Variant cas12a protein compositions and methods

Abstract

The present disclosure provides variant Cas12a proteins, nucleic acids encoding the variant proteins, and systems comprising the variant proteins or nucleic acids encoding same. The present disclosure provides methods for modifying a target nucleic acid and methods for detecting a target nucleic acid, using a variant protein.

Description

VARIANT CAs12a PROTEIN COMPOSITIONS AND METHODSCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 651 ,713 filed May 24, 2024, which application is incorporated herein by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under Grant Number Al 171110 awarded by the National Institutes of Health. The government has certain rights in the invention.INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS AN XML FILE

[0003] A Sequence Listing is provided herewith as a Sequence Listing XML, “BERK-521WO_SEQLIST.xm I” created on March 21 , 2025 and having a size of 53,171 bytes. The contents of the Sequence Listing XML are incorporated by reference herein in their entirety.I. INTRODUCTION

[0004] Bacterial adaptive immune systems employ CRISPRs (clustered regularly interspaced short palindromic repeats) and CRISPR-associated (Cas) proteins for RNA-guided nucleic acid cleavage. The CRISPR-Cas systems thereby confer adaptive immunity in bacteria and archaea via RNA-guided nucleic acid interference. To provide anti-viral immunity, processed CRISPR array transcripts (crRNAs) assemble with Cas proteincontaining surveillance complexes that recognize nucleic acids bearing sequence complementarity to the virus derived segment of the crRNAs, known as the spacer. Class 2 CRISPR-Cas are streamlined versions in which a single Cas protein bound to RNA is responsible for binding to and cleavage of a targeted sequence. The programmable nature of these minimal systems has facilitated their use as a versatile technology for genome editing.II. SUMMARY

[0005] The present disclosure provides variant Cas12a proteins, nucleic acids encoding the variant proteins, and systems comprising the variant proteins or nucleic acids encoding same. The present disclosure provides methods for modifying a targetnucleic acid, using a variant protein of the present disclosure. The present disclosure provides methods for detecting a target nucleic acid, using a variant protein of the present disclosure. Reagents, compositions, and kits / systems that find use in practicing the subject methods are provided.III. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] The following detailed description of embodiments of the invention will be better understood when read in conjunction with the appended drawings. It should be understood that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0007] FIG. 1A-1 B. Illustration of the selection of PAM-loose variants of LbCas12a selected via directed evolution. FIG. 1A. Schematic presentation of the domains of LbCas12a protein. The region in which mutants were generated in this study is shown. FIG. 1B. General illustration of mutant selection via bacteria-ccdB1 system. The crRNAs (guide RNAs) used in this selection were designed based on the arbitrary PAMs of AGCT (T), AGTC (C), TGCA (A), and TCAG (G) located in ccdB1 gene.

[0008] FIG. 2A-2B. In vitro confirmation of the newly selected variants of LbCas12a with loose PAM-requirement. FIG. 2A. Plasmid cleavage assays. Seven selected PAM- loose variant proteins of LbCas12a as well as wild-type protein were studied with each of three guide crRNAs (their corresponding PAMs are listed in parenthesis). The density of DNA bands indicated by “linearized” reflects the activity of the studied protein. FIG. 2B. A summary of in vitro dsDNA cleavage assays in 60 minutes. Eight different dsDNA targets (listed below X-axle). Relative activity (Y-axle) of each of the selected PAM-loose variant proteins as well as wild-type protein was shown as a percentage of cleaved over total (cleaved + un-cleaved) target dsDNA.

[0009] FIG. 3. Kinetic studies of the newly selected LbCas12a variants. The kinetic studies of each selected PAM-loose protein were carried out in the time course from 0 to 60 min.

[0010] FIG. 4A-4E. Genome editing by PAM-loose mutants in NPC neuronal cells. FIG. 4A and FIG. 4B are PAM-NGS logo, showing the PAM preference of LbCas12a RNPs. FIG. 4C and FIG. 4D are the images of genome editing by wild-type and PL4a mutant RNPs, respectively, demonstrating that the PL4 mutant still can use the natural PAM of TTTV. Image FIG. 4E shows that the RNP from PL4a mutant can use a simple PAM to perform genome editing.

[0011] FIG. 5A-5B. PAM-NGS logos for LbCas12a (“WT”) and for the variants PL2d, PL3, and PL4a.

[0012] FIG. 6. Depicts an alignment of wild type Cas12a proteins (SEQ ID NOs: 1-13: labeled “seq_” 1 through 13). Dots are depicted over the amino acid residues that were mutated in variants that were isolated via experiment (see experimental examples section, and also see Fig. 7).

[0013] FIG. 7 depicts SEQ ID NO: 1 (wild type LbCas12a sequence) followed by a table of residues that were mutated in the variants that were isolated via experiment (see experimental examples section). The residues that were mutated in the isolated variants are highlighted in the depicted sequence above the table.

[0014] FIG. 8A-8C Generation of PAM-relaxed Cas12a enzymatic variants.FIG. 8A. Schematic presentation of the dual-plasmid selection system used for directed evolution. Each of four crRNAs (crRNA1-4) was designed for targeting sequences flanked by a randomly designed non-canonical PAMs of AGCT, AGTC, TGCA or TCAG. FIG. 8B. Schematic presentation of the LbCas12a functional domains. The highlighted middle region (WED+PI, top panel) is the target region for mutagenesis. Overall structure (PDB: 5XUS) of LbCas12a-dsDNA-crRNA ternary complex (52) is shown on the left side of the middle panel, while mutations in three selected variants are listed in the table on right side of the middle panel. The bottom panel displays the structural positions of the mutations of D535G, S551 F and D665N in Lbm6 and the three mutated residues are labeled in red. The PAM sequence is shown in brown in the structure. FIG. 8C. In vitro kinetic studies of c / s-cleavage activity on different PAM DNAs. Cleavage efficiencies of wild-type LbCas12a (wt) and three selected variants (Lbm4, Lbm5, and Lbm6) are shown. Each data point represents the average of two independent experiments. TTTC is a canonical PAM, while others, non-canonical PAMs. Each PAM sequence is listed on the top of each panel. The observed rate constants (kobs) from these c / s-cleavage assays are also listed.

[0015] FIG. 9A-9C Nuclease activities of Lbm6 and its derivatives. FIG. 9A. Genome editing in Ai9 mouse derived neural progenitor cells (NPCs). The upper panel provides a schematic illustration of desired genome editing to turn on the tdTomato transgene in NPCs. The lower panel presents genome editing efficiencies at three target sites in NPCs. PAMs are shown on X-axis. TTTA is a canonical PAM, while others, non-canonical PAMs. Each bar represents the average of four independent experiments. FIG. 9B. Improvement of Lbm6 enzymatic cleavage activity, c / s- cleavage activity of Lbm6 is improved by introduction of the mutations from the activity-enhanced variant of Cas12a-4R, -2C or -RVQ. Introduction of RVQ mutations(G146R, R182V and E795Q) into Lbm6 makes it most active as shown by its kobs. From now on, the combined version of Lbm6 and RVQ mutations will be named Flex- Cas12a. Each data point represents the average of two independent experiments. DNA target used in this assay is DNA TO with a canonical PAM of 5’-TTTC-3’. FIG. 9C. Genome editing efficiency of Flex-Cas12a. Flex-Cas12a exhibits significantly enhanced genome editing activity which is comparable to LbCas12a-RVQ, at a locus with canonical PAM of 5’-TTTA-3’ in tdTomato NPCs. Negative control (neg Ctrl) conditions in (A and C) means cells which were not treated by any RNP. Each bar represents the average of four independent technical replicates.

[0016] FIG. 10A-10C In vitro kinetic studies and PAM depletion assays. FIG. 10A. In vitro kinetic analysis of c / s-cleavage activity. The observed kobsindicate that Flex-Cas12a exhibits cleavage activity comparable to LbCas12a-RVQ (RVQ). Each data point represents the average of two independent experiments. The Target DNA used in this assay is DNA TO with a canonical PAM of 5’-TTTC-3’. FIG. 10B. In vitro kinetic analysis of trans- cleavage activity. Due to the rapid cleavage rates, the ko sof transcleavage could not be accurately determined from these assays. Each data point represents the average of two independent experiments. FIG. 10C. Expanded PAM recognition by Flex-Cas12a. Heatmaps of NGS results from PAM depletion assays illustrate PAM preferences of LbCas12a-RVQ (RVQ) and Flex-Cas12a. Top panel presents a schematic illustration of nucleotide positions in the PAM motif. Middle panel shows the heatmap for LbCas12a-RVQ, while bottom panel depicts the heatmap for Flex-Cas12a. The heatmap represents the logw(k) of the in vitro cleavage rate constant (s-1).

[0017] FIG. 11A-11B Genome editing in Ai9 tdTomato NPCs and HEK293T-EGFP cells. FIG. 11A. Genome editing in tdTomato NPCs. The upper panel provides a schematic illustration of desired genome editing to turn on the tdTomato transgene in NPCs. The lower panels show the genome editing efficiencies at four loci by wild-type (wt), LbCas12a-RVQ (RVQ), Lbm6 and Flex-Cas12a. PAM sequence of each target is shown on top of each panel. FIG. 11 B. Genome editing in HEK293T-EGFP cells. The upper panel provides a schematic illustration of desired genome editing to turn off the EGFP transgene in HEK293T cells. The lower panels show the genome editing efficiencies of four proteins at four loci. TTTA and TTTG are canonical PAMs, while others, non-canonical PAMs. PAM sequence of each target is shown on top of each panel. The genome editing results from both cell types demonstrate that Flex-Cas12a is significantly more active than its parent version (Lbm6) and can efficiently recognizea broad range of PAM sequences for genome editing. Data are presented as mean ± SD from four independent technical replicates.

[0018] FIG. 12A-12C Genome editing at endogenous loci. FIG. 12A. Genome editing at endogenous loci in HEK293T cells. The upper panel illustrates the detection of genome editing in HEK293T cells using an anti-B2M antibody. The lower panel shows editing efficiencies at five endogenous loci in B2M gene. Flex-Cas12a can edit five endogenous loci with different PAMs, whereas LbCas12a-RVQ (RVQ) is restricted to the site with a canonical-PAM. TTTA is a canonical, while others, non-canonical PAMs. PAM sequence of each target is listed on X-axis. FIG. 12B. Genome editing analysis by next-generation sequencing (NGS). Editing efficiencies at two selected targets with a canonical PAM of 5’-TTTA-3’ and a non-canonical PAM of 5-TCAG-3’ are shown. Data presented here are generated from three independent technical replicates. Indels here represents insertions and deletions. FIG. 12C. Base editing analysis by NGS analysis. Top panel is a schematic illustration of analysis of base editing. Middle panel presents a representative data from adenine base editing (ABE). Bottom panel shows a representative data from cytosine base editing (CBE). All the data are presented as mean ± SD from three independent technical replicates.

[0019] FIG. 13A-13G Generation and analysis of PAM -relaxed variants. FIG. 13A. Target sequences for directed evolution. Left panel shows the target sequences of four crRNAs (crRNAI to 4) and right panel shows four ccdB target DNA sequences (presented as non-target strands) with randomly selected non-canonical PAMs (AGCT, AGTC, TGCA, or TCAG). B. Mutations in PAM-relaxed LbCas12a variants. Residues highlighted in bold indicate that they are localized within the PAM- interacting domain (PI). FIG. 13C. Structural presentation of the LbCas12a-dsDNA- crRNA ternary complex (PDB: 5XUS), highlighting mutation sites in Lbm4, Lbm5, and Lbm6. PI domain is colored in yellow, and WED domains are in green, PAM sequence is shown in brown, and all mutated residues are labeled in red. FIG. 13D. An SDS- PAGE gel image of the purified seven variants Lbm1-7. FIG. 13E. In vitro kinetic analysis of c / s-cleavage activity. Cleavage efficiencies of wild-type (wt) and seven variants (Lbm1 to Lbm7) are shown. Data points represent the mean of two independent experiments. Target DNAs used in these assays are same except PAM sequences. TTTC is a canonical PAM, while others, non-canonical PAMs. Each PAM sequence of DNA substrate is given on top of each panel. FIG. 13F. Plasmid cleavage assays of cccfB-containing plasmid DNA. Each designed crRNA targets a DNA sequence with a non-canonical PAM (crRNA2:AGTC, crRNA3:TGCA andcrRNA4:TCAG). Each crRNA was tested against the seven Lbm1-7 variants as well as wild-type LbCas12a (wt). FIG. 13G. In vitro cleavage assays with various synthetic DNA substrates which are derived from DNA TO with different non-canonical PAM sequences. The corresponding PAM sequences are listed on top of the gel image or on X-axis. Top panel presents a representative gel image. Lower panel quantifies cleavage efficiencies across eleven target DNAs by wt, Lbm4, Lbm5 and Lbm6, respectively. S presents substrate, and P, cleavage products.

[0020] FIG. 14A-14C DNA cleavage assays by Lbm6 and its derivatives. FIG. 14A. A gel image of in vitro cleavages by Lbm6 and its derivatives of Lbm6-4R, Lbm6-2C and Lbm6-RVQ. Target DNA used in this assay is DNA TO with a PAM of 5’-TTTC-3’). FIG. 14B. Quantification of cleavage efficiencies of each variant over time. The cleavage results indicate that Lbm6-RVQ exhibits the highest activity and is renamed Flex-Cas12a from now on. FIG. 14C. In vitro cleavage assays of DNA substrates bearing different PAMs. Lbm4-RVQ, Flex-Cas12a and LbCas12a-RVQ (RVQ) were assessed. Upper panel shows a gel image of DNA cleavage reactions. Lower panel quantifies cleavage efficiencies for thirteen target DNAs with each protein. Target DNAs used in these assays are DNA TO with different PAMs which are listed on top of the gel image or on X-axis. TTTC is a canonical PAM, while others, non-canonical PAMs. S presents substrate, and P, cleavage products.

[0021] FIG. 15A-15C Comparison of DNA cleavage activities of Flex-Cas12a to LbCas12a-RVQ (RVQ). FIG. 15A. The gel image of c / s-cleavage assays. DNA substrate used in assay is DNA TO with a canonical PAM of 5’-TTTC-3’. FIG. 15B. The gel image of trans-cleavage assays. In this assay, 45 nM unlabeled target dsDNA TO with a canonical PAM of 5’-TTTC-3’ was incubated with LbCas12a RNPs for 30 min at 37°C before addition of a labeled random ssDNA (no homology with the target DNAs or crRNAs). FIG. 15C. The gel image from the cleavage assay with a competitor DNA. In this assay with a competitor, 60 nM RNP, 10 nM labeled target DNA and 360 nM competitor DNA (pUC19 plasmid) were used. In this assay, labeled target DNA is DNA TO with a canonical PAM of 5’-TTTC-3’.

[0022] FIG. 16A-16B Genome editing in Ai9 tdTomato NPCs and HEK293-EGFP cells. FIG. 16A. Quantification of gnome editing at ten target sites in tdTomato NPCs. FIG. 16B. Quantification of genome editing at ten target sites in HEK293T-EGFP cells. PAM sequence for each target is listed on top of corresponding panel. In A and B, TTTA and TTTG are canonical PAMs, while others, non-canonical PAMs. All the editing data were quantified using flow cytometry and are presented as mean ± SDfrom four independent technical replicates, neg Ctrl means the cells not treated with any proteins. Here, wt is abbreviated from wild-type, and RVQ, LbCas12a-RVQ.

[0023] FIG. 17A-17B Off -target analysis of Flex-Cas12a. FIG. 17A. Off-target analysis of genomic DNAs isolated from the cells transfected with RNPs of LbCas12a-RVQ (RVQ) or Flex-Cas12a targeting a genomic site flanked with a 5’-TTTA-3’ PAM. Twelve off-target (OT) sites were analyzed. FIG. 17B. Off-target analysis of genomic DNAs isolated from the cells transfected with RNP of Flex-Cas12a targeting a genomic site flanked with a 5’-TCAG-3’ PAM. Ten off-target (OT) sites were analyzed. Each data point represents the average of two independent replicates. No off-target activity was detected in these assays. Neg Ctrl means genomic DNAs isolated from untreated cells.

[0024] FIG. 18A-18B Base editing activity of Flex-Cas12a. FIG. 18A. Data from ABE base editing and FIG. 18B. Data from CBE base editing. Nucleotides at each position from 3 to 14 of target sequences are listed on X-axis. PAM sequence for each target is listed on top of each corresponding panel. TTTA and TTTC are canonical PAMs, while others, non-canonical PAMs. All the data are presented as mean ± SD from three independent replicates. RVQ is abbreviated from LbCas12a-RVQ.IV. DEFINITIONS

[0025] The terms “polynucleotide” and “nucleic acid,” used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, terms “polynucleotide” and “nucleic acid” encompass single-stranded DNA; double-stranded DNA; multi-stranded DNA; single-stranded RNA; double-stranded RNA; multi-stranded RNA; genomic DNA; cDNA; DNA-RNA hybrids; and a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0026] By "hybridizable" or “complementary” or “substantially complementary" it is meant that a nucleic acid (e.g. RNA, DNA) comprises a sequence of nucleotides that enables it to non-covalently bind, i.e. form Watson-Crick base pairs and / or G / U base pairs, “anneal”, or “hybridize,” to another nucleic acid in a sequence-specific, antiparallel, manner (i.e., a nucleic acid specifically binds to a complementary nucleic acid) under the appropriate in vitro and / or in vivo conditions of temperature and solution ionic strength. Standard Watson-Crick base-pairing includes: adenine / adenosine) (A) pairing with thymidine / thymidine (T), A pairing with uracil / uridine (U), and guanine / guanosine) (G) pairing with cytosine / cytidine (C). In addition, for hybridizationbetween two RNA molecules (e.g., dsRNA), and for hybridization of a DNA molecule with an RNA molecule (e.g., when a DNA target nucleic acid base pairs with a guide RNA, etc.): G can also base pair with U. For example, G / U base-pairing is partially responsible for the degeneracy (i.e. , redundancy) of the genetic code in the context of tRNA anti-codon base-pairing with codons in mRNA. Thus, in the context of this disclosure, a G (e.g., of a protein-binding segment (e.g., dsRNA duplex) of a guide RNA molecule; of a target nucleic acid (e.g., target DNA) base pairing with a guide RNA) is considered complementary to both a U and to C. For example, when a G / U base-pair can be made at a given nucleotide position of a protein-binding segment (e.g., dsRNA duplex) of a guide RNA molecule, the position is not considered to be non-complementary, but is instead considered to be complementary.

[0027] Hybridization requires that the two nucleic acids contain complementary sequences, although mismatches between bases are possible. The conditions appropriate for hybridization between two nucleic acids depend on the length of the nucleic acids and the degree of complementarity, variables well known in the art. The greater the degree of complementarity between two nucleotide sequences, the greater the value of the melting temperature (Tm) for hybrids of nucleic acids having those sequences. Typically, the length for a hybridizable nucleic acid is 8 nucleotides or more (e.g., 10 nucleotides or more, 12 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 22 nucleotides or more, 25 nucleotides or more, or 30 nucleotides or more).

[0028] It is understood that the sequence of a polynucleotide need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. Moreover, a polynucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure, a ‘bulge’, and the like). A polynucleotide can comprise 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence complementarity to a target region within the target nucleic acid sequence to which it will hybridize. For example, an antisense nucleic acid in which 18 of 20 nucleotides of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. The remaining noncomplementary nucleotides may be clustered or interspersed with complementary nucleotides and need not be contiguous to each other or to complementary nucleotides. Percent complementarity between particular stretches of nucleic acid sequences within nucleic acids can be determined using anyconvenient method. Example methods include BLAST programs (basic local alignment search tools) and PowerBLAST programs (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656) or by using the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), e.g., using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489).

[0029] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.

[0030] "Binding" as used herein (e.g. with reference to an RNA-binding domain of a polypeptide, binding to a target nucleic acid, and the like) refers to a non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid; between a guide RNA and a target nucleic acid; and the like). While in a state of non- covalent interaction, the macromolecules are said to be “associated” or “interacting” or “binding” (e.g., when a molecule X is said to interact with a molecule Y, it is meant the molecule X binds to molecule Y in a non-covalent manner). Not all components of a binding interaction need be sequence-specific (e.g., contacts with phosphate residues in a DNA backbone), but some portions of a binding interaction may be sequencespecific. Binding interactions are generally characterized by a dissociation constant (Kd) of less than 10-6M, less than 10-7M, less than 10-8M, less than 10-9M, less than 10’10M, less than 10-11M, less than 10-12M, less than 10-13M, less than 10-14M, or less than 10'15M. "Affinity" refers to the strength of binding, increased binding affinity being correlated with a lower Kd.

[0031] By "binding domain" it is meant a protein domain that is able to bind non-covalently to another molecule. A binding domain can bind to, for example, an RNA molecule (an RNA-binding domain) and / or a protein molecule (a protein-binding domain). In the case of a protein having a protein-binding domain, it can in some cases bind to itself (to form homodimers, homotrimers, etc.) and / or it can bind to one or more regions of a different protein or proteins.

[0032] The term "conservative amino acid substitution" refers to the interchangeability in proteins of amino acid residues having similar side chains. For example, a group of amino acids having aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains consistsof serine and threonine; a group of amino acids having amide containing side chains consisting of asparagine and glutamine; a group of amino acids having aromatic side chains consists of phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains consists of lysine, arginine, and histidine; a group of amino acids having acidic side chains consists of glutamate and aspartate; and a group of amino acids having sulfur containing side chains consists of cysteine and methionine. Exemplary conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine-glycine, and asparagineglutamine. Coded amino acids (followed in parentheses by their corresponding three- letter codes and one-letter codes) include: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gin; Q), glycine (Gly; G), histidine (His; H), isoleucine (lie; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F); proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), or valine (Vai; V).

[0033] A polynucleotide or polypeptide has a certain percent "sequence identity" to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same, and in the same relative position, when comparing the two sequences. Sequence identity can be determined in a number of different ways. To determine sequence identity, sequences can be aligned using various methods and computer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, Phyre2, etc.), available over the world wide web at sites including ncbi.nlm.nili.gov / BLAST, ebi.ac.uk / Tools / msa / tcoffee / , ebi.ac.uk / Tools / msa / muscle / , mafft.cbrc.jp / alignment / software / , http: / / www.sbg.bio.ic.ac.uk / ~phyre2 / . See, e.g., Altschul et al. (1990), J. Mol. Bioi. 215:403-10.

[0034] The terms "DNA regulatory sequences," "control elements," and "regulatory elements," used interchangeably herein, refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, protein degradation signals, and the like, that provide for and / or regulate transcription of a non-coding sequence (e.g., guide RNA) or a coding sequence (e.g., protein coding) and / or regulate translation of an encoded polypeptide.

[0035] As used herein, a "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase and initiating transcription of a downstream (3' direction) coding or non-coding sequence. Eukaryotic promoters will often, but not always, contain "TATA"boxes and "CAT" boxes. Various promoters, including inducible promoters, may be used to drive the various nucleic acids (e.g., vectors) of the present disclosure.

[0036] The term "naturally-occurring" or “unmodified” or “wild type” as used herein as applied to a nucleic acid, a polypeptide, a cell, or an organism, refers to a nucleic acid, polypeptide, cell, or organism that is found in nature.

[0037] "Recombinant," as used herein, means that a particular nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction, polymerase chain reaction (PCR) and / or ligation steps resulting in a construct having a structural coding or noncoding sequence distinguishable from endogenous nucleic acids found in natural systems. DNA sequences encoding polypeptides can be assembled from cDNA fragments or from a series of synthetic oligonucleotides, to provide a synthetic nucleic acid which is capable of being expressed from a recombinant transcriptional unit contained in a cell or in a cell-free transcription and translation system. Genomic DNA comprising the relevant sequences can also be used in the formation of a recombinant gene or transcriptional unit. Sequences of non-translated DNA may be present 5' or 3' from the open reading frame, where such sequences do not interfere with manipulation or expression of the coding regions, and may indeed act to modulate production of a desired product by various mechanisms (see "DNA regulatory sequences", above). Alternatively, DNA sequences encoding RNA (e.g., guide RNA) that is not translated may also be considered recombinant. Thus, e.g., the term "recombinant" nucleic acid refers to one which is not naturally occurring, e.g., is made by the artificial combination of two otherwise separated segments of sequence through human intervention. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. Such is usually done to replace a codon with a codon encoding the same amino acid, a conservative amino acid, or a non-conservative amino acid. Alternatively, it is performed to join together nucleic acid segments of desired functions to generate a desired combination of functions. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. When a recombinant polynucleotide encodes a polypeptide, the sequence of the encoded polypeptide can be naturally occurring (“wild type”) or can be a variant (e.g., a mutant) of the naturally occurring sequence. Thus, the term "recombinant" polypeptide does not necessarily refer to a polypeptide whose sequence does not naturally occur. Instead, a “recombinant” polypeptide is encodedby a recombinant DNA sequence, but the sequence of the polypeptide can be naturally occurring (“wild type”) or non-naturally occurring (e.g., a variant, a mutant, etc.). Thus, a "recombinant" polypeptide is the result of human intervention, but may have a naturally occurring amino acid sequence.

[0038] A "vector" or “expression vector” is a replicon, such as plasmid, phage, virus, or cosmid, to which another DNA segment, i.e. an “insert”, may be attached so as to bring about the replication of the attached segment in a cell.

[0039] An “expression cassette” comprises a DNA coding sequence operably linked to a promoter. "Operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For instance, a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression (the coding sequence can also be said to be operably linked to the promoter).

[0040] The terms “recombinant expression vector,” or “DNA construct” are used interchangeably herein to refer to a DNA molecule comprising a vector and one insert. Recombinant expression vectors are usually generated for the purpose of expressing and / or propagating the insert(s), or for the construction of other recombinant nucleotide sequences. The insert(s) may or may not be operably linked to a promoter sequence and may or may not be operably linked to DNA regulatory sequences.

[0041] “Heterologous,” as used herein, refers to a nucleotide or polypeptide sequence that is not found in the native nucleic acid or protein, respectively. For example, relative to a variant Cas12a protein of the present disclosure, a heterologous polypeptide comprises an amino acid sequence from a protein other than the variant Cas12a protein. As another example, a Cas12a protein of the present disclosure can be fused to an active domain from a non-CRISPR-Cas effector protein (e.g., a histone deacetylase), and the sequence of the active domain could be considered a heterologous polypeptide (it is heterologous to the variant Cas12a protein polypeptide). As another example, a guide sequence of a guide RNA that is heterologous to a protein-binding sequence (a constant region) of a guide RNA is a guide sequence that is not found in nature together with the protein-binding sequence.

[0042] General methods in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., HaRBor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds.,Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference.

[0043] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0044] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0045] Certain ranges are presented herein with numerical values being preceded by the term "about." The term "about" is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0046] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.

[0047] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to thefiling date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0048] It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. As such, the articles “a” and “an” are used herein to refer to one or to more than one (i.e. , to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. Thus, for example, reference to “a cell” includes a plurality of such cells and reference to “the polypeptide” includes reference to one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0049] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible. For example, it is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0050] While the apparatus and method has or will be described for the sake of grammatical fluidity with functional explanations, it is to be expressly understood that the claims, unless expressly formulated under 35 U.S.C. §112, are not to be construed as necessarily limited in any way by the construction of "means" or "steps" limitations,but are to be accorded the full scope of the meaning and equivalents of the definition provided by the claims under the judicial doctrine of equivalents, and in the case where the claims are expressly formulated under 35 U.S.C. §112 are to be accorded full statutory equivalents under 35 U.S.C. §112.V. DETAILED DESCRIPTION

[0051] The present disclosure provides variant Cas12a proteins, nucleic acids encoding the variant proteins, and systems comprising the variant proteins or nucleic acids encoding same. In some cases, a variant Cas12a protein is fused to a heterologous protein. As such, also provided are fusion proteins comprising: (a) a subject variant Cas12a protein; and (b) one or more heterologous polypeptides, where a heterologous polypeptide can also be referred to as a “fusion partner.” The present disclosure provides methods for modifying a target nucleic acid, using a variant protein of the present disclosure. The present disclosure provides methods for modulating transcription from a target DNA, using a variant protein of the present disclosure fused to a heterologous protein. The present disclosure provides methods for detecting a target nucleic acid, using a variant protein of the present disclosure. Reagents, compositions, and kits / systems that find use in practicing the subject methods are provided.Compositions and MethodsVariant Cas12a protein

[0052] A wild-type type V CRISPR-Cas protein, e.g., Cas12 proteins such as Cpf1 (Cas12a) and C2c1 (Cas12b), can promiscuously cleave non-targeted single-stranded nucleic acid (e.g., single stranded DNA (ssDNA)) once activated by binding to a target nucleic acid (e.g., ssDNA or double stranded DNA (dsDNA)). For example, when a wild-type type V CRISPR-Cas effector protein (e.g., a Cas12 protein such as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas12f, Cas12g, Cas12h, or Cas12i) bound to a guide RNA (i.e., a ribonucleoprotein (RNP) complex), is guided to a target sequence of a target nucleic acid (e.g., DNA), which occurs when the guide RNA hybridizes to (binds to) the target sequence), the protein becomes an activated nuclease that promiscuously cleaves single stranded target nucleic acid (e.g., ssDNAs) (i.e., the nuclease cleaves non-target single stranded target nucleic acid, e.g., ssDNAs, i.e., ssDNAs to which the guide sequence of the guide RNA does not hybridize). When atype V CRISPR-Cas effector protein is activated and exhibits on-target cleavage of the target nucleic acid (e.g., ssDNA), such on-target cleavage is referred to as “cis” cleavage (Li et al (2018) Ce / I Research 28:491-493). When a type CRISPR-Cas effector protein is activated and exhibits cleavage of non-target single stranded nucleic acids (e.g., ssDNAs, i.e. , ssDNAs to which the guide sequence of the guide RNA does not hybridize) such non-target cleavage is referred to as “trans" cleavage.

[0053] A variant Cas12a protein of the present disclosure, when complexed with a guide RNA, binds a target nucleic acid, where the guide RNA comprises a segment that hybridizes to a complementary segment in the target nucleic acid.

[0054] A variant Cas12a protein of the present disclosure (i.e., a subject variant Cas12a protein) comprises an amino acid sequence having one or more amino acid substitutions relative to a corresponding wild-type Cas12a protein. The one or more amino acid substitutions reduce the PAM constraints of the variant Cas12a as compared to the corresponding wild type Cas12a protein. By “corresponding” wild type protein, it is meant the wild type Cas12a protein that is closest in sequence to a given variant Cas12a protein. Thus, to determine the sequence identity of a variant Cas12a protein to a corresponding wild type Cas12a protein, one would compare the sequence of the variant Cas12a protein in question with the closest known wild type Cas12a protein. In some embodiments, the corresponding wild type Cas12a protein is any one of the Cas12a proteins of SEQ ID Nos 1-13. In some embodiments, the corresponding wild type Cas12a protein is the Lachnospiraceae bacterium Cas12a (LbCas12a) protein of SEQ ID NO: 1. For example, the corresponding wild type Cas12a protein is SEQ ID NO: 1 for the variants PL2a (also referred to herein as “Lbm1”), PL2b (also referred to herein as “Lbm2”), PL2c (also referred to herein as “Lbm3”), PL2d (also referred to herein as “Lbm4”), PL3 (also referred to herein as “Lbm5”), PL4a (also referred to herein as “Lbm6”), and PL4b (also referred to herein as “Lbm7”) (SEQ ID NOs: 14-20, respectively - see FIG. 7).

[0055] In some embodiments (e.g., for any of the amino acid substitutions disclosed herein), the variant Cas12a protein comprises an amino acid sequence having 70% or more (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more) identity with any one of the wild type Cas12a proteins of SEQ ID Nos: 1-13. For example, in some embodiments, the variant Cas12a protein comprises an amino acid sequence having 80% or more (e.g., 85% or more, 90% or more, 95% or more, or 99% or more) identity with any one of the wild type Cas12a proteins of SEQ ID Nos: 1-13. For example, in some embodiments, the variant Cas12a protein comprisesan amino acid sequence having 90% or more (e g., 92% or more, 95% or more, or 99% or more) identity with any one of the wild type Cas12a proteins of SEQ ID Nos: 1-13.

[0056] In some embodiments (e.g., for any of the amino acid substitutions disclosed herein), a subject variant Cas12a protein comprises an amino acid sequence having 70% or more (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more) sequence identity with wild type LbCas12a protein of SEQ ID NO: 1. For example, in some embodiments, a subject variant Cas12a protein comprises an amino acid sequence having 80% or more (e.g., 85% or more, 90% or more, 95% or more, or 99% or more) sequence identity with the wild type LbCas12a protein of SEQ ID NO: 1. For example, in some embodiments, a subject variant Cas12a protein comprises an amino acid sequence having 90% or more (e.g., 92% or more, 95% or more, or 99% or more) sequence identity with the wild type LbCas12a protein of SEQ ID NO: 1.

[0057] In some embodiments (e.g., for any of the amino acid substitutions disclosed herein), a variant Cas12a protein comprises an amino acid sequence having 70% or more (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, or 100%) identity with any one of the variant Cas12a proteins of SEQ ID NOs: 14-20. For example, in some embodiments, a variant Cas12a protein comprises an amino acid sequence having 80% or more (e.g., 85% or more, 90% or more, 95% or more, 99% or more, or 100%) identity with any one of the variant Cas12a proteins of SEQ ID NOs: 14-20. For example, in some embodiments, a variant Cas12a protein comprises an amino acid sequence having 90% or more (e.g., 92% or more, 95% or more, 99% or more, or 100%) identity with any one of the variant Cas12a proteins of SEQ ID NOs: 14-20. For example, in some embodiments, a variant Cas12a protein comprises the amino acid sequence of any one of the variant Cas12a proteins of SEQ ID NOs: 14-20.

[0058] In some cases, the one or more amino acid substitutions of a subject variant Cas12a protein are at one or more amino acid positions corresponding to the amnio acid positions of SEQ ID NO:1 that are identified in FIG. 7. “Corresponding” in this context refers to the particular positions of SEQ ID NO: 1 identified in FIG. 7, or to equivalent amino acid positions of other wild type Cas12a proteins. In cases in which the wild type Cas12a protein is SEQ ID NO: 1 , then the corresponding amnio acid and amino acid position are identical to those of SEQ ID NO: 1. Identifying corresponding amino acid positions in orthologous proteins (e.g., Cas12a proteins from another species) iswell within the skill of one of ordinary skill in the art, e.g., using sequence alignments and / or 3 dimensional structural modeling (ideally using Cas12a proteins from multiple different species). A sequence alignment of example wild type Cas12a proteins (SEQ ID NOs: 1-13) is provided as FIG. 6.

[0059] In some embodiments, a variant Cas12a protein includes amino acid substitutions (relative to a corresponding wild type Cas12a protein) in a region spanning WED-II, PAM-interacting, and WED-III domains, corresponding to positions Y516 through N808 of SEQ ID NO: 1. In some cases, for example if the corresponding wild type Cas12a is any one of SEQ ID NOs: 2-13, the region spanning WED-II, PAM- interacting, and WED-III domains may have different starting and ending amino acid positions (i.e., different than position 516 through 808), but the region will correspond to that region of SEQ ID NO: 1. In some cases, a variant Cas12a protein includes amino acid substitutions in a region corresponding to positions Q529 through P799 of SEQ ID NO: 1. In some cases, a variant Cas12a protein includes amino acid substitutions in a region corresponding to positions Q529 through E610 of SEQ ID NO: 1. In some cases, a variant Cas12a protein includes amino acid substitutions in a region corresponding to positions Q529 through E754 of SEQ ID NO: 1. In some cases, a variant Cas12a protein includes amino acid substitutions in a region corresponding to positions D535 through D665 of SEQ ID NO: 1. In some cases, a variant Cas12a protein includes amino acid substitutions in a region corresponding to positions D535 through I765 of SEQ ID NO: 1. In some cases, a variant Cas12a protein includes amino acid substitutions in a region corresponding to positions K538 through N590 of SEQ ID NO: 1. In some cases, a variant Cas12a protein includes amino acid substitutions in a region corresponding to positions K538 through P799 of SEQ ID NO: 1. In some cases, a variant Cas12a protein includes amino acid substitutions in a region corresponding to positions K538 through E743 of SEQ ID NO: 1. For any of the above ranges in this paragraph, in some cases, the corresponding wild type Cas12a protein is the LbCas12a protein of SEQ ID NO: 1. As such, in some cases, the variant Cas12a protein comprises an amino acid sequence having 70% or more (e g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more) sequence identity with LbCas12a protein of SEQ ID NO: 1. In some cases, the variant Cas12a protein comprises an amino acid sequence having 70% or more (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more) sequence identity with the wild type Cas12a protein ofany one of SEQ ID NOs: 1-13. See above for additional examples of amino acid sequence identity.

[0060] In some embodiments, a variant Cas12a protein includes amino acid substitutions at amino acid positions corresponding to: (a) (K538 and N590); or (b) (K538, Q529, and E610); or (c) (K538, Q529, K753, and E754); or (d) (K538, Y553, D691 , K752, and P799); or (e) (K538, Y549, and E743); or any combination thereof, of SEQ ID NO: 1. For example, in some such cases, the corresponding wild type Cas12a protein is the LbCas12a protein of SEQ ID NO: 1 . As such, in some cases, the variant Cas12a protein comprises an amino acid sequence having 70% or more (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more) sequence identity with LbCas12a protein of SEQ ID NO: 1. In some cases, the variant Cas12a protein comprises an amino acid sequence having 70% or more (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more) sequence identity with the wild type Cas12a protein of any one of SEQ ID NOs: 1-13. In some cases, the variant Cas12a protein comprises an amino acid sequence having 70% or more (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, or 100%) identity with any one of the variant Cas12a proteins of SEQ ID NOs: 14-18. In some cases, the variant Cas12a protein comprises an amino acid sequence having 70% or more (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, or 100%) identity with the variant Cas12a protein of SEQ ID NO: 14. In some cases, the variant Cas12a protein comprises an amino acid sequence having 70% or more (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, or 100%) identity with the variant Cas12a protein of SEQ ID NO: 15. In some cases, the variant Cas12a protein comprises an amino acid sequence having 70% or more (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, or 100%) identity with the variant Cas12a protein of SEQ ID NO: 16. In some cases, the variant Cas12a protein comprises an amino acid sequence having 70% or more (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, or 100%) identity with the variant Cas12a protein of SEQ ID NO: 17. In some cases, the variant Cas12a protein comprises an amino acid sequence having 70% or more (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, or 100%) identity with the variant Cas12a protein of SEQ ID NO: 18. See above for additional examples of amino acid sequence identity.

[0061] In some embodiments, a variant Cas12a protein includes amino acid substitutions at amino acid positions corresponding to: (a) (D535, S551 , and D665); or (b) (D535, D573, N628, and I765); or any combination thereof, of SEQ ID NO: 1. For example, in some such cases, the corresponding wild type Cas12a protein is the LbCas12a protein of SEQ ID NO: 1. As such, in some cases, the variant Cas12a protein comprises an amino acid sequence having 70% or more (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more) sequence identity with LbCas12a protein of SEQ ID NO: 1. In some cases, the variant Cas12a protein comprises an amino acid sequence having 70% or more (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more) sequence identity with the wild type Cas12a protein of any one of SEQ ID NOs: 1-13. In some cases, the variant Cas12a protein comprises an amino acid sequence having 70% or more (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, or 100%) identity with any one of the variant Cas12a proteins of SEQ ID NOs: 19-20. In some cases, the variant Cas12a protein comprises an amino acid sequence having 70% or more (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, or 100%) identity with the variant Cas12a protein of SEQ ID NO: 19. In some cases, the variant Cas12a protein comprises an amino acid sequence having 70% or more (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, or 100%) identity with the variant Cas12a protein of SEQ ID NO: 20. See above for additional examples of amino acid sequence identity.

[0062] In some embodiments, a variant Cas12a protein includes amino acid substitutions corresponding to: (a) (K538N and N590Y); or (b) (K538N, Q529R, and E610G); or (c) (K538N, Q529R, K753R, and E754A); or (d) (K538N, Y553F, D691G, K752R, and P799R); or (e) (K538N, Y549C, and E743K); or any combination thereof, of SEQ ID NO: 1. For example, in some such cases, the corresponding wild type Cas12a protein is the LbCas12a protein of SEQ ID NO: 1. As such, in some cases, the variant Cas12a protein comprises an amino acid sequence having 70% or more (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more) sequence identity with LbCas12a protein of SEQ ID NO: 1. In some cases, the variant Cas12a protein comprises an amino acid sequence having 70% or more (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more) sequence identity with the wild type Cas12a protein of any one of SEQ ID NOs: 1-13. In some cases, the variant Cas12a protein comprises an amino acid sequence having70% or more (e g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, or 100%) identity with any one of the variant Cas12a proteins of SEQ ID NOs: 14-18. In some cases, the variant Cas12a protein comprises an amino acid sequence having 70% or more (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, or 100%) identity with the variant Cas12a protein of SEQ ID NO: 14. In some cases, the variant Cas12a protein comprises an amino acid sequence having 70% or more (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, or 100%) identity with the variant Cas12a protein of SEQ ID NO: 15. In some cases, the variant Cas12a protein comprises an amino acid sequence having 70% or more (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, or 100%) identity with the variant Cas12a protein of SEQ ID NO: 16. In some cases, the variant Cas12a protein comprises an amino acid sequence having 70% or more (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, or 100%) identity with the variant Cas12a protein of SEQ ID NO: 17. In some cases, the variant Cas12a protein comprises an amino acid sequence having 70% or more (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, or 100%) identity with the variant Cas12a protein of SEQ ID NO: 18. See above for additional examples of amino acid sequence identity.

[0063] In some embodiments, a variant Cas12a protein includes amino acid substitutions corresponding to: (a) (D535G, S551F, and D665N); or (b) (D535G, D573G, N628S, and I765T); or any combination thereof, of SEQ ID NO: 1. For example, in some such cases, the corresponding wild type Cas12a protein is the LbCas12a protein of SEQ ID NO: 1. As such, in some cases, the variant Cas12a protein comprises an amino acid sequence having 70% or more (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more) sequence identity with LbCas12a protein of SEQ ID NO: 1. In some cases, the variant Cas12a protein comprises an amino acid sequence having 70% or more (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more) sequence identity with the wild type Cas12a protein of any one of SEQ ID NOs: 1-13. In some cases, the variant Cas12a protein comprises an amino acid sequence having 70% or more (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, or 100%) identity with any one of the variant Cas12a proteins of SEQ ID NOs: 19-20. In some cases, the variant Cas12a protein comprises an amino acid sequence having 70% or more (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 99% or more,or 100%) identity with the variant Cas12a protein of SEQ ID NO: 19. In some cases, the variant Cas12a protein comprises an amino acid sequence having 70% or more (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, or 100%) identity with the variant Cas12a protein of SEQ ID NO: 20. See above for additional examples of amino acid sequence identity.

[0064] In some embodiments, a variant Cas12a protein includes amino acid substitutions at amino acid positions corresponding to: (a) (K538 and N590); or (b) (K538, Q529, and E610); or (c) (K538, Q529, K753, and E754); or (d) (K538, Y553, D691 , K752, and P799); or (e) (K538, Y549, and E743); or (f) (D535, S551 , and D665); or (g) (D535, D573, N628, and I765); or any combination thereof, of SEQ ID NO: 1. In some embodiments, a variant Cas12a protein includes amino acid substitutions corresponding to: (a) (K538N and N590Y); or (b) (K538N, Q529R, and E610G); or (c) (K538N, Q529R, K753R, and E754A); or (d) (K538N, Y553F, D691G, K752R, and P799R); or (e) (K538N, Y549C, and E743K); or (f) (D535G, S551 F, and D665N); or (g) (D535G, D573G, N628S, and I765T); or any combination thereof, of SEQ ID NO: 1. See above for examples of amino acid sequence identity.

[0065] The variants described herein can be used in combination with other variants. As would be understood by one of ordinary skill in the art, many variant forms of CRISPR-Cas effector proteins are known in the art, e.g., those harboring mutations that increase specificity (e.g., decrease off-targeting), and any convenient variant can be used (e.g., AsCas12a ultra nuclease) in combination with the variants of the present disclosure. See, e.g., Vakulskas et al., Nat Med. 2018 Aug;24(8):1216-1224; Kleinstiver et al., Nature. 2016 Jan 28;529(7587):490-5; Yuen et al., Nucleic Acids Res. 2022 Feb 22;50(3):1650-1660; Wei et al., FASEB J. 2023 Aug;37(8):e23060; Tan et al., Proc Natl Acad Sci U S A. 2019 Oct 15;116(42):20969-20976; Kleinstiver et al., Nat Biotechnol. 2019 Mar;37(3):276-282; DeWeirdt et al., Nat. Biotechnol. 2021 39, 94-104; Zhang et al., Nat Commun. 2021 Jun 23;12(1):3908. For additional examples, see Zhang et al., 2023 Genome Biol, 24, 102 [Cas12a-RVQ]; Guo et al., (2022) Nat Cell Biol, 24, 590-600 [hyperCas12a]; and Ma et al., (2022) Nucleic Acids Res, 50, 12689-12701 [iCas12a],

[0066] For example, in some cases, a subject variant Cas12a protein, in addition to including mutations that reduce PAM constraints (as discussed herein), also includes mutations corresponding to G146R, R182V and E795Q (referred to as “-RVQ”) of SEQ ID NO: 1 (which are mutations from an activity-enhanced variant of Cas12a). For example, In some cases a subject variant is a PL2a variant (also referred to as “Lbm1”) andincludes the -RVQ mutations (can therefore be referred to as “Lbm1-RVQ”), which therefore includes mutations corresponding to K538N and N590Y as well as G146R, R182V and E795Q of SEQ ID NO: 1. In some cases a subject variant is a PL2b variant (also referred to as “Lbm2”) and includes the -RVQ mutations (can therefore be referred to as “Lbm2-RVQ”), which therefore includes mutations corresponding to K538N, Q529R, and E610G as well as G146R, R182V and E795Q of SEQ ID NO: 1. In some cases a subject variant is a PL2c variant (also referred to as “Lbm3”) and includes the -RVQ mutations (can therefore be referred to as “Lbm3-RVQ”), which therefore includes mutations corresponding to K538N, Q529R, K753R, and E754A as well as G146R, R182V and E795Q of SEQ ID NO: 1. In some cases a subject variant is a PL2d variant (also referred to as “Lbm4”) and includes the -RVQ mutations (can therefore be referred to as “Lbm4-RVQ”), which therefore includes mutations corresponding to K538N, Y553F, D691G, K752R, and P799R as well as G146R, R182V and E795Q of SEQ ID NO: 1. In some cases a subject variant is a PL3 variant (also referred to as “Lbm5”) and includes the -RVQ mutations (can therefore be referred to as “Lbm5-RVQ”), which therefore includes mutations corresponding to K538N, Y549C, and E743K as well as G146R, R182V and E795Q of SEQ ID NO: 1. In some cases a subject variant is a PL4a variant (also referred to as “Lbm6”) and includes the -RVQ mutations (can therefore be referred to as “Lbm6-RVQ” - and is also referred to herein as “Flex-Cas12a” when I has SEQ ID NO: 1 but with the Lbm6 and the -RVQ mutations), which therefore includes mutations corresponding to D535G, S551 F, and D665N as well as G146R, R182V and E795Q of SEQ ID NO: 1. In some cases a subject variant is a PL4b variant (also referred to as “Lbm7”) and includes the -RVQ mutations (can therefore be referred to as “Lbm7-RVQ”), which therefore includes mutations corresponding to D535G, D573G, N628S, and I765T as well as G146R, R182V and E795Q of SEQ ID NO: 1.

[0067] As such, in some cases, the variant Cas12a protein comprises an amino acid sequence that is 70% or more (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more) identical to the LbCas12a protein of SEQ ID NO: 1 and includes mutations corresponding to K538N and N590Y as well as G146R, R182V and E795Q of SEQ ID NO: 1. In some cases, the variant Cas12a protein comprises an amino acid sequence that is 85% or more (e.g., 90% or more, 95% or more, or 99% or more) identical to the LbCas12a protein of SEQ ID NO: 1 and includes mutations corresponding to K538N and N590Y as well as G146R, R182V and E795Q of SEQ ID NO: 1. In some cases, the variant Cas12a protein comprisesan amino acid sequence that is 90% or more (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more) identical to the LbCas12a protein of SEQ ID NO: 1 and includes mutations corresponding to K538N and N590Y as well as G146R, R182V and E795Q of SEQ ID NO: 1 . In some cases, the variant Cas12a protein comprises an amino acid sequence that is 95% or more (e.g., 97% or more, 98% or more, 99% or more, or 99.5% or more) identical to the LbCas12a protein of SEQ ID NO: 1 and includes mutations corresponding to K538N and N590Y as well as G146R, R182V and E795Q of SEQ ID NO: 1 . In some cases, the variant Cas12a protein comprises the amino acid sequence of SEQ ID NO: 1, but has the mutations K538N and N590Y as well as G146R, R182V and E795Q.

[0068] In some cases, the variant Cas12a protein comprises an amino acid sequence that is 70% or more (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more) identical to the LbCas12a protein of SEQ ID NO: 1 and includes mutations corresponding to K538N, Q529R, and E610G as well as G146R, R182V and E795Q of SEQ ID NO: 1 . In some cases, the variant Cas12a protein comprises an amino acid sequence that is 85% or more (e.g., 90% or more, 95% or more, or 99% or more) identical to the LbCas12a protein of SEQ ID NO: 1 and includes mutations corresponding to K538N, Q529R, and E610G as well as G146R, R182V and E795Q of SEQ ID NO: 1 . In some cases, the variant Cas12a protein comprises an amino acid sequence that is 90% or more (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more) identical to the LbCas12a protein of SEQ ID NO: 1 and includes mutations corresponding to K538N, Q529R, and E610G as well as G146R, R182V and E795Q of SEQ ID NO: 1. In some cases, the variant Cas12a protein comprises an amino acid sequence that is 95% or more (e.g., 97% or more, 98% or more, 99% or more, or 99.5% or more) identical to the LbCas12a protein of SEQ ID NO: 1 and includes mutations corresponding to K538N, Q529R, and E610G as well as G146R, R182V and E795Q of SEQ ID NO: 1 . In some cases, the variant Cas12a protein comprises the amino acid sequence of SEQ ID NO: 1 , but has the mutations K538N, Q529R, and E610G as well as G146R, R182V and E795Q.

[0069] In some cases, the variant Cas12a protein comprises an amino acid sequence that is 70% or more (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more) identical to the LbCas12a protein of SEQ ID NO: 1 and includes mutations corresponding to K538N, Q529R, K753R, and E754A as well as G146R, R182V and E795Q of SEQ ID NO: 1. In some cases, the variant Cas12aprotein comprises an amino acid sequence that is 85% or more (e.g., 90% or more, 95% or more, or 99% or more) identical to the LbCas12a protein of SEQ ID NO: 1 and includes mutations corresponding to K538N, Q529R, K753R, and E754A as well as G146R, R182V and E795Q of SEQ ID NO: 1. In some cases, the variant Cas12a protein comprises an amino acid sequence that is 90% or more (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more) identical to the LbCas12a protein of SEQ ID NO: 1 and includes mutations corresponding to K538N, Q529R, K753R, and E754A as well as G146R, R182V and E795Q of SEQ ID NO: 1. In some cases, the variant Cas12a protein comprises an amino acid sequence that is 95% or more (e.g., 97% or more, 98% or more, 99% or more, or 99.5% or more) identical to the LbCas12a protein of SEQ ID NO: 1 and includes mutations corresponding to K538N, Q529R, K753R, and E754A as well as G146R, R182V and E795Q of SEQ ID NO: 1 . In some cases, the variant Cas12a protein comprises the amino acid sequence of SEQ ID NO: 1 , but has the mutations K538N, Q529R, K753R, and E754A as well as G146R, R182V and E795Q.

[0070] In some cases, the variant Cas12a protein comprises an amino acid sequence that is 70% or more (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more) identical to the LbCas12a protein of SEQ ID NO: 1 and includes mutations corresponding to K538N, Y553F, D691G, K752R, and P799R as well as G146R, R182V and E795Q of SEQ ID NO: 1. In some cases, the variant Cas12a protein comprises an amino acid sequence that is 85% or more (e.g., 90% or more, 95% or more, or 99% or more) identical to the LbCas12a protein of SEQ ID NO: 1 and includes mutations corresponding to K538N, Y553F, D691G, K752R, and P799R as well as G146R, R182V and E795Q of SEQ ID NO: 1. In some cases, the variant Cas12a protein comprises an amino acid sequence that is 90% or more (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more) identical to the LbCas12a protein of SEQ ID NO: 1 and includes mutations corresponding to K538N, Y553F, D691G, K752R, and P799R as well as G146R, R182V and E795Q of SEQ ID NO: 1. In some cases, the variant Cas12a protein comprises an amino acid sequence that is 95% or more (e.g., 97% or more, 98% or more, 99% or more, or 99.5% or more) identical to the LbCas12a protein of SEQ ID NO: 1 and includes mutations corresponding to K538N, Y553F, D691G, K752R, and P799R as well as G146R, R182V and E795Q of SEQ ID NO: 1. In some cases, the variant Cas12a protein comprises the amino acid sequence of SEQ ID NO: 1, but has the mutations K538N, Y553F, D691G, K752R, and P799R as well as G146R, R182V and E795Q.

[0071] In some cases, the variant Cas12a protein comprises an amino acid sequence that is 70% or more (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more) identical to the LbCas12a protein of SEQ ID NO: 1 and includes mutations corresponding to K538N, Y549C, and E743K as well as G146R, R182V and E795Q of SEQ ID NO: 1. In some cases, the variant Cas12a protein comprises an amino acid sequence that is 85% or more (e.g., 90% or more, 95% or more, or 99% or more) identical to the LbCas12a protein of SEQ ID NO: 1 and includes mutations corresponding to K538N, Y549C, and E743K as well as G146R, R182V and E795Q of SEQ ID NO: 1. In some cases, the variant Cas12a protein comprises an amino acid sequence that is 90% or more (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more) identical to the LbCas12a protein of SEQ ID NO: 1 and includes mutations corresponding to K538N, Y549C, and E743K as well as G146R, R182V and E795Q of SEQ ID NO: 1 . In some cases, the variant Cas12a protein comprises an amino acid sequence that is 95% or more (e.g., 97% or more, 98% or more, 99% or more, or 99.5% or more) identical to the LbCas12a protein of SEQ ID NO: 1 and includes mutations corresponding to K538N, Y549C, and E743K as well as G146R, R182V and E795Q of SEQ ID NO: 1. In some cases, the variant Cas12a protein comprises the amino acid sequence of SEQ ID NO: 1 , but has the mutations K538N, Y549C, and E743K as well as G146R, R182V and E795Q.

[0072] In some cases, the variant Cas12a protein comprises an amino acid sequence that is 70% or more (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more) identical to the LbCas12a protein of SEQ ID NO: 1 and includes mutations corresponding to D535G, S551 F, and D665N as well as G146R, R182V and E795Q of SEQ ID NO: 1. In some cases, the variant Cas12a protein comprises an amino acid sequence that is 85% or more (e.g., 90% or more, 95% or more, or 99% or more) identical to the LbCas12a protein of SEQ ID NO: 1 and includes mutations corresponding to D535G, S551 F, and D665N as well as G146R, R182V and E795Q of SEQ ID NO: 1. In some cases, the variant Cas12a protein comprises an amino acid sequence that is 90% or more (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more) identical to the LbCas12a protein of SEQ ID NO: 1 and includes mutations corresponding to D535G, S551 F, and D665N as well as G146R, R182V and E795Q of SEQ ID NO: 1. In some cases, the variant Cas12a protein comprises an amino acid sequence that is 95% or more (e.g., 97% or more, 98% or more, 99% or more, or 99.5% or more) identical to theLbCas12a protein of SEQ ID NO: 1 and includes mutations corresponding to D535G, S551F, and D665N as well as G146R, R182V and E795Q of SEQ ID NO: 1. In some cases, the variant Cas12a protein comprises the amino acid sequence of SEQ ID NO: 1, but has the mutations D535G, S551 F, and D665N as well as G146R, R182V and E795Q.

[0073] As such, in some cases, the variant Cas12a protein comprises an amino acid sequence that is 70% or more (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more) identical to the variant LbCas12a protein of SEQ ID NO: 28. In some cases, the variant Cas12a protein comprises an amino acid sequence that is 85% or more (e.g., 90% or more, 95% or more, or 99% or more) identical to the variant LbCas12a protein of SEQ ID NO: 28. In some cases, the variant Cas12a protein comprises an amino acid sequence that is 90% or more (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more) identical to the variant LbCas12a protein of SEQ ID NO: 28. In some cases, the variant Cas12a protein comprises an amino acid sequence that is 95% or more (e.g., 97% or more, 98% or more, 99% or more, or 99.5% or more) identical to the variant LbCas12a protein of SEQ ID NO: 28. In some cases, the variant Cas12a protein comprises the amino acid sequence of SEQ ID NO: 28.

[0074] In some cases, the variant Cas12a protein comprises an amino acid sequence that is 70% or more (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more) identical to the LbCas12a protein of SEQ ID NO: 1 and includes mutations corresponding to D535G, D573G, N628S, and I765T as well as G146R, R182V and E795Q of SEQ ID NO: 1. In some cases, the variant Cas12a protein comprises an amino acid sequence that is 85% or more (e.g., 90% or more, 95% or more, or 99% or more) identical to the LbCas12a protein of SEQ ID NO: 1 and includes mutations corresponding to D535G, D573G, N628S, and I765T as well as G146R, R182V and E795Q of SEQ ID NO: 1. In some cases, the variant Cas12a protein comprises an amino acid sequence that is 90% or more (e.g., 95% or more, 97% or more, 98% or more, 99% or more, or 99.5% or more) identical to the LbCas12a protein of SEQ ID NO: 1 and includes mutations corresponding to D535G, D573G, N628S, and I765T as well as G146R, R182V and E795Q of SEQ ID NO: 1. In some cases, the variant Cas12a protein comprises an amino acid sequence that is 95% or more (e.g., 97% or more, 98% or more, 99% or more, or 99.5% or more) identical to the LbCas12a protein of SEQ ID NO: 1 and includes mutations corresponding to D535G, D573G, N628S, and I765T as well as G146R, R182V andE795Q of SEQ ID NO: 1 . In some cases, the variant Cas12a protein comprises the amino acid sequence of SEQ ID NO: 1 , but has the mutations D535G, D573G, N628S, and I765T as well as G146R, R182V and E795Q.

[0075] For example, in some cases, a subject variant Cas12a protein, in addition to including mutations that reduce PAM constraints (as discussed herein), also includes mutations that reduce or even remove trans cleavage activity. As would be known to one of ordinary skill in the art, in some cases, a CRISPR-Cas effector protein has trans cleavage activity. For example, in some cases, once a target sequence is recognized in the target nucleic acid (e.g., target DNA or target RNA), the enzyme can become a promiscuous nuclease that cleaves, e.g., ssDNA (or ssRNA). In some cases, this activity can be harnessed for purposes of nucleic acid detection. For example, cleavage of a labeled detector oligonucleotide can be used (e.g., one that either increases or decreases in fluorescence after being cleaved by the trans cleavage activity) to detect trans cleavage activity.

[0076] However, in some cases, a variant Cas12a protein has one or more mutations that reduce or remove trans cleavage activity (see, e.g., international patent application publication No. WO2023147240, which is incorporated herein by reference for disclosure related to variant Cas12a proteins, including those with reduced trans cleavage activity). As an illustrative example, in some cases, a subject variant Cas12a protein, in addition to substitution(s) that reduces PAM constraints, includes an amino acid substitution at an amino acid position corresponding to W890 of SEQ ID NO: 1. In some cases, the amino acid at this position can be substituted with Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Tyr, or Vai. In some cases, the amino acid at this position can be substituted with Ala, Gly, Vai, lie, Leu, or Met (i.e., an amino acid with a non-polar side chain). In some cases, the amino acid at this position is substituted with Ala (i.e., W390A of SEQ ID NO: 1). Other example amnio acid positions include 879, 916, 921 , 928, 931, 941 , 958, 964, 968, 971, 1045. In some cases, a subject variant Cas12a protein includes substitutions at positions corresponding to E217, E885, W890, and 11028 of SEQ ID NO: 1. In some cases, the variant Cas12a protein exhibits less than 50% (e.g., 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 2%, or less than 1%) of the cleavage of a non-target ssDNA exhibited by the corresponding wild-type Cas12a protein.PAMs

[0077] As would be understood by one of ordinary skill in the art, a type V CRISPR-Cas effector protein (e.g., Cas12a) binds to target nucleic acid (e.g., DNA) at a target sequence defined by the region of complementarity between the guide RNA and the target nucleic acid. As is the case for many CRISPR-Cas endonucleases, site-specific binding (and / or cleavage) of a double stranded target DNA occurs at locations determined by both (i) base-pairing complementarity between the guide RNA and the target DNA; and (ii) a short motif (referred to as the protospacer adjacent motif (PAM)) in the target DNA. A variant Cas12a protein of the present disclosure recognizes a more flexible PAM when compared to a reference (e.g., the corresponding wild-type Cas12a protein).

[0078] For Cas12a proteins, the PAM is immediately 5’ of the target sequence (i.e., is positioned upstream of the targeted sequence) (5’ of the non-complementary strand of the target DNA, where the complementary strand hybridizes to the guide sequence of the guide RNA while the non-complementary strand does not directly hybridize with the guide RNA). Subject variant Cas12a proteins, also referred to herein as “PAM- loose mutants”, exhibit reduced PAM constraints compared to the PAM constraints of the corresponding wild type Cas12a protein. For example, the wild type protein of SEQ ID NO: 1 (LbCas12a) has a PAM preference of 5’-TTTV-3’, where V is A, C, or G. As such, LbCas12a can utilize the following PAMs: 5’-TTTA-3’, 5’-TTTC-3’, and 5’- TTTG-3’, which means that LbCas12a particular target sequence constraints. In contrast, variant PL2a (SEQ ID NO: 14, also see FIG. 7) has a PAM preference of 5’- YTYS-3’, where Y is C or T and S is C or G. As such, PL2a can utilize a larger number of PAMs (compared to LbCas12a): 5’-CTTC-3’, 5’-TTTC-3’, 5’-CTTG-3’, 5’- TTTG-3’, 5’-CTCC-3’, 5’-TTCC-3’, 5’-CTCG-3’, 5’-TTCG-3’. In other words, the PAM constraints of PL2a are reduced (‘loosened’) compared to the wild type Cas12a.

[0079] Example PAMs for wild type Cas12a proteinsPAM: 5’-TTTV-3’ : LbCas12a (statistically calculated from experimental results) PAM: 5’-TTTV-3’ : AsCas12aPAM: 5’-TTN-3’ : FnCas12a, PmCas12a, MbCas12a, Mb2Cas12a, Mb3Cas12a, TsCas12a, and BsCas12a*where N=(A, C, G, or T); V=(A, C, or G); Y=(C or T); K=(G or T); S=(C or G)

[0080] Example PAMs for variant Cas12a proteins (as statistically calculated from experimental results)PAM: 5’-YTYS-3’ : PL2a (SEQ ID NO: 14)PAM: 5’-YKTN-3’ : PL2b (SEQ ID NO: 15) and PL2c (SEQ ID NO: 16) PAM: 5’-YYNN-3’ : PL2d (SEQ ID NO: 17) and PL3 (SEQ ID NO: 18) PAM: 5’-TYNN-3’ : PL4a (SEQ ID NO: 19) and PL4b (SEQ ID NO: 20) *where N=(A, C, G, or T); V=(A, C, or G); Y=(C or T); K=(G or T); S=(C or G)

[0081] As such, in some embodiments, the corresponding wild type Cas12a protein (of a subject Cas12a variant protein) has a PAM preference of 5-TTTV-3', wherein V is A, C, or G. In some embodiments, a variant Cas12a protein has a PAM preference of 5'- YTYS-3', 5-YKTN-3', 5-YYNN-3', or 5'-TYNN-3', where N is (A,C,G, or T); Y is (C or T); S is (C or G); and K is (G or T). In some embodiments, a variant Cas12a protein has a PAM preference of 5 -YTYS-3'. In some embodiments, a variant Cas12a protein has a PAM preference of 5'-YKTN-3'. In some embodiments, a variant Cas12a protein has a PAM preference of 5'-YYNN-3'. In some embodiments, a variant Cas12a protein has a PAM preference of 5'-TYNN-3'.

[0082] As discovered using PAM depletion assays, the PAM can be even more flexible in some instances. For example, it was found that the PAM sequence for Lbm6 (“PI4a”) (SEQ ID NO: 19) is 5’-NYHV-3’ (where N is any base (A, C, G, or T), Y is T or C, H is A, C or T, and V is A, C or G). This is also true for the PAM for Flex-Cas12a (which is Lbm6 plus -RVQ). As such, in some embodiments, a variant Cas12a protein has a PAM preference of 5'- NYHV-3'.Fusion proteins

[0083] A Variant Cas12a protein can be fused to a heterologous protein having any desired activity such as DNA-modifying activity (e.g., nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity (e g., reverse transcriptase activity), ligase activity, helicase activity, photolyase activity or glycosylase activity); transcription modulation activity (e.g., fusion to a transcription repressor or transcription activator); an activity that modifies a protein (e.g., a histone) that is associated with target DNA (e.g., methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity,deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity or demyristoylation activity). A heterologous polypeptide can also referred to herein as a “fusion partner.”

[0084] Some activities of the heterologous protein can cause transcriptional activation (referred to as CRISPRa) while others can cause transcription repression (referred to as CRISPRi).

[0085] Examples of CRISPRi and CRISPRa fusion proteins will be known to one of ordinary skill in the art and any convenient CRISPRi or CRISPRa fusion protein can be used. A “CRISPRi fusion protein” or “CRISPRa fusion protein” includes a variant Cas12a protein linked (covalently or non-covalently) to a transcription repressing or activating protein. In some cases, the variant Cas12a protein is fused to a transcription repressing protein (and is called a CRISPRi fusion protein or simply a CRISPRi protein). In some cases, the variant Cas12a protein is fused to a transcription activating protein (and is called a CRISPRa fusion protein or simply a CRISPRa protein). In some cases, the variant Cas12a protein is linked (covalently or non- covalently) directly to the transcription repressing protein (and is called a CRISPRi fusion protein or simply a CRISPRi protein). In some cases, the variant Cas12a protein is linked (covalently or non-covalently) directly to the transcription activating protein (and is called a CRISPRa fusion protein or simply a CRISPRa protein). In some cases, the variant Cas12a protein is linked (covalently or non-covalently) indirectly to the transcription repressing or activating protein, e.g., by being linked a protein that recruits a transcription repressing or activating protein (see, e.g., Griffith et al., Cell Genom. 2023 Sep 1 ; 3(9): 100387).

[0086] In some cases, the variant Cas12a protein (of the CRISPRi or CRISPRa fusion protein) harbors a mutation that reduces the endogenous nuclease activity (e.g., in some cases renders it catalytically inactive (“dead”), e.g., a “dCas12a”). In some cases, the variant Cas12a protein is catalytically inactive (i.e., ‘dead’), which is referred to in the art as a dCas protein (e.g., dCas12a). Such a protein will not exhibit the nuclease cleavage activity of the Cas effector protein, but the fusion protein (e.g., a CRISPRi or CRISPRa fusion protein) will exhibit the activity of the protein to which the dCas protein is fused (i.e., the fusion partner - the transcription repressing or activating protein). Examples of mutations to produce a dCas effector protein will be known to one of ordinary skill in the art. As non-limiting examples, D908A and E993A of the Cas12a of SEQ ID NO: 2 have been employed.

[0087] Examples of proteins (or fragments thereof) that can be used to increase transcription (i.e., transcription activating proteins) include but are not limited to: transcriptional activators such as VP16, VP64, VP48, VP64, VP160, p65 subdomain (e.g., from NFkB), Rta, VPR (which is a fusion of VP64, p65, and Rta), and activation domain of EDLL and / or TAL activation domain (e.g., for activity in plants); histone lysine methyltransferases such as SET1A, SET1 B, MLL1 to 5, ASH1 , SYMD2, NSD1 , and the like; histone lysine demethylases such as JHDM2a / b, UTX, JMJD3, and the like; histone acetyltransferases such as GCN5, PCAF, CBP, p300, TAF1 , TIP60 / PLIP, MOZ / MYST3, MORF / MYST4, SRC1 , ACTR, P160, CLOCK, and the like; and DNA demethylases such as Ten-Eleven Translocation (TET) dioxygenase 1 (TET1CD), TET1, DI E, DML1 , DML2, ROS1 , and the like. See, e.g., Chavez et al., Nat Methods. 2015 Apr; 12(4): 326-328. In some cases, the CRISPRa system is a SAM system, which includes 3 components that form the DNA-binding complex: (1) a CRISPRa fusion protein (e.g., dCas9 fused to VP64), (2) MS2 aptamer(s) added to the guide RNA (forming a characteristic stem loop structure recognized by MS2), and (3) transcriptional activators P65 (Nuclear Factor NF-KB p65) and HSF1 (Heat Shock Factor 1) fused with an MS2-tag corresponding to the minimal aptamer-binding peptide of the MS2 coat protein. See, e.g., review articles such as Adli, Nat Commun. 2018 May 15;9(1):1911; Becirovic, Cell Mol Life Sci. 2022 Feb 12;79(2):130; and Nidhi S, et al., Int J Mol Sci. 2021 Mar 24;22(7):3327.

[0088] Examples of proteins (or fragments thereof) that can be used as heterologous proteins to decrease transcription (often referred to as CRISPRi when used with a Variant Cas12a protein) include but are not limited to: transcriptional repressors such as the Kruppel associated box (KRAB or SKD); ZIM3 KRAB; KOX1 repression domain; the Mad mSIN3 interaction domain (SID); the ERF repressor domain (ERD), the SRDX repression domain (e.g., for repression in plants), and the like; histone lysine methyltransferases such as Pr-SET7 / 8, SUV4-20H1 , RIZ1 , and the like; histone lysine demethylases such as JMJD2A / JHDM3A, JMJD2B, JMJD2C / GASC1 , JMJD2D, JARID1A / RBP2, JARID1B / PLU-1 , JARID1C / SMCX, JARID1 D / SMCY, and the like; histone lysine deacetylases such as HDAC1 , HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1 , SIRT2, HDAC11 , and the like; DNA methylases such as Hhal DNA m5c-methyltransferase (M.Hhal), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plants), ZMET2, CMT1 , CMT2 (plants), and the like; and periphery recruitment elements such as Lamin A, Lamin B, and the like.

[0089] As such, the present disclosure provides a fusion polypeptide comprising: a) a variant Cas12a protein of the present disclosure; and b) one or more heterologous polypeptides. A heterologous polypeptide to which a variant Cas12a protein of the present disclosure can be fused is referred to herein as a “fusion partner.” In some cases, a variant Cas12a protein of the present disclosure is fused to one or more heterologous polypeptides that has / have an activity of interest (e.g., a catalytic activity of interest, subcellular localization activity, etc.) to form a fusion protein.

[0090] In some cases, the fusion partner can modulate transcription (e.g., inhibit transcription, increase transcription) of a target DNA. For example, in some cases the fusion partner is a protein (or a domain from a protein) that inhibits transcription (e.g., a transcriptional repressor, a protein that functions via recruitment of transcription inhibitor proteins, modification of target DNA such as methylation, recruitment of a DNA modifier, modulation of histones associated with target DNA, recruitment of a histone modifier such as those that modify acetylation and / or methylation of histones, and the like). In some cases, the fusion partner is a protein (or a domain from a protein) that increases transcription (e.g., a transcription activator, a protein that acts via recruitment of transcription activator proteins, modification of target DNA such as demethylation, recruitment of a DNA modifier, modulation of histones associated with target DNA, recruitment of a histone modifier such as those that modify acetylation and / or methylation of histones, and the like).

[0091] In some cases, a fusion polypeptide of the present disclosure includes a heterologous polypeptide that has enzymatic activity that modifies a target nucleic acid (e.g., nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, or glycosylase activity). In some cases, the fusion partner is a reverse transcriptase. In some cases, the fusion partner is a base editor. In some cases, the fusion partner is a deaminase.

[0092] In some cases, a fusion polypeptide of the present disclosure includes a heterologous polypeptide that has enzymatic activity that modifies a polypeptide (e.g., a histone) associated with a target nucleic acid (e.g., methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity,deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity or demyristoylation activity).

[0093] Examples of proteins (or fragments thereof) that can be used in increase transcription include but are not limited to: transcriptional activators such as VP16, VP64, VP48, VP160, p65 subdomain (e.g., from NFkB), and activation domain of EDLL and / or TAL activation domain (e.g., for activity in plants); histone lysine methyltransferases such as SET1A, SET1 B, MLL1 to 5, ASH1 , SYMD2, NSD1 , and the like; histone lysine demethylases such as JHDM2a / b, UTX, JMJD3, and the like; histone acetyltransferases such as GCN5, PCAF, CBP, p300, TAF1 , TIP60 / PLIP, MOZ / MYST3, MORF / MYST4, SRC1 , ACTR, P160, CLOCK, and the like; and DNA demethylases such as Ten-Eleven Translocation (TET) dioxygenase 1 (TET1CD), TET1 , DME, DML1 , DML2, ROS1 , and the like.

[0094] Examples of proteins (or fragments thereof) that can be used in decrease transcription include but are not limited to: transcriptional repressors such as the Kruppel associated box (KRAB or SKD); KOX1 repression domain; the Mad mSIN3 interaction domain (SID); the ERF repressor domain (ERD), the SRDX repression domain (e.g., for repression in plants), and the like; histone lysine methyltransferases such as Pr-SET7 / 8, SUV4-20H1, RIZ1 , and the like; histone lysine demethylases such as JMJD2A / JHDM3A, JMJD2B, JMJD2C / GASC1 , JMJD2D, JARID1A / RBP2, JARID1 B / PLU-1 , JARID1C / SMCX, JARID1 D / SMCY, and the like; histone lysine deacetylases such as HDAC1 , HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1 , SIRT2, HDAC11 , and the like; DNA methylases such as Hhal DNA m5c-methyltransferase (M.Hhal), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plants), ZMET2, CMT1 , CMT2 (plants), and the like; and periphery recruitment elements such as Lamin A, Lamin B, and the like.

[0095] In some cases, the fusion partner has enzymatic activity that modifies the target nucleic acid (e.g., ssRNA, dsRNA, ssDNA, dsDNA). Examples of enzymatic activity that can be provided by the fusion partner include but are not limited to: nuclease activity such as that provided by a restriction enzyme (e.g., Fokl nuclease), methyltransferase activity such as that provided by a methyltransferase (e.g., Hhal DNA m5c-methyltransferase (M.Hhal), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plants), ZMET2, CMT1 , CMT2 (plants), and the like); demethylase activity such as that provided by a demethylase (e.g., Ten-Eleven Translocation (TET) dioxygenase 1(TET1CD), TET1 , DME, DML1, DML2, R0S1 , and the like) , DNA repair activity, DNA damage activity, deamination activity such as that provided by a deaminase (e.g., a cytosine deaminase enzyme such as rat APOBEC1), dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity such as that provided by an integrase and / or resolvase (e.g., Gin invertase such as the hyperactive mutant of the Gin invertase, GinH106Y; human immunodeficiency virus type 1 integrase (IN); Tn3 resolvase; and the like), transposase activity, recombinase activity such as that provided by a recombinase (e.g., catalytic domain of Gin recombinase), polymerase activity, ligase activity, helicase activity, photolyase activity, and glycosylase activity).

[0096] In some cases, the fusion partner has enzymatic activity that modifies a protein associated with the target nucleic acid (e.g., ssRNA, dsRNA, ssDNA, dsDNA) (e.g., a histone, an RNA binding protein, a DNA binding protein, and the like). Examples of enzymatic activity (that modifies a protein associated with a target nucleic acid) that can be provided by the fusion partner include but are not limited to: methyltransferase activity such as that provided by a histone methyltransferase (HMT) (e.g., suppressor of variegation 3-9 homolog 1 (SUV39H1 , also known as KMT1A), euchromatic histone lysine methyltransferase 2 (G9A, also known as KMT1C and EHMT2), SUV39H2, ESET / SETDB1, and the like, SET1A, SET1 B, MLL1 to 5, ASH1 , SYMD2, NSD1 , DOT1 L, Pr-SET7 / 8, SUV4-20H1 , EZH2, RIZ1), demethylase activity such as that provided by a histone demethylase (e.g., Lysine Demethylase 1A (KDM1A also known as LSD1), JHDM2a / b, JMJD2A / JHDM3A, JMJD2B, JMJD2C / GASC1 , JMJD2D, JARID1A / RBP2, JARID1 B / PLU-1 , JARID1C / SMCX, JARID1D / SMCY, UTX, JMJD3, and the like), acetyltransferase activity such as that provided by a histone acetylase transferase (e.g., catalytic core / fragment of the human acetyltransferase p300, GCN5, PCAF, CBP, TAF1 , TIP60 / PLIP, MOZ / MYST3, MORF / MYST4, HBO1 / MYST2, HMOF / MYST1, SRC1 , ACTR, P160, CLOCK, and the like), deacetylase activity such as that provided by a histone deacetylase (e.g., HDAC1 , HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, HDAC11 , and the like), kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity, and demyristoylation activity.

[0097] Additional examples of a suitable fusion partners are dihydrofolate reductase (DHFR) destabilization domain (e.g., to generate a chemically controllable fusion polypeptide), and a chloroplast transit peptide.

[0098] In some case, a fusion polypeptide of the present disclosure comprises: a) a Variant Cas12a protein of the present disclosure; and b) a chloroplast transit peptide. Thus, for example, a ribonucleoprotein (RNP) complex, comprising a Variant Cas12a protein of the present disclosure and a guide RNA, can be targeted to the chloroplast. In some cases, this targeting may be achieved by the presence of an N-terminal extension, called a chloroplast transit peptide (CTP) or plastid transit peptide. Chromosomal transgenes from bacterial sources must have a sequence encoding a CTP sequence fused to a sequence encoding an expressed polypeptide if the expressed polypeptide is to be compartmentalized in the plant plastid (e.g. chloroplast). Accordingly, localization of an exogenous polypeptide to a chloroplast is often 1 accomplished by means of operably linking a polynucleotide sequence encoding a CTP sequence to the 5' region of a polynucleotide encoding the exogenous polypeptide. The CTP is removed in a processing step during translocation into the plastid. Processing efficiency may, however, be affected by the amino acid sequence of the CTP and nearby sequences at the amino terminus of the peptide. Other options for targeting to the chloroplast which have been described are the maize cab-m7 signal sequence (U.S. Pat. No. 7,022,896, WO 97 / 41228) a pea glutathione reductase signal sequence (WO 97 / 41228) and the CTP described in US2009029861 .

[0099] In some cases, a fusion polypeptide of the present disclosure can comprise: a) a variant Cas12a protein of the present disclosure; and b) an endosomal escape peptide.

[0100] For examples of some of the above fusion partners (and more) used in the context of fusions with Cas9, Zinc Finger, and / or TALE proteins (for site specific target nucleic modification, modulation of transcription, and / or target protein modification, e.g., histone modification), see, e.g.: Nomura et al, J Am Chem Soc. 2007 Jul 18;129(28):8676-7; Rivenbark et al., Epigenetics. 2012 Apr;7(4):350-60; Nucleic Acids Res. 2016 Jul 8;44(12):5615-28; Gilbert et al., Cell. 2013 Jul 18;154(2):442-51 ; Kearns et al., Nat Methods. 2015 May;12(5):401-3; Mendenhall et al., Nat Biotechnol. 2013 Dec;31 (12): 1133-6; Hilton et al., Nat Biotechnol. 2015 May;33(5):510-7; Gordley et al., Proc Natl Acad Sci U S A. 2009 Mar 31 ;106(13):5053-8; Akopian et al., Proc Natl Acad Sci U S A. 2003 Jul 22;100(15):8688-91; Tan et., al., J Virol. 2006Feb; 80(4): 1939-48; Tan et al., Proc Natl Acad Sci U S A. 2003 Oct 14; 100(21): 11997- 2002; Papworth et al., Proc Natl Acad Sci U S A. 2003 Feb 18; 100(4): 1621 -6; Sanjana et al., Nat Protoc. 2012 Jan 5;7(1):171-92; Beerli et al., Proc Natl Acad Sci U S A. 1998 Dec 8; 95(25): 14628-33; Snowden et al., Curr Biol. 2002 Dec 23;12(24):2159-66; Xu et.al., Xu et al., Cell Discov. 2016 May 3;2:16009; Komor et al., Nature. 2016 Apr 20;533(7603):420-4; Chaikind et al., Nucleic Acids Res. 2016 Aug 11; Choudhury at. al., Oncotarget. 2016 Jun 23; Du et al., Cold Spring Harb Protoc. 2016 Jan 4; Pham et al., Methods Mol Biol. 2016;1358:43-57; Balboa et al., Stem Cell Reports. 2015 Sep 8;5(3):448-59; Hara et al., Sci Rep. 2015 Jun 9;5:11221 ; Piatek et al., Plant Biotechnol J. 2015 May;13(4):578-89; Hu et al., Nucleic Acids Res. 2014 Apr; 42(7):4375-90; Cheng et al., Cell Res. 2013 Oct;23(10): 1163-71 ; and Maeder et al., Nat Methods. 2013 Oct;10(10):977-9.

[0101] Additional suitable heterologous polypeptides include, but are not limited to, a polypeptide that directly and / or indirectly provides for increased or decreased transcription and / or translation of a target nucleic acid (e.g., a transcription activator or a fragment thereof, a protein or fragment thereof that recruits a transcription activator, a small molecule / drug-responsive transcription and / or translation regulator, a translation-regulating protein, etc.). Non-limiting examples of heterologous polypeptides to accomplish increased or decreased transcription include transcription activator and transcription repressor domains. In some such cases, a fusion polypeptide of the present disclosure is targeted by the guide nucleic acid (guide RNA) to a specific location (i.e. , sequence) in the target nucleic acid and exerts locusspecific regulation such as blocking RNA polymerase binding to a promoter (which selectively inhibits transcription activator function), and / or modifying the local chromatin status (e.g., when a fusion sequence is used that modifies the target nucleic acid or modifies a polypeptide associated with the target nucleic acid). In some cases, the changes are transient (e.g., transcription repression or activation). In some cases, the changes are inheritable (e.g., when epigenetic modifications are made to the target nucleic acid or to proteins associated with the target nucleic acid, e.g., nucleosomal histones).

[0102] Non-limiting examples of heterologous polypeptides for use when targeting ssRNA target nucleic acids include (but are not limited to): splicing factors (e.g., RS domains); protein translation components (e.g., translation initiation, elongation, and / or release factors; e.g., elF4G); RNA methylases; RNA editing enzymes (e.g., RNA deaminases, e.g., adenosine deaminase acting on RNA (ADAR), including A to I and / or C to Uediting enzymes); helicases; RNA-binding proteins; and the like. It is understood that a heterologous polypeptide can include the entire protein or in some cases can include a fragment of the protein (e.g., a functional domain).

[0103] The heterologous polypeptide of a subject fusion polypeptide can be any domain capable of interacting with ssRNA (which, for the purposes of this disclosure, includes intramolecular and / or intermolecular secondary structures, e.g., double-stranded RNA duplexes such as hairpins, stem-loops, etc ), whether transiently or irreversibly, directly or indirectly, including but not limited to an effector domain selected from the group comprising; Endonucleases (for example RNase III, the CRR22 DYW domain, Dicer, and PIN (PilT N-terminus) domains from proteins such as SMG5 and SMG6); proteins and protein domains responsible for stimulating RNA cleavage (for example CPSF, CstF, CFIm and CFIIm); Exonucleases (for example XRN-1 or Exonuclease T); Deadenylases (for example HNT3); proteins and protein domains responsible for nonsense mediated RNA decay (for example UPF1 , UPF2, UPF3, UPF3b, RNP S1 , Y14, DEK, REF2, and SRm160); proteins and protein domains responsible for stabilizing RNA (for example PABP); proteins and protein domains responsible for repressing translation (for example Ago2 and Ago4); proteins and protein domains responsible for stimulating translation (for example Staufen); proteins and protein domains responsible for (e.g., capable of) modulating translation (e.g., translation factors such as initiation factors, elongation factors, release factors, etc., e.g., elF4G); proteins and protein domains responsible for polyadenylation of RNA (for example PAP1 , GLD-2, and Star- PAP) ; proteins and protein domains responsible for polyuridinylation of RNA (for example Cl D1 and terminal uridylate transferase) ; proteins and protein domains responsible for RNA localization (for example from IMP1, ZBP1, She2p, She3p, and Bicaudal-D); proteins and protein domains responsible for nuclear retention of RNA (for example Rrp6); proteins and protein domains responsible for nuclear export of RNA (for example TAP, NXF1 , THO, TREX, REF, and Aly) ; proteins and protein domains responsible for repression of RNA splicing (for example PTB, Sam68, and hnRNP A1) ; proteins and protein domains responsible for stimulation of RNA splicing (for example Serine / Arginine-rich (SR) domains) ; proteins and protein domains responsible for reducing the efficiency of transcription (for example FUS (TLS)); and proteins and protein domains responsible for stimulating transcription (for example CDK7 and HIV Tat). Alternatively, the effector domain may be selected from the group comprising Endonucleases; proteins and protein domains capable of stimulating RNA cleavage; Exonucleases;Deadenylases; proteins and protein domains having nonsense mediated RNA decay activity; proteins and protein domains capable of stabilizing RNA; proteins and protein domains capable of repressing translation; proteins and protein domains capable of stimulating translation; proteins and protein domains capable of modulating translation (e.g., translation factors such as initiation factors, elongation factors, release factors, etc., e.g., elF4G); proteins and protein domains capable of polyadenylation of RNA; proteins and protein domains capable of polyuridinylation of RNA; proteins and protein domains having RNA localization activity; proteins and protein domains capable of nuclear retention of RNA; proteins and protein domains having RNA nuclear export activity; proteins and protein domains capable of repression of RNA splicing; proteins and protein domains capable of stimulation of RNA splicing; proteins and protein domains capable of reducing the efficiency of transcription ; and proteins and protein domains capable of stimulating transcription. Another suitable heterologous polypeptide is a PUF RNA-binding domain, which is described in more detail in WO2012068627, which is hereby incorporated by reference in its entirety.

[0104] Some RNA splicing factors that can be used (in whole or as fragments thereof) as heterologous polypeptides for a fusion polypeptide of the present disclosure have modular organization, with separate sequence-specific RNA binding modules and splicing effector domains. For example, members of the Serine / Arginine-rich (SR) protein family contain N-terminal RNA recognition motifs (RRMs) that bind to exonic splicing enhancers (ESEs) in pre-mRNAs and C-terminal RS domains that promote exon inclusion. As another example, the hnRNP protein hnRNP Al binds to exonic splicing silencers (ESSs) through its RRM domains and inhibits exon inclusion through a C-terminal Glycine-rich domain. Some splicing factors can regulate alternative use of splice site (ss) by binding to regulatory sequences between the two alternative sites. For example, ASF / SF2 can recognize ESEs and promote the use of intron proximal sites, whereas hnRNP Al can bind to ESSs and shift splicing towards the use of intron distal sites. One application for such factors is to generate ESFs that modulate alternative splicing of endogenous genes, particularly disease associated genes. For example, Bcl-x pre-mRNA produces two splicing isoforms with two alternative 5' splice sites to encode proteins of opposite functions. The long splicing isoform Bcl-xL is a potent apoptosis inhibitor expressed in long-lived postmitotic cells and is up-regulated in many cancer cells, protecting cells against apoptotic signals. The short isoform Bcl-xS is a pro-apoptotic isoform and expressed at high levels in cells with a high turnover rate (e.g., developing lymphocytes). The ratio of the two Bel-x splicing isoforms is regulated by multiple cco-elements that are located in either the core exon region or the exon extension region (i.e. , between the two alternative 5' splice sites). For more examples, see WO2010075303, which is hereby incorporated by reference in its entirety.

[0105] Further suitable fusion partners include, but are not limited to, proteins (or fragments thereof) that are boundary elements (e.g., CTCF), proteins and fragments thereof that provide periphery recruitment (e.g., Lamin A, Lamin B, etc.), protein docking elements (e.g., FKBP / FRB, Pil1 / Aby1, etc.).Nucleases

[0106] In some cases, a subject fusion polypeptide comprises: i) a variant Cas12a protein of the present disclosure; and ii) a heterologous polypeptide (a “fusion partner”), where the heterologous polypeptide is a nuclease. Suitable nucleases include, but are not limited to, a homing nuclease polypeptide; a Fokl polypeptide; a meganuclease polypeptide; an ARCUS nuclease; and the like. The meganuclease can be engineered from an LADLIDADG homing endonuclease (LHE). See, e.g., WO 2004 / 067736 (homing endonuclease); Urnov et al. (2005) Nature 435:646 (ZFN); Mussolino et al. (2011) Nude. Acids Res. 39:9283 (TALE nuclease); Boissel et al. (2013) Nucl. Acids Res. 42:2591 (MegaTAL).Reverse transcriptases

[0107] In some cases, a subject fusion polypeptide comprises: i) a variant Cas12a protein of the present disclosure; and ii) a heterologous polypeptide (a “fusion partner”), where the heterologous polypeptide is a reverse transcriptase polypeptide. Such a protein is sometimes referred to in the art as a primer editor. Reverse transcriptases are known in the art; see, e.g., Cote and Roth (2008) Virus Res. 134:186. Suitable reverse transcriptases include, e.g., a murine leukemia virus reverse transcriptase; a Rous sarcoma virus reverse transcriptase; a human immunodeficiency virus type I reverse transcriptase; a Moloney murine leukemia virus reverse transcriptase; a transcription xenopolymerase (RTX); avian myeloblastosis virus reverse transcriptase (AMV-RT); a Eubacterium rectale maturase reverse transcriptase (Marathon®); and the like. The reverse transcriptase fusion partner can include one or more mutations.Base editors

[0108] In some cases, a fusion polypeptide of the present disclosure comprises: i) a variant Cas12a protein of the present disclosure; and ii) one or more heterologous polypeptides (a “fusion partner”), where at least one of the one or more heterologous polypeptides is a deaminase - such a fusion polypeptide is sometimes therefore referred to as a base editor. Suitable deaminases include, e.g., an adenosine deaminase; a cytidine deaminase (e.g., an activation-induced cytidine deaminase (AID)); APOBEC3G; and the like); and the like. As such, in some cases, the fusion polypeptide is a cytosine base editor (CBE) and in some cases it is an adenine base editor (ABE).

[0109] A suitable adenosine deaminase is any enzyme that is capable of deaminating adenosine in DNA. In some cases, the deaminase is a TadA deaminase.Recombinases

[0110] In some cases, a fusion polypeptide of the present disclosure comprises: i) a variant Cas12a protein of the present disclosure; and ii) a heterologous polypeptide (a “fusion partner”), where the heterologous polypeptide is a recombinase. Suitable recombinases include, e.g., a Cre recombinase; a Hin recombinase; a Tre recombinase; a FLP recombinase; and the like.NLS

[0111] In some cases, a fusion polypeptide of the present disclosure comprises: i) a variant Cas12a protein of the present disclosure; and ii) a heterologous polypeptide (a “fusion partner”), where the heterologous polypeptide includes an amino acid sequence that provides for subcellular localization, i.e., the heterologous polypeptide contains a subcellular localization sequence (e.g., a nuclear localization signal (NLS) for targeting to the nucleus, a sequence to keep the fusion protein out of the nucleus, e.g., a nuclear export sequence (NES), a sequence to keep the fusion protein retained in the cytoplasm, a mitochondrial localization signal for targeting to the mitochondria, a chloroplast localization signal for targeting to a chloroplast, an endoplasmic reticulum (ER) retention signal, and the like). In some cases, a fusion polypeptide of the present disclosure does not include an NLS so that the protein is not targeted to the nucleus (which can be advantageous, e.g., when the target nucleic acid is an RNA that is present in the cytosol). In some cases, the heterologous polypeptide can provide a tag (i.e., the heterologous polypeptide is a detectable label)for ease of tracking and / or purification (e.g., a fluorescent protein, e.g., green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), mCherry, tdTomato, and the like; a histidine tag, e.g., a 6XHis tag; a hemagglutinin (HA) tag; a FLAG tag; a Myc tag; and the like).

[0112] In some cases, a fusion polypeptide of the present disclosure comprises: a) variant Cas12a protein of the present disclosure; and b) one or more nuclear localization signals (NLSs) (e.g., in some cases 2 or more, 3 or more, 4 or more, or 5 or more NLSs). Thus, in some cases, a fusion polypeptide of the present disclosure includes one or more NLSs (e.g., 2 or more, 3 or more, 4 or more, or 5 or more NLSs). In some cases, one or more NLSs (2 or more, 3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 50 amino acids of) the N-terminus and / or the C- terminus. In some cases, one or more NLSs (2 or more, 3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 50 amino acids of) the N-terminus. In some cases, one or more NLSs (2 or more, 3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 50 amino acids of) the C-terminus. In some cases, one or more NLSs (3 or more, 4 or more, or 5 or more NLSs) are positioned at or near (e.g., within 50 amino acids of) both the N-terminus and the C- terminus. In some cases, an NLS is positioned at the N-terminus and an NLS is positioned at the C-terminus. In some cases, a subject variant Cas12 protein is fused to 1-5 NLSs (e.g., 1-4, 1-3, 1-2, 2-5, 2-5, 2-3, 3-5, or 3-4 NLSs).

[0113] Non-limiting examples of NLSs include an NLS sequence derived from: the NLS of the SV40 virus large T-antigen, having the amino acid sequence PKKKRKV (SEQ ID NO:xx); the NLS from nucleoplasmin (e.g., the nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK (SEQ ID NO:xx)); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO:xx) or RQRRNELKRSP (SEQ ID NO:xx); the hRNPAI M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO:xx); the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO:xx) of the IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO:xx) and PPKKARED (SEQ ID NO: xx) of the myoma T protein; the sequence PQPKKKPL (SEQ ID NO:xx) of human p53; the sequence SALIKKKKKMAP (SEQ ID NO:xx) of mouse c-abl IV; the sequences DRLRR (SEQ ID NO:xx) and PKQKKRK (SEQ ID NO:xx) of the influenza virus NS1 ; the sequence RKLKKKIKKL (SEQ ID NO:xx) of the Hepatitis virus delta antigen; the sequence REKKKFLKRR (SEQ ID NO:xx) of the mouse Mx1 protein; the sequence KRKGDEVDGVDEVAKKKSKK (SEQID NO:xx) of the human poly(ADP-ribose) polymerase; and the sequence RKCLQAGMNLEARKTKK (SEQ ID NO:xx) of the steroid hormone receptors (human) glucocorticoid. In general, NLS (or multiple NLSs) are of sufficient strength to drive accumulation of the variant Cas12a protein in a detectable amount in the nucleus of a eukaryotic cell. Detection of accumulation in the nucleus may be performed by any suitable technique. For example, a detectable marker may be fused to the variant Cas12a protein such that location within a cell may be visualized. Cell nuclei may also be isolated from cells, the contents of which may then be analyzed by any suitable process for detecting protein, such as immunohistochemistry, Western blot, or enzyme activity assay. Accumulation in the nucleus may also be determined indirectly.PTDs

[0114] In some cases, a variant Cas12a protein of the present disclosure includes a "Protein Transduction Domain" or PTD (also known as a CPP - cell penetrating peptide), which refers to a polypeptide, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates traversing a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane. A PTD attached to another molecule, which can range from a small polar molecule to a large macromolecule and / or a nanoparticle, facilitates the molecule traversing a membrane, for example going from extracellular space to intracellular space, or cytosol to within an organelle. In some cases, a PTD is covalently linked to the amino terminus of a variant Cas12a protein of the present disclosure. In some cases, a PTD is covalently linked to the carboxyl terminus of a variant Cas12a protein of the present disclosure. In some cases, the PTD is inserted internally in a variant Cas12a protein (i.e., is not at the N- or C-terminus of the variant Cas12a protein) at a suitable insertion site. In some cases, a subject fusion polypeptide includes: a) a variant Cas12a protein of the present disclosure; and b) one or more PTDs (e.g., two or more, three or more, four or more PTDs). In some cases, a PTD includes a nuclear localization signal (NLS) (e.g., in some cases 2 or more, 3 or more, 4 or more, or 5 or more NLSs).

[0115] Thus, in some cases, a fusion polypeptide of the present disclosure includes one or more NLSs (e.g., 2 or more, 3 or more, 4 or more, or 5 or more NLSs). In some cases, a PTD is covalently linked to a nucleic acid (e.g., a guide RNA, a polynucleotide encoding a guide RNA, a polynucleotide encoding a fusion polypeptide, a donor polynucleotide, etc.). Examples of PTDs include but are not limited to a minimalundecapeptide protein transduction domain (corresponding to residues 47-57 of HIV-1 TAT comprising YGRKKRRQRRR; SEQ ID NO: xx); a polyarginine sequence comprising a number of arginines sufficient to direct entry into a cell (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines); a VP22 domain (Zender et al. (2002) Cancer Gene Ther. 9(6):489-96); a Drosophila Antennapedia protein transduction domain (Noguchi et al. (2003) Diabetes 52(7): 1732-1737); a truncated human calcitonin peptide (Trehin et al. (2004) Pharm. Resea rch 21 :1248-1256); polylysine (Wender et al. (2000) Proc. Natl. Acad. Sci. USA 97:13003-13008); RRQRRTSKLMKR (SEQ ID NO: xx); Transportan GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: xx);KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO: xx); and RQIKIWFQNRRMKWKK (SEQ ID NO: xx). Exemplary PTDs include but are not limited to, YGRKKRRQRRR (SEQ ID NO: xx), RKKRRQRRR (SEQ ID NO: xx); an arginine homopolymer of from 3 arginine residues to 50 arginine residues; Exemplary PTD domain amino acid sequences include, but are not limited to, any of the following: YGRKKRRQRRR (SEQ ID NO: xx); RKKRRQRR (SEQ ID NO: xx); YARAAARQARA (SEQ ID NO: xx); THRLPRRRRRR (SEQ ID NO: xx); and GGRRARRRRRR (SEQ ID NO: xx). In some cases, the PTD is an activatable CPP (ACPP) (Aguilera et al. (2009) IntegrBiol (Camb) June; 1 (5-6): 371-381). ACPPs comprise a polycationic CPP (e.g., Arg9 or “R9”) connected via a cleavable linker to a matching polyanion (e.g., Glu9 or “E9”), which reduces the net charge to nearly zero and thereby inhibits adhesion and uptake into cells. Upon cleavage of the linker, the polyanion is released, locally unmasking the polyarginine and its inherent adhesiveness, thus “activating” the ACPP to traverse the membrane.Linkers (e.g., for fusion partners)

[0116] In some embodiments, a variant Cas12a protein of the present disclosure can be fused to a fusion partner via a linker polypeptide (e.g., one or more linker polypeptides). The linker polypeptide may have any of a variety of amino acid sequences. Proteins can be joined by a spacer peptide, generally of a flexible nature, although other chemical linkages are not excluded. Suitable linkers include polypeptides of between 4 amino acids and 40 amino acids in length, or between 4 amino acids and 25 amino acids in length. These linkers can be produced by using synthetic, linker-encoding oligonucleotides to couple the proteins, or can be encoded by a nucleic acid sequence encoding the fusion protein. Peptide linkers with a degree of flexibility can be used. The linking peptides may have virtually any amino acidsequence, bearing in mind that the preferred linkers will have a sequence that results in a generally flexible peptide. The use of small amino acids, such as glycine and alanine, are of use in creating a flexible peptide. The creation of such sequences is routine to those of skill in the art. A variety of different linkers are commercially available and are considered suitable for use.

[0117] Examples of linker polypeptides include glycine polymers (G)nwhere n is an integer of at least one; glycine-serine polymers (including, for example, (GS)n, (GSGGS)n(SEQ ID NO: xx), (GGSGGS)n (SEQ ID NO: xx), (GGGGS)n (SEQ ID NO: xx), and (GGGS)n(SEQ ID NO: xx), where n is an integer of at least one; e.g., where n is an integer from 1 to 10); glycine-alanine polymers; and alanine-serine polymers. Exemplary linkers can comprise amino acid sequences including, but not limited to, GGSG (SEQ ID NO: xx), GGSGG (SEQ ID NO: xx), GSGSG (SEQ ID NO: xx), GSGGG (SEQ ID NO: xx), GGGSG (SEQ ID NO: xx), GSSSG (SEQ ID NO: xx), GGGGS (SEQ ID NO:xx), and the like. The ordinarily skilled artisan will recognize that design of a peptide conjugated to any desired element can include linkers that are all or partially flexible, such that the linker can include a flexible linker as well as one or more portions that confer less flexible structure.Detectable labels

[0118] In some cases, a variant Cas12a protein of the present disclosure, or a fusion polypeptide of the present disclosure, comprises a detectable label. Suitable detectable labels and / or moieties that can provide a detectable signal can include, but are not limited to, an enzyme, a radioisotope, a member of a specific binding pair; a fluorophore; a fluorescent protein; a quantum dot; and the like.

[0119] Suitable fluorescent proteins include, but are not limited to, green fluorescent protein (GFP) or variants thereof, a blue fluorescent protein (BFP), a cyan fluorescent (CFP), a yellow fluorescent protein (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine, GFPuv, destabilized EGFP (dEGFP), destabilised ECFP (dECFP), destabilized EYFP (dEYFP), mCFPm, Cerulean, T-Sapphire, CyPet, YPet, mKO, HcRed, t-HcRed, DsRed, DsRed2, DsRed-monomer, J-Red, dimer2, t-dimer2(12), mRFP1 , pocilloporin, Renilla GFP, Monster GFP, paGFP, Kaede protein and kindling protein, Phycobiliproteins and Phycobiliprotein conjugates including B-Phycoerythrin, R- Phycoerythrin and Allophycocyanin. Other examples of fluorescent proteins include mHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry,mCherry, mGrapel , mRaspberry, mGrape2, mPlum, Emerald, Azami green, mWasabi, T-Sapphire, Azurite, Topaz, Venus, mBanana, Kusabira Orange, mRuby and the like. See, e.g., Chudakov et al. (2010) Physiol. Rev. 90:1103; and Stepanenko et al. (2011) BioTechniques 51 :313.

[0120] Suitable enzymes include, but are not limited to, horse radish peroxidase (HRP), alkaline phosphatase (AP), beta-galactosidase (GAL), glucose-6-phosphate dehydrogenase, beta-N-acetylglucosaminidase, p-glucuronidase, invertase, xanthine oxidase, firefly luciferase, glucose oxidase (GO), and the like.Guide RNA

[0121] A nucleic acid molecule (e.g., a natural crRNA) that binds to a type V CRISPR-Cas effector protein (e.g., a Cas12a), forming a ribonucleoprotein complex (RNP), and targets the complex to a specific target sequence within a target nucleic acid (e.g., target DNA) is referred to herein as a “guide RNA.” It is to be understood that in some cases, a hybrid DNA / RNA can be made such that a guide RNA includes DNA bases in addition to RNA bases - but the term “guide RNA” is still used herein to encompass such hybrid molecules. A subject guide RNA includes a guide sequence (also referred to as a “spacer”) (that hybridizes to target sequence of a target nucleic acid, e.g., target DNA) and a constant region (e.g., a region that is adjacent to the guide sequence and binds to the Cas12a protein). A “constant region” can also be referred to herein as a “protein-binding segment” or “scaffold” or “handle.” In some cases, the constant region is 5’ of the guide sequence (i.e., the guide sequence is 3’ of the constant region). A variant Cas12a protein of the present disclosure will bind guide RNAs in substantially the same manner as a reference (e.g., a corresponding wildtype) Cas12a protein. Thus, the discussion of guide RNAs, below, pertains to guide RNAs suitable for use with a variant Cas12a protein of the present disclosure. A guide RNA that comprises: a) a guide sequence that hybridizes to a target nucleotide sequence of a target nucleic acid; and b) a constant region that binds to a variant Cas12a protein is referred to herein as a “Cas12a guide RNA,” or a “subject guide RNA” or simply a “guide.” In many instances, the guide sequence and the constant region of a Cas12a guide RNA are heterologous to one another; i.e., the guide sequence and the constant region do not occur together in nature in a guide RNA (e.g., in some cases the guide sequence hybridizes to a target sequence of a eukaryotic cell and that target sequence is not targeted by the wild type system in nature).Guide sequence

[0122] The targeting segment of a subject guide RNA includes a guide sequence (i.e., a targeting sequence), which is a nucleotide sequence that is complementary to a sequence (a target site) in a target nucleic acid. In other words, the targeting segment of a guide RNA can interact with a target nucleic acid (e.g., DNA) in a sequencespecific manner via hybridization (i.e., base pairing). The guide sequence of a guide RNA can be modified (e.g., by genetic engineering) / designed to hybridize to any desired target sequence (e.g., while taking the PAM into account, e.g., when targeting a dsDNA target) within a target nucleic acid (e.g., a eukaryotic target nucleic acid such as genomic DNA).

[0123] A guide sequence has complementarity with (hybridizes to) a target sequence of the target nucleic acid (e.g., target DNA). In some cases, the guide sequence is 15-30 nucleotides (nt) in length (e.g., 15-28, 15-26, 15-24, 15-22, 15-20, 15-18, 15-17, 16- 30, 16-28, 16-26, 16-24, 16-22, 16-20, 16-18, 16-17, 17-30, 17-28, 17-26, 17-24, 17- 22, 17-20, 17-18, 18-30, 18-28, 18-26, 18-24, 18-22, or 18-20 nt in length). In some cases, the guide sequence is 18-24 nucleotides (nt) in length. In some cases, the guide sequence is at least 15 nt long (e.g., at least 16, 18, 20, or 22 nt long). In some cases, the guide sequence is at least 17 nt long. In some cases, the guide sequence is at least 18 nt long. In some cases, the guide sequence is at least 20 nt long. In some cases, the guide sequence has a length of 17-18 nt. In some cases, the guide sequence has a length of 19 nt. In some cases, the guide sequence has a length of 20 nt. In some cases, the guide sequence has a length of 21 nt. In some cases, the guide sequence has a length of 22 nt. In some cases, the guide sequence has a length of 23 nt.

[0124] In some cases, the guide sequence has 80% or more (e.g., 85% or more, 90% or more, 95% or more, or 100% complementarity) with the target sequence of the target DNA. In some cases, the guide sequence is 100% complementary to the target sequence of the target DNA. In some cases, the target DNA includes at least 15 nucleotides (nt) of complementarity with the guide sequence of the guide RNA.Constant region

[0125] Examples of crRNA repeat sequences (also known as the constant region) for Cas12a proteins include:LbCas12a crRNA (also for PL2a, PL2b, PL2c, PL2d, PL3, PL4a, and PL4b):5’ AAUUUCUACUAAGUGUAGAU 3’ (SEQ ID NO: 21) - [spacer] AsCas12a crRNA:5’ AAUUUCUACUCUUGUAGAU 3’ (SEQ ID NO: 22) - [spacer] FnCas12a crRNA:5’ AAUUUCUACUGUUGUAGAU 3’ (SEQ ID NO: 23) - [spacer] PmCas12a crRNA:5’ AAUUUCUACUAUUGUAGAU 3’ (SEQ ID NO: 24) - [spacer] M bCas 12a / M b2Cas 12a / M b3Cas 12a crRNA:5’ AAUUUCUACUGUUUGUAGAU 3’ (SEQ ID NO: 25) - [spacer] TsCas12a crRNA5’ AAUUUCUACUGUUGUAGAU 3’ (SEQ ID NO: 26) - [spacer] BsCas12a crRNA5’ AAUUUCUACUAUUGUAGAU 3’ (SEQ ID NO: 27) - [spacer]

[0126] In some cases, the constant region of a guide RNA comprises a nucleotide sequence having 70% or more (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100%) sequence identity with the nucleotide sequence of SEQ ID NO: 21. In some cases, the constant region of a guide RNA comprises a nucleotide sequence having 85% or more (e.g., 90% or more, 95% or more, or 100%) sequence identity with the nucleotide sequence of SEQ ID NO: 21. In some cases, the constant region of a guide RNA comprises a nucleotide sequence having 95% or more (e.g., 100%) sequence identity with the nucleotide sequence of SEQ ID NO: 21. In some cases, the constant region of a guide RNA comprises the nucleotide sequence of SEQ ID NO: 21.

[0127] In some cases, the constant region of a guide RNA comprises a nucleotide sequence having 70% or more (e.g., 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100%) sequence identity with the nucleotide sequence of any one of SEQ ID NOs: 21-27. In some cases, the constant region of a guide RNA comprises a nucleotide sequence having 85% or more (e.g., 90% or more, 95% or more, or 100%) sequence identity with the nucleotide sequence of any one of SEQ ID NOs: 21-27. In some cases, the constant region of a guide RNA comprises a nucleotide sequence having 95% or more (e.g., 100%) sequence identity with the nucleotide sequence of any one of SEQ ID NOs: 21-27. In some cases, the constant region of a guide RNA comprises the nucleotide sequence of any one of SEQ ID NOs: 21-27.

[0128] In some cases, the constant region of a guide RNA is 15 or more nucleotides (nt) in length (e.g., 18 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 or more, 31 or more nt, 32 or more, 33 or more, 34 or more, or 35 or more nt in length). In some cases, the constant region of a guide RNA is 18 or more nt in length.

[0129] In some cases, the constant region of a guide RNA has a length in a range of from 12 to 100 nt (e.g., from 12 to 90, 12 to 80, 12 to 70, 12 to 60, 12 to 50, 12 to 40, 15 to100, 15 to 90, 15 to 80, 15 to 70, 15 to 60, 15 to 50, 15 to 40, 15 to 30, 15 to 20, 19 to100, 19 to 90, 19 to 80, 19 to 70, 19 to 60, 19 to 50, 19 to 40, 19 to 30, 19 to 20, 20 to100, 20 to 90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40, 20 to 30, 25 to 100, 25 to 90, 25 to 80, 25 to 70, 25 to 60, 25 to 50, 25 to 40, 25 to 30, 28 to 100, 28 to 90, 28 to 80, 28 to 70, 28 to 60, 28 to 50, 28 to 40, or 28 to 30 nt). In some cases, the constant region of a guide RNA has a length in a range of from 18-22 nt. In some cases, the constant region of a guide RNA has a length in a range of from 19-20 nt.

[0130] In some cases, the constant region of a guide RNA is truncated relative to (shorter than) the corresponding region of a corresponding wild type guide RNA. In some cases, the constant region of a guide RNA is extended relative to (longer than) the corresponding region of a corresponding wild type guide RNA.

[0131] In some cases, a subject guide RNA is 30 or more nucleotides (nt) in length (e.g., 34 or more, 40 or more, 45 or more, 50 or more, 55 or more, 60 or more, 65 or more, 70 or more, or 80 or more nt in length). In some cases, the guide RNA is 35 or more nt in length. In some cases, a subject guide RNA is 30-60 nt (e.g., 30-50, 30-45, 30-40, 35- 60, 35-50, 35-45, 35-40, 40-60, 40-50, or 40-45 nt) in length.Precursor Guide RNA array

[0132] A Type V CRISPR-Cas effector protein (e.g., a Cas12 protein such as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e) can cleave a precursor guide RNA into a mature guide RNA, e.g., by endoribonucleolytic cleavage of the precursor. A Type V CRISPR-Cas effector protein (e.g., a Cas12 protein such as Cas12a, Cas12b, Cas12c, Cas12d, Cas12e) can cleave a precursor guide RNA array (that includes more than one guide RNA arrayed in tandem) into two or more individual guide RNAs. Similarly, in some embodiments, a variant Cas12a protein of the present disclosure can cleave a precursor guide RNA into a mature guide RNA. Thus, in some cases, a precursor guide RNA array comprises two or more (e.g., 3 or more, 4 or more, 5 or more, 2, 3, 4, or 5) guide RNAs (e.g., arrayed in tandem as precursor molecules). Inother words, in some cases, two or more guide RNAs can be present on an array (a precursor guide RNA array).

[0133] In some cases, a subject guide RNA array includes 2 or more guide RNAs (e.g., 3 or more, 4 or more, 5 or more, 6 or more, or 7 or more, guide RNAs). The guide RNAs of a given array can target (i.e., can include guide sequences that hybridize to) different target sites of the same target DNA and / or can target different target DNA molecules (e.g., single nucleotide polymorphisms (SNPs), different strains of a particular virus, etc.). In some cases, each guide RNA of a precursor guide RNA array has a different guide sequence. In some cases, two or more guide RNAs of a precursor guide RNA array have the same guide sequence.

[0134] In some cases, the precursor guide RNA array comprises two or more guide RNAs that target different target sites within the same target DNA molecule. As such, in some cases as subject composition (e.g., kit) or method includes two or more guide RNAs (in the context of a precursor guide RNA array, or not in the context of a precursor guide RNA array, e.g., the guide RNAs can be mature guide RNAs).

[0135] In some cases, the precursor guide RNA array comprises two or more guide RNAs that target different target DNA molecules. As such, in some cases as subject composition (e.g., kit) or method includes two or more guide RNAs (in the context of a precursor guide RNA array, or not in the context of a precursor guide RNA array, e.g., the guide RNAs can be mature guide RNAs).

[0136] In some embodiments, a guide RNA has one or more modifications, e.g., a base modification, a backbone modification, a sugar modification, etc., to provide the nucleic acid with a new or enhanced feature (e.g., improved stability).

[0137] As would be understood to one of ordinary skill in the art, the guide RNA can be introduced into a cell as an RNA (or as a DNA / RNA hybrid) or can be introduces as a nucleic acid encoding the RNA (e.g., a DNA such as an expression vector such as a viral, plasmid, or minicircle DNA), in which case the cell transcribes the RNA from the introduced DNA. In some cases, the nucleotide sequence encoding the guide RNA is operably linked to a promoter (e.g., a Pol III promoter such as U6 or H1) . In some cases, one or more guide RNAs (e.g., 1, 2, 3, 4, 5, 6, 1-10, 1-8, 1-6, 1-5, 1-4, 1-3, 2- 10, 2-8, 2-6, 2-5, 2-4, 3-10, 3-8, 3-6, 3-5, two or more, three or more, four or more, or five or more) (or nucleotide sequences that encode said guide RNAs) can be introduced into the same cell (e.g., to target different sequences of the same target nucleic, to target different target nucleic acids, etc.).Nucleic acid modifications

[0138] In some cases, a guide RNA comprises one or more modifications, e.g., a base modification, a backbone modification, a sugar modification, etc., to provide the nucleic acid with a new or enhanced feature (e.g., improved stability). As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to the 2', the 3', or the 5' hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn, the respective ends of this linear polymeric compound can be further joined to form a circular compound, however, linear compounds are generally suitable. In addition, linear compounds may have internal nucleotide base complementarity and may therefore fold in a manner as to produce a fully or partially double-stranded compound. Within oligonucleotides, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3' to 5' phosphodiester linkage.Modified backbones and modified internucleoside linkages

[0139] Examples of suitable guide RNA modifications include modified nucleic acid backbones and non-natural internucleoside linkages. Nucleic acids having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.

[0140] Suitable modified oligonucleotide backbones containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, phosphorodiamidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage. Suitableoligonucleotides having inverted polarity comprise a single 3' to 3' linkage at the 3'- most internucleotide linkage i.e. a single inverted nucleoside residue which may be a basic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts (such as, for example, potassium or sodium), mixed salts and free acid forms are also included.

[0141] In some cases, a guide RNA comprises one or more phosphorothioate and / or heteroatom internucleoside linkages, in particular -CH2-NH-O-CH2-, -CH2-N(CH3)-O- CH2- (known as a methylene (methylimino) or MMI backbone), -CH2-O-N(CH3)-CH2-, - CH2-N(CH3)-N(CH3)-CH2- and -O-N(CH3)-CH2-CH2- (wherein the native phosphodiester internucleotide linkage is represented as -O-P(=O)(OH)-O-CH2-). MMI type internucleoside linkages are disclosed in the above referenced U.S. Pat. No. 5,489,677. Suitable amide internucleoside linkages are disclosed in t U.S. Pat. No. 5,602,240.

[0142] Also suitable are nucleic acids having morpholino backbone structures as described in, e.g., U.S. Pat. No. 5,034,506. For example, in some cases, a guide RNA comprises a 6-membered morpholino ring in place of a ribose ring. In some cases, a phosphorodiamidate or other non-phosphodiester internucleoside linkage replaces a phosphodiester linkage.

[0143] Suitable modified polynucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2component parts.Mimetics

[0144] A guide RNA can be a nucleic acid mimetic. The term "mimetic" as it is applied to polynucleotides is intended to include polynucleotides wherein only the furanose ring or both the furanose ring and the internucleotide linkage are replaced with nonfuranose groups, replacement of only the furanose ring is also referred to in the art asbeing a sugar surrogate. The heterocyclic base moiety or a modified heterocyclic base moiety is maintained for hybridization with an appropriate target nucleic acid. One such nucleic acid, a polynucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA, the sugar-backbone of a polynucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleotides are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.

[0145] One polynucleotide mimetic that has been reported to have excellent hybridization properties is a peptide nucleic acid (PNA). The backbone in PNA compounds is two or more linked aminoethylglycine units which gives PNA an amide containing backbone. The heterocyclic base moieties are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that describe the preparation of PNA compounds include, but are not limited to: U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262.

[0146] Another class of polynucleotide mimetic that has been studied is based on linked morpholino units (morpholino nucleic acid) having heterocyclic bases attached to the morpholino ring. A number of linking groups have been reported that link the morpholino monomeric units in a morpholino nucleic acid. One class of linking groups has been selected to give a non-ionic oligomeric compound. The non-ionic morpholino-based oligomeric compounds are less likely to have undesired interactions with cellular proteins. Morpholino-based polynucleotides are non-ionic mimics of oligonucleotides which are less likely to form undesired interactions with cellular proteins (Dwaine A. Braasch and David R. Corey, Biochemistry, 2002, 41(14), 4503-4510). Morpholino-based polynucleotides are disclosed in U.S. Pat. No. 5,034,506. A variety of compounds within the morpholino class of polynucleotides have been prepared, having a variety of different linking groups joining the monomeric subunits.

[0147] A further class of polynucleotide mimetic is referred to as cyclohexenyl nucleic acids (CeNA). The furanose ring normally present in a DNA / RNA molecule is replaced with a cyclohexenyl ring. CeNA DMT protected phosphoramidite monomers have been prepared and used for oligomeric compound synthesis following classical phosphoramidite chemistry. Fully modified CeNA oligomeric compounds and oligonucleotides having specific positions modified with CeNA have been prepared and studied (see Wang et al., J. Am. Chem. Soc., 2000, 122, 8595-8602). In general,the incorporation of CeNA monomers into a DNA chain increases its stability of a DNA / RNA hybrid. CeNA oligoadenylates formed complexes with RNA and DNA complements with similar stability to the native complexes. The study of incorporating CeNA structures into natural nucleic acid structures was shown by NMR and circular dichroism to proceed with easy conformational adaptation.

[0148] A further modification includes Locked Nucleic Acids (LNAs) in which the 2'-hydroxyl group is linked to the 4' carbon atom of the sugar ring thereby forming a 2'-C,4'-C- oxymethylene linkage thereby forming a bicyclic sugar moiety. The linkage can be a methylene (-CH2-), group bridging the 2' oxygen atom and the 4' carbon atom wherein n is 1 or 2 (Singh et al., Chem. Commun., 1998, 4, 455-456). LNA and LNA analogs display very high duplex thermal stabilities with complementary DNA and RNA (Tm=+3 to +10° C), stability towards 3'-exonucleolytic degradation and good solubility properties. Potent and nontoxic antisense oligonucleotides containing LNAs have been described (Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633- 5638).

[0149] The synthesis and preparation of the LNA monomers adenine, cytosine, guanine, 5- methyl-cytosine, thymine and uracil, along with their oligomerization, and nucleic acid recognition properties have been described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). LNAs and preparation thereof are also described in WO 98 / 39352 and WO 99 / 14226.Modified sugar moieties

[0150] A guide RNA can also include one or more substituted sugar moieties. Suitable polynucleotides comprise a sugar substituent group selected from: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C.sub.1 to C10 alkyl or C2to G alkenyl and alkynyl. Particularly suitable are O((CH2)nO)mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON((CH2)nCH3)2, where n and m are from 1 to about 10. Other suitable polynucleotides comprise a sugar substituent group selected from: Ci to Cw lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of anoligonucleotide, and other substituents having similar properties. A suitable modification includes 2'-methoxyethoxy (2'-O-CH2CH2OCH3, also known as 2'-O-(2- methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further suitable modification includes 2'- dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described in examples hereinbelow, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), i.e., 2'-O-CH2- O-CH2-N(CH3)2.

[0151] Other suitable sugar substituent groups include methoxy (-O-CH3), aminopropoxy (--0 CH2CH2CH2NH2), allyl (-CH2-CH=CH2), -O-allyl (-0- CH2— CH=CH2) and fluoro (F). 2'-sugar substituent groups may be in the arabino (up) position or ribo (down) position. A suitable 2'-arabino modification is 2'-F. Similar modifications may also be made at other positions on the oligomeric compound, particularly the 3' position of the sugar on the 3' terminal nucleoside or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. Oligomeric compounds may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.Base modifications and substitutions

[0152] A guide RNA may also include nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5- hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C=C-CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4- thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5- substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F- adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7- deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(1 H- pyrimido(5,4-b)(1 ,4)benzoxazin-2(3H)-one), phenothiazine cytidine (1 H-pyrimido(5,4-b)(1 ,4)benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido(5,4-(b) (1 ,4)benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindole cytidine (H- pyrido(3',2':4,5)pyrrolo(2,3-d)pyrimidin-2-one).

[0153] Heterocyclic base moieties may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7- deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991 , 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of these nucleobases are useful for increasing the binding affinity of an oligomeric compound. These include 5-substituted pyrimidines, 6- azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2- aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi et al., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are suitable base substitutions, e.g., when combined with 2'- O-methoxyethyl sugar modifications.Compositions

[0154] The present disclosure provides a composition comprising a variant Cas12a protein of the present disclosure. In some cases, a composition of the present disclosure comprises one or more of: a) a lipid; b) a buffer; c) a nuclease inhibitor; d) a protease inhibitor; e) one or more Cas12a guide RNAs, or one or more nucleic acids comprising nucleotide sequences encoding the one or more Cas12a guide RNAs; and f) a donor template DNA. The present disclosure provides a composition comprising a ribonucleoprotein (RNP) complex, where the RNP complex comprises a variant Cas12a protein of the present disclosure and a guide RNA. The present disclosure provides a composition comprising a nucleic acid comprising a nucleotide sequence encoding a variant Cas12a protein of the present disclosure, or comprising a recombinant expression vector comprising the nucleic acid.Systems

[0155] The present disclosure provides a system comprising a variant Cas12a protein of the present disclosure. A system of the present disclosure can comprise: a) a variant Cas12a protein of the present disclosure and a Cas12a guide RNA; b) a variant Cas12a protein of the present disclosure, a Cas12a guide RNA, and a donor template nucleic acid; c) a fusion polypeptide of the present disclosure and a Cas12a guide RNA; d) a fusion polypeptide of the present disclosure, a Cas12a guide RNA, and a donor template nucleic acid; e) an mRNA encoding a variant Cas12a protein of the present disclosure; and a Cas12a guide RNA; f) an mRNA encoding a variant Cas12a protein of the present disclosure, a Cas12a guide RNA, and a donor template nucleic acid; g) an mRNA encoding a fusion polypeptide of the present disclosure; and a Cas12a guide RNA; h) an mRNA encoding a fusion polypeptide of the present disclosure, a Cas12a guide RNA, and a donor template nucleic acid; i) a recombinant expression vector comprising a nucleotide sequence encoding a variant Cas12a protein of the present disclosure and a nucleotide sequence encoding a Cas12a guide RNA; j) a recombinant expression vector comprising a nucleotide sequence encoding a variant Cas12a protein of the present disclosure, a nucleotide sequence encoding a Cas12a guide RNA, and a nucleotide sequence encoding a donor template nucleic acid; k) a recombinant expression vector comprising a nucleotide sequence encoding a fusion polypeptide of the present disclosure and a nucleotide sequence encoding a Cas12a guide RNA; I) a recombinant expression vector comprising a nucleotide sequence encoding a fusion polypeptide of the present disclosure, a nucleotide sequence encoding a Cas12a guide RNA, and a nucleotide sequence encoding a donor template nucleic acid; m) a first recombinant expression vector comprising a nucleotide sequence encoding a variant Cas12a protein of the present disclosure and a second recombinant expression vector comprising a nucleotide sequence encoding a Cas12a guide RNA; n) a first recombinant expression vector comprising a nucleotide sequence encoding a variant Cas12a protein of the present disclosure, and a second recombinant expression vector comprising a nucleotide sequence encoding a Cas12a guide RNA; and a donor template nucleic acid; o) a first recombinant expression vector comprising a nucleotide sequence encoding a variant Cas12a protein of the present disclosure, and a second recombinant expression vector comprising a nucleotide sequence encoding a Cas12a guide RNA; p) a first recombinant expression vector comprising a nucleotide sequence encoding a fusion polypeptide of the present disclosure, and a second recombinant expression vector comprising a nucleotide sequence encoding a Cas12a guide RNA; and a donortemplate nucleic acid; q) a recombinant expression vector comprising a nucleotide sequence encoding a variant Cas12a protein of the present disclosure, a nucleotide sequence encoding a first Cas12a guide RNA, and a nucleotide sequence encoding a second Cas12a guide RNA; or r) a recombinant expression vector comprising a nucleotide sequence encoding a fusion polypeptide of the present disclosure, a nucleotide sequence encoding a first Cas12a guide RNA, and a nucleotide sequence encoding a second Cas12a guide RNA; or some variation of one of (a) through (r).

[0156] The present disclosure provides a ribonucleoprotein (RNP) complex, comprising a variant Cas12a protein of the present disclosure and a guide RNA.Donor nucleic acid

[0157] In some cases, a system of the present disclosure comprises a donor nucleic acid. By a “donor nucleic acid” or “donor sequence” or “donor polynucleotide” or “donor template” it is meant a nucleic acid sequence to be inserted at the site cleaved by a variant Cas12a protein (e.g., after dsDNA cleavage, after nicking a target DNA, after dual nicking a target DNA, and the like). The donor polynucleotide can contain sufficient homology to a genomic sequence at the target site, e.g. 70%, 80%, 85%, 90%, 95%, or 100% homology with the nucleotide sequences flanking the target site, e.g. within about 50 bases or less of the target site, e.g. within about 30 bases, within about 15 bases, within about 10 bases, within about 5 bases, or immediately flanking the target site, to support homology-directed repair between it and the genomic sequence to which it bears homology. Approximately 25, 50, 100, or 200 nucleotides, or more than 200 nucleotides, of sequence homology between a donor and a genomic sequence (or any integral value between 10 and 200 nucleotides, or more) can support homology-directed repair. Donor polynucleotides can be of any length, e.g. 10 nucleotides or more, 50 nucleotides or more, 100 nucleotides or more, 250 nucleotides or more, 500 nucleotides or more, 1000 nucleotides or more, 5000 nucleotides or more, etc.

[0158] The donor sequence is typically not identical to the genomic sequence that it replaces. Rather, the donor sequence may contain at least one or more single base changes, insertions, deletions, inversions or rearrangements with respect to the genomic sequence, so long as sufficient homology is present to support homology- directed repair (e.g., for gene correction, e.g., to convert a disease-causing base pair or a non disease-causing base pair). In some embodiments, the donor sequence comprises a non-homologous sequence flanked by two regions of homology, suchthat homology-directed repair between the target DNA region and the two flanking sequences results in insertion of the non-homologous sequence at the target region. Donor sequences may also comprise a vector backbone containing sequences that are not homologous to the DNA region of interest and that are not intended for insertion into the DNA region of interest. Generally, the homologous region(s) of a donor sequence will have at least 50% sequence identity to a genomic sequence with which recombination is desired. In certain embodiments, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 99.9% sequence identity is present. Any value between 1% and 100% sequence identity can be present, depending upon the length of the donor polynucleotide.

[0159] The donor sequence may comprise certain sequence differences as compared to the genomic sequence, e.g. restriction sites, nucleotide polymorphisms, selectable markers (e.g., drug resistance genes, fluorescent proteins, enzymes etc.), etc., which may be used to assess for successful insertion of the donor sequence at the cleavage site or in some cases may be used for other purposes (e.g., to signify expression at the targeted genomic locus). In some cases, if located in a coding region, such nucleotide sequence differences will not change the amino acid sequence, or will make silent amino acid changes (i.e., changes which do not affect the structure or function of the protein). Alternatively, these sequences differences may include flanking recombination sequences such as FLPs, loxP sequences, or the like, that can be activated at a later time for removal of the marker sequence.

[0160] In some cases, the donor sequence is provided to the cell as single-stranded DNA. In some cases, the donor sequence is provided to the cell as double-stranded DNA. It may be introduced into a cell in linear or circular form. If introduced in linear form, the ends of the donor sequence may be protected (e.g., from exonucleolytic degradation) by any convenient method and such methods are known to those of skill in the art. For example, one or more dideoxynucleotide residues can be added to the 3' terminus of a linear molecule and / or self-complementary oligonucleotides can be ligated to one or both ends. See, for example, Chang et al. (1987) Proc. Natl. Acad Sci USA 84:4959-4963; Nehls et al. (1996) Science 272:886-889. Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, addition of terminal amino group(s) and the use of modified internucleotide linkages such as, for example, phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyribose residues. As an alternative to protecting the termini of a linear donor sequence, additional lengths of sequence may be included outside of the regions ofhomology that can be degraded without impacting recombination. A donor sequence can be introduced into a cell as part of a vector molecule having additional sequences such as, for example, replication origins, promoters and genes encoding antibiotic resistance.Nucleic Acids

[0161] The present disclosure provides one or more nucleic acids comprising one or more of: a donor nucleic acid, a nucleotide sequence encoding variant Cas12a protein of the present disclosure, a Cas12a guide RNA, and a nucleotide sequence encoding a Cas12a guide RNA. The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a variant Cas12a protein of the present disclosure. The present disclosure provides a nucleic acid comprising a nucleotide sequence encoding a fusion polypeptide comprising: a) a variant Cas12a protein of the present disclosure; and b) a heterologous polypeptide (a fusion partner). The present disclosure provides a recombinant expression vector that comprises a nucleotide sequence encoding a variant Cas12a protein of the present disclosure. The present disclosure provides a recombinant expression vector that comprises a nucleotide sequence encoding a fusion polypeptide of the present disclosure. The present disclosure provides a recombinant expression vector that comprises: a) a nucleotide sequence encoding a variant Cas12a protein of the present disclosure; and b) a nucleotide sequence encoding a Cas12a guide RNA(s). The present disclosure provides a recombinant expression vector that comprises: a) a nucleotide sequence encoding a fusion polypeptide of the present disclosure; and b) a nucleotide sequence encoding a Cas12a guide RNA(s). In some cases, the nucleotide sequence encoding the variant Cas12a protein of the present disclosure and / or the nucleotide sequence encoding the Cas12a guide RNA and / or the nucleotide sequence encoding the fusion polypeptide is operably linked to a promoter that is operable in a cell type of choice (e.g., a prokaryotic cell, a eukaryotic cell, a plant cell, an animal cell, a mammalian cell, a primate cell, a rodent cell, a human cell, etc.). Various nucleic acid and expression vectors are described below in the context of a variant Cas12a protein of the present disclosure; these descriptions apply equally to a fusion polypeptide of the present disclosure.

[0162] In some cases, a nucleotide sequence encoding a variant Cas12a protein of the present disclosure, or a fusion polypeptide of the present disclosure, is codon optimized. This type of optimization can entail a mutation of a variant Cas12a protein-encoding nucleotide sequence to mimic the codon preferences of the intended host organism or cell while encoding the same protein. Thus, the codons can be changed, but the encoded protein remains unchanged. For example, if the intended target cell was a human cell, a human codon-optimized variant Cas12a protein-encoding nucleotide sequence could be used. As another non-limiting example, if the intended host cell were a mouse cell, then a mouse codon-optimized variant Cas12a proteinencoding nucleotide sequence could be generated. As another non-limiting example, if the intended host cell were a plant cell, then a plant codon-optimized variant Cas12a protein-encoding nucleotide sequence could be generated. As another nonlimiting example, if the intended host cell were an insect cell, then an insect codon- optimized variant Cas12a protein-encoding nucleotide sequence could be generated.

[0163] The present disclosure provides one or more recombinant expression vectors that include (in separate recombinant expression vectors in some cases, and in the same recombinant expression vector in some cases): (i) a nucleotide sequence of a donor template nucleic acid (where the donor template comprises a nucleotide sequence having homology to a target sequence of a target nucleic acid (e.g., a target genome)); (ii) a nucleotide sequence that encodes a Cas12a guide RNA that hybridizes to a target sequence of the target locus of the targeted genome (e.g., operably linked to a promoter that is operable in a target cell such as a eukaryotic cell); and (iii) a nucleotide sequence encoding a variant Cas12a protein of the present disclosure (e.g., operably linked to a promoter that is operable in a target cell such as a eukaryotic cell). The present disclosure provides one or more recombinant expression vectors that include (in separate recombinant expression vectors in some cases, and in the same recombinant expression vector in some cases): (i) a nucleotide sequence of a donor template nucleic acid (where the donor template comprises a nucleotide sequence having homology to a target sequence of a target nucleic acid (e.g., a target genome)); and (ii) a nucleotide sequence that encodes a Cas12a guide RNA that hybridizes to a target sequence of the target locus of the targeted genome (e.g., operably linked to a promoter that is operable in a target cell such as a eukaryotic cell). The present disclosure provides one or more recombinant expression vectors that include (in separate recombinant expression vectors in some cases, and in the same recombinant expression vector in some cases): (i) a nucleotide sequence that encodes a Cas12a guide RNA that hybridizes to a target sequence of the target locus of the targeted genome (e.g., operably linked to a promoter that is operable in a target cell such as a eukaryotic cell); and (ii) anucleotide sequence encoding a variant Cas12a protein of the present disclosure (e.g., operably linked to a promoter that is operable in a target cell such as a eukaryotic cell).

[0164] Suitable expression vectors include viral expression vectors (e.g. viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:25432549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO 94 / 12649, WO 93 / 03769; WO 93 / 19191 ; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655); adeno-associated virus (AAV) (see, e.g., Ali et al., Hum Gene Ther 9:81 86, 1998, Flannery et al., PNAS 94:69166921 , 1997; Bennett et al., Invest Opthalmol Vis Sci 38:28572863, 1997; Jomary et al., Gene Ther 4:683 690, 1997, Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali et al., Hum Mol Genet 5:591 594, 1996; Srivastava in WO 93 / 09239, Samulski et al., J. Vir. (1989) 63:3822-3828; Mendelson et al., Virol. (1988) 166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al., PNAS 94:10319 23, 1997; Takahashi et al., J Virol 73:7812 7816, 1999); a retroviral vector (e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like. In some cases, a recombinant expression vector of the present disclosure is a recombinant adeno-associated virus (AAV) vector. In some cases, a recombinant expression vector of the present disclosure is a recombinant lentivirus vector. In some cases, a recombinant expression vector of the present disclosure is a recombinant retroviral vector.

[0165] Depending on the host / vector system utilized, any of a number of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector.

[0166] In some cases, a nucleotide sequence encoding a Cas12a guide RNA is operably linked to a control element, e.g., a transcriptional control element, such as a promoter. In some embodiments, a nucleotide sequence encoding a variant Cas12a protein of the present disclosure or a fusion polypeptide of the present disclosure is operably linked to a control element, e.g., a transcriptional control element, such as a promoter.

[0167] The transcriptional control element can be a promoter. In some cases, the promoter is a constitutively active promoter. In some cases, the promoter is a regulatablepromoter. In some cases, the promoter is an inducible promoter. In some cases, the promoter is a tissue-specific promoter. In some cases, the promoter is a cell typespecific promoter. In some cases, the transcriptional control element (e.g., the promoter) is functional in a targeted cell type or targeted cell population. For example, in some cases, the transcriptional control element can be functional in eukaryotic cells, e.g., hematopoietic stem cells (e.g., mobilized peripheral blood (mPB) CD34(+) cell, bone marrow (BM) CD34(+) cell, etc ).

[0168] Non-limiting examples of eukaryotic promoters (promoters functional in a eukaryotic cell) include EF1a, those from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, long terminal repeats (LTRs) from retrovirus, and mouse metallothionein-l. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art. The expression vector may also contain a ribosome binding site for translation initiation and a transcription terminator. The expression vector may also include appropriate sequences for amplifying expression. The expression vector may also include nucleotide sequences encoding protein tags (e.g., 6xHis tag, hemagglutinin tag, fluorescent protein, etc.) that can be fused to variant Cas12a protein of the present disclosure, thus resulting in a fusion polypeptide.

[0169] In some cases, a nucleotide sequence encoding a Cas12a guide RNA and / or a variant Cas12a protein of the present disclosure, or a fusion polypeptide of the present disclosure, is operably linked to an inducible promoter. In some cases, a nucleotide sequence encoding a Cas12a guide RNA and / or a variant Cas12a protein of the present disclosure is operably linked to a constitutive promoter.

[0170] A promoter can be a constitutively active promoter (i.e. , a promoter that is constitutively in an active / ”ON” state), it may be an inducible promoter (i.e., a promoter whose state, active / ”ON” or inactive / “OFF”, is controlled by an external stimulus, e.g., the presence of a particular temperature, compound, or protein.), it may be a spatially restricted promoter (i.e., transcriptional control element, enhancer, etc.)(e.g., tissue specific promoter, cell type specific promoter, etc.), and it may be a temporally restricted promoter (i.e., the promoter is in the “ON” state or “OFF” state during specific stages of embryonic development or during specific stages of a biological process, e.g., hair follicle cycle in mice).

[0171] Suitable promoters can be derived from viruses and can therefore be referred to as viral promoters, or they can be derived from any organism, including prokaryotic or eukaryotic organisms. Suitable promoters can be used to drive expression by anyRNA polymerase (e.g., pol I, pol II, pol III). Exemplary promoters include, but are not limited to the SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), a rous sarcoma virus (RSV) promoter, a human U6 small nuclear promoter (U6) (Miyagishi et al., Nature Biotechnology 20, 497 - 500 (2002)), an enhanced U6 promoter (e.g., Xia et al., Nucleic Acids Res. 2003 Sep 1 ;31 (17)), a human H1 promoter (H 1), and the like.

[0172] In some cases, a nucleotide sequence encoding a Cas12a guide RNA is operably linked to (under the control of) a promoter operable in a eukaryotic cell (e.g., a U6 promoter, an enhanced U6 promoter, an H1 promoter, and the like). As would be understood by one of ordinary skill in the art, when expressing an RNA (e.g., a guide RNA) from a nucleic acid (e.g., an expression vector) using a U6 promoter (e.g., in a eukaryotic cell), or another Pollll promoter, the RNA may need to be mutated if there are several Ts in a row (coding for Us in the RNA). This is because a string of Ts (e.g., 5 Ts) in DNA can act as a terminator for polymerase III (Pol III). Thus, in order to ensure transcription of a guide RNA in a eukaryotic cell it may sometimes be necessary to modify the sequence encoding the guide RNA to eliminate runs of Ts. In some cases, a nucleotide sequence encoding a variant Cas12a protein of the present disclosure is operably linked to a promoter operable in a eukaryotic cell (e.g., a CMV promoter, an EF1a promoter, an estrogen receptor- regulated promoter, and the like).

[0173] Examples of inducible promoters include, but are not limited toT7 RNA polymerase promoter, T3 RNA polymerase promoter, Isopropyl-beta-D-thiogalactopyranoside (IPTG)-regulated promoter, lactose induced promoter, heat shock promoter, Tetracycline-regulated promoter, Steroid-regulated promoter, Metal-regulated promoter, estrogen receptor-regulated promoter, etc. Inducible promoters can therefore be regulated by molecules including, but not limited to, doxycycline; estrogen and / or an estrogen analog; IPTG; etc.

[0174] Inducible promoters suitable for use include any inducible promoter described herein or known to one of ordinary skill in the art. Examples of inducible promoters include, without limitation, chemically / biochemically-regulated and physically-regulated promoters such as alcohol-regulated promoters, tetracycline-regulated promoters (e.g., anhydrotetracycline (aTc)-responsive promoters and other tetracyclineresponsive promoter systems, which include a tetracycline repressor protein (tetR), a tetracycline operator sequence (tetO) and a tetracycline transactivator fusion protein(tTA)), steroid-regulated promoters (e.g., promoters based on the rat glucocorticoid receptor, human estrogen receptor, moth ecdysone receptors, and promoters from the steroid / retinoid / thyroid receptor superfamily), metal-regulated promoters (e.g., promoters derived from metallothionein (proteins that bind and sequester metal ions) genes from yeast, mouse and human), pathogenesis-regulated promoters (e.g., induced by salicylic acid, ethylene or benzothiadiazole (BTH)), temperature / heat- inducible promoters (e.g., heat shock promoters), and light-regulated promoters (e.g., light responsive promoters from plant cells).

[0175] In some cases, the promoter is a spatially restricted promoter (i.e., cell type specific promoter, tissue specific promoter, etc.) such that in a multi-cellular organism, the promoter is active (i.e., “ON”) in a subset of specific cells. Spatially restricted promoters may also be referred to as enhancers, transcriptional control elements, control sequences, etc. Any convenient spatially restricted promoter may be used as long as the promoter is functional in the targeted host cell (e.g., eukaryotic cell; prokaryotic cell).

[0176] In some cases, the promoter is a reversible promoter. Suitable reversible promoters, including reversible inducible promoters are known in the art. Such reversible promoters may be isolated and derived from many organisms, e.g., eukaryotes and prokaryotes. Modification of reversible promoters derived from a first organism for use in a second organism, e.g., a first prokaryote and a second a eukaryote, a first eukaryote and a second a prokaryote, etc., is well known in the art. Such reversible promoters, and systems based on such reversible promoters but also comprising additional control proteins, include, but are not limited to, alcohol regulated promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, promoters responsive to alcohol transactivator proteins (AlcR), etc.), tetracycline regulated promoters, (e.g., promoter systems including TetActivators, TetON, TetOFF, etc.), steroid regulated promoters (e.g., rat glucocorticoid receptor promoter systems, human estrogen receptor promoter systems, retinoid promoter systems, thyroid promoter systems, ecdysone promoter systems, mifepristone promoter systems, etc.), metal regulated promoters (e.g., metallothionein promoter systems, etc.), pathogenesis-related regulated promoters (e.g., salicylic acid regulated promoters, ethylene regulated promoters, benzothiadiazole regulated promoters, etc.), temperature regulated promoters (e.g., heat shock inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter, etc.), light regulated promoters, synthetic inducible promoters, and the like.

[0177] Methods of introducing a nucleic acid (e.g., a nucleic acid comprising a donor polynucleotide sequence, one or more nucleic acids encoding a variant Cas12a protein of the present disclosure (or a fusion polypeptide of the present disclosure) and / or a Cas12a guide RNA, and the like) into a host cell are known in the art, and any convenient method can be used to introduce a nucleic acid (e.g., an expression construct) into a cell. Suitable methods include e.g., viral infection, transfection, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)- mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, and the like.

[0178] Introducing the recombinant expression vector into cells can occur in any culture media and under any culture conditions that promote the survival of the cells. Introducing the recombinant expression vector into a target cell can be carried out in vivo or ex vivo. Introducing the recombinant expression vector into a target cell can be carried out in vitro.

[0179] In some cases, a variant Cas12a protein of the present disclosure can be provided as RNA. The RNA can be provided by direct chemical synthesis or may be transcribed in vitro from a DNA (e.g., encoding the variant Cas12a protein). Once synthesized, the RNA may be introduced into a cell by any of the well-known techniques for introducing nucleic acids into cells (e.g., microinjection, electroporation, transfection, etc.).

[0180] Nucleic acids may be provided to the cells using well-developed transfection techniques; see, e.g. Angel and Yanik (2010) PLoS ONE 5(7): e11756, and the commercially available TransMessenger® reagents from Qiagen, Stemfect™ RNA Transfection Kit from Stemgent, and TranslT®-mRNA Transfection Kit from Mirus Bio LLC. See also Beumer et al. (2008) PNAS 105(50): 19821 -19826.

[0181] Vectors may be provided directly to a target host cell. In other words, the cells are contacted with vectors comprising the subject nucleic acids (e.g., recombinant expression vectors having the donor template sequence and encoding a Cas12a guide RNA; recombinant expression vectors encoding a variant Cas12a protein of the present disclosure (or a fusion polypeptide of the present disclosure); etc.) such that the vectors are taken up by the cells. Methods for contacting cells with nucleic acid vectors that are plasmids, include electroporation, calcium chloride transfection, microinjection, and lipofection are well known in the art. For viral vector delivery, cells can be contacted with viral particles comprising the subject viral expression vectors.

[0182] Retroviruses, for example, lentiviruses, are suitable for use in methods of the present disclosure. Commonly used retroviral vectors are “defective”, i.e. unable to produce viral proteins required for productive infection. Rather, replication of the vector requires growth in a packaging cell line. To generate viral particles comprising nucleic acids of interest, the retroviral nucleic acids comprising the nucleic acid are packaged into viral capsids by a packaging cell line. Different packaging cell lines provide a different envelope protein (ecotropic, amphotropic or xenotropic) to be incorporated into the capsid, this envelope protein determining the specificity of the viral particle for the cells (ecotropic for murine and rat; amphotropic for most mammalian cell types including human, dog and mouse; and xenotropic for most mammalian cell types except murine cells). The appropriate packaging cell line may be used to ensure that the cells are targeted by the packaged viral particles. Methods of introducing subject vector expression vectors into packaging cell lines and of collecting the viral particles that are generated by the packaging lines are well known in the art. Nucleic acids can also introduced by direct micro-injection (e.g., injection of RNA).

[0183] Vectors used for providing the nucleic acids encoding Cas12a guide RNA and / or a variant Cas12a protein of the present disclosure (or a fusion polypeptide of the present disclosure) to a target host cell can include suitable promoters for driving the expression, that is, transcriptional activation, of the nucleic acid of interest. In other words, in some cases, the nucleic acid of interest will be operably linked to a promoter. This may include ubiquitously acting promoters, for example, the CMV-p- actin promoter, or inducible promoters, such as promoters that are active in particular cell populations or that respond to the presence of drugs such as tetracycline. By transcriptional activation, it is intended that transcription will be increased above basal levels in the target cell by 10 fold, by 100 fold, more usually by 1000 fold. In addition, vectors used for providing a nucleic acid encoding a Cas12a guide RNA and / or a variant Cas12a protein of the present disclosure to a cell may include nucleic acid sequences that encode for selectable markers in the target cells, so as to identify cells that have taken up the Cas12a guide RNA and / or variant Cas12a protein.

[0184] A nucleic acid comprising a nucleotide sequence encoding a variant Cas12a protein of the present disclosure, or a fusion polypeptide of the present disclosure (a fusion polypeptide comprising: a) a variant Cas12a protein of the present disclosure; and b) a heterologous polypeptide), is in some cases an RNA. Thus, a fusion protein of the present disclosure can be introduced into cells as RNA. Methods of introducing RNA into cells are known in the art and may include, for example, direct injection,transfection, or any other method used for the introduction of DNA. A variant Cas12a protein of the present disclosure may instead be provided to cells as a polypeptide. Such a polypeptide may optionally be fused to a polypeptide domain that increases solubility of the product. The domain may be linked to the polypeptide through a defined protease cleavage site, e.g. a TEV sequence, which is cleaved by TEV protease. The linker may also include one or more flexible sequences, e.g. from 1 to 10 glycine residues. In some embodiments, the cleavage of the fusion protein is performed in a buffer that maintains solubility of the product, e.g. in the presence of from 0.5 to 2 M urea, in the presence of polypeptides and / or polynucleotides that increase solubility, and the like. Domains of interest include endosomolytic domains, e.g. influenza HA domain; and other polypeptides that aid in production, e.g. IF2 domain, GST domain, GRPE domain, and the like. The polypeptide may be formulated for improved stability. For example, the peptides may be PEGylated, where the polyethyleneoxy group provides for enhanced lifetime in the blood stream.

[0185] Additionally, or alternatively, a variant Cas12a protein of the present disclosure may be fused to a polypeptide permeant domain to promote uptake by the cell. A number of permeant domains are known in the art and may be used in the non-integrating polypeptides of the present disclosure, including peptides, peptidomimetics, and nonpeptide carriers. For example, a permeant peptide may be derived from the third alpha helix of Drosophila melanogaster transcription factor Antennapaedia, referred to as penetratin, which comprises the amino acid sequence RQIKIWFQNRRMKWKK (SEQ ID NO:xx). As another example, the permeant peptide comprises the HIV-1 tat basic region amino acid sequence, which may include, for example, amino acids 49- 57 of naturally-occurring tat protein. Other permeant domains include poly-arginine motifs, for example, the region of amino acids 34-56 of HIV-1 rev protein, nonaarginine, octa-arginine, and the like. (See, for example, Futaki et al. (2003) Curr Protein Pept Sci. 2003 Apr; 4(2): 87-9 and 446; and Wender et al. (2000) Proc. Natl. Acad. Sci. U.S.A 2000 Nov. 21 ; 97(24): 13003-8; published U.S. Patent applications 20030220334; 20030083256; 20030032593; and 20030022831 , herein specifically incorporated by reference for the teachings of translocation peptides and peptoids). The nona-arginine (R9) sequence can be used. The site at which the fusion is made may be selected in order to optimize the biological activity, secretion or binding characteristics of the polypeptide. The optimal site will be determined by routine experimentation.

[0186] A variant Cas12a protein of the present disclosure may be produced in vitro or by eukaryotic cells or by prokaryotic cells, and it may be further processed by unfolding, e.g. heat denaturation, dithiothreitol reduction, etc. and may be further refolded, using methods known in the art.

[0187] Modifications of interest that do not alter primary sequence include chemical derivatization of polypeptides, e.g., acylation, acetylation, carboxylation, amidation, etc. Also included are modifications of glycosylation, e.g. those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g. by exposing the polypeptide to enzymes which affect glycosylation, such as mammalian glycosylating or deglycosylating enzymes. Also encompassed are sequences that have phosphorylated amino acid residues, e.g. phosphotyrosine, phosphoserine, or phosphothreonine.

[0188] Also suitable for inclusion in embodiments of the present disclosure are nucleic acids (e.g., encoding a Cas12a guide RNA, encoding a fusion protein of the present disclosure, etc.) and proteins (e.g., a variant Cas12a protein of the present disclosure; a fusion protein of the present disclosure) that have been modified using ordinary molecular biological techniques and synthetic chemistry so as to improve their resistance to proteolytic degradation, to change the target sequence specificity, to optimize solubility properties, to alter protein activity (e.g., transcription modulatory activity, enzymatic activity, etc.) or to render them more suitable. Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, e.g. D-amino acids or non-naturally occurring synthetic amino acids. D-amino acids may be substituted for some or all of the amino acid residues.

[0189] A variant Cas12a protein of the present disclosure may be prepared by in vitro synthesis, using conventional methods as known in the art. Various commercial synthetic apparatuses are available, for example, automated synthesizers by Applied Biosystems, Inc., Beckman, etc. By using synthesizers, naturally occurring amino acids may be substituted with unnatural amino acids. The particular sequence and the manner of preparation will be determined by convenience, economics, purity required, and the like.

[0190] If desired, various groups may be introduced into the peptide during synthesis or during expression, which allow for linking to other molecules or to a surface. Thus, cysteines can be used to make thioethers, histidines for linking to a metal ion complex, carboxyl groups for forming amides or esters, amino groups for forming amides, and the like.

[0191] A variant Cas12a protein of the present disclosure may also be isolated and purified in accordance with conventional methods of recombinant synthesis. A lysate may be prepared of the expression host and the lysate purified using high performance liquid chromatography (HPLC), exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique. For the most part, the compositions which are used will comprise 20% or more by weight of the desired product, more usually 75% or more by weight, preferably 95% or more by weight, and for therapeutic purposes, usually 99.5% or more by weight, in relation to contaminants related to the method of preparation of the product and its purification. Usually, the percentages will be based upon total protein. Thus, in some cases, a variant Cas12a protein of the present disclosure, or a fusion polypeptide of the present disclosure, is at least 80% pure, at least 85% pure, at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure (e.g., free of contaminants, non-variant Cas12a proteins or other macromolecules, etc.).

[0192] To induce cleavage or any desired modification to a target nucleic acid (e.g., genomic DNA), or any desired modification to a polypeptide associated with target nucleic acid, the Cas12a guide RNA and / or the variant Cas12a protein of the present disclosure and / or the donor template sequence, whether they be introduced as nucleic acids or polypeptides, are provided to the cells for about 30 minutes to about 24 hours, e.g., 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 16 hours, 18 hours, 20 hours, or any other period from about 30 minutes to about 24 hours, which may be repeated with a frequency of about every day to about every 4 days, e.g., every 1.5 days, every 2 days, every 3 days, or any other frequency from about every day to about every four days. The agent(s) may be provided to the subject cells one or more times, e.g. one time, twice, three times, or more than three times, and the cells allowed to incubate with the agent(s) for some amount of time following each contacting event e.g. 16-24 hours, after which time the media is replaced with fresh media and the cells are cultured further.

[0193] In cases in which two or more different targeting complexes are provided to the cell (e.g., two different Cas12a guide RNAs that are complementary to different sequences within the same or different target nucleic acid), the complexes may be provided simultaneously (e.g. as two polypeptides and / or nucleic acids), or delivered simultaneously. Alternatively, they may be provided consecutively, e.g. the targeting complex being provided first, followed by the second targeting complex, etc. or vice versa.

[0194] To improve the delivery of a DNA vector into a target cell, the DNA can be protected from damage and its entry into the cell facilitated, for example, by using lipoplexes and polyplexes. Thus, in some cases, a nucleic acid of the present disclosure (e.g., a recombinant expression vector of the present disclosure) can be covered with lipids in an organized structure like a micelle or a liposome. When the organized structure is complexed with DNA it is called a lipoplex. There are three types of lipids, anionic (negatively charged), neutral, or cationic (positively charged). Lipoplexes that utilize cationic lipids have proven utility for gene transfer. Cationic lipids, due to their positive charge, naturally complex with the negatively charged DNA. Also, as a result of their charge, they interact with the cell membrane. Endocytosis of the lipoplex then occurs, and the DNA is released into the cytoplasm. The cationic lipids also protect against degradation of the DNA by the cell.

[0195] Complexes of polymers with DNA are called polyplexes. Most polyplexes consist of cationic polymers and their production is regulated by ionic interactions. One large difference between the methods of action of polyplexes and lipoplexes is that polyplexes cannot release their DNA load into the cytoplasm, so to this end, cotransfection with endosome-lytic agents (to lyse the endosome that is made during endocytosis) such as inactivated adenovirus must occur. However, this is not always the case; polymers such as polyethylenimine have their own method of endosome disruption as does chitosan and trimethylchitosan.

[0196] Dendrimers, a highly branched macromolecule with a spherical shape, may be also be used to genetically modify stem cells. The surface of the dendrimer particle may be functionalized to alter its properties. In particular, it is possible to construct a cationic dendrimer (i.e. , one with a positive surface charge). When in the presence of genetic material such as a DNA plasmid, charge complementarity leads to a temporary association of the nucleic acid with the cationic dendrimer. On reaching its destination, the dendrimer-nucleic acid complex can be taken up into a cell by endocytosis.

[0197] In some cases, a nucleic acid of the disclosure (e.g., an expression vector) includes an insertion site for a guide sequence of interest. For example, a nucleic acid can include an insertion site for a guide sequence of interest, where the insertion site is immediately adjacent to a nucleotide sequence encoding the portion of a Cas12a guide RNA that does not change when the guide sequence is changed to hybridized to a desired target sequence (e.g., sequences that contribute to the type V CRISPR- Cas polypeptide-binding aspect of the guide RNA, e.g., the sequences that contributeto the dsRNA duplex(es) of the Cas12a guide RNA - this portion of the guide RNA can also be referred to as the 'scaffold’ or ‘constant region’ of the guide RNA). Thus, in some cases, a subject nucleic acid (e.g., an expression vector) includes a nucleotide sequence encoding a Cas12a guide RNA, except that the portion encoding the guide sequence portion of the guide RNA is an insertion sequence (an insertion site). An insertion site is any nucleotide sequence used for the insertion of a desired sequence. “Insertion sites” for use with various technologies are known to those of ordinary skill in the art and any convenient insertion site can be used. An insertion site can be for any method for manipulating nucleic acid sequences. For example, in some cases the insertion site is a multiple cloning site (MCS) (e.g., a site including one or more restriction enzyme recognition sequences), a site for ligation independent cloning, a site for recombination-based cloning (e.g., recombination based on att sites), a nucleotide sequence recognized by a CRISPR-Cas (e.g. Cas9) based technology, and the like.

[0198] An insertion site can be any desirable length, and can depend on the type of insertion site (e.g., can depend on whether (and how many) the site includes one or more restriction enzyme recognition sequences, whether the site includes a target site for a CRISPR-Cas protein, etc.). In some cases, an insertion site of a subject nucleic acid is 3 or more nucleotides (nt) in length (e.g., 5 or more, 8 or more, 10 or more, 15 or more, 17 or more, 18 or more, 19 or more, 20 or more or 25 or more, or 30 or more nt in length). In some cases, the length of an insertion site of a subject nucleic acid has a length in a range of from 2 to 50 nucleotides (nt) (e.g., from 2 to 40 nt, from 2 to 30 nt, from 2 to 25 nt, from 2 to 20 nt, from 5 to 50 nt, from 5 to 40 nt, from 5 to 30 nt, from 5 to 25 nt, from 5 to 20 nt, from 10 to 50 nt, from 10 to 40 nt, from 10 to 30 nt, from 10 to 25 nt, from 10 to 20 nt, from 17 to 50 nt, from 17 to 40 nt, from 17 to 30 nt, from 17 to 25 nt). In some cases, the length of an insertion site of a subject nucleic acid has a length in a range of from 5 to 40 nt.Modified Host Cells (genetically modified host cells)

[0199] The present disclosure provides a modified cell comprising a variant Cas12a protein of the present disclosure and / or a nucleic acid comprising a nucleotide sequence encoding a variant Cas12a protein of the present disclosure. The description, below, of modified host cells that comprise a variant Cas12a protein of the present disclosure and / or a nucleic acid comprising a nucleotide sequence encoding a variant Cas12a protein of the present disclosure apply equally to a modified host cell comprising afusion polypeptide of the present disclosure and / or a nucleic acid comprising a nucleotide sequence encoding a fusion polypeptide of the present disclosure. Thus, for example, the present disclosure provides a modified host cell comprising a fusion polypeptide of the present disclosure and / or a nucleic acid comprising a nucleotide sequence encoding a fusion polypeptide of the present disclosure. The present disclosure provides a modified cell comprising a variant Cas12a protein of the present disclosure, where the modified cell is a cell that does not normally comprise a variant Cas12a protein of the present disclosure. The present disclosure provides a modified cell (e.g., a genetically modified cell) comprising nucleic acid comprising a nucleotide sequence encoding a variant Cas12a protein of the present disclosure. The present disclosure provides a genetically modified cell that is genetically modified with an mRNA comprising a nucleotide sequence encoding a variant Cas12a protein of the present disclosure. The present disclosure provides a genetically modified cell that is genetically modified with a recombinant expression vector comprising a nucleotide sequence encoding a variant Cas12a protein of the present disclosure. The present disclosure provides a genetically modified cell that is genetically modified with a recombinant expression vector comprising: a) a nucleotide sequence encoding a variant Cas12a protein of the present disclosure; and b) a nucleotide sequence encoding a Cas12a guide RNA of the present disclosure. The present disclosure provides a genetically modified cell that is genetically modified with a recombinant expression vector comprising: a) a nucleotide sequence encoding a variant Cas12a protein of the present disclosure e; b) a nucleotide sequence encoding a Cas12a guide RNA of the present disclosure; and c) a nucleotide sequence encoding a donor template.

[0200] A cell that serves as a recipient for a variant Cas12a protein of the present disclosure and / or a nucleic acid comprising a nucleotide sequence encoding a variant Cas12a protein of the present disclosure and / or a Cas12a guide RNA of the present disclosure, can be any of a variety of cells, including, e.g., in vitro cells; in vivo cells; ex vivo cells; primary cells; cancer cells; animal cells; plant cells; algal cells; fungal cells; etc. A cell that serves as a recipient for variant Cas12a protein of the present disclosure and / or a nucleic acid comprising a nucleotide sequence encoding a variant Cas12a protein of the present disclosure and / or a guide RNA of the present disclosure is referred to as a “host cell” or a “target cell.” A host cell or a target cell can be a recipient of a Cas12a system of the present disclosure. A host cell or a target cell can be a recipient of a ribonucleoprotein (RNP) of the present disclosure, where theRNP comprises: i) a variant Cas12a protein of the present disclosure; and ii) a Cas12a guide RNA. A host cell or a target cell can be a recipient of a single component of a system of the present disclosure.

[0201] Non-limiting examples of cells (target cells) include: a prokaryotic cell, eukaryotic cell, a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a protozoa cell, a cell from a plant (e.g., cells from plant crops, fruits, vegetables, grains, soy bean, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin, hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, angiosperms, ferns, clubmosses, hornworts, liverworts, mosses, dicotyledons, monocotyledons, etc.), an algal cell, (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C. agardh, and the like), seaweeds (e.g. kelp) a fungal cell (e.g., a yeast cell, a cell from a mushroom), an animal cell, a cell from an invertebrate animal (e.g., fruit fly, cnidarian, echinoderm, nematode, etc.), a cell from a vertebrate animal (e.g., fish, amphibian, reptile, bird, mammal), a cell from a mammal (e.g., an ungulate (e.g., a pig, a cow, a goat, a sheep); a rodent (e.g., a rat, a mouse); a non-human primate; a human; a feline (e.g., a cat); a canine (e.g., a dog); etc.), and the like. In some cases, the cell is a cell that does not originate from a natural organism (e.g., the cell can be a synthetically made cell; also referred to as an artificial cell).

[0202] A cell can be an in vitro cell (e.g., established cultured cell line). A cell can be an ex vivo cell (cultured cell from an individual). A cell can be an in vivo cell (e.g., a cell in an individual). A cell can be an isolated cell. A cell can be a cell inside of an organism. A cell can be an organism. A cell can be a cell in a cell culture (e.g., in vitro cell culture). A cell can be one of a collection of cells. A cell can be a prokaryotic cell or derived from a prokaryotic cell. A cell can be a bacterial cell or can be derived from a bacterial cell. A cell can be an archaeal cell or derived from an archaeal cell. A cell can be a eukaryotic cell or derived from a eukaryotic cell. A cell can be a plant cell or derived from a plant cell. A cell can be an animal cell or derived from an animal cell. A cell can be an invertebrate cell or derived from an invertebrate cell. A cell can be a vertebrate cell or derived from a vertebrate cell. A cell can be a mammalian cell or derived from a mammalian cell. A cell can be a rodent cell or derived from a rodent cell. A cell can be a human cell or derived from a human cell. A cell can be a microbe cell or derived from a microbe cell. A cell can be a fungi cell or derived from a fungi cell. A cell can be an insect cell. A cell can be an arthropod cell. A cell can be a protozoan cell. A cell can be a helminth cell.

[0203] Suitable cells include a stem cell (e.g. an embryonic stem (ES) cell, an induced pluripotent stem (iPS) cell; a germ cell (e.g., an oocyte, a sperm, an oogonia, a spermatogonia, etc.); a somatic cell, e.g. a fibroblast, an oligodendrocyte, a glial cell, a hematopoietic cell, a neuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell, etc.

[0204] Suitable cells include human embryonic stem cells, fetal cardiomyocytes, myofibroblasts, mesenchymal stem cells, cardiomyocytes, adipocytes, totipotent cells, pluripotent cells, blood stem cells, myoblasts, neurons, astrocytes, islet cells, beta cells, alpha cells, delta cells, adult stem cells, bone marrow cells, mesenchymal cells, embryonic stem cells, parenchymal cells, epithelial cells, endothelial cells, mesothelial cells, fibroblasts, osteoblasts, chondrocytes, exogenous cells, endogenous cells, stem cells, hematopoietic stem cells, bone-marrow derived progenitor cells, myocardial cells, skeletal cells, fetal cells, undifferentiated cells, multi-potent progenitor cells, unipotent progenitor cells, monocytes, cardiac myoblasts, skeletal myoblasts, macrophages, capillary endothelial cells, xenogeneic cells, allogeneic cells, and postnatal stem cells.

[0205] In some cases, the cell is an immune cell, a neuron, an epithelial cell, and endothelial cell, or a stem cell. In some cases, the immune cell is a T cell, a B cell, a monocyte, a natural killer cell, a dendritic cell, or a macrophage. In some cases, the immune cell is a cytotoxic T cell. In some cases, the immune cell is a helper T cell. In some cases, the immune cell is a regulatory T cell (Treg).

[0206] In some cases, the cell is a stem cell. Stem cells include adult stem cells. Adult stem cells are also referred to as somatic stem cells.

[0207] Adult stem cells are resident in differentiated tissue, but retain the properties of selfrenewal and ability to give rise to multiple cell types, usually cell types typical of the tissue in which the stem cells are found. Numerous examples of somatic stem cells are known to those of skill in the art, including muscle stem cells; hematopoietic stem cells; epithelial stem cells; neural stem cells; mesenchymal stem cells; mammary stem cells; intestinal stem cells; mesodermal stem cells; endothelial stem cells; olfactory stem cells; neural crest stem cells; and the like.

[0208] Stem cells of interest include mammalian stem cells, where the term “mammalian” refers to any animal classified as a mammal, including humans; non-human primates; domestic and farm animals; and zoo, laboratory, sports, or pet animals, such as dogs, horses, cats, cows, mice, rats, rabbits, etc. In some cases, the stem cell is a humanstem cell. In some cases, the stem cell is a rodent (e.g., a mouse; a rat) stem cell. In some cases, the stem cell is a non-human primate stem cell.

[0209] Stem cells can express one or more stem cell markers, e.g., SOX9, KRT19, KRT7, LGR5, CA9, FXYD2, CDH6, CLDN18, TSPAN8, BPIFB1 , OLFM4, CDH17, and PPARGC1A.

[0210] In some cases, the stem cell is a hematopoietic stem cell (HSC). HSCs are mesoderm-derived cells that can be isolated from bone marrow, blood, cord blood, fetal liver and yolk sac. HSCs are characterized as CD34+ and CD3-. HSCs can repopulate the erythroid, neutrophil-macrophage, megakaryocyte and lymphoid hematopoietic cell lineages in vivo. In vitro, HSCs can be induced to undergo at least some self-renewing cell divisions and can be induced to differentiate to the same lineages as is seen in vivo. As such, HSCs can be induced to differentiate into one or more of erythroid cells, megakaryocytes, neutrophils, macrophages, and lymphoid cells.

[0211] In other instances, the stem cell is a neural stem cell (NSC). Neural stem cells (NSCs) can differentiate into neurons, and glia (including oligodendrocytes, and astrocytes). A neural stem cell is a multipotent stem cell which is capable of multiple divisions, and under specific conditions can produce daughter cells which are neural stem cells, or neural progenitor cells that can be neuroblasts or glioblasts, e.g., cells committed to become one or more types of neurons and glial cells respectively. Methods of obtaining NSCs are known in the art.

[0212] In other instances, the stem cell is a mesenchymal stem cell (MSC). MSCs originally derived from the embryonal mesoderm and isolated from adult bone marrow, can differentiate to form muscle, bone, cartilage, fat, marrow stroma, and tendon. Methods of isolating MSC are known in the art; and any known method can be used to obtain MSC. See, e.g., U.S. Pat. No. 5,736,396, which describes isolation of human MSC.

[0213] A cell is in some cases a plant cell. A plant cell can be a cell of a monocotyledon. A cell can be a cell of a dicotyledon.

[0214] In some cases, the cell is a plant cell. For example, the cell can be a cell of a major agricultural plant, e.g., Barley, Beans (Dry Edible), Canola, Corn, Cotton (Pima), Cotton (Upland), Flaxseed, Hay (Alfalfa), Hay (Non-Alfalfa), Oats, Peanuts, Rice, Sorghum, Soybeans, Sugarbeets, Sugarcane, Sunflowers (Oil), Sunflowers (Non-Oil), Sweet Potatoes , Tobacco (Burley), Tobacco (Flue-cured), Tomatoes, Wheat (Durum), Wheat (Spring), Wheat (Winter), and the like. As another example, the cell isa cell of a vegetable crops which include but are not limited to, e.g., alfalfa sprouts, aloe leaves, arrow root, arrowhead, artichokes, asparagus, bamboo shoots, banana flowers, bean sprouts, beans, beet tops, beets, bittermelon, bok choy, broccoli, broccoli rabe (rappini), brussels sprouts, cabbage, cabbage sprouts, cactus leaf (nopales), calabaza, cardoon, carrots, cauliflower, celery, chayote, Chinese artichoke (crosnes), Chinese cabbage, Chinese celery, Chinese chives, choy sum, chrysanthemum leaves (tung ho), collard greens, corn stalks, corn-sweet, cucumbers, daikon, dandelion greens, dasheen, dau mue (pea tips), donqua (winter melon), eggplant, endive, escarole, fiddle head ferns, field cress, frisee, gai choy (Chinese mustard), gailon, galanga (siam, thai ginger), garlic, ginger root, gobo, greens, hanover salad greens, huauzontle, Jerusalem artichokes, jicama, kale greens, kohlrabi, lamb's quarters (quilete), lettuce (bibb), lettuce (boston), lettuce (boston red), lettuce (green leaf), lettuce (iceberg), lettuce (lolla rossa), lettuce (oak leaf - green), lettuce (oak leaf - red), lettuce (processed), lettuce (red leaf), lettuce (romaine), lettuce (ruby romaine), lettuce (russian red mustard), linkok, Io bok, long beans, lotus root, mache, maguey (agave) leaves, malanga, mesculin mix, mizuna, moap (smooth luffa), moo, moqua (fuzzy squash), mushrooms, mustard, nagaimo, okra, ong choy, onions green, opo (long squash), ornamental corn, ornamental gourds, parsley, parsnips, peas, peppers (bell type), peppers, pumpkins, radicchio, radish sprouts, radishes, rape greens, rape greens, rhubarb, romaine (baby red), rutabagas, salicornia (sea bean), sinqua (angled / ridged luffa), spinach, squash, straw bales, sugarcane, sweet potatoes, swiss chard, tamarindo, taro, taro leaf, taro shoots, tatsoi, tepeguaje (guaje), tindora, tomatillos, tomatoes, tomatoes (cherry), tomatoes (grape type), tomatoes (plum type), tumeric, turnip tops greens, turnips, water chestnuts, yampi, yams (names), yu choy, yuca (cassava), and the like.

[0215] A cell is in some cases an arthropod cell. For example, the cell can be a cell of a suborder, a family, a sub-family, a group, a sub-group, or a species of, e.g., Chelicerata, Myriapodia, Hexipodia, Arachnida, Insecta, Archaeognatha, Thysanura, Palaeoptera, Ephemeroptera, Odonata, Anisoptera, Zygoptera, Neoptera, Exopterygota, Plecoptera , Embioptera , Orthoptera, Zoraptera , Dermaptera, Dictyoptera, Notoptera, Grylloblattidae, Mantophasmatidae, Phasmatodea , Blattaria, Isoptera, Mantodea, Parapneuroptera, Psocoptera, Thysanoptera, Phthiraptera, Hemiptera, Endopterygota or Holometabola , Hymenoptera , Coleoptera, Strepsiptera, Raphidioptera, Megaloptera, Neuroptera , Mecoptera , Siphonaptera, Diptera, Trichoptera, or Lepidoptera.

[0216] A cell is in some cases an insect cell. For example, in some cases, the cell is a cell of a mosquito, a grasshopper, a true bug, a fly, a flea, a bee, a wasp, an ant, a louse, a moth, or a beetle.Kits

[0217] The present disclosure provides a kit comprising a system of the present disclosure, or a component of a system of the present disclosure.

[0218] A kit of the present disclosure can comprise: a) a variant Cas12a protein of the present disclosure and a Cas12a guide RNA; b) a variant Cas12a protein of the present disclosure, a Cas12a guide RNA, and a donor template nucleic acid; c) a fusion polypeptide of the present disclosure and a Cas12a guide RNA; d) a fusion polypeptide of the present disclosure, a Cas12a guide RNA, and a donor template nucleic acid; e) an mRNA encoding a variant Cas12a protein of the present disclosure; and a Cas12a guide RNA; f) an mRNA encoding a variant Cas12a protein of the present disclosure, a Cas12a guide RNA, and a donor template nucleic acid; g) an mRNA encoding a fusion polypeptide of the present disclosure; and a Cas12a guide RNA; h) an mRNA encoding a fusion polypeptide of the present disclosure, a Cas12a guide RNA, and a donor template nucleic acid; i) a recombinant expression vector comprising a nucleotide sequence encoding a variant Cas12a protein of the present disclosure and a nucleotide sequence encoding a Cas12a guide RNA; j) a recombinant expression vector comprising a nucleotide sequence encoding a variant Cas12a protein of the present disclosure, a nucleotide sequence encoding a Cas12a guide RNA, and a nucleotide sequence encoding a donor template nucleic acid; k) a recombinant expression vector comprising a nucleotide sequence encoding a fusion polypeptide of the present disclosure and a nucleotide sequence encoding a Cas12a guide RNA; I) a recombinant expression vector comprising a nucleotide sequence encoding a fusion polypeptide of the present disclosure, a nucleotide sequence encoding a Cas12a guide RNA, and a nucleotide sequence encoding a donor template nucleic acid; m) a first recombinant expression vector comprising a nucleotide sequence encoding aa variant Cas12a protein of the present disclosure and a second recombinant expression vector comprising a nucleotide sequence encoding a Cas12a guide RNA; n) a first recombinant expression vector comprising a nucleotide sequence encoding a variant Cas12a protein of the present disclosure, and a second recombinant expression vector comprising a nucleotide sequence encoding a Cas12a guide RNA; and a donor template nucleic acid; o) a first recombinant expression vector comprising a nucleotide sequence encoding a variant Cas12a protein of the present disclosure, and a second recombinant expression vector comprising a nucleotide sequence encoding a Cas12a guide RNA; p) a first recombinant expression vector comprising a nucleotide sequence encoding a fusionpolypeptide of the present disclosure, and a second recombinant expression vector comprising a nucleotide sequence encoding a Cas12a guide RNA; and a donor template nucleic acid; q) a recombinant expression vector comprising a nucleotide sequence encoding a variant Cas12a protein of the present disclosure, a nucleotide sequence encoding a first Cas12a guide RNA, and a nucleotide sequence encoding a second Cas12a guide RNA; or r) a recombinant expression vector comprising a nucleotide sequence encoding a fusion polypeptide of the present disclosure, a nucleotide sequence encoding a first Cas12a guide RNA, and a nucleotide sequence encoding a second Cas12a guide RNA; or some variation of one of (a) through (r).

[0219] A kit of the present disclosure can comprise: a) a component, as described above, of a system of the present disclosure, or can comprise a system of the present disclosure; and b) one or more additional reagents, e.g., i) a buffer; ii) a protease inhibitor; iii) a nuclease inhibitor; iv) a reagent required to develop or visualize a detectable label; v) a positive and / or negative control target DNA; vi) a positive and / or negative control Cas12a guide RNA; and the like. A kit of the present disclosure can comprise: a) a component, as described above, of a system of the present disclosure, or can comprise a system of the present disclosure; and b) a therapeutic agent.

[0220] A kit of the present disclosure can comprise a recombinant expression vector comprising: a) an insertion site for inserting a nucleic acid comprising a nucleotide sequence encoding a portion of a Cas12a guide RNA that hybridizes to a target nucleotide sequence in a target nucleic acid; and b) a nucleotide sequence encoding the variant Cas12a protein-binding portion of a Cas12a guide RNA. A kit of the present disclosure can comprise a recombinant expression vector comprising: a) an insertion site for inserting a nucleic acid comprising a nucleotide sequence encoding a portion of a Cas12a guide RNA that hybridizes to a target nucleotide sequence in a target nucleic acid; b) a nucleotide sequence encoding the variant Cas12a proteinbinding portion of a Cas12a guide RNA; and c) a nucleotide sequence encoding a variant Cas12a protein of the present disclosure.Utility / Methods

[0221] A variant Cas12a protein of the present disclosure, or a fusion polypeptide of the present disclosure, finds use in a variety of methods (e.g., in combination with a Cas12a guide RNA and in some cases further in combination with a donor template). For example, a variant Cas12a protein of the present disclosure can be used to (i) modify (e.g., cleave, e.g., nick; methylate; base edit; etc.) target nucleic acid (DNA orRNA; single stranded or double stranded); (ii) modulate transcription of a target nucleic acid; (iii) label a target nucleic acid; (iv) bind a target nucleic acid (e.g., for purposes of isolation, labeling, imaging, tracking, etc.); (v) modify a polypeptide (e.g., a histone) associated with a target nucleic acid; and the like. Thus, the present disclosure provides a method of modifying a target nucleic acid. In some cases, the modification is cleavage, and in some such cases the cleavage leans to editing of the target nucleic acid, e.g., gene editing. In some cases, a method of the present disclosure for modifying a target nucleic acid comprises contacting the target nucleic acid with: a) a variant Cas12a protein (or fusion polypeptide) of the present disclosure; and b) one or more (e.g., two) Cas12a guide RNAs. In some cases, a method of the present disclosure for modifying a target nucleic acid comprises contacting the target nucleic acid with: a) a variant Cas12a protein of the present disclosure; b) a Cas12a guide RNA; and c) a donor nucleic acid (e.g., a donor template). In some cases, the contacting step is carried out in a cell in vitro. In some cases, the contacting step is carried out in a cell in vivo. In some cases, the contacting step is carried out in a cell ex vivo.

[0222] Because a method that uses a variant Cas12a protein of the present disclosure includes binding of the variant Cas12a protein to a particular region in a target nucleic acid (by virtue of being targeted there by an associated Cas12a guide RNA), the methods can generally referred to herein as methods of guiding a variant Cas12a to a target nucleic acid or methods of binding (e.g., a method of binding a target nucleic acid). However, it is to be understood that in some cases, while a method of binding may result in nothing more than binding of the target nucleic acid, in other cases, the method can have different final results (e.g., the method can result in modification of the target nucleic acid, e.g., cleavage / methylation / etc., modulation of transcription from the target nucleic acid; modulation of translation of the target nucleic acid; genome editing; modulation of a protein associated with the target nucleic acid; isolation of the target nucleic acid; etc.).

[0223] For examples of suitable methods, see, for example, Jinek et al., Science. 2012 Aug 17;337(6096):816-21 ; Chylinski et al., RNA Biol. 2013 May; 10(5):726-37; Ma et al., Biomed Res Int. 2013;2013:270805; Hou et al., Proc Natl Acad Sci U S A. 2013 Sep 24; 110(39): 15644-9; Jinek et al., Elife. 2013;2:e00471; Pattanayak et al., Nat Biotechnol. 2013 Sep;31(9):839-43; Qi et al, Cell. 2013 Feb 28; 152(5): 1173-83; Wang et al., Cell. 2013 May 9;153(4):910-8; Auer et al., Genome Res. 2013 Oct 31 ; Chen et al., Nucleic Acids Res. 2013 Nov 1 ;41(20):e19; Cheng et al., Cell Res. 2013Oct;23(10):1163-71; Cho et al., Genetics. 2013 Nov; 195(3): 1177-80; DiCarlo et al., Nucleic Acids Res. 2013 Apr;41(7):4336-43; Dickinson et al., Nat Methods. 2013 Oct; 10(10): 1028-34; Ebina et al., Sci Rep. 2013;3:2510; Fujii et al, Nucleic Acids Res. 2013 Nov 1 ;41 (20):e187; Hu et al., Cell Res. 2013 Nov;23(11):1322-5; Jiang et al., Nucleic Acids Res. 2013 Nov 1 ;41(20):e188; Larson et al., Nat Protoc. 2013 Nov;8(11):2180-96; Mali et. at., Nat Methods. 2013 Oct; 10(10):957-63; Nakayama et al., Genesis. 2013 Dec;51(12):835-43; Ran et al., Nat Protoc. 2013 Nov;8(11):2281- 308; Ran et al., Cell. 2013 Sep 12;154(6):1380-9; Upadhyay et al., G3 (Bethesda). 2013 Dec 9;3(12):2233-8; Walsh et al., Proc Natl Acad Sci U S A. 2013 Sep 24; 110(39): 15514-5; Xie et al., Mol Plant. 2013 Oct 9; Yang et al., Cell. 2013 Sep 12;154(6):1370-9; and U.S. patents and patent applications: 8,906,616; 8,895,308; 8,889,418; 8,889,356; 8,871 ,445; 8,865,406; 8,795,965; 8,771 ,945; 8,697,359; 20140068797; 20140170753; 20140179006; 20140179770; 20140186843; 20140186919; 20140186958; 20140189896; 20140227787; 20140234972; 20140242664; 20140242699; 20140242700; 20140242702; 20140248702; 20140256046; 20140273037; 20140273226; 20140273230; 20140273231 ; 20140273232; 20140273233; 20140273234; 20140273235; 20140287938; 20140295556; 20140295557; 20140298547; 20140304853; 20140309487; 20140310828; 20140310830; 20140315985; 20140335063; 20140335620; 20140342456; 20140342457; 20140342458; 20140349400; 20140349405; 20140356867; 20140356956; 20140356958; 20140356959; 20140357523; 20140357530; 20140364333; and 20140377868; each of which is hereby incorporated by reference in its entirety.

[0224] For example, the present disclosure provides (but is not limited to) methods of cleaving a target nucleic acid; methods of editing a target nucleic acid; methods of modulating transcription from a target nucleic acid; methods of isolating a target nucleic acid, methods of binding a target nucleic acid, methods of imaging a target nucleic acid, methods of modifying a target nucleic acid, and the like.

[0225] As used herein, the terms / phrases “contact a target nucleic acid” and “contacting a target nucleic acid”, for example, with a variant Cas12a protein of the present disclosure or with a fusion polypeptide of the present disclosure, etc., encompass all methods for contacting the target nucleic acid. For example, a variant Cas12a protein of the present disclosure can be provided to a cell as protein, RNA (encoding the variant Cas12a protein), or DNA (encoding the variant Cas12a protein); while a Cas12a guide RNA can be provided as a guide RNA or as a nucleic acid encoding theguide RNA. As such, when, for example, performing a method in a cell (e.g., inside of a cell in vitro, inside of a cell in vivo, inside of a cell ex vivo), a method that includes contacting the target nucleic acid encompasses the introduction into the cell of any or all of the components in their active / final state (e.g., in the form of a protein(s) for variant Cas12a protein; in the form of a protein for a fusion polypeptide; in the form of an RNA in some cases for the guide RNA), and also encompasses the introduction into the cell of one or more nucleic acids encoding one or more of the components (e.g., nucleic acid(s) comprising nucleotide sequence(s) encoding a variant Cas12a protein or a fusion polypeptide comprising a variant Cas12a protein, nucleic acid(s) comprising nucleotide sequence(s) encoding guide RNA(s), nucleic acid comprising a nucleotide sequence encoding a donor template, and the like). Because the methods can also be performed in vitro outside of a cell, a method that includes contacting a target nucleic acid, (unless otherwise specified) encompasses contacting outside of a cell in vitro, inside of a cell in vitro, inside of a cell in vivo, inside of a cell ex vivo, etc.

[0226] In some cases, a method of the present disclosure for modifying a target nucleic acid comprises contacting a target nucleic acid with a variant Cas12a protein of the present disclosure, or with a fusion polypeptide of the present disclosure. In some cases, a method of the present disclosure for modifying a target nucleic acid comprises contacting a target nucleic acid with a variant Cas12a protein of the present disclosure and a Cas12a guide RNA. In some cases, a method of the present disclosure for modifying a target nucleic acid comprises contacting a target nucleic acid with a variant Cas12a protein of the present disclosure, a first Cas12a guide RNA, and a second Cas12a guide RNA In some cases, a method of the present disclosure for modifying a target nucleic acid comprises contacting a target nucleic acid with a variant Cas12a protein of the present disclosure and a Cas12a guide RNA and a donor DNA template.Introducing components into a target cell

[0227] A guide RNA (or a nucleic acid comprising a nucleotide sequence encoding the guide RNA) and / or a variant Cas12a protein of the present disclosure (or a nucleic acid comprising a nucleotide sequence encoding the variant Cas12a protein), and optionally also a donor template nucleic acid, can be introduced into a host cell by any of a variety of well-known methods. As a non-limiting example, a guide RNA and / or a variant Cas12a protein of the present disclosure can be combined with a lipid. As a non-limiting example, a guide RNA, a variant Cas12a protein of the presentdisclosure, and a donor template nucleic acid, can be combined with a lipid. As another non-limiting example, a guide RNA and / or variant Cas12a protein of the present disclosure can be combined with a particle, or formulated into a particle. As another non-limiting example, a guide RNA, a variant Cas12a protein of the present disclosure, and a donor template nucleic acid, can be combined with a particle, or formulated into a particle.

[0228] Methods of introducing a nucleic acid and / or protein into a host cell are known in the art, and any convenient method can be used to introduce a subject nucleic acid (e.g., an expression construct / vector) into a target cell (e.g., prokaryotic cell, eukaryotic cell, plant cell, animal cell, mammalian cell, human cell, and the like). Suitable methods include, e.g., viral infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro injection, nanoparticle-mediated nucleic acid delivery (see, e.g., Panyam et al. Adv Drug Deliv Rev. 2012 Sep 13. pii: S0169-409X(12)00283-9. doi: 10.1016 / j.addr.2O12.09.023 ), and the like.

[0229] A guide RNA can be introduced, e.g., as a DNA molecule encoding the guide RNA, or can be provided directly as an RNA molecule (or a hybrid molecule when applicable). In some cases, a variant Cas12a protein of the present disclosure is provided as a nucleic acid (e.g., an mRNA, a DNA, a plasmid, an expression vector, a viral vector, a minicircle, etc.) that encodes the protein. In some cases, the variant Cas12a protein is provided directly as a protein (e.g., without an associated guide RNA or with an associate guide RNA, i.e. , as a ribonucleoprotein complex - RNP). Like a guide RNA, a variant Cas12a protein of the present disclosure can be introduced into a cell (provided to the cell) by any convenient method; such methods are known to those of ordinary skill in the art. As an illustrative example, a variant Cas12a protein of the present disclosure can be injected directly into a cell (e.g., with or without a guide RNA or nucleic acid encoding a guide RNA). As another example, a pre-formed complex of a variant Cas12a protein of the present disclosure and a guide RNA (an RNP) can be introduced into a cell (e.g., eukaryotic cell) (e.g., via injection, via nucleofection; via a protein transduction domain (PTD) conjugated to one or more components, e.g., conjugated to the variant Cas12a protein, conjugated to a guide RNA; etc.).

[0230] In some cases, a nucleic acid (e.g., a guide RNA; a nucleic acid comprising a nucleotide sequence encoding a variant Cas12a protein of the present disclosure; a nucleic acid comprising a nucleotide sequence encoding a guide RNA; a donor template nucleic acid; etc.) and / or a polypeptide (e.g., a variant Cas12a protein of the present disclosure) is delivered to a cell (e.g., a target host cell) in a particle, or associated with a particle. The terms “particle” and “nanoparticle” can be used interchangeably, as appropriate.

[0231] This can be achieved, e.g., using particles or lipid envelopes, e.g., a ribonucleoprotein (RNP) complex can be delivered via a particle, e.g., a delivery particle comprising lipid or lipidoid and hydrophilic polymer, e.g., a cationic lipid and a hydrophilic polymer, for instance wherein the cationic lipid comprises 1,2-dioleoyl-3-trimethylammonium- propane (DOTAP) or 1 ,2-ditetradecanoyl-sn-glycero-3-phosphocholine (DMPC) and / or wherein the hydrophilic polymer comprises ethylene glycol or polyethylene glycol (PEG); and / or wherein the particle further comprises cholesterol (e.g., particle from formulation 1=DOTAP 100, DMPC 0, PEG 0, Cholesterol 0; formulation number 2=DOTAP 90, DMPC 0, PEG 10, Cholesterol 0; formulation number 3=DOTAP 90, DMPC 0, PEG 5, Cholesterol 5).

[0232] A variant Cas12a protein of the present disclosure (or an mRNA or a DNA comprising a nucleotide sequence encoding the protein) and / or a guide RNA (or a nucleic acid such as one or more expression vectors encoding the guide RNA) may be delivered simultaneously using particles or lipid envelopes. For example, a biodegradable coreshell structured nanoparticle with a poly (P-amino ester) (PBAE) core enveloped by a phospholipid bilayer shell can be used. In some cases, particles / nanoparticles based on self assembling bioadhesive polymers are used; such particles / nanoparticles may be applied to oral delivery of peptides, intravenous delivery of peptides and nasal delivery of peptides, e.g., to the brain. Other embodiments, such as oral absorption and ocular delivery of hydrophobic drugs are also contemplated. A molecular envelope technology, which involves an engineered polymer envelope which is protected and delivered to the site of the disease, can be used. Doses of about 5 mg / kg can be used, with single or multiple doses, depending on various factors, e.g., the target tissue.

[0233] Lipidoid compounds (e.g., as described in US patent publication 20110293703) are also useful in the administration of polynucleotides, and can be used. In one aspect, aminoalcohol lipidoid compounds are combined with an agent to be delivered to a cell or a subject to form microparticles, nanoparticles, liposomes, or micelles. Theaminoalcohol lipidoid compounds may be combined with other aminoalcohol lipidoid compounds, polymers (synthetic or natural), surfactants, cholesterol, carbohydrates, proteins, lipids, etc. to form the particles. These particles may then optionally be combined with a pharmaceutical excipient to form a pharmaceutical composition.

[0234] A poly(beta-amino alcohol) (PBAA) can be used, sugar-based particles may be used, for example GalNAc, as described with reference to WO2014118272 (incorporated herein by reference) and Nair, J K et al., 2014, Journal of the American Chemical Society 136 (49), 16958-16961). In some cases, lipid nanoparticles (LNPs) are used. Spherical Nucleic Acid (SNA™) constructs and other nanoparticles (particularly gold nanoparticles) can be used to a target cell. See, e.g., Cutler et al., J. Am. Chem. Soc. 2011 133:9254-9257, Hao et al., Small. 2011 7:3158-3162, Zhang et al., ACS Nano.2011 5:6962-6970, Cutler et al., J. Am. Chem. Soc. 2012 134:1376-1391 , Young et al., Nano Lett. 2012 12:3867-71 , Zheng et al., Proc. Natl. Acad. Sci. USA. 2012 109:11975-80, Mirkin, Nanomedicine 2012 7:635-638 Zhang et al., J. Am. Chem. Soc.2012 134:16488-1691 , Weintraub, Nature 2013 495:S14-S16, Choi et al., Proc. Natl. Acad. Sci. USA. 2013 110(19): 7625-7630, Jensen et al., Sci. Transl. Med. 5, 209ra152 (2013) and Mirkin, et al., Small, 10:186-192. Semi-solid and soft nanoparticles are also suitable for delivery. An exosome can be used for delivery. Exosomes are endogenous nano-vesicles that transport RNAs and proteins, and which can deliver RNA to the brain and other target organs. Supercharged proteins can be used for delivery to a cell. Supercharged proteins are a class of engineered or naturally occurring proteins with unusually high positive or negative net theoretical charge. Both supernegatively and superpositively charged proteins exhibit the ability to withstand thermally or chemically induced aggregation. Superpositively charged proteins are also able to penetrate mammalian cells. Associating cargo with these proteins, such as plasmid DNA, RNA, minicircles, or other proteins, can facilitate the functional delivery of these macromolecules into mammalian cells both in vitro and in vivo. Cell Penetrating Peptides (CPPs) can be used for delivery. CPPs typically have an amino acid composition that either contains a high relative abundance of positively charged amino acids such as lysine or arginine or has sequences that contain an alternating pattern of polar / charged amino acids and non-polar, hydrophobic amino acids.Target nucleic acids and target cells of interest

[0235] A variant Cas12a protein of the present disclosure, or a fusion polypeptide of the present disclosure, when bound to a Cas12a guide RNA, can bind to a target nucleic acid, and in some cases, can bind to and modify a target nucleic acid. A target nucleic acid can be any nucleic acid (e.g., DNA, RNA), can be double stranded or single stranded, can be any type of nucleic acid (e.g., a chromosome (genomic DNA), derived from a chromosome, chromosomal DNA, plasmid, viral, extracellular, intracellular, mitochondrial, chloroplast, linear, circular, etc.) and can be from any organism (e.g., as long as the Cas12a guide RNA comprises a nucleotide sequence that hybridizes to a target sequence in a target nucleic acid, such that the target nucleic acid can be targeted).

[0236] A target nucleic acid can be DNA or RNA. A target nucleic acid can be double stranded (e.g., dsDNA, dsRNA) or single stranded (e.g., ssRNA, ssDNA). In some cases, a target nucleic acid is single stranded. In some cases, a target nucleic acid is a single stranded RNA (ssRNA). In some cases, a target ssRNA (e.g., a target cell ssRNA, a viral ssRNA, etc.) is selected from: mRNA, rRNA, tRNA, non-coding RNA (ncRNA), long non-coding RNA (IncRNA), and microRNA (miRNA). In some cases, a target nucleic acid is a single stranded DNA (ssDNA) (e.g., a viral DNA). As noted above, in some cases, a target nucleic acid is single stranded.

[0237] A target nucleic acid can be located anywhere, for example, outside of a cell in vitro, inside of a cell in vitro; inside of a cell in vivo; inside of a cell ex vivo; or inside of an organelle (e.g., mitochondrion; nucleus; etc.) within a cell that is in vitro, in vivo, or ex vivo. Suitable target cells (which can comprise target nucleic acids such as genomic DNA) include, but are not limited to: a bacterial cell; an archaeal cell; a cell of a singlecell eukaryotic organism; a plant cell; an algal cell, e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C. agardh, and the like; a fungal cell (e.g., a yeast cell); an animal cell; a cell from an invertebrate animal (e.g. fruit fly, a cnidarian, an echinoderm, a nematode, etc.); a cell of an insect (e.g., a mosquito; a bee; an agricultural pest; etc.); a cell of an arachnid (e.g., a spider; a tick; etc.); a cell from a vertebrate animal (e.g., a fish, an amphibian, a reptile, a bird, a mammal); a cell from a mammal (e.g., a cell from a rodent; a cell from a human; a cell of a non-human mammal; a cell of a rodent (e.g., a mouse, a rat); a cell of a lagomorph (e.g., a rabbit); a cell of an ungulate (e.g., a cow, a horse, a camel, a llama, a vicuna, a sheep, a goat, etc.); a cell of a marine mammal (e.g., a whale, a seal, an elephant seal, a dolphin, a sea lion; etc.) and thelike. Any type of cell may be of interest (e.g. a stem cell, e.g. an embryonic stem (ES) cell, an induced pluripotent stem (iPS) cell, a germ cell (e.g., an oocyte, a sperm, an oogonia, a spermatogonia, etc.), an adult stem cell, a somatic cell, e.g. a fibroblast, a hematopoietic cell, a neuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell; an in vitro or in vivo embryonic cell of an embryo at any stage, e.g., a 1-cell, 2- cell, 4-cell, 8-cell, etc. stage zebrafish embryo; etc.).

[0238] Cells may be from established cell lines or they may be primary cells, where “primary cells”, “primary cell lines”, and “primary cultures” are used interchangeably herein to refer to cells and cells cultures that have been derived from a subject and allowed to grow in vitro for a limited number of passages, i.e. splittings, of the culture. For example, primary cultures are cultures that may have been passaged 0 times, 1 time, 2 times, 4 times, 5 times, 10 times, or 15 times, but not enough times go through the crisis stage. Typically, the primary cell lines are maintained for fewer than 10 passages in vitro. Target cells can be unicellular organisms and / or can be grown in culture. If the cells are primary cells, they may be harvest from an individual by any convenient method. For example, leukocytes may be conveniently harvested by apheresis, leukocytopheresis, density gradient separation, etc., while cells from tissues such as skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestine, stomach, etc. can be conveniently harvested by biopsy.

[0239] In some of the above applications, the subject methods may be employed to induce target nucleic acid cleavage, target nucleic acid modification, and / or to bind target nucleic acids (e.g., for visualization, for collecting and / or analyzing, etc.) in mitotic or post-mitotic cells in vivo and / or ex vivo and / or in vitro (e.g., to disrupt production of a protein encoded by a targeted mRNA, to cleave or otherwise modify target DNA, to genetically modify a target cell, and the like). Because the guide RNA provides specificity by hybridizing to target nucleic acid, a mitotic and / or post-mitotic cell of interest in the disclosed methods may include a cell from any organism (e.g. a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a plant cell, an algal cell, e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C. agardh, and the like, a fungal cell (e.g., a yeast cell), an animal cell, a cell from an invertebrate animal (e.g. fruit fly, cnidarian, echinoderm, nematode, etc.), a cell from a vertebrate animal (e.g., fish, amphibian, reptile, bird, mammal), a cell from a mammal, a cell from a rodent, a cell from a human, etc.). In some cases, a variant Cas12a protein of the present disclosure (and / or nucleic acid encoding the protein such as DNA and / or RNA),and / or Cas12a guide RNA (and / or a DNA encoding the guide RNA), and / or donor template, and / or RNP can be intrduced into an individual (i.e. , the target cell can be in vivo) (e.g., a mammal, a rat, a mouse, a pig, a primate, a non-human primate, a human, etc.). In some case, such an administration can be for the purpose of treating and / or preventing a disease, e.g., by editing the genome of targeted cells.

[0240] Plant cells include cells of a monocotyledon, and cells of a dicotyledon. The cells can be root cells, leaf cells, cells of the xylem, cells of the phloem, cells of the cambium, apical meristem cells, parenchyma cells, collenchyma cells, sclerenchyma cells, and the like. Plant cells include cells of agricultural crops such as wheat, corn, rice, sorghum, millet, soybean, etc. Plant cells include cells of agricultural fruit and nut plants, e.g., plant that produce apricots, oranges, lemons, apples, plums, pears, almonds, etc.

[0241] Additional examples of target cells are listed above in the section titled “Modified cells.” Non-limiting examples of cells (target cells) include: a prokaryotic cell, eukaryotic cell, a bacterial cell, an archaeal cell, a cell of a single-cell eukaryotic organism, a protozoa cell, a cell from a plant (e.g., cells from plant crops, fruits, vegetables, grains, soy bean, corn, maize, wheat, seeds, tomatoes, rice, cassava, sugarcane, pumpkin, hay, potatoes, cotton, cannabis, tobacco, flowering plants, conifers, gymnosperms, angiosperms, ferns, clubmosses, hornworts, liverworts, mosses, dicotyledons, monocotyledons, etc.), an algal cell, (e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C. agardh, and the like), seaweeds (e.g. kelp) a fungal cell (e.g., a yeast cell, a cell from a mushroom), an animal cell, a cell from an invertebrate animal (e.g., fruit fly, cnidarian, echinoderm, nematode, etc.), a cell from a vertebrate animal (e.g., fish, amphibian, reptile, bird, mammal), a cell from a mammal (e.g., an ungulate (e.g., a pig, a cow, a goat, a sheep); a rodent (e.g., a rat, a mouse); a non-human primate; a human; a feline (e.g., a cat); a canine (e.g., a dog); etc.), and the like. In some cases, the cell is a cell that does not originate from a natural organism (e.g., the cell can be a synthetically made cell; also referred to as an artificial cell).

[0242] A cell can be an in vitro cell (e.g., established cultured cell line). A cell can be an ex vivo cell (cultured cell from an individual). A cell can be an in vivo cell (e.g., a cell in an individual). A cell can be an isolated cell. A cell can be a cell inside of an organism. A cell can be an organism. A cell can be a cell in a cell culture (e.g., in vitro cell culture). A cell can be one of a collection of cells. A cell can be a prokaryotic cell orderived from a prokaryotic cell. A cell can be a bacterial cell or can be derived from a bacterial cell. A cell can be an archaeal cell or derived from an archaeal cell. A cell can be a eukaryotic cell or derived from a eukaryotic cell. A cell can be a plant cell or derived from a plant cell. A cell can be an animal cell or derived from an animal cell. A cell can be an invertebrate cell or derived from an invertebrate cell. A cell can be a vertebrate cell or derived from a vertebrate cell. A cell can be a mammalian cell or derived from a mammalian cell. A cell can be a rodent cell or derived from a rodent cell. A cell can be a human cell or derived from a human cell. A cell can be a microbe cell or derived from a microbe cell. A cell can be a fungi cell or derived from a fungi cell. A cell can be an insect cell. A cell can be an arthropod cell. A cell can be a protozoan cell. A cell can be a helminth cell.

[0243] Suitable cells include a stem cell (e.g. an embryonic stem (ES) cell, an induced pluripotent stem (iPS) cell; a germ cell (e.g., an oocyte, a sperm, an oogonia, a spermatogonia, etc.); a somatic cell, e.g. a fibroblast, an oligodendrocyte, a glial cell, a hematopoietic cell, a neuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell, etc.

[0244] Suitable cells include human embryonic stem cells, fetal cardiomyocytes, myofibroblasts, mesenchymal stem cells, cardiomyocytes, adipocytes, totipotent cells, pluripotent cells, blood stem cells, myoblasts, adult stem cells, bone marrow cells, mesenchymal cells, embryonic stem cells, parenchymal cells, epithelial cells, endothelial cells, mesothelial cells, fibroblasts, neurons, astrocytes, islet cells, alpha cells, beta cells delta cells, osteoblasts, chondrocytes, exogenous cells, endogenous cells, stem cells, hematopoietic stem cells, bone-marrow derived progenitor cells, myocardial cells, skeletal cells, fetal cells, undifferentiated cells, multi-potent progenitor cells, unipotent progenitor cells, monocytes, cardiac myoblasts, skeletal myoblasts, macrophages, capillary endothelial cells, xenogenic cells, allogenic cells, and post-natal stem cells.

[0245] In some cases, the cell is an immune cell, a neuron, an epithelial cell, and endothelial cell, or a stem cell. In some cases, the immune cell is a T cell, a B cell, a monocyte, a natural killer cell, a dendritic cell, or a macrophage. In some cases, the immune cell is a cytotoxic T cell. In some cases, the immune cell is a helper T cell. In some cases, the immune cell is a regulatory T cell (Treg).

[0246] In some cases, the cell is a stem cell. Stem cells include adult stem cells. Adult stem cells are also referred to as somatic stem cells.

[0247] Adult stem cells are resident in differentiated tissue, but retain the properties of selfrenewal and ability to give rise to multiple cell types, usually cell types typical of the tissue in which the stem cells are found. Numerous examples of somatic stem cells are known to those of skill in the art, including muscle stem cells; hematopoietic stem cells; epithelial stem cells; neural stem cells; mesenchymal stem cells; mammary stem cells; intestinal stem cells; mesodermal stem cells; endothelial stem cells; olfactory stem cells; neural crest stem cells; and the like.

[0248] Stem cells of interest include mammalian stem cells, where the term “mammalian” refers to any animal classified as a mammal, including humans; non-human primates; domestic and farm animals; and zoo, laboratory, sports, or pet animals, such as dogs, horses, cats, cows, mice, rats, rabbits, etc. In some cases, the stem cell is a human stem cell. In some cases, the stem cell is a rodent (e.g., a mouse; a rat) stem cell. In some cases, the stem cell is a non-human primate stem cell.

[0249] Stem cells can express one or more stem cell markers, e.g., SOX9, KRT19, KRT7, LGR5, CA9, FXYD2, CDH6, CLDN18, TSPAN8, BPIFB1 , OLFM4, CDH17, and PPARGC1A.

[0250] In some cases, the stem cell is a hematopoietic stem cell (HSC). HSCs are mesoderm-derived cells that can be isolated from bone marrow, blood, cord blood, fetal liver and yolk sac. HSCs are characterized as CD34+ and CD3-. HSCs can repopulate the erythroid, neutrophil-macrophage, megakaryocyte and lymphoid hematopoietic cell lineages in vivo. In vitro, HSCs can be induced to undergo at least some self-renewing cell divisions and can be induced to differentiate to the same lineages as is seen in vivo. As such, HSCs can be induced to differentiate into one or more of erythroid cells, megakaryocytes, neutrophils, macrophages, and lymphoid cells.

[0251] In other cases, the stem cell is a neural stem cell (NSC). Neural stem cells (NSCs) are capable of differentiating into neurons, and glia (including oligodendrocytes, and astrocytes). A neural stem cell is a multipotent stem cell which is capable of multiple divisions, and under specific conditions can produce daughter cells which are neural stem cells, or neural progenitor cells that can be neuroblasts or glioblasts, e.g., cells committed to become one or more types of neurons and glial cells respectively. Methods of obtaining NSCs are known in the art.

[0252] In other cases, the stem cell is a mesenchymal stem cell (MSC). MSCs originally derived from the embryonal mesoderm and isolated from adult bone marrow, can differentiate to form muscle, bone, cartilage, fat, marrow stroma, and tendon.Methods of isolating MSC are known in the art; and any known method can be used to obtain MSC. See, e.g., U.S. Pat. No. 5,736,396, which describes isolation of human MSC.

[0253] A cell is in some cases a plant cell. A plant cell can be a cell of a monocotyledon. A cell can be a cell of a dicotyledon.

[0254] In some cases, the cell is a plant cell. For example, the cell can be a cell of a major agricultural plant, e.g., Barley, Beans (Dry Edible), Canola, Corn, Cotton (Pima), Cotton (Upland), Flaxseed, Hay (Alfalfa), Hay (Non-Alfalfa), Oats, Peanuts, Rice, Sorghum, Soybeans, Sugarbeets, Sugarcane, Sunflowers (Oil), Sunflowers (Non-Oil), Sweet Potatoes , Tobacco (Burley), Tobacco (Flue-cured), Tomatoes, Wheat (Durum), Wheat (Spring), Wheat (Winter), and the like. As another example, the cell is a cell of a vegetable crops which include but are not limited to, e.g., alfalfa sprouts, aloe leaves, arrow root, arrowhead, artichokes, asparagus, bamboo shoots, banana flowers, bean sprouts, beans, beet tops, beets, bittermelon, bok choy, broccoli, broccoli rabe (rappini), brussels sprouts, cabbage, cabbage sprouts, cactus leaf (nopales), calabaza, cardoon, carrots, cauliflower, celery, chayote, Chinese artichoke (crosnes), Chinese cabbage, Chinese celery, Chinese chives, choy sum, chrysanthemum leaves (tung ho), collard greens, corn stalks, corn-sweet, cucumbers, daikon, dandelion greens, dasheen, dau mue (pea tips), donqua (winter melon), eggplant, endive, escarole, fiddle head ferns, field cress, frisee, gai choy (Chinese mustard), gailon, galanga (siam, thai ginger), garlic, ginger root, gobo, greens, hanover salad greens, huauzontle, Jerusalem artichokes, jicama, kale greens, kohlrabi, lamb's quarters (quilete), lettuce (bibb), lettuce (boston), lettuce (boston red), lettuce (green leaf), lettuce (iceberg), lettuce (lolla rossa), lettuce (oak leaf - green), lettuce (oak leaf - red), lettuce (processed), lettuce (red leaf), lettuce (romaine), lettuce (ruby romaine), lettuce (russian red mustard), linkok, Io bok, long beans, lotus root, mache, maguey (agave) leaves, malanga, mesculin mix, mizuna, moap (smooth luffa), moo, moqua (fuzzy squash), mushrooms, mustard, nagaimo, okra, ong choy, onions green, opo (long squash), ornamental corn, ornamental gourds, parsley, parsnips, peas, peppers (bell type), peppers, pumpkins, radicchio, radish sprouts, radishes, rape greens, rape greens, rhubarb, romaine (baby red), rutabagas, salicornia (sea bean), sinqua (angled / ridged luffa), spinach, squash, straw bales, sugarcane, sweet potatoes, swiss chard, tamarindo, taro, taro leaf, taro shoots, tatsoi, tepeguaje (guaje), tindora, tomatillos, tomatoes, tomatoes (cherry), tomatoes (grapetype), tomatoes (plum type), tumeric, turnip tops greens, turnips, water chestnuts, yampi, yams (names), yu choy, yuca (cassava), and the like.

[0255] A cell is in some cases an arthropod cell. For example, the cell can be a cell of a suborder, a family, a sub-family, a group, a sub-group, or a species of, e.g., Chelicerata, Myriapodia, Hexipodia, Arachnida, Insecta, Archaeognatha, Thysanura, Palaeoptera, Ephemeroptera, Odonata, Anisoptera, Zygoptera, Neoptera, Exopterygota, Plecoptera , Embioptera , Orthoptera, Zoraptera , Dermaptera, Dictyoptera, Notoptera, Grylloblattidae, Mantophasmatidae, Phasmatodea , Blattaria, Isoptera, Mantodea, Parapneuroptera, Psocoptera, Thysanoptera, Phthiraptera, Hemiptera, Endopterygota or Holometabola , Hymenoptera , Coleoptera, Strepsiptera, Raphidioptera, Megaloptera, Neuroptera , Mecoptera , Siphonaptera, Diptera, Trichoptera, or Lepidoptera.

[0256] A cell is in some cases an insect cell. For example, in some cases, the cell is a cell of a mosquito, a grasshopper, a true bug, a fly, a flea, a bee, a wasp, an ant, a louse, a moth, or a beetle.Detection Methods

[0257] The present disclosure provides methods of detecting a nucleic acid (e.g., a target nucleic acid) in a sample. The methods comprise contacting a sample that may comprise the target nucleic acid with a variant Cas12a protein of the present disclosure.

[0258] In some cases, as noted above, a variant Cas12a protein of the present disclosure can promiscuously cleave non-targeted single stranded DNA (ssDNA) once activated by detection of a target DNA (double or single stranded). Once a variant Cas12a protein of the present disclosure is activated by a guide RNA, which occurs when the guide RNA hybridizes to a target sequence of a target DNA (i.e. , the sample includes the targeted DNA), the variant Cas12a protein becomes a nuclease that promiscuously cleaves ssDNAs (i.e., the nuclease cleaves non-target ssDNAs, i.e., ssDNAs to which the guide sequence of the guide RNA does not hybridize). Thus, when the target DNA is present in the sample (e.g., in some cases above a threshold amount), the result is cleavage of ssDNAs in the sample, which can be detected using any convenient detection method (e.g., using a labeled single stranded detector DNA). Cleavage of non-target nucleic acid is referred to as “trans cleavage.” In some cases, a variant Cas12a protein of the present disclosure mediates trans cleavage of ssDNA, but not ssRNA.

[0259] Provided are compositions and methods for detecting a target DNA (double stranded or single stranded) in a sample. In some cases, a detector DNA is used that is single stranded (ssDNA) and does not hybridize with the guide sequence of the guide RNA (i.e. , the detector ssDNA is a non-target ssDNA). Such methods can include(a) contacting the sample with: (i) a variant Cas12a protein of the present disclosure; (ii) a guide RNA comprising: a region that binds to the variant Cas12a protein, and a guide sequence that hybridizes with the target DNA; and (iii) a detector DNA that is single stranded and does not hybridize with the guide sequence of the guide RNA; and (b) measuring a detectable signal produced by cleavage of the single stranded detector DNA by the variant Cas12a protein, thereby detecting the target DNA. As noted above, once a variant Cas12a protein of the present disclosure is activated by a guide RNA, which occurs when the sample includes a target DNA to which the guide RNA hybridizes (i.e., the sample includes the targeted target DNA), the variant Cas12a protein is activated and functions as an endoribonuclease that non- specifically cleaves ssDNAs (including non-target ssDNAs) present in the sample. Thus, when the targeted target DNA is present in the sample (e.g., in some cases above a threshold amount), the result is cleavage of ssDNA (including non-target ssDNA) in the sample, which can be detected using any convenient detection method (e.g., using a labeled detector ssDNA).

[0260] Also provided are compositions and methods for cleaving single stranded DNAs (ssDNAs) (e.g., non-target ssDNAs). Such methods can include contacting a population of nucleic acids, wherein said population comprises a target DNA and a plurality of non-target ssDNAs, with: (i) a variant Cas12a protein of the present disclosure; and (ii) a guide RNA comprising: a region that binds to the variant Cas12a protein and a guide sequence that hybridizes with the target DNA, wherein the variant Cas12a protein cleaves non-target ssDNAs of said plurality. Such a method can be used, e.g., to cleave foreign ssDNAs (e.g., viral DNAs) in a cell.

[0261] The contacting step of a subject method can be carried out in a composition comprising divalent metal ions. The contacting step can be carried out in an acellular environment, e.g., outside of a cell. The contacting step can be carried out inside a cell. The contacting step can be carried out in a cell in vitro. The contacting step can be carried out in a cell ex vivo. The contacting step can be carried out in a cell in vivo.

[0262] The guide RNA can be provided as RNA or as a nucleic acid encoding the guide RNA (e.g., a DNA such as a recombinant expression vector). The variant Cas12a protein can be provided as a protein or as a nucleic acid encoding the protein (e.g., anmRNA, a DNA such as a recombinant expression vector). In some cases, two or more (e.g., 3 or more, 4 or more, 5 or more, or 6 or more) guide RNAs can be provided by (e.g., using a precursor guide RNA array, which can be cleaved by the variant Cas12a protein into individual (“mature”) guide RNAs).

[0263] In some cases (e.g., when contacting with a guide RNA and a variant Cas12a protein of the present disclosure, the sample is contacted for 2 hours or less (e.g., 1 .5 hours or less, 1 hour or less, 40 minutes or less, 30 minutes or less, 20 minutes or less, 10 minutes or less, or 5 minutes or less, or 1 minute or less) prior to the measuring step. For example, in some cases the sample is contacted for 40 minutes or less prior to the measuring step. In some cases, the sample is contacted for 20 minutes or less prior to the measuring step. In some cases, the sample is contacted for 10 minutes or less prior to the measuring step. In some cases, the sample is contacted for 5 minutes or less prior to the measuring step. In some cases, the sample is contacted for 1 minute or less prior to the measuring step. In some cases, the sample is contacted for from 50 seconds to 60 seconds prior to the measuring step. In some cases, the sample is contacted for from 40 seconds to 50 seconds prior to the measuring step. In some cases, the sample is contacted for from 30 seconds to 40 seconds prior to the measuring step. In some cases, the sample is contacted for from 20 seconds to 30 seconds prior to the measuring step. In some cases, the sample is contacted for from 10 seconds to 20 seconds prior to the measuring step.

[0264] A method of the present disclosure for detecting a target DNA (single-stranded or double-stranded) in a sample can detect a target DNA with a high degree of sensitivity. In some cases, a method of the present disclosure can be used to detect a target DNA present in a sample comprising a plurality of DNAs (including the target DNA and a plurality of non-target DNAs), where the target DNA is present at one or more copies per 107non-target DNAs (e.g., one or more copies per 106non-target DNAs, one or more copies per 105non-target DNAs, one or more copies per 104non- target DNAs, one or more copies per 103non-target DNAs, one or more copies per 102non-target DNAs, one or more copies per 50 non-target DNAs, one or more copies per 20 non-target DNAs, one or more copies per 10 non-target DNAs, or one or more copies per 5 non-target DNAs). In some cases, a method of the present disclosure can be used to detect a target DNA present in a sample comprising a plurality of DNAs (including the target DNA and a plurality of non-target DNAs), where the target DNA is present at one or more copies per 1018non-target DNAs (e.g., one or more copies per 1015non-target DNAs, one or more copies per 1012non-targetDNAs, one or more copies per 109non-target DNAs, one or more copies per 106nontarget DNAs, one or more copies per 105non-target DNAs, one or more copies per 104non-target DNAs, one or more copies per 103non-target DNAs, one or more copies per 102non-target DNAs, one or more copies per 50 non-target DNAs, one or more copies per 20 non-target DNAs, one or more copies per 10 non-target DNAs, or one or more copies per 5 non-target DNAs).

[0265] In some cases, a method of the present disclosure can detect a target DNA present in a sample, where the target DNA is present at from one copy per 107non-target DNAs to one copy per 10 non-target DNAs (e.g., from 1 copy per 107non-target DNAs to 1 copy per 102non-target DNAs, from 1 copy per 107non-target DNAs to 1 copy per 103non-target DNAs, from 1 copy per 107non-target DNAs to 1 copy per 104non-target DNAs, from 1 copy per 107non-target DNAs to 1 copy per 105non-target DNAs, from 1 copy per 107non-target DNAs to 1 copy per 106non-target DNAs, from 1 copy per106non-target DNAs to 1 copy per 10 non-target DNAs, from 1 copy per 106non- target DNAs to 1 copy per 102non-target DNAs, from 1 copy per 106non-target DNAs to 1 copy per 103non-target DNAs, from 1 copy per 106non-target DNAs to 1 copy per 104non-target DNAs, from 1 copy per 106non-target DNAs to 1 copy per 105non- target DNAs, from 1 copy per 105non-target DNAs to 1 copy per 10 non-target DNAs, from 1 copy per 105non-target DNAs to 1 copy per 102non-target DNAs, from 1 copy per 105non-target DNAs to 1 copy per 103non-target DNAs, or from 1 copy per 105non-target DNAs to 1 copy per 104non-target DNAs).

[0266] In some cases, a method of the present disclosure can detect a target DNA present in a sample, where the target DNA is present at from one copy per 1018non-target DNAs to one copy per 10 non-target DNAs (e.g., from 1 copy per 1018non-target DNAs to 1 copy per 102non-target DNAs, from 1 copy per 1015non-target DNAs to 1 copy per 102non-target DNAs, from 1 copy per 1012non-target DNAs to 1 copy per 102non- target DNAs, from 1 copy per 109non-target DNAs to 1 copy per 102non-target DNAs, from 1 copy per 107non-target DNAs to 1 copy per 102non-target DNAs, from 1 copy per 107non-target DNAs to 1 copy per 103non-target DNAs, from 1 copy per107non-target DNAs to 1 copy per 104non-target DNAs, from 1 copy per 107non- target DNAs to 1 copy per 105non-target DNAs, from 1 copy per 107non-target DNAs to 1 copy per 106non-target DNAs, from 1 copy per 106non-target DNAs to 1 copy per 10 non-target DNAs, from 1 copy per 106non-target DNAs to 1 copy per 102non- target DNAs, from 1 copy per 106non-target DNAs to 1 copy per 103non-target DNAs, from 1 copy per 106non-target DNAs to 1 copy per 104non-target DNAs, from1 copy per 106non-target DNAs to 1 copy per 105non-target DNAs, from 1 copy per105non-target DNAs to 1 copy per 10 non-target DNAs, from 1 copy per 105non- target DNAs to 1 copy per 102non-target DNAs, from 1 copy per 105non-target DNAs to 1 copy per 103non-target DNAs, or from 1 copy per 105non-target DNAs to 1 copy per 104non-target DNAs).

[0267] In some cases, a method of the present disclosure can detect a target DNA present in a sample, where the target DNA is present at from one copy per 107non-target DNAs to one copy per 100 non-target DNAs (e.g., from 1 copy per 107non-target DNAs to 1 copy per 102non-target DNAs, from 1 copy per 107non-target DNAs to 1 copy per 103non-target DNAs, from 1 copy per 107non-target DNAs to 1 copy per 104non-target DNAs, from 1 copy per 107non-target DNAs to 1 copy per 105non-target DNAs, from 1 copy per 107non-target DNAs to 1 copy per 106non-target DNAs, from 1 copy per106non-target DNAs to 1 copy per 100 non-target DNAs, from 1 copy per 106non- target DNAs to 1 copy per 102non-target DNAs, from 1 copy per 106non-target DNAs to 1 copy per 103non-target DNAs, from 1 copy per 106non-target DNAs to 1 copy per 104non-target DNAs, from 1 copy per 106non-target DNAs to 1 copy per 105non- target DNAs, from 1 copy per 105non-target DNAs to 1 copy per 100 non-target DNAs, from 1 copy per 105non-target DNAs to 1 copy per 102non-target DNAs, from 1 copy per 105non-target DNAs to 1 copy per 103non-target DNAs, or from 1 copy per 105non-target DNAs to 1 copy per 104non-target DNAs).

[0268] In some cases, the threshold of detection, for a subject method of detecting a target DNA in a sample, is 10 nM or less. The term “threshold of detection” is used herein to describe the minimal amount of target DNA that must be present in a sample in order for detection to occur. Thus, as an illustrative example, when a threshold of detection is 10 nM, then a signal can be detected when a target DNA is present in the sample at a concentration of 10 nM or more. In some cases, a method of the present disclosure has a threshold of detection of 5 nM or less. In some cases, a method of the present disclosure has a threshold of detection of 1 nM or less. In some cases, a method of the present disclosure has a threshold of detection of 0.5 nM or less. In some cases, a method of the present disclosure has a threshold of detection of 0.1 nM or less. In some cases, a method of the present disclosure has a threshold of detection of 0.05 nM or less. In some cases, a method of the present disclosure has a threshold of detection of 0.01 nM or less. In some cases, a method of the present disclosure has a threshold of detection of 0.005 nM or less. In some cases, a method of the present disclosure has a threshold of detection of 0.001 nM or less. In some cases, a methodof the present disclosure has a threshold of detection of 0.0005 nM or less. In some cases, a method of the present disclosure has a threshold of detection of 0.0001 nM or less. In some cases, a method of the present disclosure has a threshold of detection of 0.00005 nM or less. In some cases, a method of the present disclosure has a threshold of detection of 0.00001 nM or less. In some cases, a method of the present disclosure has a threshold of detection of 10 pM or less. In some cases, a method of the present disclosure has a threshold of detection of 1 pM or less. In some cases, a method of the present disclosure has a threshold of detection of 500 fM or less. In some cases, a method of the present disclosure has a threshold of detection of 250 fM or less. In some cases, a method of the present disclosure has a threshold of detection of 100 fM or less. In some cases, a method of the present disclosure has a threshold of detection of 50 fM or less. In some cases, a method of the present disclosure has a threshold of detection of 500 aM (attomolar) or less. In some cases, a method of the present disclosure has a threshold of detection of 250 aM or less. In some cases, a method of the present disclosure has a threshold of detection of 100 aM or less. In some cases, a method of the present disclosure has a threshold of detection of 50 aM or less. In some cases, a method of the present disclosure has a threshold of detection of 10 aM or less. In some cases, a method of the present disclosure has a threshold of detection of 1 aM or less.

[0269] In some cases, the threshold of detection (for detecting the target DNA in a subject method), is in a range of from 500 fM to 1 nM (e.g., from 500 fM to 500 pM, from 500 fM to 200 pM, from 500 fM to 100 pM, from 500 fM to 10 pM, from 500 fM to 1 pM, from 800 fM to 1 nM, from 800 fM to 500 pM, from 800 fM to 200 pM, from 800 fM to 100 pM, from 800 fM to 10 pM, from 800 fM to 1 pM, from 1 pM to 1 nM, from 1 pM to 500 pM, from 1 pM to 200 pM, from 1 pM to 100 pM, or from 1 pM to 10 pM) (where the concentration refers to the threshold concentration of target DNA at which the target DNA can be detected). In some cases, a method of the present disclosure has a threshold of detection in a range of from 800 fM to 100 pM. In some cases, a method of the present disclosure has a threshold of detection in a range of from 1 pM to 10 pM. In some cases, a method of the present disclosure has a threshold of detection in a range of from 10 fM to 500 fM, e.g., from 10 fM to 50 fM, from 50 fM to 100 fM, from 100 fM to 250 fM, or from 250 fM to 500 fM.

[0270] In some cases, the minimum concentration at which a target DNA can be detected in a sample is in a range of from 500 fM to 1 nM (e.g., from 500 fM to 500 pM, from 500 fM to 200 pM, from 500 fM to 100 pM, from 500 fM to 10 pM, from 500 fM to 1 pM,from 800 fM to 1 nM, from 800 fM to 500 pM, from 800 fM to 200 pM, from 800 fM to 100 pM, from 800 fM to 10 pM, from 800 fM to 1 pM, from 1 pM to 1 nM, from 1 pM to 500 pM, from 1 pM to 200 pM, from 1 pM to 100 pM, or from 1 pM to 10 pM). In some cases, the minimum concentration at which a target DNA can be detected in a sample is in a range of from 800 fM to 100 pM. In some cases, the minimum concentration at which a target DNA can be detected in a sample is in a range of from 1 pM to 10 pM.

[0271] In some cases, the threshold of detection (for detecting the target DNA in a subject method), is in a range of from 1 aM to 1 nM (e.g., from 1 aM to 500 pM, from 1 aM to 200 pM, from 1 aM to 100 pM, from 1 aM to 10 pM, from 1 aM to 1 pM, from 100 aM to 1 nM, from 100 aM to 500 pM, from 100 aM to 200 pM, from 100 aM to 100 pM, from 100 aM to 10 pM, from 100 aM to 1 pM, from 250 aM to 1 nM, from 250 aM to 500 pM, from 250 aM to 200 pM, from 250 aM to 100 pM, from 250 aM to 10 pM, from 250 aM to 1 pM, from 500 aM to 1 nM, from 500 aM to 500 pM, from 500 aM to 200 pM, from 500 aM to 100 pM, from 500 aM to 10 pM, from 500 aM to 1 pM, from 750 aM to 1 nM, from 750 aM to 500 pM, from 750 aM to 200 pM, from 750 aM to 100 pM, from 750 aM to 10 pM, from 750 aM to 1 pM, from 1 fM to 1 nM, from 1 fM to 500 pM, from 1 fM to 200 pM, from 1 fM to 100 pM, from 1 fM to 10 pM, from 1 fM to 1 pM, from 500 fM to 500 pM, from 500 fM to 200 pM, from 500 fM to 100 pM, from 500 fM to 10 pM, from 500 fM to 1 pM, from 800 fM to 1 nM, from 800 fM to 500 pM, from 800 fM to 200 pM, from 800 fM to 100 pM, from 800 fM to 10 pM, from 800 fM to 1 pM, from 1 pM to 1 nM, from 1 pM to 500 pM, from 1 pM to 200 pM, from 1 pM to 100 pM, or from 1 pM to 10 pM) (where the concentration refers to the threshold concentration of target DNA at which the target DNA can be detected). In some cases, a method of the present disclosure has a threshold of detection in a range of from 1 aM to 800 aM. In some cases, a method of the present disclosure has a threshold of detection in a range of from 50 aM to 1 pM. In some cases, a method of the present disclosure has a threshold of detection in a range of from 50 aM to 500 fM.

[0272] In some cases, the minimum concentration at which a target DNA can be detected in a sample is in a range of from 1 aM to 1 nM (e.g., from 1 aM to 500 pM, from 1 aM to 200 pM, from 1 aM to 100 pM, from 1 aM to 10 pM, from 1 aM to 1 pM, from 100 aM to 1 nM, from 100 aM to 500 pM, from 100 aM to 200 pM, from 100 aM to 100 pM, from 100 aM to 10 pM, from 100 aM to 1 pM, from 250 aM to 1 nM, from 250 aM to 500 pM, from 250 aM to 200 pM, from 250 aM to 100 pM, from 250 aM to 10 pM, from 250 aM to 1 pM, from 500 aM to 1 nM, from 500 aM to 500 pM, from 500 aM to 200 pM, from 500 aM to 100 pM, from 500 aM to 10 pM, from 500 aM to 1 pM, from 750aM to 1 nM, from 750 aM to 500 pM, from 750 aM to 200 pM, from 750 aM to 100 pM, from 750 aM to 10 pM, from 750 aM to 1 pM, from 1 fM to 1 nM, from 1 fM to 500 pM, from 1 fM to 200 pM, from 1 fM to 100 pM, from 1 fM to 10 pM, from 1 fM to 1 pM, from 500 fM to 500 pM, from 500 fM to 200 pM, from 500 fM to 100 pM, from 500 fM to 10 pM, from 500 fM to 1 pM, from 800 fM to 1 nM, from 800 fM to 500 pM, from 800 fM to 200 pM, from 800 fM to 100 pM, from 800 fM to 10 pM, from 800 fM to 1 pM, from 1 pM to 1 nM, from 1 pM to 500 pM, from 1 pM to 200 pM, from 1 pM to 100 pM, or from 1 pM to 10 pM). In some cases, the minimum concentration at which a target DNA can be detected in a sample is in a range of from 1 aM to 500 pM. In some cases, the minimum concentration at which a target DNA can be detected in a sample is in a range of from 100 aM to 500 pM.

[0273] In some cases, a subject composition or method exhibits an attomolar (aM) sensitivity of detection. In some cases, a subject composition or method exhibits a femtomolar (fM) sensitivity of detection. In some cases, a subject composition or method exhibits a picomolar (pM) sensitivity of detection. In some cases, a subject composition or method exhibits a nanomolar (nM) sensitivity of detection.Target DNA

[0274] A target nucleic acid (e.g., target DNA) can be single stranded (e.g., ssDNA) or double stranded (e.g., dsDNA). When the target DNA is single stranded, there is no preference or requirement for a PAM sequence in the target DNA. However, when the target DNA is dsDNA, a PAM is usually present adjacent to the target sequence of the target DNA (e.g., see discussion of the PAM elsewhere herein). The source of the target DNA can be the same as the source of the sample, e.g., as described below.

[0275] The source of the target DNA can be any source. In some cases, the target DNA is a viral DNA (e.g., a genomic DNA of a DNA virus). As such, subject method can be for detecting the presence of a viral DNA amongst a population of nucleic acids (e.g., in a sample). A subject method can also be used for the cleavage of non-target ssDNAs in the present of a target DNA. For example, if a method takes place in a cell, a subject method can be used to promiscuously cleave non-target ssDNAs in the cell (ssDNAs that do not hybridize with the guide sequence of the guide RNA) when a particular target DNA is present in the cell (e.g., when the cell is infected with a virus and viral target DNA is detected).

[0276] Examples of possible target DNAs include, but are not limited to, viral DNAs such as: a papovavirus (e.g., human papillomavirus (HPV), polyomavirus); a hepadnavirus(e.g., Hepatitis B Virus (HBV)); a herpesvirus (e.g., herpes simplex virus (HSV), varicella zoster virus (VZV), epstein-barr virus (EBV), cytomegalovirus (CMV), herpes lymphotropic virus, Pityriasis Rosea, kaposi’s sarcoma-associated herpesvirus); an adenovirus (e.g., atadenovirus, aviadenovirus, ichtadenovirus, mastadenovirus, siadenovirus); a poxvirus (e.g., smallpox, vaccinia virus, cowpox virus, monkeypox virus, orf virus, pseudocowpox, bovine papular stomatitis virus; tanapox virus, yaba monkey tumor virus; molluscum contagiosum virus (MCV)); a parvovirus (e.g., adeno- associated virus (AAV), Parvovirus B19, human bocavirus, bufavirus, human parv4 G1); Geminiviridae; Nanoviridae; Phycodnaviridae; and the like. In some cases, the target DNA is parasite DNA. In some cases, the target DNA is bacterial DNA, e.g., DNA of a pathogenic bacterium.Samples

[0277] A subject sample includes nucleic acid (e.g., a plurality of nucleic acids). The term “plurality” is used herein to mean two or more. Thus, in some cases, a sample includes two or more (e.g., 3 or more, 5 or more, 10 or more, 20 or more, 50 or more, 100 or more, 500 or more, 1 ,000 or more, or 5,000 or more) nucleic acids (e.g., DNAs). A subject method can be used as a very sensitive way to detect a target DNA present in a sample (e.g., in a complex mixture of nucleic acids such as DNAs). In some cases, the sample includes 5 or more DNAs (e.g., 10 or more, 20 or more, 50 or more, 100 or more, 500 or more, 1 ,000 or more, or 5,000 or more DNAs) that differ from one another in sequence. In some cases, the sample includes 10 or more, 20 or more, 50 or more, 100 or more, 500 or more, 103or more, 5 x 103or more, 104or more, 5 x 104or more, 105or more, 5 x 105or more, 106or more 5 x 106or more, or 107or more, DNAs. In some cases, the sample comprises from 10 to 20, from 20 to 50, from 50 to 100, from 100 to 500, from 500 to 103, from 103to 5 x 103, from 5 x 103to 104, from 104to 5 x 104, from 5 x 104to 105, from 105to 5 x 105, from 5 x 105to 106, from 106to 5 x 106, or from 5 x 106to 107, or more than 107, DNAs. In some cases, the sample comprises from 5 to 107DNAs (e.g., that differ from one another in sequence)(e.g., from 5 to 106, from 5 to 105, from 5 to 50,000, from 5 to 30,000, from 10 to 106, from 10 to 105, from 10 to 50,000, from 10 to 30,000, from 20 to 10s, from 20 to 105, from 20 to 50,000, or from 20 to 30,000 DNAs). In some cases, the sample includes 20 or more DNAs that differ from one another in sequence. In some cases, the sample includes DNAs from a cell lysate (e.g., a eukaryotic cell lysate, a mammalian cell lysate, a human cell lysate, a prokaryotic cell lysate, a plant celllysate, and the like). For example, in some cases the sample includes DNA from a cell such as a eukaryotic cell, e.g., a mammalian cell such as a human cell.

[0278] The term “sample” is used herein to mean any sample that includes DNA (e.g., in order to determine whether a target DNA is present among a population of DNAs). The sample can be derived from any source, e.g., the sample can be a synthetic combination of purified DNAs; the sample can be a cell lysate, an DNA-enriched cell lysate, or DNAs isolated and / or purified from a cell lysate. The sample can be from a patient (e.g., for the purpose of diagnosis). The sample can be from permeabilized cells. The sample can be from crosslinked cells. The sample can be in tissue sections. The sample can be from tissues prepared by crosslinking followed by delipidation and adjustment to make a uniform refractive index. Examples of tissue preparation by crosslinking followed by delipidation and adjustment to make a uniform refractive index have been described in, for example, Shah et al., Development (2016) 143, 2862-2867 doi:10.1242 / dev.138560.

[0279] A “sample” can include a target DNA and a plurality of non-target DNAs. In some cases, the target DNA is present in the sample at one copy per 10 non-target DNAs, one copy per 20 non-target DNAs, one copy per 25 non-target DNAs, one copy per 50 non-target DNAs, one copy per 100 non-target DNAs, one copy per 500 non-target DNAs, one copy per 103non-target DNAs, one copy per 5 x 103non-target DNAs, one copy per 104non-target DNAs, one copy per 5 x 104non-target DNAs, one copy per 105non-target DNAs, one copy per 5 x 105non-target DNAs, one copy per 106non- target DNAs, or less than one copy per 106non-target DNAs. In some cases, the target DNA is present in the sample at from one copy per 10 non-target DNAs to 1 copy per 20 non-target DNAs, from 1 copy per 20 non-target DNAs to 1 copy per 50 non-target DNAs, from 1 copy per 50 non-target DNAs to 1 copy per 100 non-target DNAs, from 1 copy per 100 non-target DNAs to 1 copy per 500 non-target DNAs, from 1 copy per 500 non-target DNAs to 1 copy per 103non-target DNAs, from 1 copy per 103non-target DNAs to 1 copy per 5 x 103non-target DNAs, from 1 copy per 5 x 103non-target DNAs to 1 copy per 104non-target DNAs, from 1 copy per 104non-target DNAs to 1 copy per 105non-target DNAs, from 1 copy per 105non-target DNAs to 1 copy per 106non-target DNAs, or from 1 copy per 106non-target DNAs to 1 copy per 107non-target DNAs.

[0280] Suitable samples include but are not limited to saliva, blood, serum, plasma, urine, aspirate, and biopsy samples. Thus, the term “sample” with respect to a patient encompasses blood and other liquid samples of biological origin, solid tissue samplessuch as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof. The definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents; washed; or enrichment for certain cell populations, such as cancer cells. The definition also includes sample that have been enriched for particular types of molecules, e.g., DNAs. The term “sample” encompasses biological samples such as a clinical sample such as blood, plasma, serum, aspirate, cerebral spinal fluid (CSF), and also includes tissue obtained by surgical resection, tissue obtained by biopsy, cells in culture, cell supernatants, cell lysates, tissue samples, organs, bone marrow, and the like. A “biological sample” includes biological fluids derived therefrom (e.g., cancerous cell, infected cell, etc.), e.g., a sample comprising DNAs that is obtained from such cells (e.g., a cell lysate or other cell extract comprising DNAs).

[0281] A sample can comprise, or can be obtained from, any of a variety of cells, tissues, organs, or acellular fluids. Suitable sample sources include eukaryotic cells, bacterial cells, and archaeal cells. Suitable sample sources include single-celled organisms and multi-cellular organisms. Suitable sample sources include single-cell eukaryotic organisms; a plant or a plant cell; an algal cell, e.g., Botryococcus braunii, Chlamydomonas reinhardtii, Nannochloropsis gaditana, Chlorella pyrenoidosa, Sargassum patens, C. agardh, and the like; a fungal cell (e.g., a yeast cell); an animal cell, tissue, or organ; a cell, tissue, or organ from an invertebrate animal (e.g. fruit fly, cnidarian, echinoderm, nematode, an insect, an arachnid, etc.); a cell, tissue, fluid, or organ from a vertebrate animal (e.g., fish, amphibian, reptile, bird, mammal); a cell, tissue, fluid, or organ from a mammal (e.g., a human; a non-human primate; an ungulate; a feline; a bovine; an ovine; a caprine; etc.). Suitable sample sources include nematodes, protozoans, and the like. Suitable sample sources include parasites such as helminths, malarial parasites, etc.

[0282] Suitable sample sources include a cell, tissue, or organism of any of the six kingdoms, e.g., Bacteria (e.g., Eubacteria); Archaebacteria; Protista; Fungi; Plantae; and Animalia. Suitable sample sources include plant-like members of the kingdom Protista, including, but not limited to, algae (e.g., green algae, red algae, glaucophytes, cyanobacteria); fungus-like members of Protista, e.g., slime molds, water molds, etc.; animal-like members of Protista, e.g., flagellates (e.g., Euglena), amoeboids (e.g., amoeba), sporozoans (e.g., Apicomplexa, Myxozoa, Microsporidia), and ciliates (e.g., Paramecium). Suitable sample sources include members of the kingdom Fungi, including, but not limited to, members of any of the phyla:Basidiomycota (club fungi; e g., members of Agaricus, Amanita, Boletus, Cantherellus, etc.); Ascomycota (sac fungi, including, e.g., Saccharomyces); Mycophycophyta (lichens); Zygomycota (conjugation fungi); and Deuteromycota. Suitable sample sources include members of the kingdom Plantae, including, but not limited to, members of any of the following divisions: Bryophyta (e.g., mosses), Anthocerotophyta (e.g., hornworts), Hepaticophyta (e.g., liverworts), Lycophyta (e.g., club mosses), Sphenophyta (e.g., horsetails), Psilophyta (e.g., whisk ferns), Ophioglossophyta, Pterophyta (e.g., ferns), Cycadophyta, Gingkophyta, Pinophyta, Gnetophyta, and Magnoliophyta (e.g., flowering plants). Suitable sample sources include members of the kingdom Animalia, including, but not limited to, members of any of the following phyla: Porifera (sponges); Placozoa; Orthonectida (parasites of marine invertebrates); Rhombozoa; Cnidaria (corals, anemones, jellyfish, sea pens, sea pansies, sea wasps); Ctenophora (comb jellies); Platyhelminthes (flatworms); Nemertina (ribbon worms); Ngathostomulida (jawed worms)p Gastrotricha; Rotifera; Priapulida; Kinorhyncha; Loricifera; Acanthocephala; Entoprocta; Nemotoda; Nematomorpha; Cycliophora; Mollusca (mollusks); Sipuncula (peanut worms); Annelida (segmented worms); Tardigrada (water bears); Onychophora (velvet worms); Arthropoda (including the subphyla: Chelicerata, Myriapoda, Hexapoda, and Crustacea, where the Chelicerata include, e.g., arachnids, Merostomata, and Pycnogonida, where the Myriapoda include, e.g., Chilopoda (centipedes), Diplopoda (millipedes), Paropoda, and Symphyla, where the Hexapoda include insects, and where the Crustacea include shrimp, krill, barnacles, etc.; Phoronida; Ectoprocta (moss animals); Brachiopoda; Echinodermata (e.g. starfish, sea daisies, feather stars, sea urchins, sea cucumbers, brittle stars, brittle baskets, etc.); Chaetognatha (arrow worms); Hemichordata (acorn worms); and Chordata. Suitable members of Chordata include any member of the following subphyla: Urochordata (sea squirts; including Ascidiacea, Thaliacea, and Larvacea); Cephalochordata (lancelets); Myxini (hagfish); and Vertebrata, where members of Vertebrata include, e.g., members of Petromyzontida (lampreys), Chondrichthyces (cartilaginous fish), Actinopterygii (ray- finned fish), Actinista (coelocanths), Dipnoi (lungfish), Reptilia (reptiles, e.g., snakes, alligators, crocodiles, lizards, etc.), Aves (birds); and Mammalian (mammals). Suitable plants include any monocotyledon and any dicotyledon.

[0283] Suitable sources of a sample include cells, fluid, tissue, or organ taken from an organism; from a particular cell or group of cells isolated from an organism; etc. For example, where the organism is a plant, suitable sources include xylem, the phloem,the cambium layer, leaves, roots, etc. Where the organism is an animal, suitable sources include particular tissues (e.g., lung, liver, heart, kidney, brain, spleen, skin, fetal tissue, etc.), or a particular cell type (e.g., neuronal cells, epithelial cells, endothelial cells, astrocytes, macrophages, glial cells, islet cells, T lymphocytes, B lymphocytes, etc.).

[0284] In some cases, the source of the sample is a (or is suspected of being a diseased cell, fluid, tissue, or organ. In some cases, the source of the sample is a normal (nondiseased) cell, fluid, tissue, or organ. In some cases, the source of the sample is a (or is suspected of being) a pathogen-infected cell, tissue, or organ. For example, the source of a sample can be an individual who may or may not be infected - and the sample could be any biological sample (e.g., blood, saliva, biopsy, plasma, serum, bronchoalveolar lavage, sputum, a fecal sample, cerebrospinal fluid, a fine needle aspirate, a swab sample (e.g., a buccal swab, a cervical swab, a nasal swab), interstitial fluid, synovial fluid, nasal discharge, tears, buffy coat, a mucous membrane sample, an epithelial cell sample (e.g., epithelial cell scraping), etc.) collected from the individual. In some cases, the sample is a cell-free liquid sample. In some cases, the sample is a liquid sample that can comprise cells. Pathogens include viruses, fungi, helminths, protozoa, malarial parasites, Plasmodium parasites, Toxoplasma parasites, Schistosoma parasites, and the like. “Helminths” include roundworms, heartworms, and phytophagous nematodes (Nematoda), flukes (Tematoda), Acanthocephala, and tapeworms (Cestoda). Protozoan infections include infections from Giardia spp., Trichomonas spp., African trypanosomiasis, amoebic dysentery, babesiosis, balantidial dysentery, Chaga's disease, coccidiosis, malaria and toxoplasmosis. Examples of pathogens such as parasitic / protozoan pathogens include, but are not limited to: Plasmodium falciparum, Plasmodium vivax, Trypanosoma cruzi and Toxoplasma gondii. Fungal pathogens include, but are not limited to: Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, and Candida albicans. Pathogenic viruses include, e.g., human immunodeficiency virus (e.g., HIV); influenza virus; dengue; West Nile virus; herpes virus; yellow fever virus; Hepatitis C Virus; Hepatitis A Virus; Hepatitis B Virus; papillomavirus; and the like. Pathogenic viruses can include DNA viruses such as: a papovavirus (e.g., human papillomavirus (HPV), polyomavirus); a hepadnavirus (e.g., Hepatitis B Virus (HBV)); a herpesvirus (e.g., herpes simplex virus (HSV), varicella zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes lymphotropic virus, Pityriasis Rosea, Kaposi’ssarcoma-associated herpesvirus); an adenovirus (e.g., atadenovirus, aviadenovirus, ichtadenovirus, mastadenovirus, siadenovirus); a poxvirus (e.g., smallpox, vaccinia virus, cowpox virus, monkeypox virus, orf virus, pseudocowpox, bovine papular stomatitis virus; tanapox virus, yaba monkey tumor virus; molluscum contagiosum virus (MCV)); a parvovirus (e.g., adeno-associated virus (AAV), Parvovirus B19, human bocavirus, bufavirus, human parv4 G1); Geminiviridae; Nanoviridae; Phycodnaviridae; and the like. Pathogens can include, e.g., DNAviruses (e.g.: a papovavirus (e.g., human papillomavirus (HPV), polyomavirus); a hepadnavirus (e.g., Hepatitis B Virus (HBV)); a herpesvirus (e.g., herpes simplex virus (HSV), varicella zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes lymphotropic virus, Pityriasis Rosea, Kaposi’s sarcoma-associated herpesvirus); an adenovirus (e.g., atadenovirus, aviadenovirus, ichtadenovirus, mastadenovirus, siadenovirus); a poxvirus (e.g., smallpox, vaccinia virus, cowpox virus, monkeypox virus, orf virus, pseudocowpox, bovine papular stomatitis virus; tanapox virus, yaba monkey tumor virus; molluscum contagiosum virus (MCV)); a parvovirus (e.g., adeno- associated virus (AAV), Parvovirus B19, human bocavirus, bufavirus, human parv4 G1); Geminiviridae; Nanoviridae; Phycodnaviridae; and the like, Mycobacterium tuberculosis, Streptococcus agalactiae, methicillin-resistant Staphylococcus aureus, Legionella pneumophila, Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis, Pneumococcus, Cryptococcus neoformans, Histoplasma capsulatum, Hemophilus influenzae B, Treponema pallidum, Lyme disease spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae, Brucella abortus, rabies virus, influenza virus, cytomegalovirus, herpes simplex virus I, herpes simplex virus II, human serum parvo-like virus, respiratory syncytial virus, varicellazoster virus, hepatitis B virus, hepatitis C virus, measles virus, adenovirus, human T- cell leukemia viruses, Epstein-Barr virus, murine leukemia virus, mumps virus, vesicular stomatitis virus, Sindbis virus, lymphocytic choriomeningitis virus, wart virus, blue tongue virus, Sendai virus, feline leukemia virus, Reovirus, polio virus, simian virus 40, mouse mammary tumor virus, dengue virus, rubella virus, West Nile virus, Plasmodium falciparum, Plasmodium vivax, Toxoplasma gondii, Trypanosoma rangeli, Trypanosoma cruzi, Trypanosoma rhodesiense, Trypanosoma brucei, Schistosoma mansoni, Schistosoma japonicum, Babesia bovis, Eimeria tenella, Onchocerca volvulus, Leishmania tropica, Mycobacterium tuberculosis, Trichinella spiralis, Theileria parva, Taenia hydatigena, Taenia ovis, Taenia saginata,Echinococcus granulosus, Mesocestoides corti, Mycoplasma arthritidis, M. hyorhinis, M. orale, M. arginini, Acholeplasma laidlawii, M. salivarium and M. pneumoniae.Measuring a detectable signal

[0285] In some cases, a subject method includes a step of measuring (e.g., measuring a detectable signal produced by variant Cas12a protein-mediated ssDNA cleavage). Because a variant Cas12a protein of the present disclosure cleaves non-targeted ssDNA once activated, which occurs when a guide RNA hybridizes with a target DNA in the presence of a variant Cas12a protein, a detectable signal can be any signal that is produced when ssDNA is cleaved. For example, in some cases, the step of measuring can include one or more of: gold nanoparticle-based detection (e.g., see Xu et al., Angew Chem Int Ed Engl. 2007;46(19):3468-70; and Xia et al., Proc Natl Acad Sci U S A. 2010 Jun 15;107(24):10837-41), fluorescence polarization, colloid phase transition / dispersion (e.g., Baksh et al., Nature. 2004 Jan 8;427(6970): 139-41), electrochemical detection, semiconductor-based sensing (e.g., Rothberg et al., Nature. 2011 Jul 20;475(7356):348-52; e.g., one could use a phosphatase to generate a pH change after ssDNA cleavage reactions, by opening 2’-3’ cyclic phosphates, and by releasing inorganic phosphate into solution), and detection of a labeled detector ssDNA (see elsewhere herein for more details). The readout of such detection methods can be any convenient readout. Examples of possible readouts include but are not limited to: a measured amount of detectable fluorescent signal; a visual analysis of bands on a gel (e.g., bands that represent cleaved product versus uncleaved substrate), a visual or sensor based detection of the presence or absence of a color (i.e., color detection method), and the presence or absence of (or a particular amount of) an electrical signal.

[0286] The measuring can in some cases be quantitative, e.g., in the sense that the amount of signal detected can be used to determine the amount of target DNA present in the sample. The measuring can in some cases be qualitative, e.g., in the sense that the presence or absence of detectable signal can indicate the presence or absence of targeted DNA (e.g., virus, SNP, etc.). In some cases, a detectable signal will not be present (e.g., above a given threshold level) unless the targeted DNA(s) (e.g., virus, SNP, etc.) is present above a particular threshold concentration. In some cases, the threshold of detection can be titrated by modifying the amount of variant Cas12a protein, guide RNA, sample volume, and / or detector ssDNA (if one is used). As such, for example, as would be understood by one of ordinary skill in the art, a number ofcontrols can be used if desired in order to set up one or more reactions, each set up to detect a different threshold level of target DNA, and thus such a series of reactions could be used to determine the amount of target DNA present in a sample (e.g., one could use such a series of reactions to determine that a target DNA is present in the sample ‘at a concentration of at least X’).

[0287] Examples of uses of a detection method of the present disclosure include, e.g., single nucleotide polymorphism (SNP) detection, cancer screening, detection of bacterial infection, detection of antibiotic resistance, detection of viral infection, and the like. The compositions and methods of this disclosure can be used to detect any DNA target. For example, any virus that integrates nucleic acid material into the genome can be detected because a subject sample can include cellular genomic DNA - and the guide RNA can be designed to detect integrated nucleotide sequence.

[0288] In some cases, a method of the present disclosure can be used to determine the amount of a target DNA in a sample (e.g., a sample comprising the target DNA and a plurality of non-target DNAs). Determining the amount of a target DNA in a sample can comprise comparing the amount of detectable signal generated from a test sample to the amount of detectable signal generated from a reference sample. Determining the amount of a target DNA in a sample can comprise: measuring the detectable signal to generate a test measurement; measuring a detectable signal produced by a reference sample to generate a reference measurement; and comparing the test measurement to the reference measurement to determine an amount of target DNA present in the sample.

[0289] For example, in some cases, a method of the present disclosure for determining the amount of a target DNA in a sample comprises: a) contacting the sample (e.g., a sample comprising the target DNA and a plurality of non-target DNAs) with: (i) a guide RNA that hybridizes with the target DNA, (ii) a variant Cas12a protein of the present disclosure that cleaves RNAs present in the sample, and (iii) a detector ssDNA; b) measuring a detectable signal produced by variant Cas12a protein-mediated ssDNA cleavage (e.g., cleavage of the detector ssDNA), generating a test measurement; c) measuring a detectable signal produced by a reference sample to generate a reference measurement; and d) comparing the test measurement to the reference measurement to determine an amount of target DNA present in the sample.

[0290] As another example, in some cases, a method of the present disclosure for determining the amount of a target DNA in a sample comprises: a) contacting the sample (e.g., a sample comprising the target DNA and a plurality of non-target DNAs)with: i) a precursor guide RNA array comprising two or more guide RNAs each of which has a different guide sequence; (ii) a variant Cas12a protein of the present disclosure that cleaves the precursor guide RNA array into individual guide RNAs, and also cleaves RNAs of the sample; and (iii) a detector ssDNA; b) measuring a detectable signal produced by variant Cas12a protein- mediated ssDNA cleavage (e.g., cleavage of the detector ssDNA), generating a test measurement; c) measuring a detectable signal produced by each of two or more reference samples to generate two or more reference measurements; and d) comparing the test measurement to the reference measurements to determine an amount of target DNA present in the sample.Amplification of nucleic acids in the sample

[0291] In some embodiments, sensitivity of a subject composition and / or method (e.g., for detecting the presence of a target DNA, such as viral DNA or a SNR, in cellular genomic DNA) can be increased by coupling detection with nucleic acid amplification. In some cases, the nucleic acids in a sample are amplified prior to contact with a variant Cas12a protein of the present disclosure that cleaved ssDNA (e.g., amplification of nucleic acids in the sample can begin prior to contact with a variant Cas12a protein of the present disclosure). In some cases, the nucleic acids in a sample are amplified simultaneously with contact with a variant Cas12a protein of the present disclosure. For example, in some cases, a subject method includes amplifying nucleic acids of a sample (e.g., by contacting the sample with amplification components) prior to contacting the amplified sample with a variant Cas12a protein of the present disclosure. In some cases, a subject method includes contacting a sample with amplification components at the same time (simultaneous with) that the sample is contacted with a variant Cas12a protein of the present disclosure. If all components are added simultaneously (amplification components and detection components such as a variant Cas12a protein of the present disclosure, a guide RNA, and a detector DNA), it is possible that the trans-cleavage activity of the variant Cas12a protein will begin to degrade the nucleic acids of the sample at the same time the nucleic acids are undergoing amplification. However, even if this is the case, amplifying and detecting simultaneously can still increase sensitivity compared to performing the method without amplification.

[0292] In some cases, specific sequences (e.g., sequences of a virus, sequences that include a SNR of interest) are amplified from the sample, e.g., using primers. As such,a sequence to which the guide RNA will hybridize can be amplified in order to increase sensitivity of a subject detection method - this could achieve biased amplification of a desired sequence in order to increase the number of copies of the sequence of interest present in the sample relative to other sequences present in the sample. As one illustrative example, if a subject method is being used to determine whether a given sample includes a particular virus (or a particular SNP), a desired region of viral sequence (or non-viral genomic sequence) can be amplified, and the region amplified will include the sequence that would hybridize to the guide RNA if the viral sequence (or SNP) were in fact present in the sample.

[0293] As noted, in some cases the nucleic acids are amplified (e.g., by contact with amplification components) prior to contacting the amplified nucleic acids with a variant Cas12a protein of the present disclosure. In some cases, amplification occurs for 10 seconds or more, (e.g., 30 seconds or more, 45 seconds or more, 1 minute or more, 2 minutes or more, 3 minutes or more, 4 minutes or more, 5 minutes or more, 7.5 minutes or more, 10 minutes or more, etc.) prior to contact with a variant Cas12a protein of the present disclosure. In some cases, amplification occurs for 2 minutes or more (e.g., 3 minutes or more, 4 minutes or more, 5 minutes or more, 7.5 minutes or more, 10 minutes or more, etc.) prior to contact with a variant Cas12a protein of the present disclosure. In some cases, amplification occurs for a period of time in a range of from 10 seconds to 60 minutes (e.g., 10 seconds to 40 minutes, 10 seconds to 30 minutes, 10 seconds to 20 minutes, 10 seconds to 15 minutes, 10 seconds to 10 minutes, 10 seconds to 5 minutes, 30 seconds to 40 minutes, 30 seconds to 30 minutes, 30 seconds to 20 minutes, 30 seconds to 15 minutes, 30 seconds to 10 minutes, 30 seconds to 5 minutes, 1 minute to 40 minutes, 1 minute to 30 minutes, 1 minute to 20 minutes, 1 minute to 15 minutes, 1 minute to 10 minutes, 1 minute to 5 minutes, 2 minutes to 40 minutes, 2 minutes to 30 minutes, 2 minutes to 20 minutes, 2 minutes to 15 minutes, 2 minutes to 10 minutes, 2 minutes to 5 minutes, 5 minutes to 40 minutes, 5 minutes to 30 minutes, 5 minutes to 20 minutes, 5 minutes to 15 minutes, or 5 minutes to 10 minutes). In some cases, amplification occurs for a period of time in a range of from 5 minutes to 15 minutes. In some cases, amplification occurs for a period of time in a range of from 7 minutes to 12 minutes.

[0294] In some cases, a sample is contacted with amplification components at the same time as contact with a variant Cas12a protein of the present disclosure. In some such cases, the variant Cas12a protein is inactive at the time of contact and is activated once nucleic acids in the sample have been amplified.

[0295] Various amplification methods and components will be known to one of ordinary skill in the art and any convenient method can be used (see, e.g., Zanoli and Spoto, Biosensors (Basel). 2013 Mar; 3(1): 18-43; Gill and Ghaemi, Nucleosides, Nucleotides, and Nucleic Acids, 2008, 27: 224-243; Craw and Balachandrana, Lab Chip, 2012, 12, 2469-2486; which are herein incorporated by reference in their entirety). Nucleic acid amplification can comprise polymerase chain reaction (PCR), reverse transcription PCR (RT-PCR), quantitative PCR (qPCR), reverse transcription qPCR (RT-qPCR), nested PCR, multiplex PCR, asymmetric PCR, touchdown PCR, random primer PCR, hemi-nested PCR, polymerase cycling assembly (PCA), colony PCR, ligase chain reaction (LCR), digital PCR, methylation specific-PCR (MSP), coamplification at lower denaturation temperature-PCR (COLD-PCR), allele-specific PCR, intersequence-specific PCR (ISS-PCR), whole genome amplification (WGA), inverse PCR, and thermal asymmetric interlaced PCR (TAIL-PCR).

[0296] In some cases, the amplification is isothermal amplification. The term "isothermal amplification" indicates a method of nucleic acid (e.g., DNA) amplification (e.g., using enzymatic chain reaction) that can use a single temperature incubation thereby obviating the need for a thermal cycler. Isothermal amplification is a form of nucleic acid amplification which does not rely on the thermal denaturation of the target nucleic acid during the amplification reaction and hence may not require multiple rapid changes in temperature. Isothermal nucleic acid amplification methods can therefore be carried out inside or outside of a laboratory environment. By combining with a reverse transcription step, these amplification methods can be used to isothermally amplify RNA.

[0297] Examples of isothermal amplification methods include but are not limited to: loop- mediated isothermal Amplification (LAMP), helicase-dependent Amplification (HDA), recombinase polymerase amplification (RPA), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), transcription mediated amplification (TMA), nicking enzyme amplification reaction (NEAR), rolling circle amplification (RCA), multiple displacement amplification (MDA), Ramification (RAM), circular helicase-dependent amplification (cHDA), single primer isothermal amplification (SPIA), signal mediated amplification of RNA technology (SMART), selfsustained sequence replication (3SR), genome exponential amplification reaction (GEAR) and isothermal multiple displacement amplification (IMDA).

[0298] In some cases, the amplification is recombinase polymerase amplification (RPA) (see, e.g., U.S. Patent Nos. 8,030,000; 8,426,134; 8,945,845; 9,309,502; and 9,663,820,which are hereby incorporated by reference in their entirety). Recombinase polymerase amplification (RPA) uses two opposing primers (much like PCR) and employs three enzymes - a recombinase, a single-stranded DNA-binding protein (SSB) and a strand-displacing polymerase. The recombinase pairs oligonucleotide primers with homologous sequence in duplex DNA, SSB binds to displaced strands of DNA to prevent the primers from being displaced, and the strand displacing polymerase begins DNA synthesis where the primer has bound to the target DNA. Adding a reverse transcriptase enzyme to an RPA reaction can facilitate detection RNA as well as DNA, without the need for a separate step to produce cDNA. One example of components for an RPA reaction is as follows (see, e.g., U.S. patent Nos. 8,030,000; 8,426,134; 8,945,845; 9,309,502; 9,663,820): 50mM Tris pH 8.4, 80mM Potassium actetate, 10mM Magnesium acetate, 2 mM dithiothreitol (DTT), 5% PEG compound (Carbowax-20M), 3mM ATP, 30 mM Phosphocreatine, 100 ng / pl creatine kinase, 420 ng / pl gp32, 140 ng / pl UvsX, 35 ng / pl UvsY, 2000M dNTPs, 300 nM each oligonucleotide, 35 ng / pl Bsu polymerase, and a nucleic acid-containing sample).

[0299] In a transcription mediated amplification (TMA), an RNA polymerase is used to make RNA from a promoter engineered in the primer region, and then a reverse transcriptase synthesizes cDNA from the primer. A third enzyme, e.g., Rnase H can then be used to degrade the RNA target from cDNA without the heat-denatured step. This amplification technique is similar to Self-Sustained Sequence Replication (3SR) and Nucleic Acid Sequence Based Amplification (NASBA), but varies in the enzymes employed. For another example, helicase-dependent amplification (HDA) utilizes a thermostable helicase (Tte-UvrD) rather than heat to unwind dsDNA to create singlestrands that are then available for hybridization and extension of primers by polymerase. For yet another example, a loop mediated amplification (LAMP) employs a thermostable polymerase with strand displacement capabilities and a set of four or more specific designed primers. Each primer is designed t...

Claims

1. CLAIMSWhat is claimed is:

1. A variant Cas12a protein, comprising an amino acid sequence having amino acid substitutions relative to a corresponding wild type Cas12a protein in a region spanning WED-II, PAM-interacting, and WED-III domains, corresponding to positions Y516 through N808 of SEQ ID NO: 1 , wherein said amino acid substitutions cause reduced PAM constraints as compared to PAM constraints of the corresponding wild type Cas12a protein.

2. The variant Cas12a protein of claim 1 , wherein said corresponding wild type Cas12a protein is any one of the Cas12a proteins of SEQ ID NOs: 1-13.

3. The variant Cas12a protein of claim 1 , wherein said corresponding wild type Cas12a protein is the Lachnospiraceae bacterium Cas12a protein of SEQ ID NO: 1.

4. The variant Cas12a protein of any one of claims 1-3, wherein said amino acid substitutions comprise: (1) a substitution at an amino acid position corresponding to K538 of SEQ ID NO: 1 , and (2) substitutions at amino acid positions corresponding to:(a) N590; or(b) Q529, and E610; or(c) Q529, K753, and E754; or(d) Y553, D691 , K752, and P799; or(e) Y549, and E743; or(f) any combination thereof of SEQ ID NO: 1.

5. The variant Cas12a protein of any one of claims 1-3, wherein said amino acid substitutions comprise: (1) a substitution corresponding to K538N of SEQ ID NO: 1, and (2) substitutions corresponding to:(a) N590Y; or(b) Q529R, and E610G; or(c) Q529R, K753R, and E754A; or(d) Y553F, D691G, K752R, and P799R; or(e) Y549C, and E743K; or(f) any combination thereof of SEQ ID NO: 1.

6. The variant Cas12a protein of any one of claims 1-3, wherein said amino acid substitutions comprise: (1) a substitution at an amino acid position corresponding to D535 of SEQ ID NO: 1 , and (2) substitutions at amino acid positions corresponding to:(a) S551 and D665; or(b) D573, N628, and I765 of SEQ ID NO: 1.

7. The variant Cas12a protein of any one of claims 1-3, wherein said amino acid substitutions comprise: (1) a substitution corresponding to D535G of SEQ ID NO: 1 , and (2) substitutions corresponding to:(a) S551F and D665N; or(b) D573G, N628S, and I765T of SEQ ID NO: 1.

8. The variant Cas12a protein of any one of claims 1-3, wherein said amino acid substitutions are at amino acid positions corresponding to:(a) K538 and N590; or(b) K538, Q529, and E610; or(c) K538, Q529, K753, and E754; or(d) K538, Y553, D691 , K752, and P799; or(e) K538, Y549, and E743; or(f) D535, S551 , and D665; or(g) D535, D573, N628, and I765; or(h) any combination thereof of SEQ ID NO: 1.

9. The variant Cas12a protein of any one of claims 1-3, wherein said amino acid substitutions correspond to:(a) K538N and N590Y; or(b) K538N, Q529R, and E610G; or(c) K538N, Q529R, K753R, and E754A; or(d) K538N, Y553F, D691G, K752R, and P799R; or(e) K538N, Y549C, and E743K; or(f) D535G, S551 F, and D665N; or(g) D535G, D573G, N628S, and I765T; or(h) any combination thereof of SEQ ID NO: 1.

10. The variant Cas12a protein of any one of claims 1-9, wherein said amino acid substitutions correspond to D535G, S551F, and D665N of SEQ ID NO: 1.11 . The variant Cas12a protein of any one of claims 1-10, further comprising amino acid substitutions that correspond to G146R, R182V and E795Q of SEQ ID NO: 1.

12. The variant Cas12a protein of any one of claims 1-11 , wherein said corresponding wild type Cas12a protein has a PAM preference of 5'-TTTV-3', wherein V is A, C, or G.

13. The variant Cas12a protein of any one of claims 1-12, wherein the variant Cas12a protein has a PAM preference of 5'-YTYS-3', 5'-YKTN-3', 5'-YYNN-3', or 5'- TYNN-3', wherein: N is A,C,G, or T; Y is C or T; S is C or G; and K is G or T.

14. The variant Cas12a protein of any one of claims 1-12, wherein the variant Cas12a protein has a PAM preference of 5'- NYHV -3', wherein: N is A,C,G, or T; Y is C or T; H is A, C or T; and V is A, C, or G.

15. The variant Cas12a protein of any one of claims 1-14, wherein the variant Cas12a protein comprises an amino acid sequence having 80% or more sequence identity with any one of the wild type Cas12a proteins of SEQ ID NOs: 1-13.

16. The variant Cas12a protein of any one of claims 1-14, wherein the variant Cas12a protein comprises an amino acid sequence having 80% or more sequence identity with the wild type Cas12a protein of SEQ ID NO: 1.

17. The variant Cas12a protein of any one of claims 1-14, wherein the variant Cas12a protein comprises an amino acid sequence having 90% or more sequence identity with the wild type Cas12a protein of SEQ ID NO: 1.

18. The variant Cas12a protein of any one of claims 1-14, wherein the variant Cas12a protein comprises an amino acid sequence having 80% or more sequence identity with any one of the variant Cas12a proteins of SEQ ID NOs: 14-20 and 28.

19. The variant Cas12a protein of any one of claims 1-14, wherein the variant Cas12a protein comprises an amino acid sequence having 90% or more sequence identity with any one of the variant Cas12a proteins of SEQ ID NOs: 14-20 and 28.

20. The variant Cas12a protein of any one of claims 1-19, wherein the variant Cas12a protein is fused to one or more heterologous protein sequences.21 . The variant Cas12a protein of claim 20, wherein said one or more heterologous protein sequences includes one or more NLS sequences.

22. A nucleic acid comprising a nucleotide sequence encoding the variant Cas12a protein of any one of claims 1-21.

23. The nucleic acid of claim 20, wherein the nucleotide sequence encoding the variant Cas12a protein is codon optimized for expression in a eukaryotic cell.

24. The nucleic acid of any one of claim 22 or claim 23, wherein the nucleotide sequence encoding the variant Cas12a protein is operably linked to a promoter.

25. The nucleic acid of claim 24, wherein the promoter is an inducible promoter.

26. The nucleic acid of claim 24 or claim 25, wherein the promoter is functional in a eukaryotic cell.

27. The nucleic acid of any one of claims 22-26, wherein the nucleic acid is a recombinant expression vector.

28. The nucleic acid of claim 27, wherein the recombinant expression vector is a minicircle, plasmid, or viral vector.

29. A composition comprising: the variant Cas12a protein of any one of claims 1-21 , and (a) a lipid; (b) a buffer; (c) a nuclease inhibitor; (d) a protease inhibitor; (e) one or more Cas12a guide RNAs; (f) a donor template DNA; or (g) any combination thereof.

30. A system comprising: (a) the variant Cas12a protein of any one of claims 1-21 or the nucleic acid of any one of claims 19-25; and (b) a Cas12a guide RNA or a nucleic acid encoding the Cas12a guide RNA.31 . The system of claim 30, comprising two or more of said Cas12a guide RNAs or one or more nucleic acids encoding the two or more Cas12a guide RNAs.

32. The system of claim 30 or claim 31 , wherein the Cas12a guide RNA(s) comprises one or more base modifications, one or more a sugar modifications, one or more backbone modifications, or any combination thereof.

33. The system of any one of claims 30-32, further comprising a donor polynucleotide.

34. A ribonucleoprotein complex (RNP), comprising: the variant Cas12a protein of any one of claims 1-21 and a Cas12a guide RNA.

35. A modified host cell, comprising the variant Cas12a protein of any one of claims 1-18 and / or the nucleic acid of any one of claims 22-28.

36. The modified host cell of claim 35, further comprising a Cas12a guide RNA or a nucleic acid encoding the Cas12a guide RNA.

37. The modified host cell of claim 35 or claim 36, wherein the cell is a eukaryotic cell.

38. The modified host cell of claim 37, wherein the eukaryotic cell is a plant cell, a fungal cell, an algal cell, a mammalian cell, an insect cell, an arachnid cell, a bird cell, a reptile cell, an amphibian cell, an invertebrate cell, a rodent cell, a mouse cell, a rat cell, a primate cell, a non-human primate cell, or a human cell.

39. The modified host cell of claim 37 or claim 38, wherein the cell is in vitro or ex vivo.

40. The modified host cell of claim 35 or claim 36, wherein the host cell is a prokaryotic cell.41 . A method of modifying a target nucleic acid, the method comprising contacting the target nucleic acid with:(a) the variant Cas12a protein of any one of claims 1-21 ; and(b) a Cas12a guide RNA that comprises a guide sequence that hybridizes to a target sequence of the target nucleic acid, wherein said contacting results in modification of the target nucleic acid by the variant Cas12a protein.

42. The method of claim 41 , wherein said modification comprises cleavage of the target nucleic acid.

43. The method of claim 41 or claim 42, wherein the target nucleic acid is genomic DNA.

44. The method of any one of claims 41-43, wherein said contacting takes place inside of a cell in vitro.

45. The method of any one of claims 41-43, wherein said contacting takes place inside of a cell ex vivo.

46. The method of any one of claims 41-43, wherein said contacting takes place inside of a cell in vivo.

47. The method of any one of claims 41-43, wherein said contacting takes place inside of cell and comprises introducing into the cell: (a) the variant Cas12a protein or a nucleic acid encoding the variant Cas12a protein; (b) the Cas12a guide RNA or a nucleic acid encoding the Cas12a guide RNA; (c) an RNP comprising the variant Cas12a protein and the Cas12a guide RNA; or (d) any combination thereof.

48. The method of any one of claims 44-47, wherein the cell is a eukaryotic cell.

49. The method of claim 48, wherein the eukaryotic cell is a plant cell, a fungal cell, an algal cell, a mammalian cell, a reptile cell, an insect cell, an avian cell, a fish cell, a parasite cell, an arthropod cell, a cell of an invertebrate, a cell of a vertebrate, a rodent cell, a mouse cell, a rat cell, a primate cell, a non-human primate cell, and a human cell50. The method of any one of claims 44-49, wherein said contacting results in genome editing.

51. The method of any one of claims 44-50, wherein said contacting further comprises: introducing a DNA donor template into the cell.

52. The method of any one of claims 44-51, wherein said modification comprises homology-directed repair.

53. A method of detecting a target nucleic acid in a sample, the method comprising:(a) contacting the sample with:(i) a variant Cas12a protein of any one of claims 1-21 ;(ii) a Cas12a guide RNA comprising: a region that binds to the variant Cas12a protein, and a guide sequence that hybridizes with the target nucleic acid; and(iii) a detector nucleic acid that is single stranded and does not hybridize with the guide sequence of the guide RNA; and(b) measuring a detectable signal produced by cleavage of the single stranded detector nucleic acid by the variant Cas12a protein, thereby detecting the target nucleic acid.

54. The method of claim 53, wherein the target nucleic acid is bacterial DNA, a disease-associated DNA, a DNA comprising a single nucleotide polymorphism, or a viral DNA.

55. The method of claim 53, wherein the target nucleic acid is viral DNA.

56. The method of claim 55, wherein the target nucleic acid is papovavirus, human papillomavirus (HPV), hepadnavirus, Hepatitis B Virus (HBV), herpesvirus, varicella zoster virus (VZV), Epstein-Barr virus (EBV), Kaposi’s sarcoma-associated herpesvirus, adenovirus, poxvirus, or parvovirus DNA.

57. The method of claim 53, wherein the target nucleic acid is from a human cell.

58. The method of claim 53, wherein the target nucleic acid is human fetal or cancer cell DNA.

59. The method of any one of claims 53-58, wherein the sample comprises a cell lysate.

60. The method of any one of claims 53-58, wherein the sample comprises cells.

61. The method of any one of claims 53-60, wherein the sample is a blood, serum, plasma, urine, aspirate, or biopsy sample.

62. The method of any one of claims 53-61, further comprising determining an amount of the target nucleic acid present in the sample.

63. The method of any one of claims 53-62, wherein said measuring comprises one or more of: visual based detection, sensor-based detection, color detection, gold nanoparticle-based detection, fluorescence polarization, colloid phase transition / dispersion, electrochemical detection, and semiconductor-based sensing.

64. The method of any one of claims 53-63, wherein the labeled detector nucleic acid is DNA.

65. The method of any one of claims 53-63, wherein the labeled detector nucleic acid is RNA.

66. The method of any one of claims 53-65, wherein the labeled detector nucleic acid comprises a modified nucleobase, a modified sugar moiety, and / or a modified nucleic acid linkage.

67. The method of any one of claims 53-66, wherein the detectable signal is detectable in less than 45 minutes.

68. The method of any one of claims 53-66, wherein the detectable signal is detectable in less than 30 minutes.

69. The method of any one of claims 53-68, further comprising amplifying the target nucleic acid in the sample by loop-mediated isothermal amplification (LAMP), helicasedependent amplification (HDA), recombinase polymerase amplification (RPA), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), transcription mediated amplification (TMA), nicking enzyme amplification reaction (NEAR), rolling circle amplification (RCA), multiple displacement amplification (MDA), Ramification (RAM), circular helicase-dependent amplification (cHDA), single primer isothermal amplification (SPIA), signal mediated amplification of RNA technology (SMART), self-sustained sequence replication (3SR), genome exponential amplification reaction (GEAR), or isothermal multiple displacement amplification (IMDA).

70. The method of any one of claim 53-69, wherein the label of the labeled detector nucleic acid comprises a fluorescence-emitting dye pair.

71. The method of claim 70, wherein the fluorescence-emitting dye pair is a fluorescence resonance energy transfer (FRET) pair.

72. The method of claim 70, wherein the fluorescence-emitting dye pair is a quencher / fluor pair.

73. The method according to any one of claims 53-69, wherein the labeled detector nucleic acid comprises two or more fluorescence-emitting dye pairs.

74. The method of claim 73, wherein said two or more fluorescence-emitting dye pairs include a fluorescence resonance energy transfer (FRET) pair and a quencher / fluor pair.

75. The method of any one of claims 41-74, wherein the target nucleic acid is a target DNA.

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