NO:63. In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D836 substitution, wherein the position of the D836 substitution is defined with respect to the amino acid numbering of SEQ ID NO:62 (corresponding to amino acid 908 of SEQ ID NO:121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise an E963 substitution, wherein the position of the E963 substitution is defined with respect to the amino acid numbering of SEQ ID NO:62 (corresponding to amino acid 993 of SEQ ID NO:121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a R1172 substitution, wherein the position of the R1172 substitution is defined with respect to the amino acid numbering of SEQ ID NO:62 (corresponding to amino acid 1226 of SEQ ID NO:121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D1211 substitution, wherein the position of the D1211 substitution is defined with respect to the amino acid numbering of SEQ ID NO:62 (corresponding to amino acid 1263 of SEQ ID NO:121). In some embodiments, a ZXHQ Type V Cas protein is catalytically inactive, for example due to a R1172 substitution in combination with a D836 substitution, a E963 substitution, and / or D1211 substitution. 6.2.12. ZQKH Type V Cas Proteins

[0119] In one aspect, the disclosure provides ZQKH Type V Cas proteins. ZQKH Type V Cas proteins can be further classified as Type V-A Cas proteins. The ZQKH Type V Cas proteins typically comprise an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:67. In some embodiments, the ZQKH Type V Cas proteins comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:67. In some embodiments, a ZQKH Type V Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:67.

[0120] Exemplary ZQKH Type V Cas protein sequences and nucleotide sequences encoding exemplary ZQKH Type V Cas proteins are set forth in Table 1L. TABLE 1L ZQKH Type V Cas Sequences Name Sequence SEQ ID NO. Wildtype amino acid sequence AYQVVKCLINDYCQNEIIAPQLQKVSCDNTWIVKLREFQEAANWEAQKIIQQDLIGIINK KLPKKFNSKALIEAIPDYLQGKSKEDLQRMLSGIHDYEIKVKNQNLQVAWNNGLEDFC NLCYQQFRGFSGYLDALSENLKFLFSGRKNGIAYRIVYQNLVTFERNRRAYESLILINE 67 TABLE 1L ZQKH Type V Cas Sequences Name Sequence SEQ ID NO. (without N-terminal methionine) TFRVQDEALLLNYSSSLTQEGINTYNERIGQLVKNLKEFGDTDRSFRNWHRRFKKLN KQILSPRVAPPWLARAYRSDEEMVMSLQSFLDEFNPLKPRLKQLIANLESYDEHIYYF RKSLSLLSVTLRNDYKALDEELSIPQEQANCRSLSLSWIPFRQELINEIERIIDSSYTDIE KCLASASEYLNTERAKRNDYRLDNTVSFTIKKLMDVFLSLYRAVKPLTGTGEEEDRNE DFYDEFTTIWDVLQYVQKLYNAVFAWLNKKPYENNSYPAYLDEFTLLKNWKEKAAYI KRNGKFYFIMFNGIDEQDIIEHRGDSAILYHVESQSPDRIKANLTKQFVFSKKANAGKG RPNPSKAKFVRDNPEFQADWERVKTEAYKVAGNTEALAHAIRYFQRCLQSHPDYNR FPFNFRPANDYTSLDDFVDSIKDKLFMMEETAINWSYVRQLAEEGTIYLFKLYNKDYA KNRVGGSKPNLHTLYWEAMFSSENLRENNIKLEEPKLFYREVATNRDGELNMRLIPH RYATDQLELHVPIHLNVNATASSDINMMVLDAIREGSIENVIGIDRGERNLLYYSVLRL SDGEIVDQKSLNITFNDVDYHAKLSTKEEEIHDEQREWKAKTSIRKLKEGYLSQAIHQL TSLIVKYHAVVVLEDLSEDFYSKRQKINKQIYQIFEKRLIEKLSYFVDKDAAEGQAGNIY SALQLSSPNLVRKDNKKIFQNGIVFFVPPEYTSAIDPVTGFCNLFDKNRVRNICELLYR FENICYNRKNDRFEFTWDYRNVMTYTRLEQDNISHLWTACSLGNRIEWSGSERNKN RRCEIVNLTQSMKVLFEKHGIQYQTGKDVREAVCSIRNNDFKKELKRLFFLMLSLRNS IVDGKVKKDYILSPVQNQRGSFFDSREYEELDNPKLPKCGDANGAYNIARKGILTIRKL ENGNEKALTLDEWVISTQKGNIRM Wildtype amino acid sequence (with N-terminal methionine) MAYQVVKCLINDYCQNEIIAPQLQKVSCDNTWIVKLREFQEAANWEAQKIIQQDLIGII NKKLPKKFNSKALIEAIPDYLQGKSKEDLQRMLSGIHDYEIKVKNQNLQVAWNNGLED FCNLCYQQFRGFSGYLDALSENLKFLFSGRKNGIAYRIVYQNLVTFERNRRAYESLILI NETFRVQDEALLLNYSSSLTQEGINTYNERIGQLVKNLKEFGDTDRSFRNWHRRFKK LNKQILSPRVAPPWLARAYRSDEEMVMSLQSFLDEFNPLKPRLKQLIANLESYDEHIY YFRKSLSLLSVTLRNDYKALDEELSIPQEQANCRSLSLSWIPFRQELINEIERIIDSSYT DIEKCLASASEYLNTERAKRNDYRLDNTVSFTIKKLMDVFLSLYRAVKPLTGTGEEED RNEDFYDEFTTIWDVLQYVQKLYNAVFAWLNKKPYENNSYPAYLDEFTLLKNWKEKA AYIKRNGKFYFIMFNGIDEQDIIEHRGDSAILYHVESQSPDRIKANLTKQFVFSKKANA GKGRPNPSKAKFVRDNPEFQADWERVKTEAYKVAGNTEALAHAIRYFQRCLQSHPD YNRFPFNFRPANDYTSLDDFVDSIKDKLFMMEETAINWSYVRQLAEEGTIYLFKLYNK DYAKNRVGGSKPNLHTLYWEAMFSSENLRENNIKLEEPKLFYREVATNRDGELNMR LIPHRYATDQLELHVPIHLNVNATASSDINMMVLDAIREGSIENVIGIDRGERNLLYYSV LRLSDGEIVDQKSLNITFNDVDYHAKLSTKEEEIHDEQREWKAKTSIRKLKEGYLSQAI HQLTSLIVKYHAVVVLEDLSEDFYSKRQKINKQIYQIFEKRLIEKLSYFVDKDAAEGQA GNIYSALQLSSPNLVRKDNKKIFQNGIVFFVPPEYTSAIDPVTGFCNLFDKNRVRNICE LLYRFENICYNRKNDRFEFTWDYRNVMTYTRLEQDNISHLWTACSLGNRIEWSGSER NKNRRCEIVNLTQSMKVLFEKHGIQYQTGKDVREAVCSIRNNDFKKELKRLFFLMLSL RNSIVDGKVKKDYILSPVQNQRGSFFDSREYEELDNPKLPKCGDANGAYNIARKGILT IRKLENGNEKALTLDEWVISTQKGNIRM 68 Expression construct (with N-terminal methionine, V5-tag and C-terminal NLS) aa sequence MGSGAYQVVKCLINDYCQNEIIAPQLQKVSCDNTWIVKLREFQEAANWEAQKIIQQDL IGIINKKLPKKFNSKALIEAIPDYLQGKSKEDLQRMLSGIHDYEIKVKNQNLQVAWNNG LEDFCNLCYQQFRGFSGYLDALSENLKFLFSGRKNGIAYRIVYQNLVTFERNRRAYE SLILINETFRVQDEALLLNYSSSLTQEGINTYNERIGQLVKNLKEFGDTDRSFRNWHR RFKKLNKQILSPRVAPPWLARAYRSDEEMVMSLQSFLDEFNPLKPRLKQLIANLESYD EHIYYFRKSLSLLSVTLRNDYKALDEELSIPQEQANCRSLSLSWIPFRQELINEIERIIDS SYTDIEKCLASASEYLNTERAKRNDYRLDNTVSFTIKKLMDVFLSLYRAVKPLTGTGE EEDRNEDFYDEFTTIWDVLQYVQKLYNAVFAWLNKKPYENNSYPAYLDEFTLLKNWK EKAAYIKRNGKFYFIMFNGIDEQDIIEHRGDSAILYHVESQSPDRIKANLTKQFVFSKKA NAGKGRPNPSKAKFVRDNPEFQADWERVKTEAYKVAGNTEALAHAIRYFQRCLQSH PDYNRFPFNFRPANDYTSLDDFVDSIKDKLFMMEETAINWSYVRQLAEEGTIYLFKLY NKDYAKNRVGGSKPNLHTLYWEAMFSSENLRENNIKLEEPKLFYREVATNRDGELN MRLIPHRYATDQLELHVPIHLNVNATASSDINMMVLDAIREGSIENVIGIDRGERNLLYY SVLRLSDGEIVDQKSLNITFNDVDYHAKLSTKEEEIHDEQREWKAKTSIRKLKEGYLS QAIHQLTSLIVKYHAVVVLEDLSEDFYSKRQKINKQIYQIFEKRLIEKLSYFVDKDAAEG QAGNIYSALQLSSPNLVRKDNKKIFQNGIVFFVPPEYTSAIDPVTGFCNLFDKNRVRNI CELLYRFENICYNRKNDRFEFTWDYRNVMTYTRLEQDNISHLWTACSLGNRIEWSGS ERNKNRRCEIVNLTQSMKVLFEKHGIQYQTGKDVREAVCSIRNNDFKKELKRLFFLML SLRNSIVDGKVKKDYILSPVQNQRGSFFDSREYEELDNPKLPKCGDANGAYNIARKGI LTIRKLENGNEKALTLDEWVISTQKGNIRMSRKRTADGSEFESPKKKRKVGSGKPIPN PLLGLDST 69 Wildtype coding sequence (with N-terminal methionine ATGGCATACCAAGTGGTTAAATGCCTAATCAACGACTATTGCCAGAATGAAATCAT TGCACCTCAATTGCAGAAAGTTTCCTGTGATAACACTTGGATTGTAAAACTTCGCG AGTTTCAAGAGGCTGCCAATTGGGAAGCCCAAAAAATTATCCAGCAAGATCTTAT TGGTATCATAAACAAGAAACTTCCTAAAAAGTTCAATAGCAAGGCATTGATAGAAG CCATTCCTGACTATTTACAAGGCAAGTCTAAAGAAGATCTGCAACGTATGTTGAGT 70 TABLE 1L ZQKH Type V Cas Sequences Name Sequence SEQ ID NO. and stop codon) GGTATACATGACTATGAGATTAAGGTAAAAAATCAGAACCTTCAGGTGGCTTGGA ATAATGGGTTAGAAGAI I I I I G IAACC I C I GO IAI CAACAAI I IAGAGGAI I I I CT GGCTATCTTGACGCTTTATCTGAGAACCTGAAATTTCTATTCTCGGGCAGAAAAAA TGGTATAGCCTATAGAATAGTGTATCAGAACCTTGTTACATTTGAGAGGAATAGGA GAGCTTATGAATCCCTAATATTAATAAATGAGAC Illi AGGGTACAAGATGAGGCT CTACTTCTTAATTACTCCAGTAGTCTGACCCAAGAAGGTATCAACACCTATAATGA ACGAATAGGGCAACTTGTCAAAAATCTGAAAGAATTTGGCGATACAGACAGATCT TTCAGAAACTGGCATCGCCGATTCAAGAAACTGAACAAGCAAATCCTAAGCCCTC GTGTTGCTCCACCTTGGTTGGCACGCGCCTACAGAAGCGATGAAGAGATGGTGA TGTCGCTACAGTC Illi CTCGACGAGTTCAATCCATTAAAACCTCGTTTGAAGCAA CTTATTGCTAATCTGGAATCTTACGATGAGCATATCTATTACTTCCGCAAGTCTCT TTCTCTATTATCGGTGACCTTGAGGAATGATTATAAGGCACTTGATGAAGAACTCT CAATACCACAAGAACAGGCCAATTGCAGAAGTTTAAGCCTTTCGTGGATTCCGTT TCGCCAAGAATTGATAAACGAAATAGAACGAATTATTGACAGTTCATATACAGACA TAGAGAAGTGTCTTGCCTCTGCCTCGGAATATCTGAACACGGAGAGAGCAAAAC GGAACGACTATCGTCTAGATAATACTGTGTCTTTCACAATCAAGAAACTGATGGA CGTATTCCTGTCATTGTATCGTGCGGTGAAGCCTCTGACTGGAACAGGAGAGGA GGAGGATCGAAACGAGGACTTCTATGATGAGTTTACAACAATCTGGGATGTGCTT CAATATGTACAAAAACTTTATAATGCAGTTTTTGCATGGCTGAACAAGAAGCCTTA TGAGAACAACAGCTATCCTGCCTATTTGGACGAGTTTACACTTCTTAAAAACTGGA AGGAGAAAGCCGCGTATATAAAACGGAATGGGAAGTTCTAI I I IATCATGTTCAAT GGTATTGATGAACAAGACATTATCGAGCATCGAGGTGATTCTGCAATCTTGTATC ATGTGGAAAGTCAATCCCCCGATAGGATTAAGGCAAATCTCACCAAACAATTTGT TTTTTCCAAAAAAGCAAATGCAGGAAAGGGGCGACCAAATCCTTCTAAAGCCAAA TTCGTGCGTGACAATCCAGAATTCCAAGCTGACTGGGAACGTGTGAAAACTGAAG CATATAAAGTAGCTGGAAACACAGAAGCGCTTGCTCATGCCATTCGATA Illi CAA CGCTGCCTTCAATCACATCCTGACTATAATAGGTTTCCGTTCAA Illi AGACCAGC GAATGACTACACTAGTTTAGATGA Illi GTTGACTCCATTAAAGACAAATTGTTTAT GATGGAAGAAACTGCTATTAACTGGTCGTATGTGAGGCAATTAGCAGAAGAAGGA ACAATTTACTTGTTTAAACTCTACAATAAAGATTATGCCAAGAATAGAGTTGGCGG GTCTAAACCCAACTTGCATACGCTCTATTGGGAGGCGATGTTCAGCTCTGAGAAC CTTCGTGAAAATAATATAAAGTTGGAGGAACCCAAACTCTTCTATCGTGAAGTTGC AACTAACCGTGATGGTGAATTGAATATGCGCTTGATACCTCACAGATATGCAACA GACCAACTTGAGCTGCATGTTCCAATTCACTTAAATGTGAATGCAACCGCTTCAA GCGATATAAATATGATGGTGTTGGATGCAATACGAGAAGGGAGTATTGAAAATGT CATTGGTATTGACCGTGGAGAGAGGAACCTTCTCTACTATTCAGTCTTGCGGTTG TCAGATGGTGAAATTGTTGACCAAAAAAGTTTGAATATTACTTTCAATGATGTTGA CTACCACGCCAAACTGTCGACTAAAGAGGAGGAAATCCATGACGAACAAAGAGA ATGGAAAGCAAAAACAAGTATTCGGAAACTGAAAGAAGGATACCTTAGTCAAGCT ATCCACCAACTAACATCGCTGATTGTCAAGTACCATGCTGTGGTAGTGCTAGAAG ACTTATCAGAGGACTTCTATTCGAAGCGCCAGAAGATAAACAAGCAAATCTATCA GATATTTGAAAAAAGGCTGATAGAAAAACTGAGTTA Illi GTCGATAAGGATGCTG CAGAAGGTCAGGCAGGCAATATATATTCAGCATTGCAGTTGTCAAGCCCCAACTT GGTGAGGAAAGATAATAAAAAAATCTTTCAGAACGGCATCGTCTTTTTTGTGCCAC CTGAATATACAAGTGCCATTGACCCTGTAACAGGGTTCTGCAATCTCTTTGACAA GAATCGGGTAAGAAATATTTGCGAACTTCTCTACAGATTTGAAAACATCTGCTATA ATAGGAAAAATGACCGATTTGAGTTCACATGGGACTATCGTAATGTTATGACTTAT ACGCGTCTGGAGCAGGACAATATTTCACATCTTTGGACAGCATGCTCTTTAGGAA ACAGGATTGAATGGTCTGGTAGCGAACGTAATAAAAACAGAAGGTGCGAAATTGT AAACCTTACGCAATCTATGAAAG Illi GTTTGAAAAACATGGTATCCAATACCAAA CAGGAAAAGATGTAAGGGAGGCTGTATGCAGCATAAGAAACAACGA Illi AAAAA AGAATTGAAGCGCCTGTTCTTCTTGATGTTATCTTTAAGGAATAGCATTGTTGATG GAAAAGTGAAAAAAGACTATATATTATCCCCCGTTCAGAACCAACGAGGCAGTTT TTTCGATAGTAGAGAATATGAAGAGTTGGACAATCCAAAACTCCCTAAATGTGGA GATGCAAATGGCGCATATAATATTGCAAGGAAAGGGATACTGACAATTAGAAAGT TGGAAAATGGCAATGAAAAGGCATTAACCCTTGATGAGTGGGTTATTTCTACGCA AAAAGGGAATATACGCATGTAA Codon optimized coding sequence (no N-terminal GCCTACCAGGTGGTGAAATGCCTGATTAACGACTACTGCCAGAACGAGATCATC GCCCCTCAGCTGCAAAAGGTGAGCTGCGACAATACCTGGATCGTGAAGCTCAGA GAGTTCCAGGAGGCCGCAAACTGGGAAGCCCAGAAGATCATCCAGCAGGACCT GATCGGCATTATCAATAAGAAACTGCCTAAGAAATTCAACTCTAAGGCCCTGATC GAGGCTATACCTGATTACCTCCAGGGCAAGAGCAAGGAAGATCTGCAGAGAATG CTGTCCGGCATCCACGACTATGAGATCAAGGTGAAGAACCAGAACCTGCAGGTA 71 TABLE 1L ZQKH Type V Cas Sequences Name Sequence SEQ ID NO. methionine, no stop codon) GCTTGGAACAATGGCCTGGAAGATTTCTGTAACTTGTGCTACCAACAATTTAGAG GCTTTTCCGGCTACCTTGATGCTCTGTCAGAAAATCTGAAGTTCCTGTTCAGCGG CAGAAAAAACGGCATCGCCTACAGGATCGTCTACCAGAACCTGGTGACCTTCGA GCGGAACCGGAGAGCTTACGAGAGCCTGATCCTGATCAACGAGACATTTAGAGT GCAGGACGAGGCCCTGCTGCTCAACTACTCTAGCTCTCTGACACAGGAGGGAAT CAACACGTACAACGAGCGGATCGGCCAGCTGGTGAAGAACCTGAAGGAGTTCG GCGACACCGACCGGAGCTTTCGGAACTGGCACAGACGGTTCAAGAAACTGAACA AGCAGATCCTGAGCCCTAGAGTGGCCCCTCCTTGGCTGGCTCGTGCCTACAGAA GCGATGAGGAAATGGTGATGAGCCTGCAGAGCTTCCTGGATGAGTTCAACCCTC TGAAACCTAGACTCAAACAGCTGATCGCCAATCTGGAGTCCTACGACGAGCACAT CTACTACTTCAGAAAGTCCCTGTCTCTGCTGTCAGTGACACTGAGGAACGACTAT AAGGCACTGGATGAAGAGCTGAGCATCCCTCAGGAGCAGGCCAACTGCAGATCT CTTAGCCTGAGCTGGATTCCTTTCAGACAGGAACTGATCAACGAGATCGAGAGAA TCATCGATAGCAGCTACACAGACATTGAGAAGTGCCTGGCCAGCGCCTCCGAGT ACCTGAACACCGAGAGAGCCAAGAGAAACGACTACCGGCTAGATAATACCGTGT CCTTCACCATCAAGAAGCTGATGGACGTGTTCCTGAGCCTGTACCGCGCCGTGA AGCCTCTGACCGGAACAGGCGAAGAGGAGGACAGAAATGAAGATTTCTACGACG AGTTCACCACCATCTGGGATGTGCTGCAATACGTGCAGAAGCTGTACAACGCTGT TTTCGCCTGGCTGAACAAGAAGCCCTACGAGAACAATAGCTACCCTGCCTACCTG GATGAATTTACCCTGCTGAAGAACTGGAAGGAAAAGGCCGCCTACATCAAGAGG AATGGAAAATTCTACTTCATCATGTTCAACGGCATCGACGAGCAGGATATCATCG AACACAGAGGAGATTCTGCCATCCTGTACCATGTGGAAAGCCAGAGCCCTGATA GAATCAAGGCCAATCTGACCAAGCAGTTCGTGTTCAGCAAGAAAGCCAATGCCG GCAAGGGCCGGCCCAATCCCAGCAAGGCCAAGTTCGTGAGAGATAACCCCGAG TTTCAGGCCGACTGGGAGCGGGTGAAAACCGAGGCCTACAAGGTGGCCGGAAA CACCGAGGCCCTGGCCCACGCCATCAGATACTTCCAAAGATGCCTGCAAAGCCA CCCCGATTATAATCGGTTCCCCTTCAACTTCAGACCTGCCAACGACTACACATCT CTGGATGACTTCGTGGACAGCATCAAGGACAAGCTGTTCATGATGGAAGAAACC GCCATCAACTGGAGTTATGTGAGACAGCTGGCCGAAGAAGGCACAATCTACCTG TTCAAGCTGTATAACAAAGACTACGCCAAGAACCGGGTGGGCGGCAGCAAGCCT AACCTGCACACCCTGTACTGGGAGGCCATGTTCAGCTCTGAGAATCTGAGAGAA AACAACATCAAACTGGAAGAACCCAAACTGTTCTACAGAGAGGTGGCCACAAACC GGGACGGCGAGCTGAACATGAGACTGATCCCCCACAGATACGCCACCGACCAG CTGGAACTGCACGTGCCTATCCACCTGAATGTGAACGCCACAGCCAGCAGCGAC ATCAACATGATGGTCCTTGATGCCATCCGGGAAGGATCTATTGAGAACGTGATCG GCATCGACCGGGGAGAACGGAACCTGCTGTACTACAGCGTCCTGCGACTGTCC GACGGCGAGATCGTGGACCAGAAGAGCCTGAATATCACCTTTAACGATGTGGAC TACCACGCAAAGTTGTCTACCAAGGAGGAAGAAATCCATGATGAGCAGAGAGAG TGGAAAGCCAAGACCTCCATCAGAAAGCTGAAGGAAGGTTACCTGTCTCAGGCT ATCCACCAGCTGACCAGCCTGATCGTGAAGTACCACGCTGTGGTAGTGCTGGAA GATCTGAGCGAAGATTTCTACAGCAAGCGGCAGAAAATCAACAAGCAGATCTACC AGATTTTCGAGAAAAGACTTATCGAGAAGCTGAGCTACTTTGTGGACAAAGACGC CGCCGAGGGCCAGGCAGGCAACATCTACAGCGCCCTGCAGCTGAGCTCCCCAA ATCTGGTGAGAAAGGACAACAAGAAGATCTTCCAGAACGGCATCGTGTTCTTCGT GCCACCTGAGTACACGAGTGCGATTGACCCCGTGACCGGCTTCTGCAACCTGTT TGACAAGAACAGAGTGCGCAATATCTGTGAGCTGCTCTACAGATTCGAAAACATT TGCTACAACAGAAAGAATGACCGGTTTGAGTTCACATGGGACTATAGAAACGTGA TGACCTACACCAGACTTGAGCAGGACAACATCTCTCACCTGTGGACCGCTTGTAG CCTCGGCAACCGGATCGAGTGGAGCGGCTCTGAAAGAAATAAGAACAGAAGATG CGAGATCGTGAACCTGACACAAAGCATGAAGGTCCTGTTTGAGAAGCACGGCAT CCAGTACCAGACCGGCAAGGACGTGCGGGAAGCTGTGTGTAGTATCAGAAACAA CGACTTTAAGAAAGAACTGAAGAGACTGTTTTTCCTGATGCTGAGCCTGCGTAAC AGCATCGTGGATGGAAAGGTGAAAAAGGACTACATCCTGAGCCCAGTGCAAAAC CAGCGGGGTAGC Illi TCGACTCCAGAGAATATGAAGAACTGGACAACCCGAAG TTGCCTAAGTGCGGGGACGCCAACGGCGCCTACAACATCGCCAGAAAAGGAATC CTGACAATCAGAAAGCTGGAGAACGGCAACGAGAAAGCCCTGACCCTGGACGAA TGGGTGATCAGCACCCAGAAGGGCAACATCAGAATG Expression construct (with N-terminal methionine and stop codon, ATGggctccggaGCCTACCAGGTGGTGAAATGCCTGATTAACGACTACTGCCAGAAC GAGATCATCGCCCCTCAGCTGCAAAAGGTGAGCTGCGACAATACCTGGATCGTG AAGCTCAGAGAGTTCCAGGAGGCCGCAAACTGGGAAGCCCAGAAGATCATCCAG CAGGACCTGATCGGCATTATCAATAAGAAACTGCCTAAGAAATTCAACTCTAAGG CCCTGATCGAGGCTATACCTGATTACCTCCAGGGCAAGAGCAAGGAAGATCTGC AGAGAATGCTGTCCGGCATCCACGACTATGAGATCAAGGTGAAGAACCAGAACC 72 TABLE 1L ZQKH Type V Cas Sequences Name Sequence SEQ ID NO. includes V5-tag and C-terminal NLS) TGCAGGTAGCTTGGAACAATGGCCTGGAAGATTTCTGTAACTTGTGCTACCAACA ATTTAGAGGCTTTTCCGGCTACCTTGATGCTCTGTCAGAAAATCTGAAGTTCCTGT TCAGCGGCAGAAAAAACGGCATCGCCTACAGGATCGTCTACCAGAACCTGGTGA CCTTCGAGCGGAACCGGAGAGCTTACGAGAGCCTGATCCTGATCAACGAGACAT TTAGAGTGCAGGACGAGGCCCTGCTGCTCAACTACTCTAGCTCTCTGACACAGG AGGGAATCAACACGTACAACGAGCGGATCGGCCAGCTGGTGAAGAACCTGAAG GAGTTCGGCGACACCGACCGGAGCTTTCGGAACTGGCACAGACGGTTCAAGAAA CTGAACAAGCAGATCCTGAGCCCTAGAGTGGCCCCTCCTTGGCTGGCTCGTGCC TACAGAAGCGATGAGGAAATGGTGATGAGCCTGCAGAGCTTCCTGGATGAGTTC AACCCTCTGAAACCTAGACTCAAACAGCTGATCGCCAATCTGGAGTCCTACGACG AGCACATCTACTACTTCAGAAAGTCCCTGTCTCTGCTGTCAGTGACACTGAGGAA CGACTATAAGGCACTGGATGAAGAGCTGAGCATCCCTCAGGAGCAGGCCAACTG CAGATCTCTTAGCCTGAGCTGGATTCCTTTCAGACAGGAACTGATCAACGAGATC GAGAGAATCATCGATAGCAGCTACACAGACATTGAGAAGTGCCTGGCCAGCGCC TCCGAGTACCTGAACACCGAGAGAGCCAAGAGAAACGACTACCGGCTAGATAAT ACCGTGTCCTTCACCATCAAGAAGCTGATGGACGTGTTCCTGAGCCTGTACCGC GCCGTGAAGCCTCTGACCGGAACAGGCGAAGAGGAGGACAGAAATGAAGATTTC TACGACGAGTTCACCACCATCTGGGATGTGCTGCAATACGTGCAGAAGCTGTAC AACGCTG Illi CGCCTGGCTGAACAAGAAGCCCTACGAGAACAATAGCTACCCTG CCTACCTGGATGAATTTACCCTGCTGAAGAACTGGAAGGAAAAGGCCGCCTACAT CAAGAGGAATGGAAAATTCTACTTCATCATGTTCAACGGCATCGACGAGCAGGAT ATCATCGAACACAGAGGAGATTCTGCCATCCTGTACCATGTGGAAAGCCAGAGC CCTGATAGAATCAAGGCCAATCTGACCAAGCAGTTCGTGTTCAGCAAGAAAGCCA ATGCCGGCAAGGGCCGGCCCAATCCCAGCAAGGCCAAGTTCGTGAGAGATAAC CCCGAGTTTCAGGCCGACTGGGAGCGGGTGAAAACCGAGGCCTACAAGGTGGC CGGAAACACCGAGGCCCTGGCCCACGCCATCAGATACTTCCAAAGATGCCTGCA AAGCCACCCCGATTATAATCGGTTCCCCTTCAACTTCAGACCTGCCAACGACTAC ACATCTCTGGATGACTTCGTGGACAGCATCAAGGACAAGCTGTTCATGATGGAAG AAACCGCCATCAACTGGAGTTATGTGAGACAGCTGGCCGAAGAAGGCACAATCT ACCTGTTCAAGCTGTATAACAAAGACTACGCCAAGAACCGGGTGGGCGGCAGCA AGCCTAACCTGCACACCCTGTACTGGGAGGCCATGTTCAGCTCTGAGAATCTGA GAGAAAACAACATCAAACTGGAAGAACCCAAACTGTTCTACAGAGAGGTGGCCA CAAACCGGGACGGCGAGCTGAACATGAGACTGATCCCCCACAGATACGCCACC GACCAGCTGGAACTGCACGTGCCTATCCACCTGAATGTGAACGCCACAGCCAGC AGCGACATCAACATGATGGTCCTTGATGCCATCCGGGAAGGATCTATTGAGAAC GTGATCGGCATCGACCGGGGAGAACGGAACCTGCTGTACTACAGCGTCCTGCG ACTGTCCGACGGCGAGATCGTGGACCAGAAGAGCCTGAATATCACCTTTAACGA TGTGGACTACCACGCAAAGTTGTCTACCAAGGAGGAAGAAATCCATGATGAGCA GAGAGAGTGGAAAGCCAAGACCTCCATCAGAAAGCTGAAGGAAGGTTACCTGTC TCAGGCTATCCACCAGCTGACCAGCCTGATCGTGAAGTACCACGCTGTGGTAGT GCTGGAAGATCTGAGCGAAGATTTCTACAGCAAGCGGCAGAAAATCAACAAGCA GATCTACCAGA Illi CGAGAAAAGACTTATCGAGAAGCTGAGCTACTTTGTGGAC AAAGACGCCGCCGAGGGCCAGGCAGGCAACATCTACAGCGCCCTGCAGCTGAG CTCCCCAAATCTGGTGAGAAAGGACAACAAGAAGATCTTCCAGAACGGCATCGT GTTCTTCGTGCCACCTGAGTACACGAGTGCGATTGACCCCGTGACCGGCTTCTG CAACCTGTTTGACAAGAACAGAGTGCGCAATATCTGTGAGCTGCTCTACAGATTC GAAAACATTTGCTACAACAGAAAGAATGACCGGTTTGAGTTCACATGGGACTATA GAAACGTGATGACCTACACCAGACTTGAGCAGGACAACATCTCTCACCTGTGGA CCGCTTGTAGCCTCGGCAACCGGATCGAGTGGAGCGGCTCTGAAAGAAATAAGA ACAGAAGATGCGAGATCGTGAACCTGACACAAAGCATGAAGGTCCTGTTTGAGA AGCACGGCATCCAGTACCAGACCGGCAAGGACGTGCGGGAAGCTGTGTGTAGT ATCAGAAACAACGACTTTAAGAAAGAACTGAAGAGACTGTTTTTCCTGATGCTGA GCCTGCGTAACAGCATCGTGGATGGAAAGGTGAAAAAGGACTACATCCTGAGCC CAGTGCAAAACCAGCGGGGTAGCTTTTTCGACTCCAGAGAATATGAAGAACTGG ACAACCCGAAGTTGCCTAAGTGCGGGGACGCCAACGGCGCCTACAACATCGCCA GAAAAGGAATCCTGACAATCAGAAAGCTGGAGAACGGCAACGAGAAAGCCCTGA CCCTGGACGAATGGGTGATCAGCACCCAGAAGGGCAACATCAGAATGtctagaAAG CGGACAGCAGACGGCTCCGAATTTGAAAGCCCTAAGAAAAAGAGAAAGGTGggat ccGGCAAACCTATCCCCAATCCCCTGCTGGGCCTGGACAGCACCTGA

[0121] In some embodiments a ZQKH Type V Cas protein comprises an amino acid sequence of SEQ ID NO:67, SEQ ID NO:68, or SEQ ID NO:69. In some embodiments, a ZQKH Type V Cas protein has nickase activity, for example resulting from one or more amino acid substitutions relative to the sequence of SEQ ID NO:67, SEQ ID NO:68, or SEQ ID NO:69. In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D744 substitution, wherein the position of the D744 substitution is defined with respect to the amino acid numbering of SEQ ID NO:68 (corresponding to amino acid 908 of SEQ ID NO:121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise an E831 substitution, wherein the position of the E831 substitution is defined with respect to the amino acid numbering of SEQ ID NO:68 (corresponding to amino acid 993 of SEQ ID NO:121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a R1048 substitution, wherein the position of the R1048 substitution is defined with respect to the amino acid numbering of SEQ ID NO:68 (corresponding to amino acid 1226 of SEQ ID NO:121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D1091 substitution, wherein the position of the D1091 substitution is defined with respect to the amino acid numbering of SEQ ID NO:68 (corresponding to amino acid 1263 of SEQ ID NO:121). In some embodiments, a ZQKH Type V Cas protein is catalytically inactive, for example due to a R1048 substitution in combination with a D744 substitution, a E831 substitution, and / or D1091 substitution. 6.2.13. ZRGM Type V Cas Protein

[0122] In one aspect, the disclosure provides ZRGM Type V Cas proteins. ZRGM Type V Cas proteins can be further classified as Type V-A Cas proteins. The ZRGM Type V Cas proteins typically comprise an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:73. In some embodiments, the ZRGM Type V Cas proteins comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:73. In some embodiments, a ZRGM Type V Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:73.

[0123] Exemplary ZRGM Type V Cas protein sequences and nucleotide sequences encoding exemplary ZRGM Type V Cas proteins are set forth in Table 1M. TABLE1M ZRGM Type V Cas Sequences Name Sequence SEQ ID NO. Wildtype amino acid sequence (without N-terminal methionine) ERMYEEFRNCYSVRKTLSFKAIPTEETKKHLQLQWEVLGDEIRFENYDKMKMVLDQ LHQSYISRKLDNIGEENQKKIVEILEKLVLVMKKIDTTHQKDKEKAQNQLQSLQASLR KEIGMFFPKNEWQQLQGKNVFKKDGVLSEYNISEENKKNIQCYDGFMTFFKKYNET RANIYSTEEKSTAITFRIVNDNLPKYVRNADNYEQIKKLIPEALEEVEKTYPNLTNYFSI KNYLKYWSQKGIETYNTVIGEINKQVNLVVQQRKDSKFRKYKMQVLYQQILSDREE QSFVYQQDQEVFAAVNELAELVNGSAFNEAIELLKSPNINENEIFIPYAKLAEVSIKMK MGWNGLEEAFINDLQQQYPKKDHEKLVQKLKKEKKVFSLNEIKDVVMKIEHEEDWK FVSLLDCVEDYQKQLTETRDAYVEYAKTYAGSTGTSLQGNDVAPIKAFLDSCLQLVR WCKLFEYSDLYGNRDKIFYGGAESIILALDSLISVYNKTRNYVTMRPGQARKMHLMF NYPEFGDGFSNSKVDSYGTILLREGKKYYLAVIKKGIKVLLEDTINENDSYERLSYML FPDVKKMIPKCSISTKKVKEHFENSDDDYTIRKGESYAKELLVKKEDYDLYFVNLYDD KKMFQKDYLSKTGDKKGYRQALERWIRFCIRFLQAYKSTKDYDLSELEPISNFRSLD EFYDKLDTLLYKIEWKTISREQIKQMESSGQLFLFELYNKDFSEHAKGKKNLFTLYW EQIFCEENLKQPVIKLCGGAEMFYRKVAIQKKYVHKKDSILVDKTYVDQNGVRKTLP DTIYKEWSDFMNKKITSVSQEASKYKGLVNCHEAKYDITKDKRYTEDQFEFHVPITL NYSALGKGQLNDSVLDCLCQKEKYNVIGIDRGERNLLAYCVVNQDGQILEQGTFNKI VGGNKQEVDYKQKLQEKEVNRQQARKEWKNIGKIKELKNGYLSQVIYQLTQMMVK 73 TABLE1M ZRGM Type V Cas Sequences Name Sequence SEQID NO. YDAIVVMEDLNVGFKRGRFKVERQVYQKFEKALIDKLNYLVTKKDENQYGIEGSVSN AYQLTEKIKSFKDIGKQNGMIFYVPAGYTSKIDPTTGFVDVLNRTGLTNAKARKAFFE NFDDINYSKEDNMFAFSFDYSKFKTFQEMHRKKWTVYTNGKKYIYSKKERKEKQID VTELMKEELRKVGITEYDNLYSQITNVEDDKEHADFWKSLQFVFDRTMQLRSSQIDN GEDNLEDKIISPVKNAEGVFYESNGNYGDTSQPADADTNGAFHIARKGLLLAENVKK TGRGANGKWNSSVKNISNKDWFAFVQK Wildtype amino acid sequence (with N-terminal methionine) MERMYEEFRNCYSVRKTLSFKAIPTEETKKHLQLQWEVLGDEIRFENYDKMKMVLD QLHQSYISRKLDNIGEENQKKIVEILEKLVLVMKKIDTTHQKDKEKAQNQLQSLQASL RKEIGMFFPKNEWQQLQGKNVFKKDGVLSEYNISEENKKNIQCYDGFMTFFKKYNE TRANIYSTEEKSTAITFRIVNDNLPKYVRNADNYEQIKKLIPEALEEVEKTYPNLTNYF SIKNYLKYWSQKGIETYNTVIGEINKQVNLVVQQRKDSKFRKYKMQVLYQQILSDRE EQSFVYQQDQEVFAAVNELAELVNGSAFNEAIELLKSPNINENEIFIPYAKLAEVSIKM KMGWNGLEEAFINDLQQQYPKKDHEKLVQKLKKEKKVFSLNEIKDVVMKIEHEEDW KFVSLLDCVEDYQKQLTETRDAYVEYAKTYAGSTGTSLQGNDVAPIKAFLDSCLQLV RWCKLFEYSDLYGNRDKIFYGGAESIILALDSLISVYNKTRNYVTMRPGQARKMHLM FNYPEFGDGFSNSKVDSYGTILLREGKKYYLAVIKKGIKVLLEDTINENDSYERLSYM LFPDVKKMIPKCSISTKKVKEHFENSDDDYTIRKGESYAKELLVKKEDYDLYFVNLYD DKKMFQKDYLSKTGDKKGYRQALERWIRFCIRFLQAYKSTKDYDLSELEPISNFRSL DEFYDKLDTLLYKIEWKTISREQIKQMESSGQLFLFELYNKDFSEHAKGKKNLFTLY WEQIFCEENLKQPVIKLCGGAEMFYRKVAIQKKYVHKKDSILVDKTYVDQNGVRKTL PDTIYKEWSDFMNKKITSVSQEASKYKGLVNCHEAKYDITKDKRYTEDQFEFHVPIT LNYSALGKGQLNDSVLDCLCQKEKYNVIGIDRGERNLLAYCVVNQDGQILEQGTFN KIVGGNKQEVDYKQKLQEKEVNRQQARKEWKNIGKIKELKNGYLSQVIYQLTQMMV KYDAIVVMEDLNVGFKRGRFKVERQVYQKFEKALIDKLNYLVTKKDENQYGIEGSVS NAYQLTEKIKSFKDIGKQNGMIFYVPAGYTSKIDPTTGFVDVLNRTGLTNAKARKAFF ENFDDINYSKEDNMFAFSFDYSKFKTFQEMHRKKWTVYTNGKKYIYSKKERKEKQI DVTELMKEELRKVGITEYDNLYSQITNVEDDKEHADFWKSLQFVFDRTMQLRSSQID NGEDNLEDKIISPVKNAEGVFYESNGNYGDTSQPADADTNGAFHIARKGLLLAENVK KTGRGANGKWNSSVKNISNKDWFAFVQK 74 Expression construct (with N-terminal methionine, V5-tag and C-terminal NLS) aa sequence MGSGERMYEEFRNCYSVRKTLSFKAIPTEETKKHLQLQWEVLGDEIRFENYDKMK MVLDQLHQSYISRKLDNIGEENQKKIVEILEKLVLVMKKIDTTHQKDKEKAQNQLQSL QASLRKEIGMFFPKNEWQQLQGKNVFKKDGVLSEYNISEENKKNIQCYDGFMTFFK KYNETRANIYSTEEKSTAITFRIVNDNLPKYVRNADNYEQIKKLIPEALEEVEKTYPNL TNYFSIKNYLKYWSQKGIETYNTVIGEINKQVNLVVQQRKDSKFRKYKMQVLYQQIL SDREEQSFVYQQDQEVFAAVNELAELVNGSAFNEAIELLKSPNINENEIFIPYAKLAE VSIKMKMGWNGLEEAFINDLQQQYPKKDHEKLVQKLKKEKKVFSLNEIKDVVMKIEH EEDWKFVSLLDCVEDYQKQLTETRDAYVEYAKTYAGSTGTSLQGNDVAPIKAFLDS CLQLVRWCKLFEYSDLYGNRDKIFYGGAESIILALDSLISVYNKTRNYVTMRPGQAR KMHLMFNYPEFGDGFSNSKVDSYGTILLREGKKYYLAVIKKGIKVLLEDTINENDSYE RLSYMLFPDVKKMIPKCSISTKKVKEHFENSDDDYTIRKGESYAKELLVKKEDYDLYF VNLYDDKKMFQKDYLSKTGDKKGYRQALERWIRFCIRFLQAYKSTKDYDLSELEPIS NFRSLDEFYDKLDTLLYKIEWKTISREQIKQMESSGQLFLFELYNKDFSEHAKGKKNL FTLYWEQIFCEENLKQPVIKLCGGAEMFYRKVAIQKKYVHKKDSILVDKTYVDQNGV RKTLPDTIYKEWSDFMNKKITSVSQEASKYKGLVNCHEAKYDITKDKRYTEDQFEFH VPITLNYSALGKGQLNDSVLDCLCQKEKYNVIGIDRGERNLLAYCVVNQDGQILEQG TFNKIVGGNKQEVDYKQKLQEKEVNRQQARKEWKNIGKIKELKNGYLSQVIYQLTQ MMVKYDAIVVMEDLNVGFKRGRFKVERQVYQKFEKALIDKLNYLVTKKDENQYGIE GSVSNAYQLTEKIKSFKDIGKQNGMIFYVPAGYTSKIDPTTGFVDVLNRTGLTNAKA RKAFFENFDDINYSKEDNMFAFSFDYSKFKTFQEMHRKKWTVYTNGKKYIYSKKER KEKQIDVTELMKEELRKVGITEYDNLYSQITNVEDDKEHADFWKSLQFVFDRTMQLR SSQIDNGEDNLEDKIISPVKNAEGVFYESNGNYGDTSQPADADTNGAFHIARKGLLL AENVKKTGRGANGKWNSSVKNISNKDWFAFVQKSRKRTADGSEFESPKKKRKVG SGKPIPNPLLGLDST 75 Wildtype coding sequence (with N-terminal methionine and stop codon) ATGGAGAGAATGTACGAAGAATTTAGAAATTGTTATTCAGTACGAAAAACATTGT CATTTAAGGCAATCCCAACAGAGGAAACAAAAAAACATTTACAATTACAATGGGA AGTGTTGGGGGATGAGATACG Illi GAAAAC IAI GA IAAAAI GAAAAI GG I I I I G GATCAACTTCATCAATCATATATTTCGAGAAAATTAGATAATATAGGAGAAGAAAA TCAAAAAAAGATAGTTGAAATCTTAGAGAAACTCGTATTAGTTATGAAAAAGATA GATACTACGCATCAAAAGGATAAAGAGAAAGCGCAAAATCAGCTTCAATCGTTA CAAGCTTCATTAAGGAAAGAAATAGGAATGTTTTTTCCTAAAAACGAATGGCAAC AATTACAGGGAAAAAATGTATTTAAGAAGGATGGGGTACTAAGCGAGTATAACAT TTCGGAAGAGAATAAGAAAAATATTCAATGTTATGATGGI I I IATGACATTCTTTA AAAAATATAATGAAACTAGAGCAAATATATATAGTACAGAGGAAAAAAGCACGGC AATCAC Illi CGAATTGTGAATGATAATCTTCCAAAATATGTGAGAAATGCGGAT 76 TABLE1M ZRGM Type V Cas Sequences Name Sequence SEQID NO. AATTACGAACAGATTAAAAAATTAATTCCTGAAGCTCTTGAAGAAGTAGAAAAAA CATACCCAAATTTGACGAATTATTTCTCGATTAAAAACTATTTGAAGTATTGGAGT CAGAAGGGGATTGAAACATACAATACTGTTATTGGAGAAATAAATAAGCAGGTTA ATCTTGTAGTACAACAAAGAAAAGATTCGAAATTTAGAAAATACAAGATGCAGGT GTTGTATCAACAAATTCTAAGTGATAGAGAGGAACAGTC Illi GTGTATCAACAG GATCAGGAAGTTTTTGCTGCTGTTAATGAACTTGCAGAACTTGTGAACGGTAGT GCTTTTAACGAGGCAATTGAATTGTTGAAATCACCTAATATTAACGAAAATGAGA TATTTATTCCCTATGCAAAATTAGCAGAAGTATCCATAAAAATGAAAATGGGATG GAATGGATTAGAGGAGGCTTTTATAAACGATTTGCAACAGCAGTATCCAAAGAA GGATCATGAAAAATTGGTGCAAAAATTAAAAAAAGAGAAAAAAGTTTTTTCTTTGA ATGAAATTAAAGATGTTGTTATGAAAATTGAACATGAAGAAGATTGGAAATTTGTT AGTTTGCTGGATTGTGTTGAGGATTATCAAAAACAGTTGACAGAGACAAGAGAT GCATATGTGGAATATGCAAAAACTTATGCAGGTTCAACCGGTACATCATTACAAG GAAATGATGTAGCACCGATAAAAGCAT I I I IAGATAGTTGTTTGCAATTGGTACG ATGGTGTAAGTTGTTTGAATATTCTGATTTGTATGGAAATCGAGATAAAATA Illi ATGGAGGAGCAGAGTCGATTATACTTGCATTAGATTCCTTAATATCTGTGTATAA TAAAACAAGAAATTATGTGACTATGCGACCGGGGCAGGCTAGAAAAATGCATTT AATGTTTAATTATCCGGAATTCGGTGATGGCTTTAGTAATAGTAAAGTGGATTCT TATGGTACGA Illi GCTTCGTGAAGGAAAGAAATATTATTTAGCTGTTATTAAAAA AGGCATAAAAGTCTTGCTGGAAGATACCATAAATGAAAATGACAGTTATGAACGT TTGAGTTATATGTTGTTTCCTGATGTAAAAAAAATGATACCGAAATGTTCTATTAG TACGAAGAAAGTTAAAGAACA Illi GAAAATTCGGATGATGATTATACGATTCGT AAAGGTGAATCTTATGCAAAAGAATTACTTGTGAAAAAAGAAGATTATGACCTTT ACTTTGTAAATCTTTATGATGATAAGAAGATGTTTCAAAAGGACTATTTGAGTAAA ACTGGAGATAAAAAAGGATATAGACAGGCGTTAGAACGCTGGATACGTTTTTGC ATTCGATTTTTACAAGCTTATAAGAGTACAAAGGATTATGATCTCAGTGAATTAGA GCCAATTTCGAAI I I I CG I I CO I I AGA I GAG Illi ATGATAAATTGGATACTTTGT TATACAAGATAGAGTGGAAAACAATTTCAAGAGAACAAATTAAGCAAATGGAGTC ATCTGGTCAGTTGTTTTTATTTGAATTATATAACAAAGATTTCTCTGAACATGCAA AAGGAAAGAAAAATTTATTTACATTGTATTGGGAACAGA Illi CTGTGAAGAGAA TTTAAAACAGCCAGTGATTAAACTTTGTGGCGGGGCAGAGATG Illi ATCGTAAG GTTGCCATTCAAAAAAAATATGTACATAAAAAAGACTCCA Illi GGTGGATAAAA CGTATGTGGATCAGAATGGAGTCAGAAAAACACTTCCGGATACTATATATAAAGA GTGGTCGGA Illi ATGAATAAAAAGATAACATCTGTCAGCCAGGAGGCAAGTAA ATATAAAGGTTTGGTTAATTGTCATGAGGCAAAATATGATATTACAAAAGATAAAA GATATACGGAAGATCAATTTGAGTTTCATGTGCCAATTACTTTAAATTATTCAGCA TTAGGAAAAGGGCAATTAAATGATAGTGTTCTGGATTGTCTATGTCAGAAAGAAA AATATAATGTGATAGGAATTGACCGTGGAGAAAGAAACTTGTTGGCTTACTGTGT CGTAAATCAAGATGGACAGAI I I IAGAACAAGGGACATTTAATAAGATTGTAGGT GGAAATAAACAGGAAGTAGATTACAAACAGAAGTTACAGGAGAAAGAAGTAAAT CGACAACAAGCAAGAAAAGAGTGGAAAAATATTGGAAAAATTAAAGAATTAAAGA ACGGTTATTTGTCTCAGGTTATTTATCAACTGACGCAAATGATGGTAAAATATGA TGCTATTGTTGTTATGGAAGATTTGAATGTTGGCTTTAAACGTGGTCGATTTAAG GTGGAACGACAGGTTTACCAGAAATTTGAAAAAGCGCTGATTGACAAATTAAATT ATTTAGTAACTAAAAAAGATGAAAATCAATATGGAATAGAGGGTAGCGTAAGCAA TGCATATCAACTGACAGAAAAAATCAAATCATTTAAAGATATTGGCAAACAAAAC GGGATGATAI I I IATGTGCCAGCGGGATATACCTCTAAAATAGATCCTACAACAG GATTTGTGGATGTGCTAAATCGAACAGGATTAACAAATGCCAAAGCCAGAAAAG CGTTCTTTGAAAA Illi GATGATATTAACTATTCAAAAGAAGATAATATGTTTGCC I I I I C I I I I GATTATAGCAAGTTTAAGACATTTCAAGAAATGCATAGAAAAAAATG GACAGTTTACACAAATGGTAAAAAGTACATTTATTCAAAAAAAGAACGAAAAGAA AAACAAATTGATGTTACTGAGTTGATGAAAGAAGAATTGAGAAAAGTAGGAATTA CAGAGTATGATAATCTTTATTCGCAAATTACTAATGTGGAAGATGATAAAGAACA TGCAGATTTTTGGAAATCTTTACAGTTTGTATTTGATAGAACGATGCAGTTGAGA AGTAGTCAAATTGACAATGGAGAGGATAATCTTGAGGATAAGATTATATCTCCGG TGAAAAATGCAGAGGGTGTAI I I IATGAATCAAATGGAAATTATGGTGACACTTC ACAACCTGCAGATGCAGATACAAATGGTGC Illi CATATTGCAAGGAAGGGATT ACTACTTGCAGAAAATGTGAAAAAAACAGGTAGAGGAGCAAATGGAAAATGGAA TTCTTCTGTAAAAAATATTTCTAATAAGGATTGGTTTGCATTTGTTCAAAAATAA Codon optimized coding sequence (no N-terminal GAACGGATGTACGAGGAGTTCAGAAACTGCTACTCCGTGCGGAAAACACTGTC CTTCAAAGCCATCCCTACCGAGGAGACAAAGAAGCACCTGCAGCTGCAGTGGG AAGTGCTCGGCGACGAGATTAGATTTGAGAATTATGATAAGATGAAAATGGTGC TGGACCAGCTGCACCAGTCTTACATCAGCCGGAAGCTGGACAACATCGGCGAG GAGAACCAGAAAAAGATTGTAGAAATCCTGGAGAAGCTGGTGCTGGTGATGAAG 77 TABLE1M ZRGM Type V Cas Sequences Name Sequence SEQID NO. methionine, no stop codon) AAGATCGATACAACCCACCAGAAGGACAAGGAGAAGGCCCAGAATCAACTGCA GAGCCTGCAGGCTTCCCTGCGGAAGGAAATTGGTATGTTTTTCCCAAAGAACGA GTGGCAGCAGCTGCAGGGCAAAAACGTGTTCAAGAAGGACGGCGTTCTCAGCG AATACAACATCAGCGAGGAAAACAAGAAGAACATCCAGTGTTACGACGGCTTTA TGACCTTCTTCAAGAAGTACAACGAGACACGGGCCAATATCTATTCTACGGAGG AAAAGAGCACCGCCATCACCTTCAGGATCGTGAATGATAATCTGCCTAAGTATG TGCGAAACGCTGACAACTACGAGCAGATAAAGAAGCTGATCCCCGAAGCTCTG GAAGAAGTCGAAAAGACCTATCCTAATCTGACCAACTACTTCAGCATCAAGAACT ATCTGAAGTACTGGAGCCAGAAGGGGATCGAAACATACAACACCGTGATCGGC GAGATCAACAAGCAGGTGAACCTGGTGGTCCAACAGAGAAAGGACAGCAAGTT CAGGAAGTACAAAATGCAGGTGCTGTACCAGCAGATCCTATCCGACAGAGAGG AGCAGAGCTTCGTGTACCAGCAGGACCAGGAGGTGTTCGCCGCCGTGAACGAG CTGGCCGAGCTGGTGAATGGCAGCGCCTTCAATGAAGCTATCGAATTGCTGAAA AGCCCAAACATCAACGAGAATGAGA Illi CATCCCCTACGCCAAGCTCGCCGAG GTGTCTATCAAGATGAAAATGGGATGGAACGGCCTGGAGGAGGCCTTCATCAA CGATCTGCAGCAACAATACCCCAAGAAAGACCACGAAAAATTGGTTCAGAAGCT GAAGAAAGAGAAGAAGGTGTTTAGCCTGAATGAAATCAAGGATGTGGTCATGAA GATCGAACACGAGGAAGATTGGAAATTCGTGAGCCTGCTGGACTGCGTGGAGG ATTACCAGAAGCAGCTTACAGAGACAAGAGATGCCTACGTGGAGTACGCTAAGA CATACGCCGGCAGCACAGGCACCAGCCTGCAGGGCAACGACGTGGCCCCTAT CAAGGCCTTCCTGGACTCCTGCCTGCAACTGGTGCGGTGGTGCAAGCTGTTCG AGTACAGCGACCTGTACGGCAACAGAGACAAGATCTTCTACGGAGGCGCCGAG AGCATCATCCTGGCCCTGGATAGCCTGATTTCCGTGTACAACAAAACCAGAAAC TACGTGACCATGCGGCCTGGCCAGGCCAGAAAAATGCACCTGATGTTCAACTAC CCCGAGTTTGGCGACGGCTTCAGCAACAGCAAAGTGGATTCTTACGGCACCAT CCTGCTGAGAGAAGGCAAGAAGTACTACCTGGCTGTGATCAAGAAGGGCATCA AAGTGCTGCTGGAGGACACCATTAACGAGAATGACTCTTACGAGCGGCTGTCCT ACATGCTGTTCCCCGACGTGAAAAAGATGATCCCTAAGTGCAGCATCAGTACCA AGAAGGTGAAAGAGCATTTCGAGAACAGCGACGACGACTACACCATCAGAAAG GGCGAGAGCTATGCCAAGGAGCTGCTGGTGAAGAAGGAAGATTACGACCTGTA TTTCGTGAACCTGTACGACGACAAAAAGATGTTCCAGAAAGACTACCTGAGCAA AACCGGCGACAAGAAGGGATACAGACAGGCCCTGGAGAGGTGGATCAGATTCT GCATCAGATTCCTGCAGGCTTACAAGTCTACAAAGGATTATGACCTGTCTGAACT GGAACCTATCAGCAACTTCAGAAGCCTGGACGAGTTCTACGATAAGCTGGACAC CCTACTGTACAAGATCGAGTGGAAAACCATCTCCAGAGAGCAGATCAAGCAAAT GGAATCCTCTGGCCAGCTCTTCCTGTTCGAGTTGTACAACAAGGACTTCTCTGA ACACGCCAAGGGAAAGAAGAACCTGTTCACCCTGTACTGGGAGCAAATTTTTTG TGAAGAGAACCTGAAGCAGCCTGTGATCAAGCTGTGCGGCGGAGCCGAGATGT TCTACAGAAAGGTTGCCATCCAGAAAAAGTACGTGCACAAGAAGGACAGCATCC TGGTAGACAAGACCTACGTGGATCAGAACGGCGTTCGCAAGACCCTGCCTGAT ACCATCTACAAGGAATGGTCCGACTTCATGAACAAAAAGATCACCAGCGTGTCC CAAGAAGCCTCTAAATACAAGGGCCTGGTGAACTGTCACGAGGCCAAGTACGA CATCACCAAGGACAAGAGATACACCGAAGATCAATTCGAATTTCACGTGCCAAT CACACTGAACTACAGCGCCCTCGGAAAAGGTCAGCTGAACGACAGCGTGCTGG ACTGCCTGTGTCAGAAAGAGAAGTACAACGTGATTGGAATCGACCGGGGAGAA AGAAACCTGCTGGCCTACTGCGTGGTGAACCAGGATGGCCAGATCCTGGAACA GGGCACCTTCAACAAGATCGTGGGCGGCAATAAGCAGGAGGTGGACTATAAGC AGAAACTGCAGGAGAAGGAGGTGAATAGACAGCAGGCCAGGAAGGAGTGGAA GAACATCGGCAAGATCAAGGAGTTGAAAAACGGCTACCTGAGCCAAGTAATCTA CCAGCTGACACAGATGATGGTGAAGTACGATGCCATCGTGGTGATGGAAGATCT GAACGTGGGCTTTAAGAGAGGCAGATTCAAGGTTGAGCGGCAGGTGTACCAGA AGTTCGAAAAGGCTCTGATCGATAAGCTGAATTATCTGGTCACCAAGAAGGACG AGAACCAATACGGGATCGAGGGCAGCGTTTCGAATGCCTACCAGCTGACCGAG AAAATCAAGAGCTTCAAAGACATCGGAAAACAGAACGGCATGATCTTCTACGTG CCTGCTGGCTATACAAGCAAAATCGACCCTACGACCGGATTCGTCGATGTGCTG AACAGAACCGGCCTGACAAACGCCAAGGCTAGAAAAGCCTTCTTCGAGAA Illi GACGACATCAACTACTCTAAGGAGGACAACATGTTCGCCTTCAGCTTCGATTAC AGCAAGTTCAAGACCTTTCAGGAAATGCATAGAAAAAAGTGGACAGTGTACACA AACGGAAAAAAATACATCTACAGCAAGAAGGAACGGAAGGAAAAGCAGATAGAC GTGACCGAACTGATGAAAGAAGAGCTGAGAAAGGTGGGCATAACCGAGTACGA CAACCTCTACAGCCAGATCACCAACGTGGAAGATGATAAGGAGCACGCCGACTT TTGGAAGTCTCTGCAGTTCGTGTTCGACAGAACAATGCAGCTGAGAAGCAGCCA GATCGACAACGGCGAGGACAATCTGGAAGATAAGATCATTTCACCTGTGAAAAA CGCCGAGGGCGTGTTCTATGAAAGCAACGGCAACTACGGCGATACGAGCCAGC TABLE1M ZRGM Type V Cas Sequences Name Sequence SEQID NO. CCGCCGACGCGGACACCAACGGCGCCTTCCACATCGCGCGGAAGGGCCTGCT GCTCGCCGAGAATGTGAAGAAAACCGGAAGAGGCGCCAATGGCAAATGGAATA GCAGCGTGAAGAACATCTCTAACAAGGATTGGTTCGCCTTTGTGCAGAAA Expression construct (with N-terminal methionine and stop codon, includes V5-tag and C-terminal NLS) ATGggctccggaGAACGGATGTACGAGGAGTTCAGAAACTGCTACTCCGTGCGGAA AACACTGTCCTTCAAAGCCATCCCTACCGAGGAGACAAAGAAGCACCTGCAGCT GCAGTGGGAAGTGCTCGGCGACGAGATTAGATTTGAGAATTATGATAAGATGAA AATGGTGCTGGACCAGCTGCACCAGTCTTACATCAGCCGGAAGCTGGACAACA TCGGCGAGGAGAACCAGAAAAAGATTGTAGAAATCCTGGAGAAGCTGGTGCTG GTGATGAAGAAGATCGATACAACCCACCAGAAGGACAAGGAGAAGGCCCAGAA TCAACTGCAGAGCCTGCAGGCTTCCCTGCGGAAGGAAATTGGTATGT Illi CCC AAAGAACGAGTGGCAGCAGCTGCAGGGCAAAAACGTGTTCAAGAAGGACGGCG TTCTCAGCGAATACAACATCAGCGAGGAAAACAAGAAGAACATCCAGTGTTACG ACGGCTTTATGACCTTCTTCAAGAAGTACAACGAGACACGGGCCAATATCTATTC TACGGAGGAAAAGAGCACCGCCATCACCTTCAGGATCGTGAATGATAATCTGCC TAAGTATGTGCGAAACGCTGACAACTACGAGCAGATAAAGAAGCTGATCCCCGA AGCTCTGGAAGAAGTCGAAAAGACCTATCCTAATCTGACCAACTACTTCAGCAT CAAGAACTATCTGAAGTACTGGAGCCAGAAGGGGATCGAAACATACAACACCGT GATCGGCGAGATCAACAAGCAGGTGAACCTGGTGGTCCAACAGAGAAAGGACA GCAAGTTCAGGAAGTACAAAATGCAGGTGCTGTACCAGCAGATCCTATCCGACA GAGAGGAGCAGAGCTTCGTGTACCAGCAGGACCAGGAGGTGTTCGCCGCCGT GAACGAGCTGGCCGAGCTGGTGAATGGCAGCGCCTTCAATGAAGCTATCGAAT TGCTGAAAAGCCCAAACATCAACGAGAATGAGA Illi CATCCCCTACGCCAAGC TCGCCGAGGTGTCTATCAAGATGAAAATGGGATGGAACGGCCTGGAGGAGGCC TTCATCAACGATCTGCAGCAACAATACCCCAAGAAAGACCACGAAAAATTGGTT CAGAAGCTGAAGAAAGAGAAGAAGGTGTTTAGCCTGAATGAAATCAAGGATGTG GTCATGAAGATCGAACACGAGGAAGATTGGAAATTCGTGAGCCTGCTGGACTG CGTGGAGGATTACCAGAAGCAGCTTACAGAGACAAGAGATGCCTACGTGGAGT ACGCTAAGACATACGCCGGCAGCACAGGCACCAGCCTGCAGGGCAACGACGT GGCCCCTATCAAGGCCTTCCTGGACTCCTGCCTGCAACTGGTGCGGTGGTGCA AGCTGTTCGAGTACAGCGACCTGTACGGCAACAGAGACAAGATCTTCTACGGA GGCGCCGAGAGCATCATCCTGGCCCTGGATAGCCTGATTTCCGTGTACAACAA AACCAGAAACTACGTGACCATGCGGCCTGGCCAGGCCAGAAAAATGCACCTGA TGTTCAACTACCCCGAGTTTGGCGACGGCTTCAGCAACAGCAAAGTGGATTCTT ACGGCACCATCCTGCTGAGAGAAGGCAAGAAGTACTACCTGGCTGTGATCAAG AAGGGCATCAAAGTGCTGCTGGAGGACACCATTAACGAGAATGACTCTTACGAG CGGCTGTCCTACATGCTGTTCCCCGACGTGAAAAAGATGATCCCTAAGTGCAGC ATCAGTACCAAGAAGGTGAAAGAGCATTTCGAGAACAGCGACGACGACTACACC ATCAGAAAGGGCGAGAGCTATGCCAAGGAGCTGCTGGTGAAGAAGGAAGATTA CGACCTGTATTTCGTGAACCTGTACGACGACAAAAAGATGTTCCAGAAAGACTA CCTGAGCAAAACCGGCGACAAGAAGGGATACAGACAGGCCCTGGAGAGGTGG ATCAGATTCTGCATCAGATTCCTGCAGGCTTACAAGTCTACAAAGGATTATGACC TGTCTGAACTGGAACCTATCAGCAACTTCAGAAGCCTGGACGAGTTCTACGATA AGCTGGACACCCTACTGTACAAGATCGAGTGGAAAACCATCTCCAGAGAGCAGA TCAAGCAAATGGAATCCTCTGGCCAGCTCTTCCTGTTCGAGTTGTACAACAAGG ACTTCTCTGAACACGCCAAGGGAAAGAAGAACCTGTTCACCCTGTACTGGGAGC AAATTTTTTGTGAAGAGAACCTGAAGCAGCCTGTGATCAAGCTGTGCGGCGGAG CCGAGATGTTCTACAGAAAGGTTGCCATCCAGAAAAAGTACGTGCACAAGAAGG ACAGCATCCTGGTAGACAAGACCTACGTGGATCAGAACGGCGTTCGCAAGACC CTGCCTGATACCATCTACAAGGAATGGTCCGACTTCATGAACAAAAAGATCACC AGCGTGTCCCAAGAAGCCTCTAAATACAAGGGCCTGGTGAACTGTCACGAGGC CAAGTACGACATCACCAAGGACAAGAGATACACCGAAGATCAATTCGAATTTCA CGTGCCAATCACACTGAACTACAGCGCCCTCGGAAAAGGTCAGCTGAACGACA GCGTGCTGGACTGCCTGTGTCAGAAAGAGAAGTACAACGTGATTGGAATCGAC CGGGGAGAAAGAAACCTGCTGGCCTACTGCGTGGTGAACCAGGATGGCCAGAT CCTGGAACAGGGCACCTTCAACAAGATCGTGGGCGGCAATAAGCAGGAGGTGG ACTATAAGCAGAAACTGCAGGAGAAGGAGGTGAATAGACAGCAGGCCAGGAAG GAGTGGAAGAACATCGGCAAGATCAAGGAGTTGAAAAACGGCTACCTGAGCCA AGTAATCTACCAGCTGACACAGATGATGGTGAAGTACGATGCCATCGTGGTGAT GGAAGATCTGAACGTGGGCTTTAAGAGAGGCAGATTCAAGGTTGAGCGGCAGG TGTACCAGAAGTTCGAAAAGGCTCTGATCGATAAGCTGAATTATCTGGTCACCA AGAAGGACGAGAACCAATACGGGATCGAGGGCAGCGTTTCGAATGCCTACCAG CTGACCGAGAAAATCAAGAGCTTCAAAGACATCGGAAAACAGAACGGCATGATC TTCTACGTGCCTGCTGGCTATACAAGCAAAATCGACCCTACGACCGGATTCGTC GATGTGCTGAACAGAACCGGCCTGACAAACGCCAAGGCTAGAAAAGCCTTCTTC 78 TABLE1M ZRGM Type V Cas Sequences Name Sequence SEQ ID NO. GAGAA Illi GACGACATCAACTACTCTAAGGAGGACAACATGTTCGCCTTCAGC TTCGATTACAGCAAGTTCAAGACCTTTCAGGAAATGCATAGAAAAAAGTGGACA GTGTACACAAACGGAAAAAAATACATCTACAGCAAGAAGGAACGGAAGGAAAAG CAGATAGACGTGACCGAACTGATGAAAGAAGAGCTGAGAAAGGTGGGCATAAC CGAGTACGACAACCTCTACAGCCAGATCACCAACGTGGAAGATGATAAGGAGC ACGCCGAC Illi GGAAGTCTCTGCAGTTCGTGTTCGACAGAACAATGCAGCTGA GAAGCAGCCAGATCGACAACGGCGAGGACAATCTGGAAGATAAGATCATTTCAC CTGTGAAAAACGCCGAGGGCGTGTTCTATGAAAGCAACGGCAACTACGGCGAT ACGAGCCAGCCCGCCGACGCGGACACCAACGGCGCCTTCCACATCGCGCGGA AGGGCCTGCTGCTCGCCGAGAATGTGAAGAAAACCGGAAGAGGCGCCAATGG CAAATGGAATAGCAGCGTGAAGAACATCTCTAACAAGGATTGGTTCGCCTTTGT GCAGAAAtctagaAAGCGGACAGCAGACGGCTCCGAATTTGAAAGCCCTAAGAAA AAGAGAAAGGTGggatccGGCAAACCTATCCCCAATCCCCTGCTGGGCCTGGACA GCACCTGA

[0124] In some embodiments a ZRGM Type V Cas protein comprises an amino acid sequence of SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:75. In some embodiments, a ZRGM Type V Cas protein has nickase activity, for example resulting from one or more amino acid substitutions relative to the sequence of SEQ ID NO:73, SEQ ID NO:74, or SEQ ID NO:75. In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D890 substitution, wherein the position of the D890 substitution is defined with respect to the amino acid numbering of SEQ ID NO:74 (corresponding to amino acid 908 of SEQ ID NO:121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise an E980 substitution, wherein the position of the E980 substitution is defined with respect to the amino acid numbering of SEQ ID NO:74 (corresponding to amino acid 993 of SEQ ID NO:121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a R1194 substitution, wherein the position of the R1194 substitution is defined with respect to the amino acid numbering of SEQ ID NO:74 (corresponding to amino acid 1226 of SEQ ID NO:121). In some embodiments, the one or more amino acid substitutions providing nickase activity comprise a D1237 substitution, wherein the position of the D1237 substitution is defined with respect to the amino acid numbering of SEQ ID NO:74 (corresponding to amino acid 1263 of SEQ ID NO:121). In some embodiments, a ZRGM Type V Cas protein is catalytically inactive, for example due to a R1194 substitution in combination with a D890 substitution, a E980 substitution, and / or D1237 substitution. 6.2.14. ZTAE Type V Cas Protein

[0125] In one aspect, the disclosure provides ZTAE Type V Cas proteins. ZTAE Type V Cas proteins can be further classified as Type V-A Cas proteins. The ZTAE Type V Cas proteins typically comprise an amino acid sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO:79. In some embodiments, the ZTAE Type V Cas proteins comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:79. In some embodiments, a ZTAE Type V Cas protein comprises an amino acid sequence that is identical to SEQ ID NO:79.

[0126] Exemplary ZTAE Type V Cas protein sequences and nucleotide sequences encoding exemplary ZTAE Type V Cas proteins are set forth in Table 1N. TABLE1N ZTAE Type V Cas Sequences Name Sequence SEQ ID NO. Wildtype amino acid sequence (without N-terminal methionine) SFESFTNVYPVSKTLRFELRPVGATAEKLKESGILEHDTKRGKEYATLKDLLDEQHKE LLADALKPERVKNALKPNSGKSKKDKLVEENYITEDGEIRWETLAAAMEAFRAGEVE KNVLEAIQTQFRKLIVTILKADERYPGLTASTPSAVIKTLLKQDVHPEAVETFAKFACYF TGFQENRKNIYAEEKQATAVATRVVHDNFAKFHTQSKIIGVIKNKYPEILQSVEMELM DELGGMKITDIFSINSYSKWMTQEGIDFINKIIGGYSPSVGVKVRGLNEFINLYRQQHE EANADRRNLAKMPMLFKQILSDISTRSFIPVMFENDAELKDSIEAFLTGLNDFELNAQK FNVVVALGNLFQKIVPCEGIFLDAALMEKVSKTATGDWSLLAQSMEAYAETAFTRAK DRDAWLRKNYYSLSELSQVPILKNTDEGMLKFELSAYWSGEKMESFVKGIMDAELA MKPVLASIGQKTEEVRLRDRIDDVVKIKGYLDSIQNFLHHLKPFCAPTELNRDADFYS DFDALYNQLVLVIPLYNCVRNYVTQKVTEVQKLRLKFDAPTLADGWDANKENDNKAV LFEKDGLYFLGILNPNLKAKDRPVFEHESNVTKKSCYRKIVYKLLPGPNKMLPKVFFA DSNRTLYHPSKSLLDRYHNGEYKKGDSFDIKFCHELIDYFKASISIHPDWKEFGFQFS ATKTYESIDGFYREVEEQGYKVNFAFVRADLIDKYVESGSLFLFQLYNKDFSCASSGK PNLHTLYWKSLFAKENLDEPILKLCGGAELFFRPVAIQKPYVHTLGEKLVNRRLGEHG KGEAIPERVHKELVDYYNHRVSVLSHDGKAFKDKVVVRDVAHSITKDRRFSEAKFFF HVPIMFNRTASKSAKFNDKVVDYLKTTQNVNVIGLDRGERNLIYLTMVNLHGKLIEQR SFNLVNGVDYHSKLDLREKERMDARVNWENIGGIKDLKTGYLSAVVHEIAKMMVTNN AIVVLEDLNFGFKRGRFKVEKQVYQKFEKMLIDKLNFLMFKECNQAALGGVRRAYQL TDKFVSFEKLGKQTGFLFYVPAGYTSKIDPTTGFTNLFNTKKCTNAEGRKVFFEAMN SIIYDGSRKSFAFSFDYGNPVFRASQTSFKKEWTVYSADTRIVYNRGEKTVNTIHPTQI LHDALCALGIDVHDGLNVLNVVRETPADKIHAKFFSDLFYAFDRTLQMRNSVSGTDE DYIQSPVLNATGEFFDSRKADSTLPQDADANGAYHIALKGLLLLQRMKDIGSDIKLDLS IKHEDWFAFAQKRCQR 79 Wildtype amino acid sequence (with N-terminal methionine) MSFESFTNVYPVSKTLRFELRPVGATAEKLKESGILEHDTKRGKEYATLKDLLDEQHK ELLADALKPERVKNALKPNSGKSKKDKLVEENYITEDGEIRWETLAAAMEAFRAGEV EKNVLEAIQTQFRKLIVTILKADERYPGLTASTPSAVIKTLLKQDVHPEAVETFAKFACY FTGFQENRKNIYAEEKQATAVATRVVHDNFAKFHTQSKIIGVIKNKYPEILQSVEMELM DELGGMKITDIFSINSYSKWMTQEGIDFINKIIGGYSPSVGVKVRGLNEFINLYRQQHE EANADRRNLAKMPMLFKQILSDISTRSFIPVMFENDAELKDSIEAFLTGLNDFELNAQK FNVVVALGNLFQKIVPCEGIFLDAALMEKVSKTATGDWSLLAQSMEAYAETAFTRAK DRDAWLRKNYYSLSELSQVPILKNTDEGMLKFELSAYWSGEKMESFVKGIMDAELA MKPVLASIGQKTEEVRLRDRIDDVVKIKGYLDSIQNFLHHLKPFCAPTELNRDADFYS DFDALYNQLVLVIPLYNCVRNYVTQKVTEVQKLRLKFDAPTLADGWDANKENDNKAV LFEKDGLYFLGILNPNLKAKDRPVFEHESNVTKKSCYRKIVYKLLPGPNKMLPKVFFA DSNRTLYHPSKSLLDRYHNGEYKKGDSFDIKFCHELIDYFKASISIHPDWKEFGFQFS ATKTYESIDGFYREVEEQGYKVNFAFVRADLIDKYVESGSLFLFQLYNKDFSCASSGK PNLHTLYWKSLFAKENLDEPILKLCGGAELFFRPVAIQKPYVHTLGEKLVNRRLGEHG KGEAIPERVHKELVDYYNHRVSVLSHDGKAFKDKVVVRDVAHSITKDRRFSEAKFFF HVPIMFNRTASKSAKFNDKVVDYLKTTQNVNVIGLDRGERNLIYLTMVNLHGKLIEQR SFNLVNGVDYHSKLDLREKERMDARVNWENIGGIKDLKTGYLSAVVHEIAKMMVTNN AIVVLEDLNFGFKRGRFKVEKQVYQKFEKMLIDKLNFLMFKECNQAALGGVRRAYQL TDKFVSFEKLGKQTGFLFYVPAGYTSKIDPTTGFTNLFNTKKCTNAEGRKVFFEAMN SIIYDGSRKSFAFSFDYGNPVFRASQTSFKKEWTVYSADTRIVYNRGEKTVNTIHPTQI LHDALCALGIDVHDGLNVLNVVRETPADKIHAKFFSDLFYAFDRTLQMRNSVSGTDE DYIQSPVLNATGEFFDSRKADSTLPQDADANGAYHIALKGLLLLQRMKDIGSDIKLDLS IKHEDWFAFAQKRCQR 80 Expression construct (with N-terminal methionine, V5-tag and C-terminal NLS) aa sequence MGSGSFESFTNVYPVSKTLRFELRPVGATAEKLKESGILEHDTKRGKEYATLKDLLDE QHKELLADALKPERVKNALKPNSGKSKKDKLVEENYITEDGEIRWETLAAAMEAFRA GEVEKNVLEAIQTQFRKLIVTILKADERYPGLTASTPSAVIKTLLKQDVHPEAVETFAKF ACYFTGFQENRKNIYAEEKQATAVATRVVHDNFAKFHTQSKIIGVIKNKYPEILQSVEM ELMDELGGMKITDIFSINSYSKWMTQEGIDFINKIIGGYSPSVGVKVRGLNEFINLYRQ QHEEANADRRNLAKMPMLFKQILSDISTRSFIPVMFENDAELKDSIEAFLTGLNDFELN AQKFNVVVALGNLFQKIVPCEGIFLDAALMEKVSKTATGDWSLLAQSMEAYAETAFT RAKDRDAWLRKNYYSLSELSQVPILKNTDEGMLKFELSAYWSGEKMESFVKGIMDA ELAMKPVLASIGQKTEEVRLRDRIDDVVKIKGYLDSIQNFLHHLKPFCAPTELNRDADF YSDFDALYNQLVLVIPLYNCVRNYVTQKVTEVQKLRLKFDAPTLADGWDANKENDNK AVLFEKDGLYFLGILNPNLKAKDRPVFEHESNVTKKSCYRKIVYKLLPGPNKMLPKVF FADSNRTLYHPSKSLLDRYHNGEYKKGDSFDIKFCHELIDYFKASISIHPDWKEFGFQ FSATKTYESIDGFYREVEEQGYKVNFAFVRADLIDKYVESGSLFLFQLYNKDFSCASS GKPNLHTLYWKSLFAKENLDEPILKLCGGAELFFRPVAIQKPYVHTLGEKLVNRRLGE HGKGEAIPERVHKELVDYYNHRVSVLSHDGKAFKDKVVVRDVAHSITKDRRFSEAKF FFHVPIMFNRTASKSAKFNDKVVDYLKTTQNVNVIGLDRGERNLIYLTMVNLHGKLIE QRSFNLVNGVDYHSKLDLREKERMDARVNWENIGGIKDLKTGYLSAVVHEIAKMMVT 81 TABLE1N ZTAE Type V Cas Sequences Name Sequence SEQ ID NO. NNAIVVLEDLNFGFKRGRFKVEKQVYQKFEKMLIDKLNFLMFKECNQAALGGVRRAY QLTDKFVSFEKLGKQTGFLFYVPAGYTSKIDPTTGFTNLFNTKKCTNAEGRKVFFEAM NSIIYDGSRKSFAFSFDYGNPVFRASQTSFKKEWTVYSADTRIVYNRGEKTVNTIHPT QILHDALCALGIDVHDGLNVLNVVRETPADKIHAKFFSDLFYAFDRTLQMRNSVSGTD EDYIQSPVLNATGEFFDSRKADSTLPQDADANGAYHIALKGLLLLQRMKDIGSDIKLDL SIKHEDWFAFAQKRCQRSRKRTADGSEFESPKKKRKVGSGKPIPNPLLGLDST Wildtype coding sequence (with N-terminal methionine and stop codon) ATGAG Illi GAA rCATTCACTAACGTTTATCCCGTTTCCAAGACTTTGCGCTTTGA GCTGAGGCCCGTTGGTGCAACTGCAGAGAAGCTTAAGGAAAGTGGTATCCTTGA GCATGATACGAAACGAGGTAAGGAATATGCGACTCTCAAGGATCTGCTTGATGAG CAACATAAGGAGTTACTTGCTGACGCCCTAAAACCTGAACGTGTGAAGAATGCGC TTAAGCCCAATAGTGGTAAGAGTAAAAAAGATAAATTGGTTGAAGAGAATTACATT ACGGAAGACGGGGAGATTCGATGGGAAACTCTTGCGGCTGCGATGGAGGCATTT CGCGCCGGTGAGGTAGAGAAAAATGTGCTTGAAGCAATACAGACGCAATTTAGA AAGCTGATTGTAACGATACTGAAGGCGGATGAGCGGTATCCGGGACTGACAGCT TCAACGCCTTCGGCTGTCATTAAGACTCTTCTTAAGCAGGATGTTCATCCAGAAG CAGTAGAGACATTTGCAAAATTTGCCTGTTA Illi ACCGG Illi CAGGAAAATCGG AAGAATATCTATGCGGAAGAAAAGCAAGCAACTGCAGTTGCAACGCGAGTTGTTC ATGATAATTTCGCAAAGTTCCATACACAATCGAAAATAATAGGTGTCATAAAGAAT AAATATCCAGAAATCCTTCAGTCGGTAGAAATGGAATTGATGGACGAATTAGGTG GGATGAAAATCACTGATATC Illi CTATCAACAGCTATTCCAAATGGATGACGCAA GAAGGGATAGACTTTATTAATAAGATTATAGGTGGCTATAGCCCATCTGTTGGTGT GAAGGTGCGTGGTCTGAACGAGTTCATTAATCTTTATCGGCAGCAGCATGAAGAG GCAAATGCAGATCGGCGGAATCTCGCAAAAATGCCGATGCTGTTTAAACAAATTT TAAGTGATATTTCGACACGATCATTCATTCCGGTGATGTTTGAAAATGATGCGGAA CTAAAGGATTCAATAGAAGCATTCTTGACAGGTCTGAATGA Illi GAGTTGAATGC TCAGAAGTTTAACGTTGTCGTTGCATTAGGTAATC Illi CCAAAAAATTGTGCCTT GCGAAGGTA Illi CTTGGATGCAGCATTGATGGAAAAAGTTTCGAAGACGGCTAC AGGAGATTGGAGTCTTCTTGCTCAGTCGATGGAGGCGTATGCAGAGACAGCATT CACAAGAGCAAAAGACCGAGACGCATGGCTAAGGAAAAATTATTATTCGCTGTCC GAGCTGAGCCAAGTTCCGA Illi GAAGAACACTGATGAAGGAATGTTGAAGTTTG AACTATCTGCCTATTGGTCAGGCGAAAAGATGGAAAG Illi GTTAAAGGAATCAT GGATGCTGAATTGGCAATGAAACCAGTTCTTGCCAGCATTGGTCAGAAAACCGAA GAGGTGCGTCTTCGTGATCGGATTGACGATGTCGTAAAAATCAAGGGATATCTTG ATTCAATTCAGAAI Illi I AOA ICACC IAAAACCG I I I I GTGCTCCAACTGAATTGA ATCGTGATGCGGATTTTTATTCTGACTTTGACGCATTGTATAATCAGCTTGTACTG GTTATACCGCTTTATAACTGTGTCCGCAATTACGTGACACAGAAAGTGACAGAGG TTCAGAAACTGAGGCTAAAGTTTGATGCCCCTACATTGGCGGACGGATGGGACG CGAATAAAGAAAATGATAATAAGGCAGTTCTGTTTGAAAAGGACGGGCTATA Illi CTTGGAATCCTGAATCCTAACCTGAAGGCGAAAGATCGTCCAGTCTTTGAGCATG AAAGTAATGTTACAAAGAAATCTTGTTATCGCAAGATTGTCTATAAACTTTTGCCA GGACCAAATAAAATGCTTCCCAAGGTCTTTTTTGCTGATTCCAATAGGACACTGTA CCATCCTTCCAAGTCGTTGCTGGATCGTTATCACAACGGTGAATACAAGAAAGGC GATTCATTCGACATCAAATTCTGTCATGAATTGATTGATTAI I I IAAAGCCTCGATT AGTATTCACCCCGATTGGAAGGAATTCGGTTTCCAATTCAGTGCGACAAAAACAT ATGAGAGCATTGATGGTTTTTATCGTGAGGTTGAGGAGCAAGGATATAAAGTTAA I I I I GO I I I I GTAAGGGCGGATTTAATTGACAAATATGTGGAAAGTGG...

Claims

1. A Type V Cas protein comprising an amino acid sequence having at least 90% sequence identity to:(a)     the amino acid sequence of a WED-I domain of a reference protein sequence;(b)     the amino acid sequence of a REC1 domain of a reference protein sequence;(c)     the amino acid sequence of a REC2 domain of a reference protein sequence;(d)     the amino acid sequence of a WED-II domain of a reference protein sequence;(e)     the amino acid sequence of a PI domain of a reference protein sequence;(f)     the amino acid sequence of a WED-HI domain of a reference protein sequence;(g)     the amino acid sequence of a RuvC-l domain of a reference protein sequence;(h)     the amino acid sequence of a BH domain of a reference protein sequence;(i)      the amino acid sequence of a RuvC-ll domain of a reference protein sequence;(j)     the amino acid sequence of a NUC domain of a reference protein sequence;(k)     the amino acid sequence of a RuvC-lll domain of a reference protein sequence;or(I) the amino acid sequence of the full length of a reference protein sequence; wherein the reference protein sequence is SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NQ:20, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:49, SEQ ID NQ:50, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:79, SEQ ID NQ:80, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NQ:103, SEQ ID NQ:104, SEQ ID NQ:109, SEQ ID NQ:110, SEQ ID NO:115, or SEQ ID NO:116.

2. The Type V Cas protein of claim 1, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, or is identical to the full length of the reference protein sequence.

3. The Type V Cas protein of claim 1, wherein the amino acid sequence of the Type V Cas protein comprises an amino acid sequence that is identical to the full length of the reference protein sequence.

4. The Type V Cas protein of any one of claims 1 to 3, which is a fusion protein.

5. The Type V Cas protein of claim 4, which comprises one or more nuclear localizationsignals, and which optionally comprises an N-terminal nuclear localization signal and / or a C-terminal nuclear localization signal.

6. The Type V Cas protein of claim 5, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence KRTADGSEFESPKKKRKV (SEQ ID NO:122), PKKKRKV (SEQ ID NO:123), PKKKRRV (SEQ ID NO:124), KRPAATKKAGQAKKKK (SEQ IDNO:125), YGRKKRRQRRR (SEQ ID NO:126), RKKRRQRRR (SEQ ID NO:127), PAAKRVKLD (SEQ ID NO:128), RQRRNELKRSP (SEQ ID NO:129), VSRKRPRP (SEQ ID NQ:130), PPKKARED (SEQ ID N0:131), PQPKKKPL (SEQ ID NO:132), SALIKKKKKMAP (SEQ ID NO:133), PKQKKRK (SEQ ID NO:134), RKLKKKIKKL (SEQ ID NO:135), REKKKFLKRR (SEQ ID NO:136), KRKGDEVDGVDEVAKKKSKK (SEQ ID NO:137), RKCLQAGMNLEARKTKK (SEQ ID NO:138), NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO:139), RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO:140), or SSDDEATADSQHAAPPKKKRKV (SEQ ID NO:178).

7. The Type V Cas protein of claim 5, wherein the amino acid sequence of one or more of the nuclear localization signals comprises the amino acid sequence GRSSDDEATADSQHAAPPKKKRKV (SEQ ID NO:180).

8. The Type V Cas protein of any one of claims 5 to 7, comprising a C-terminal nuclear localization signal.

9. The Type V Cas protein of any one of claims 4 to 8, which comprises a fusion partner which is a DNA, RNA or protein modification enzyme, optionally wherein the DNA, RNA or protein modification enzyme is an adenosine deaminase, a cytidine deaminase, a reverse transcriptase, a guanosyl transferase, a DNA methyltransferase, a RNA methyltransferase, a DNA demethylase, a RNA demethylase, a dioxygenase, a polyadenylate polymerase, a pseudouridine synthase, an acetyltransferase, a deacetylase, a ubiquitin-ligase, a deubiquitinase, a kinase, a phosphatase, a NEDD8-ligase, a de-NEDDylase, a SUMO-ligase, a deSUMOylase, a histone deacetylase, a histone acetyltransferase, a histone methyltransferase, or a histone demethylase.

10. The Type V Cas protein of any one of claims 4 to 9, which comprises (a) a fusion partner which is an adenosine deaminase, optionally wherein the amino acid sequence of the adenosine deaminase comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO:143, optionally wherein the adenosine deaminase is the adenosine deaminase moiety contained in the adenine base editor ABE8e; (b) a fusion partner which is a cytodine deaminase; or (c) a fusion partner which is a reverse transcriptase.

11. The Type V Cas protein of any one of claims 4 to 10, which comprises a tag, e.g., a SV5 tag, optionally wherein the SV5 tag comprises the amino acid sequence GKPIPNPLLGLDST (SEQ ID NO:141).

12. A Type V Cas protein whose amino acid sequence is identical to a Type V Cas protein of any one of claims 1 to 11 except for one or more amino acid substitutions relative to the reference sequence that provide nickase activity, optionally wherein the one or more amino acid substitutions comprise a substiution (e.g., alanine substitution) at a position corresponding to position D908 of Cas12a, E993 of Cas12a, R1226 of Cas12a, or D1263 of Cas12a (e.g., as shown in Table 5), or a combination thereof.

13. A guide RNA (gRNA) molecule:(a) for editing a human B2Mgene comprising a spacer whose nucleotide sequence comprises 15 or more consecutive nucleotides of a reference sequence or comprises a nucleotide sequence that is at least 85% identical to the reference sequence, wherein the reference sequence is selected from SEQ ID NOs: 164-168;(b) for editing a human TRAC gene comprising a spacer whose nucleotide sequence comprises 15 or more consecutive nucleotides of a reference sequence or comprises a nucleotide sequence that is at least 85% identical to the reference sequence, wherein the reference sequence is selected from SEQ ID NOs: 169-173;(c) for editing a human PD1 gene comprising a spacer whose nucleotide sequence comprises 15 or more consecutive nucleotides of a reference sequence or comprises a nucleotide sequence that is at least 85% identical to the reference sequence, wherein the reference sequence is selected from SEQ ID NOs:174-177;(d) for editing a human B2Mgene comprising a spacer whose nucleotide sequence comprises 15 or more consecutive nucleotides of a reference sequence or comprises a nucleotide sequence that is at least 85% identical to the reference sequence, wherein the reference sequence is selected from SEQ ID NOs: 181-183;(e) for editing a human TRAC gene comprising a spacer whose nucleotide sequence comprises 15 or more consecutive nucleotides of a reference sequence or comprises a nucleotide sequence that is at least 85% identical to the reference sequence, wherein the reference sequence is selected from SEQ ID NOs:184-192;(f) for editing a human AAVS1 genomic sequence comprising a spacer whose nucleotide sequence comprises 15 or more consecutive nucleotides of a reference sequence or comprises a nucleotide sequence that is at least 85% identical to the reference sequence, wherein the reference sequence is selected from SEQ ID NOs:193-196;(g) for editing a human EMX1 genomic sequence comprising a spacer whose nucleotide sequence comprises 15 or more consecutive nucleotides of a reference sequence or comprises a nucleotide sequence that is at least 85% identical to the reference sequence, wherein the reference sequence is selected from SEQ ID NOs:197-198;(h) for editing a human BCL11A genomic sequence comprising a spacer whose nucleotide sequence comprises 15 or more consecutive nucleotides of a reference sequence or comprises a nucleotide sequence that is at least 85% identical to the reference sequence, wherein the reference sequence is selected from SEQ ID NOs:199-202;(i) for editing a human PCSK9 genomic sequence comprising a spacer whose nucleotide sequence comprises 15 or more consecutive nucleotides of a reference sequence or -212-comprises a nucleotide sequence that is at least 85% identical to the reference sequence, wherein the reference sequence is selected from SEQ ID N0s:203-204; or(j) for editing a human VEGF genomic sequence comprising a spacer whose nucleotide sequence comprises 15 or more consecutive nucleotides of a reference sequence or comprises a nucleotide sequence that is at least 85% identical to the reference sequence, wherein the reference sequence is SEQ ID NO:205.

14. A gRNA comprising a spacer and a crRNA scaffold, wherein:(a) the spacer is positioned 3’ to the crRNA scaffold; and(b) the nucleotide sequence of the crRNA scaffold comprises a nucleotide sequence that is at least 50% identical to a reference scaffold sequence, wherein the reference scaffold sequence is selected from SEQ ID NOS:211-213.

15. A gRNA comprising a spacer and a crRNA scaffold, wherein:(a) the spacer is positioned 3’ to the crRNA scaffold; and(b) the nucleotide sequence of the crRNA scaffold comprises a nucleotide sequence that is at least 50% identical to a reference scaffold sequence, wherein the reference scaffold sequence is selected from SEQ ID NOS:144-163.

16. The gRNA of any one of claims 14 to 15, which comprises a spacer that is 15 to 30 nucleotides in length, 18 to 30 nucleotides in length, 20 to 28 nucleotides in length, 22 to 26 nucleotides in length, 23 to 25 nucleotides in length, 22 to 25 nucleotides in length, 15 to 25 nucleotides in length, 16 to 24 nucleotides in length, 17 to 23 nucleotides in length, 18 to 22 nucleotides in length, 19 to 21 nucleotides in length, 25 nucleotides in length, 24 nucleotides in length, 23 nucleotides in length, 22 nucleotides in length, 21 nucleotides in length, or 20 nucleotides in length.

17. A system comprising the Type V Cas protein of any one of claims 1 to 12 and a guide RNA (gRNA) comprising a spacer sequence, optionally wherein the gRNA is a gRNA according to any one of claims 13 to 16.

18. A nucleic acid encoding the Type V Cas protein of any one of claims 1 to 12, optionally wherein the nucleotide sequence encoding the Type V Cas protein is operably linked to a promoter that is heterologous to the Type V Cas protein.

19. The nucleic acid of claim 18, wherein the nucleotide sequence encoding the Type V Cas protein is codon optimized for expression in human cells.

20. The nucleic acid of claim 18 or claim 19, which is a plasmid or a viral genome, optionally an adeno-associated virus (AAV) genome, e.g., an AAV2, AAV5, AAV7m8, AAV8, AAV9, AAVrh8r, or AAVrhIO genome.

21. The nucleic acid of any one of claims 18 to 20, further encoding a gRNA22.    A nucleic acid encoding the gRNA of any one of claims 13 to 16.

23. A nucleic acid encoding the Type V Cas protein and gRNA of the system of claim 17.

24. A plurality of nucleic acids comprising separate nucleic acids encoding the Type V Cas protein and gRNA of the system of claim 17.

25. A particle comprising a Type V Cas protein according to any one of claims 1 to 12, a gRNA according to any one of claims 13 to 16, a system according to claim 17, a nucleic acid according to any one of claims 18 to 23, or a plurality of nucleic acids according to claim 24.

26. A pharmaceutical composition comprising a Type V Cas protein according to any one of claims 1 to 12, a gRNA according to any one of claims 13 to 16, a system according to claim 17, a nucleic acid according to any one of claims 18 to 23, a plurality of nucleic acids according to claim 24, or a particle according to claim 25 and at least one pharmaceutically acceptable excipient.

27. A Type V Cas protein according to any one of claims 1 to 12, a gRNA according to any one of claims 13 to 16, a system according to claim 17, a nucleic acid according to any one of claims 18 to 23, a plurality of nucleic acids according to claim 24, a particle according to claim 25, or a pharmaceutical composition according to claim 26 for use in a method of editing a human genomic sequence.

28. The Type V Cas protein, gRNA, system, nucleic acid, a plurality of nucleic acids, particle, or pharmaceutical composition for use according to claim 27, wherein the human genomic sequence is a TRAC, B2M, or PD1, genomic sequence, optionally wherein the human genomic sequence is in a T cell.

29. An ex vivo human cell comprising a Type V Cas protein according to any one of claims 1 to 12, a gRNA according to any one of claims 13 to 16, a system according to claim 17, a nucleic acid according to any one of claims 18 to 23, a plurality of nucleic acids according to claim 24, or a particle according to claim 25.

30. A Type V Cas protein according to any one of claims 1 to 12, the gRNA according to any one of claims 13 to 16, or the system of claim 17 for use in a nucleic acid detection assay.

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