Cas12a nickase proteins and uses thereof

Abstract

The present disclosure relates to novel Cas12a nickase proteins. Systems and methods of their use are also disclosed.

Description

CAS12A NICKASE PROTEINS AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims to the benefit of U.S. Provisional Application No. 63 / 600,545, filed November 17, 2023, the entirety of which is incorporated herein by reference. BACKGROUND

[0002] Type V CRISPR / Cas12a effector proteins (also referred to as Cpf1 effector proteins) have been described as an alternative to Type II CRISPR / Cas9 effector proteins for genome editing applications (Zetsche et al., Cell 163:759-771 (2015); Shmakov et al., Mol Cell 60(3):385-97 (2015); Kleinstiver et al., Nat Biotechnol 34 (8):869-74 (2016); Kim et al., Nat Biotechnol 34(8):863-8 (2016)). Cas12a effector proteins possess a number of potentially advantageous properties that include, but are not limited to: recognition of T-rich protospacer-adjacent motif (PAM) sequences, greater genome-wide specificity in human cells compared to, e.g., the prototypical Streptococcus pyogenes Cas9 (SpCas9), endoribonuclease activity that processes pre-crRNAs and simplifies the simultaneous targeting of multiple sites (multiplexing), staggered cutting activity that generates a 5’ DNA overhang (rather than a blunt double-strand break as observed with SpCas9), development of a 5’ pseudoknot, and cleavage of the protospacer DNA sequence on the end most distal from the PAM (compared with cleavage at the PAM proximal end of the protospacer as is observed with Cas9 effector proteins). While Cas12a effector proteins provide a useful alternative to Cas9 effector proteins, there is still a need for novel Cas12a effector proteins, in particular Cas12a nickase proteins. SUMMARY

[0003] The present disclosure provides strategies, systems, compositions, and methods related to novel Cas12a effector proteins for altering a cell, e.g., altering a structure, e.g., altering a sequence, of a target nucleic acid of a cell. For example, in some embodiments, the present disclosure provides novel Cas12a effector proteins that introduce single-strand breaks (or “nicks”) in a non-target nucleic acid sequence (i.e., PAM containing strand) whereas nicks of the target nucleic acid sequence is attenuated. The present disclosure provides, inter alia, Cas12a effector proteins comprising mutations. In some embodiments, the present disclosure provides for engineered Cas12a effector proteins thatcomprise mutations at one or more target strand cleavage attenuation residues. In some embodiments, Cas12a effector proteins comprise mutations within the alpha helical lid domain (or the core lid domain). In some embodiments, Cas12a effector proteins comprise mutations within the bridge helix domain. In some embodiments, Cas12a effector proteins comprise mutations within the RuvC domain. In some embodiments, Cas12a effector proteins comprise mutations within the REC lobe domain. In some embodiments, Cas12a effector proteins comprise mutations within the PAM-interacting (PI) domain. In some embodiments, a Cas12a effector protein is a Cas12a nickase effector protein comprising 1, 2, 3, 4, 5, 6, 7, or 8 of the amino acid substitutions corresponding to positions M537, H800, F870, W958, F999, K1000, K1002, and R1003 in AsCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase effector protein comprising 1, 2, 3, 4, 5, 6, 7, or 8 of the amino acid substitutions corresponding to M537R, H800A, F870L, W958A, F999G, K1000G, K1002G, and R1003G in AsCas12a.

[0004] Furthermore, the present disclosure provides for engineered Cas12a effector proteins and systems that may further comprise guide RNA molecules comprising a spacer region that is at least 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 nucleotides in length (also referred to herein interchangeably as an “extended spacer region”). In some embodiments, the present disclosure describes that there may be a synergistic effect between the use of the Cas12a effector proteins disclosed herein with guide RNAs comprising an extended spacer region toward increased nicking specificity of the non-target strand over the target strand.

[0005] Among other things, the present disclosure also provides strategies, systems, compositions, and methods related to engineered Cas12a effector proteins and variants thereof that are fused to one or more heterologous protein domains (or “fusion proteins”). The present disclosure provides Cas12a nickase proteins comprising mutations within the core lid and bridge helix regions of the Cas12a effector protein. For example, in some embodiments, Cas12a effector proteins may be fused to one or more heterologous protein domains such as a deaminase or catalytic domain for base editing. As another example, in some embodiments, Cas12a effector proteins may be fused to one or more heterologous protein domains such as a polymerase domain for prime editing. The fusion proteins provided herein exhibit increased activity(ies) compared to fusion proteins known in the art.

[0006] The disclosed Cas12a effector proteins, and related strategies, systems, compositions, and methods, present several advantages compared to other Cas12a effector proteins known in the art. For example, in some embodiments, the described Cas12a effectorproteins, and related strategies, systems, compositions, and method, create a single strand break in the non-target strand with higher nicking specificity compared to other Cas12a effector proteins known in the art. Use of these systems and compositions disclosed herein provides for the incorporation of small DNA changes afforded to by non-target strand nicking activity. Thus, in some embodiments, the described Cas12a effector proteins, and related strategies, systems, compositions, and methods, alter the genomes of a plurality of cells at a higher rate compared to other Cas12a effector proteins known in the art. BRIEF DESCRIPTION OF THE DRAWING

[0007] The teachings described herein will be more fully understood from the following description of various exemplary embodiments, when read together with the accompanying drawing. It should be understood that the drawing described below is for illustration purposes only and is not intended to limit the scope of the present teachings in any way.

[0008] Figure 1A shows a graph of insertion / deletion (“INDEL”) rate (%) of an exemplary target gene (Exemplary Target Gene 1) by exemplary Cas12a effector proteins described herein.

[0009] Figure 1B shows a graph of insertion / deletion (“INDEL”) rate (%) of an exemplary target gene (Exemplary Target Gene 1) by exemplary Cas12a effector proteins described herein.

[0010] Figures 2A-2B show gel analysis (Figure 2B) of the cleavage activity of a match-site substrate (Figure 2A) by exemplary AsCas12a effector proteins described herein.

[0011] Figure 3A demonstrates cleavage activity over time of either a target strand (“TS”) or non-target strand (“NTS”) with an AsCas12a effector protein comprising M537R, H800A, and F870L (AsCas12a-MHF) mutations.

[0012] Figure 3B demonstrates cleavage activity over time of either a target strand (“TS”) or non-target strand (“NTS”) with an AsCas12a effector protein comprising M537R, H800A, F870L, and W958A (AsCas12a-MHFW) mutations.

[0013] Figure 3C demonstrates cleavage activity over time of either a target strand (“TS”) or non-target strand (“NTS”) with an AsCas12a effector protein comprising M537R, H800A, F870L, and R1226A (AsCas12a-MHFR) mutations.

[0014] Figure 3D demonstrates cleavage activity over time of either a target strand (“TS”) or non-target strand (“NTS”) with an AsCas12a effector protein comprising M537R, H800A, F870L, K1000G, and K1002G (AsCas12a-MHFKK) mutations.

[0015] Figure 3E demonstrates cleavage activity over time of either a target strand (“TS”) or non-target strand (“NTS”) with an AsCas12a effector protein comprising M537R, H800A, F870L, F999G, K1000G, K1002G, and R1003G (AsCas12a-MHFFKKR) mutations.

[0016] Figure 3F demonstrates cleavage activity over time of a target strand (“TS”) or non-target strand (“NTS”) with an AsCas12a effector protein comprising M537R, H800A, F870L, W958A, F999G, K1000G, K1002G, and R1003G (AsCas12a-MHFWFKKR) mutations.

[0017] Figure 4A shows a graph of insertion / deletion (“INDEL”) rate (%) of an exemplary target gene (Exemplary Target Gene 1) using guide RNAs with different spacer lengths and either AsCas12a effector protein with M537R, H800A, and F870L mutations (AsCas12a-MHF) or AsCas12a with M537R, H800A, F870L, and W958A mutations (AsCas12a-MHFW).

[0018] Figure 4B shows a graph of insertion / deletion (“INDEL”) rate (%) of an exemplary target gene (Exemplary Target Gene 2) using guide RNAs with different spacer lengths and either AsCas12a effector protein with M537R, H800A, and F870L mutations (AsCas12a-MHF) or AsCas12a with M537R, H800A, F870L, and W958A mutations (AsCas12a-MHFW).

[0019] Figure 5A demonstrates cleavage activity over time of either a target strand (“TS”) or non-target strand (“NTS”) via an AsCas12a effector protein comprising M537R, H800A, F870L, and W958A (AsCas12a-MHFW) mutations complexed with a guide RNA having a spacer region that is 26 nucleotides in length.

[0020] Figure 5B demonstrates cleavage activity over time of either a target strand (“TS”) or non-target strand (“NTS”) via an AsCas12a effector protein comprising M537R, H800A, F870L, F999G, K1000G, K1002G, and R1003G (AsCas12a-MHFFKKR) mutations complexed with a guide RNA having a spacer region that is 26 nucleotides in length.

[0021] Figure 5C demonstrates cleavage activity over time either a target strand (“TS”) or non-target strand (“NTS”) via an AsCas12a effector protein comprising M537R, H800A, F870L, W958A, F999G, K1000G, K1002G, and R1003G (AsCas12a-MHFWFKKR)mutations complexed with a guide RNA having a spacer region that is 26 nucleotides in length. DETAILED DESCRIPTION Definitions and Abbreviations

[0022] Unless otherwise specified, each of the following terms have the meaning set forth in this section.

[0023] The indefinite articles “a” and “an” refer to at least one of the associated noun and are used interchangeably with the terms “at least one” and “one or more.” The conjunctions “or” and “and / or” are used interchangeably as non-exclusive disjunctions.

[0024] The terms “CRISPR / Cas effector protein”, “Cas effector protein”, and “Cas protein” are generally used interchangeably and at all points of reference herein refer by analogy to new CRISPR / Cas effector proteins further described in this application, unless otherwise apparent, such as by specific reference to Cas12a or Cpf1. In some embodiments, a Cas effector protein is part of a fusion protein comprising one or more heterologous protein domains (e.g., about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains in addition to the Cas effector protein). In some embodiments, one or more heterologous protein domains comprises a reverse transcriptase domain. In some embodiments, a Cas effector protein is a nuclease. In some embodiments, a Cas effector protein is a nickase. In some embodiments, a Cas effector protein is engineered (e.g., made by hand of man). In some embodiments, a Cas effector protein is a variant Cas effector protein.

[0025] The terms “CRISPR / Cas nuclease” or “Cas nuclease” as used herein refer to any Cas effector protein with DNA nuclease activity, e.g., a Cas12a nuclease protein that exhibits specific association (or “targeting”) to a DNA target site, e.g., within a genomic sequence in a cell in the presence of a guide molecule. The strategies, systems, and methods disclosed herein can use any combination of CRISPR / Cas nuclease disclosed herein.

[0026] The terms “CRISPR / Cas nickase” or “Cas nickase” as used herein refer to any Cas effector protein with DNA nickase activity, e.g., a Cas12a nickase protein that exhibits specific association (or “targeting”) to a DNA target site, e.g., within a genomic sequence in a cell in the presence of a guide molecule. In some embodiments, a Cas nickase exhibits specific association and / or cleavage of the target strand (also interchangeably referred toherein as “the non-PAM containing strand”). In some embodiments, a Cas nickase exhibits specific association and / or cleavage of the non-target strand (also interchangeably referred to herein as “the PAM containing strand”). In some embodiments, a Cas nickase refers to a Cas nuclease that exhibits at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% nickase activity. The strategies, systems, and methods disclosed herein can use any combination of Cas nickases disclosed herein.

[0027] The terms “alpha helical lid domain” or “core lid domain” as used herein refer to a region that resides within the RuvC nuclease domain of a Cas12 effector protein. As known by those skilled in the art, the alpha helical lid domain both plays a role in anchoring the formation of the CRISPR RNA (crRNA)-target strand hybrid complex (also interchangeably referred to herein as the “crRNA:TS hybrid complex”), and occlusion of the RuvC nuclease domain in Cas12 effector proteins, particularly of importance within Cas12a proteins. It is also an insight of the present disclosure that the alpha helical lid domain plays a role in attenuation (or reduction) of target strand cleavage as described herein. In some embodiments, a Cas12a nickase comprises one or more mutations within the RuvC domain. In some embodiments, a Cas12a nickase comprises one or more mutations within the alpha helical lid domain. In some such embodiments, a Cas12a nickase further comprises one or more mutations within other domains adjacent or not adjacent to the RuvC domain. In some embodiments, a Cas12a nickase comprises one or more mutations not in its alpha helical lid domain. In some such embodiments, a Cas12a nickase comprising said mutation(s) exhibits increased nicking specificity and / or nicking activity of the non-target strand when compared to Cas12a effector proteins known in the art. In some embodiments, the alpha helical lid domain refers to positions 995-1008 of SEQ ID NO: 11. In some embodiments, the alpha helical lid domain comprises at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO: 1. In some embodiments, the alpha helical lid domain comprises a sequence according to SEQ ID NO: 1. In some embodiments, the alpha helical lid domain refers to positions 1006-1018 of SEQ ID NO: 62. In some embodiments, the alpha helical lid domain comprises at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94%identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO: 2. In some embodiments, the alpha helical lid domain comprises a sequence according to SEQ ID NO: 2. In some embodiments, the alpha helical lid domain refers to positions 925-937 of SEQ ID NO: 71. In some embodiments, the alpha helical lid domain comprises at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO: 3. In some embodiments, the alpha helical lid domain comprises a sequence according to SEQ ID NO: 3. SEQ ID NO: 1 - Exemplary AsCas12a alpha helical lid domain sequence LNFGFKSKRTGIAE SEQ ID NO: 2 - Exemplary FnCas12a alpha helical lid domain sequence EDLNFGFKRGRFK SEQ ID NO: 3 - Exemplary LbCas12a alpha helical lid domain sequence EDLNSGFKNSRVK

[0028] The term “bridge helix” as used herein refers to a region that resides within the RuvC nuclease domain of a Cas12 effector protein. As known by those skilled in the art, the bridge helix domain is located between the RuvC-I and RuvC-II motifs. The RuvC-I and RuvC-II motifs are located between the REC and NUC lobes of a Cas12 effector protein, e.g., a Cas12a effector protein. In some embodiments, a Cas12a effector protein comprises one or more mutations within the RuvC domain. In some embodiments, a Cas12a nickase comprises one or more mutations within the RuvC domain. In some embodiments, a Cas12a nickase comprises one or more mutations in its bridge helix. In some embodiments, a Cas12a nickase comprises one or more mutations just outside of its bridge helix. In some embodiments, the bridge helix refers to positions 940-958 of SEQ ID NO: 11. In some embodiments, the bridge helix comprises at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, and or at least 100% identity to SEQ ID NO: 4. In some embodiments, the bridge helix comprises a polypeptide sequence according to SEQ ID NO: 4. In some embodiments, the bridge helix comprises a sequence according to SEQ ID NO: 4.In some other embodiments, the bridge helix refers to positions 953-971 of SEQ ID NO: 62. In some embodiments, the bridge helix comprises at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO: 5. In some embodiments, the bridge helix comprises a sequence according to SEQ ID NO: 5. In some embodiments, the bridge helix refers to positions 872-890 of SEQ ID NO: 71. In some embodiments, the bridge helix comprises at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO: 6. In some embodiments, the bridge helix comprises a sequence according to SEQ ID NO: 6. SEQ ID NO: 4 - Exemplary AsCas12a bridge helix sequence YQKKLDNREKERVAARQAW SEQ ID NO: 5 - Exemplary FnCas12a bridge helix sequence YHDKLAAIEKDRDSARKDW SEQ ID NO: 6 - Exemplary LbCas12a bridge helix sequence YHSLLDKKEKERFEARQNW

[0029] The term “correspond to” may be used to designate the position or identity of a structural element in a compound or composition through comparison with an appropriate reference compound or composition. For example, in some embodiments, a Cas12a effector protein, polypeptide, or polynucleotide encoding thereof (e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide) may be identified as “corresponding to” a residue in an appropriate reference Cas12a effector protein, polypeptide, or polynucleotide encoding thereof. For example, those of skill in the art appreciate that residues in a provided polypeptide or polynucleotide sequence are often designated (e.g., numbered or labeled) according to the scheme of a related reference sequence (even if, e.g., such designation does not reflect literal numbering of the provided Cas12a effector protein sequence). By way of illustration, if a reference sequence includes a particular amino acid motif at positions 999-1003, and a second related sequence includes the same motif at positions 971-1018, the motif positions of the second related sequence can be said to “correspond to” positions 999-1003 of the reference sequence. For example, positions 537,800, 870, 958, 999, 1000, 1002, and 1003 of AsCas12a may correspond to positions 602, 843, 879, 971, 1012, 1013, 1016, 1018 of FnCas12a. Furthermore, for example, positions 537, 800, 870, 958, 999, 1000, 1002, and 1003 of AsCas12a may correspond to positions 527, 759, 795, 890, 931, 932, 935, and 937 of LbCas12a. Those of skill in the art appreciate that corresponding positions can be readily identified, e.g., by alignment of sequences, and that such alignment is commonly accomplished by any of a variety of known tools, strategies, and / or algorithms, including without limitation software programs such as, for example, BLAST, CS-BLAST, CUDASW++, DIAMOND, FASTA, GGSEARCH / GLSEARCH, Genoogle, HMMER, Hhpred / Hhsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI- BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHI-LS, SWIMM, or SWIPE. Two sequences can be identified as corresponding if they are identical or if they share substantial identity (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity) over a length of (e.g., at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500 or more) units (e.g., nucleotides or amino acids).

[0030] The term “endogenous,” as used herein in the context of nucleic acids refers to a native nucleic acid (e.g., a gene, a protein coding sequence) in its natural location, e.g., within the genome of a cell.

[0031] The term “exogenous,” as used herein in the context of nucleic acids refers to a nucleic acid (whether native or non-native) that has been artificially introduced into a man- made construct (e.g., a knock-in cassette, or a donor template) or into the genome of a cell using, for example, gene editing or genetic engineering techniques, e.g., HDR based integration techniques.

[0032] The term “fuse” or “fused” refers to the covalent linkage between two polypeptides in a fusion protein. The polypeptides may be fused via a peptide bond, either directly to each other or via a linker. The term “fusion protein” refers to a protein having at least two polypeptides covalently linked, either directly or via a linker (e.g., an amino acid linker). The polypeptides forming a fusion protein may be linked C-terminus to N-terminus, C-terminus to C-terminus, N-terminus to N-terminus, or N-terminus to C-terminus. The polypeptides of the fusion protein may be in any order and may include more than one of either or both of the constituent polypeptides. In some embodiments, a fusion protein may be a protein developed from a fusion gene that is created through adjoining of two or more genesoriginally coding for separate proteins. In some embodiments, a fusion protein comprises a Cas effector protein. In some embodiments, a fusion protein comprises a Cas effector protein and a polymerase. In some embodiments, a fusion protein comprises a Cas effector protein and a reverse transcriptase. In some embodiments, a fusion protein comprises a Cas effector protein and a DNA polymerase. In some embodiments, a fusion protein comprises a Cas effector protein and a deaminase. Translation of this fusion gene may result in a single or multiple polypeptides with functional properties derived from each of the original proteins.

[0033] The term “guide molecule” or “guide RNA” or “gRNA” or “gRNA molecule” when used in reference to a CRISPR / Cas effector system is any nucleic acid that promotes the specific association (or “targeting”) of a Cas effector protein, e.g., a Cas12a effector protein to a DNA target site such as within a genomic sequence in a cell. While guide molecules are typically RNA molecules it is well known in the art that chemically modified RNA molecules including DNA / RNA hybrid molecules can be used as guide molecules. It is well understood that these chemical modifications of the guide RNA molecules include, but are not limited to, 2’-Fluorination (“2’-F”) and 2’-O-methylation (“2’-OMe”). In some embodiments, guide RNAs further comprise a scaffold and / or linker. In some embodiments, the guide RNA comprises a spacer sequence that is partially or completely complementary to a DNA target site and that is 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 nucleotides in length.

[0034] The term “linker” is used to refer to that portion of a multi-element agent that connects different elements to one another. For example, those of ordinary skill in the art appreciate that a polypeptide whose structure includes two or more functional or organizational domains often includes a stretch of amino acids between such domains that links them to one another. In some embodiments, a polypeptide comprising a linker element has an overall structure of the general form S1-L-S2, wherein S1 and S2 may be the same or different and represent two domains associated with one another by the linker (L). In some embodiments, a polypeptide linker is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids in length. In some embodiments, a linker is characterized in that it tends not to adopt a rigid three-dimensional structure, but rather provides flexibility to the polypeptide. A variety of different linker elements that can appropriately be used when engineering polypeptides (e.g., fusion polypeptides) are known in the art (see e.g., Holliger etal., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) and Poljak et al., Structure 2:1121-1123 (1994), the contents of each of which are hereby incorporated by reference herein in entirety).

[0035] The term “nickase activity” as used herein refers to non-target strand cleavage activity or target strand cleavage activity of a Cas12 effector protein described herein. In some embodiments, nickase activity refers to a fraction or percentage of double-stranded nucleic acid molecules exhibiting single-stranded cleavage (i.e., not double-stranded cleavage) of a non-target strand or target strand at a target site following incubation with a complex comprising a Cas12 effector protein (or a nucleic acid sequence encoding a Cas12 effector protein) and a gRNA molecule. In some embodiments, nickase activity refers to a fraction or percentage of double-stranded nucleic acid molecules exhibiting single-stranded cleavage of a non-target strand or target strand at a target site following incubation with a complex comprising (1) a fusion protein comprising a Cas12 effector protein (or a nucleic acid sequence encoding a fusion protein) and (2) a gRNA molecule. In some embodiments, a complex is a ribonucleoprotein (“RNP”) complex comprising a Cas12 effector protein. In some embodiments, a complex is a ribonucleoprotein (“RNP”) complex comprising a Cas12 effector protein and a gRNA molecule. In some embodiments, a complex is a ribonucleoprotein (“RNP”) complex comprising a Cas12a effector protein and a gRNA molecule. In some embodiments, a complex is a ribonucleoprotein (“RNP”) complex comprising a Cas12a nickase and a gRNA molecule. In some embodiments, a complex is a ribonucleoprotein (“RNP”) complex comprising a fusion protein comprising a Cas12 effector protein and a gRNA molecule. In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and at least 100% of double-stranded nucleic acid molecules exhibit cleavage of a non-target strand (and not the target strand) or target strand (and not the non-target strand) following incubation with a Cas12 effector protein (or a nucleic acid sequence encoding a Cas12 effector protein) and a gRNA molecule. In some embodiments, a nickase described herein cleaves a target strand of a double-stranded DNA molecule at a rate that is less than a rate of cleavage of the non-target strand of the double-stranded DNA molecule. In some embodiments, a nickase described herein cleaves a target strand of a double-stranded DNA molecule at a rate that is less than 100%, less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%,less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.05%, less than 0.01%, less than 0.001% and less than 0.0001% of a rate of cleavage of the non-target strand of the double-stranded DNA molecule. In some embodiments, cleavage of the target strand of a DNA molecule cannot be detected using conventional techniques following incubation of a mixture of double-stranded DNA molecules with a nickase described herein, e.g. following incubation of at least 10 seconds, at least 30 seconds, at least 100 seconds, at least 500 seconds, at least 1000 seconds, at least 1500 seconds, at least 2000 seconds, at least 2500 seconds, at least 3000 seconds, at least 3500 seconds, at least 4000 seconds, at least 4500 seconds, at least 5000 seconds, or greater than 5000 seconds. In some embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and at least 100% of initially double-stranded DNA molecules in a mixture comprise an uncleaved target strand following incubation with a nickase described herein.

[0036] The term “non-target strand” (including, but not limited to, “PAM containing strand”, and “complementary strand”) as used herein refers to a strand of a double stranded nucleic acid comprising the protospacer adjacent motif (PAM). In some embodiments, a Cas effector protein may cleave a non-target strand upon interaction with a genomic site. In some such embodiments, a non-target strand may get cleaved before or after cleaving of a target strand. In some embodiments, a non-target strand may get preferentially cleaved when compared to non-target strand cleavage activity or lack thereof.

[0037] The term “nuclease” as used herein refers to any protein that catalyzes the cleavage of phosphodiester bonds. In some embodiments the nuclease is a DNA nuclease. In some embodiments, the nuclease is a nickase. In some embodiments, the nuclease is a nickase which causes a single-strand break in either the target or non-target strand of the double-stranded DNA. In some embodiments the nuclease cleaves both strands of double- stranded DNA, e.g., genomic DNA in a cell. In some embodiments the nuclease binds a specific target site within the double-stranded DNA that overlaps with or is adjacent to the location of the resulting break. In some embodiments, the nuclease causes a double-strand break that contains overhangs ranging from zero (blunt ends) to 22 nucleotides in both 3’ and5’ orientations. As discussed herein, Cas nucleases are exemplary nucleases that can be used in accordance with the strategies, systems, and methods of the present disclosure.

[0038] The terms “nucleic acid”, “polynucleotide”, “nucleotide sequence”, “nucleic acid molecule”, and “nucleic acid sequence” are generally used interchangeably and refer to any compound and / or substance that is or can be incorporated into a polynucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is or can be incorporated into a polynucleotide chain via a phosphodiester linkage. As will be clear from context, in some embodiments, “nucleic acid” refers to an individual nucleic acid residue (e.g., a nucleotide and / or nucleoside); in some embodiments, “nucleic acid” refers to a polynucleotide chain comprising individual nucleic acid residues. In some embodiments, a “nucleic acid” is or comprises RNA; in some embodiments, a “nucleic acid” is or comprises DNA. In some embodiments, a nucleic acid is, comprises, or consists of one or more natural nucleic acid residues. In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. For example, in some embodiments, a nucleic acid is, comprises, or consists of one or more “peptide nucleic acids”, which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present invention. Alternatively, or additionally, in some embodiments, a nucleic acid has one or more phosphorothioate and / or 5’-N-phosphoramidite linkages rather than phosphodiester bonds. In some embodiments, a nucleic acid is, comprises, or consists of one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxy guanosine, and deoxycytidine). In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo- pyrimidine, 3 -methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl- uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5- propynyl-uridine, C5 -propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7- deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, 2- thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, a nucleic acid comprises one or more modified sugars (e.g., 2’-fluororibose, ribose, 2’-deoxyribose, arabinose, and hexose) as compared with those in natural nucleic acids. In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or protein. In some embodiments, a nucleic acidincludes one or more introns. In some embodiments, nucleic acids are prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis. In some embodiments, a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues long. In some embodiments, a nucleic acid is partly or wholly single-stranded; in some embodiments, a nucleic acid is partly or wholly double stranded. In some embodiments a nucleic acid has a nucleotide sequence comprising at least one element that encodes, or is the complement of a sequence that encodes, a polypeptide. In some embodiments, a nucleic acid has enzymatic activity.

[0039] As used herein the term “identity” or “sequence identity” refers to overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be “substantially identical” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% identical. Calculation of percent identity of two nucleic acid or polypeptide sequences, for example, can be performed by aligning two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In some embodiments, a length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or substantially 100% of length of a reference sequence; nucleotides at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as a corresponding position in the second sequence, then the two molecules (i.e., first and second) are identical at that position. Percent identity between two sequences is a function of the number of identical positions shared by the two sequences being compared, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of thetwo sequences. Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4: 11-17, which is herein incorporated by reference in its entirety), which has been incorporated into the ALIGN program (version 2.0). In some embodiments, nucleic acid sequence comparisons made with the ALIGN program use a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In some embodiments, sequence identity refers to a measure of similarity between two nucleotide sequences. In some other embodiments, sequence identity refers to a measure of similarity between two amino acid sequences. In some embodiments, sequence identity accounts for a measure of similarity between the Cas12 effector proteins described herein and reference sequences. In some embodiments, reference sequences may be other Cas12 effector proteins. In some embodiments, reference sequences may be a wild-type (non-mutated) variant of Cas12 effector proteins. It is appreciated by those skilled in the art, that sequence identity encompasses and acknowledges degeneracy within the nucleotide sequence (e.g., also interchangeably referred to as “genetic code”) toward encoding amino acids that may be part of the amino acid sequence being described herein.

[0040] Conventional IUPAC notation is used in nucleotide sequences presented herein, as shown in Table 1, below (see also Cornish-Bowden, Nucleic Acids Res. 13(9):3021-30 (1985), incorporated by reference herein). It should be noted, however, that “T” denotes “Thymine or Uracil” in those instances where a sequence may be encoded by either DNA or RNA, for example in certain CRISPR / Cas guide molecules. Table 1. IUPAC nucleic acid notation Character Base A Adenine

[0041] The term “operably linked” refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A control element “operably linked” to a functional element is associated in such a way that expression and / or activity of the functional element is achieved under conditions compatible with the control element. In some embodiments, “operably linked” control elements are contiguous (e.g., covalently linked) with coding elements of interest; in some embodiments, control elements act in trans to the functional element of interest. In some embodiments, “operably linked” refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. In some embodiments, for example, a functional linkage may include transcriptional control. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences can be contiguous with each other and, e.g., where necessary to join two protein coding regions, are in the same reading frame.

[0042] The terms “orthologue” (also referred to as “ortholog” herein) and “homologue” (also referred to as “homolog” herein) are known in the art. By means of further guidance, a “homologue” of a protein as used herein is a protein of the same species which performs the same or a similar function as the protein it is a homologue of. Homologous proteins may but need not be structurally related or are only partially structurally related. An “orthologue” of a protein as used herein is a protein of a different species which performs the same or a similar function as the protein it is an orthologue of. Orthologous proteins may but need not be structurally related or are only partially structurally related. Homologs and orthologs may be identified by homology modelling (see, e.g., Greer, Science 228:1055 (1985) and Blundell et al., Eur J Biochem 172:513 (1988)) or “structural BLAST” (Dey et al., Protein Sci.22(4):359-66 (2013)). See also Shmakov et al., Mol Cell60(3):385-97 (2015) for application in the field of CRISPR / Cas loci. Homologous proteins may but need not be structurally related or are only partially structurally related.

[0043] The term “polypeptide” refers to any polymeric chain of residues (e.g., amino acids) that are typically linked by peptide bonds. In some embodiments, a polypeptide has an amino acid sequence that occurs in nature. In some embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, a polypeptide has an amino acid sequence that is engineered in that it is designed and / or produced through action of the hand of man. In some embodiments, a polypeptide may comprise or consist of natural amino acids, non-natural amino acids, or both. In some embodiments, a polypeptide may include one or more pendant groups or other modifications, e.g., modifying or attached to one or more amino acid side chains, at a polypeptide’s N-terminus, at a polypeptide’s C- terminus, or any combination thereof. In some embodiments, such pendant groups or modifications may be acetylation, amidation, lipidation, methylation, pegylation, etc., including combinations thereof. In some embodiments, polypeptides may contain L-amino acids, D-amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art. In some embodiments, useful modifications may be or include, e.g., terminal acetylation, amidation, methylation, etc. In some embodiments, a protein may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof.

[0044] The terms “prevent,” “preventing,” and “prevention” as used herein refer to the prevention of a disease in a mammal, e.g., in a human, including (a) avoiding or precluding the disease; (b) affecting the predisposition toward the disease; or (c) preventing or delaying the onset of at least one symptom of the disease.

[0045] The term “reference” describes a native or wild-type or non-mutated sequence. In some embodiments, a reference sequence is a wild-type polypeptide sequence of a Cas12a effector protein. In some embodiments, a reference sequence is the wild-type nucleic acid sequence encoding a Cas12a effector protein. In some embodiments, a reference sequence of a Cas12a effector protein is mutated to improve activity (e.g., nicking activity, e.g., specificity of nicking activity) of a variant Cas12a effector protein compared to a reference Cas12a effector protein. In some embodiments, activity (e.g., nicking activity, e.g., specificity of nicking activity) can be compared between a variant (or mutated) sequence and a reference sequence. In some embodiments, a reference sequence of a Cas12a effector protein is mutated to improve efficiency (e.g., on-target specificity) of a variant Cas12aeffector protein compared to a reference Cas12a effector protein. In some embodiments, efficiency (e.g., on-target specificity) can be compared between a variant (or mutated) sequence and a reference sequence. In some embodiments, a reference sequence is a reference gRNA sequence. In some embodiments, a reference gRNA sequence is a gRNA sequence comprising a standard (or conventional) number of nucleotides. In some embodiments, a reference sequence is a reference gRNA spacer sequence. In some embodiments, a reference gRNA spacer sequence is from about 17 nucleotides to about 21 nucleotides in length.

[0046] As used herein, the term “recombinant” is intended to refer to polypeptides that are designed, engineered, prepared, expressed, created, manufactured, and / or or isolated by recombinant means, such as polypeptides expressed using a recombinant expression construct transfected into a host cell; polypeptides isolated from a recombinant, combinatorial human polypeptide library; polypeptides isolated from an animal (e.g., a mouse, rabbit, sheep, fish, etc.) that is transgenic for or otherwise has been manipulated to express a gene or genes, or gene components that encode and / or direct expression of the polypeptide or one or more component(s), portion(s), element(s), or domain(s) thereof; and / or polypeptides prepared, expressed, created or isolated by any other means that involves splicing or ligating selected nucleic acid sequence elements to one another, chemically synthesizing selected sequence elements, and / or otherwise generating a nucleic acid that encodes and / or directs expression of a polypeptide or one or more component(s), portion(s), element(s), or domain(s) thereof. In some embodiments, one or more of such selected sequence elements is found in nature. In some embodiments, one or more of such selected sequence elements is designed in silico. In some embodiments, one or more such selected sequence elements results from mutagenesis (e.g., in vivo or in vitro) of a known sequence element, e.g., from a natural or synthetic source such as, for example, in the germline of a source organism of interest (e.g., of a human, a mouse, etc.).

[0047] The term “spacer” as used herein refers to a series of nucleotide bases (also called “nucleotides”) that form a component of the guide RNA molecule, which may or may not be adjacent to the “scaffold” region of the guide RNA molecule. In some embodiments, the spacer may refer to a region within the guide RNA molecule that defines the genomic target to be modified. In some embodiments, the spacer of the guide RNA undergoes base pairing (e.g., Watson-Crick base pairing) with the complementary strand to the DNA protospacer. In some such embodiments, the spacer may anneal to the target strand (TS) ofthe double-stranded DNA. In some such embodiments, the spacer:TS complex may induce or prevent kinking of the TS required for interaction with the Cas12 effector protein. In some embodiments, the spacer is 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 nucleotides in length. In some embodiments, the spacer is an extended spacer as described herein.

[0048] The term “subject” as used herein means a human or non-human animal. In some embodiments a human subject can be any age (e.g., an infant, child, young adult, or adult). In some embodiments a human subject may be at risk of or suffer from a disease or may be in need of alteration of a gene or a combination of specific genes. Alternatively, in some embodiments, a subject may be a non-human animal, which may include, but is not limited to, a mammal. In some embodiments, a non-human animal is a non-human primate, a rodent (e.g., a mouse, rat, hamster, guinea pig, etc.), a rabbit, a dog, a cat, and so on. In some embodiments, the non-human animal subject is livestock, e.g., a cow, a horse, a sheep, a goat, etc. In some embodiments, the non-human animal subject is poultry, e.g., a chicken, a turkey, a duck, etc.

[0049] The term “target strand” (including, but not limited to, “non-PAM containing strand”, and “non-complementary strand”) as used herein refers to a strand of a double- stranded nucleic acid that does not comprise the protospacer adjacent motif (PAM). In some embodiments, a target strand may anneal to a guide RNA molecule, which may be alone or a part of a complex with a Cas effector protein. In some such embodiments, a Cas effector protein may nick the target strand upon interaction with the guide RNA. In some such embodiments, a target strand may get nicked before or after nicking of the non-target strand. In some embodiments, a target strand may get preferentially nicked when compared to non- target strand nicking activity or lack thereof. In some embodiments, cleavage activity of the target strand may get attenuated by introduction of mutations within target strand cleavage attenuation residue(s).

[0050] The term “target strand cleavage attenuation residue” as used herein refers to any residue that plays a role in conformational transitions of the CRISPR RNA (crRNA)- DNA hybrid complex after non-target strand cleavage to direct a target strand into the RuvC domain of a Cas12a effector protein and result in target strand cleavage. As understood in the art, Cas12a effector proteins have one RuvC domain, which under physiological conditions is first occupied by the non-target strand to be cleaved before conformational changes occur. In such embodiments, the cleaved non-target stand is moved out of the RuvC domain so that thetarget strand enters into the RuvC active site for cleavage. In some embodiments, one or more substitutions of target strand cleavage attenuation residue(s) may result in attenuated target strand cleavage. In some embodiments, one or more substitutions of target strand cleavage attenuation residue(s) can result in a reduction in the rate of target strand cleavage as compared to the rate of non-target strand cleavage of a double-stranded DNA molecule. In some embodiments, one or more substitutions of target strand cleavage attenuation residue(s) can result in a rate of cleavage of the target strand of a double-stranded DNA molecule that is less than 100%, less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.05%, less than 0.01%, less than 0.001% and less than 0.0001% of a rate of cleavage of the non-target strand of the double-stranded DNA molecule. In some embodiments, one or more substitutions of target strand cleavage attenuation residue(s) can result in an increase in the ratio of non-target strand: target strand cleavage (e.g., see Examples) following incubation with a mixture of double-stranded DNA molecules relative to a Cas12a effector protein having no substitutions in a target strand cleavage attenuation residue. In some embodiments, a Cas12a effector protein having no substitutions in a target strand cleavage attenuation residue exhibits a ratio of non-target strand:target strand (NTS / TS) cleavage of about 1. In some embodiments, one or more substitutions of target strand cleavage attenuation residue(s) can result in a ratio of NTS / TS cleavage of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, at least 500, at least 1000, at least 2000, at least 3000, at least 4000, at least 5000, at least 6000, at least 7000, at least 8000, at least 9000, at least 10,000 at least 15,000, at least 20,000, at least 25,000, at least 30,0000, at least 35,000, at least 40,000, at least 45,000, at least 50,000, at least 55,000, at least 60,000, or greater than 60,000.

[0051] In some embodiments, a target strand cleavage attenuation residue is a residue within the RuvC domain of a Cas12a effector protein. In some embodiments, a target strand cleavage attenuation residue is a residue within the alpha helical lid domain (also referred to herein as the core lid domain) of a Cas12a effector protein. In some embodiments, a target strand cleavage attenuation residue is a residue not in (e.g., outside of) the alpha helical liddomain of a Cas12a effector protein. In some embodiments, a target strand cleavage attenuation residue is a residue adjacent to the alpha helical lid domain of a Cas12a effector protein. In some embodiments, a target strand cleavage attenuation residue is a residue within the bridge helix domain of a Cas12a effector protein. In some embodiments, a target strand cleavage attenuation residue is a residue adjacent to the bridge helix domain of a Cas12a effector protein. In some embodiments, a target strand cleavage attenuation residue is a residue that resides within the bridge helix domain of a Cas12a effector protein. In some embodiments, a target strand cleavage attenuation residue is a positively charged surface residue of a Cas12a effector protein. In some embodiments, a target strand cleavage attenuation residue is a residue that attenuates double-stranded cleavage activity of a Cas12a effector protein. In some embodiments, increasing the number of amino acid substitutions at target strand cleavage attenuation residues in a Cas12a effector polypeptide sequence relative to a wild-type Cas12a effector polypeptide sequence increases attenuation of double-stranded cleavage activity of a Cas12a effector protein relative to a wild-type Cas12a effector protein. In some embodiments, “increased attenuation” of double-stranded cleavage activity refers to a reduction in the rate of target strand cleavage relative to non-target strand cleavage of a double-stranded DNA molecule, e.g., such that the rate of cleavage of the target strand is less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, less than 0.05%, less than 0.01%, less than 0.001% and less than 0.0001% of a rate of cleavage of the non-target strand of the double-stranded DNA molecule.

[0052] In some embodiments, a target strand cleavage attenuation residue is at a position corresponding to W958 of SEQ ID NO: 11. In some embodiments, a target strand cleavage attenuation residue is at a position corresponding to F999 of SEQ ID NO: 11. In some embodiments, a target strand cleavage attenuation residue is at a position corresponding to K1000 of SEQ ID NO: 11. In some embodiments, a target strand cleavage attenuation residue is at a position corresponding to S1001 of SEQ ID NO: 11. In some embodiments, a target strand cleavage attenuation residue is at a position corresponding to K1002 of SEQ ID NO: 11. In some embodiments, a target strand cleavage attenuation residue is at a position corresponding to R1003 of SEQ ID NO: 11.

[0053] In some embodiments, a target strand cleavage attenuation residue is at a position corresponding to W971 of SEQ ID NO: 62. In some embodiments, a target strand cleavage attenuation residue is at a position corresponding to F1012 of SEQ ID NO: 62. In some embodiments, a target strand cleavage attenuation residue is at a position corresponding to K1013 of SEQ ID NO: 62. In some embodiments, a target strand cleavage attenuation residue is at a position corresponding to R1014 of SEQ ID NO: 62. In some embodiments, a target strand cleavage attenuation residue is at a position corresponding to K1018 of SEQ ID NO: 62. In some embodiments, a target strand cleavage attenuation residue is at a position corresponding to R1016 of SEQ ID NO: 62.

[0054] In some embodiments, a target strand cleavage attenuation residue is at a position corresponding to W890 of SEQ ID NO: 71. In some embodiments, a target strand cleavage attenuation residue is at a position corresponding to F931 of SEQ ID NO: 71. In some embodiments, a target strand cleavage attenuation residue is at a position corresponding to K932 of SEQ ID NO: 71. In some embodiments, a target strand cleavage attenuation residue is at a position corresponding to N933 of SEQ ID NO: 71. In some embodiments, a target strand cleavage attenuation residue is at a position corresponding to K937 of SEQ ID NO: 71. In some embodiments, a target strand cleavage attenuation residue is at a position corresponding to R385 of SEQ ID NO: 71.

[0055] The terms “treatment,” “treat,” and “treating,” as used herein refer to a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progress, ameliorate, reduce severity of, prevent or delay the recurrence of a disease, disorder, or condition or one or more symptoms thereof, and / or improve one or more symptoms of a disease, disorder, or condition as described herein. In some embodiments, a condition includes an injury. In some embodiments, an injury may be acute or chronic (e.g., tissue damage from an underlying disease or disorder that causes, e.g., secondary damage such as tissue injury). In some embodiments, treatment may be administered to a subject after one or more symptoms have developed and / or after a disease has been diagnosed. Treatment may be administered in the absence of symptoms, e.g., to prevent or delay onset of a symptom or inhibit onset or progression of a disease. For example, in some embodiments, treatment may be administered to a susceptible subject prior to the onset of symptoms (e.g., in light of genetic or other susceptibility factors). In some embodiments, treatment may also be continued after symptoms have resolved, for example to prevent or delay their recurrence. In someembodiments, treatment results in improvement and / or resolution of one or more symptoms of a disease, disorder or condition.

[0056] The term “variant” as used herein refers to an entity such as a polypeptide or polynucleotide that shows significant structural identity with a reference entity but differs structurally from the reference entity in the presence or level of one or more chemical moieties as compared with the reference entity. In many embodiments, a variant also differs functionally from its reference entity. In general, whether a particular entity is properly considered to be a “variant” of a reference entity is based on its degree of structural identity with the reference entity. As used herein, the terms “functional variant” refer to a variant that confers the same function as the reference entity. It is to be understood that a functional variant need not be functionally equivalent to the reference entity as long as it confers the same function as the reference entity. CRISPR / Cas Effector Systems

[0057] CRISPR / Cas effector systems according to the present disclosure comprise, but are not limited to Cas12a effector proteins that exhibit predominantly nickase activity and effector proteins derived or obtained therefrom. In functional terms, CRISPR / Cas effector systems are defined as comprising a Cas effector protein that: (A) interacts with (e.g., complexes with) a gRNA molecule; and (B) together with the gRNA molecule, associate with, and optionally alter, cleave or modify, a target region of a DNA that includes (1) a sequence complementary to the targeting domain of the gRNA and, optionally, (2) an additional sequence referred to as a “protospacer adjacent motif,” or “PAM,” which is described in greater detail below. As the following examples will illustrate, Cas effector proteins can be defined, in broad terms, by their PAM specificity and cleavage activity, even though variations may exist between individual Cas effector proteins that share the same PAM specificity or cleavage activity. Skilled artisans will appreciate that some aspects of the present disclosure relate to systems and methods that can be implemented using any suitable Cas effector proteins having a certain PAM specificity and / or cleavage activity (i.e., nickase activity). For this reason, unless otherwise specified, the term Cas effector proteins should be understood as a generic term, and not limited to any species (e.g., Acidaminococcus sp., Lachnospiraceae bacterium, etc.) or variation (e.g., full-length vs. truncated or split; naturally-occurring PAM specificity vs. engineered PAM specificity, etc.) of Cas effector proteins.

[0058] In general, a Cas effector protein can be delivered to the cell as a protein or a nucleic acid encoding the protein, e.g., a DNA molecule or mRNA molecule. The protein or nucleic acid can be combined with other delivery agents, e.g., lipids or polymers in a lipid or polymer nanoparticle and targeting agents such as antibodies or other binding agents with specificity for the cell. The DNA molecule can be a nucleic acid vector, such as a viral genome or circular double-stranded DNA, e.g., a plasmid. Nucleic acid vectors encoding a Cas effector protein can include other coding or non-coding elements. For example, a Cas effector protein can be delivered as part of a viral genome (e.g., in an AAV, adenoviral or lentiviral genome) that includes certain genomic backbone elements (e.g., inverted terminal repeats, in the case of an AAV genome).

[0059] Cas effector proteins described herein have activities and properties that can be useful in a variety of applications, but the skilled artisan will appreciate that Cas effector proteins can also be modified in certain instances, to alter cleavage activity, PAM specificity, or other structural or functional features.

[0060] For example, a CRISPR / Cas effector system may comprise a nickase or fusion protein as described herein. In some embodiments, a CRISPR / Cas effector system comprises a nickase. In some such embodiments, a nickase may nick either the target strand or a non- target strand to create indels in the genome of a cell, as those described herein. In some embodiments, a nickase preferentially nicks the non-target strand relative to the target strand to reduce the rate of insertions / deletions (“INDEL rate”) in the genome of a cell. It is contemplated that the Cas effector proteins described herein can be delivered to a cell in vitro, in vivo, or ex vivo.

[0061] Cas effector proteins may also optionally include a tag, such as, but not limited to, a nuclear localization signal, to facilitate movement of the Cas effector protein into the nucleus of a cell. In some embodiments, the Cas effector protein can incorporate C- and / or N-terminal nuclear localization signals. Nuclear localization sequences are known in the art. In some embodiments, the nuclear localization sequence is a c-Myc sequence. In some other embodiments, the nuclear localization sequence is a linker-SV40 sequence. In some embodiments, the Cas effector protein may incorporate multiple nuclear localization sequences (NLS), e.g., at one or both termini (e.g., two c-Myc sequences at the C-terminus). Exemplary nuclear localization sequences are shown in SEQ ID NO: 7, 8, 9, or 10. SEQ ID NO: 7: Exemplary NLS (2x Linker-c-Myc NLS)GGSPAAKRVKLDGGSPAAKRVKLD SEQ ID NO: 8: Exemplary NLS (c-Myc NLS) PAAKRVKLD SEQ ID NO: 9: Exemplary NLS (Linker-SV40 NLS) GRSSDDEATADSQHAAPPKKKRKV SEQ ID NO: 10: Exemplary NLS (SV40 NLS) PKKKRKV

[0062] In some embodiments, CRISPR / Cas effector systems and methods of their use are described in US Publication No.2019 / 0062735 A1, the disclosure of which is incorporated by reference herein in its entirety. In some embodiments, CRISPR / Cas effector systems and methods of their use are described in International Publication No. WO 2023 / 097316 A1, the disclosure of which is incorporated by reference herein in its entirety. Cas12a Effector Proteins and variants thereof

[0063] The present disclosure describes the use of Cas12a effector proteins, derived from a Cas12a locus denoted as subtype V-A, and variants thereof. Such effector proteins are also referred to herein as Cas12a effector proteins. The subtype V-A loci encompasses Cas1, Cas2, a distinct gene denoted Cas12a and a CRISPR array. Cpf1 (CRISPR-associated protein Cpf1, subtype PREFRAN) (Cas12a) is a large protein (about 1300 amino acids) that contains a RuvC-like nuclease domain homologous to the corresponding domain of Cas9 along with a counterpart to the characteristic arginine-rich cluster of Cas9. However, Cas12a lacks the HNH nuclease domain that is present in all Cas9 proteins, and the RuvC-like domain is contiguous in the Cas12a sequence, in contrast to Cas9 where it contains long inserts including the HNH domain. Accordingly, in particular embodiments, a Cas12a effector protein comprises a RuvC-like nuclease domain and lacks an HNH nuclease domain.

[0064] A crystal structure of Acidaminococcus sp. Cas12a in complex with crRNA and a dsDNA target including a TTTN PAM sequence has been solved by Yamano et al., Cell 165(4):949-962 (2016). Cas12a has two lobes: a REC (recognition) lobe, and a NUC (nuclease) lobe. The REC lobe includes REC1 and REC2 domains, which lack similarity to any known protein structures. The NUC lobe, meanwhile, includes three RuvC domains (RuvC-I, -II and -III) and a bridge helix (BH) domain. The Cas12a REC lobe lacks an HNH domain and includes other domains that also lack similarity to known protein structures: astructurally unique PAM-interacting (PI) domain, three Wedge (WED) domains (WED-I, -II and -III), and a nuclease (Nuc) domain.

[0065] Crystal structures of Cas12a from Francisella novicida (FnCas12a) further revealed that upon recognition of a short protospacer adjacent motif (PAM) by Cas12a, the CRISPR RNA (crRNA) binds a DNA strand (i.e., the target strand, also interchangeably referred to herein as “TS” or “non-PAM containing strand”), and subsequently forms a crRNA:TS hybrid complex that displaces the non-target strand (Saha et al. An Alpha-helical Lid Guides the Target DNA toward Catalysis in CRISPR-Cas12a. bioRxiv; 2022. DOI: 10.1101 / 2022.09.05.506663). This greater three strand complex involving the crRNA, target strand, and non-target strand is understood by those in the art as formation of an R-loop structure. Furthermore, it is well understood that R-loop formation triggers the double- stranded cleavage activity of the sole RuvC nuclease domain of Cas12a, thus acting as a molecular scissor (Strohkendl et al., Structural Basis of Cas12a R-loop Propagation on Pathway to DNA Cleavage. bioRxiv; 2023. DOI: 10.1101 / 2023.03.13.532460).

[0066] Within the RuvC domain, there resides a core lid domain (also interchangeably referred to herein as the “alpha helical lid”) which is recognized as playing a role in anchoring the crRNA:TS complex through formation of salt bridge interactions with its numerous positively charged amino acids. As provided herein, it is also an insight of the present disclosure that the alpha helical lid domain plays a role in positioning the crRNA:DNA hybrid complex such that target strand cleavage activity is attenuated (e.g., relative to non-target strand cleavage activity). Moreover, the present disclosure provides, among other things, variant Cas12a nickase proteins that comprise one or more mutations within the alpha helical lid domain in conjunction with one or more mutations within the greater RuvC domain, including within the REC lobe and bridge helix. In some embodiments, the Cas12a nickase proteins described herein provide a desirable alternative to other Cas effector proteins in terms of efficient, programmable small DNA alterations and to instigate potential large DNA insertions and deletions.

[0067] In some embodiments, a Cas12a effector protein may be from an organism of a genus which includes, but is not limited to Acidaminococcus sp, Lachnospiraceae bacterium, or Francisella tularensis subsp. Novicida. In some embodiments, a Cas12a effector protein may be an organism of a species which includes but is not limited to Acidaminococcus sp. BV3L6 (AsCas12a); or Lachnospiraceae bacterium ND2006 (LbCas12a). In some embodiments, a Cas12a effector protein as referred to herein has asequence identity of at least 80%, more preferably at least 85%, even more preferably at least 90%, such as for instance at least 95%, such as for instance at least 97%, such as for instance at least 98%, such as for instance at least 99%, such as for instance at least 99.1%, such as for instance at least 99.2%, such as for instance at least 99.3%, such as for instance at least 99.4%, such as for instance at least 99.5%, such as for instance at least 99.6%, such as for instance at least 99.7%, such as for instance at least 99.8%, such as for instance at least 99.9% with one or more of the Cas12a sequences disclosed herein. In further embodiments, the Cas12a effector protein as referred to herein has a sequence identity of at least 80%, more preferably at least 85%, even more preferably at least 90%, such as for instance at least 95%, such as for instance at least 97%, such as for instance at least 98%, such as for instance at least 99%, such as for instance at least 99.1%, such as for instance at least 99.2%, such as for instance at least 99.3%, such as for instance at least 99.4%, such as for instance at least 99.5%, such as for instance at least 99.6%, such as for instance at least 99.7%, such as for instance at least 99.8%, such as for instance at least 99.9% with a wild-type FnCas12a, AsCas12a, or LbCas12a polypeptide sequence. In some embodiments, a Cas12a effector protein as referred to herein has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mutations relative to a wild-type FnCas12a, AsCas12a, or LbCas12a polypeptide sequence. In some embodiments, a Cas12a effector protein as referred to herein has less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 relative to a wild-type FnCas12a, AsCas12a, or LbCas12a polypeptide sequence. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 11. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 12. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 13. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 14. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 15. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 16. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 17. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 18. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 19. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 20. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 21. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 22. In some embodiments, a Cas12a effector protein comprises anamino acid sequence of SEQ ID NO: 23. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 24. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 25. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 26. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 27. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 28. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 29. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 30. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 31. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 32. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 33. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 34. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 35. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 36. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 37. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 38. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 39. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 40. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 41. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 42. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 43. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 44. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 45. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 46. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 47. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 48. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 49. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 50. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 51. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 52. In someembodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 53. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 54. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 55. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 56. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 57. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 58. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 59. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 60. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 61. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 62. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 63. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 64. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 65. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 66. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 67. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 68. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 69. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 70. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 71. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 72. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 73. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 74. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 75. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 76. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 77. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 78. In some embodiments, a Cas12a effector protein comprises an amino acid sequence of SEQ ID NO: 79.

[0068] In some embodiments, a Cas12a effector protein has a sequence identity of at least 60%, more particularly at least 70%, at least 80%, more preferably at least 85%, evenmore preferably at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% with AsCas12a, FnCas12a, or LbCas12a. In some embodiments, a Cas12a effector protein as referred to herein has a sequence identity of at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% with a wild-type AsCas12a, FnCas12a, or LbCas12a. In some embodiments, a Cas12a effector protein has less than 60% sequence identity with AsCas12a. In some embodiments, a Cas12a effector protein has less than 60% sequence identity with FnCas12a. In some embodiments, a Cas12a effector protein has less than 60% sequence identity with LbCas12a. A skilled person will understand that this includes truncated forms of a Cas12a effector protein whereby the sequence identity is determined over the length of the truncated form.

[0069] Cas12a effector proteins may also refer to Cas12a nucleases, Cas12a nickases, and / or dead Cas12a effector proteins, and related variants thereof as described herein. In some embodiments, Cas12a effector proteins are fused to one or more heterologous protein domains (“fusion proteins”) as described herein.

[0070] The foregoing list of modifications is intended to be exemplary in nature, and the skilled artisan will appreciate, in view of the instant disclosure, that other modifications may be possible or desirable in certain applications. For brevity, therefore, exemplary systems, methods and compositions of the present disclosure are presented with reference to particular Cas effector proteins, but it should be understood that the Cas effector proteins used may be modified in ways that do not alter their operating principles. Such modifications are within the scope of the present disclosure.

[0071] In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1 or 2 of the amino acid substitutions corresponding to positions K1000 and S1001 in AsCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1 or 2 of the amino acid substitutions corresponding to K1000G and S1001G in AsCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, 4, or 5 of the amino acid substitutions corresponding to positions M537, H800, F870, K1000, and S1001 in AsCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, 4, or 5 of the amino acid substitutions corresponding to M537R, H800A, F870L, K1000G, and S1001G in AsCas12a.

[0072] In some embodiments, a Cas12a effector protein is an AsCas12a nickase variant comprising 1 or 2 of the amino acid substitutions corresponding to positions K1000 and S1001 in AsCas12a. In some embodiments, a Cas12a effector protein is a AsCas12a nickase variant comprising 1 or 2 of the amino acid substitutions corresponding to K1000G and S1001G in AsCas12a. In some embodiments, a Cas12a effector protein is a AsCas12a nickase variant comprising 1, 2, 3, 4, or 5 of the amino acid substitutions corresponding to positions M537, H800, F870, K1000, and S1001 in AsCas12a. In some embodiments, a Cas12a effector protein is a AsCas12a nickase variant comprising 1, 2, 3, 4, or 5 of the amino acid substitutions corresponding to M537R, H800A, F870L, K1000G, and S1001G in AsCas12a.

[0073] In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1 or 2 of the amino acid substitutions corresponding to positions K1000 and K1002 in AsCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1 or 2 of the amino acid substitutions corresponding to K1000G and K1002G in AsCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, 4, or 5 of the amino acid substitutions corresponding to positions M537, H800, F870, K1000, and K1002 in AsCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, 4, or 5 of the amino acid substitutions corresponding to M537R, H800A, F870L, K1000G, and K1002G in AsCas12a.

[0074] In some embodiments, a Cas12a effector protein is an AsCas12a nickase variant comprising 1 or 2 of the amino acid substitutions corresponding to positions K1000 and S1001 in AsCas12a. In some embodiments, a Cas12a effector protein is a AsCas12a nickase variant comprising 1 or 2 of the amino acid substitutions corresponding to K1000G and S1001G in AsCas12a. In some embodiments, a Cas12a effector protein is a AsCas12a nickase variant comprising 1, 2, 3, 4, or 5 of the amino acid substitutions corresponding to positions M537, H800, F870, K1000, and K1002 in AsCas12a. In some embodiments, a Cas12a effector protein is a AsCas12a nickase variant comprising 1, 2, 3, 4, or 5 of the amino acid substitutions corresponding to M537R, H800A, F870L, K1000G, and K1002G in AsCas12a.

[0075] In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising an amino acid substitution corresponding to position R1226 in AsCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising an amino acid substitution corresponding to R1226A in AsCas12a. In some embodiments, aCas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to positions M537, H800, F870, and R1226 in AsCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to M537R, H800A, F870L, and R1226A in AsCas12a.

[0076] In some embodiments, a Cas12a effector protein is an AsCas12a nickase variant comprising an amino acid substitution corresponding to position R1226 in AsCas12a. In some embodiments, a Cas12a effector protein is a AsCas12a nickase variant comprising an amino acid substitution corresponding to R1226A in AsCas12a. In some embodiments, a Cas12a effector protein is a AsCas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to positions M537, H800, F870, and R1226 in AsCas12a. In some embodiments, a Cas12a effector protein is a AsCas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to M537R, H800A, F870L, and R1226A in AsCas12a.

[0077] In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising an amino acid substitution corresponding to position W958 in AsCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising an amino acid substitution corresponding to W958A in AsCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to positions M537, H800, F870, and W958 in AsCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to M537R, H800A, F870L, and W958A in AsCas12a.

[0078] In some embodiments, a Cas12a effector protein is an AsCas12a nickase variant comprising an amino acid substitution corresponding to s position W958 in AsCas12a. In some embodiments, a Cas12a effector protein is a AsCas12a nickase variant comprising an amino acid substitution corresponding to W958A in AsCas12a. In some embodiments, a Cas12a effector protein is a AsCas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to positions M537, H800, F870, and W958 in AsCas12a. In some embodiments, a Cas12a effector protein is a AsCas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to M537R, H800A, F870L, and W958A in AsCas12a.

[0079] In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising an amino acid substitution corresponding to position W382 in AsCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising an amino acid substitution corresponding to W382A in AsCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to positions M537, H800, F870, and W382 in AsCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to M537R, H800A, F870L, and W382A in AsCas12a.

[0080] In some embodiments, a Cas12a effector protein is an AsCas12a nickase variant comprising an amino acid substitution corresponding to position W382 in AsCas12a. In some embodiments, a Cas12a effector protein is a AsCas12a nickase variant comprising an amino acid substitution corresponding to W382A in AsCas12a. In some embodiments, a Cas12a effector protein is a AsCas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to positions M537, H800, F870, and W382 in AsCas12a. In some embodiments, a Cas12a effector protein is a AsCas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to M537R, H800A, F870L, and W382A in AsCas12a.

[0081] In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising an amino acid substitution corresponding to position F999 in AsCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising an amino acid substitution corresponding to F999G in AsCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to positions M537, H800, F870, and F999 in AsCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to M537R, H800A, F870L, and F999G in AsCas12a.

[0082] In some embodiments, a Cas12a effector protein is an AsCas12a nickase variant comprising an amino acid substitution corresponding to position F999 in AsCas12a. In some embodiments, a Cas12a effector protein is a AsCas12a nickase variant comprising an amino acid substitution corresponding to F999G in AsCas12a. In some embodiments, a Cas12a effector protein is a AsCas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to positions M537, H800, F870, and F999 in AsCas12a. Insome embodiments, a Cas12a effector protein is a AsCas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to M537R, H800A, F870L, and F999G in AsCas12a.

[0083] In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, or 3 of the amino acid substitutions corresponding to positions K1000, K1002, and R1003 in AsCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, or 3 of the amino acid substitutions corresponding to K1000G, K1002G, and R1003G in AsCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, 4, 5, or 6 of the amino acid substitutions corresponding to positions M537, H800, F870, K1000, K1002, and R1003 in AsCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, 4, 5, or 6 of the amino acid substitutions corresponding to M537R, H800A, F870L, K1000G, K1002G, and R1003G in AsCas12a.

[0084] In some embodiments, a Cas12a effector protein is an AsCas12a nickase variant comprising 1, 2, or 3 of the amino acid substitutions corresponding to positions K1000, K1002, and R1003 in AsCas12a. In some embodiments, a Cas12a effector protein is a AsCas12a nickase variant comprising 1, 2, or 3 of the amino acid substitutions corresponding to K1000G, K1002G, and R1003G in AsCas12a. In some embodiments, a Cas12a effector protein is a AsCas12a nickase variant comprising 1, 2, 3, 4, 5, or 6 of the amino acid substitutions corresponding to positions M537, H800, F870, K1000, K1002, and R1003 in AsCas12a. In some embodiments, a Cas12a effector protein is a AsCas12a nickase variant comprising 1, 2, 3, 4, 5, or 6 of the amino acid substitutions corresponding to M537R, H800A, F870L, K1000G, K1002G, and R1003G in AsCas12a.

[0085] In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to positions F999, K1000, K1002, and R1003 in AsCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to F999G, K1000G, K1002G, and R1003G in AsCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, 4, 5, 6, or 7 of the amino acid substitutions corresponding to positions M537, H800, F870, F999, K1000, K1002, and R1003 in AsCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, 4, 5, 6, or 7 of the amino acid substitutionscorresponding to M537R, H800A, F870L, F999G, K1000G, K1002G, and R1003G in AsCas12a.

[0086] In some embodiments, a Cas12a effector protein is an AsCas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to positions F999, K1000, K1002, and R1003 in AsCas12a. In some embodiments, a Cas12a effector protein is a AsCas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to F999G, K1000G, K1002G, and R1003G in AsCas12a. In some embodiments, a Cas12a effector protein is a AsCas12a nickase variant comprising 1, 2, 3, 4, 5, 6, or 7 of the amino acid substitutions corresponding to positions M537, H800, F870, F999, K1000, K1002, and R1003 in AsCas12a. In some embodiments, a Cas12a effector protein is a AsCas12a nickase variant comprising 1, 2, 3, 4, 5, 6, or 7 of the amino acid substitutions corresponding to M537R, H800A, F870L, F999G, K1000G, K1002G, and R1003G in AsCas12a.

[0087] In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, 4, or 5 of the amino acid substitutions corresponding to positions W958, F999, K1000, K1002, and R1003 in AsCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, 4, or 5 of the amino acid substitutions corresponding to W958A, F999G, K1000G, K1002G, and R1003G in AsCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, 4, 5, 6, 7, or 8 of the amino acid substitutions corresponding to positions M537, H800, F870, W958, F999, K1000, K1002, and R1003 in AsCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, 4, 5, 6, 7, or 8 of the amino acid substitutions corresponding to M537R, H800A, F870L, W958A, F999G, K1000G, K1002G, and R1003G in AsCas12a.

[0088] In some embodiments, a Cas12a effector protein is an AsCas12a nickase variant comprising 1, 2, 3, 4, or 5 of the amino acid substitutions corresponding to positions W958, F999, K1000, K1002, and R1003 in AsCas12a. In some embodiments, a Cas12a effector protein is an AsCas12a nickase variant comprising 1, 2, 3, 4, or 5 of the amino acid substitutions corresponding to W958A, F999G, K1000G, K1002G, and R1003G in AsCas12a. In some embodiments, a Cas12a effector protein is an AsCas12a nickase variant comprising 1, 2, 3, 4, 5, 6, 7, or 8 of the amino acid substitutions corresponding to positions M537, H800, F870, W958, F999, K1000, K1002, and R1003 in AsCas12a. In some embodiments, a Cas12a effector protein is an AsCas12a nickase variant comprising 1, 2, 3, 4,5, 6, 7, or 8 of the amino acid substitutions corresponding to M537R, H800A, F870L, W958A, F999G, K1000G, K1002G, and R1003G in AsCas12a.

[0089] In some embodiments, a Cas12a effector protein amino acid sequence comprises an amino acid sequence having at least about 90%, 95%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% identity to an AsCas12a nickase variant sequence described herein (e.g., SEQ ID NOs: 7-39).

[0090] In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1 or 2 of the amino acid substitutions corresponding to positions K1013 and R1014 in FnCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1 or 2 of the amino acid substitutions corresponding to K1013G and R1014G in FnCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, 4, or 5 of the amino acid substitutions corresponding to positions N602, H843, F879, K1013, and R1014 in FnCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, 4, or 5 of the amino acid substitutions corresponding to N602R, H843A, F879L, K1013G, and R1014G in FnCas12a.

[0091] In some embodiments, a Cas12a effector protein is a FnCas12a nickase variant comprising 1 or 2 of the amino acid substitutions corresponding to positions K1013 and R1014 in FnCas12a. In some embodiments, a Cas12a effector protein is a FnCas12a nickase variant comprising 1 or 2 of the amino acid substitutions corresponding to K1013G and R1014G in FnCas12a. In some embodiments, a Cas12a effector protein is a FnCas12a nickase variant comprising 1, 2, 3, 4, or 5 of the amino acid substitutions corresponding to positions N602, H843, F879, K1013, and R1014 in FnCas12a. In some embodiments, a Cas12a effector protein is a FnCas12a nickase variant comprising 1, 2, 3, 4, or 5 of the amino acid substitutions corresponding to N602R, H843A, F879L, K1013G, and R1014G in FnCas12a.

[0092] In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1 or 2 of the amino acid substitutions corresponding to positions K1013 and K1018 in FnCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1 or 2 of the amino acid substitutions corresponding to K1013G and K1018G in FnCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, 4, or 5 of the amino acid substitutions corresponding to positions N602, H843, F879, K1013, and K1018 in FnCas12a. In some embodiments, a Cas12aeffector protein is a Cas12a nickase variant comprising 1, 2, 3, 4, or 5 of the amino acid substitutions corresponding to N602R, H843A, F879L, K1013G, and K1018G in FnCas12a.

[0093] In some embodiments, a Cas12a effector protein is a FnCas12a nickase variant comprising 1 or 2 of the amino acid substitutions corresponding to positions K1013 and K1018 in FnCas12a. In some embodiments, a Cas12a effector protein is a FnCas12a nickase variant comprising 1 or 2 of the amino acid substitutions corresponding to K1013G and K1018 in FnCas12a. In some embodiments, a Cas12a effector protein is a FnCas12a nickase variant comprising 1, 2, 3, 4, or 5 of the amino acid substitutions corresponding to positions N602, H843, F879, K1013, and K1018 in FnCas12a. In some embodiments, a Cas12a effector protein is a FnCas12a nickase variant comprising 1, 2, 3, 4, or 5 of the amino acid substitutions corresponding to N602R, H843A, F879L, K1013G, and K1018G in FnCas12a.

[0094] In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising an amino acid substitution corresponding to position R1218 in FnCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising an amino acid substitution corresponding to R1218A in FnCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to positions N602, H843, F879, and R1218A in FnCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to N602R, H843A, F879L, and R1218A in FnCas12a.

[0095] In some embodiments, a Cas12a effector protein is a FnCas12a nickase variant comprising an amino acid substitution corresponding to position R1218 in FnCas12a. In some embodiments, a Cas12a effector protein is a FnCas12a nickase variant comprising an amino acid substitution corresponding to R1218A in FnCas12a. In some embodiments, a Cas12a effector protein is a FnCas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to positions N602, H843, F879, and R1218 in FnCas12a. In some embodiments, a Cas12a effector protein is a FnCas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to N602R, H843A, F879L, and R1218A in FnCas12a.

[0096] In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising an amino acid substitution corresponding to position W971 in FnCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising anamino acid substitution corresponding to W971A in FnCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to positions N602, H843, F879, and W971 in FnCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to N602R, H843A, F879L, and W971A in FnCas12a.

[0097] In some embodiments, a Cas12a effector protein is a FnCas12a nickase variant comprising an amino acid substitution corresponding to position W971 in FnCas12a. In some embodiments, a Cas12a effector protein is a FnCas12a nickase variant comprising an amino acid substitution corresponding to W971A in FnCas12a. In some embodiments, a Cas12a effector protein is a FnCas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to positions N602, H843, F879, and W971 in FnCas12a. In some embodiments, a Cas12a effector protein is a FnCas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to N602R, H843A, F879L, and W971A in FnCas12a.

[0098] In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising an amino acid substitution corresponding to position Y410 in FnCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising an amino acid substitution corresponding to Y410A in FnCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to positions N602, H843, F879, and Y410 in FnCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to N602R, H843A, F879L, and Y410A in FnCas12a.

[0099] In some embodiments, a Cas12a effector protein is a FnCas12a nickase variant comprising an amino acid substitution corresponding to position Y410 in FnCas12a. In some embodiments, a Cas12a effector protein is a FnCas12a nickase variant comprising an amino acid substitution corresponding Y410A in FnCas12a. In some embodiments, a Cas12a effector protein is a FnCas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to positions N602, H843, F879, and Y410 in FnCas12a. In some embodiments, a Cas12a effector protein is a FnCas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to N602R, H843A, F879L, and Y410A in FnCas12a.

[0100] In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising an amino acid substitution corresponding to position F1012 in FnCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising an amino acid substitution corresponding to F1012G in FnCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to positions N602, H843, F879, and F1012 in FnCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to N602R, H843A, F879L, and F1012G in FnCas12a.

[0101] In some embodiments, a Cas12a effector protein is a FnCas12a nickase variant comprising an amino acid substitution corresponding to position F1012 in FnCas12a. In some embodiments, a Cas12a effector protein is a FnCas12a nickase variant comprising an amino acid substitution corresponding to F1012G in FnCas12a. In some embodiments, a Cas12a effector protein is a FnCas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to positions N602, H843, F879, and F1012 in FnCas12a. In some embodiments, a Cas12a effector protein is a FnCas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to N602R, H843A, F879L, and F1012G in FnCas12a.

[0102] In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, or 3 of the amino acid substitutions corresponding to positions K1013, K1018, and R1016 in FnCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, or 3 of the amino acid substitutions corresponding to K1013G, K1018G, and R1016G in FnCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, 4, 5, or 6 of the amino acid substitutions corresponding to positions N602, H843, F879, K1013, K1018, and R1016 in FnCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, 4, 5, or 6 of the amino acid substitutions corresponding to N602R, H843A, F879L, K1013G, K1018G, and R1016G in FnCas12a.

[0103] In some embodiments, a Cas12a effector protein is a FnCas12a nickase variant comprising 1, 2, or 3 of the amino acid substitutions corresponding to positions K1013, K1018, and R1016 in FnCas12a. In some embodiments, a Cas12a effector protein is a FnCas12a nickase variant comprising 1, 2, or 3 of the amino acid substitutions corresponding to K1013G, K1018G, and R1016G in FnCas12a. In some embodiments, a Cas12a effectorprotein is a FnCas12a nickase variant comprising 1, 2, 3, 4, 5, or 6 of the amino acid substitutions corresponding to positions N602, H843, F879, K1013, K1018, and R1016 in FnCas12a. In some embodiments, a Cas12a effector protein is a FnCas12a nickase variant comprising 1, 2, 3, 4, 5, or 6 of the amino acid substitutions corresponding to N602R, H843A, F879L, K1013G, K1018G, and R1016G in FnCas12a.

[0104] In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to positions F1012, K1013, K1018, and R1016 in FnCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to F1012G, K1013G, K1018G, and R1016G in FnCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, 4, 5, 6, or 7 of the amino acid substitutions corresponding to positions N602, H843, F879, F1012, K1013, K1018, and R1016 in FnCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, 4, 5, 6, or 7 of the amino acid substitutions corresponding to N602R, H843A, F879L, F1012G, K1013G, K1018G, and R1016G in FnCas12a.

[0105] In some embodiments, a Cas12a effector protein is a FnCas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to positions F1012, K1013, K1018, and R1016 in FnCas12a. In some embodiments, a Cas12a effector protein is a FnCas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to F1012G, K1013G, K1018G, and R1016G in FnCas12a. In some embodiments, a Cas12a effector protein is a FnCas12a nickase variant comprising 1, 2, 3, 4, 5, 6, or 7 of the amino acid substitutions corresponding to positions N602, H843, F879, F1012, K1013, K1018, and R1016 in FnCas12a. In some embodiments, a Cas12a effector protein is a FnCas12a nickase variant comprising 1, 2, 3, 4, 5, 6, or 7 of the amino acid substitutions corresponding to N602R, H843A, F879L, F1012G, K1013G, K1018G, and R1016G in FnCas12a.

[0106] In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, 4, or 5 of the amino acid substitutions corresponding to positions W971, F1012, K1013, K1018, and R1016 in FnCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, 4, or 5 of the amino acid substitutions corresponding to W971A, F1012G, K1013G, K1018G, and R1016G in FnCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, 4, 5,6, 7, or 8 of the amino acid substitutions corresponding to positions N602, H843, F879, W971, F1012, K1013, K1018, and R1016 in FnCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, 4, 5, 6, 7, or 8 of the amino acid substitutions corresponding to N602R, H843A, F879L, W971A, F1012G, K1013G, K1018G, and R1016G in FnCas12a.

[0107] In some embodiments, a Cas12a effector protein is a FnCas12a nickase variant comprising 1, 2, 3, 4, or 5 of the amino acid substitutions corresponding to positions W971, F1012, K1013, K1018, and R1016 in FnCas12a. In some embodiments, a Cas12a effector protein is a FnCas12a nickase variant comprising 1, 2, 3, 4, or 5 of the amino acid substitutions corresponding to W971A, F1012G, K1013G, K1018G, and R1016G in FnCas12a. In some embodiments, a Cas12a effector protein is a FnCas12a nickase variant comprising 1, 2, 3, 4, 5, 6, 7, or 8 of the amino acid substitutions corresponding to positions N602, H843, F879, W971, F1012, K1013, K1018, and R1016 in FnCas12a. In some embodiments, a Cas12a effector protein is a FnCas12a nickase variant comprising 1, 2, 3, 4, 5, 6, 7, or 8 of the amino acid substitutions corresponding to N602R, H843A, F879L, W971A, F1012G, K1013G, K1018G, and R1016G in FnCas12a.

[0108] In some embodiments, a Cas12a effector protein amino acid sequence comprises an amino acid sequence having at least about 90%, 95%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% identity to an FnCas12a nickase variant sequence described herein (e.g., SEQ ID NOs: 43-48).

[0109] In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1 or 2 of the amino acid substitutions corresponding to positions K932 and N933 in LbCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1 or 2 of the amino acid substitutions corresponding to K932G and N933G in LbCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, 4, or 5 of the amino acid substitutions corresponding to positions N527, H759, E795, K932, and N933 in LbCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, 4, or 5 of the amino acid substitutions corresponding to N527R, H759A, E795L, K932G, and N933G in LbCas12a.

[0110] In some embodiments, a Cas12a effector protein is a LbCas12a nickase variant comprising 1 or 2 of the amino acid substitutions corresponding to positions K932 and N933 in LbCas12a. In some embodiments, a Cas12a effector protein is a LbCas12a nickase variantcomprising 1 or 2 of the amino acid substitutions corresponding to K932G and N933G in LbCas12a. In some embodiments, a Cas12a effector protein is a LbCas12a nickase variant comprising 1, 2, 3, 4, or 5 of the amino acid substitutions corresponding to positions N527, H759, E795, K932, and N933 in LbCas12a. In some embodiments, a Cas12a effector protein is a LbCas12a nickase variant comprising 1, 2, 3, 4, or 5 of the amino acid substitutions corresponding to N527R, H759A, E795L, K932G, and N933G in LbCas12a.

[0111] In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1 or 2 of the amino acid substitutions corresponding to positions K932 and K937 in LbCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1 or 2 of the amino acid substitutions corresponding to K932G and K937G in LbCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, 4, or 5 of the amino acid substitutions corresponding to positions N527, H759, E795, K932, and K937 in LbCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, 4, or 5 of the amino acid substitutions corresponding to N527R, H759A, E795L, K932G, and K937G in LbCas12a.

[0112] In some embodiments, a Cas12a effector protein is a LbCas12a nickase variant comprising 1 or 2 of the amino acid substitutions corresponding to positions K932 and K937 in LbCas12a. In some embodiments, a Cas12a effector protein is a LbCas12a nickase variant comprising 1 or 2 of the amino acid substitutions corresponding to K932G and K937G in LbCas12a. In some embodiments, a Cas12a effector protein is a LbCas12a nickase variant comprising 1, 2, 3, 4, or 5 of the amino acid substitutions corresponding to positions N527, H759, E795, K932, and K937 in LbCas12a. In some embodiments, a Cas12a effector protein is a LbCas12a nickase variant comprising 1, 2, 3, 4, or 5 of the amino acid substitutions corresponding to N527R, H759A, E795L, K932G, and K937G in LbCas12a.

[0113] In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising an amino acid substitution corresponding to position R1138 in LbCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising an amino acid substitution corresponding to R1138A in LbCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to positions N527, H759, E795, and R1138 in LbCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to N527R, H759A, E795L, and R1138A in LbCas12a.

[0114] In some embodiments, a Cas12a effector protein is a LbCas12a nickase variant comprising an amino acid substitution corresponding to position R1138 in LbCas12a. In some embodiments, a Cas12a effector protein is a LbCas12a nickase variant comprising an amino acid substitution corresponding to R1138A in LbCas12a. In some embodiments, a Cas12a effector protein is a LbCas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to positions N527, H759, E795, and R1138 in LbCas12a. In some embodiments, a Cas12a effector protein is a LbCas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to N527R, H759A, E795L, and R1138A in LbCas12a.

[0115] In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising an amino acid substitution corresponding to position W890 in LbCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising an amino acid substitution corresponding to W890A in LbCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to positions N527, H759, E795, and W890 in LbCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to N527R, H759A, E795L, and W890A in LbCas12a.

[0116] In some embodiments, a Cas12a effector protein is a LbCas12a nickase variant comprising an amino acid substitution corresponding to position W890 in LbCas12a. In some embodiments, a Cas12a effector protein is a LbCas12a nickase variant comprising an amino acid substitution corresponding to W890A in LbCas12a. In some embodiments, a Cas12a effector protein is a LbCas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to positions N527, H759, E795, and W890 in LbCas12a. In some embodiments, a Cas12a effector protein is a LbCas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to N527R, H759A, E795L, and W890A in LbCas12a.

[0117] In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising an amino acid substitution corresponding to position W355 in LbCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising an amino acid substitution corresponding to W355A in LbCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to positions N527, H759, E795, and W355 in LbCas12a. In someembodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to N527R, H759A, E795L, and W355A in LbCas12a.

[0118] In some embodiments, a Cas12a effector protein is a LbCas12a nickase variant comprising an amino acid substitution corresponding to position W355 in LbCas12a. In some embodiments, a Cas12a effector protein is a LbCas12a nickase variant comprising an amino acid substitution corresponding to W355A in LbCas12a. In some embodiments, a Cas12a effector protein is a LbCas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to positions N527, H759, E795, and W355 in LbCas12a. In some embodiments, a Cas12a effector protein is a LbCas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to N527R, H759A, E795L, and W355A in LbCas12a.

[0119] In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising an amino acid substitution corresponding to position F931 in LbCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising an amino acid substitution corresponding to F931G in LbCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to positions N527, H759, E795, and F931 in LbCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to N527R, H759A, E795L, and F931G in LbCas12a.

[0120] In some embodiments, a Cas12a effector protein is a LbCas12a nickase variant comprising an amino acid substitution corresponding to position F931 in LbCas12a. In some embodiments, a Cas12a effector protein is a LbCas12a nickase variant comprising an amino acid substitution corresponding to F931G in LbCas12a. In some embodiments, a Cas12a effector protein is a LbCas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to positions N527, H759, E795, and F931 in LbCas12a. In some embodiments, a Cas12a effector protein is a LbCas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to N527R, H759A, E795L, and F931G in LbCas12a.

[0121] In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, or 3 of the amino acid substitutions corresponding to positions K932, K937,and R935 in LbCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, or 3 of the amino acid substitutions corresponding to K932G, K937G, and R935G in LbCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, 4, 5, or 6 of the amino acid substitutions corresponding to positions N527, H759, E795, K932, K937, and R935 in LbCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, 4, 5, or 6 of the amino acid substitutions corresponding to N527R, H759A, E795L, K932G, K937G, and R935G in LbCas12a.

[0122] In some embodiments, a Cas12a effector protein is a LbCas12a nickase variant comprising 1, 2, or 3 of the amino acid substitutions corresponding to positions K932, K937, and R935 in LbCas12a. In some embodiments, a Cas12a effector protein is a LbCas12a nickase variant comprising 1, 2, or 3 of the amino acid substitutions corresponding to K932G, K937G, and R935G in LbCas12a. In some embodiments, a Cas12a effector protein is a LbCas12a nickase variant comprising 1, 2, 3, 4, 5, or 6 of the amino acid substitutions corresponding to positions N527, H759, E795, K932, K937, and R935 in LbCas12a. In some embodiments, a Cas12a effector protein is a LbCas12a nickase variant comprising 1, 2, 3, 4, 5, or 6 of the amino acid substitutions corresponding to N527R, H759A, E795L, K932G, K937G, and R935G in LbCas12a.

[0123] In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to positions F931, K932, K937, and R935 in LbCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to F931G, K932G, K937G, and R935G in LbCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, 4, 5, 6, or 7 of the amino acid substitutions corresponding to positions N527, H759, E795, F931, K932, K937, and R935 in LbCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, 4, 5, 6, or 7 of the amino acid substitutions corresponding to N527R, H759A, E795L, F931G, K932G, K937G, and R935G in LbCas12a.

[0124] In some embodiments, a Cas12a effector protein is a LbCas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to positions F931, K932, K937, and R935 in LbCas12a. In some embodiments, a Cas12a effector protein is a LbCas12a nickase variant comprising 1, 2, 3, or 4 of the amino acid substitutions corresponding to F931G, K932G, K937G, and R935G in LbCas12a. In some embodiments, aCas12a effector protein is a LbCas12a nickase variant comprising 1, 2, 3, 4, 5, 6, or 7 of the amino acid substitutions corresponding to positions N527, H759, E795, F931, K932, K937, and R935 in LbCas12a. In some embodiments, a Cas12a effector protein is a LbCas12a nickase variant comprising 1, 2, 3, 4, 5, 6, or 7 of the amino acid substitutions corresponding to N527R, H759A, E795L, F931G, K932G, K937G, and R935G in LbCas12a.

[0125] In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, 4, or 5 of the amino acid substitutions corresponding to positions W890, F931, K932, K937, and R935 in LbCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, 4, or 5 of the amino acid substitutions corresponding to W890A, F931G, K932G, K937G, and R935G in LbCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, 4, 5, 6, 7, or 8 of the amino acid substitutions corresponding to positions N527, H759, E795, W890, F931, K932, K937, and R935 in LbCas12a. In some embodiments, a Cas12a effector protein is a Cas12a nickase variant comprising 1, 2, 3, 4, 5, 6, 7, or 8 of the amino acid substitutions corresponding to N527R, H759A, E795L, W890A, F931G, K932G, K937G, and R935G in LbCas12a.

[0126] In some embodiments, a Cas12a effector protein is a LbCas12a nickase variant comprising 1, 2, 3, 4, or 5 of the amino acid substitutions corresponding to positions W890, F931, K932, K937, and R935 in LbCas12a. In some embodiments, a Cas12a effector protein is a LbCas12a nickase variant comprising 1, 2, 3, 4, or 5 of the amino acid substitutions corresponding to W890A, F931G, K932G, K937G, and R935G in LbCas12a. In some embodiments, a Cas12a effector protein is a LbCas12a nickase variant comprising 1, 2, 3, 4, 5, 6, 7, or 8 of the amino acid substitutions corresponding to positions N527, H759, E795, W890, F931, K932, K937, and R935 in LbCas12a. In some embodiments, a Cas12a effector protein is a LbCas12a nickase variant comprising 1, 2, 3, 4, 5, 6, 7, or 8 of the amino acid substitutions corresponding to N527R, H759A, E795L, W890A, F931G, K932G, K937G, and R935G in LbCas12a.

[0127] In some embodiments, a Cas12a effector protein amino acid sequence comprises an amino acid sequence having at least about 90%, 95%, 97%, 98%, 99% or 100% identity to an LbCas12a nickase variant sequence described herein (e.g., SEQ ID NOs: 52- 57).

[0128] Other suitable modifications of a Cas12a amino acid sequence (or polypeptide sequence) are known to those of ordinary skill in the art. Some exemplary amino acid sequences of wild-type Cas12a effector proteins and variants thereof are provided below: SEQ ID NO: 11 – Exemplary AsCas12a wild-type amino acid sequence MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKR HAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDIS TAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQIL SDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLE TISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFV KNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQ IYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKM LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDK FFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVL ENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILH FKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYP ANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRN SEQ ID NO: 12 – Exemplary AsCas12a wild-type amino acid sequence MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKR HAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDIS TAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQIL SDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLE TISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFV KNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQ IYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKM LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDK FFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVL ENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILH FKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYP ANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRNGGSPAAKRVKLDGGSPAAKRVKLD SEQ ID NO: 13 – Exemplary AsCas12a wild-type amino acid sequence MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKR HAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDIS TAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQIL SDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLE TISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFV KNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQ IYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKM LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDK FFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVL ENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILH FKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYP ANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRNGGSPAAKRVKLDGGSPAAKRVKLDHHHHHH SEQ ID NO: 14 – Exemplary AsCas12a nickase variant amino acid sequence MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKR HAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDIS TAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQIL SDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLE TISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQRPTLASGWDVNKEKNNGAILFV KNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQ IYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAARLGEKM LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDK FLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAASVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVL ENLNFGGGSGGTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILH FKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYP ANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRNSEQ ID NO: 15 – Exemplary AsCas12a nickase variant amino acid sequence MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKR HAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDIS TAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQIL SDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLE TISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQRPTLASGWDVNKEKNNGAILFV KNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQ IYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAARLGEKM LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDK FLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAASVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVL ENLNFGGGSGGTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILH FKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYP ANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRNGGSPAAKRVKLDGGSPAAKRVKLD SEQ ID NO: 16 – Exemplary AsCas12a nickase variant amino acid sequence MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKR HAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDIS TAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQIL SDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLE TISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQRPTLASGWDVNKEKNNGAILFV KNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQ IYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAARLGEKM LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDK FLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAASVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVL ENLNFGGGSGGTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILH FKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYP ANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRNGGSPAAKRVKLDGGSPAAKRVKLDHHHHHH SEQ ID NO: 17 – Exemplary AsCas12a nickase variant amino acid sequenceMTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKR HAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDIS TAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQIL SDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLE TISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQRPTLASGWDVNKEKNNGAILFV KNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQ IYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAARLGEKM LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDK FLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVL ENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILH FKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYP ANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRN SEQ ID NO: 18 – Exemplary AsCas12a nickase variant amino acid sequence MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKR HAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDIS TAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQIL SDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLE TISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQRPTLASGWDVNKEKNNGAILFV KNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQ IYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAARLGEKM LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDK FLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVL ENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILH FKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYP ANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRNGGSPAAKRVKLDGGSPAAKRVKLD SEQ ID NO: 19 – Exemplary AsCas12a nickase variant amino acid sequence MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKR HAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQIL SDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLE TISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQRPTLASGWDVNKEKNNGAILFV KNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQ IYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAARLGEKM LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDK FLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVL ENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILH FKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYP ANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRNGGSPAAKRVKLDGGSPAAKRVKLDHHHHHH SEQ ID NO: 20 – Exemplary AsCas12a nickase variant amino acid sequence MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKR HAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDIS TAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQIL SDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLE TISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQRPTLASGWDVNKEKNNGAILFV KNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQ IYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAARLGEKM LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDK FLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVL ENLNFGFGGKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILH FKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYP ANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRN SEQ ID NO: 21 – Exemplary AsCas12a nickase variant amino acid sequence MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKR HAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDIS TAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQIL SDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQRPTLASGWDVNKEKNNGAILFV KNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQ IYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAARLGEKM LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDK FLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVL ENLNFGFGGKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILH FKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYP ANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRNGGSPAAKRVKLDGGSPAAKRVKLD SEQ ID NO: 22 – Exemplary AsCas12a nickase variant amino acid sequence MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKR HAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDIS TAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQIL SDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLE TISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQRPTLASGWDVNKEKNNGAILFV KNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQ IYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAARLGEKM LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDK FLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVL ENLNFGFGGKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILH FKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYP ANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRNGGSPAAKRVKLDGGSPAAKRVKLDHHHHHH SEQ ID NO: 23 – Exemplary AsCas12a nickase variant amino acid sequence MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKR HAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDIS TAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQIL SDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLE TISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQRPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQ IYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAARLGEKM LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDK FLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVL ENLNFGFGSGRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILH FKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYP ANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRN SEQ ID NO: 24 – Exemplary AsCas12a nickase variant amino acid sequence MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKR HAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDIS TAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQIL SDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLE TISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQRPTLASGWDVNKEKNNGAILFV KNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQ IYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAARLGEKM LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDK FLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVL ENLNFGFGSGRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILH FKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYP ANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRNGGSPAAKRVKLDGGSPAAKRVKLD SEQ ID NO: 25 – Exemplary AsCas12a nickase variant amino acid sequence MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKR HAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDIS TAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQIL SDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLE TISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQRPTLASGWDVNKEKNNGAILFV KNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAARLGEKM LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDK FLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVL ENLNFGFGSGRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILH FKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYP ANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRNGGSPAAKRVKLDGGSPAAKRVKLDHHHHHH SEQ ID NO: 26 – Exemplary AsCas12a nickase variant amino acid sequence MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKR HAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDIS TAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQIL SDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLE TISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQRPTLASGWDVNKEKNNGAILFV KNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQ IYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAARLGEKM LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDK FLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVL ENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILH FKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYP ANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMGNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRN SEQ ID NO: 27 – Exemplary AsCas12a nickase variant amino acid sequence MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKR HAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDIS TAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQIL SDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLE TISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQRPTLASGWDVNKEKNNGAILFV KNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQ IYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAARLGEKM LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDK FLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVL ENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILH FKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYP ANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMGNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRNGGSPAAKRVKLDGGSPAAKRVKLD SEQ ID NO: 28 – Exemplary AsCas12a nickase variant amino acid sequence MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKR HAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDIS TAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQIL SDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLE TISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQRPTLASGWDVNKEKNNGAILFV KNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQ IYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAARLGEKM LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDK FLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVL ENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILH FKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYP ANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMGNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRNGGSPAAKRVKLDGGSPAAKRVKLDHHHHHH SEQ ID NO: 29 – Exemplary AsCas12a nickase variant amino acid sequence MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKR HAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDIS TAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQIL SDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLE TISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQRPTLASGWDVNKEKNNGAILFV KNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQ IYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAARLGEKM LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDK FLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAASVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVL ENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYP ANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRN SEQ ID NO: 30 – Exemplary AsCas12a nickase variant amino acid sequence MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKR HAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDIS TAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQIL SDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLE TISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQRPTLASGWDVNKEKNNGAILFV KNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQ IYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAARLGEKM LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDK FLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAASVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVL ENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILH FKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYP ANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRNGGSPAAKRVKLDGGSPAAKRVKLD SEQ ID NO: 31 – Exemplary AsCas12a nickase variant amino acid sequence MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKR HAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDIS TAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQIL SDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLE TISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQRPTLASGWDVNKEKNNGAILFV KNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQ IYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAARLGEKM LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDK FLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAASVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVL ENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILH FKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYP ANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRNGGSPAAKRVKLDGGSPAAKRVKLDHHHHHH SEQ ID NO: 32 – Exemplary AsCas12a nickase variant amino acid sequence MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKR HAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDIS TAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQIL SDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLE TISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQRPTLASGWDVNKEKNNGAILFV KNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQ IYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAARLGEKM LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDK FLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVL ENLNFGGKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILH FKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYP ANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRN SEQ ID NO: 33 – Exemplary AsCas12a nickase variant amino acid sequence MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKR HAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDIS TAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQIL SDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLE TISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQRPTLASGWDVNKEKNNGAILFV KNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQ IYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAARLGEKM LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDK FLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVL ENLNFGGKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILH FKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYP ANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRNGGSPAAKRVKLDGGSPAAKRVKLDSEQ ID NO: 34 – Exemplary AsCas12a nickase variant amino acid sequence MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKR HAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDIS TAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQIL SDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLE TISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQRPTLASGWDVNKEKNNGAILFV KNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQ IYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAARLGEKM LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDK FLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVL ENLNFGGKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILH FKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYP ANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRNGGSPAAKRVKLDGGSPAAKRVKLDHHHHHH SEQ ID NO: 35 – Exemplary AsCas12a nickase variant amino acid sequence MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKR HAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDIS TAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQIL SDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLE TISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQRPTLASGWDVNKEKNNGAILFV KNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQ IYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAARLGEKM LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDK FLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVL ENLNFGFGSGGTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILH FKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYP ANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRNSEQ ID NO: 36 – Exemplary AsCas12a nickase variant amino acid sequence MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKR HAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDIS TAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQIL SDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLE TISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQRPTLASGWDVNKEKNNGAILFV KNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQ IYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAARLGEKM LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDK FLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVL ENLNFGFGSGGTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILH FKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYP ANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRNGGSPAAKRVKLDGGSPAAKRVKLD SEQ ID NO: 37 – Exemplary AsCas12a nickase variant amino acid sequence MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKR HAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDIS TAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQIL SDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLE TISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQRPTLASGWDVNKEKNNGAILFV KNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQ IYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAARLGEKM LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDK FLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVL ENLNFGFGSGGTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILH FKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYP ANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRNGGSPAAKRVKLDGGSPAAKRVKLDHHHHHHSEQ ID NO: 38 – Exemplary AsCas12a nickase variant amino acid sequence MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKR HAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDIS TAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQIL SDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLE TISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQRPTLASGWDVNKEKNNGAILFV KNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQ IYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAARLGEKM LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDK FLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVL ENLNFGGGSGGTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILH FKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYP ANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRN SEQ ID NO: 39 – Exemplary AsCas12a nickase variant amino acid sequence MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKR HAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDIS TAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQIL SDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLE TISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQRPTLASGWDVNKEKNNGAILFV KNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQ IYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAARLGEKM LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDK FLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVL ENLNFGGGSGGTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILH FKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYP ANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRNGGSPAAKRVKLDGGSPAAKRVKLDSEQ ID NO: 40 – Exemplary AsCas12a nickase variant amino acid sequence MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKR HAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDIS TAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQIL SDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLE TISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQRPTLASGWDVNKEKNNGAILFV KNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQ IYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAARLGEKM LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDK FLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVL ENLNFGGGSGGTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILH FKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYP ANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRNGGSPAAKRVKLDGGSPAAKRVKLDHHHHHH SEQ ID NO: 41 – Exemplary AsCas12a nickase variant amino acid sequence MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKR HAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDIS TAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQIL SDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLE TISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFV KNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQ IYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKM LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDK FLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAASVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVL ENLNFGGGSGGTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILH FKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYP ANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRNSEQ ID NO: 42 – Exemplary AsCas12a nickase variant amino acid sequence MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKR HAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDIS TAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQIL SDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLE TISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFV KNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQ IYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKM LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDK FLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAASVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVL ENLNFGGGSGGTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILH FKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYP ANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRNGGSPAAKRVKLDGGSPAAKRVKLD SEQ ID NO: 43 – Exemplary AsCas12a nickase variant amino acid sequence MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKR HAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDIS TAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQIL SDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLE TISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFV KNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQ IYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKM LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDK FLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAASVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVL ENLNFGGGSGGTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILH FKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYP ANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRNGGSPAAKRVKLDGGSPAAKRVKLDHHHHHHSEQ ID NO: 44 – Exemplary AsCas12a nickase variant amino acid sequence MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKR HAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDIS TAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQIL SDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLE TISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFV KNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQ IYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKM LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDK FLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAASVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVL ENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILH FKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYP ANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRN SEQ ID NO: 45 – Exemplary AsCas12a nickase variant amino acid sequence MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKR HAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDIS TAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQIL SDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLE TISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFV KNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQ IYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKM LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDK FLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAASVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVL ENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILH FKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYP ANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRNGGSPAAKRVKLDGGSPAAKRVKLDSEQ ID NO: 46 – Exemplary AsCas12a nickase variant amino acid sequence MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKR HAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDIS TAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQIL SDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLE TISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFV KNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQ IYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKM LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDK FLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAASVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVL ENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILH FKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYP ANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRNGGSPAAKRVKLDGGSPAAKRVKLDHHHHHH SEQ ID NO: 47 – Exemplary AsCas12a nickase variant amino acid sequence MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKR HAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDIS TAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQIL SDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLE TISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFV KNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQ IYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKM LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDK FLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVL ENLNFGFGGKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILH FKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYP ANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRNSEQ ID NO: 48 – Exemplary AsCas12a nickase variant amino acid sequence MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKR HAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDIS TAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQIL SDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLE TISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFV KNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQ IYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKM LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDK FLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVL ENLNFGFGGKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILH FKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYP ANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRNGGSPAAKRVKLDGGSPAAKRVKLD SEQ ID NO: 49 – Exemplary AsCas12a nickase variant amino acid sequence MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKR HAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDIS TAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQIL SDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLE TISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFV KNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQ IYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKM LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDK FLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVL ENLNFGFGGKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILH FKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYP ANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRNGGSPAAKRVKLDGGSPAAKRVKLDHHHHHHSEQ ID NO: 50 – Exemplary AsCas12a nickase variant amino acid sequence MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKR HAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDIS TAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQIL SDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLE TISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFV KNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQ IYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKM LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDK FLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVL ENLNFGFGSGRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILH FKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYP ANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRN SEQ ID NO: 51 – Exemplary AsCas12a nickase variant amino acid sequence MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKR HAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDIS TAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQIL SDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLE TISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFV KNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQ IYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKM LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDK FLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVL ENLNFGFGSGRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILH FKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYP ANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRNGGSPAAKRVKLDGGSPAAKRVKLDSEQ ID NO: 52 – Exemplary AsCas12a nickase variant amino acid sequence MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKR HAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDIS TAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQIL SDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLE TISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFV KNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQ IYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKM LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDK FLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVL ENLNFGFGSGRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILH FKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYP ANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRNGGSPAAKRVKLDGGSPAAKRVKLDHHHHHH SEQ ID NO: 53 – Exemplary AsCas12a nickase variant amino acid sequence MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKR HAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDIS TAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQIL SDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLE TISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFV KNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQ IYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKM LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDK FLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVL ENLNFGFGSGGTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILH FKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYP ANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRNSEQ ID NO: 54 – Exemplary AsCas12a nickase variant amino acid sequence MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKR HAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDIS TAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQIL SDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLE TISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFV KNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQ IYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKM LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDK FLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVL ENLNFGFGSGGTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILH FKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYP ANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRNGGSPAAKRVKLDGGSPAAKRVKLD SEQ ID NO: 55 – Exemplary AsCas12a nickase variant amino acid sequence MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKR HAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDIS TAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQIL SDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLE TISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFV KNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQ IYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKM LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDK FLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVL ENLNFGFGSGGTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILH FKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYP ANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRNGGSPAAKRVKLDGGSPAAKRVKLDHHHHHHSEQ ID NO: 56 – Exemplary AsCas12a nickase variant amino acid sequence MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKR HAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDIS TAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQIL SDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLE TISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFV KNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQ IYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKM LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDK FLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVL ENLNFGGGSGGTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILH FKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYP ANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRN SEQ ID NO: 57 – Exemplary AsCas12a nickase variant amino acid sequence MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKR HAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDIS TAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQIL SDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLE TISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFV KNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQ IYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKM LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDK FLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVL ENLNFGGGSGGTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILH FKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYP ANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRNGGSPAAKRVKLDGGSPAAKRVKLDSEQ ID NO: 58 – Exemplary AsCas12a nickase variant amino acid sequence MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKR HAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDIS TAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQIL SDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLE TISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFV KNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQ IYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKM LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDK FLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVL ENLNFGGGSGGTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILH FKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYP ANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRNGGSPAAKRVKLDGGSPAAKRVKLDHHHHHH SEQ ID NO: 59 – Exemplary catalytically dead AsCas12a (dAsCas12a) variant amino acid sequence MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKR HAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDIS TAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQIL SDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLE TISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQRPTLASGWDVNKEKNNGAILFV KNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQ IYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAARLGEKM LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDK FLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIARGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVL ANLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILH FKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYP ANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRNSEQ ID NO: 60 – Exemplary catalytically dead AsCas12a (dAsCas12a) variant amino acid sequence MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKR HAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDIS TAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQIL SDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLE TISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQRPTLASGWDVNKEKNNGAILFV KNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQ IYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAARLGEKM LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDK FLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIARGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVL ANLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILH FKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYP ANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRNGGSPAAKRVKLDGGSPAAKRVKLD SEQ ID NO: 61 – Exemplary catalytically dead AsCas12a (dAsCas12a) variant amino acid sequence MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYA DQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKR HAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDIS TAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQ LLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQIL SDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLE TISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEA FKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEF SARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQRPTLASGWDVNKEKNNGAILFV KNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQ THTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRD FLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQ IYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAARLGEKM LNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDK FLFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIARGERNLIYITVIDSTGKILEQR SLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVL ANLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAK MGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILH FKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYP ANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSP VRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYI QELRNGGSPAAKRVKLDGGSPAAKRVKLDHHHHHHSEQ ID NO: 62 – Exemplary FnCas12a wild-type amino acid sequence MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFI EEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFN QNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENR KNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDY KTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQ INDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKE TLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPS KKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNK DNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKD EHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAI LFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFY NPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDT QRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTL YWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLI KDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSIDRGERHLAYYTLVD GKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEI AKLVIEYNAIVVFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRA YQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKIC YNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLL KDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNF FDSRQAPKNMPQDADANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN SEQ ID NO: 63 – Exemplary FnCas12a wild-type amino acid sequence MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFI EEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFN QNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENR KNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDY KTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQ INDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKE TLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPS KKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNK DNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKD EHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAI LFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFY NPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDT QRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTL YWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLI KDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSIDRGERHLAYYTLVD GKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEI AKLVIEYNAIVVFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRA YQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKIC YNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLL KDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNF FDSRQAPKNMPQDADANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNNGG SPAAKRVKLDGGSPAAKRVKLD SEQ ID NO: 64 – Exemplary FnCas12a wild-type amino acid sequence MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFI EEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENR KNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDY KTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQ INDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKE TLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPS KKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNK DNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKD EHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAI LFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFY NPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDT QRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTL YWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLI KDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSIDRGERHLAYYTLVD GKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEI AKLVIEYNAIVVFEDLNFGFKRGRFKVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRA YQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKIC YNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLL KDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNF FDSRQAPKNMPQDADANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNNGG SPAAKRVKLDGGSPAAKRVKLDHHHHHH SEQ ID NO: 65 – Exemplary FnCas12a nickase variant amino acid sequence MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFI EEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFN QNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENR KNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDY KTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQ INDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKE TLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPS KKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNK DNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKD EHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFERSTLANGWDKNKEPDNTAI LFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFY NPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDT QRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTL YWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITAPAKEAIANKNKDNPKKESVFEYDLI KDKRFTEDKFLFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSIDRGERHLAYYTLVD GKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDAKKINNIKEMKEGYLSQVVHEI AKLVIEYNAIVVFEDLNFGGGRGGFGVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRA YQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKIC YNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLL KDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNF FDSRQAPKNMPQDADANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN SEQ ID NO: 66 – Exemplary FnCas12a nickase variant amino acid sequence MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFI EEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFN QNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENR KNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDY KTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQ INDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPS KKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNK DNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKD EHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFERSTLANGWDKNKEPDNTAI LFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFY NPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDT QRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTL YWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITAPAKEAIANKNKDNPKKESVFEYDLI KDKRFTEDKFLFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSIDRGERHLAYYTLVD GKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDAKKINNIKEMKEGYLSQVVHEI AKLVIEYNAIVVFEDLNFGGGRGGFGVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRA YQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKIC YNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLL KDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNF FDSRQAPKNMPQDADANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNNGG SPAAKRVKLDGGSPAAKRVKLD SEQ ID NO: 67 – Exemplary FnCas12a nickase variant amino acid sequence MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFI EEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFN QNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENR KNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDY KTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQ INDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKE TLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPS KKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNK DNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKD EHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFERSTLANGWDKNKEPDNTAI LFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFY NPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDT QRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTL YWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITAPAKEAIANKNKDNPKKESVFEYDLI KDKRFTEDKFLFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSIDRGERHLAYYTLVD GKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDAKKINNIKEMKEGYLSQVVHEI AKLVIEYNAIVVFEDLNFGGGRGGFGVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRA YQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKIC YNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLL KDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNF FDSRQAPKNMPQDADANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNNGG SPAAKRVKLDGGSPAAKRVKLDHHHHHH SEQ ID NO: 68 – Exemplary FnCas12a nickase variant amino acid sequence MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFI EEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFN QNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENR KNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDY KTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQ INDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKE TLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPS KKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNK DNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAI LFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFY NPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDT QRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTL YWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLI KDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSIDRGERHLAYYTLVD GKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDAKKINNIKEMKEGYLSQVVHEI AKLVIEYNAIVVFEDLNFGGGRGGFGVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRA YQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKIC YNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLL KDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNF FDSRQAPKNMPQDADANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN SEQ ID NO: 69 – Exemplary FnCas12a nickase variant amino acid sequence MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFI EEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFN QNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENR KNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDY KTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQ INDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKE TLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPS KKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNK DNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKD EHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAI LFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFY NPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDT QRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTL YWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLI KDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSIDRGERHLAYYTLVD GKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDAKKINNIKEMKEGYLSQVVHEI AKLVIEYNAIVVFEDLNFGGGRGGFGVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRA YQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKIC YNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLL KDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNF FDSRQAPKNMPQDADANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN GGSPAAKRVKLDGGSPAAKRVKLD SEQ ID NO: 70 – Exemplary FnCas12a nickase variant amino acid sequence MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFI EEILSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFN QNLIDAKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENR KNVYSSNDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDY KTSEVNQRVFSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQ INDKTLKKYKMSVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKE TLSLLFDDLKAQKLDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPS KKEQELIAKKTEKAKYLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNK DNLAQISIKYQNQGKKDLLQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKD EHFYLVFEECYFELANIVPLYNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAI LFIKDDKYYLGVMNKKNNKIFDDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFY NPSEDILRIRNHSTHTKNGSPQKGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDT QRYNSIDEFYREVENQGYKLTFENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEAELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLI KDKRFTEDKFFFHCPITINFKSSGANKFNDEINLLLKEKANDVHILSIDRGERHLAYYTLVD GKGNIIKQDTFNIIGNDRMKTNYHDKLAAIEKDRDSARKDAKKINNIKEMKEGYLSQVVHEI AKLVIEYNAIVVFEDLNFGGGRGGFGVEKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRA YQLTAPFETFKKMGKQTGIIYYVPAGFTSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKIC YNLDKGYFEFSFDYKNFGDKAAKGKWTIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLL KDYSIEYGHGECIKAAICGESDKKFFAKLTSVLNTILQMRNSKTGTELDYLISPVADVNGNF FDSRQAPKNMPQDADANGAYHIGLKGLMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN GGSPAAKRVKLDGGSPAAKRVKLDHHHHHH SEQ ID NO: 71 – Exemplary LbCas12a wild-type amino acid sequence MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAEDYKGVKKLLDRYYLSFI NDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKEIAKAFKGNEGYKSLFKKDII ETILPEFLDDKDEIALVNSFNGFTTAFTGFFDNRENMFSEEAKSTSIAFRCINENLTRYISN MDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGEFFNFVLTQEGIDVYNAIIGGFVTES GEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVLSDRESLSFYGEGYTSDEEVLEVFRNTLNK NSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKDIFGEWNVIRDKWNAEYDDIHLK KKAVVTEKYEDDRRKSFKKIGSFSLEQLQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEK LFDADFVLEKSLKKNDAVVAIMKDLLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDI LLKVDHIYDAIRNYVTQKPYSKDKFKLYFQNPQFMGGWDKDKETDYRATILRYGSKYYLAIM DKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGT FKKGDMFNLNDCHKLIDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSF ESASKKEVDKLVEEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAEL FMRRASLKKEELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYELHIPIAINKCP KNIFKINTEVRVLLKHDDNPYVIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRI KTDYHSLLDKKEKERFEARQNWTSIENIKELKAGYISQVVHKICELVEKYDAVIALEDLNSG FKNSRVKVEKQVYQKFEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQNGF IFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALDYKNFSR TDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGINYQQGDIRALLC EQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLISPVKNSDGIFYDSRNYEAQENAILPKN ADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTSVKHKRPAATKKAGQA KKKKGSYPYDVPDYAYPYDVPDYAYPYDVPDYA SEQ ID NO: 72 – Exemplary LbCas12a wild-type amino acid sequence MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAEDYKGVKKLLDRYYLSFI NDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKEIAKAFKGNEGYKSLFKKDII ETILPEFLDDKDEIALVNSFNGFTTAFTGFFDNRENMFSEEAKSTSIAFRCINENLTRYISN MDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGEFFNFVLTQEGIDVYNAIIGGFVTES GEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVLSDRESLSFYGEGYTSDEEVLEVFRNTLNK NSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKDIFGEWNVIRDKWNAEYDDIHLK KKAVVTEKYEDDRRKSFKKIGSFSLEQLQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEK LFDADFVLEKSLKKNDAVVAIMKDLLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDI LLKVDHIYDAIRNYVTQKPYSKDKFKLYFQNPQFMGGWDKDKETDYRATILRYGSKYYLAIM DKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGT FKKGDMFNLNDCHKLIDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSF ESASKKEVDKLVEEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAEL FMRRASLKKEELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYELHIPIAINKCP KNIFKINTEVRVLLKHDDNPYVIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRI KTDYHSLLDKKEKERFEARQNWTSIENIKELKAGYISQVVHKICELVEKYDAVIALEDLNSG FKNSRVKVEKQVYQKFEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQNGF IFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALDYKNFSRTDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGINYQQGDIRALLC EQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLISPVKNSDGIFYDSRNYEAQENAILPKN ADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTSVKHKRPAATKKAGQA KKKKGSYPYDVPDYAYPYDVPDYAYPYDVPDYAGGSPAAKRVKLDGGSPAAKRVKLD SEQ ID NO: 73 – Exemplary LbCas12a wild-type amino acid sequence MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAEDYKGVKKLLDRYYLSFI NDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKEIAKAFKGNEGYKSLFKKDII ETILPEFLDDKDEIALVNSFNGFTTAFTGFFDNRENMFSEEAKSTSIAFRCINENLTRYISN MDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGEFFNFVLTQEGIDVYNAIIGGFVTES GEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVLSDRESLSFYGEGYTSDEEVLEVFRNTLNK NSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKDIFGEWNVIRDKWNAEYDDIHLK KKAVVTEKYEDDRRKSFKKIGSFSLEQLQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEK LFDADFVLEKSLKKNDAVVAIMKDLLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDI LLKVDHIYDAIRNYVTQKPYSKDKFKLYFQNPQFMGGWDKDKETDYRATILRYGSKYYLAIM DKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGT FKKGDMFNLNDCHKLIDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSF ESASKKEVDKLVEEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAEL FMRRASLKKEELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYELHIPIAINKCP KNIFKINTEVRVLLKHDDNPYVIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRI KTDYHSLLDKKEKERFEARQNWTSIENIKELKAGYISQVVHKICELVEKYDAVIALEDLNSG FKNSRVKVEKQVYQKFEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQNGF IFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALDYKNFSR TDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGINYQQGDIRALLC EQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLISPVKNSDGIFYDSRNYEAQENAILPKN ADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTSVKHKRPAATKKAGQA KKKKGSYPYDVPDYAYPYDVPDYAYPYDVPDYAGGSPAAKRVKLDGGSPAAKRVKLDHHHHH H SEQ ID NO: 74 – Exemplary LbCas12a nickase variant amino acid sequence MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAEDYKGVKKLLDRYYLSFI NDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKEIAKAFKGNEGYKSLFKKDII ETILPEFLDDKDEIALVNSFNGFTTAFTGFFDNRENMFSEEAKSTSIAFRCINENLTRYISN MDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGEFFNFVLTQEGIDVYNAIIGGFVTES GEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVLSDRESLSFYGEGYTSDEEVLEVFRNTLNK NSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKDIFGEWNVIRDKWNAEYDDIHLK KKAVVTEKYEDDRRKSFKKIGSFSLEQLQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEK LFDADFVLEKSLKKNDAVVAIMKDLLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDI LLKVDHIYDAIRNYVTQKPYSKDKFKLYFQRPQFMGGWDKDKETDYRATILRYGSKYYLAIM DKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGT FKKGDMFNLNDCHKLIDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSF ESASKKEVDKLVEEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAEL FMRRASLKKEELVVAPANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYLLHIPIAINKCP KNIFKINTEVRVLLKHDDNPYVIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRI KTDYHSLLDKKEKERFEARQNATSIENIKELKAGYISQVVHKICELVEKYDAVIALEDLNSG GGNSGVGVEKQVYQKFEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQNGF IFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALDYKNFSR TDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGINYQQGDIRALLC EQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLISPVKNSDGIFYDSRNYEAQENAILPKN ADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTSVKHKRPAATKKAGQA KKKKGSYPYDVPDYAYPYDVPDYAYPYDVPDYASEQ ID NO: 75 – Exemplary LbCas12a nickase variant amino acid sequence MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAEDYKGVKKLLDRYYLSFI NDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKEIAKAFKGNEGYKSLFKKDII ETILPEFLDDKDEIALVNSFNGFTTAFTGFFDNRENMFSEEAKSTSIAFRCINENLTRYISN MDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGEFFNFVLTQEGIDVYNAIIGGFVTES GEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVLSDRESLSFYGEGYTSDEEVLEVFRNTLNK NSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKDIFGEWNVIRDKWNAEYDDIHLK KKAVVTEKYEDDRRKSFKKIGSFSLEQLQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEK LFDADFVLEKSLKKNDAVVAIMKDLLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDI LLKVDHIYDAIRNYVTQKPYSKDKFKLYFQRPQFMGGWDKDKETDYRATILRYGSKYYLAIM DKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGT FKKGDMFNLNDCHKLIDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSF ESASKKEVDKLVEEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAEL FMRRASLKKEELVVAPANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYLLHIPIAINKCP KNIFKINTEVRVLLKHDDNPYVIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRI KTDYHSLLDKKEKERFEARQNATSIENIKELKAGYISQVVHKICELVEKYDAVIALEDLNSG GGNSGVGVEKQVYQKFEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQNGF IFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALDYKNFSR TDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGINYQQGDIRALLC EQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLISPVKNSDGIFYDSRNYEAQENAILPKN ADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTSVKHKRPAATKKAGQA KKKKGSYPYDVPDYAYPYDVPDYAYPYDVPDYAGGSPAAKRVKLDGGSPAAKRVKLD SEQ ID NO: 76 – Exemplary LbCas12a nickase variant amino acid sequence MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAEDYKGVKKLLDRYYLSFI NDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKEIAKAFKGNEGYKSLFKKDII ETILPEFLDDKDEIALVNSFNGFTTAFTGFFDNRENMFSEEAKSTSIAFRCINENLTRYISN MDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGEFFNFVLTQEGIDVYNAIIGGFVTES GEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVLSDRESLSFYGEGYTSDEEVLEVFRNTLNK NSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKDIFGEWNVIRDKWNAEYDDIHLK KKAVVTEKYEDDRRKSFKKIGSFSLEQLQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEK LFDADFVLEKSLKKNDAVVAIMKDLLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDI LLKVDHIYDAIRNYVTQKPYSKDKFKLYFQRPQFMGGWDKDKETDYRATILRYGSKYYLAIM DKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGT FKKGDMFNLNDCHKLIDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSF ESASKKEVDKLVEEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAEL FMRRASLKKEELVVAPANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYLLHIPIAINKCP KNIFKINTEVRVLLKHDDNPYVIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRI KTDYHSLLDKKEKERFEARQNATSIENIKELKAGYISQVVHKICELVEKYDAVIALEDLNSG GGNSGVGVEKQVYQKFEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQNGF IFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALDYKNFSR TDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGINYQQGDIRALLC EQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLISPVKNSDGIFYDSRNYEAQENAILPKN ADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTSVKHKRPAATKKAGQA KKKKGSYPYDVPDYAYPYDVPDYAYPYDVPDYAGGSPAAKRVKLDGGSPAAKRVKLDHHHHH H SEQ ID NO: 77 – Exemplary LbCas12a nickase variant amino acid sequence MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAEDYKGVKKLLDRYYLSFI NDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKEIAKAFKGNEGYKSLFKKDIIETILPEFLDDKDEIALVNSFNGFTTAFTGFFDNRENMFSEEAKSTSIAFRCINENLTRYISN MDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGEFFNFVLTQEGIDVYNAIIGGFVTES GEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVLSDRESLSFYGEGYTSDEEVLEVFRNTLNK NSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKDIFGEWNVIRDKWNAEYDDIHLK KKAVVTEKYEDDRRKSFKKIGSFSLEQLQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEK LFDADFVLEKSLKKNDAVVAIMKDLLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDI LLKVDHIYDAIRNYVTQKPYSKDKFKLYFQMPQFMGGWDKDKETDYRATILRYGSKYYLAIM DKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGT FKKGDMFNLNDCHKLIDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSF ESASKKEVDKLVEEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAEL FMRRASLKKEELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYFLHIPIAINKCP KNIFKINTEVRVLLKHDDNPYVIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRI KTDYHSLLDKKEKERFEARQNATSIENIKELKAGYISQVVHKICELVEKYDAVIALEDLNSG GGNSGVGVEKQVYQKFEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQNGF IFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALDYKNFSR TDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGINYQQGDIRALLC EQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLISPVKNSDGIFYDSRNYEAQENAILPKN ADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTSVKHKRPAATKKAGQA KKKKGSYPYDVPDYAYPYDVPDYAYPYDVPDYA SEQ ID NO: 78 – Exemplary LbCas12a nickase variant amino acid sequence MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAEDYKGVKKLLDRYYLSFI NDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKEIAKAFKGNEGYKSLFKKDII ETILPEFLDDKDEIALVNSFNGFTTAFTGFFDNRENMFSEEAKSTSIAFRCINENLTRYISN MDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGEFFNFVLTQEGIDVYNAIIGGFVTES GEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVLSDRESLSFYGEGYTSDEEVLEVFRNTLNK NSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKDIFGEWNVIRDKWNAEYDDIHLK KKAVVTEKYEDDRRKSFKKIGSFSLEQLQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEK LFDADFVLEKSLKKNDAVVAIMKDLLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDI LLKVDHIYDAIRNYVTQKPYSKDKFKLYFQMPQFMGGWDKDKETDYRATILRYGSKYYLAIM DKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGT FKKGDMFNLNDCHKLIDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSF ESASKKEVDKLVEEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAEL FMRRASLKKEELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYFLHIPIAINKCP KNIFKINTEVRVLLKHDDNPYVIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRI KTDYHSLLDKKEKERFEARQNATSIENIKELKAGYISQVVHKICELVEKYDAVIALEDLNSG GGNSGVGVEKQVYQKFEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQNGF IFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALDYKNFSR TDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGINYQQGDIRALLC EQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLISPVKNSDGIFYDSRNYEAQENAILPKN ADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTSVKHKRPAATKKAGQA KKKKGSYPYDVPDYAYPYDVPDYAYPYDVPDYAGGSPAAKRVKLDGGSPAAKRVKLD SEQ ID NO: 79 – Exemplary LbCas12a nickase variant amino acid sequence MSKLEKFTNCYSLSKTLRFKAIPVGKTQENIDNKRLLVEDEKRAEDYKGVKKLLDRYYLSFI NDVLHSIKLKNLNNYISLFRKKTRTEKENKELENLEINLRKEIAKAFKGNEGYKSLFKKDII ETILPEFLDDKDEIALVNSFNGFTTAFTGFFDNRENMFSEEAKSTSIAFRCINENLTRYISN MDIFEKVDAIFDKHEVQEIKEKILNSDYDVEDFFEGEFFNFVLTQEGIDVYNAIIGGFVTES GEKIKGLNEYINLYNQKTKQKLPKFKPLYKQVLSDRESLSFYGEGYTSDEEVLEVFRNTLNK NSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKDIFGEWNVIRDKWNAEYDDIHLK KKAVVTEKYEDDRRKSFKKIGSFSLEQLQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSLKKNDAVVAIMKDLLDSVKSFENYIKAFFGEGKETNRDESFYGDFVLAYDI LLKVDHIYDAIRNYVTQKPYSKDKFKLYFQMPQFMGGWDKDKETDYRATILRYGSKYYLAIM DKKYAKCLQKIDKDDVNGNYEKINYKLLPGPNKMLPKVFFSKKWMAYYNPSEDIQKIYKNGT FKKGDMFNLNDCHKLIDFFKDSISRYPKWSNAYDFNFSETEKYKDIAGFYREVEEQGYKVSF ESASKKEVDKLVEEGKLYMFQIYNKDFSDKSHGTPNLHTMYFKLLFDENNHGQIRLSGGAEL FMRRASLKKEELVVHPANSPIANKNPDNPKKTTTLSYDVYKDKRFSEDQYFLHIPIAINKCP KNIFKINTEVRVLLKHDDNPYVIGIDRGERNLLYIVVVDGKGNIVEQYSLNEIINNFNGIRI KTDYHSLLDKKEKERFEARQNATSIENIKELKAGYISQVVHKICELVEKYDAVIALEDLNSG GGNSGVGVEKQVYQKFEKMLIDKLNYMVDKKSNPCATGGALKGYQITNKFESFKSMSTQNGF IFYIPAWLTSKIDPSTGFVNLLKTKYTSIADSKKFISSFDRIMYVPEEDLFEFALDYKNFSR TDADYIKKWKLYSYGNRIRIFRNPKKNNVFDWEEVCLTSAYKELFNKYGINYQQGDIRALLC EQSDKAFYSSFMALMSLMLQMRNSITGRTDVDFLISPVKNSDGIFYDSRNYEAQENAILPKN ADANGAYNIARKVLWAIGQFKKAEDEKLDKVKIAISNKEWLEYAQTSVKHKRPAATKKAGQA KKKKGSYPYDVPDYAYPYDVPDYAYPYDVPDYAGGSPAAKRVKLDGGSPAAKRVKLDHHHHH H

[0129] Cas12a effector proteins can be, in some embodiments, size-optimized or truncated, for instance via one or more deletions that reduce the size of the effector protein while still retaining gRNA association, target and PAM recognition, and cleavage activities (e.g., non-target strand cleavage activity). In some embodiments, Cas12a effector proteins are bound, covalently or non-covalently, to another polypeptide, nucleotide, or other structure, optionally by means of a linker. Exemplary bound effector proteins and linkers are described by Guilinger et al., Nature Biotech 32:577-582 (2014), the contents of which is hereby incorporated by reference herein in its entirety.

[0130] Additional suitable Cas12a effector proteins and variants thereof will be apparent to the skilled artisan based on the present. Moreover, a number of amino acid sequences of wild-type Cas12a effector protein orthologues are provided in US Publication No.2021 / 0079366 A1, the disclosure of which is hereby incorporated herein by reference in its entirety. Exemplary suitable Cas12a effector proteins may include, but are not limited to, those provided in Tables 2-5. In some embodiments, an AsCas12a effector protein may comprise any of the combinations of amino acid substitutions present in Tables 2-3. In some embodiments, an FnCas12a effector protein may comprise any of the combinations of amino acid substitutions present in Tables 2 and 4. In some embodiments, an LbCas12a effector protein may comprise any of the combinations of amino acid substitutions present in Tables 2 and 5.

[0131] A skilled person would understand that other Cas12a effector proteins and orthologues thereof can be used in accordance with the embodiments described by the present disclosure.Table 2. Exemplary CRISPR / Cas12a effector proteinsTable 3. Exemplary AsCas12a effector proteins with substitutions relative to SEQ ID NO: 4 M537R H800A F870L W382A W958A F999G K1000G S1001G K1002G R1003G R1226A6A6A6ATable 4. Exemplary FnCas12a effector proteins with substitutions relative to SEQ ID NO: 40 N602R H843A F879L Y410A W971A F1012G K1013G R1014G R1016G K1018G R1218G1 X8G8G8GTable 5. Exemplary LbCas12a effector proteins with substitutions relative to SEQ ID NO: 49 8G8G8G8GFusion Proteins for Base Editing

[0132] In one aspect, the present disclosure provides Cas12a effector proteins fused to one or more heterologous protein domains (“fusion proteins”). In some embodiments, fusion proteins as described herein are for base editing. In some embodiments, one or more heterologous protein domains comprise or are deaminase domains and / or polypeptides. Any deaminase domain and / or polypeptide useful for base editing may be used in a fusion protein of the present disclosure. A cytosine base editor (CBE), as used herein, comprises a cytosine deaminase. An adenine base editor (ABE), as used herein, comprises an adenine deaminase. Cytosine deaminase

[0133] In some embodiments, a deaminase comprises or is a cytosine deaminase or a cytidine deaminase. A “cytosine deaminase” and “cytidine deaminase” as used herein refer to a polypeptide or domain thereof that catalyzes or is capable of catalyzing cytosine deamination in that the polypeptide or domain catalyzes or is capable of catalyzing the removal of an amine group from a cytosine base. Thus, a cytosine deaminase may result in conversion of cytosine to a thymidine (through a uracil intermediate), causing a C to T conversion, or a G to A conversion in the complementary strand in the genome. Thus, in some embodiments, a cytosine deaminase encoded by a polynucleotide of the presentdisclosure generates a C to T conversion in the sense (e.g., “+”; template) strand of the target nucleic acid and / or a G to A conversion in antisense (e.g., complementary) strand of the target nucleic acid. In some embodiments, a cytosine deaminase encoded by a polynucleotide of the present disclosure generates a C to T, G, or A conversion in the complementary strand in the genome.

[0134] In some embodiments, a cytosine deaminase may be any known or later identified cytosine deaminase from any organism (see, e.g., U.S. Patent No.10,167,457 and Thuronyi et al. Continuous evolution of base editors with expanded target compatibility and improved activity. Nat. Biotechnol.2019 Sep;37(9):1070-1079. doi: 10.1038 / s41587-019- 0193-0. Epub 2019 Jul 22., each of which is incorporated by reference herein for its disclosure of cytosine deaminases). Cytosine deaminases can catalyze the hydrolytic deamination of cytidine or deoxycytidine to uridine or deoxyuridine, respectively. Thus, in some embodiments, a deaminase or deaminase domain may be a cytidine deaminase domain, catalyzing the hydrolytic deamination of cytosine to uracil. In some embodiments, a cytosine deaminase may be a variant of a naturally-occurring cytosine deaminase, including, but not limited to, a primate (e.g., a human, monkey, chimpanzee, gorilla), a dog, a cow, a rat, or a mouse cytosine deaminase. Thus, in some embodiments, an cytosine deaminase useful with the invention may be about 70% to about 100% identical to a wild-type cytosine deaminase (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, and any range or value therein, to a naturally occurring cytosine deaminase).

[0135] In some embodiments, a cytosine deaminase useful with the invention may be an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase. In some embodiments, a cytosine deaminase may be an APOBEC1 deaminase, an APOBEC2 deaminase, an APOBEC3A deaminase, an APOBEC3B deaminase, an APOBEC3C deaminase, an APOBEC3D deaminase, an APOBEC3F deaminase, an APOBEC3G deaminase, an APOBEC3H deaminase, an APOBEC4 deaminase, a human activation induced deaminase (hAID), an rAPOBEC1, FERNY, and / or a CDA1, optionally a pmCDA1, an atCDA1 (e.g., At2gl9570), and evolved versions of the same. Evolved deaminases are disclosed in, for example, U.S. Patent No.10,113,163, Gaudelli et al., Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 2017 Nov 23;551(7681):464-471. doi: 10.1038 / nature24644. Epub 2017 Oct 25) and Thuronyi et al.Continuous evolution of base editors with expanded target compatibility and improved activity. Nat. Biotechnol.2019 Sep;37(9):1070-1079. doi: 10.1038 / s41587-019-0193-0. Epub 2019 Jul 22., each of which are incorporated by reference herein for their disclosure of deaminases and evolved deaminases. In some embodiments, a cytosine deaminase may be an APOBEC1 deaminase having the amino acid sequence of SEQ ID NO: 81. In some embodiments, a cytosine deaminase may be an APOBEC3A deaminase having the amino acid sequence of SEQ ID NO: 82. In some embodiments, a cytosine deaminase may be a CDA1 deaminase, optionally a CDA1 having the amino acid sequence of SEQ ID NO: 83. In some embodiments, a cytosine deaminase may be a FERNY deaminase, optionally a FERNY having the amino acid sequence of SEQ ID NO: 84. In some embodiments, a cytosine deaminase may be an rAPOBEC1 deaminase, optionally an rAPOBEC1 deaminase having the amino acid sequence of SEQ ID NO: 85. In some embodiments, a cytosine deaminase may be an hAID deaminase, optionally an hAID having the amino acid sequence of SEQ ID NO: 86 or SEQ ID NO: 87. In some embodiments, a cytosine deaminase may be about 70% to about 100% identical (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical) to the amino acid sequence of a naturally occurring cytosine deaminase (e.g., “evolved deaminases”) (see, e.g., SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90). In some embodiments, a cytosine deaminase useful with the invention may be about 70% to about 99.5% identical (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical) to the amino acid sequence of any one of SEQ ID NOs: 81-90 (e.g., at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence of any one of SEQ ID NOs: 81-90). In some embodiments, a polynucleotide encoding a cytosine deaminase may be codon optimized for expression in a mammal and the codon optimized polynucleotide may be about 70% to 99.5% identical to the reference polynucleotide.

[0136] In some embodiments a cytosine base editor (CBE) of the present disclosure comprises a cytidine deaminase fused to a Cas12a nickase tethered to one (BE3) or two (BE4) monomers of uracil glycosylase inhibitor (UGI). In some embodiments the cytidine deaminase is PpAPOBEC1, e.g., having the following sequence:MTSEKGPSTGDPTLRRRIESWEFDVFYDPRELRKETCLLYEIKWGMSRKIWRSSGKNTTNHV EVNFIKKFTSERRFHSSISCSITWFLSWSPCWECSQAIREFLSQHPGVTLVIYVARLFWHMD QRNRQGLRDLVNSGVTIQIMRASEYYHCWRNFVNYPPGDEAHWPQYPPLWMMLYALELHCII LSLPPCLKISRRWQNHLAFFRLHLQNCHYQTIPPHILLATGLIHPSVTWR (SEQ ID NO: 80).

[0137] In some embodiments the cytosine base editor comprises PpAPOBEC1 fused to a Cas12a nickase tethered to two (BE4) monomers of uracil glycosylase inhibitor (UGI), e.g., as described in Yu et al., Nat. Comm.11: 2052, 2020 and WO2020160517A1 (the entire contents of each of which are incorporated herein by reference). Exemplary Cytosine Deaminase Sequences SSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVE VNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADP RNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIIL GLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK (SEQ ID NO: 81) MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTSVKMDQHRGFLHNQAKNL LCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGEVRAFLQENTHVRLRI FAARIYDYDPLYKEALQMLRDAGAQVSIMTYDEFKHCWDTFVDHQGCPFQPWDGLDEHSQAL SGRLRAILQNQGN (SEQ ID NO: 82) MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGERRACFWGYAVNKPQSGTE RGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKILEWYNQELRGNGHTLKIWAC KLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCRKIFIQSSHNQLNENRWLEKTLKRAEK RRSELSIMIQVKILHTTKSPAV (SEQ ID NO: 83) FERNYDPRELRKETYLLYEIKWGKSGKLWRHWCQNNRTQHAEVYFLENIFNARRFNPSTHCS ITWYLSWSPCAECSQKIVDFLKEHPNVLEIYVARLYYHEDERNRQGLRDLVNSGVTIRIMDL PDYNYCWKTFVSDQGGDEDYWPGHFAPWIKQYSLKL (SEQ ID NO: 84) MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHV EVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHAD PRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCII LGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK (SEQ ID NO: 85) MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLRNKNGCHVELLFL RYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGNPNLSLRIFTARLYFCEDRKAEPE GLRRLHRAGVQIAIMTFKDYFYCWNTFVENHERTFKAWEGLHENSVRLSRQLRRILLPLYEV DDLRDAFRTLGL (SEQ ID NO: 86) MDSLLMNRREFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLRNKNGCHVELLFL RYISDWDLDPGRCYRVTWFISWSPCYDCARHVADFLRGNPNLSLRIFTARLYFCEDRKAEPE GLRRLHRAGVQIAIMTFKDYFYCWNTFVENHGRTFKAWEGLHENSVRLSRQLRRILLPLYEV DDLRDAFRTCT (SEQ ID NO: 87) TDAEYVRIHEKLDIYTFKKQFSNNKKSVSHRCYVLFELKRRGERRACFWGYAVNKPQSGTER GIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKILEWYNQELRGNGHTLKIWVCKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCRKIFIQSSHNQLNENRWLEKTLKRAEKR RSELSIMFQVKILHTTKSPAV (SEQ ID NO: 88) SSKTGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVE VNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPNVTLFIYIARLYHLANP RNRQGLRDLISSGVTIQIMTEQESGYCWHNFVNYSPSNESHWPRYPHLWVRLYVLELYCIIL GLPPCLNILRRKQSQLTSFTIALQSCHYQRLPPHILWATGLK (SEQ ID NO: 89) SFERNYDPRELRKETYLLYEIKWGKSGKLWRHWCQNNRTQHAEVYFLENIFNARRFNPSTHC SITWYLSWSPCAECSQKIVDFLKEHPNVNLEIYVARLYYPENERNRQGLRDLVNSGVTIRIM DLPDYNYCWKTFVSDQGGDEDYWPGHFAPWIKQYSLKL (SEQ ID NO: 90) Adenine deaminase

[0138] In some embodiments, a deaminase comprises or is an adenine deaminase or an adenosine deaminase. An “adenine deaminase” and “adenosine deaminase” as used herein refer to a polypeptide or domain thereof that catalyzes or is capable of catalyzing the hydrolytic deamination (e.g., removal of an amine group from adenine) of adenine or adenosine. In some embodiments, an adenine deaminase may catalyze the hydrolytic deamination of adenosine or deoxy adenosine to inosine or deoxyinosine, respectively. In some embodiments, an adenine deaminase may catalyze the hydrolytic deamination of adenine or adenosine in DNA. In some embodiments, an adenine deaminase encoded by a nucleic acid may generate an A to G conversion in the sense (e.g., “+”; template) strand of the target nucleic acid or a T to C conversion in the antisense (e.g.,plementary) strand of the target nucleic acid.

[0139] An adenine deaminase may be any known or later identified adenine deaminase from any organism (see, e.g., U.S. Patent No.10,113,163, which is incorporated by reference herein for its disclosure of adenine deaminases).

[0140] In some embodiments, an adenine deaminase may be a variant of a naturally- occurring adenine deaminase. Thus, in some embodiments, an adenine deaminase may be about 70% to 100% identical to a wild-type adenine deaminase (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, and any range or value therein, to a naturally occurring adenine deaminase). In some embodiments, an adenine deaminase does not occur in nature and may be referred to as an engineered, mutated or evolved adenine deaminase. Thus, for example, an engineered, mutated or evolved adenine deaminase polypeptide or an adenine deaminase domain may be about 70% to 99.9% identical to a naturally occurring adenine deaminase polypeptide / domain (e.g.,about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical, and any range or value therein, to a naturally occurring adenine deaminase polypeptide or adenine deaminase domain). In some embodiments, the adenosine deaminase may be from a bacterium, (e.g., Escherichia coli, Staphylococcus aureus, Haemophilus influenzae, Caulobacter crescentus). In some embodiments, a polynucleotide encoding an adenine deaminase polypeptide / domain may be codon optimized for expression in a mammal and the codon optimized polynucleotide may be about 70% to 99.5% identical to the reference polynucleotide.

[0141] In some embodiments, an adenine deaminase domain may be a wild-type tRNA-specific adenosine deaminase domain, e.g., a tRNA-specific adenosine deaminase (TadA) and / or a mutated / evolved adenosine deaminase domain, e.g., mutated / evolved tRNA- specific adenosine deaminase domain (TadA*). In some embodiments, a TadA domain may be from E. coli. In some embodiments, a TadA may be modified, e.g., truncated, missing one or more N-terminal and / or C-terminal amino acids relative to a full-length TadA (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 N-terminal and / or C terminal amino acid residues may be missing relative to a full length TadA. In some embodiments, a TadA polypeptide or TadA domain does not comprise an N-terminal methionine. In some embodiments, a wild-type E. coli TadA comprises the amino acid sequence of SEQ ID NO: 94. In some embodiments, a mutated / evolved E. coli TadA* comprises the amino acid sequence of SEQ ID NOs: 91-93 (e.g., SEQ ID NOs: 91, 92, or 93). In some embodiments, a polynucleotide encoding a TadA / TadA* may be codon optimized for expression in a mammal. In some embodiments, an adenine deaminase may comprise all or a portion of an amino acid sequence of any one of SEQ ID NOs: 95-105.

[0142] In some embodiment an adenine base editor of the present disclosure comprises an adenosine deaminase with one or more mutations to reduce RNA editing activity. In some embodiments, the base editor comprises an engineered E. coli TadA, e.g., with the mutations found in ABEs 0.1, 0.2, 1.1 , 1.2, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 2.10, 2.11, 2.12, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 4.1, 4.2, 4.3, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 5.10, 5.11, 5.12, 5.13, 5.14, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 7.10, ABEmax as described in Gaudelli et al., Nature 551(7681): 464-471, 2017 and Koblan et al., Nat. Biotechnol.36(9):843-846, 2018 (the entire contents of each of whichare incorporated herein by reference) or any of the ABE8s variants, e.g., ABE8.17-m, described in Gaudelli et al., Nat. Biotechnol.38:892-900, 2020 and US20210130805A1 (the entire contents of each of which are incorporated herein by reference). The mutations can include substitution with any other amino acid other than the wild-type amino acid. In some embodiments the substitution is with alanine or glycine. For example, the engineered E.coli TadA sequence present in ABE7.10 (TadA*7.10) is as follows: SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMAL RQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPG MNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 91).

[0143] In ABE7.10, a wild-type E.coli TadA sequence is fused to this engineered E.coli TadA sequence using a 32 amino acid linker, forming a heterodimer, the sequence of which is as follows: MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMA LRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHP GMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTDSGGSSGGSSGSETPGTSES ATPESSGGSSGGSSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIG LHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTG AAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 92).

[0144] As a further example, ABE8.17-m comprises a monomeric construct containing TadA*7.10 with V82S and Q154R mutations (TadA*8.17) as follows: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA LRQGGLVMQNYRLIDATLYSTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYP GMNHRVEITEGILADECAALLCYFFRMPRRVFNAQKKAQSSTD (SEQ ID NO: 93).

[0145] In some embodiments an adenine base editor (ABE) of the present disclosure comprises an adenosine deaminase fused to a Cas12a nickase, e.g., a Cas12a nickase fused to a wild-type E.coli TadA, e.g., of SEQ ID NO: 94: MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMA LRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHP GMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD (SEQ ID NO: 94) Exemplary Adenine Deaminase Sequences SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMAL RQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPG MNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD (SEQ ID NO: 95) SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMAL RQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPG MNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 96)SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVLNNRVIGEGWNRSIGLHDPTAHAEIMAL RQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPG MNHRVEITEGILADECAALLCYFFRMRRQVFNAQKKAQSSTD (SEQ ID NO: 97) SEVEFSHEYWMRHALTLAKRALDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMAL RQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPG MNHRVEITEGILADECNALLCYFFRMRRQVFNAQKKAQSSTD (SEQ ID NO: 98) SEVEFSHEYWMRHALTLAKRALDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMAL RQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPG MNHRVEITEGILADECNALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 99) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMAL RQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPG MNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD (SEQ ID NO: 100) SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMAL RQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYPG MNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN (SEQ ID NO: 101) SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMAL RQGGLVMQNYRLYDATLYSTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHPG MNHRVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTD (SEQ ID NO: 102) SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMAL RQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPG MNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTDSGGSSGGSSGSETPGTSESA TPESSGGSSGGSSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGL HDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGA AGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 103) MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA LRQGGLVMQNYRLYDATLYSTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHHP GMNHRVEITEGILADECAALLCRFFRMPRRVFNAQKKAQSSTD (SEQ ID NO: 104) MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA LRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNSKRGAAGSLMNVLNYP GMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSIN (SEQ ID NO: 105)

[0146] In some embodiments, a nucleic acid of the present disclosure may further encode a glycosylase inhibitor (e.g., a uracil glycosylase inhibitor (UGI) such as uracil-DNA glycosylase inhibitor). Thus, in some embodiments, a nucleic acid encoding a Cas12a effector protein and a cytosine deaminase and / or adenine deaminase may further encode a glycosylase inhibitor, optionally wherein the glycosylase inhibitor may be codon optimized for expression in a mammal. In some embodiments, present disclosure provides fusionproteins comprising a Cas12a effector protein and a UGI and / or one or more polynucleotides encoding the same, optionally wherein the one or more polynucleotides may be codon optimized for expression in a mammal. In some embodiments, the present disclosure provides fusion proteins comprising a Cas12a effector protein, a deaminase domain (e.g., an adenine deaminase domain and / or a cytosine deaminase domain) and a UGI and / or one or more polynucleotides encoding the same, optionally wherein the one or more polynucleotides may be codon optimized for expression in a mammal. In some embodiments, the invention provides fusion proteins, wherein a Cas12a effector protein, a deaminase domain, and / or a UGI may be fused to any combination of peptide tags and affinity polypeptides as described herein, which may thereby recruit the deaminase domain and / or UGI to the Cas12a effector protein and to a target nucleic acid. In some embodiments, a guide nucleic acid may be linked to a recruiting RNA motif and one or more of the deaminase domain and / or UGI may be fused to an affinity polypeptide that is capable of interacting with the recruiting RNA motif, thereby recruiting the deaminase domain and UGI to a target nucleic acid.

[0147] A “uracil glycosylase inhibitor” or “UGI” may be any protein or polypeptide or domain thereof that is capable of inhibiting a uracil-DNA glycosylase base-excision repair enzyme. In some embodiments, a UGI comprises a wild-type UGI or a fragment thereof. In some embodiments, a UGI is about 70% to about 100% identical (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical and any range or value therein) to the amino acid sequence of a naturally occurring UGI. In some embodiments, a UGI may comprise the amino acid sequence of: TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAP EYKPWALVIQDSNGENKIKML (SEQ ID NO: 106) or a polypeptide having about 70% to about 99.5% identity to the amino acid sequence of SEQ ID NO: 106 (e.g., at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence of SEQ ID NO: 106). For example, in some embodiments, a UGI may comprise a fragment of the amino acid sequence of SEQ ID NO: 106 that is 100% identical to a portion of consecutive nucleotides (e.g., 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 consecutive nucleotides; e.g., about 10, 15, 20, 25, 30, 35, 40, 45, to about 50, 55, 60, 65, 70, 75, 80 consecutive nucleotides) of the amino acid sequence of SEQ ID NO: 106. In some embodiments, a UGI may be a variant of a known UGI (e.g., SEQ ID NO: 106) havingabout 70% to about 99.5% identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% identity, and any range or value therein) to the known UGI. In some embodiments, a polynucleotide encoding a UGI may be codon optimized for expression in a mammal and the codon optimized polynucleotide may be about 70% to about 99.5% identical to the reference polynucleotide. Fusion Proteins for Prime Editing

[0148] In one aspect, the present disclosure provides Cas12a effector proteins fused to one or more heterologous protein domains (“fusion proteins”). In some embodiments, fusion proteins as described herein are for prime editing. In some embodiments, one or more heterologous protein domains comprise or are polymerase domains and / or polypeptides. In some embodiments, one or more heterologous protein domains comprise or are reverse transcriptase domains and / or polypeptides. In some embodiments, one or more heterologous protein domains comprise or are DNA polymerase domains and / or polypeptides.

[0149] Any polymerase domain and / or polypeptide useful for prime editing may be used in a fusion protein of the present disclosure. Reverse transcriptase

[0150] In some embodiments, one or more heterologous protein domains comprise or are reverse transcriptase domains and / or polypeptides. In some embodiments, a reverse transcriptase is or comprises a M-MMLV reverse transcriptase, or variant thereof. In some embodiments, a reverse transcriptase is or comprises a HIV-1 reverse transcriptase, or variant thereof. In some embodiments, a reverse transcriptase is or comprises a AMV reverse transcriptase, or variant thereof. In some embodiments, a reverse transcriptase is or comprises a telomerase reverse transcriptase, or variant thereof.

[0151] In some embodiments, one or more heterologous protein domains comprise or are error prone reverse transcriptase domains and / or polypeptides. As used herein, the term “error-prone” reverse transcriptase refers to a reverse transcriptase that occurs naturally or which has been derived from another reverse transcriptase (e.g., a wild type M-MLV reverse transcriptase) which has an error rate that is less than the error rate of wild type M-MLV reverse transcriptase.

[0152] As an example, the error rate of wild type M-MLV reverse transcriptase is reported to be in the range of one error in 15,000 (higher) to 27,000 (lower). An error rate of1 in 15,000 corresponds with an error rate of 6.7 x 10-5. An error rate of 1 in 27,000 corresponds with an error rate of 3.7 x 10-5. See Boutabout et al. (2001) “DNA synthesis fidelity by the reverse transcriptase of the yeast retrotransposon Ty1,” Nucleic Acids Res 29(11):2217–2222, which is incorporated herein by reference in its entirety. Thus, for purposes of this application, the term “error prone” refers to those reverse transcriptases that have an error rate that is greater than one error in 15,000 nucleobase incorporation (6.7 x 10-5or higher), e.g., 1 error in 14,000 nucleobases (7.14 x 10-5or higher), 1 error in 13,000 nucleobases or fewer (7.7 x 10-5or higher), 1 error in 12,000 nucleobases or fewer (7.7 x 10-5or higher), 1 error in 11,000 nucleobases or fewer (9.1 x 10-5or higher), 1 error in 10,000 nucleobases or fewer (1 x 10-4or 0.0001 or higher), 1 error in 9,000 nucleobases or fewer (0.00011 or higher), 1 error in 8,000 nucleobases or fewer (0.00013 or higher) 1 error in 7,000 nucleobases or fewer (0.00014 or higher), 1 error in 6,000 nucleobases or fewer (0.00016 or higher), 1 error in 5,000 nucleobases or fewer (0.0002 or higher), 1 error in 4,000 nucleobases or fewer (0.00025 or higher), 1 error in 3,000 nucleobases or fewer (0.00033 or higher), 1 error in 2,000 nucleobase or fewer (0.00050 or higher), or 1 error in 1,000 nucleobases or fewer (0.001 or higher), or 1 error in 500 nucleobases or fewer (0.002 or higher), or 1 error in 250 nucleobases or fewer (0.004 or higher). DNA polymerase

[0153] In some embodiments, one or more heterologous protein domains comprise or are DNA polymerase domains and / or polypeptides. In some embodiments, a DNA polymerase is or comprises a T7 DNA polymerase, Pol I polymerase, Pol γ polymerase, Pol θ polymerase, or a Pol ν polymerase. In some embodiments, a DNA polymerase is or comprises a Pol II polymerase, Pol B polymerase, Pol ζ polymerase, Pol α polymerase, Pol δ polymerase, or Pol ε polymerase. In some embodiments, a DNA polymerase is or comprises a Pol III polymerase. In some embodiments, a DNA polymerase is a Pol D polymerase. In some embodiments, a DNA polymerase is or comprises a Pol β polymerase, Pol σ polymerase, Pol λ polymerase, Pol μ polymerase, or terminal deoxynucleotidyl transferase. In some embodiments, a polymerase is or comprises a Pol ι polymerase, Pol κ polymerase, Pol η polymerase, Pol IV polymerase, or Pol V polymerase. Guide RNA (gRNA) molecules

[0154] A gRNA molecule or gRNA for use in a CRISPR / Cas12a genome editing system generally includes a targeting domain and a complementarity domain (alternatelyreferred to as a “handle”). It should also be noted that, in gRNAs for use with Cas12a, the targeting domain is usually present at or near the 3’ end, rather than the 5’ end as in connection with Cas9 gRNAs (the handle is at or near the 5’ end of a Cas12a gRNA).

[0155] Those of skill in the art will appreciate, however, that although structural differences may exist between gRNAs from different prokaryotic species, the principles by which gRNAs operate are generally consistent. Because of this consistency of operation, gRNAs can be defined, in broad terms, by their targeting domain sequences, and skilled artisans will appreciate that a given targeting domain sequence can be incorporated in any suitable gRNA, including a unimolecular or chimeric gRNA, or a gRNA that includes one or more chemical modifications and / or sequential modifications (substitutions, additional nucleotides, truncations, etc.). Thus, for economy of presentation in this disclosure, gRNAs may be described solely in terms of their targeting domain sequences.

[0156] More generally, skilled artisans will appreciate that some aspects of the present disclosure relate to systems, methods and compositions that can be implemented using multiple Cas effector proteins. For this reason, unless otherwise specified, the term gRNA should be understood to encompass any suitable gRNA that can be used with any CRISPR / Cas effector system, and not only those gRNAs that are compatible with a particular species of Cas12a. By way of illustration, the term gRNA can, in some embodiments, include a gRNA for use with any Cas effector protein occurring in a Class 2 CRISPR system, such as a Type V CRISPR system, or a Cas effector protein derived or adapted therefrom.

[0157] In some embodiments a method or system of the present disclosure may use more than one gRNA. In some embodiments, two or more gRNAs may be used to create two or more double strand breaks in the genome of a cell. In some embodiments, two or more gRNAs may be used to create two or more nicks in the genome of a cell.

[0158] In some embodiments using more than one gRNA, a double-strand break may be caused by a dual-gRNA paired “nickase” strategy. gRNA design

[0159] Methods for selection and validation of target nucleic acid sequences as well as off-target analyses have been described previously, e.g., in Fu et al., Nat Biotechnol 32(3):279-84 (2014), Heigwer et al., Nat methods 11(2):122-3 (2014); Bae et al., Bioinformatics 30(10):1473-5 (2014); and Xiao et al. Bioinformatics 30(8):1180-1182 (2014). As a non-limiting example, gRNA design may involve the use of a software tool tooptimize the choice of potential target nucleic acid sequences corresponding to a user’s target nucleic acid sequence, e.g., to minimize total off-target activity across the genome. While off-target activity is not limited to cleavage, the cleavage efficiency at each off-target nucleic acid sequence can be predicted, e.g., using an experimentally-derived weighting scheme. These and other guide selection methods are described in detail in Park et al., Bioinformatics 34(6):1077-1079 (2018), the disclosure of which is hereby incorporated herein by reference in its entirety. gRNA modifications

[0160] In some embodiments, gRNAs as used herein may be modified or unmodified gRNAs. In some embodiments, gRNAs as used herein may be modified for increased activity compared to unmodified gRNAs. In some embodiments, a gRNA may include one or more modifications. In some embodiments, the one or more modifications may include a phosphorothioate linkage modification, a phosphorodithioate linkage modification, a 2’-O- methyl modification, or combinations thereof. In some embodiments, the one or more modifications may be at the 5’ end of the gRNA, at the 3’ end of the gRNA, or combinations thereof.

[0161] In some embodiments, the spacer region of a gRNA is at least 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 nucleic acids in length. In some embodiments, a spacer region comprises an “extended” stretch of nucleic acids as compared to a reference gRNA spacer region (also interchangeably referred to herein as an “extended spacer region”). In some embodiments, the 3’ end of the gRNA includes an additional stretch of nucleotides (e.g., at least 1 nucleotide, at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides) as compared to the 3’ end of a reference gRNA. In some embodiments, a gRNA used herein comprises an extended spacer region that is at least 1 nucleotide, at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, at least 11 nucleotides, at least 12 nucleotides, at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotide longer thana reference gRNA spacer region (e.g., that is about 17 to 21 nucleotides in length). It can be appreciated by those in the art that a gRNA with an extended spacer region may result in potentially protecting the target strand from nicking. In some embodiments, a gRNA with an extended spacer region may have synergistic effects with Cas12a effector proteins described herein. In some embodiments, an extended spacer region may result in 1) inhibiting target strand nicking and / or 2) further stabilization of a 3’ overhang RNA / DNA complex. In some embodiments, the extended spacer region comprises modified nucleotides. In some such embodiments, modified nucleotides include but are not limited to 2’-fluorinated or 2-O- methylated nucleotides.

[0162] In some embodiments, a gRNA used herein includes one or more or a stretch of deoxyribonucleic acid (DNA) bases, also referred to herein as a “DNA extension.” In some embodiments, a gRNA used herein includes a DNA extension at the 5’ end of the gRNA. In some embodiments, the DNA extension may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 DNA bases long. For example, in some embodiments, the DNA extension may be 1, 2, 3, 4, 5, 10, 15, 20, or 25 DNA bases long. In some embodiments, the DNA extension may include one or more DNA bases selected from adenine (A), guanine (G), cytosine (C), or thymine (T). In some embodiments, the DNA extension includes the same DNA bases. For example, the DNA extension may include a stretch of adenine (A) bases. In some embodiments, the DNA extension may include a stretch of thymine (T) bases. In some embodiments, the DNA extension includes a combination of different DNA bases.

[0163] Exemplary suitable 5’ extensions for Cas12a guide RNAs are provided in Table 6 below: Table 6. Exemplary Cas12a gRNA 5’ Extensions SEQ ID NO: 5’ extension sequence 5’ modification N / A rCrUrUrUrU +5 RNAS S S S S S llas a chemical modification, e.g., one or more phosphorothioate linkage modifications, one or more phosphorodithioate linkage modifications, one or more 2’-O-methyl modifications, or one or more additional suitable chemical gRNA modification disclosed herein, orcombinations thereof. In some embodiments, the one or more modifications may be at the 5’ end of the gRNA, at the 3’ end of the gRNA, or combinations thereof.

[0165] Without wishing to be bound by theory, it is contemplated that any DNA extension may be used with any gRNA disclosed herein, so long as it does not hybridize to the target nucleic acid being targeted by the gRNA and it also exhibits an increase in editing at the target nucleic acid site relative to a gRNA which does not include such a DNA extension.

[0166] In some embodiments, a gRNA used herein includes one or more or a stretch of ribonucleic acid (RNA) bases, also referred to herein as an “RNA extension”. In some embodiments, a gRNA used herein includes an RNA extension at the 5’ end of the gRNA, the 3’ end of the gRNA, or a combination thereof. In some embodiments, the RNA extension may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 RNA bases long. For example, in some embodiments, the RNA extension may be 1, 2, 3, 4, 5, 10, 15, 20, or 25 RNA bases long. Exemplary suitable 5’ extensions for Cas12a guide RNAs are provided in Table 6 above. In some embodiments, the RNA extension may include one or more RNA bases selected from adenine (rA), guanine (rG), cytosine (rC), or uracil (rU), in which the “r” represents RNA, 2’-hydroxy. In some embodiments, the RNA extension includes the same RNA bases. For example, the RNA extension may include a stretch of adenine (rA) bases. In some embodiments, the RNA extension includes a combination of different RNA bases. In some embodiments, a gRNA used herein includes an RNA extension as well as one or more phosphorothioate linkage modifications, one or more phosphorodithioate (PS2) linkage modifications, one or more 2’-O-methyl modifications, one or more additional suitable gRNA modification, e.g., chemical modification, disclosed herein, or combinations thereof. In some embodiments, the one or more modifications may be at the 5’ end of the gRNA, at the 3’ end of the gRNA, or combinations thereof. In some embodiments, a gRNA including a RNA extension may comprise a sequence set forth herein.

[0167] It is contemplated that gRNAs used herein may also include an RNA extension and a DNA extension. In some embodiments, the RNA extension and DNA extension may both be at the 5’ end of the gRNA, the 3’ end of the gRNA, or a combination thereof. In some embodiments, the RNA extension is at the 5’ end of the gRNA and the DNA extensionis at the 3’ end of the gRNA. In some embodiments, the RNA extension is at the 3’ end of the gRNA and the DNA extension is at the 5’ end of the gRNA.

[0168] In some embodiments, a gRNA which includes a modification, e.g., a DNA extension at the 5’ end and / or a chemical modification as disclosed herein, is complexed with a Cas effector protein, e.g., a Cas12a effector protein, to form an RNP, which is then employed to edit a target cell, e.g., a pluripotent stem cell or a progeny thereof.

[0169] Certain exemplary modifications discussed in this section can be included at any position within a gRNA sequence including, without limitation at or near the 5’ end (e.g., within 1-10, 1-5, or 1-2 nucleotides of the 5’ end) and / or at or near the 3’ end (e.g., within 1- 10, 1-5, or 1-2 nucleotides of the 3’ end). In some cases, modifications are positioned within functional motifs, such as a stem loop structure of a Cas12a gRNA, and / or a targeting domain of a gRNA.

[0170] As one example, the 5’ end of a gRNA can include a eukaryotic mRNA cap structure or cap analog (e.g., a G(5’)ppp(5’)G cap analog, a m7G(5’)ppp(5’)G cap analog, or a 3’-O-Me-m7G(5’)ppp(5’)G anti-reverse cap analog (ARCA)), as shown below: The cap ornthesis of the gRNA.

[0171] Along similar lines, the 5’ end of the gRNA can lack a 5’ triphosphate group. For instance, in vitro transcribed gRNAs can be phosphatase-treated (e.g., using calf intestinal alkaline phosphatase) to remove a 5’ triphosphate group.

[0172] Another common modification involves the addition, at the 3’ end of a gRNA, of a plurality (e.g., 1-10, 10-20, or 25-200) of adenine (A) residues referred to as a polyA tract. The polyA tract can be added to a gRNA during chemical or enzymatic synthesis, using a polyadenosine polymerase (e.g., E. coli Poly(A)Polymerase).

[0173] Guide RNAs can be modified at a 3’ terminal U ribose. For example, the two terminal hydroxyl groups of the U ribose can be oxidized to aldehyde groups and a concomitant opening of the ribose ring to afford a modified nucleoside as shown below:wherein “U” can be an unmodified or modified uridine.

[0174] The 3’ terminal U ribose can be modified with a 2’3’ cyclic phosphate as shown below: wherein “U” can be an unmodified or modified uridine.

[0175] Guide RNAs can contain 3’ nucleotides that can be stabilized against degradation, e.g., by incorporating one or more of the modified nucleotides described herein. In some embodiments, uridines can be replaced with modified uridines, e.g., 5-(2- amino)propyl uridine, and 5-bromo uridine, or with any of the modified uridines described herein; adenosines and guanosines can be replaced with modified adenosines and guanosines, e.g., with modifications at the 8-position, e.g., 8-bromo guanosine, or with any of the modified adenosines or guanosines described herein.

[0176] In some embodiments, sugar-modified ribonucleotides can be incorporated into a gRNA, e.g., wherein the 2’ OH-group is replaced by a group selected from H, -OR, -R (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), halo, -SH, -SR (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), amino (wherein amino can be, e.g., NH2, alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); or cyano (-CN). In some embodiments, the phosphate backbone can be modified as described herein, e.g., with a phosphothioate (PhTx) group. In some embodiments, one or more of the nucleotides of the gRNA can each independently be a modified or unmodified nucleotide including, but not limited to 2’-sugar modified, such as, 2’-O-methyl, 2’-O-methoxyethyl, or 2’-Fluoro modified including, e.g., 2’-F or 2’-O-methyl, adenosine (A), 2’-F or 2’-O-methyl, cytidine (C), 2’-F or 2’-O-methyl, uridine (U), 2’-F or 2’-O-methyl, thymidine (T), 2’-F or 2’-O-methyl, guanosine (G), 2’-O-methoxyethyl-5-methyluridine (Teo), 2’-O-methoxyethyladenosine (Aeo), 2’-O- methoxyethyl-5-methylcytidine (m5Ceo), and any combinations thereof.

[0177] Guide RNAs can also include “locked” nucleic acids (LNA) in which the 2’ OH-group can be connected, e.g., by a C1-6 alkylene or C1-6 heteroalkylene bridge, to the 4’ carbon of the same ribose sugar. Any suitable moiety can be used to provide such bridges, including without limitation methylene, propylene, ether, or amino bridges; O-amino (wherein amino can be, e.g., NH2, alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino) and aminoalkoxy or O(CH2)n-amino (wherein amino can be, e.g., NH2, alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino).

[0178] In some embodiments, a gRNA can include a modified nucleotide which is multicyclic (e.g., tricyclo; and “unlocked” forms, such as glycol nucleic acid (GNA) (e.g., R- GNA or S-GNA, where ribose is replaced by glycol units attached to phosphodiester bonds), or threose nucleic acid (TNA, where ribose is replaced with α-L-threofuranosyl-(3’→2’)).

[0179] Generally, gRNAs include the sugar group ribose, which is a 5-membered ring having an oxygen. Exemplary modified gRNAs can include, without limitation, replacement of the oxygen in ribose (e.g., with sulfur (S), selenium (Se), or alkylene, such as, e.g., methylene or ethylene); addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ring contraction of ribose (e.g., to form a 4-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g., to form a 6- or 7-membered ring having an additional carbon or heteroatom, such as for example, anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino that also has a phosphoramidate backbone). Although the majority of sugar analog alterations are localized to the 2’ position, other sites are amenable to modification, including the 4’ position. In some embodiments, a gRNA comprises a 4’-S, 4’-Se or a 4’-C-aminomethyl-2’-O-Me modification.

[0180] In some embodiments, deaza nucleotides, e.g., 7-deaza-adenosine, can be incorporated into a gRNA. In some embodiments, O- and N-alkylated nucleotides, e.g., N6- methyl adenosine, can be incorporated into a gRNA. In some embodiments, one or more or all of the nucleotides in a gRNA are deoxynucleotides.

[0181] In some embodiments, a bifunctional cross-linker is used to link a 5’ end of a first gRNA fragment and a 3’ end of a second gRNA fragment, and the 3’ or 5’ ends of thegRNA fragments to be linked are modified with functional groups that react with the reactive groups of the cross-linker. In general, these modifications comprise one or more of amine, sulfhydryl, carboxyl, hydroxyl, alkene (e.g., a terminal alkene), azide and / or another suitable functional group. Multifunctional (e.g., bifunctional) cross-linkers are also generally known in the art, and may be either heterofunctional or homofunctional, and may include any suitable functional group, including without limitation isothiocyanate, isocyanate, acyl azide, an NHS ester, sulfonyl chloride, tosyl ester, tresyl ester, aldehyde, amine, epoxide, carbonate (e.g., Bis(p-nitrophenyl) carbonate), aryl halide, alkyl halide, imido ester, carboxylate, alkyl phosphate, anhydride, fluorophenyl ester, HOBt ester, hydroxymethyl phosphine, O- methylisourea, DSC, NHS carbamate, glutaraldehyde, activated double bond, cyclic hemiacetal, NHS carbonate, imidazole carbamate, acyl imidazole, methylpyridinium ether, azlactone, cyanate ester, cyclic imidocarbonate, chlorotriazine, dehydroazepine, 6-sulfo- cytosine derivatives, maleimide, aziridine, TNB thiol, Ellman’s reagent, peroxide, vinylsulfone, phenylthioester, diazoalkanes, diazoacetyl, epoxide, diazonium, benzophenone, anthraquinone, diazo derivatives, diazirine derivatives, psoralen derivatives, alkene, phenyl boronic acid, etc. In some embodiments, a first gRNA fragment comprises a first reactive group and the second gRNA fragment comprises a second reactive group. For example, the first and second reactive groups can each comprise an amine moiety, which are crosslinked with a carbonate-containing bifunctional crosslinking reagent to form a urea linkage. In other instances, (a) the first reactive group comprises a bromoacetyl moiety and the second reactive group comprises a sulfhydryl moiety, or (b) the first reactive group comprises a sulfhydryl moiety and the second reactive group comprises a bromoacetyl moiety, which are crosslinked by reacting the bromoacetyl moiety with the sulfhydryl moiety to form a bromoacetyl-thiol linkage. These and other cross-linking chemistries are known in the art, and are summarized in the literature, including by Greg T. Hermanson, Bioconjugate Techniques, 3rdEd.2013, published by Academic Press.

[0182] Additional suitable gRNA modifications will be apparent to those of ordinary skill in the art based on the present disclosure. Suitable gRNA modifications include, for example, those described herein and in PCT Publication Nos. WO2019070762A1, WO2016089433A1, WO2016164356A1, or WO2017053729A1, the entire contents of each of which are incorporated herein by reference.Exemplary gRNAs

[0183] Non-limiting examples of guide RNAs suitable for certain embodiments embraced by the present disclosure are provided herein. Those of ordinary skill in the art will be able to envision suitable guide RNA sequences for a specific CRISPR effector protein, e.g., a Cas12a effector protein, from the disclosure of the targeting domain sequence, either as a DNA or RNA sequence. For example, a guide RNA comprising a targeting sequence consisting of RNA nucleotides would include the RNA sequence corresponding to the targeting domain sequence provided as a DNA sequence and contain uracil instead of thymidine nucleotides. Suitable gRNA scaffold sequences are known to those of ordinary skill in the art. For a Cas12a, for example, a suitable scaffold sequence comprises a sequence selected from Table 7, or a pair of sequences selected from Table 8. In Table 7, it is understood that a “modulator sequence” listed herein may constitute the nucleotide sequence of a modulator nucleic acid. Alternatively, additional nucleotide sequences can be comprised in the modulator nucleic acid 5’ and / or 3’ to a “modulator sequence” listed herein. In the consensus PAM sequences of Table 7 and Table 8, N represents A, C, G or T. Where the PAM sequence is preceded by “5’,” it means that the PAM is located immediately upstream of the target nucleotide sequence when using the non-target strand (i.e., the strand not hybridized with the spacer sequence) as the coordinate. Table 7. Exemplary Single gRNA Scaffold Sequences Exemplary Cas12a Protein Scaffold Sequence PAMExemplary Modul Targeter Stem Cas12a Protein ator Sequence Sequence PAM N N, , . .,disclosed herein, will be apparent to the skilled artisan based on the present disclosure in view of the general knowledge in the art.

[0185] It will be understood that the exemplary gRNAs disclosed herein are provided to illustrate non-limiting embodiments embraced by the present disclosure. Additional suitable gRNA sequences will be apparent to the skilled artisan based on the present disclosure, and the disclosure is not limited in this respect. Systems and Methods for Editing the Genome of a Cell

[0186] In one aspect the present disclosure provides systems for editing the genome of a cell. In some embodiments, a Cas12a effector protein causes a double-strand break. In some embodiments a Cas12a effector protein causes a single-strand break, e.g., in some embodiments, a Cas12a effector protein is a nickase.

[0187] Genome editing systems and methods comprising a Cas12a effector protein can be implemented (e.g., administered or delivered to a cell or a subject) in a variety of ways, and different implementations may be suitable for distinct applications. For instance, a genome editing system is implemented. In some embodiments, as a protein / RNA complex (a ribonucleoprotein, or RNP). In some embodiments, a genome editing system and / or method is implemented as one or more nucleic acids encoding a Cas12a effector protein and guide RNA components described herein (optionally with one or more additional components). In some embodiments, a genome editing system and / or method is implemented as one or more vectors comprising such nucleic acids, for instance a viral vector such as an adeno-associated virus. In some embodiments, a genome editing system and / or method is implemented as a combination of any of the foregoing. Additional or modified implementations that operate according to the principles set forth herein will be apparent to the skilled artisan and are within the scope of this disclosure.

[0188] In some embodiments, genome editing systems and / or methods may be capable of target disruption, such as target mutation or alteration, such as leading to gene knockout. In some embodiments, genome editing systems and / or methods may involve replacement of particular target sites, such as leading to target correction. In some embodiments, genome editing systems and / or methods may involve removal of particular target sites, such as leading to target deletion. In some embodiments, genome editing systems and methods comprise a Cas12a effector protein comprising a Cas12a dual nickase for homology directed repair (HDR). In some embodiments, genome editing systems and / ormethods may involve modulation of target site functionality, such as target site activity or accessibility, leading for instance to (transcriptional and / or epigenetic) gene or genomic region activation or gene or genomic region silencing.

[0189] The present disclosure further provides a method of altering a cell, e.g., altering the structure, e.g., altering the sequence, of a target nucleic acid of a cell, comprising contacting the cell with: (a) a gRNA molecule as described herein and (b) a Cas12a effector protein is a nickase as described herein, and optionally, (c) a second gRNA molecule as described herein. In another aspect, disclosed herein is a method of treating a subject (e.g., a subject suffering from a disease, e.g., a cancer), e.g., altering the structure, e.g., sequence, of a target nucleic acid of the subject, comprising contacting the subject (or a cell from the subject) with: (a) a gRNA as described herein; and (b) a Cas12a effector protein as described herein, and optionally, (c) a second gRNA molecule as described herein.

[0190] In some embodiments, the contacting comprises delivering to the cell a Cas12a effector protein of (b) as a protein or an mRNA, and a nucleic acid molecule which encodes (a) and optionally (c). In some embodiments, the contacting comprises delivering to the cell a Cas12a effector protein or fusion protein of (b) as a protein or an mRNA, the gRNA of (a) as an RNA, and optionally the second gRNA of (c), as an RNA.

[0191] In some embodiments, (a) and (b) are present on one nucleic acid molecule, e.g., one vector, e.g., one viral vector, e.g., an AAV vector. Exemplary AAV vectors that may be used in any of the described compositions and methods include an AAV1 vector, a modified AAV1 vector, an AAV2 vector, a modified AAV2 vector, an AAV3 vector, an AAV4 vector, a modified AAV4 vector, an AAV5 vector, a modified AAV5 vector, a modified AAV3 vector, an AAV6 vector, a modified AAV6 vector, an AAV7 vector, a modified AAV7 vector, an AAV8 vector, an AAV5 vector, an AAV.rh10 vector, a modified AAV.rh10 vector, an AAV.rh32 / 33 vector, a modified AAV.rh32 / 33 vector, an AAV.rh43 vector, a modified AAV.rh43 vector, an AAV.rh64R1 vector, and a modified AAV.rh64R1 vector. In some embodiments, (a) is present on a first nucleic acid molecule, e.g., a first vector, e.g., a first viral vector, e.g., a first AAV vector; and (b) is present on a second nucleic acid molecule, e.g., a second vector, e.g., a second vector, e.g., a second AAV vector. The first and second nucleic acid molecules may be AAV vectors.

[0192] In some embodiments, (a) and (c) are present on one nucleic acid molecule, e.g., one vector, e.g., one viral vector, e.g., one AAV vector. In some embodiments, (a) and(c) are on different vectors. For example, (a) may be present on a first nucleic acid molecule, e.g., a first vector, e.g., a first viral vector, e.g., a first AAV vector; and (c) may be present on a second nucleic acid molecule, e.g., a second vector, e.g., a second vector, e.g., a second AAV vector. In some embodiments, the first and second nucleic acid molecules are AAV vectors.

[0193] In some embodiments, (a), (b), and (c) are present on one nucleic acid molecule, e.g., one vector, e.g., one viral vector, e.g., an AAV vector. In some embodiments, the nucleic acid molecule is an AAV vector. In some embodiments, one of (a), (b), and (c) is present on a first nucleic acid molecule, e.g., a first vector, e.g., a first viral vector, e.g., a first AAV vector; and a second and third of (a), (b), and (c) is encoded on a second nucleic acid molecule, e.g., a second vector, e.g., a second vector, e.g., a second AAV vector. The first and second nucleic acid molecule may be AAV vectors.

[0194] In some embodiments, (a) is present on a first nucleic acid molecule, e.g., a first vector, e.g., a first viral vector, a first AAV vector; and (b) and (c) are present on a second nucleic acid molecule, e.g., a second vector, e.g., a second vector, e.g., a second AAV vector. The first and second nucleic acid molecule may be AAV vectors.

[0195] In some embodiments, (b) is present on a first nucleic acid molecule, e.g., a first vector, e.g., a first viral vector, e.g., a first AAV vector; and (a) and (c) are present on a second nucleic acid molecule, e.g., a second vector, e.g., a second vector, e.g., a second AAV vector. The first and second nucleic acid molecule may be AAV vectors.

[0196] In some embodiments, (c) is present on a first nucleic acid molecule, e.g., a first vector, e.g., a first viral vector, e.g., a first AAV vector; and (b) and (a) are present on a second nucleic acid molecule, e.g., a second vector, e.g., a second vector, e.g., a second AAV vector. The first and second nucleic acid molecule may be AAV vectors.

[0197] In some embodiments, each of (a), (b) and (c) are present on different nucleic acid molecules, e.g., different vectors, e.g., different viral vectors, e.g., different AAV vector. For example, (a) may be on a first nucleic acid molecule, (b) on a second nucleic acid molecule, and (c) on a third nucleic acid molecule. The first, second and third nucleic acid molecule may be AAV vectors.

[0198] AAV vectors may be formulated as AAV particles as described herein. In some embodiments, AAV particles comprise (i) an AAV polynucleotide construct (e.g., a recombinant AAV polynucleotide construct), and (ii) a capsid comprising capsid proteins. Insome embodiments, an AAV polynucleotide construct comprises a polynucleotide sequence encoding a Cas12a effector protein or a characteristic portion thereof. In some embodiments, an AAV polynucleotide construct comprises a polynucleotide sequence encoding a gRNA molecule or a characteristic portion thereof.

[0199] In certain embodiments, the contacting comprises delivering to the cell the gRNA of (a) as an RNA, optionally the second gRNA of (c) as an RNA, and a nucleic acid composition that encodes a Cas12a effector protein or fusion protein of (b).

[0200] In some embodiments, a gRNA molecule as described herein and a Cas12a effector protein or fusion protein as described herein or a nucleic acid encoding the Cas12a effector protein or fusion protein, and optionally the gRNA molecule, and further, optionally, a second gRNA molecule, as described herein can be delivered to a cell via a lipid-based system. A lipid-based system can comprise any components and / or structures known in the art. In some embodiments, a lipid-based system is or comprises a lipid nanoparticle (LNP).

[0201] A Cas12a effector protein or fusion protein can be delivered to the cell as a protein or a nucleic acid encoding the protein, e.g., a DNA molecule or mRNA molecule. The guide molecule can be delivered as an RNA molecule or encoded by a DNA molecule. A Cas12a effector protein or fusion protein can also be delivered with a guide molecule as a ribonucleoprotein (RNP) and introduced into the cell via nucleofection (electroporation).

[0202] In some embodiments, the method of altering a cell, e.g., altering the structure, e.g., altering the sequence, of a target nucleic acid of a cell, comprising altering one or more target genes expressed by target cells as described herein. In some embodiments, the method of altering a cell comprises altering two or more target genes expressed by target cells as described herein. In some embodiments, the method of altering a cell comprises altering three or more target genes expressed by target cells as described herein. In some embodiments, the method of altering a cell comprises altering four or more target genes expressed by target cells as described herein. In some embodiments, the method of altering a cell comprises altering five or more target genes expressed by target cells as described herein. In some embodiments, the method of altering a cell comprises altering six or more target genes expressed by target cells as described herein. In certain embodiments, the method of altering a cell comprises altering seven or more target genes expressed by target cells as described herein. In certain embodiments, the method of altering a cell comprises altering each of a target gene as described herein.

[0203] In some embodiments, a contacting step comprises contacting the cell with a nucleic acid composition as described herein. In some embodiments, a contacting step comprises contacting the cell with a composition as described herein. In some embodiments, the composition is a ribonucleoprotein composition.

[0204] In some embodiments, a nucleic acid composition further comprises (c) a third nucleotide sequence that encodes a second gRNA molecule comprising a targeting domain that is complementary with a target domain from a target cell. In some embodiments, a second gRNA targets the same target position as the first gRNA molecule.

[0205] The presently disclosed subject matter further provides a reaction mixture comprising a, gRNA molecule as described herein, a nucleic acid composition as described herein, or a composition as described herein, and a cell, e.g., a cell from a subject who would benefit from one or more alteration at one or more cell target positions in the one or more target genes.

[0206] The presently disclosed subject matter further provides a kit comprising, (a) a gRNA molecule as described herein, or a nucleic acid composition that encodes the gRNA, and one or more of the following: (b) a Cas12a effector protein as described herein; (c) a second gRNA molecule as described herein.

[0207] Additionally, the presently disclosed subject matter provides a gRNA molecule as described herein for use in treating a disease, e.g., a cancer, in a subject. In some embodiments, the gRNA molecule is used in combination with (b) a Cas12a effector protein.

[0208] The presently disclosed subject matter further provides use of a gRNA molecule as described herein in the manufacture of a medicament for treating a disease, e.g., a cancer, in a subject. In certain embodiments, the medicament further comprises (b) a Cas12a effector protein.

[0209] A skilled person will understand that modulation of target site functionality may involve a CRISPR effector protein variant (such as for instance generation of a catalytically inactive or dead CRISPR effector) and / or functionalization (such as for instance fusion of the CRISPR effector with a heterologous functional domain, such as a deaminase), as described herein.

[0210] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0211] Throughout this specification, unless the context requires otherwise, the words “comprise”, “comprises” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. By “consisting of is meant including, and limited to, whatever follows the phrase “consisting of:” Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of” is meant including any elements listed after the phrase and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially” of indicates that the listed elements are required or mandatory, but that no other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

[0212] These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.

[0213] The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and / or listed in the Application Data Sheet are incorporated herein by reference, in their entirety. The contents of database entries, e.g., NCBI nucleotide or protein database entries provided herein, are incorporated herein in their entirety. Where database entries are subject to change over time, the contents as of the filing date of the present application are incorporated herein by reference. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments.

[0214] The disclosure is further illustrated by the following examples. The examples are provided for illustrative purposes only. They are not to be construed as limiting the scope or content of the disclosure in any way.EXAMPLES Example 1: Cas12a effector proteins reduce indel rate and increase attenuation of target strand cleavage activity

[0215] The present example describes a set of exemplary Cas12a effector proteins comprising one or more substitutions (or mutations) at certain residues that resulted in increased attenuation of target strand cleavage activity. Further, the present example demonstrates that the exemplary Cas12a effector proteins described herein reduced rates of insertions and / or deletions (INDELs).

[0216] First, exemplary AsCas12a effector proteins having one or more substitutions within target strand cleavage attenuation residues were assessed for their rate of indel formation (see Table 9). All experiments were performed in an M537R / H800A / F870L (“MHF”) background. An AsCas12a effector protein with only MHF background mutations, a catalytically dead Cas12a effector protein (“dAsCas12a”) with MHF and E993A mutations, and a negative (no Cas12a effector protein) were used as controls. Table 9. Exemplary AsCas12a effector proteins Exemplary AsCas12a Location of target s

[0217] Rates of editing for each AsCas12a effector protein with a guide RNA targeting an exemplary target gene (Exemplary Target Gene 1) were measured using Illumina-sequencing, with the reported values being the percentage of the total reads that contain an INDEL in the sequencing amplicon [INDEL percentage (%)]. AsCas12a effector proteins with mutations within target strand cleavage attenuation residues (AsCas12a- MHFKK, AsCas12a-FKKR, and AsCas12a-WFKKR) all exhibit reduced INDEL percentage (Figures 1A-1B). Notably, increasing the number of mutated target strand cleavage attenuation residues (e.g., W958A, F999, K1000, K1002, and R1003) reduced INDEL rates. Particularly, AsCas12a-MHFWFKKR, which includes amino acid substitutions in the alpha helical lid domain and the bridge helix domain, nullified INDEL formation similar to dAsCas12a-MHFDE (Figures 1A-1B). Accordingly, the present example demonstrates that Cas12a effector proteins comprising substitutions in target strand cleavage attenuation residues reduce the rate of INDEL formation.

[0218] Next, a subset of these exemplary Cas12a effector proteins were assessed for non-target strand (NTS) and target strand (TS) cleavage using a double stranded DNA match site substrate as shown in Figure 2A. Briefly, a double stranded DNA (dsDNA) match site substrate, an exemplary Cas12a effector protein (AsCas12a-MHF, AsCas12a-MHFFKKR, and AsCas12a-MHFWFKKR), and a guide RNA targeting a sequence within the match site substrate were incubated together in a series of experiments to assess cleavage of the match site substrate. Subsequently, the products were run on a denaturing urea gel so that the Cy5 (red)-labeled non-target strand (NTS) and cleaved products can be distinguished from the Alexa-488 (green)-labeled target strand (TS) and cleaved products. These results confirmed that AsCas12a-MHFWFKKR had increased attenuation of target strand cleavage activity, Thus, as a result, the AsCas12a-MHFWFKKR almost exclusively cleaves the non-target strand relative to cleavage of the target strand in comparison to other Cas12a effector proteins (Figure 2B).

[0219] Accordingly, the present example provides Cas12a effector proteins having one or more amino acid substitutions within one or more target strand cleavage attenuation residues of a Cas12a effector protein that exhibited attenuation of target strand cleavage. Moreover, the present example provides Cas12a effector proteins having one or more amino acid substitutions within one or more target strand cleavage attenuation residues thatexhibited reduced INDEL formation relative to Cas12a effector proteins without one or more amino acid substitutions within one or more target strand cleavage attenuation residues.

[0220] Moreover, the present example demonstrates that increasing the number of mutated target strand cleavage residues increased attenuation of target strand cleavage relative to a Cas12a effector protein without (or with less) mutated target strand cleavage strand residues. For instance, the present example shows that increasing the number of amino acid substitutions within the alpha helical lid domain and / or bridge helix domain of a Cas12a effector protein exhibited increased attenuation of target strand cleavage relative to a Cas12a effector protein without mutated target strand cleavage strand residues. The present example also demonstrates that increasing the number of amino acid substitutions within the alpha helical lid domain and / or bridge helix domain of a Cas12a effector protein exhibited reduced INDEL formation relative to Cas12a effector proteins without one or more amino acid substitutions (or with less amino acid substitutions) within one or more target strand cleavage attenuation residues.

[0221] A skilled person would understand that other Cas12a effector proteins and orthologues thereof can be used in accordance with the embodiments described by the present disclosure. Example 2: Cas12a effector proteins with attenuated target strand cleavage activity

[0222] The present example provides for a series of exemplary Cas12a effector proteins with attenuated target strand cleavage. Furthermore, the present example also demonstrates increased non-target strand (NTS) cleavage relative to target strand (TS) cleavage. Cas12a effector proteins described herein comprise one or more mutations in target strand cleavage attenuation residues which increased attenuation of target strand cleavage activity relative to Cas12a effector proteins without mutations in target strand cleavage attenuation residues. Notably, the present example shows a combinatorial and synergistic effect in attenuation of target strand cleavage activity by increasing the number of mutated target strand cleavage residues in Cas12a effector proteins.

[0223] First, exemplary AsCas12a effector proteins shown in Table 10 were fused to a C-terminal 6x His tag and expressed overnight in E. coli using the IPTG-inducible pET28a system. Bacterial pellets were collected, lysed by a microfluidizer, and the resulting lysate clarified by ultracentrifugation. Exemplary AsCas12a effector proteins were affinity purifiedfrom bacterial lysates via Ni-NTA agarose resin in a gravity flow column, eluted with 250 mM imidazole, and dialyzed overnight prior to being tested in vitro. Table 10. Exemplary AsCas12a effector proteins s

[0224] Next, exemplary AsCas12a effector proteins were assessed for their non-target strand (NTS) and target strand cleavage (TS) activity using a double stranded DNA template comprising a quencher and fluorophore on either the NTS or TS strand. Addition of one or more amino acid substitutions in the alpha helical lid domain (e.g., AsCas12a-MHFKK, AsCas12a-MHFFKKR, and AsCas12a-MHFWFKKR) resulted in increased non-target strand (NTS) cleavage activity relative to target strand (TS) cleavage activity (as measured by relative fluorescent units (RFU)) when compared to the double-stranded cleavage activity of the control AsCas12a-MHF and nicking activity of AsCas12a-MHFW (Figures 3A-3F). Furthermore, addition of a bridge helix mutation (W958A) in combination with alpha helical mutations (e.g., F999G, K1000G, K1001G, and R1003G) demonstrated enhanced non-target strand nicking activity relative to other exemplary Cas12a effector proteins (Figures 3F).

[0225] Accordingly, the present example demonstrates that Cas12a effector proteins having one or more amino acid substitutions in target strand cleavage attenuation residues asdescribed herein have increased non-target strand nicking activity when compared to Cas12a effector proteins without amino acid substitutions in target strand cleavage attenuation residues. Moreover, the present example also demonstrates that Cas12a effector proteins having substitutions in the alpha helical domain and / or bridge helix domain exhibit robust attenuation of target strand cleavage activity compared to other Cas12a effector proteins.

[0226] A skilled person would understand that other Cas12a effector proteins and orthologues thereof can be used in accordance with the embodiments described by the present disclosure. Example 3: Guide RNAs comprising an extended spacer region increase attenuation of target strand cleavage activity of exemplary Cas12a effector proteins

[0227] The present example demonstrates that extending the spacer region (also interchangeably referred to herein as an “extended spacer region”) of the guide RNA molecule increases attenuation of target strand cleavage activity.

[0228] In order to evaluate synergistic effects between a Cas12a effector protein and the spacer length of the guide RNA on cleavage activity, AsCas12a-MHF and AsCas12a- MHFW (see Table 10) were introduced with guide RNAs comprising different spacer lengths targeting either Exemplary Target gene 1 (Figure 4A) or Exemplary Target Gene 2 (Figure 4B). AsCas12a-MHFW exhibited reduced indel percentage in comparison to AsCas12a-MHF, which further decreases upon extension of the guide RNA spacer region regardless of target gene (Figures 4A-4B). Thus, the present example demonstrates that a guide RNA having an extended spacer region (i.e., a spacer that is longer than 21 nucleotides) effectively reduces INDEL formation.

[0229] Next, kinetic assessment of Cas12a effector proteins (AsCas12a-MHFW, AsCas12a-MHFFKKR, and AsCas12a-MHFWFKKR as shown in Table 10) was conducted using the double stranded DNA template described in Example 2 in combination with a guide RNA having an extended spacer region that is 26 nucleotides in length (Figures 5A- 5C). Non-target strand (NTS) cleavage of each of the Cas12a effector proteins was substantially higher than target strand (TS) cleavage in the presence of a guide RNA comprising an extended spacer region (Figure 5A-5C). Furthermore, AsCas12a- MHFWFKKR which has four amino acid substitutions in the alpha helical lid domain (F999G, K1000G, K1001G, and R1003G) and an amino acid substitution in the bridge helix domain (W958A) results in almost exclusive non-target strand nicking activity whencomplexed with a guide RNA with an extended spacer region (Figure 5C). Moreover, AsCas12a-MHFWFKKR demonstrate increased attenuation of target strand cleavage activity relative to other exemplary Cas12a proteins without or with less mutated target strand cleavage attenuation residues.

[0230] Further assessment of target strand vs. non-target strand nicking activity of Cas12a effector proteins with a series of guide RNAs having (1) a spacer region that is 21 nucleotides in length, (2) an extended spacer region that is 26 nucleotides in length, or (3) an extended spacer region that is 26 nucleotides in length and a 2’-fluoro modification was conducted. Comparison of non-target strand and target strand nicking activity of Cas12a effector proteins comprising nickase mutations (e.g., W958A, K1000G, K1002G, and R1003G) showed enhanced non-target strand cleavage over target strand cleavage when in combination with a guide RNA having an extended spacer region of 26 nucleotides in length (Table 11) when compared to control AsCas12a-MHF. Further, gRNAs having a 2-fluoro (2’ F) modification and an extended spacer region further increased specificity for the non-target strand over target strand for Cas12a effector proteins. Table 11. Average Ratio of Non-Target Strand (NTS) to Target Strand (TS) Cleavage Average NTS / TS )orthologues thereof can be used in accordance with the embodiments described by the present disclosure.REFERENCES Bae et al., Bioinformatics 30(10):1473-5 (2014) Blundell et al., Eur J Biochem 172:513 (1988) Boutabout et al. “DNA synthesis fidelity by the reverse transcriptase of the yeast retrotransposon Ty1,” Nucleic Acids Res 29(11):2217–2222 (2001) Cabios, 4: 11-17 (1989) Cornish-Bowden, Nucleic Acids Res.13(9):3021-30 (1985) Dey et al., Protein Sci.22(4):359-66 (2013) Fu et al., Nat Biotechnol 32(3):279-84 (2014) Greer, Science 228:1055 (1985) Guilinger et al., Nature Biotech 32:577-582 (2014) Heigwer et al., Nat methods 11(2):122-3 (2014) Hermanson, Greg T., Bioconjugate Techniques, 3rdEd. (2013) Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) Kim et al., Nat Biotechnol 34(8):863-8 (2016) Kleinstiver et al., Nat Biotechnol 34 (8):869-74 (2016) Poljak et al., Structure 2:1121-1123 (1994) Saha et al. An Alpha-helical Lid Guides the Target DNA toward Catalysis in CRISPR- Cas12a. bioRxiv. DOI: 10.1101 / 2022.09.05.506663 (2022) Strohkendl et al., Structural Basis of Cas12a R-loop Propagation on Pathway to DNA Cleavage. bioRxiv. DOI: 10.1101 / 2023.03.13.532460 (2023) Shmakov et al., Mol Cell 60(3):385-97 (2015) Xiao et al. Bioinformatics 30(8):1180-1182 (2014) Yamano et al., Cell 165(4):949-962 (2016) Zetsche et al., Cell 163:759-771 (2015) CERTAIN EMBODIMENTS

[0232] Embodiment 1. A Cas12a nickase protein comprising a polypeptide sequence with one or more amino acid ...

Claims

CLAIMS We claim:

1. A Cas12a nickase protein comprising: a polypeptide sequence with one or more amino acid substitutions relative to a polypeptide sequence of a naturally occurring Cas12a effector protein, wherein: (i) the one or more amino acid substitutions are in target strand cleavage attenuation residues; and (ii) at least one amino acid substitution of the one or more amino acid substitutions is in its alpha helical lid domain and / or at least one amino acid substitution of the one or more amino acid substitutions is not in its alpha helical lid domain.

2. The Cas12a nickase protein of claim 1, wherein at least one amino acid substitution is in its bridge helix domain.

3. The Cas12a nickase protein of claim 1 or 2, wherein the naturally occurring Cas12a effector protein has a polypeptide sequence of any one of SEQ ID NOs: 11, 62, and 71.

4. The Cas12a nickase protein of any one of the preceding claims, wherein the polypeptide sequence comprises at least one amino acid substitution in its alpha helical lid domain at a position corresponding to F999 of SEQ ID NO: 11, F1012 of SEQ ID NO: 62, or F931 of SEQ ID NO:

71.

5. The Cas12a nickase protein of any one of the preceding claims, wherein the polypeptide sequence comprises at least one amino acid substitution in its alpha helical lid domain corresponding to F999G of SEQ ID NO: 11, F1012G of SEQ ID NO: 62, or F931G of SEQ ID NO:

71.

6. The Cas12a nickase protein of any one of the preceding claims, wherein the polypeptide sequence comprises at least one amino acid substitution in its alpha helical liddomain at a position corresponding to K1000 of SEQ ID NO: 11, K1013 of SEQ ID NO: 62, K932 of SEQ ID NO:

71.

7. The Cas12a nickase protein of any one of the preceding claims, wherein the polypeptide sequence comprises at least one amino acid substitution in its alpha helical lid domain corresponding to K1000G of SEQ ID NO: 11, K1013G of SEQ ID NO: 62, or K932G of SEQ ID NO:

71.

8. The Cas12a nickase protein of any one of the preceding claims, wherein the polypeptide sequence comprises at least one amino acid substitution in its alpha helical lid domain at a position corresponding to S1001 of SEQ ID NO: 11, R1014 of SEQ ID NO: 62, or N933 of SEQ ID NO:

71.

9. The Cas12a nickase protein of any one of the preceding claims, wherein the polypeptide sequence comprises at least one amino acid substitution in its alpha helical lid domain corresponding to S1001G of SEQ ID NO: 11, R1014G of SEQ ID NO: 62, or N933G of SEQ ID NO:

71.

10. The Cas12a nickase protein of any one of the preceding claims, wherein the polypeptide sequence comprises at least one amino acid substitution in its alpha helical lid domain at a position corresponding to K1002 of SEQ ID NO: 11, K1018 of SEQ ID NO: 62, or K937 of SEQ ID NO:

71.

11. The Cas12a nickase protein of any one of the preceding claims, wherein the polypeptide sequence comprises at least one amino acid substitution in its alpha helical lid domain corresponding to K1002G of SEQ ID NO: 11, K1018G of SEQ ID NO: 62, or K937G of SEQ ID NO:

71.

12. The Cas12a nickase protein of any one of the preceding claims, wherein the polypeptide sequence comprises at least one amino acid substitution in its alpha helical liddomain at a position corresponding to R1003 of SEQ ID NO: 11, R1016 of SEQ ID NO: 62, or R935 of SEQ ID NO:

71.

13. The Cas12a nickase protein of any one of the preceding claims, wherein the polypeptide sequence comprises at least one amino acid substitution in its alpha helical lid domain corresponding to R1003G of SEQ ID NO: 11, R1016G of SEQ ID NO: 62, or R935G of SEQ ID NO:

71.

14. The Cas12a nickase protein of any one of the preceding claims, wherein the polypeptide sequence comprises at least one amino acid substitution that is not in its alpha helical lid domain at a position corresponding to W382 of SEQ ID NO: 11, Y410 of SEQ ID NO: 62, or W355 of SEQ ID NO:

71.

15. The Cas12a nickase protein of any one of the preceding claims, wherein the polypeptide sequence comprises at least one amino acid substitution that is not in its alpha helical lid domain corresponding to W382A of SEQ ID NO: 11, Y410A of SEQ ID NO: 62, or W355A of SEQ ID NO:

71.

16. The Cas12a nickase protein of any one of the preceding claims, wherein the polypeptide sequence comprises at least one amino acid substitution that is not in its alpha helical lid domain (e.g., in its bridge helix domain) at a position corresponding to W958 of SEQ ID NO: 11, W971 of SEQ ID NO: 62, or W890 of SEQ ID NO:

71.

17. The Cas12a nickase protein of any one of the preceding claims, wherein the polypeptide sequence comprises at least one amino acid substitution that is not in its alpha helical lid domain corresponding to W958A of SEQ ID NO: 11, W971A of SEQ ID NO: 62, or W890A of SEQ ID NO:

71.

18. The Cas12a nickase protein of any one of the preceding claims, wherein the polypeptide sequence comprises at least one amino acid substitution that is not in its alphahelical lid domain at a position corresponding to R1226 of SEQ ID NO: 11, R1218 of SEQ ID NO: 62, or R1138 of SEQ ID NO:

71.

19. The Cas12a nickase protein of any one of the preceding claims, wherein the polypeptide sequence comprises at least one amino acid substitution that is not in its alpha helical lid domain corresponding to R1226A of SEQ ID NO: 11, R1218A of SEQ ID NO: 62, or R1138A of SEQ ID NO:

71.

20. The Cas12a nickase protein of any one of the preceding claims, wherein the polypeptide sequence has: (i) at least one amino acid substitution in its alpha helical lid domain at a position corresponding to K1000, and / or S1001 in SEQ ID NO: 11; (ii) at least one amino acid substitution in its alpha helical lid domain at a position corresponding to K1013 and / or R1014 in SEQ ID NO: 62; or (iii) at least one amino acid substitution in its alpha helical lid domain at a position corresponding to K932 and / or N933 in SEQ ID NO:

71.

21. The Cas12a nickase protein of any one of the preceding claims, wherein the polypeptide sequence has: (i) at least one amino acid substitution in its alpha helical lid domain corresponding to K1000G and / or S1001G in SEQ ID NO: 11; (ii) at least one amino acid substitution in its alpha helical lid domain corresponding to K1013G and / or R1014G in SEQ ID NO: 62; or (iii) at least one amino acid substitution in its alpha helical lid domain corresponding to K932G and / or N933G in SEQ ID NO:

71.

22. The Cas12a nickase protein of any one of the preceding claims, wherein the polypeptide sequence has:(i) at least one amino acid substitution in its alpha helical lid domain at a position corresponding to K1000 and / or K1002 in SEQ ID NO: 11; (ii) at least one amino acid substitution in its alpha helical lid domain at a position corresponding to K1013 and / or K1018 in SEQ ID NO: 62; or (iii) at least one amino acid substitution in its alpha helical lid domain at a position corresponding to K932 and / or K937 in SEQ ID NO:

71.

23. The Cas12a nickase protein of any one of the preceding claims, wherein the polypeptide sequence has: (i) at least one amino acid substitution in its alpha helical lid domain corresponding to K1000G and / or K1002G in SEQ ID NO: 11; (ii) at least one amino acid substitution in its alpha helical lid domain corresponding to K1013G and / or K1018G in SEQ ID NO: 62; or (iii) at least one amino acid substitution in its alpha helical lid domain corresponding to K932G and / or K937G in SEQ ID NO:

71.

24. The Cas12a nickase protein of any one of the preceding claims, wherein the polypeptide sequence has: (i) at least one amino acid substitution in its alpha helical lid domain at a position corresponding to K1000, K1002, and / or R1003 in SEQ ID NO: 11; (ii) at least one amino acid substitution in its alpha helical lid domain at a position corresponding to K1013, K1018, and / or R1016 in SEQ ID NO: 62; or (iii) at least one amino acid substitution in its alpha helical lid domain at a position corresponding to K932, K937, and / or R935 in SEQ ID NO:

71.

25. The Cas12a nickase protein of any one of the preceding claims, wherein the polypeptide sequence has: (i) at least one amino acid substitution in its alpha helical lid domain corresponding to K1000G, K1002G, and / or R1003G in SEQ ID NO: 11;(ii) at least one amino acid substitution in its alpha helical lid domain corresponding to K1013G, K1018G, and / or R1016G in SEQ ID NO: 62; or (iii) at least one amino acid substitution in its alpha helical lid domain corresponding to K932G, K937G, and / or R935G in SEQ ID NO:

71.

26. The Cas12a nickase protein of any one of the preceding claims, wherein the polypeptide sequence has: (i) at least one amino acid substitution in its alpha helical lid domain at a position corresponding to F999, K1000, K1002, and / or R1003 in SEQ ID NO: 11; (ii) at least one amino acid substitution in its alpha helical lid domain at a position corresponding to F1012, K1013, K1018, and / or R1016 in SEQ ID NO: 62; or (iii) at least one amino acid substitution in its alpha helical lid domain at a position corresponding to F931, K932, K937, and / or R935 in SEQ ID NO:

71.

27. The Cas12a nickase protein of any one of the preceding claims, wherein the polypeptide sequence has: (i) at least one amino acid substitution in its alpha helical lid domain corresponding to F999G, K1000G, K1002G, and / or R1003G in SEQ ID NO: 11; (ii) at least one amino acid substitution in its alpha helical lid domain corresponding to F1012G, K1013G, K1018G, and / or R1016G in SEQ ID NO: 62; or (iii) at least one amino acid substitution in its alpha helical lid domain corresponding to F931G, K932G, K937G, and / or R935G in SEQ ID NO: 71; or 28. The Cas12a nickase protein of any one of the preceding claims, wherein the polypeptide sequence has: (i) at least one amino acid substitution in its alpha helical lid domain at a position corresponding to F999, K1000, K1002, and / or R1003 in SEQ ID NO: 11 and at least one amino acid substitution that is not in its alpha helical lid domain (e.g., in its bridge helix domain) at a position corresponding to W958 in SEQ ID NO: 11;(ii) at least one amino acid substitution in its alpha helical lid domain at a position corresponding to F1012, K1013, K1018, and / or R1016 in SEQ ID NO: 62 and at least one amino acid substitution that is not in its alpha helical lid domain (e.g., in its bridge helix domain) at a position corresponding to W971 in SEQ ID NO: 62; or (iii) at least one amino acid substitution in its alpha helical lid domain at a position corresponding to F931, K932, K937, and / or R935 in SEQ ID NO: 71 and at least one amino acid substitution that is not in its alpha helical lid domain (e.g., in its bridge helix domain) at a position corresponding to W890 in SEQ ID NO:

71.

29. The Cas12a nickase protein of any one of the preceding claims, wherein the polypeptide sequence has: (i) at least one amino acid substitution in its alpha helical lid domain corresponding to F999G, K1000G, K1002G, and / or R1003G in SEQ ID NO: 11 and at least one amino acid substitution that is not in its alpha helical lid domain (e.g., in its bridge helix domain) corresponding to W958A in SEQ ID NO: 11; (ii) at least one amino acid substitution in its alpha helical lid domain corresponding to F1012G, K1013G, K1018G, and / or R1016G in SEQ ID NO: 62 and at least one amino acid substitution that is not in its alpha helical lid domain (e.g., in its bridge helix domain) corresponding to W971A in SEQ ID NO: 62; or (iii) at least one amino acid substitution in its alpha helical lid domain corresponding to F931G, K932G, K937G, and / or R935G in SEQ ID NO: 71 and at least one amino acid substitution that is not in its alpha helical lid domain (e.g., in its bridge helix domain) corresponding to W890A in SEQ ID NO:

71.

30. A Cas12a nickase comprising a polypeptide sequence, wherein the polypeptide sequence comprises: (i) at least one amino acid substitution that is not in its alpha helical lid domain (e.g., in its bridge helix domain) at a position corresponding to W958 of SEQ ID NO: 11; (ii) at least one amino acid substitution that is not in its alpha helical lid domain (e.g., in its bridge helix domain) at a position corresponding to W971 of SEQ ID NO: 62; or(iii) at least one amino acid substitution that is not in its alpha helical lid domain (e.g., in its bridge helix domain) at a position corresponding to W890 of SEQ ID NO:

71.

31. A Cas12a nickase comprising a polypeptide sequence, wherein the polypeptide sequence comprises: (i) at least one amino acid substitution that is not in its alpha helical lid domain (e.g., in its bridge helix domain) corresponding to W958A of SEQ ID NO: 11; (ii) at least one amino acid substitution that is not in its alpha helical lid domain (e.g., in its bridge helix domain) corresponding to W971A of SEQ ID NO: 62; or (iii) at least one amino acid substitution that is not in its alpha helical lid domain (e.g., in its bridge helix domain) corresponding to W890A of SEQ ID NO:

71.

32. A Cas12a nickase comprising a polypeptide sequence, wherein the polypeptide sequence comprises: (i) at least one amino acid substitution in its alpha helical lid domain at a position corresponding to K1000 and / or S1001 in SEQ ID NO: 11; (ii) at least one amino acid substitution in its alpha helical lid domain at a position corresponding to K1013 and / or R1014 in SEQ ID NO: 62; or (iii) at least one amino acid substitution in its alpha helical lid domain at a position corresponding to K932 and / or N933 in SEQ ID NO:

71.

33. A Cas12a nickase comprising a polypeptide sequence, wherein the polypeptide sequence comprises: (i) at least one amino acid substitution in its alpha helical lid domain corresponding to K1000G and / or S1001G in SEQ ID NO: 11; (ii) at least one amino acid substitution in its alpha helical lid domain corresponding to K1013G and / or R1014G in SEQ ID NO: 62; or (iii) at least one amino acid substitution in its alpha helical lid domain corresponding to K932G and / or N933G in SEQ ID NO: 71.

34. A Cas12a nickase comprising a polypeptide sequence, wherein the polypeptide sequence comprises: (i) at least one amino acid substitution in its alpha helical lid domain at a position corresponding to K1000 and / or K1002 in SEQ ID NO: 11; (ii) at least one amino acid substitution in its alpha helical lid domain at a position corresponding to K1013 and / or K1018 in SEQ ID NO: 62; or (iii) at least one amino acid substitution in its alpha helical lid domain at a position corresponding to K932 and / or K937 in SEQ ID NO:

71.

35. A Cas12a nickase comprising a polypeptide sequence, wherein the polypeptide sequence comprises: (i) at least one amino acid substitution in its alpha helical lid domain corresponding to K1000G and / or K1002G in SEQ ID NO: 11; (ii) at least one amino acid substitution in its alpha helical lid domain corresponding to K1013G and / or K1018G in SEQ ID NO: 62; or (iii) at least one amino acid substitution in its alpha helical lid domain corresponding to K932G and / or K937G in SEQ ID NO:

71.

36. A Cas12a nickase comprising a polypeptide sequence, wherein the polypeptide sequence comprises: (i) at least one amino acid substitution in its alpha helical lid domain at a position corresponding to K1000, K1002, and / or R1003 in SEQ ID NO: 11; (ii) at least one amino acid substitution in its alpha helical lid domain at a position corresponding to K1013, K1018, and / or R1016 in SEQ ID NO: 62; or (iii) at least one amino acid substitution in its alpha helical lid domain at a position corresponding to K932, K937, and / or R935 in SEQ ID NO: 71.

37. A Cas12a nickase comprising a polypeptide sequence, wherein the polypeptide sequence comprises: (i) at least one amino acid substitution in its alpha helical lid domain corresponding to K1000G, K1002G, and / or R1003G in SEQ ID NO: 11; (ii) at least one amino acid substitution in its alpha helical lid domain corresponding to K1013G, K1018G, and / or R1016G in SEQ ID NO: 62; or (iii) at least one amino acid substitution in its alpha helical lid domain corresponding to K932G, K937G, and / or R935G in SEQ ID NO:

71.

38. A Cas12a nickase comprising a polypeptide sequence, wherein the polypeptide sequence comprises: (i) at least one amino acid substitution in its alpha helical lid domain at a position corresponding to F999, K1000, K1002, and / or R1003 in SEQ ID NO: 11; (ii) at least one amino acid substitution in its alpha helical lid domain at a position corresponding to F1012, K1013, K1018, and / or R1016 in SEQ ID NO: 62; or (iii) at least one amino acid substitution in its alpha helical lid domain at a position corresponding to F931, K932, K937, and / or R935 in SEQ ID NO:

71.

39. A Cas12a nickase comprising a polypeptide sequence, wherein the polypeptide sequence comprises: (i) at least one amino acid substitution in its alpha helical lid domain corresponding to F999G, K1000G, K1002G, and / or R1003G in SEQ ID NO: 11; (ii) at least one amino acid substitution in its alpha helical lid domain corresponding to F1012G, K1013G, K1018G, and / or R1016G in SEQ ID NO: 62; or (iii) at least one amino acid substitution in its alpha helical lid domain corresponding to F931G, K932G, K937G, and / or R935G in SEQ ID NO:

71.

40. A Cas12a nickase comprising a polypeptide sequence, wherein the polypeptide sequence comprises:(i) at least one amino acid substitution in its alpha helical lid domain at a position corresponding to F999, K1000, K1002, and / or R1003 in SEQ ID NO: 11 and at least one amino acid substitution that is not in its alpha helical lid domain (e.g., in its bridge helix domain) at a position corresponding to W958 in SEQ ID NO: 11; (ii) at least one amino acid substitution in its alpha helical lid domain at a position corresponding to F1012, K1013, K1018, and / or R1016 in SEQ ID NO: 62 and at least one amino acid substitution that is not in its alpha helical lid domain (e.g., in its bridge helix domain) at a position corresponding to W971 in SEQ ID NO: 62; or (iii) at least one amino acid substitution in its alpha helical lid domain at a position corresponding to F931, K932, K937, and / or R935 in SEQ ID NO: 71 and at least one amino acid substitution that is not in its alpha helical lid domain (e.g., in its bridge helix domain) at a position corresponding to W890 in SEQ ID NO:

71.

41. A Cas12a nickase comprising a polypeptide sequence, wherein the polypeptide sequence comprises: (i) at least one amino acid substitution in its alpha helical lid domain corresponding to F999G, K1000G, K1002G, and / or R1003G in SEQ ID NO: 11 and at least one amino acid substitution that is not in its alpha helical lid domain (e.g., in its bridge helix domain) corresponding to W958A in SEQ ID NO: 11; (ii) at least one amino acid substitution in its alpha helical lid domain corresponding to F1012G, K1013G, K1018G, and / or R1016G in SEQ ID NO: 62 and at least one amino acid substitution that is not in its alpha helical lid domain (e.g., in its bridge helix domain) corresponding to W971A in SEQ ID NO: 62; or (iii) at least one amino acid substitution in its alpha helical lid domain corresponding to F931G, K932G, K937G, and / or R935G in SEQ ID NO: 71 and at least one amino acid substitution that is not in its alpha helical lid domain (e.g., in its bridge helix domain) corresponding to W890A in SEQ ID NO:

71.

42. The Cas12a nickase protein of any one of the preceding claims, wherein the polypeptide sequence comprises at least one amino acid substitution that is not in its alpha helical lid domain at a position corresponding to:(i) M537, H800, and / or F870 of SEQ ID NO: 11; (ii) N602, H843, and / or F879 of SEQ ID NO: 62; or (iii) N527, H759, E795 of SEQ ID NO:

71.

43. The Cas12a nickase protein of any one of the preceding claims, wherein the polypeptide sequence comprises at least one amino acid substitution that is not in its alpha helical lid domain corresponding to: (i) M537R, H800A, and / or F870L of SEQ ID NO: 11; (ii) N602R, H843A, and / or F879L of SEQ ID NO: 62; or (iii) N527R, H759A, and / or E795L of SEQ ID NO:

71.

44. The Cas12a nickase protein of any one of the preceding claims, wherein the polypeptide sequence is at least 80% identical to the polypeptide sequence according to any one of SEQ ID NOs: 11-61; at least 80% identical to the polypeptide sequence according to any one of SEQ ID NOs: 62-70; or at least 80% identical to the polypeptide sequence according to any one of SEQ ID NOs: 71-79.

45. The Cas12a nickase protein of any one of the preceding claims, further comprising one or more nuclear localization sequences.

46. The Cas12a nickase protein of claim 45, wherein the nuclear localization sequence comprises one or more c-myc nuclear localization sequences.

47. The Cas12a nickase protein of claim 45, wherein the nuclear localization sequence comprises a sequence according to any one of SEQ ID NOs: 7-10.

48. The Cas12a nickase protein of any one of the preceding claims, further comprising a poly-histidine tag sequence.

49. The Cas12a nickase protein of any one of the preceding claims, further comprising a linker sequence.

50. The Cas12a nickase protein of any one of the preceding claims, wherein the Cas12a nickase protein cleaves a non-target strand of a double stranded DNA (dsDNA) molecule.

51. The Cas12a nickase protein of any one of the preceding claims, wherein the Cas12a nickase protein exhibits attenuated target strand cleavage activity relative to non-target strand cleavage activity of a double stranded DNA (dsDNA) molecule.

52. The Cas12a nickase protein of any one of the preceding claims, wherein the Cas12a nickase protein exhibits a nickase activity of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least and at least 100% cleavage of the non-target strand of the dsDNA molecule.

53. A fusion protein comprising a Cas12a nickase protein according to any one of claims 1-52.

54. The fusion protein of claim 53, further comprising a polymerase.

55. The fusion protein of claim 53 or 54, further comprising a reverse transcriptase.

56. The fusion protein of claim 53 or 54, further comprising a DNA polymerase.

57. A polynucleotide encoding a Cas12a nickase protein according to any one of claims 1-52 or a fusion protein according to any one of claims 53-56.

58. The polynucleotide according to claim 57, wherein the polynucleotide is codon- optimized for expression in a host cell.

59. The polynucleotide of claim 57 or 58, further comprising a nucleic acid sequence encoding a gRNA molecule.

60. A recombinant vector comprising a polynucleotide of claim 57 or 58.

61. The recombinant vector of claim 60, wherein the polynucleotide is operably linked to a promoter.

62. The recombinant vector of claim 60 or 61, further comprising a nucleic acid sequence encoding a gRNA molecule.

63. The recombinant vector of claim 62, wherein the gRNA comprises a spacer that is at least 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 nucleotides in length.

64. An AAV particle comprising the polynucleotide of any one of claims 57-59 or recombinant vector of any one of claims 60-63.

65. A lipid nanoparticle (LNP) comprising the polynucleotide of any one of claims 57-59 or recombinant vector of any one of claims 60-63.

66. A cell comprising the Cas12a nickase protein of any one of claims 1-52 or the fusion protein according to any one of claims 53-56.

67. A cell comprising the polynucleotide of any one of any one of claims 57-59.

68. A cell comprising the recombinant vector of any one of claims 60-63.

69. A CRISPR / Cas12a effector system comprising: (a) a polynucleotide of any one of claims 57-59, or a recombinant vector of any one of claims 60-63; and (b) a polynucleotide or a recombinant vector comprising a nucleic acid sequence encoding a gRNA molecule.

70. The CRISPR / Cas12a effector system of claim 69, wherein gRNA comprises a spacer that is at least 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 nucleotides in length.

71. The CRISPR / Cas12a effector system of claim 69 or 70, further comprising (c) a cell for expression of the polynucleotide or recombinant vector of (a) and (b).

72. The cell of any one of claims 66-68 or the CRISPR / Cas12a effector system of any one of claims 69-71, wherein the cell is a prokaryotic cell or a eukaryotic cell.

73. A composition (e.g., a pharmaceutical composition) comprising the Cas12a nickase protein of any one of claims 1-52 and a gRNA molecule, the fusion protein according to any one of claims 53-56 and a gRNA molecule, the polynucleotide of any one of claims 57-59, the recombinant vector of any one of claims 60-63, the AAV particle of claim 64, the LNP of claim 65, or the cell of any one of claims 66-68.

74. A composition comprising a ribonucleoprotein (RNP) complex comprising the Cas12a nickase protein of any one of claims 1-52 and a gRNA molecule or the fusion protein according to any one of claims 53-56 and a gRNA molecule.

75. The composition of claim 74, wherein the gRNA molecule comprises one or more chemically modified nucleotides.

76. The composition of claim 75, wherein the one or more chemically modified nucleotides comprises a 2’-F.

77. The composition of any one of claims 74-76, wherein the gRNA molecule comprises a spacer that is at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 nucleotides in length.

78. A method of genetically engineering a population of cells, the method comprising: expressing in the cells, or contacting the cells with, the Cas12a nickase protein of any one of claims 1-52 and a gRNA molecule or the fusion protein according to any one of claims 53-56 and a gRNA molecule, whereby genomes of at least a plurality of the cells are altered.

79. A method of editing a population of double stranded DNA (dsDNA) molecules, the method comprising: contacting the dsDNA molecules with the Cas12a nickase protein any one of claims 1-52 and a gRNA molecule or the fusion protein according to any one of claims 53-56 and a gRNA molecule, whereby a plurality of the dsDNA molecules are edited.

80. The method of claims 78-79, wherein the Cas12a nickase protein and gRNA molecule or the fusion protein and a gRNA molecule are administered as a ribonucleoprotein (RNP).

81. A method of treatment comprising: introducing the Cas12a nickase protein of any one of claims 1-52 and a gRNA molecule or the fusion protein according to any one of claims 53-56 and a gRNA molecule, the polynucleotide of any one of claims 57-59, the recombinant vector of any one of claims60-63, the AAV particle of claim 64, the LNP of claim 65, or the composition of any one of claims 73-77 into a subject.

82. Use of the Cas12a nickase protein of any one of claims 1-52 and a gRNA molecule or the fusion protein according to any one of claims 53-56 and a gRNA molecule, the polynucleotide of any one of claims 57-59, the recombinant vector of any one of claims 60- 63, the AAV particle of claim 64, the LNP of claim 65, the CRISPR / Cas12a effector system of any one of claims 69-72, or the composition of any one of claims 73-77 into a subject for genetically engineering a population of cells, whereby genomes of at least a plurality of the cells are altered.

83. Use of the Cas12a nickase protein of any one of claims 1-52 and a gRNA molecule or the fusion protein according to any one of claims 53-56 and a gRNA molecule, the polynucleotide of any one of claims 57-59, the recombinant vector of any one of claims 60- 63, the AAV particle of claim 64, the LNP of claim 65, the CRISPR / Cas12a effector system of any one of claims 69-72, or the composition of any one of claims 73-77 into a subject for editing a population of double stranded DNA (dsDNA) molecules, whereby a plurality of the dsDNA molecules are edited.

84. Use of the Cas12a nickase protein any one of claims 1-52 and a gRNA molecule or the fusion protein according to any one of claims 53-56 and a gRNA molecule, the polynucleotide of any one of claims 57-59, the recombinant vector of any one of claims 60- 63, the AAV particle of claim 64, the LNP of claim 65, the CRISPR / Cas12a effector system of any one of claims 69-72, or the composition of any one of claims 73-77 into a subject for treatment of a subject.

85. Use of the Cas12a nickase protein any one of claims 1-52 and a gRNA molecule or the fusion protein according to any one of claims 53-56 and a gRNA molecule, the polynucleotide of any one of claims 57-59, the recombinant vector of any one of claims 60- 63, the AAV particle of claim 64, the LNP of claim 65, the CRISPR / Cas12a effector systemof any one of claims 69-72, or the composition of any one of claims 73-77 into a subject in the manufacture of a medicament for the treatment of a subject.

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