CAS12A endonuclease variants and methods of use

Abstract

The present disclosure provides endonuclease variants with improved properties, such as higher activity and / or lower promiscuous single-stranded DNase activity, as compared to the corresponding wild-type endonuclease.

Description

[Technical field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 63 / 297,182, filed January 6, 2022, and U.S. Provisional Patent Application No. 63 / 297,189, filed January 6, 2022, which are incorporated by reference in their entireties herein.

[0002] Reference to Electronic Sequence Listing The contents of the electronic sequence listing (T085570001WO00-SEQ-HJD.xml, size: 634,084 bytes, and creation date: January 6, 2023) are incorporated herein by reference in their entirety. [Background technology]

[0003] Prokaryotes have developed an adaptive immune system called clustered regularly interspaced short palindromic repeats (CRISPR) that associates with Cas proteins to constitute an adaptive immune system capable of combating attacks by foreign mobile genetic elements such as plasmids and phages. CRISPR-Cas systems are classified into two classes (classes 1 and 2) and subdivided into six types (types I–VI). Class 1 (types I, III, and IV) systems use multiple Cas proteins for CRISPR ribonucleoprotein effector nucleases, while class 2 (types II, V, and VI) systems use a single Cas protein. Class 2 type V is further classified into four subtypes (VA, VB, VC, and VU). Currently, VC and VU are largely unexplored, and no structural information is available on these systems. VA encodes the protein Cas12a (also known as Cpf1), and several high-resolution structures of Cas12a have recently shed light on its mechanism of action. Summary of the Invention [Problem to be solved by the invention]

[0004] CRISPR-Cas12a, class 2 type V, is an RNA-guided endonuclease that has been exploited as a genome editing tool. For these enzymes to be widely used for gene and epigenetic editing, certain properties need to be improved. [Means for solving the problem]

[0005] The present disclosure provides, in some embodiments, mutant Cas12a endonucleases with improved properties, such as high activity and low promiscuous single-stranded DNA degradation activity. The widespread use of wild-type Cas12a has been limited, in part, due to its low editing efficiency and its promiscuous single-stranded DNA degradation activity compared to Cas9. The lid region, which is involved in the checkpoint for accurate target recognition, is responsible for this promiscuous ssDNA degradation activity exhibited by all wild-type Cas12a orthologs. Surprisingly, the data described herein demonstrate that certain modifications to the lid region of Cas12a can affect not only the promiscuous single-stranded deoxyribonuclease (ssDNase) activity, but also the target cleavage activity (both double-stranded and single-stranded cleavage activity).

[0006] In some embodiments, the engineered mutant endonucleases exhibit more efficient cleavage activity compared to their wild-type reference Cas12a endonucleases.In other embodiments, the engineered mutant endonucleases of the present disclosure exhibit low or no promiscuous single-stranded DNase activity.Also provided herein are mutant Cas12a endonucleases that, in some embodiments, exhibit a preference for cleavage of one strand of double-stranded DNA over the other strand.

[0007] From the structural study of LbCas12a ND2006 endonuclease, applicant has identified a specific domain involved in a subset of catalytic events, referred to herein as "LID-hub domain".For example, certain substitutions at positions K932, N933, and V936 increase cleavage efficiency ("high activity"), and certain substitutions at positions K932, N933, V936, Q944, F983, and M986 reduce promiscuous ssDNase activity.Certain amino acid substitutions near the LID and LID-hub domains also affect activity (e.g., V938 or Q941).

[0008] Furthermore, Applicants have also unexpectedly demonstrated that modification of the LID-stabilizing charge network (defined by its three-dimensional structure to include at least positions E835, R836, R935, and / or K940, with reference to the numbering of the amino acid positions of LbCas12a ND2006) shifts Cas12a cleavage preference (e.g., away from double-stranded DNA cleavage activity).

[0009] In some embodiments, the mutant Cas12a endonuclease comprises an amino acid mutation at one or more amino acid positions within the lid region. For example, in some embodiments, the mutant Cas12a endonuclease comprises one or more mutations at amino acid positions corresponding to positions 925-937 of Lachnospiraceae ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutant Cas12a endonuclease comprises one or more mutations at amino acid positions corresponding to positions 936-948 of Lachnospiraceae COE1 (e.g., SEQ ID NO: 47).

[0010] As used herein, the term "mutant Cas12a endonuclease" is interchangeable with the term "Cas12a mutant."

[0011] Some aspects relate to engineered mutant Cas12a endonucleases comprising a polypeptide sequence comprising a mutation at an amino acid position corresponding to position E95, E125, N256, R747, H759, N813, K932, N933, S934, V936, S982, or K984, with reference to the numbering of the amino acid positions of LbCas12a ND2006. In some embodiments, any one or more of the aforementioned mutant Cas12a endonucleases exhibit high activity.

[0012] Other aspects relate to engineered mutant Cas12a endonucleases comprising a polypeptide sequence comprising a mutation at an amino acid position corresponding to position E95R, E95Y, E125A, E125W, N256A, R747Y, H759V, H759D, N813R, N813H, K932L, N933E, N933V, S934Q, V936E, V936M, V936K, S982N, or K984R, with reference to the numbering of amino acid positions of LbCas12a ND2006. In some embodiments, any one or more of the aforementioned mutant Cas12a endonucleases exhibit high activity.

[0013] Some aspects relate to engineered mutant Cas12a endonucleases comprising a polypeptide sequence comprising a mutation at an amino acid position corresponding to position N256, I831, K932, N933, S934, V936, Q944, S982, F983, K984, M986, or T988, with reference to the numbering of amino acid positions of LbCas12a ND2006. In some embodiments, any one or more of the aforementioned mutant Cas12a endonucleases exhibit high activity.

[0014] Other embodiments relate to engineered mutant Cas12a endonucleases comprising a polypeptide sequence comprising a mutation at an amino acid position corresponding to position N256K, I831A, I831Y, K932A, K932F, K932H, K932M, K932N, K932Q, K932R, K932S, K932T, K932W, K932Y, N933L, S934W, V936G, Q944D, Q944E, Q944K, Q944M, S982T, S982W, F983G, F983L, K984F, M986G, M986L, M986S, or T988F, with reference to the numbering of the amino acid positions of LbCas12a ND2006. In some embodiments, any one or more of the aforementioned mutant Cas12a endonucleases exhibit high activity.

[0015] Still other embodiments include, with reference to the numbering of amino acid positions of LbCas12a ND2006, positions K932F and F983L, K932F and T988F, K932R and Q944D, K932R and F983L, K932R and T988F, K932Y and F983L, K932Y and T988F, N933L and Q944M, V936G and Q944D, V936G and S982W ...3L, V932G and S983L, V932G and S988F, The present invention relates to engineered mutant Cas12a endonucleases comprising a polypeptide sequence comprising mutations at amino acid positions corresponding to 36G and M986G, V936G and T988F, Q944D and S982W, Q944D and F983L, Q944D and T988F, S982W and F983L, S982W and T988F, or F983G and M986G. In some embodiments, any one or more of the aforementioned mutant Cas12a endonucleases exhibit high activity.

[0016] Some aspects relate to engineered mutant Cas12a endonucleases comprising a polypeptide sequence comprising a mutation at an amino acid position corresponding to position N813, I831, K932, N933, S934, V936, Q944, S982, F983, K984, M986, or T988, with reference to the numbering of amino acid positions of LbCas12a ND2006. In some embodiments, any one or more of the aforementioned mutant Cas12a endonucleases exhibit low ssDNase activity (or no ssDNase activity), e.g., exhibit low promiscuous ssDNase activity (or no promiscuous ssDNase activity).

[0017] Other embodiments relate to engineered mutant Cas12a endonucleases comprising a polypeptide sequence comprising a mutation at an amino acid position corresponding to position N813H, N813R, N813W, I831A, I831Y, K932A, K932F, K932H, K932M, K932N, K932Q, K932R, K932S, K932T, K932W, K932Y, N933E, N933L, S934K, S934Q, V936E, V936G, Q944D, Q944E, Q944K, S982W, F983G, F983L, K984F, M986F, M986G, or T988F, with reference to the numbering of the amino acid positions of LbCas12a ND2006. In some embodiments, any one or more of the aforementioned mutant Cas12a endonucleases exhibit low (or no) ssDNase activity, e.g., low (or no) promiscuous ssDNase activity.

[0018] Yet another aspect relates to an engineered mutant Cas12a endonuclease comprising a polypeptide sequence comprising mutations at amino acid positions corresponding to positions N933L and Q944M, or F983G and M986G, with reference to the numbering of amino acid positions of LbCas12a ND2006. In some embodiments, any one or more of the aforementioned mutant Cas12a endonucleases exhibit low (or no) ssDNase activity, e.g., low (or no) promiscuous ssDNase activity.

[0019] In some embodiments, the engineered mutant Cas12a endonuclease is fused to an effector protein.

[0020] In some embodiments, the engineered mutant Cas12a endonucleases provided herein comprise an amino acid sequence that has at least 85%, at least 90%, or at least 95% but less than 100% identity to the amino acid sequence of a wild-type Cas12a endonuclease selected from Acidaminococcus species, Lachnospira genus, and Francisella species.

[0021] In some embodiments, the engineered mutant Cas12a endonuclease further comprises no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions relative to a wild-type reference Cas12a endonuclease. In some embodiments, the mutant Cas12a endonuclease further comprises no more than 5 additional amino acid substitutions relative to a wild-type reference Cas12a endonuclease.

[0022] The present disclosure also provides an engineered mutant Cas12a endonuclease comprising a polypeptide sequence comprising the amino acid sequence of any one of SEQ ID NOs: 48-119 and 367-387, or an ortholog thereof.

[0023] Also provided herein are polynucleotides encoding the engineered mutant Cas12a endonucleases of the present disclosure.

[0024] Further provided herein is a cell comprising (a) an engineered mutant Cas12a endonuclease of the present disclosure or a polynucleotide endonuclease of the present disclosure, and (b) a guide RNA or a polynucleotide encoding the guide RNA.

[0025] Some aspects herein relate to methods comprising introducing into a cell (a) an engineered mutant Cas12a endonuclease of the disclosure or a polynucleotide of the disclosure, and optionally (b) a guide RNA or a polynucleotide encoding the guide RNA.

[0026] The present disclosure also provides uses of the engineered mutant Cas12a endonucleases of the present disclosure to cleave nucleic acids.

[0027] In some embodiments, a method for introducing a double-stranded break into a target nucleic acid comprises introducing into a cell containing the target nucleic acid (a) an engineered mutant Cas12a endonuclease of the present disclosure and (b) a guide RNA, and incubating the cell to generate a double-stranded break in the target nucleic acid.

[0028] In other embodiments, a method for introducing a double-stranded break into a target nucleic acid comprises introducing into a cell containing the target nucleic acid (a) an engineered mutant Cas12a endonuclease of the present disclosure and (b) a guide RNA, and incubating the cell to allow a double-stranded break to occur in the target nucleic acid.

[0029] In some embodiments, off-target single-stranded nucleic acid cleavage in the cells is reduced compared to off-target single-stranded nucleic acid cleavage in a control cell comprising a wild-type Cas12a endonuclease and a guide RNA.

[0030] In some embodiments, a method for introducing a single-stranded break into a target nucleic acid comprises introducing into a cell containing the target nucleic acid (a) an engineered mutant Cas12a endonuclease of the present disclosure and (b) a guide RNA, and incubating the cell to generate a single-stranded break in the target nucleic acid.

[0031] Some embodiments relate to an engineered polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 48-119 and 367-387, or a variant thereof having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of any one of SEQ ID NOs: 48-119 and 367-387, wherein the engineered polypeptide may be an endonuclease that exhibits high activity, low activity, and / or low ssDNase activity (e.g., low promiscuous ssDNase activity) relative to a naturally occurring Cas12a endonuclease (e.g., SEQ ID NO: 1).

[0032] Some aspects relate to a fusion protein comprising the engineered mutant Cas12a endonuclease of any one of the preceding aspects or embodiments, and a base editing enzyme.

[0033] Some embodiments relate to engineered polypeptides comprising the amino acid sequence of any one of SEQ ID NOs: 163-185 and 388-408, or a variant thereof having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of any one of SEQ ID NOs: 163-185 and 388-408.

[0034] Further aspects relate to fusion proteins comprising engineered mutant Cas12a endonucleases comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of any one of SEQ ID NOs: 367-387.

[0035] In some embodiments, the base editing enzyme can convert a purine to a different purine, or a pyrimidine to a different pyrimidine.

[0036] In some embodiments, the base editing enzyme comprises a deaminase, guanine oxidase, or guanine methyltransferase.

[0037] In some embodiments, the deaminase is a cytidine deaminase or an adenosine deaminase.

[0038] In some embodiments, the deaminase comprises a rAPOBEC1 polypeptide, an evoAPOBEC1 polypeptide, a hAPOBEC3A polypeptide, an evoCDA polypeptide, an evoFERNY polypeptide, or a TadA polypeptide.

[0039] In some embodiments, the fusion protein further comprises a uracil glycosylase inhibitor (UGI).

[0040] In some embodiments, the fusion protein further comprises one or more nuclear localization signals (NLS), which may be selected from an SV40 NLS, a nucleoprotein (NP) NLS, and a bipartite (BP) NLS.

[0041] In some embodiments, the fusion protein further comprises uracil DNA glycosylase (UNG), optionally human UNG (hUNG) or Escherichia coli UNG (eUNG).

[0042] In some embodiments, the fusion protein further comprises an N-methylpurine glycosylase (MPG), which may be located at or near the N-terminus or C-terminus of the fusion protein.

[0043] In some embodiments, the fusion protein further comprises one or more linkers.

[0044] In some embodiments, the linker comprises the sequence SGSETPGTSESATPES (SEQ ID NO: 203).

[0045] In some embodiments, the linker comprises the sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 204).

[0046] In some embodiments, the fusion protein further comprises a DNA binding domain (DBD).

[0047] In some embodiments, the DBD is a Rad51 DBD.

[0048] Some aspects relate to a polynucleotide encoding the fusion protein of any one of the preceding aspects or embodiments.

[0049] Another aspect relates to a cell comprising a target nucleic acid comprising a target strand and a non-target strand, a guide RNA (gRNA) that binds to the target strand or a nucleic acid encoding the gRNA, and a fusion protein of any one of the preceding aspects or embodiments or a polynucleotide of any one of the preceding aspects or embodiments.

[0050] In some embodiments, the cells are human cells.

[0051] In some embodiments, the human cell is derived from a human subject, and the human subject has a disease, disorder or condition associated with the target nucleic acid.

[0052] In some embodiments, the cell is a plant cell.

[0053] Some aspects relate to a method comprising introducing into a cell (a) a fusion protein of any one of the preceding aspects or embodiments or a polynucleotide of any one of the preceding aspects or embodiments, and, optionally, (b) a guide RNA or a polynucleotide encoding the guide RNA.

[0054] Some aspects relate to a method of gene editing comprising: (i) contacting a target nucleic acid sequence with a fusion protein of any one of the preceding aspects or embodiments and a guide RNA, wherein the target nucleic acid comprises a target nucleobase; and (ii) modifying the target nucleobase.

[0055] In some embodiments, the target nucleic acid is a target double-stranded DNA nucleic acid.

[0056] In some embodiments, the guide RNA directs the fusion protein to bind to a specific segment of the target nucleic acid and to approach the target nucleobase.

[0057] In some embodiments, the fusion protein cleaves the target nucleic acid.

[0058] In some embodiments, the fusion protein comprises a cytidine deaminase and the target nucleobase is cytidine. In some embodiments, the fusion protein comprises a guanine oxidase and the target nucleobase is guanosine. In some embodiments, the fusion protein comprises a guanosine methyltransferase and the target nucleobase is guanosine.

[0059] In some embodiments, the method is performed in a cell, suitably a human cell or a plant cell.

[0060] In some embodiments, the method is performed in vitro or ex vivo, hi other embodiments, the method is performed in vivo.

[0061] In some embodiments, the target nucleic acid is a gene that contains a nucleobase mutation compared to a wild-type gene.

[0062] In some embodiments, the gene comprising the nucleobase mutation is associated with a disease or disorder. [Brief description of the drawings]

[0063] [Figure 1] Figures 1A-1X show alignments of various wild-type Cas12a endonuclease amino acid sequences using the Clustal Omega online multiple sequence alignment program (from top to bottom, SEQ ID NOs: 29, 39, 12, 36, 8, 2, 43, 18, 37, 6, 46, 14, 28, 38, 45, 20, 13, 5, 21, 7, 42, 24, 40, 16, 35, 19, 41, 3, 10, 30, 1, 44, 22, 11, 27, 31, 4, 25, 26, 34, 32-33, 9, 17, and 23). [Diagram 2] 2A-2D show experimental data for various examples of hyperactive Cas12a endonuclease mutants of the present disclosure. [Diagram 3] 3A-3E show experimental data for various examples of low activity Cas12a endonuclease mutants of the present disclosure. [Figure 4] 4A-4C show experimental data for various examples of low activity Cas12a endonuclease mutants of the present disclosure. [Diagram 5] FIG. 5 shows experimental data for various examples of Cas12a endonuclease mutants of the present disclosure that have low promiscuous ssDNase activity. [Figure 6] Figures 6A-6C provide graphs of data comparing the percent (%) of total reads with C to T nucleotide edits at genomic positions corresponding to guide RNA (gRNA) positions C8, C9, C10, C11, and C13 using the LbBEv2 base editor in U2OS cells. [Figure 7]Figures 7A-7D provide graphs of data comparing the percent (%) of total reads with C to T nucleotide edits at genomic positions corresponding to gRNA positions C8 and C10 using the LbBEv2, LbBEv3, LbBEv4, or LbBEv5 base editors in U2OS cells. [Figure 8] Figures 8A-8D provide graphs of data comparing the percent (%) of total reads with C to T nucleotide edits at genomic positions corresponding to gRNA positions C9, C10, and C15 using LbBEv2, LbBEv3, LbBEv4, or LbBEv5 base editors in U2OS cells. [Figure 9] Figures 9A-9F provide data showing increased efficiency and specificity of base editing by the LbBEv5 C to T base editor containing TBN04 (LbCas12a) compared to the LbBEv5 base editor containing an inactive LbCas12a in U2OS cells (SEQ ID NOs: 214-263 from top to bottom). [Figure 10] Figures 10A-10F provide data showing increased efficiency and specificity of base editing by the LbBEv5 C to T base editor. [Figure 11] Figures 11A-11F provide data showing the efficiency and specificity of base editing by the LbABE8e A to G base editor (from top to bottom, SEQ ID NOs: 316-365). [Figure 12] Figures 12A-12C provide data showing increased efficiency and specificity of base editing by A to G base editors, including mutant Cas12a. [Figure 13] Figures 13A-13B provide data showing the ability of base editors including N-methylpurine glycosylase (MPG) to perform A to C base editing (SEQ ID NOs: 409 and 366). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0064] I. Cas12a endonuclease Provided herein are mutants of class II type V CRISPR-Cas12a endonucleases. An "endonuclease" is an enzyme capable of cleaving phosphodiester bonds within a polynucleotide strand. Some endonucleases are specific (i.e., they recognize a given nucleotide sequence that indicates the cleavage site), while some are non-specific. The present disclosure provides specific mutant Cas12a endonucleases. Endonucleases can cleave both strands of a double-stranded polynucleotide, or endonucleases may demonstrate a preference for cleaving one strand over the other strand of a double-stranded polynucleotide.

[0065] The recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)-Cpf1 system, now reclassified as Cas12a, is a DNA editing platform similar to the widely used CRISPR-Cas9 system. The Cas12a system exhibits several distinct features, such as higher specificity than the CRISPR-Cas9 system and smaller gene size encoding the nuclease and the matching CRISPR guide RNA (crRNA), respectively, which can mitigate the off-target and delivery issues described for the Cas9 system. However, the Cas12a system exhibits reduced gene editing efficiency compared to Cas9. Many of the mutant Cas12a endonucleases provided herein exhibit increased gene editing efficiency compared to wild-type Cas12a systems characterized to date.

[0066] RNA sequencing of small RNA molecules extracted from Francisella novicida U112 cultures containing a Cas12a-based CRISPR locus showed that the mature crRNA of Cas12a is 42–44 nucleotides (nt) long, with the first 19 / 20 nt corresponding to the repeat sequence and the remaining 23–25 nt corresponding to the spacer sequence. Cas12a processes its own pre-crRNA into mature crRNA without the need for tracrRNA, making it an intrinsic effector protein with both endoribonuclease and endonuclease activities. After the pre-crRNA is transcribed during the expression phase, Cas12a cleaves it 4 nt upstream of the hairpin structure formed by the CRISPR repeats, generating an intermediate crRNA molecule that is further processed in vivo into the mature crRNA.

[0067] Type V (Cas12a) CRISPR-Cas systems possess a characteristic Ruv-C-like nuclease domain that has been shown to be related to the TnpB protein encoded by IS605 family transposons. Crystallographic and cryo-EM data reveal that Cas12a adopts a bilobed structure formed by the REC and Nuc lobes. The REC lobe is composed of the REC1 and REC2 domains, while the Nuc lobe is composed of the RuvC domain, the PAM-interacting (PI) domain, and the WED domain, as well as a bridge helix (BH). The RuvC endonuclease domain of this effector protein is composed of three discontinuous parts (RuvC I-III). The RNase site for processing its own crRNA is located in the WED-III subdomain, and the DNase site is located at the interface between the RuvC and Nuc domains. These structural studies also show that only the 5' repeat region of the crRNA is involved in the assembly of the binary complex. The 19 / 20nt repeat region forms a pseudoknot structure by intramolecular base pairing. The crRNA is stabilized through interactions with the WED, RuvC, REC2 domains of the endonuclease, and two hydrated Mg2+ ions. This binary interference complex is then involved in the recognition and degradation of foreign DNA. See Paul, B. & Montoya, G. et al. Biomedical Journal 2020; 43(1): 8-17.

[0068] Recognition of protospacer adjacent motifs (PAMs) is a critical initial step to identify DNA molecules that are candidates for degradation because PAMs allow CRISPR-Cas systems to distinguish between their own genomic DNA and invading nucleic acids. Cas12a employs a multi-step quality control procedure to ensure accurate and precise recognition of the target spacer sequence. The WED II-III domain, the REC1 domain, and the AM-interacting domain are responsible for initiating PAM recognition and hybridization of the DNA target with crRNA. After the WED and REC1 domains recognize the dsDNA, the conserved loop-lysine helix-loop (LKL) region of the PI domain, which contains three conserved lysines (K667, K671, and K677 in FnCas12a), inserts a helix into the PAM duplex with the help of two conserved prolines in the LKL region. Structural studies have shown that the helix is ​​inserted at a 45° angle to the longitudinal axis of the dsDNA, facilitating the unwinding of the helical dsDNA. The critical location of three conserved lysines on the dsDNA initiates the uncoupling of the Watson-Crick interactions between the base pairs of the dsDNA after the PAM. Dissociation of the target dsDNA allows hybridization of the crRNA with the strand containing the PAM, i.e., the target strand, while the uncoupled DNA strand, i.e., the non-target strand (NTS), is guided to the DNase site by the PAM-interacting domain. Cas12a has been shown to efficiently target spacer sequences following the 5'T-rich PAM sequence. The PAMs of LbCas12a and AsCas12a have the sequence 5'-TTTN-3', while the PAM of FnCas12a has the sequence 5'-TTN-3' and is located upstream of the 5' end of the non-target strand [26, 31, 34]. In addition to the canonical 5'-TTTN-3'PAM, Cas12a has also been shown to exhibit relaxed PAM recognition for non-optimal C-containing PAM sequences by forming altered interactions with the target DNA duplex. See Paul, B. & Montoya, G. et al.

[0069] Exemplary, non-limiting wild-type Cas12a protein sequences are provided in Table 1.

[0070] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12] [Table 1-13]

[0071] Guide RNA (crRNA) Guide RNA (gRNA) is an RNA that functions to guide an RNA or DNA targeting enzyme to a specific target. Targeting requires a gRNA that is complementary to the target site and a 5' protospacer adjacent motif (PAM) on the DNA strand opposite the target sequence. gRNAs for Cas12a endonucleases are relatively short, in some embodiments, about 35-50 nucleotides long (e.g., 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides long). In some embodiments, the gRNA is about 40-44 nucleotides long. The portion of the gRNA that base pairs with the protospacer may be about 15-30 nucleotides long (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides long). In some embodiments, the portion of the gRNA that base pairs with the protospacer is about 20-24 nucleotides in length, for example about 21 nucleotides in length. There is also a constant portion that binds to Cas12a, which is about 15-25 nucleotides in length. In some embodiments, the constant portion that binds to Cas12a is about 20 nucleotides in length.

[0072] In the case of Cas12a endonuclease, the target sequence to which the gRNA binds should be adjacent to a PAM sequence, e.g., TTTV, where V can represent A, C, or G. The "V" of TTTV is typically immediately adjacent to the 5'-most non-target strand of the protospacer element. The PAM sequence may vary depending on the mutant Cas12a endonuclease.

[0073] II. Mutant Cas12a endonucleases Provided herein, in some embodiments, are engineered mutant Cas12a endonucleases that have altered activity compared to wild-type Cas12 endonuclease. As used herein, "mutant Cas12a endonuclease" refers to a non-naturally occurring endonuclease obtained by mutation of a wild-type (e.g., naturally occurring) Cas12a gene, such as a Cas12a gene from Table 1 (any one of SEQ ID NOs: 1-47). Mutants of other wild-type Cas12 genes are contemplated herein. Thus, the mutant Cas12a endonucleases provided herein are "engineered."

[0074] Mutations contemplated herein include, but are not limited to, substitutions, additions and deletions with respect to amino acid sequence. An amino acid "substitution" is a single amino acid change relative to a reference amino acid sequence. For example, in the context of the LbCas12a ND2006 amino acid sequence of SEQ ID NO: 1 in Figures 1A-1X, a substitution at position E95 would include any amino acid other than E at position 95 (counting from the methionine (M) start codon), such as E95R (E is replaced by R) and E95Y (E is replaced by Y).

[0075] Mutant Cas12a endonucleases provided herein, in some embodiments, exhibit high or low promiscuous single-stranded deoxyribonuclease (DNase) activity, as described in more detail elsewhere herein.

[0076] The activity of mutant Cas12a endonucleases (e.g., high activity and / or promiscuous single-stranded DNase activity) can be evaluated using any method known in the art. In some embodiments, the activity of mutant Cas12a endonucleases is determined using a gel-based assay. In some embodiments, the activity of mutant Cas12a endonucleases is determined using a fluorophore and / or a fluorophore-quencher system. In some embodiments, the activity of mutant Cas12a endonucleases can be evaluated using short labeled oligonucleotides that measure the activity of Cas9 and CasΦ, respectively (see, for example, Jinek et al., Science, 2012, 337(6096): 816-821 and Pausche et al., Science, 2020, 396(6501): 333-337). In some embodiments, short oligonucleotides labeled with fluorophores are used to assess cleavage on both strands (see, e.g., Stella et al., Cell, 2018, 175: 1856-1871). In some embodiments, nickase activity is determined using optical tweezers (see, e.g., Paul et al., bioRxiv, 2021, doi.org / 10.1101 / 2021.06.09.447528). In some embodiments, longer oligonucleotides labeled with fluorophores are used to assess Cas12a cleavage on both strands (see, e.g., Yamano et al., Cell, 2016, 165(4): 494-962; Cofsky et al., eLife, 2020, 9:e55143). In some embodiments, single-stranded DNA labeled with a quencher-fluorophore is used to assess ssDNase activity (see, e.g., Chen et al., Science, 2018, 360(6387):436-439).In some embodiments, mutant Cas12a endonuclease activity is assessed using single molecule fluorescence resonance energy transfer (FRET) (see, e.g., Son et al., PNAS, 2021, 118(49): e2113747118). Other methods are also contemplated herein.

[0077] In embodiments where an amino acid substitution is exemplified (e.g., E95R), the present disclosure contemplates alternative substitutions with "equivalent" charge, polarity, and / or chemical class (as defined by the amino acid side chain). Table 2 provides the 20 naturally occurring amino acids with descriptions of the corresponding charge, polarity, and chemical class. For example, arginine has the same charge as histidine and lysine, the same polarity as asparagine, glutamine, serine, threonine, tyrosine, aspartic acid, glutamic acid, arginine, histidine, and lysine, and the same chemical class / side chain as histidine and lysine. Thus, using the E95R substitution as an example, E95H and E95K are examples of amino acid substitutions with equivalent charge; E95N, E95Q, E95S, E95T, E95Y, E95D, E95E, E95H and E95K are examples of amino acid substitutions with equivalent polarity; E95H and E95K are examples of amino acid substitutions with equivalent chemical class. In some embodiments, a given amino acid substitution is equivalent in charge, polarity, and chemical class. Again, using the E95R substitution as an example, E95H and E95K are examples of amino acid substitutions with equivalent charge (i.e., positive), equivalent polarity (i.e., polar), and equivalent chemical class (i.e., basic).

[0078] [Table 2]

[0079] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising a mutation at an amino acid position corresponding to position E95, E125, N256, R747, H759, N813, K932, N933, S934, V936, S982, or K984, with reference to the numbering of the amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO:1), and the mutant Cas12a endonuclease may exhibit increased activity. In other embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising a mutation at an amino acid position corresponding to N256, I831, K932, N933, S934, V936, Q944, S982, F983, K984, M986, or T988, with reference to the numbering of the amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO:1), and the mutant Cas12a endonuclease may exhibit reduced activity. In yet other embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising a mutation at an amino acid position corresponding to position N813, I831, K932, N933, S934, V936, Q944, S982, F983, K984, M986, or T988, with reference to the numbering of the amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO:1), and the mutant Cas12a endonuclease may exhibit reduced promiscuous ssDNase activity (or may not exhibit promiscuous ssDNase activity). With reference to the numbering of the amino acid positions of LbCas12a ND2006, it should be understood that mutant Cas12a endonucleases containing mutations at amino acid positions corresponding to particular positions encompass mutants of LbCas12a ND2006 (e.g., SEQ ID NO: 1), as well as mutants of Cas12a orthologues of LbCas12a ND2006, including, but not limited to, mutants of any one of the Cas12a endonucleases of Table 1 (e.g., SEQ ID NOs: 2-47). Identification of such "corresponding" amino acid positions can be readily performed by aligning any Cas12a endonuclease amino acid sequence to those examples provided herein, in particular to the LbCas12a ND2006 sequence, such as the amino acid sequence of SEQ ID NO: 1 shown in Figures 1A-1X.

[0080] FIG. 1A, for example, shows an alignment of various Cas12a homologs, highlighting that mutant Cas12a endonucleases comprising a mutation at an amino acid position corresponding to E95 with reference to the numbering of the amino acid positions of LbCas12a ND2006, mutant Cas12a endonucleases comprising a mutation at position I96 with reference to the numbering of the amino acid positions of AsCas12a BV3L6, and mutant Cas12a endonucleases comprising a mutation at position K99 with reference to the numbering of the amino acid positions of FnCas12a.

[0081] A mutant Cas12a endonuclease of the present disclosure may share a certain percent identity with a wild-type Cas12a endonuclease. For example, a mutant Cas12a endonuclease may include any one or more mutations (e.g., amino acid substitutions) described herein and may include an amino acid sequence that has at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%) identity to the amino acid sequence of any one of the Cas12a endonucleases in Table 1 (e.g., SEQ ID NOs: 1-47), their orthologs, or other wild-type Cas12a protein sequences.

[0082] In some embodiments, the mutant Cas12a endonuclease comprises a mutation at an amino acid position corresponding to position E95, E125, N256, R747, H759, N813, K932, N933, S934, V936, S982, or K984, with reference to the numbering of the amino acid positions of LbCas12a, and comprises a polypeptide sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%) identity to the amino acid sequence of any one of the Cas12a endonucleases in Table 1 (e.g., SEQ ID NOs: 1-47), their orthologs, or other wild-type Cas12a protein sequences. In some embodiments, any one or more of the aforementioned mutant Cas12a endonucleases exhibit high activity.

[0083] In some embodiments, the mutant Cas12a endonuclease comprises a mutation at an amino acid position corresponding to position E95R, E95Y, E125A, E125W, N256A, R747Y, H759V, H759D, N813R, N813H, K932L, N933E, N933V, S934Q, V936E, V936M, V936K, S982N, or K984R, with reference to the numbering of the amino acid positions of LbCas12a, and comprises a polypeptide sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%) identity to the amino acid sequence of any one of the Cas12a endonucleases of Table 1 (e.g., SEQ ID NOs: 1-47), their orthologs, or other wild-type Cas12a protein sequences. In some embodiments, any one or more of the aforementioned mutant Cas12a endonucleases exhibit high activity.

[0084] In some embodiments, the mutant Cas12a endonuclease comprises a mutation at an amino acid position corresponding to position N256, I831, K932, N933, S934, V936, Q944, S982, F983, K984, M986, or T988, with reference to the numbering of the amino acid positions of LbCas12a, and comprises a polypeptide sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%) identity to the amino acid sequence of any one of the Cas12a endonucleases in Table 1 (e.g., SEQ ID NOs: 1-47), their orthologs, or other wild-type Cas12a protein sequences. In some embodiments, any one or more of the aforementioned mutant Cas12a endonucleases exhibit reduced activity.

[0085] In some embodiments, the mutant Cas12a endonuclease is at positions N256K, I831A, I831Y, K932A, K932F, K932H, K932M, K932N, K932Q, K932R, K932S, K932T, K932W, K932Y, N933L, S934W, V936G, Q944D, Q944E, Q944K, Q944M, S982T, S982W, F983G, F983L, K983Q, K983R, K983Q ... The mutant Cas12a endonucleases include a polypeptide sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%) identity to the amino acid sequence of any one of the Cas12a endonucleases in Table 1 (e.g., SEQ ID NOs: 1-47), their orthologs, or other wild-type Cas12a protein sequences. In some embodiments, any one or more of the aforementioned mutant Cas12a endonucleases exhibit reduced activity.

[0086] In some embodiments, the mutant Cas12a endonuclease is located at positions K932F and F983L, K932F and T988F, K932R and Q944D, K932R and F983L, K932R and T988F, K932Y and F983L, K932Y and T988F, N933L and Q944M, V936G and Q944D, V936G and S982W, V936G and M986G, V936G and T988F, Q944D and S982W, Q944D and and F983L, Q944D and T988F, S982W and F983L, S982W and T988F, or F983G and M986G, and a polypeptide sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%) identity to the amino acid sequence of any one of the Cas12a endonucleases in Table 1 (e.g., SEQ ID NOs: 1-47), their orthologs, or other wild-type Cas12a protein sequences. In some embodiments, any one or more of the foregoing mutant Cas12a endonucleases exhibit reduced activity.

[0087] In some embodiments, the mutant Cas12a endonuclease comprises a mutation at an amino acid position corresponding to position N813, I831, K932, N933, S934, V936, Q944, S982, F983, K984, M986, or T988, with reference to the numbering of the amino acid positions of LbCas12a, and comprises a polypeptide sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%) identity to the amino acid sequence of any one of the Cas12a endonucleases in Table 1 (e.g., SEQ ID NOs: 1-47), their orthologs, or other wild-type Cas12a protein sequences. In some embodiments, any one or more of the foregoing mutant Cas12a endonucleases exhibit reduced ssDNase activity (or do not exhibit reduced ssDNase activity).

[0088] In some embodiments, the mutant Cas12a endonuclease is at positions N813H, N813R, N813W, I831A, I831Y, K932A, K932F, K932H, K932M, K932N, K932Q, K932R, K932S, K932T, K932W, K932Y, N933E, N933L, S934K, S934Q, V936E, V936G, Q944D, Q944E, Q944K, S982W, F982H ... The mutant Cas12a endonucleases include a polypeptide sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%) identity to the amino acid sequence of any one of the Cas12a endonucleases in Table 1 (e.g., SEQ ID NOs: 1-47), their orthologs, or other wild-type Cas12a protein sequences. In some embodiments, any one or more of the foregoing mutant Cas12a endonucleases exhibit reduced ssDNase activity (or do not exhibit reduced ssDNase activity).

[0089] In some embodiments, the mutant Cas12a endonuclease comprises mutations at amino acid positions corresponding to positions N933L and Q944M, or F983G and M986G, with reference to the numbering of the amino acid positions of LbCas12a, and comprises a polypeptide sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%) identity to the amino acid sequence of any one of the Cas12a endonucleases in Table 1 (e.g., SEQ ID NOs: 1-47), its orthologs, or other wild-type Cas12a protein sequences. In some embodiments, any one or more of the foregoing mutant Cas12a endonucleases exhibit reduced ssDNase activity (or do not exhibit reduced ssDNase activity).

[0090] "Identity" refers to the relationship between two or more sequences (e.g., amino acid sequences or nucleotide sequences) as determined by comparing the sequences to each other. Identity also refers to the degree of sequence relatedness between sequences as determined by the number of matches between strings of amino acids or strings of nucleotides. Identity is a measure of the percentage of identical matches between the smaller of two or more sequences with gap alignment (if any) as addressed by a particular mathematical model or computer program (e.g., "algorithm"). The identity of related polypeptides and polynucleotides can be readily calculated by known methods. For example, "percent (%) identity" as applied to a protein or gene, such as the Cas12a endonuclease described herein, is defined as the percentage of residues (amino acid or nucleic acid residues) of a first protein or gene sequence that are identical to the residues of a second protein or gene sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity.

[0091] Methods and computer programs for alignment are well known in the art. Identity is dependent on the calculation of percent identity, but it is understood that the value may vary due to gaps and penalties introduced in the calculation. In general, a particular protein or gene variant has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, but less than 100% sequence identity with its particular wild-type, native, or reference sequence, as determined by sequence alignment programs and parameters described herein and known to those skilled in the art. Such tools for alignment include, but are not limited to, those in the BLAST suite (Altschul, SF, et al. Nucleic Acids Res. 1997;25:3389-3402) and those based on the Smith-Waterman algorithm (Smith, TF & Waterman, MSJ Mol. Biol. 1981;147:195-197). A popular global alignment technique based on dynamic programming is the Needleman-Wunsch algorithm (Needleman, SB & Wunsch, CDJ Mol. Biol. 1920;48:443-453). The Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) has also been developed, which is said to generate global alignments of nucleotide and amino acid sequences faster than other optimal global alignment methods, including the Needleman-Wunsch algorithm.

[0092] Alignments of non-limiting examples of wild-type Cas12a endonuclease sequences are provided in Figures 1A-1X.

[0093] A Cas12 "homolog" refers to a Cas12a endonuclease that has at least some sequence identity (e.g., at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% identity) to a wild-type reference Cas12a endonuclease and exhibits at least one activity exhibited by the wild-type reference Cas12a endonuclease (e.g., cleavage of double-stranded or single-stranded polynucleotides, binding to crRNA, etc.). For example, wild-type Cas12a endonuclease exhibits promiscuous ssDNase activity, cleaves approximately 14 bp from the PAM, and possesses RNase activity that self-processes pre-crRNA. Additionally, its cleavage activity results in a 5' staggered overhang, and its PAM site is 3' to the target binding site. In contrast, wild-type Cas9 endonuclease does not exhibit promiscuous ssDNase activity, cleaves approximately 3-4 bp away from the PAM, and does not possess RNase activity to self-process the pre-crRNA (it requires accessory proteins to mediate processing of the pre-crRNA). Furthermore, the cleavage activity of Cas9 results in blunt ends, and its PAM site is 5' to the target binding site.

[0094] "Ortholog" of Cas12a refers to Cas12a genes (and proteins encoded by them) that are inferred to be derived from the same ancestral sequence separated by a speciation event; when a species splits into two separate species, the resulting copies of a single gene in the two species are said to be orthologs. Orthologs, or orthologous genes, are genes in different species that originate from a single gene of the last common ancestor by vertical descent. Cas12a orthologs can be identified and characterized based on sequence similarity with the Cas12a system of the present invention, for example, as described for the type II system. For example, orthologs of Cas12a include the Cas12a endonucleases in Table 1.

[0095] III. Hyperactive variants of Cas12a endonuclease Some aspects of the present disclosure relate to high activity mutant Cas12a endonucleases, i.e. mutant Cas12a endonucleases that exhibit high activity. As used herein, "high activity" refers to the polynucleotide cleavage activity of mutant endonucleases that is at least 10% higher than the polynucleotide cleavage activity of wild-type endonuclease or other reference endonucleases. High activity mutant Cas12a endonucleases have a higher reaction rate or initiate a cleavage reaction faster than the corresponding wild-type Cas12a endonuclease. In some embodiments, high activity mutant Cas12a endonucleases exhibit cleavage activity that is at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% higher than the polynucleotide cleavage activity of wild-type endonuclease or other reference endonucleases. See, for example, Zhang, L. et al. Nat Commun. 2021 Jun 23;12(1):3908.

[0096] In some embodiments, the mutant Cas12a endonuclease (a) comprises a mutation at an amino acid position corresponding to position E95, E125, N256, R747, H759, N813, K932, N933, S934, V936, S982, or K984, with reference to the numbering of the amino acid positions of LbCas12a, and (b) exhibits high activity, and optionally the mutant Cas12a endonuclease has at least 85%, at least 90%, at least 95%, or at least 98% identity to a wild-type reference Cas12a endonuclease.

[0097] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising a mutation at an amino acid position corresponding to position E95, with reference to the numbering of the amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutation is E95R or E95Y. In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position E95, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position E95, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit high activity.

[0098] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising a mutation at an amino acid position corresponding to position E125, with reference to the numbering of the amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutation is E125A or E125W. In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position E125, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position E125, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit high activity.

[0099] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising a mutation at an amino acid position corresponding to position N256, with reference to the numbering of the amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutation is N256A. In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position N256, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position N256, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit high activity.

[0100] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising a mutation at an amino acid position corresponding to position R747, with reference to the numbering of the amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutation is R747Y. In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position R747, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position R747, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit high activity.

[0101] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising a mutation at an amino acid position corresponding to position H759, with reference to the numbering of the amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutation is H759V or H759D. In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position H759, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position H759, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit high activity.

[0102] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising a mutation at an amino acid position corresponding to position N813, with reference to the numbering of the amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutation is N813R or N813H. In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position N813, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position N813, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit high activity.

[0103] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising a mutation at an amino acid position corresponding to position K932, with reference to the numbering of the amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutation is K932L. In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position K932, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position K932, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit high activity.

[0104] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising a mutation at an amino acid position corresponding to position N933, with reference to the numbering of the amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutation is N933E or N933V. In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position N933, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position N933, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit high activity.

[0105] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising a mutation at an amino acid position corresponding to position S934, with reference to the numbering of the amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutation is S934Q. In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position S934, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position S934, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit high activity.

[0106] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising a mutation at an amino acid position corresponding to position V936, with reference to the numbering of the amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutation is V936E, V936M, or V936K. In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position V936, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position V936, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit high activity.

[0107] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising a mutation at an amino acid position corresponding to position S982, with reference to the numbering of the amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutation is S982N. In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position S982, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position S982, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit high activity.

[0108] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising a mutation at an amino acid position corresponding to position K984, with reference to the numbering of the amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutation is K984R. In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position K984, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position K984, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit high activity.

[0109] Further engineered mutant Cas12a endonucleases with high activity In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of E95 (e.g., E95R or E95Y) and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of E95 (e.g., E95R or E95Y) and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of E95 (e.g., E95R or E95Y) and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of E95 (e.g., E95R or E95Y) and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of E95 (e.g., E95R or E95Y) and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing mutant Cas12a endonucleases may exhibit high activity.

[0110] In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of E125 (e.g., E125A or E125W) and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of E125 (e.g., E125A or E125W) and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of E125 (e.g., E125A or E125W) and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of E125 (e.g., E125A or E125W) and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of E125 (e.g., E125A or E125W) and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing mutant Cas12a endonucleases may exhibit high activity.

[0111] In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of N256 (e.g., N256A) and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of N256 (e.g., N256A) and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of N256 (e.g., N256A) and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of N256 (e.g., N256A) and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution at N256 (e.g., N256A) and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the above mutant Cas12a endonucleases may exhibit high activity.

[0112] In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of R747 (e.g., R747Y) and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of R747 (e.g., R747Y) and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of R747 (e.g., R747Y) and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of R747 (e.g., R747Y) and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of R747 (e.g., R747Y) and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing mutant Cas12a endonucleases may exhibit high activity.

[0113] In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of H759 (e.g., H759V or H759D) and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of H759 (e.g., H759V or H759D) and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of H759 (e.g., H759V or H759D) and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of H759 (e.g., H759V or H759D) and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of H759 (e.g., H759V or H759D) and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing mutant Cas12a endonucleases may exhibit high activity.

[0114] In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of N813 (e.g., N813R or N813H) and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of N813 (e.g., N813R or N813H) and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of N813 (e.g., N813R or N813H) and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of N813 (e.g., N813R or N813H) and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of N813 (e.g., N813R or N813H) and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing mutant Cas12a endonucleases may exhibit high activity.

[0115] In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K932 (e.g., K932L) and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K932 (e.g., K932L) and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K932 (e.g., K932L) and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K932 (e.g., K932L) and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K932 (e.g., K932L) and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing mutant Cas12a endonucleases may exhibit high activity.

[0116] In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of N933 (e.g., N933E or N933V) and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of N933 (e.g., N933E or N933V) and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of N933 (e.g., N933E or N933V) and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of N933 (e.g., N933E or N933V) and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of N933 (e.g., N933E or N933V) and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing mutant Cas12a endonucleases may exhibit high activity.

[0117] In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of S934Q (e.g., S934Q) and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of S934Q (e.g., S934Q) and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of S934Q (e.g., S934Q) and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of S934Q (e.g., S934Q) and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of S934Q (e.g., S934Q) and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing mutant Cas12a endonucleases may exhibit high activity.

[0118] In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of V936 (e.g., V936E, V936M, or V936K) and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of V936 (e.g., V936E, V936M, or V936K) and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of V936 (e.g., V936E, V936M, or V936K) and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of V936 (e.g., V936E, V936M, or V936K) and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of V936 (e.g., V936E, V936M, or V936K) and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing mutant Cas12a endonucleases may exhibit high activity.

[0119] In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of S982 (e.g., S982N) and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of S982 (e.g., S982N) and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of S982 (e.g., S982N) and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of S982 (e.g., S982N) and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of S982 (e.g., S982N) and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing mutant Cas12a endonucleases may exhibit high activity.

[0120] In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K984 (e.g., K984R) and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K984 (e.g., K984R) and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K984 (e.g., K984R) and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K984 (e.g., K984R) and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K984 (e.g., K984R) and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing mutant Cas12a endonucleases may exhibit high activity.

[0121] IV. Cas12a endonuclease low activity mutants Another aspect of the present disclosure provides a low activity mutant Cas12a endonuclease, i.e., a mutant Cas12a endonuclease that exhibits low activity. As used herein, "low activity" refers to the polynucleotide cleavage activity of the mutant endonuclease that is at least 10% lower than the polynucleotide cleavage activity of the wild-type endonuclease or other reference endonuclease. In some embodiments, the low activity mutant Cas12a endonuclease exhibits a cleavage activity that is at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% lower than the polynucleotide cleavage activity of the wild-type endonuclease or other reference endonuclease.

[0122] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising a mutation at an amino acid position corresponding to position N256, with reference to the numbering of the amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutation is N256K. In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position N256, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position N256, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit reduced activity.

[0123] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising a mutation at an amino acid position corresponding to position I831, with reference to the numbering of the amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutation is I831A or I831Y. In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position I831, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position I831, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit reduced activity.

[0124] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising a mutation at an amino acid position corresponding to position K932, with reference to the numbering of amino acid positions in LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutation is K932A, K932F, K932H, K932M, K932N, K932Q, K932R, K932S, K932T, K932W, or K932Y. In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position K932 with reference to the numbering of the amino acid positions of the LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1) and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position K932 with reference to the numbering of the amino acid positions of the LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1) and has no more than 1, 2, 3, 4, or 5 additional substitutions with respect to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the above mutant Cas12a endonucleases may exhibit reduced activity.

[0125] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising a mutation at an amino acid position corresponding to position N933, with reference to the numbering of the amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutation is N933L. In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position N933, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position N933, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit reduced activity.

[0126] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising a mutation at an amino acid position corresponding to position S934, with reference to the numbering of the amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutation is S934W. In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position S934, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position S934, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit reduced activity.

[0127] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising a mutation at an amino acid position corresponding to position V936, with reference to the numbering of the amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutation is V936G. In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position V936, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position V936, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit reduced activity.

[0128] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising a mutation at an amino acid position corresponding to position Q944, with reference to the numbering of the amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutation is Q944D, Q944E, Q944K, or Q944M. In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position Q944, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position Q944, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit reduced activity.

[0129] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising a mutation at an amino acid position corresponding to position S982, with reference to the numbering of the amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutation is S982T or S982W. In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position S982, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position S982, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit reduced activity.

[0130] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising a mutation at an amino acid position corresponding to position F983, with reference to the numbering of the amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutation is F983G or F983L. In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position F983, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position F983, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit reduced activity.

[0131] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising a mutation at an amino acid position corresponding to position K984, with reference to the numbering of the amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutation is K984F. In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position K984, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position K984, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit reduced activity.

[0132] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising a mutation at an amino acid position corresponding to position M986, with reference to the numbering of the amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutation is M986G, M986L, or M986S. In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position M986, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position M986, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit reduced activity.

[0133] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising a mutation at an amino acid position corresponding to position T988, with reference to the numbering of the amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutation is T988F. In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position T988, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position T988, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit reduced activity.

[0134] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising mutations at amino acid positions corresponding to positions K932 and F983, with reference to the numbering of amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutations are K932F and F983L. In some embodiments, the mutant LbCas12a endonuclease comprises mutations at positions K932 and F983, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises mutations at positions K932 and F983, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit reduced activity.

[0135] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising mutations at amino acid positions corresponding to positions K932 and T988, with reference to the numbering of the amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutations are K932F and T988F. In some embodiments, the mutant LbCas12a endonuclease comprises mutations at positions K932 and T988, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises mutations at positions K932 and T988, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit reduced activity.

[0136] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising mutations at amino acid positions corresponding to positions K932 and Q944, with reference to the numbering of amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutations are K932R and Q944D. In some embodiments, the mutant LbCas12a endonuclease comprises mutations at positions K932 and Q944, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises mutations at positions K932 and Q944, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit reduced activity.

[0137] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising mutations at amino acid positions corresponding to positions K932 and F983, with reference to the numbering of amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutations are K932R and F983L. In some embodiments, the mutant LbCas12a endonuclease comprises mutations at positions K932 and F983, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises mutations at positions K932 and F983, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit reduced activity.

[0138] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising mutations at amino acid positions corresponding to positions K932 and T988, with reference to the numbering of the amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutations are K932R and T988F. In some embodiments, the mutant LbCas12a endonuclease comprises mutations at positions K932 and T988, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises mutations at positions K932 and T988, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit reduced activity.

[0139] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising mutations at amino acid positions corresponding to positions K932 and F983, with reference to the numbering of amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutations are K932Y and F983L. In some embodiments, the mutant LbCas12a endonuclease comprises mutations at positions K932 and F983, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises mutations at positions K932 and F983, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit reduced activity.

[0140] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising mutations at amino acid positions corresponding to positions K932 and T988, with reference to the numbering of the amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutations are K932Y and T988F. In some embodiments, the mutant LbCas12a endonuclease comprises mutations at positions K932 and T988, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises mutations at positions K932 and T988, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit reduced activity.

[0141] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising mutations at amino acid positions corresponding to positions N933 and Q944, with reference to the numbering of amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutations are N933L and Q944M. In some embodiments, the mutant LbCas12a endonuclease comprises mutations at positions N933 and Q944, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises mutations at positions N933 and Q944, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit reduced activity.

[0142] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising mutations at amino acid positions corresponding to positions V936 and Q944, with reference to the numbering of amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutations are V936G and Q944D. In some embodiments, the mutant LbCas12a endonuclease comprises mutations at positions V936 and Q944, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises mutations at positions V936 and Q944, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit reduced activity.

[0143] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising mutations at amino acid positions corresponding to positions V936 and S982, with reference to the numbering of amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutations are V936G and S982W. In some embodiments, the mutant LbCas12a endonuclease comprises mutations at positions V936 and S982, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises mutations at positions V936 and S982, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit reduced activity.

[0144] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising mutations at amino acid positions corresponding to positions V936 and M986, with reference to the numbering of amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutations are V936G and M986G. In some embodiments, the mutant LbCas12a endonuclease comprises mutations at positions V936 and M986, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises mutations at positions V936 and M986, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit reduced activity.

[0145] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising mutations at amino acid positions corresponding to positions V936 and T988, with reference to the numbering of the amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutations are V936G and T988F. In some embodiments, the mutant LbCas12a endonuclease comprises mutations at positions V936 and T988, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises mutations at positions V936 and T988, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit reduced activity.

[0146] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising mutations at amino acid positions corresponding to positions Q944 and S982, with reference to the numbering of the amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutations are Q944D and S982W. In some embodiments, the mutant LbCas12a endonuclease comprises mutations at positions Q944 and S982, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises mutations at positions Q944 and S982, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit reduced activity.

[0147] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising mutations at amino acid positions corresponding to positions Q944 and F983, with reference to the numbering of amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutations are Q944D and F983L. In some embodiments, the mutant LbCas12a endonuclease comprises mutations at positions Q944 and F983, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises mutations at positions Q944 and F983, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit reduced activity.

[0148] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising mutations at amino acid positions corresponding to positions Q944 and T988, with reference to the numbering of the amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutations are Q944D and T988F. In some embodiments, the mutant LbCas12a endonuclease comprises mutations at positions Q944 and T988, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises mutations at positions Q944 and T988, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit reduced activity.

[0149] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising mutations at amino acid positions corresponding to positions S982 and F983, with reference to the numbering of amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutations are S982W and F983L. In some embodiments, the mutant LbCas12a endonuclease comprises mutations at positions S982 and F983, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises mutations at positions S982 and F983, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit reduced activity.

[0150] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising mutations at amino acid positions corresponding to positions S982 and T988, with reference to the numbering of the amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutations are S982W and T988F. In some embodiments, the mutant LbCas12a endonuclease comprises mutations at positions S982 and T988, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises mutations at positions S982 and T988, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit reduced activity.

[0151] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising mutations at amino acid positions corresponding to positions F983 and M986, with reference to the numbering of amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutations are F983G and M986G. In some embodiments, the mutant LbCas12a endonuclease comprises mutations at positions F983 and M986, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises mutations at positions F983 and M986, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit reduced activity.

[0152] Further engineered mutant Cas12a endonucleases with reduced activity In some embodiments, the mutant LbCas12a endonuclease comprises a substitution at N256 (e.g., N256K) and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution at N256 (e.g., N256K) and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution at N256 (e.g., N256K) and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution at N256 (e.g., N256K) and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution at N256 (e.g., N256K) and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing mutant Cas12a endonucleases may exhibit reduced activity.

[0153] In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of I831 (e.g., I831A or I831Y) and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of I831 (e.g., I831A or I831Y) and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of I831 (e.g., I831A or I831Y) and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of I831 (e.g., I831A or I831Y) and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of I831 (e.g., I831A or I831Y) and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing mutant Cas12a endonucleases may exhibit reduced activity.

[0154] In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K932 (e.g., K932A, K932F, K932H, K932M, K932N, K932Q, K932R, K932S, K932T, K932W, or K932Y) and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K932 (e.g., K932A, K932F, K932H, K932M, K932N, K932Q, K932R, K932S, K932T, K932W, or K932Y) and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K932 (e.g., K932A, K932F, K932H, K932M, K932N, K932Q, K932R, K932S, K932T, K932W, or K932Y) and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K932 (e.g., K932A, K932F, K932H, K932M, K932N, K932Q, K932R, K932S, K932T, K932W, or K932Y) and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K932 (e.g., K932A, K932F, K932H, K932M, K932N, K932Q, K932R, K932S, K932T, K932W, or K932Y) and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing mutant Cas12a endonucleases may exhibit reduced activity.

[0155] In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of N933 (e.g., N933L) and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of N933 (e.g., N933L) and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of N933 (e.g., N933L) and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of N933 (e.g., N933L) and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of N933 (e.g., N933L) and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing mutant Cas12a endonucleases may exhibit reduced activity.

[0156] In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of S934 (e.g., S934W) and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of S934 (e.g., S934W) and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of S934 (e.g., S934W) and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of S934 (e.g., S934W) and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of S934 (e.g., S934W) and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing mutant Cas12a endonucleases may exhibit reduced activity.

[0157] In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of V936 (e.g., V936G) and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of V936 (e.g., V936G) and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of V936 (e.g., V936G) and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of V936 (e.g., V936G) and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of V936 (e.g., V936G) and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing mutant Cas12a endonucleases may exhibit reduced activity.

[0158] In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of Q944 (e.g., Q944D, Q944E, Q944K, or Q944M) and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of Q944 (e.g., Q944D, Q944E, Q944K, or Q944M) and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of Q944 (e.g., Q944D, Q944E, Q944K, or Q944M) and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of Q944 (e.g., Q944D, Q944E, Q944K, or Q944M) and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of Q944 (e.g., Q944D, Q944E, Q944K, or Q944M) and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing mutant Cas12a endonucleases may exhibit reduced activity.

[0159] In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of S982 (e.g., S982T or S982W) and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of S982 (e.g., S982T or S982W) and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of S982 (e.g., S982T or S982W) and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of S982 (e.g., S982T or S982W) and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution at S982 (e.g., S982T or S982W) and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing mutant Cas12a endonucleases may exhibit reduced activity.

[0160] In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of F983 (e.g., F983G or F983L) and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of F983 (e.g., F983G or F983L) and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of F983 (e.g., F983G or F983L) and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of F983 (e.g., F983G or F983L) and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of F983 (e.g., F983G or F983L) and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing mutant Cas12a endonucleases may exhibit reduced activity.

[0161] In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K984 (e.g., K984F) and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K984 (e.g., K984F) and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K984 (e.g., K984F) and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K984 (e.g., K984F) and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K984 (e.g., K984F) and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing mutant Cas12a endonucleases may exhibit reduced activity.

[0162] In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of M986 (e.g., M986G, M986L, or M986S) and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of M986 (e.g., M986G, M986L, or M986S) and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of M986 (e.g., M986G, M986L, or M986S) and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of M986 (e.g., M986G, M986L, or M986S) and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of M986 (e.g., M986G, M986L, or M986S) and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing mutant Cas12a endonucleases may exhibit reduced activity.

[0163] In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of T988 (e.g., T988F) and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of T988 (e.g., T988F) and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of T988 (e.g., T988F) and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of T988 (e.g., T988F) and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of T988 (e.g., T988F) and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing mutant Cas12a endonucleases may exhibit reduced activity.

[0164] In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K932F and F983L and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K932F and F983L and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K932F and F983L and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K932F and F983L and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K932F and F983L and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the above mutant Cas12a endonucleases may exhibit reduced activity.

[0165] In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K932F and T988F and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K932F and T988F and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K932F and T988F and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K932F and T988F and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K932F and T988F and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the above mutant Cas12a endonucleases may exhibit reduced activity.

[0166] In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K932R and Q944D and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K932R and Q944D and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K932R and Q944D and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K932R and Q944D and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K932R and Q944D and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the above mutant Cas12a endonucleases may exhibit reduced activity.

[0167] In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K932R and F983L and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K932R and F983L and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K932R and F983L and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K932R and F983L and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K932R and F983L and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the above mutant Cas12a endonucleases may exhibit reduced activity.

[0168] In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K932R and T988F and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K932R and T988F and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K932R and T988F and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K932R and T988F and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K932R and T988F and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the above mutant Cas12a endonucleases may exhibit reduced activity.

[0169] In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K932Y and F983L and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K932Y and F983L and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K932Y and F983L and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K932Y and F983L and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K932Y and F983L and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the above mutant Cas12a endonucleases may exhibit reduced activity.

[0170] In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K932Y and T988F and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K932Y and T988F and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K932Y and T988F and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K932Y and T988F and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K932Y and T988F and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the above mutant Cas12a endonucleases may exhibit reduced activity.

[0171] In some embodiments, the mutant LbCas12a endonuclease comprises N933L and Q944M substitutions and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises N933L and Q944M substitutions and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises N933L and Q944M substitutions and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises N933L and Q944M substitutions and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises N933L and Q944M substitutions and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the above mutant Cas12a endonucleases may exhibit reduced activity.

[0172] In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of V936G and Q944D and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of V936G and Q944D and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of V936G and Q944D and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of V936G and Q944D and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of V936G and Q944D and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the above mutant Cas12a endonucleases may exhibit reduced activity.

[0173] In some embodiments, the mutant LbCas12a endonuclease comprises a V936G and S982W substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a V936G and S982W substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a V936G and S982W substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a V936G and S982W substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a V936G and S982W substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the above mutant Cas12a endonucleases may exhibit reduced activity.

[0174] In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of V936G and M986G and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of V936G and M986G and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of V936G and M986G and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of V936G and M986G and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of V936G and M986G and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the above mutant Cas12a endonucleases may exhibit reduced activity.

[0175] In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of V936G and T988F and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of V936G and T988F and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of V936G and T988F and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of V936G and T988F and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of V936G and T988F and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the above mutant Cas12a endonucleases may exhibit reduced activity.

[0176] In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of Q944D and S982W and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of Q944D and S982W and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of Q944D and S982W and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of Q944D and S982W and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of Q944D and S982W and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the above mutant Cas12a endonucleases may exhibit reduced activity.

[0177] In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of Q944D and F983L and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of Q944D and F983L and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of Q944D and F983L and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of Q944D and F983L and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of Q944D and F983L and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the above mutant Cas12a endonucleases may exhibit reduced activity.

[0178] In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of Q944D and T988F and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of Q944D and T988F and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of Q944D and T988F and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of Q944D and T988F and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of Q944D and T988F and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the above mutant Cas12a endonucleases may exhibit reduced activity.

[0179] In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of S982W and F983L and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of S982W and F983L and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of S982W and F983L and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of S982W and F983L and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of S982W and F983L and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the above mutant Cas12a endonucleases may exhibit reduced activity.

[0180] In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of S982W and T988F and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of S982W and T988F and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of S982W and T988F and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of S982W and T988F and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of S982W and T988F and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the above mutant Cas12a endonucleases may exhibit reduced activity.

[0181] In some embodiments, the mutant LbCas12a endonuclease comprises substitutions of F983G and M986G and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises substitutions of F983G and M986G and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises substitutions of F983G and M986G and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises substitutions of F983G and M986G and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises substitutions of F983G and M986G and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the above mutant Cas12a endonucleases may exhibit reduced activity.

[0182] V. Cas12a endonuclease low promiscuous ssDNase mutant In addition to highly specific double-stranded DNA (dsDNA) cleavage, Cas12a has also been shown to exhibit promiscuous ssDNA degradation activity when activated by single-stranded DNA (ssDNA) complementary to the crRNA guide as well as dsDNA complementary to the crRNA guide. This activity is exhibited by all Cas12a orthologs, and degrades any available ssDNA molecule into single-stranded / double-stranded nucleotides. Comparison of Cas12a structures before, during, and after cleavage reveals the structural changes that lead to such promiscuous activity. This action is carried out by the lid region, which is involved in a checkpoint for accurate target recognition. Before the formation of the crRNA-DNA hybrid, the lid occludes the cleft where the catalytic residues reside. Once the hybrid is formed, the lid changes conformation to form an α-helix and interacts with the crRNA of the hybrid assembly, dissociating the polar interaction to make the catalytic pocket available. In the post-cleavage R-loop structure, this region appears disordered, indicating that the distal portion of the dsDNA substrate is accessible to the catalytic site after dissociation from the complex. Thus, the catalytic cleft is open and can indiscriminately cleave ssDNA. This molecular mechanism may explain how ssDNA molecules are degraded by Cas12a after being activated by the presence of RNA-DNA hybrids. Furthermore, recent studies have reported nonspecific nicking of target sequences with mismatches in the distal regions of the target DNA, suggesting that this could be problematic in potential applications. See Paul, B. & Montoya, G. et al.

[0183] Some mutant Cas12a endonucleases provided herein surprisingly exhibit low or no promiscuous ssDNA degradation activity, also referred to as promiscuous single-stranded deoxyribonuclease (ssDNase) activity. This activity was unexpected, in part, because the mutations made in the wild-type parent enzyme were outside the lid region, i.e., the region believed to be responsible for promiscuous ssDNase activity. As used herein, "low ssDNase activity" refers to a promiscuous ssDNA degradation activity of the mutant endonuclease that is at least 10% lower than the promiscuous ssDNA degradation activity of the wild-type endonuclease or other reference endonucleases. In some embodiments, the mutant Cas12a endonuclease exhibits promiscuous ssDNase activity that is at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% less than the promiscuous ssDNase activity of the wild-type endonuclease or another reference endonuclease. In some embodiments, the mutant Cas12a endonuclease does not exhibit (is not measurable) promiscuous ssDNase activity.

[0184] In some embodiments, the mutant Cas12a endonuclease (a) comprises a mutation at an amino acid position corresponding to position N813, I831, K932, N933, S934, V936, Q944, S982, F983, K984, M986, or T988, with reference to the numbering of the amino acid positions of LbCas12a, and (b) exhibits low activity (or no activity) as a single promiscuous ssDNase, and optionally the mutant Cas12a endonuclease has at least 85%, at least 90%, at least 95%, or at least 98% identity to a wild-type reference Cas12a endonuclease.

[0185] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising a mutation at an amino acid position corresponding to position N813, with reference to the numbering of the amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutation is N813H, N813R, or N813W. In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position N813, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position N813, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit (or not exhibit) reduced promiscuous ssDNase activity.

[0186] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising a mutation at an amino acid position corresponding to position I831, with reference to the numbering of the amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutation is I831A or I831Y. In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position I831, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position I831, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit (or not exhibit) reduced promiscuous ssDNase activity.

[0187] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising a mutation at an amino acid position corresponding to position K932, with reference to the numbering of amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutation is K932A, K932F, K932H, K932M, K932N, K932Q, K932R, K932S, K932T, K932W, or K932Y. In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position K932 with reference to the numbering of the amino acid positions of the LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1) and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position K932 with reference to the numbering of the amino acid positions of the LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1) and has no more than 1, 2, 3, 4, or 5 additional substitutions with respect to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit reduced (or no) promiscuous ssDNase activity.

[0188] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising a mutation at an amino acid position corresponding to position N933, with reference to the numbering of the amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutation is N933E or N933L. In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position N933, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position N933, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit (or not exhibit) reduced promiscuous ssDNase activity.

[0189] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising a mutation at an amino acid position corresponding to position S934, with reference to the numbering of the amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutation is S934K or S934Q. In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position S934, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position S934, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit (or not exhibit) reduced promiscuous ssDNase activity.

[0190] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising a mutation at an amino acid position corresponding to position V936, with reference to the numbering of the amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutation is V936E or V936G. In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position V936, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position V936, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit (or not exhibit) reduced promiscuous ssDNase activity.

[0191] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising a mutation at an amino acid position corresponding to position Q944, with reference to the numbering of the amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutation is Q944D, Q944E, or Q944K. In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position Q944, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position Q944, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing mutant Cas12a endonucleases may exhibit (or not exhibit) reduced promiscuous ssDNase activity.

[0192] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising a mutation at an amino acid position corresponding to position S982, with reference to the numbering of the amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutation is S982W. In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position S982, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position S982, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing mutant Cas12a endonucleases may exhibit (or not exhibit) reduced promiscuous ssDNase activity.

[0193] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising a mutation at an amino acid position corresponding to position F983, with reference to the numbering of the amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutation is F983G or F983L. In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position F983, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position F983, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit (or not exhibit) reduced promiscuous ssDNase activity.

[0194] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising a mutation at an amino acid position corresponding to position K984, with reference to the numbering of the amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutation is K984F. In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position K984, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position K984, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit (or not exhibit) reduced promiscuous ssDNase activity.

[0195] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising a mutation at an amino acid position corresponding to position M986, with reference to the numbering of the amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutation is M986F or M986G. In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position M986, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position M986, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing mutant Cas12a endonucleases may exhibit (or not exhibit) reduced promiscuous ssDNase activity.

[0196] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising a mutation at an amino acid position corresponding to position T988, with reference to the numbering of the amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutation is T988F. In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position T988, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position T988, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing mutant Cas12a endonucleases may exhibit (or not exhibit) reduced promiscuous ssDNase activity.

[0197] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising mutations at amino acid positions corresponding to positions N933 and Q944, with reference to the numbering of amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutations are N933L and Q944M. In some embodiments, the mutant LbCas12a endonuclease comprises mutations at positions N933 and Q944, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises mutations at positions N933 and Q944, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit (or not exhibit) reduced promiscuous ssDNase activity.

[0198] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising mutations at amino acid positions corresponding to positions F983 and M986, with reference to the numbering of amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutations are F983G and M986G. In some embodiments, the mutant LbCas12a endonuclease comprises mutations at positions F983 and M986, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises mutations at positions F983 and M986, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit (or not exhibit) reduced promiscuous ssDNase activity.

[0199] Further engineered mutant Cas12a endonucleases with low ssDNase activity In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of N813 (e.g., N813H, N813R, or N813W) and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of N813 (e.g., N813H, N813R, or N813W) and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of N813 (e.g., N813H, N813R, or N813W) and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of N813 (e.g., N813H, N813R, or N813W) and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of N813 (e.g., N813H, N813R, or N813W) and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing mutant Cas12a endonucleases may exhibit (or not exhibit) reduced ssDNase activity.

[0200] In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of I831 (e.g., I831A or I831Y) and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of I831 (e.g., I831A or I831Y) and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of I831 (e.g., I831A or I831Y) and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of I831 (e.g., I831A or I831Y) and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of I831 (e.g., I831A or I831Y) and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing mutant Cas12a endonucleases may exhibit reduced (or no) ssDNase activity.

[0201] In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K932 (e.g., K932A, K932F, K932H, K932M, K932N, K932Q, K932R, K932S, K932T, K932W, or K932Y) and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K932 (e.g., K932A, K932F, K932H, K932M, K932N, K932Q, K932R, K932S, K932T, K932W, or K932Y) and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K932 (e.g., K932A, K932F, K932H, K932M, K932N, K932Q, K932R, K932S, K932T, K932W, or K932Y) and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K932 (e.g., K932A, K932F, K932H, K932M, K932N, K932Q, K932R, K932S, K932T, K932W, or K932Y) and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K932 (e.g., K932A, K932F, K932H, K932M, K932N, K932Q, K932R, K932S, K932T, K932W, or K932Y) and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing mutant Cas12a endonucleases may exhibit reduced (or no) ssDNase activity.

[0202] In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of N933 (e.g., N933E or N933L) and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of N933 (e.g., N933E or N933L) and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of N933 (e.g., N933E or N933L) and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of N933 (e.g., N933E or N933L) and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of N933 (e.g., N933E or N933L) and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing mutant Cas12a endonucleases may exhibit reduced (or no) ssDNase activity.

[0203] In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of S934 (e.g., S934K or S934Q) and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of S934 (e.g., S934K or S934Q) and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of S934 (e.g., S934K or S934Q) and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of S934 (e.g., S934K or S934Q) and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of S934 (e.g., S934K or S934Q) and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing mutant Cas12a endonucleases may exhibit reduced (or no) ssDNase activity.

[0204] In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of V936 (e.g., V936E or V936G) and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of V936 (e.g., V936E or V936G) and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of V936 (e.g., V936E or V936G) and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of V936 (e.g., V936E or V936G) and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of V936 (e.g., V936E or V936G) and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing mutant Cas12a endonucleases may exhibit reduced (or no) ssDNase activity.

[0205] In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of Q944 (e.g., Q944D, Q944E, or Q944K) and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of Q944 (e.g., Q944D, Q944E, or Q944K) and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of Q944 (e.g., Q944D, Q944E, or Q944K) and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of Q944 (e.g., Q944D, Q944E, or Q944K) and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of Q944 (e.g., Q944D, Q944E, or Q944K) and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing mutant Cas12a endonucleases may exhibit (or not exhibit) reduced ssDNase activity.

[0206] In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of S982 (e.g., S982W) and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of S982 (e.g., S982W) and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of S982 (e.g., S982W) and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of S982 (e.g., S982W) and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of S982 (e.g., S982W) and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing mutant Cas12a endonucleases may exhibit reduced (or no) ssDNase activity.

[0207] In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of F983 (e.g., F983G or F983L) and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of F983 (e.g., F983G or F983L) and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of F983 (e.g., F983G or F983L) and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of F983 (e.g., F983G or F983L) and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of F983 (e.g., F983G or F983L) and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing mutant Cas12a endonucleases may exhibit reduced (or no) ssDNase activity.

[0208] In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K984 (e.g., K984F) and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K984 (e.g., K984F) and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K984 (e.g., K984F) and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K984 (e.g., K984F) and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of K984 (e.g., K984F) and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing mutant Cas12a endonucleases may exhibit reduced (or no) ssDNase activity.

[0209] In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of M986 (e.g., M986F or M986G) and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of M986 (e.g., M986F or M986G) and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of M986 (e.g., M986F or M986G) and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of M986 (e.g., M986F or M986G) and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of M986 (e.g., M986F or M986G) and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing mutant Cas12a endonucleases may exhibit reduced (or no) ssDNase activity.

[0210] In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of T988 (e.g., T988F) and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of T988 (e.g., T988F) and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of T988 (e.g., T988F) and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of T988 (e.g., T988F) and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises a substitution of T988 (e.g., T988F) and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing mutant Cas12a endonucleases may exhibit reduced (or no) ssDNase activity.

[0211] In some embodiments, the mutant LbCas12a endonuclease comprises N933L and Q944M substitutions and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises N933L and Q944M substitutions and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises N933L and Q944M substitutions and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises N933L and Q944M substitutions and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises N933L and Q944M substitutions and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit reduced (or no) ssDNase activity.

[0212] In some embodiments, the mutant LbCas12a endonuclease comprises substitutions of F983G and M986G and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises substitutions of F983G and M986G and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises substitutions of F983G and M986G and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises substitutions of F983G and M986G and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mutant LbCas12a endonuclease comprises substitutions of F983G and M986G and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the aforementioned mutant Cas12a endonucleases may exhibit reduced (or no) ssDNase activity.

[0213] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7]

Table 3-8

Table 3-9

Table 3-10

Table 3-11

Table 3-12

Table 3-13

Table 3-14

Table 3-15

Table 3-16

Table 3-17

Table 3-18

Table 3-19

Table 3-20

[0214] VI. Polynucleotides The present disclosure provides, in some embodiments, a polynucleotide encoding a mutant Cas12a endonuclease. A nucleic acid comprises a polymer of nucleotides (nucleotide monomers). Thus, a nucleic acid is also referred to as a polynucleotide. A nucleic acid may be or include, for example, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA, including LNA with β-D-ribo configuration, α-LNA (diastereomer of LNA) with α-L-ribo configuration, 2'-amino-LNA with 2'-amino functionalization, and 2'-amino-α-LNA with 2'-amino functionalization), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA) and / or chimeras and / or combinations thereof.

[0215] In some embodiments, the polynucleotide encoding the mutant Cas12a endonuclease is an RNA, such as an mRNA. A messenger RNA (mRNA) is an RNA that encodes (at least one) protein (naturally occurring, non-naturally occurring, or modified polymer of amino acids) and can be translated to produce the encoded protein in vitro, in vivo, in situ, or ex vivo. The mRNA provided herein typically comprises an open reading frame (ORF) encoding the mutant Cas12a endonuclease. In some embodiments, the mRNA also comprises an ORF encoding a crRNA or multiple crRNAs. In some embodiments, the mRNA further comprises a 5' cap, a 5' untranslated region (UTR), a 3' UTR, and a poly(A) tail.

[0216] An ORF is a continuous stretch of DNA or RNA that begins with a start codon (e.g., methionine (ATG or AUG)) and ends with a stop codon (e.g., TAA, TAG or TGA, or UAA, UAG or UGA). ORFs typically encode proteins. It will be understood that the sequences disclosed herein may further comprise additional elements, such as 5' and / or 3' UTRs, but that such elements, unlike ORFs, are not necessarily required to be present in the RNA (e.g., mRNA) of the present disclosure.

[0217] In some embodiments, the ORF encoding the mutant Cas12a endonuclease of the present disclosure is codon-optimized. Codon optimization methods are known in the art. Any one or more open reading frames of the mutant Cas12a endonuclease provided herein may be codon-optimized. Codon optimization can be used in some embodiments to match the codon frequency in target and host organisms to ensure proper folding; bias GC content to increase RNA (e.g., mRNA) stability or reduce secondary structure; minimize tandem repeat codons or base runs that may impair gene assembly or expression; customize transcriptional and translational control regions; insert or remove protein transport sequences; remove / add post-translational modification sites (glycosylation sites) in encoded proteins; add, remove or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and RNA (e.g., mRNA) degradation sites; adjust translation rates so that various domains of proteins are properly folded; or reduce or eliminate problematic secondary structures in polynucleotides. Codon optimization tools, algorithms and services are known in the art, non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA), and / or proprietary methods. In some embodiments, the open reading frame sequence is optimized using an optimization algorithm.

[0218] "5' untranslated region" (UTR) refers to the region of an mRNA immediately upstream (i.e., 5') from the start codon (i.e., the first codon of an mRNA transcript that is translated by a ribosome) that does not code for a polypeptide. "3' untranslated region" (UTR) refers to the region of an mRNA immediately downstream (i.e., 3') from the stop codon (i.e., the codon of an mRNA transcript that signals the end of translation) that does not code for a polypeptide. If an RNA transcript is produced, the 5' UTR may include a promoter sequence.

[0219] In some embodiments, the RNA (e.g., mRNA) comprises a 5'-end cap. 5'-capping of polynucleotides can be completed simultaneously during the in vitro transcription reaction, for example, using the following chemical RNA cap analogs to generate a 5'-guanosine cap structure according to the manufacturer's protocol: 3'-O-Me-m7G(5')ppp(5')G [ARCA cap]; G(5')ppp(5')A; G(5')ppp(5')G; m7G(5')ppp(5')A; m7G(5')ppp(5')G (New England BioLabs, Ipswich, MA). 5'-capping of modified RNA (e.g., mRNA) can be completed post-transcriptionally, for example, using Vaccinia Virus Capping Enzyme to generate a "Cap 0" structure: m7G(5')ppp(5')G (New England BioLabs, Ipswich, MA). The cap 1 structure may be generated using both the Vaccinia Virus Capping Enzyme and a 2'-O methyltransferase: m7G(5')ppp(5')G-2'-O-methyl. The cap 2 structure may be generated from cap 1 structure followed by 2'-O-methylation of the 5'-front terminal nucleotide using a 2'-O methyltransferase. The cap 3 structure may be generated from cap 2 structure followed by 2'-O-methylation of the 5'-front terminal nucleotide using a 2'-O methyltransferase. The enzymes may be from recombinant sources. Other cap analogs may be used.

[0220] A "poly(A) tail" is a region of an mRNA downstream, e.g., directly downstream (i.e., 3') of the 3' UTR, that contains multiple consecutive adenosine monophosphates. A poly(A) tail can contain 10-300 adenosine monophosphates. A poly(A) tail can contain up to about 400 adenine nucleotides in some cases. For example, a poly(A) tail can contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 adenosine monophosphates. In some embodiments, a poly(A) tail contains 50-250 adenosine monophosphates. In relevant biological environments (e.g., intracellular, in vivo), poly(A) tails function, for example, in the cytoplasm, to protect mRNAs from enzymatic degradation and aid in transcription termination and / or transport and translation of mRNAs from the nucleus. In some embodiments, the length of the 3'-poly(A) tail may be an essential factor for the stability of individual mRNAs. In some embodiments, the poly(A) tail has a length of about 50, about 100, about 150, about 200, about 250, about 300, about 350, or about 400 nucleotides. In some embodiments, the poly(A) tail has a length of 100 nucleotides.

[0221] VII. Fusion Proteins Some embodiments relate to fusion proteins comprising any one or more of the mutant Cas12a endonucleases provided herein and one or more effector proteins (e.g., proteins such as enzymes that regulate biological activity). Non-limiting examples include proteins that exhibit deaminase activity (e.g., adenosine deaminase and / or cytidine deaminase), reverse transcriptase, endonuclease (e.g., FokI), exonuclease activity (e.g., T5 exonuclease), methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitination activity, adenylation activity, deadenylation activity, sumoylation activity, desumoylation activity, ribosylation activity, deribosylation activity, myristoylation activity, or demyristoylation activity.

[0222] Provided herein, in some embodiments, are base editing fusion proteins comprising one or more base editing enzymes. A "base editing enzyme" is an enzyme that can convert a target nucleobase or base pair to a different nucleobase or base pair (e.g., converting adenine to thymine (A to T), cytosine to thymine (C to T), adenine to guanine (A to G), cytosine to guanine (C to G)) without requiring the creation and / or repair of a double-stranded break in a polynucleotide strand. The base editing enzyme may be specific for DNA bases or specific for RNA bases. Either base editing enzyme may be utilized in the fusion proteins described herein.

[0223] The base editing enzyme may be able to convert adenine to guanine, adenine to thymine, adenine to uracil, adenine to cytosine, guanine to adenine, guanine to thymine, guanine to uracil, guanine to cytosine, thymine to adenine, thymine to guanine, thymine to uracil, thymine to cytosine, uracil to adenine, uracil to guanine, uracil to thymine, uracil to cytosine, cytosine to adenine, cytosine to guanine, cytosine to thymine, or cytosine to uracil. In some embodiments, the base editing enzyme can convert a standard nucleobase (e.g., A, C, G, T, U) to a modified nucleobase (e.g., hypoxanthine, xanthine 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, 5-hydroxymethylcytosine). In some embodiments, the base editing enzyme can convert a modified nucleobase (e.g., hypoxanthine, xanthine 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, 5-hydroxymethylcytosine) to a standard nucleobase (e.g., A, C, G, T, U). In some embodiments, the base editing enzyme can convert a modified nucleobase to a different modified nucleobase.

[0224] In some embodiments, the base editing enzyme converts the target base pair. For example, in some embodiments, the base editing enzyme converts a CG base pair to a TA base pair. In some embodiments, the base editing enzyme converts an AT base pair to a GC base pair.

[0225] In some embodiments, the base editing enzyme is a deaminase (e.g., cytidine deaminase or adenosine deaminase) that can remove an amino group from a molecule. Cytidine deaminase can remove an amino group from cytidine, and adenosine deaminase can remove an amino group from adenosine. The cytidine deaminase may be an apolipoprotein B mRNA editing enzyme complex (APOBEC1) family protein. In some embodiments, the deaminase is an APOBEC1 polypeptide, an APOBEC2 polypeptide, an APOBEC3 polypeptide, an APOBEC3A polypeptide, an APOBEC3B polypeptide, an APOBEC3C polypeptide, an APOBEC3D polypeptide, an APOBEC3E polypeptide, an APOBEC3F polypeptide, an APOBEC3G deaminase polypeptide, an APOBEC3H polypeptide, an APOBEC4 polypeptide, or an activation-induced deaminase (AID). In some embodiments, the adenosine deaminase is a TadA polypeptide.

[0226] In some embodiments, the base editing enzyme is an oxidase (e.g., guanine oxidase) that can oxidize a certain nucleobase. For example, guanine oxidase functions to oxidize a certain guanine nucleobase in a target gene to form 8-oxoguanine (8-oxo-G). 8-oxo-G induces the steric rotation of the nucleobase around the glycosidic bond, and base pairs in the Hoogsteen orientation of 8-oxo-G. When the mismatch of 8-oxo-G / cytosine pairing is recognized in cells, the cytosine is naturally repaired to adenine. After further replication or mismatch repair, 8-oxo-G is converted to thymine, thereby resulting in the conversion of guanine to thymine. In some embodiments, the guanine oxidase is a wild-type guanine oxidase, a xanthine dehydrogenase (XHD), a cytochrome P450 enzyme (e.g., CYP1A2, CYP2A6, or CYP3A6), a TET-oxidase (e.g., TET1, TET1-CD, TET2, or TET3), an alpha-ketoglutarate-dependent hydroxylase (e.g., AlkB). In some embodiments, the xanthine dehydrogenase is Streptomyces cyanogenus xanthine dehydrogenase, C. capitata xanthine dehydrogenase, N. crassa xanthine dehydrogenase, M. hansupus xanthine dehydrogenase, E. cloacae xanthine dehydrogenase, S. snoursei xanthine dehydrogenase, S. albulus xanthine dehydrogenase, S. himastatinicus xanthine dehydrogenase, or S. lividans xanthine dehydrogenase.

[0227] In some embodiments, the base editing enzyme is a methyltransferase (e.g., guanine methyltransferase) that can methylate a certain nucleobase. For example, guanine methyltransferase functions to methylate a certain guanine nucleobase in a target gene to form N2,N2-dimethyl-guanine or N-methyl-guanine. These methylated bases disrupt the hydrogen bond interaction with paired cytosine. When cells recognize mismatch pairing, cytosine is naturally repaired to adenine. After further replication or mismatch repair, methylated guanine is converted to thymine, thereby resulting in the conversion of guanine to thymine. In some embodiments, the guanine methyltransferase is wild-type RlmA, E. coli RlmA, human TrmTIOA, E. coli TrmD, M. Jannaschii Trm5b, P. Abyssi Trm5a, Trm5c from Archaea, or Staphylococcus scirui Cfr.

[0228] In some embodiments, the base editing enzyme (e.g., deaminase) is a base editing enzyme derived from a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse deaminase. In some embodiments, the base editing enzyme is a human base editing enzyme (e.g., a human deaminase, e.g., a hAPOBEC polypeptide). In some embodiments, the base editing enzyme is a rat base editing enzyme (e.g., a rat deaminase, e.g., a rAPOBEC1 polypeptide). In some embodiments, the base editing enzyme (e.g., a deaminase) is an evolved variant of a wild-type base editing enzyme. For example, in some embodiments, the base editing enzyme is an evolved APOBEC polypeptide (e.g., an evoAPOBEC1 polypeptide), an evolved cytidine deaminase polypeptide (e.g., an evoCDA polypeptide), or an evolved FERNY polypeptide (e.g., an evoFERNY polypeptide). In some embodiments, the base editing enzyme is described in Thuronyi, et. al. Nat Biotechnol. 2019 Sep; 37(9): 1070-1079, the contents of which are incorporated herein by reference. In some embodiments, the base editing enzyme is described in U.S. Patent Publication No. 20200172931, the contents of which are incorporated herein by reference.

[0229] Exemplary, non-limiting sequences of base editing enzymes are provided in Table 4. In some embodiments, the base editing enzyme comprises the amino acid sequence of any one of SEQ ID NOs: 27-69. In some embodiments, the base editing enzyme comprises an amino acid sequence that includes one or more mutations (e.g., amino acid substitutions) relative to the amino acid sequence of any one of SEQ ID NOs: 27-69 and has at least 70% (e.g., at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%) identity to the amino acid sequence of any one of the base editing enzymes in Table 4 (e.g., SEQ ID NOs: 120-162), its orthologs, or other base editing enzyme sequences.

[0230] [Table 4-1]

Table 4-2

Table 4-3

Table 4-4

Table 4-5

Table 4-6

Table 4-7

[0231] In some embodiments, the fusion protein comprises one or more mutant Cas12a endonucleases, one or more base editing enzymes, and one or more additional proteins (e.g., proteins such as enzymes that modulate a biological activity). Non-limiting examples of additional protein elements include a polypeptide having uracil glycosylase inhibitor (UGI) activity, a polypeptide having uracil DNA glycosylase activity, a DNA binding domain (e.g., Rad51 DNA binding domain), a reverse transcriptase, an endonuclease (e.g., FokI), a polypeptide having exonuclease activity (e.g., T5 exonuclease), a polypeptide having methyltransferase activity, a polypeptide having demethylase activity, a polypeptide having acetyltransferase activity, a polypeptide having deacetylase activity, a polypeptide having kinase activity, a polypeptide having phosphatase activity, a polypeptide having ubiquitin ligase activity, a polypeptide having deubiquitination activity, a polypeptide having adenylation activity, a polypeptide having deadenylation activity, a polypeptide having SUMOylation activity, a polypeptide having deSUMOylation activity, a polypeptide having ribosylation activity, a polypeptide having deribosylation activity, a polypeptide having myristoylation activity, or a polypeptide having demyristoylation activity.

[0232] The fusion protein may comprise any one of the mutant Cas12a endonucleases described herein (e.g., any one of SEQ ID NOs: 48-119 and 367-387, preferably any one of SEQ ID NOs: 367-387). In some embodiments, the fusion protein comprises any one of the base editing enzymes described herein.

[0233] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising a mutation at an amino acid position corresponding to position R833, with reference to the numbering of the amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutation is R833L, R833K, or R833M. In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position R833, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position R833, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO:1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO:1).

[0234] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising a mutation at an amino acid position corresponding to position K932, with reference to the numbering of the amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutation is K932E or K932G. In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position K932, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position K932, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO:1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO:1).

[0235] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising a mutation at an amino acid position corresponding to position Q944, with reference to the numbering of the amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutation is Q944K. In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position Q944, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position Q944, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO:1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO:1).

[0236] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising a mutation at an amino acid position corresponding to position K940, with reference to the numbering of the amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutation is K940G. In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position K940, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease comprises a mutation at position K940, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO:1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO:1).

[0237] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising mutations at amino acid positions corresponding to positions K932, N933, V936 and S929, with reference to the numbering of amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutations are K932G, N933G, V936G and S929G. In some embodiments, the mutant LbCas12a endonuclease comprises mutations at positions K932, N933, V936 and S929, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease contains mutations at positions K932, N933, V936 and S929, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO:1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO:1).

[0238] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising mutations at amino acid positions corresponding to positions K940 and Q944, with reference to the numbering of amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutations are K940G and Q944K. In some embodiments, the mutant LbCas12a endonuclease comprises mutations at positions K940 and Q944, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease contains mutations at positions K940 and Q944, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO:1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO:1).

[0239] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising mutations at amino acid positions corresponding to positions R836 and Q944, with reference to the numbering of amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutations are R836G and Q944K. In some embodiments, the mutant LbCas12a endonuclease comprises mutations at positions R836 and Q944, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease contains mutations at positions R836 and Q944, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO:1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO:1).

[0240] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising mutations at amino acid positions corresponding to positions R833, E835, and Y943, with reference to the numbering of amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutations are R833M, E835D, and Y943T. In some embodiments, the mutant LbCas12a endonuclease comprises mutations at positions R833, E835, and Y943, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease contains mutations at positions R833, E835, and Y943, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO:1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO:1).

[0241] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising mutations at amino acid positions corresponding to positions R836, Q944, and R935, with reference to the numbering of the amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutations are R836G, Q944K, and R935G. In some embodiments, the mutant LbCas12a endonuclease comprises mutations at positions R836, Q944, and R935, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease contains mutations at positions R836, Q944, and R935, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO:1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO:1).

[0242] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising mutations at amino acid positions corresponding to positions R833, E835, Y943, and R935, with reference to the numbering of amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutations are R833M, E835D, Y943T, and R935G. In some embodiments, the mutant LbCas12a endonuclease comprises mutations at positions R833, E835, Y943, and R935, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease contains mutations at positions R833, E835, Y943, and R935, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO:1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO:1).

[0243] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising mutations at amino acid positions corresponding to positions R833, E835, Y943, and Q941, with reference to the numbering of amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutations are R833M, E835D, Y943T, and Q941K. In some embodiments, the mutant LbCas12a endonuclease comprises mutations at positions R833, E835, Y943, and Q941, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease contains mutations at positions R833, E835, Y943, and Q941, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO:1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO:1).

[0244] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising mutations at amino acid positions corresponding to positions R833, E835, and E125, with reference to the numbering of amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutations are R833M, E835D, and E125A. In some embodiments, the mutant LbCas12a endonuclease comprises mutations at positions R833, E835, and E125, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease contains mutations at positions R833, E835, and E125, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO:1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO:1).

[0245] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising mutations at amino acid positions corresponding to positions Y943, Q944, K932, N933, and E125, with reference to the numbering of amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutations are Y943F, Q944K, K932G, N933G, and E125A. In some embodiments, the mutant LbCas12a endonuclease contains mutations at positions Y943, Q944, K932, N933, and E125, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO:1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO:1). In some embodiments, the mutant LbCas12a endonuclease contains mutations at positions Y943, Q944, K932, N933, and E125, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO:1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO:1).

[0246] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising mutations at amino acid positions corresponding to positions R836, Q944, R935, and E125, with reference to the numbering of amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutations are R836G, Q944K, R935G, and E125A. In some embodiments, the mutant LbCas12a endonuclease comprises mutations at positions R836, Q944, R935, and E125, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). In some embodiments, the mutant LbCas12a endonuclease contains mutations at positions R836, Q944, R935, and E125, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO:1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO:1).

[0247] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising mutations at amino acid positions corresponding to positions R833, E835, Y943, R935, and E125, with reference to the numbering of amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutations are R833M, E835D, Y943T, R935G, and E125A. In some embodiments, the mutant LbCas12a endonuclease contains mutations at positions R833, E835, Y943, R935, and E125, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO:1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO:1). In some embodiments, the mutant LbCas12a endonuclease contains mutations at positions R833, E835, Y943, R935, and E125, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO:1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO:1).

[0248] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising mutations at amino acid positions corresponding to positions R833, E835, Y943, Q941, and E125, with reference to the numbering of amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutations are R833M, E835D, Y943T, Q941K, and E125A. In some embodiments, the mutant LbCas12a endonuclease contains mutations at positions R833, E835, Y943, Q941, and E125, with reference to the numbering of amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO:1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO:1). In some embodiments, the mutant LbCas12a endonuclease contains mutations at positions R833, E835, Y943, Q941, and E125, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO:1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO:1).

[0249] In some embodiments, the mutant Cas12a endonuclease comprises a polypeptide sequence comprising mutations at amino acid positions corresponding to positions D832, Y943, Q944, K932, N933, and E125, with reference to the numbering of amino acid positions of LbCas12a ND2006 (e.g., SEQ ID NO: 1). In some embodiments, the mutations are D832A, Y943F, Q944K, K932G, N933G, and E125A. In some embodiments, the mutant LbCas12a endonuclease contains mutations at positions D832, Y943, Q944, K932, N933, and E125, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO:1), and has at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO:1). In some embodiments, the mutant LbCas12a endonuclease contains mutations at positions D832, Y943, Q944, K932, N933, and E125, with reference to the numbering of the amino acid positions of LbCas12a ND2006 endonuclease (e.g., SEQ ID NO:1), and has no more than 1, 2, 3, 4, or 5 additional substitutions relative to the wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO:1).

[0250] In some embodiments, the fusion protein comprises a mutant Cas12a endonuclease located at or near the N-terminus of the protein. In some embodiments, the fusion protein comprises a mutant Cas12a endonuclease located at or near the C-terminus of the protein. In some embodiments, the fusion protein comprises a base editing enzyme located at or near the N-terminus of the protein. In some embodiments, the fusion protein comprises a base editing enzyme located at or near the C-terminus of the protein. In some embodiments, the fusion protein comprises an N-terminal mutant Cas12a endonuclease and a C-terminal base editing enzyme (i.e., the mutant Cas12a endonuclease is located closer to the N-terminus of the protein than the base editing enzyme). In some embodiments, the fusion protein comprises an N-terminal base editing enzyme and a C-terminal mutant Cas12a endonuclease (i.e., the base editing enzyme is located closer to the N-terminus of the protein than the mutant Cas12a endonuclease).

[0251] The fusion protein may include one or more nuclear localization signals (NLS). In some embodiments, the fusion protein includes one, two, three, four, five, or more NLSs. An NLS is an amino acid sequence that directs the fusion protein to be imported into the cell nucleus by nuclear transport. In some embodiments, the NLS is a positively charged amino acid sequence that includes several lysine and / or arginine amino acids. In some embodiments, the fusion protein includes an NLS located at or near the N-terminus of the protein. In some embodiments, the fusion protein includes an NLS located at or near the C-terminus of the protein. In some embodiments, the fusion protein includes an NLS located at or near the N-terminus of the protein and an NLS located at or near the C-terminus of the protein.

[0252] Non-limiting examples of NLSs include SV40 NLS, nucleoprotein (NP) NLS, and bipartite (BP) NLS. In some embodiments, the SV40 NLS comprises the amino acid sequence of PKKKRKV (SEQ ID NO: 193). In some embodiments, the nucleoprotein NLS comprises the amino acid sequence of KRPAATKKAGQAKKKK (SEQ ID NO: 194). In some embodiments, the bipartite NLS comprises the amino acid sequence of KRTADGSEFESPKKKRKV (SEQ ID NO: 195). In some embodiments, the fusion protein comprises an SV40 NLS located at or near the N-terminus of the protein, and / or an SV40 NLS located at or near the C-terminus of the protein. In some embodiments, the fusion protein comprises an SV40 NLS located at or near the N-terminus of the protein, an SV40 NLS located at or near the C-terminus of the protein, and an NP NLS located at or near the C-terminus of the protein. In some embodiments, the fusion protein comprises an NP NLS located at or near the N-terminus of the protein, an NP NLS located at or near the C-terminus of the protein, and an SV40 NLS located at or near the C-terminus of the protein. In some embodiments, the fusion protein comprises a BP NLS located at or near the N-terminus of the protein, an SV40 NLS located at or near the C-terminus of the protein, and an NP NLS located at or near the C-terminus of the protein. In some embodiments, the fusion protein comprises a BP NLS located at or near the N-terminus of the protein, and a BP NLS located at or near the C-terminus of the protein. In some embodiments, the fusion protein comprises a BP NLS located at or near the N-terminus of the protein, and an NP NLS located at or near the C-terminus of the protein.

[0253] The fusion protein may include one or more linkers. The linkers for use in the fusion proteins of the present disclosure are generally amino acid linkers. In some embodiments, the linker functions to provide separation between different protein elements of the fusion protein (e.g., mutant Cas12a endonuclease and base editing enzyme). In some embodiments, the presence of a linker between two protein elements of the fusion protein provides flexibility between the two elements of the fusion protein (e.g., allows each protein to fold and perform its function, e.g., enzymatic function). In some embodiments, the fusion protein includes a linker between the mutant Cas12a endonuclease and the base editing enzyme.

[0254] In some embodiments, the linker is a flexible linker. In some embodiments, the linker is a flexible linker comprising serine and / or glycine amino acids. In some embodiments, the linker is an amino acid sequence, and a majority of the amino acids of the linker are serine and / or glycine amino acids. In some embodiments, the linker comprises an amino acid sequence of (GS)n (SEQ ID NO: 196), (GGS)n (SEQ ID NO: 197), (GSS)n (SEQ ID NO: 198), (GGSS)n (SEQ ID NO: 199), (SGGGS)n (SEQ ID NO: 200) or (SGGS)n (SEQ ID NO: 201), where n is 1 to 10. In some embodiments, the linker comprises an amino acid sequence of GSSGGSGGSGGSGS (SEQ ID NO: 202). In some embodiments, the linker comprises an amino acid sequence of SGSETPGTSESATPES (SEQ ID NO: 203). In other embodiments, the linker comprises an amino acid sequence of SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 204). In some embodiments, the linker comprises the amino acid sequence of SGGSGGSGGS (SEQ ID NO: 205). In some embodiments, the linker comprises the amino acid sequence of GGGGGGS (SEQ ID NO: 206), GSSGGSGGSGGS (SEQ ID NO: 207), or SGGS (SEQ ID NO: 208).

[0255] In various embodiments, the base editor fusion protein further comprises a base excision repair inhibitor ("iBER") that binds covalently or non-covalently to the mutated nucleobase and prevents its removal during subsequent mismatch repair. The use of iBER in the base editor fusion protein may increase base editing efficiency over deamination-oxidation and other strategies. In certain embodiments, the iBER is an 8-oxo-guanine glycosylase (OGG or OGGI) inhibitor ("OGG inhibitor"), a thymine-DNA glycosylase (TDG) inhibitor, a uracil-DNA glycosylase (UDG) inhibitor, or a methyl-CpG binding domain 4 (MBD4) inhibitor. In certain embodiments, the iBER comprises a catalytically inactive OGG that binds to 8-oxo-inosine and prevents its excision during subsequent mismatch repair.

[0256] The fusion protein may include one or more uracil glycosylase inhibitors (UGI). In some embodiments, the fusion protein includes one, two, three, four, or five UGI polypeptides. In some embodiments, the UGI is a polypeptide that can inhibit uracil-DNA glycosylase base excision repair enzymes (e.g., from the uracil base excision repair (UBER) pathway). In some embodiments, the UGI can inhibit the repair mechanism, such that the UGI polypeptide increases the efficiency of C to T conversion. In some embodiments, the UGI polypeptide reduces the rate of non-C to T conversion. The UGI polypeptide may be a wild-type UGI polypeptide or a mutant UGI polypeptide. In some embodiments, the UGI is UGI from Bacillus subtilis bacteriophage PBS1. In some embodiments, the UGI polypeptide comprises the amino acid sequence of TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVML LTSDAPEYKPWALVIQDSNGENKIKML (SEQ ID NO: 209). In some embodiments, the UGI polypeptide comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 97% identity to SEQ ID NO: 209.

[0257] The fusion protein may comprise one or more DNA glycosylases. In some embodiments, the fusion protein comprises one, two, three, four, or five DNA glycosylases. In some embodiments, the fusion protein comprises uracil DNA glycosylase (UNG). In some embodiments, the fusion protein comprises N-methylpurine glycosylase (MPG).

[0258] In some embodiments, N-methylpurine glycosylase (MPG) functions in the recognition and repair of base pairs containing deoxyinosine.Hypoxanthine (the nucleobase of deoxyinosine) is recognized and removed by MPG, resulting in an abasic site (AP site).The abasic site is then processed by the base excision repair pathway.Thus, MPG is useful in some embodiments for A to C or A to T conversion, especially when combined with adenosine deaminase (e.g., TadA deaminase).

[0259] In some embodiments, the fusion protein comprises MPG and an adenosine deaminase (e.g., TadA deaminase). In such embodiments, the adenosine deaminase converts adenine to inosine, and MPG subsequently removes the inosine to generate an abasic site. The abasic site can then be processed (e.g., to place a cytosine at that position). In some embodiments, the fusion protein comprises MPG and a cytidine deaminase (e.g., APOBEC1 deaminase).

[0260] In some embodiments, the MPG polypeptide comprises the amino acid sequence of SEQ ID NO: 210. In some embodiments, the MPG polypeptide comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 97% identity to SEQ ID NO: 210. VTPALQMKKPKQFCRRMGQKKQRPARAGQPHSSSDAAQAPAEQPHSSSDAAQAPCPRERCLGPPTTPGPYRSIYFSSPKGHLTRLGLEFFDQPAVPLARAFLGQVLVRRLPNGTELRGRIVETEAYLGPEDEAAHSRGGRQTPRNRGMFMKPGTLYVYIIYGMYFCMNISSQGDGACVLLRALEPLEGLETMRQLRSTLRKGTASRVLKDRELCSGPSKLCQALAINKSFDQRDLAQDEAVWLERGPLEPSEPAVVAAARVGVGHAGEWARKPLRFYVRGSPWVSVVDRVAEQDTQA (SEQ ID NO: 210)

[0261] In some embodiments, UNG is a polypeptide that can recognize and remove uracil from DNA strands. UNG polypeptide can remove unwanted uracil bases from DNA molecules by cleaving N-glycosidic bonds and initiating base excision repair (BER) pathway. In some embodiments, UNG can increase the efficiency of C to G conversion. UNG polypeptide can be human UNG (hUNG) or E. coli UNG (eUNG). In some embodiments, hUNG polypeptide is mitochondrial UNG1 or nuclear UNG2A. UNG polypeptide can be wild type UNG polypeptide or mutant UNG polypeptide. In some embodiments, the UNG polypeptide comprises the amino acid sequence of ANELTWHDVLAEEKQQPYFLNTLQTVASERQSGVTIYPPQKDVFNAFRFTELGDVKVVILGQDPYHGPGQAHGLAFSVRPGIAIPPSLLNMYKELENTIPGFTRPNHGYLESWARQGVLLLNTVLTVRAGQAHSHASLGWETFTDKVISLINQHREGVVFLLWGSHAQKKGAIIDKQRHHVLKAPHPSPLSAHRGFFGCNHFVLANQWLEQRGETPIDWMPVLPAESE (SEQ ID NO: 211). In some embodiments, the UNG polypeptide comprises the amino acid sequence of IGQKTLYSFFSPSPARKRHAPSPEPAVQGTGVAGVPEESGDAAAIPAKKAPAGQEEPGTPPSSPLSAEQLDRIQRNKAAALLRLAARNVPVGFGESWKKHLSGEFGKPYFIKLMGFVAEERKHYTVYPPPHQVFTWTQMCDIKDVKVVILGQDPYHGPNQAHGLCFSVQRPVPPPPPSLENIYKELSTDIEDFVHPGHGDLSGWAKQGVLLLNAVLTVRAHQANSHKERGWEQFTDAVVSWLNQNSNGLVFLLWGSYAQKKGSAIDRKRHHVLQTAHPSPLSVYRGFFGCRHFSKTNELLQKSGKKPIDWKEL (SEQ ID NO: 212).In some embodiments, the UNG polypeptide comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 97% identity to SEQ ID NO:211 or 212.

[0262] The fusion protein may comprise one or more DNA binding domains (DBDs). In some embodiments, the fusion protein comprises 1, 2, 3, 4, or 5 DBDs. In some embodiments, the DBD is a DBD that recognizes a sequence-specific single-stranded DNA molecule. In some embodiments, the DBD is a DBD that recognizes a non-sequence-specific single-stranded DNA molecule. In some embodiments, the DBD is a DBD that recognizes a sequence-specific double-stranded DNA molecule. In some embodiments, the DBD is a DBD that recognizes a non-sequence-specific double-stranded DNA molecule. In some embodiments, the DBD comprises a Rad51 DNA binding domain (DBD). The DBD may be a wild-type DBD polypeptide or a mutant DBD polypeptide. In some embodiments, the DBD polypeptide comprises the amino acid sequence of MAMQMQLEANADTSVEEESFGPQPISRLEQ CGINANDVKKLEEAGFHTVEAVAYAPKKELINIKGISEAKADKILAEAAKLVPMGFTTATEFHQRRSEIIQITTGSKELDKLLQ (SEQ ID NO: 213). In some embodiments, the DBD polypeptide comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 97% identity to SEQ ID NO:213.

[0263] Fusion proteins can be designed to be specific for a particular type of enzymatic conversion. For example, a particular fusion protein is designed for the C to T conversion, the A to G conversion, or the C to G conversion.

[0264] In some embodiments, a fusion protein comprising a mutant Cas12a endonuclease, a cytidine deaminase (e.g., APOBEC1), and a UGI is a C to T base editor (i.e., enzymatically converts C to T). In some embodiments, a fusion protein comprising a mutant Cas12a endonuclease, a cytidine deaminase (e.g., APOBEC1), a UGI, and a Rad51 DBD is a C to T base editor (i.e., enzymatically converts C to T). In some embodiments, a fusion protein comprising a mutant Cas12a endonuclease and a base editing enzyme (e.g., TadA) is an A to G base editor (i.e., enzymatically converts A to G). In some embodiments, a fusion protein comprising a mutant Cas12a endonuclease, an adenosine deaminase (e.g., TadA), and a Rad51 DBD is an A to G base editor (i.e., enzymatically converts A to G). In some embodiments, a fusion protein comprising a mutant Cas12a endonuclease and a cytidine deaminase (e.g., APOBEC1) is a C to G base editor (i.e., enzymatically converts C to G). In some embodiments, a fusion protein comprising a mutant Cas12a endonuclease, a cytidine deaminase (e.g., APOBEC1), and UNG is a C to G base editor (i.e., enzymatically converts C to G).

[0265] Exemplary, non-limiting fusion protein sequences are provided in Table 5.

[0266] [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4]

Table 5-5

Table 5-6

Table 5-7

Table 5-8

Table 5-9

Table 5-10

Table 5-11

Table 5-12

Table 5-13

Table 5-14

Table 5-15

Table 5-16

[0267] In some embodiments, the fusion protein comprises the amino acid sequence of any one of SEQ ID NOs: 163-185. In some embodiments, the fusion protein comprises an amino acid sequence that includes any one or more mutations (e.g., amino acid substitutions) described herein and has at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%) identity to the amino acid sequence of any one of the fusion proteins of Table 5 (e.g., SEQ ID NOs: 163-185).

[0268] VIII.Cells Aspects of the present disclosure relate to cells comprising any of the mutant Cas12a endonucleases described herein. Additionally, as described below, the mutant Cas12a endonucleases described herein may be used to modify cells.

[0269] The cell may be a eukaryotic cell or a prokaryotic cell. Non-limiting examples of eukaryotic cells include animal cells, plant cells, and fungal cells. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell (e.g., a human primary cell or a human immortalized cell). In some embodiments, the cell is a stem cell, such as an adult stem cell or an induced pluripotent stem cell (iPSC). Non-human cells are also provided herein. For example, the cell can be selected from a non-human primate cell, a porcine cell, a bovine cell, a canine cell, a feline cell, or a rodent cell (e.g., a rat or mouse cell).

[0270] A variety of human cell types are contemplated, including, but not limited to, immune cells (e.g., T cells (e.g., NKT cells, CD4+ T cells, CD8+ T cells, regulatory T cells, artificial T cells, e.g., CAR-T, TCR)), B cells, NK cells, tumor infiltrating lymphocytes, etc.), neuronal cells, cardiovascular cells, epidermal cells, and metabolic cells. The cells may be cancerous or non-cancerous. In some embodiments, the cells are tumor cells. Other cell types are also contemplated herein.

[0271] IX.How to use The Cas12a endonuclease provided herein has many uses, many of which are known in the art.The mutant Cas12a endonuclease of the present disclosure can be used in some cases to improve their use.Some non-limiting examples of such uses include genome editing, biotechnology, diagnosis and agricultural advances.

[0272] A. Genome Editing In some embodiments, mutant Cas12a endonucleases are used for genome editing. Genome editing is a type of genetic engineering in which DNA is inserted into, deleted from, or replaced in the genome of an organism. The application of CRISPR-Cas systems as molecular tools for genome editing utilizes their ability to generate double-strand breaks (DSBs) at specific genomic loci and entirely relies on the DNA repair mechanisms of host cells to repair the damage caused by these systems. Repair mechanisms can be either of the following processes: homology-directed repair (HDR) or non-homologous end joining (NHEJ). HDR utilizes template DNA (unbroken sister chromatid or homologous chromosome) that is homologous to the break site to repair DSBs, whereas NHEJ is based on directly joining the broken ends of DSBs, making NHEJ the more error-prone mechanism of the two. Thus, HDR can be used to provide foreign template DNA to introduce user-defined changes into the host genome. NHEJ can be applied to disrupt genes, whereas HDR allows for the introduction of new genetic information or the direct modification of the sequence of specific loci.

[0273] Central to CRISPR-mediated genome engineering today is Cas9, with applications including, but not limited to, gene knockout and precise genome editing. Despite rapid progress in genome editing with Cas9, challenges still exist, such as the possibility of off-target effects and difficulties in delivery of ribonucleoprotein particles. Cas12a presents an alternative molecular genome editing tool due to substantial differences from Cas9. The use of Cas12a in genome editing of various cell types has been explored in several studies to date. A comparative study of gene suppression by catalytically-dead Cas9 (SpdCas9) from Streptococcus pyogenes and catalytically-dead Cas12a (EedCas12a) from Eubacterium eligens revealed that the latter exhibited higher gene suppression in the template strand of the target DNA than SpdCas9. The pre-crRNA processing activity of Cas12a was also shown to make it an attractive candidate for multiplexed gene regulation, which is tedious when attempted with Cas9. This automated processing of unique crRNAs has been used to simultaneously modify multiple genetic elements to generate constitutive, conditional, inducible, orthogonal, and multiplexed genome engineering of endogenous targets using multiple CRISPR RNAs delivered on a single plasmid.

[0274] The viability of this approach has been further established by other studies, where multiplex gene regulation by Cas12a has been successfully observed in bacteria, plants, and mammalian cells. Cas12a may also serve as a solution in cell types where the use of Cas9 is toxic, such as some industrial strains of Streptomyces.

[0275] As shown in rice, targeted mutagenesis in plants can also be achieved by co-expressing Cas12a and its cognate crRNA in vivo. Furthermore, it was shown that mutagenesis was more efficient using pre-crRNA with full-length direct repeats rather than mature crRNA. Efficient mutagenesis by delivery of preassembled ribonucleoprotein (RNP) particles was also observed in soybean and wild tobacco. RNPs were assembled from recombinantly expressed Cas12a and in vitro transcribed or chemically synthesized crRNA.

[0276] Successful gene editing of mammalian cells using Cas12a has included the correction of the causative mutation of Duchenne muscular dystrophy (DMD) in patient-derived induced pluripotent stem cells (iPSCs) and in mdx mice, a well-known research model of DMD. After Cas12a-mediated gene editing, dystrophin expression was restored in iPSCs, and correction of pathophysiological features of muscular dystrophy was observed in mdx mice. Delivery of an adenoviral vector carrying an AsCas12a expression cassette successfully induced the mutation in primary human hepatocytes in humanized mice with chimeric livers. Cas12a-mediated genome editing has also been used to engineer a rat model mimicking human atherosclerosis, and this system may have applications in understanding early atherosclerosis.

[0277] See Paul, B. & Montoya, G. et al. Biomedical Journal 2020; 43(1): 8-17, incorporated herein in its entirety.

[0278] B. Biotechnology In some embodiments, mutant Cas12a endonucleases are used in bioengineering. Currently, huge efforts are underway to redesign all these tools for biomedical and biotechnological applications. However, recent studies have envisioned the possibility of using CRISPR-Cas nucleases to bioengineer smart materials, such as hydrogels. These water-filled polymers are encapsulated by DNA. Cas12a has been used to specifically degrade the DNA scaffolding of DNA hydrogels, opening the possibility of turning this smart cutter into a programmable device that delivers DNA-encapsulated hydrogel cargo at a specific time and place. The cleavage properties of Cas12a make it an ideal candidate to facilitate controlled delivery of cargo. See Paul, B. & Montoya, G. et al. Biomedical Journal 2020; 43(1): 8-17, incorporated herein in its entirety.

[0279] C. Detection and Quantification of Nucleic Acids (e.g., Diagnostics) In some embodiments, mutant Cas12a endonucleases are used to detect and / or quantify nucleic acids. For example, mutants can be used as in vitro diagnostic tools for pathogenic (e.g., bacterial or viral) nucleic acids, or to identify biomarkers indicative of diseases such as cancer (e.g., for identifying and quantifying single CpG methylation sites, see, e.g., van Dongen, JE et al. Biosensors and Bioelectronics 2021; 194(15): 113624).

[0280] In some embodiments, mutant Cas12a endonucleases are used with Specific Enhancer for Detection of PCR-amplified Nucleic Acids (SENA) methods that combine the trans-cleavage activity of Cas12a with the sensitivity provided by real-time PCR. See, e.g., Huang W, et al. EBioMedicine 2020; 61: 103036.

[0281] In some embodiments, mutant Cas12a endonucleases are used with DNA endonuclease-targeted CRISPR transreporter (DETECTR) technology that simultaneously reverse-transcription and isothermal amplification using loop-mediated amplification (RT-LAMP) on RNA extracted from biological samples. See, for example, Broughton JP, et al. Nature Biotechnology 2020; 38: 870-874.

[0282] In some embodiments, mutant Cas12a endonucleases are used with 1-hour low-cost versatile high-efficiency system (HOLMES) technology. See, e.g., Li, L. et al. ACS Synth Biol. 2019; 8(10): 2228-2237.

[0283] Other nucleic acid detection methods are also contemplated herein.

[0284] D. Agriculture In some embodiments, mutant Cas12a endonuclease is used for agricultural advancement applications.Cas12a editing is widely used in many crops, including rice, wheat, corn, soybean, cotton, tomato, citrus, tobacco, and model plant Arabidopsis.Currently, three Cas12a genome editing systems, AsCas12a, FnCas12a, and LbCas12a, have been demonstrated in plants with various efficiencies.

[0285] Rice is one of the most studied crops due to its agronomic importance, small genome size, ease of transformation and available genetic resources making it ideal as a flagship genome for grass crops. These factors also make it an ideal testing ground for developing genome editing technologies. Codon-optimized FnCas12a binary vectors were utilized for targeted mutagenesis in rice (OsDL, OsALS, OsNCED1, OsAO1) and tobacco (NtPDS and NtSTF1) with targeted mutation frequencies averaging 47.2% and 28.2%, respectively. Using LbCas12a nuclease, two rice endogenous genes, OsPDS and OsBEL, were targeted with mutation frequencies of 21.4% and 41.2%, respectively. An independent study targeting the disruption of OsPDS with LbCas12a similarly yielded a high editing frequency of 32.3%. It was also shown that pre-crRNA was more efficient at generating mutants than mature crRNA in rice. However, the opposite results were observed in HEK293T cells. In addition to these proof-of-concept experiments, LbCas12a was also used to generate loss-of-function alleles of OsEPFL9, which regulates stomatal density. These lines showed 8-fold increased water use efficiency in T2 generation plants. See Bandyopadhyay, A. et al. Front. Plant Sci. 2020.

[0286] Additional Embodiments Additional embodiments are described in the following numbered paragraphs:

[0287] 1. An engineered mutant Cas12a endonuclease comprising a polypeptide sequence comprising a mutation at an amino acid position corresponding to position E95, E125, V245, N260, Y277, R747, H759, I765, F810, N813, T814, I831, T870, G902, K960, S982, K984, or T988, with reference to the numbering of the amino acid positions of LbCas12a ND2006.

[0288] 2. The engineered mutant Cas12a endonuclease of paragraph 1, wherein the mutant Cas12a endonuclease exhibits high activity, low promiscuous single-stranded deoxyribonuclease (DNase) activity, targeted nickase activity (or preference for cleaving one strand of dsDNA over the other), or protospacer adjacent motif (PAM) nickase activity.

[0289] 3. The engineered mutant Cas12a endonuclease of paragraph 1 or 2, wherein the mutation is an amino acid substitution.

[0290] 4. The engineered mutant Cas12a endonuclease of paragraph 3, wherein the amino acid substitutions are amino acids of equivalent charge, polarity and / or chemical class.

[0291] 5. The engineered mutant Cas12a endonuclease of any one of paragraphs 1 to 4, wherein (a) the polypeptide sequence comprises a mutation at an amino acid position corresponding to position E95, E125, V245, Y277, R747, H759, I765, F810, or T814, with reference to the numbering of the amino acid positions of LbCas12a ND2006; and (b) the mutant Cas12a endonuclease exhibits high activity.

[0292] 6. The engineered mutant Cas12a endonuclease of paragraph 5, wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position E95, with reference to the numbering of the amino acid positions of LbCas12a ND2006, and wherein the polypeptide sequence may have at least 90% identity to a wild-type reference Cas12a endonuclease, optionally a wild-type reference Cas12a endonuclease in Table 1.

[0293] 7. The engineered mutant Cas12a endonuclease of paragraph 6, wherein the mutation is a substitution of a polar, positively charged, and / or basic amino acid at position E95, preferably E95R or E95H, more preferably E95R, with reference to the numbering of the amino acid positions of LbCas12a ND2006.

[0294] 8. The engineered mutant Cas12a endonuclease of paragraph 6, wherein the mutation is a polar, uncharged, and / or aromatic amino acid substitution at position E95, preferably E95Y, with reference to the numbering of the amino acid positions of LbCas12a ND2006.

[0295] 9. The engineered mutant Cas12a endonuclease of paragraph 5, wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position E125, with reference to the numbering of the amino acid positions of LbCas12a ND2006, and wherein the polypeptide sequence may have at least 90% identity to a wild-type reference Cas12a endonuclease, optionally a wild-type reference Cas12a endonuclease of Table 1.

[0296] 10. The engineered mutant Cas12a endonuclease of paragraph 9, wherein the mutation is a non-polar, uncharged and / or aliphatic amino acid substitution at position E125, preferably E125A, E125G, E125I, E125L, E125P, E125V, more preferably E125A, with reference to the numbering of the amino acid positions of LbCas12a ND2006.

[0297] 14. The engineered mutant Cas12a endonuclease of paragraph 5, wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position V245, with reference to the numbering of the amino acid positions of LbCas12a ND2006, and wherein the polypeptide sequence may have at least 90% identity to a wild-type reference Cas12a endonuclease, optionally a wild-type reference Cas12a endonuclease of Table 1.

[0298] 15. The engineered mutant Cas12a endonuclease of paragraph 14, wherein the mutation is a non-polar, uncharged and / or aliphatic amino acid substitution at position V245, preferably V245I, V245A, V245G, V245L, or V245P, more preferably V245I, with reference to the numbering of the amino acid positions of LbCas12a ND2006.

[0299] 16. The engineered mutant Cas12a endonuclease of paragraph 14, wherein the mutation is a polar, uncharged and / or aromatic amino acid substitution at position V245, preferably V245Y, with reference to the numbering of the amino acid positions of LbCas12a ND2006.

[0300] 21. The engineered mutant Cas12a endonuclease of paragraph 5, wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position Y277, with reference to the numbering of the amino acid positions of LbCas12a ND2006, and wherein the polypeptide sequence may have at least 90% identity to a wild-type reference Cas12a endonuclease, optionally a wild-type reference Cas12a endonuclease of Table 1.

[0301] 22. The engineered mutant Cas12a endonuclease of paragraph 21, wherein the mutation is a polar, uncharged and / or hydroxy amino acid substitution at position Y277, preferably Y277S or Y277T, more preferably Y277S, with reference to the numbering of the amino acid positions of LbCas12a ND2006.

[0302] 29. The engineered mutant Cas12a endonuclease of paragraph 5, wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position R747, with reference to the numbering of the amino acid positions of LbCas12a ND2006, and wherein the polypeptide sequence may have at least 90% identity to a wild-type reference Cas12a endonuclease, optionally a wild-type reference Cas12a endonuclease of Table 1.

[0303] 30. The engineered mutant Cas12a endonuclease of paragraph 29, wherein the mutation is a polar, uncharged and / or aromatic amino acid substitution at position R747, preferably R747Y, with reference to the numbering of the amino acid positions of LbCas12a ND2006.

[0304] 31. The engineered mutant Cas12a endonuclease of paragraph 5, wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position H759, with reference to the numbering of the amino acid positions of LbCas12a ND2006, and wherein the polypeptide sequence may have at least 90% identity to a wild-type reference Cas12a endonuclease, optionally a wild-type reference Cas12a endonuclease of Table 1.

[0305] 32. The engineered mutant Cas12a endonuclease of paragraph 31, wherein the mutation is a non-polar, uncharged and / or aliphatic amino acid substitution at position H759, preferably H759V, H759A, H759G, H759I, H759L, or H759P, more preferably H759V, with reference to the numbering of the amino acid positions of LbCas12a ND2006.

[0306] 33. The engineered mutant Cas12a endonuclease of paragraph 31, wherein the mutation is a polar, negatively charged and / or acidic amino acid substitution at position H759, preferably H759D, with reference to the numbering of the amino acid positions of LbCas12a ND2006.

[0307] 34. The engineered mutant Cas12a endonuclease of paragraph 5, wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position I765, with reference to the numbering of the amino acid positions of LbCas12a ND2006, and wherein the polypeptide sequence may have at least 90% identity to a wild-type reference Cas12a endonuclease, optionally a wild-type reference Cas12a endonuclease of Table 1.

[0308] 35. The engineered mutant Cas12a endonuclease of paragraph 34, wherein the mutation is a polar, uncharged and / or aromatic amino acid substitution at position I765, preferably I765Y, with reference to the numbering of the amino acid positions of LbCas12a ND2006.

[0309] 36. The engineered mutant Cas12a endonuclease of paragraph 5, wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position F810, with reference to the numbering of the amino acid positions of LbCas12a ND2006, and wherein the polypeptide sequence may have at least 90% identity to a wild-type reference Cas12a endonuclease, optionally a wild-type reference Cas12a endonuclease of Table 1.

[0310] 37. The engineered mutant Cas12a endonuclease of paragraph 36, wherein the mutation is a substitution of a non-polar, uncharged and / or aromatic amino acid at position F810, preferably F810W, with reference to the numbering of the amino acid positions of LbCas12a ND2006.

[0311] 38. The engineered mutant Cas12a endonuclease of paragraph 36, wherein the mutation is a substitution of a polar, charged and / or acidic amino acid at position F810, preferably F810Q, with reference to the numbering of the amino acid positions of LbCas12a ND2006.

[0312] 39. The engineered mutant Cas12a endonuclease of paragraph 5, wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position T814, with reference to the numbering of the amino acid positions of LbCas12a ND2006, and wherein the polypeptide sequence may have at least 90% identity to a wild-type reference Cas12a endonuclease, optionally a wild-type reference Cas12a endonuclease of Table 1.

[0313] 40. The engineered mutant Cas12a endonuclease of paragraph 39, wherein the mutation is a substitution of a non-polar, uncharged and / or aromatic amino acid at position T814, preferably T814E, with reference to the numbering of the amino acid positions of LbCas12a ND2006.

[0314] 41. The engineered mutant Cas12a endonuclease of paragraph 39, wherein the mutation is a substitution of a polar, positively charged and / or basic amino acid at position T814, preferably T814R, T814H or T814K, more preferably T814R, with reference to the numbering of the amino acid positions of LbCas12a ND2006.

[0315] 42. An engineered mutant Cas12a endonuclease described in any one of paragraphs 1 to 4, wherein (a) the polypeptide sequence comprises a mutation at an amino acid position corresponding to position N813, I831, T870, G902, S982, K984, or T988, with reference to the numbering of the amino acid positions of LbCas12a ND2006, and (b) the mutant Cas12a endonuclease exhibits reduced promiscuous single-stranded deoxyribonuclease (ssDNase) activity.

[0316] 43. The engineered mutant Cas12a endonuclease of paragraph 42, wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position N813, with reference to the numbering of the amino acid positions of LbCas12a ND2006, and wherein the polypeptide sequence may have at least 90% identity to a wild-type reference Cas12a endonuclease, optionally a wild-type reference Cas12a endonuclease of Table 1.

[0317] 44. The engineered mutant Cas12a endonuclease of paragraph 43, wherein the mutation is a substitution of a non-polar, uncharged and / or aromatic amino acid at position N813, preferably N813W, with reference to the numbering of the amino acid positions of LbCas12a ND2006.

[0318] 45. The engineered mutant Cas12a endonuclease of paragraph 43, wherein the mutation is a substitution of a polar, positively charged and / or basic amino acid at position N813, preferably N813R, N813H or N813K, more preferably N813R or N813H, with reference to the numbering of the amino acid positions of LbCas12a ND2006.

[0319] 46. ​​The engineered mutant Cas12a endonuclease of paragraph 42, wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position I831, with reference to the numbering of the amino acid positions of LbCas12a ND2006, and wherein the polypeptide sequence has at least 90% identity to a wild-type reference Cas12a endonuclease, optionally a wild-type reference Cas12a endonuclease in Table 1.

[0320] 47. The engineered mutant Cas12a endonuclease of paragraph 46, wherein the mutation is a non-polar, uncharged and / or aliphatic amino acid substitution at position I831, preferably I831A, I831G, I831L, I831P, or I831V, more preferably I831A, with reference to the numbering of the amino acid positions of LbCas12a ND2006.

[0321] 48. The engineered mutant Cas12a endonuclease of paragraph 46, wherein the mutation is a polar, uncharged and / or aromatic amino acid substitution at position I831, preferably I831Y, with reference to the numbering of the amino acid positions of LbCas12a ND2006.

[0322] 49. The engineered mutant Cas12a endonuclease of paragraph 42, wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position T870, with reference to the numbering of the amino acid positions of LbCas12a ND2006, and wherein the polypeptide sequence has at least 90% identity to a wild-type reference Cas12a endonuclease, optionally a wild-type reference Cas12a endonuclease in Table 1.

[0323] 50. The engineered mutant Cas12a endonuclease of paragraph 47, wherein the mutation is a polar, uncharged and / or aromatic amino acid substitution at position T870, preferably T870Y, with reference to the numbering of the amino acid positions of LbCas12a ND2006.

[0324] 51. The engineered mutant Cas12a endonuclease of paragraph 47, wherein the mutation is a substitution of a non-polar, uncharged and / or aromatic amino acid at position T870, preferably T870F or T870W, more preferably T870F, with reference to the numbering of the amino acid positions of LbCas12a ND2006.

[0325] 52. The engineered mutant Cas12a endonuclease of paragraph 42, wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position G902, with reference to the numbering of the amino acid positions of LbCas12a ND2006, and wherein the polypeptide sequence may have at least 90% identity to a wild-type reference Cas12a endonuclease, optionally a wild-type reference Cas12a endonuclease of Table 1.

[0326] 53. The engineered mutant Cas12a endonuclease of paragraph 52, wherein the mutation is a polar, positively charged and / or basic amino acid substitution at position G902, preferably G902R, G902H or G902K, more preferably G902R, with reference to the numbering of the amino acid positions of LbCas12a ND2006.

[0327] 54. The engineered mutant Cas12a endonuclease of paragraph 52, wherein the mutation is a substitution of a non-polar, uncharged and / or aromatic amino acid at position G902, preferably G902W or G902F, more preferably G902W, with reference to the numbering of the amino acid positions of LbCas12a ND2006.

[0328] 55. The engineered mutant Cas12a endonuclease of paragraph 42, wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position S982, with reference to the numbering of the amino acid positions of LbCas12a ND2006, and wherein the polypeptide sequence may have at least 90% identity to a wild-type reference Cas12a endonuclease, optionally a wild-type reference Cas12a endonuclease of Table 1.

[0329] 56. The engineered mutant Cas12a endonuclease of paragraph 55, wherein the mutation is a substitution of a non-polar, uncharged and / or aromatic amino acid at position S982, preferably S982F or S982W, more preferably S982W, with reference to the numbering of the amino acid positions of LbCas12a ND2006.

[0330] 57. The engineered mutant Cas12a endonuclease of paragraph 42, wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position K984, with reference to the numbering of the amino acid positions of LbCas12a ND2006, and wherein the polypeptide sequence may have at least 90% identity to a wild-type reference Cas12a endonuclease, optionally a wild-type reference Cas12a endonuclease of Table 1.

[0331] 58. The engineered mutant Cas12a endonuclease of paragraph 57, wherein the mutation is a substitution of a non-polar, uncharged and / or aromatic amino acid at position K984, preferably K984F or K984W, more preferably K984F, with reference to the numbering of the amino acid positions of LbCas12a ND2006.

[0332] 59. The engineered mutant Cas12a endonuclease of paragraph 57, wherein the mutation is a substitution of a polar, positively charged and / or basic amino acid at position K984, preferably K984R, K984H or K984K, more preferably K984R, with reference to the numbering of the amino acid positions of LbCas12a ND2006.

[0333] 60. The engineered mutant Cas12a endonuclease of paragraph 42, wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position T988, with reference to the numbering of the amino acid positions of LbCas12a ND2006, and wherein the polypeptide sequence may have at least 90% identity to a wild-type reference Cas12a endonuclease, optionally a wild-type reference Cas12a endonuclease of Table 1.

[0334] 61. The engineered mutant Cas12a endonuclease of paragraph 60, wherein the mutation is a substitution of a non-polar, uncharged and / or aromatic amino acid at position T988, preferably T988F or T988W, more preferably T988F, with reference to the numbering of the amino acid positions of LbCas12a ND2006.

[0335] 62. An engineered mutant Cas12a endonuclease according to any one of paragraphs 1 to 4, wherein (a) the polypeptide sequence comprises a mutation at an amino acid position corresponding to position N260 or G902, with reference to the numbering of the amino acid positions of LbCas12a ND2006, and (b) the mutant Cas12a endonuclease exhibits targeted nickase activity (or a preference for cleaving one strand of dsDNA over the other strand).

[0336] 63. The engineered mutant Cas12a endonuclease of paragraph 62, wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position N260, with reference to the numbering of the amino acid positions of LbCas12a, and wherein the polypeptide sequence may have at least 90% identity to a wild-type reference Cas12a endonuclease, optionally a wild-type reference Cas12a endonuclease in Table 1.

[0337] 64. The engineered mutant Cas12a endonuclease of paragraph 63, wherein the mutation is a substitution of a polar, positively charged and / or basic amino acid at position N260, preferably N260R, N260H or N260K, more preferably N260R, with reference to the numbering of the amino acid positions of LbCas12a ND2006.

[0338] 65. The engineered mutant Cas12a endonuclease of paragraph 62, wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position G902, with reference to the numbering of the amino acid positions of LbCas12a ND2006, and wherein the polypeptide sequence may have at least 90% identity to a wild-type reference Cas12a endonuclease, optionally a wild-type reference Cas12a endonuclease of Table 1.

[0339] 66. The engineered mutant Cas12a endonuclease of paragraph 65, wherein the mutation is a substitution of a non-polar, uncharged and / or aromatic amino acid at position G902, preferably G902W or G902F, more preferably G902W, with reference to the numbering of the amino acid positions of LbCas12a ND2006.

[0340] 67. The engineered mutant Cas12a endonuclease of any one of paragraphs 1 to 4, wherein (a) the polypeptide sequence comprises a mutation at an amino acid position corresponding to position K960, with reference to the numbering of the amino acid positions of LbCas12a ND2006, and (b) the mutant Cas12a endonuclease exhibits PAM nickase activity (or a preference for cleaving one strand of dsDNA over the other strand).

[0341] 68. The engineered mutant Cas12a endonuclease of paragraph 67, wherein the mutation is a polar, negatively charged and / or amide amino acid substitution at position K960, preferably K960E, with reference to the numbering of the amino acid positions of LbCas12a ND2006, and wherein the polypeptide sequence may have at least 90% identity to a wild-type reference Cas12a endonuclease, optionally to a wild-type reference Cas12a endonuclease of Table 1.

[0342] 69. The engineered mutant Cas12a endonuclease of any one of the preceding paragraphs, comprising an amino acid sequence having at least 85%, at least 90%, or at least 95% but less than 100% identity to the amino acid sequence of a wild-type Cas12a endonuclease selected from Acidaminococcus species, Lachnospira species, and Francisella species.

[0343] 70. The engineered mutant Cas12a endonuclease of any one of the preceding paragraphs, further comprising no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions relative to a wild-type reference Cas12a endonuclease.

[0344] 71. An engineered mutant Cas12a endonuclease comprising a polypeptide sequence comprising the amino acid sequence of any one of SEQ ID NOs: 48 to 119.

[0345] 72. A polynucleotide encoding a mutant Cas12a endonuclease according to any one of the preceding paragraphs.

[0346] 73. A cell comprising (a) a mutant Cas12a endonuclease according to any one of the preceding paragraphs or a polynucleotide according to paragraph 72 and (b) a guide RNA or a polynucleotide encoding a guide RNA.

[0347] 74. A method comprising introducing into a cell (a) a mutant Cas12a endonuclease according to any one of the preceding paragraphs or a polynucleotide according to paragraph 72, and, optionally, (b) a guide RNA or a polynucleotide encoding the guide RNA.

[0348] 75. Use of a mutant Cas12a endonuclease according to any one of the preceding paragraphs to cleave a nucleic acid.

[0349] 76. A method for introducing a double-strand break into a target nucleic acid, comprising: Introducing into a cell containing a target nucleic acid (a) a mutant Cas12a endonuclease according to any one of paragraphs 5 to 41 and (b) a guide RNA; incubating the cells to cause a double-stranded break in the target nucleic acid; A method comprising:

[0350] 77. A method for introducing a double-strand break into a target nucleic acid, comprising: Introducing into a cell containing a target nucleic acid (a) a mutant Cas12a endonuclease according to any one of paragraphs 42 to 61 and (b) a guide RNA; incubating the cells to cause a double-stranded break in the target nucleic acid; A method comprising:

[0351] 78. The method of paragraph 77, wherein off-target single-stranded nucleic acid cleavage in the cell is reduced compared to off-target single-stranded nucleic acid cleavage in a control cell comprising a wild-type Cas12a endonuclease and a guide RNA.

[0352] 79. A method for introducing a single-stranded break into a target nucleic acid, comprising: introducing into a cell containing the target nucleic acid (a) a mutant Cas12a endonuclease described in any one of paragraphs 62 to 68; and (b) a guide RNA; incubating the cells to produce a single-stranded break in the target nucleic acid; A method comprising:

[0353] Further embodiments Further embodiments are described in the following numbered paragraphs:

[0354] 1. An engineered mutant Cas12a endonuclease comprising a polypeptide sequence comprising a mutation at an amino acid position corresponding to position R833, E835, R836, S929, F931, K932, N933, S934, R935, V936, K937, V938, K940, Q941, Y943, Q944, F983, or M986, with reference to the numbering of the amino acid positions of LbCas12a ND2006.

[0355] 2. An engineered mutant Cas12a endonuclease according to paragraph 1, which exhibits high activity, low promiscuous single-stranded deoxyribonuclease (DNase) activity, or targeted nickase activity (or preference for cleaving one strand of dsDNA over the other).

[0356] 3. The engineered mutant Cas12a endonuclease of paragraph 1 or 2, wherein the mutation is an amino acid substitution.

[0357] 4. The engineered mutant Cas12a endonuclease of paragraph 3, wherein the amino acid substitutions are amino acids of equivalent charge, polarity, and / or chemical class.

[0358] 5. The engineered mutant Cas12a endonuclease of any one of paragraphs 1 to 4, wherein (a) the polypeptide sequence comprises a mutation at an amino acid position corresponding to position K932, N933, or V936, with reference to the numbering of the amino acid positions of LbCas12a ND2006, and (b) the engineered mutant Cas12a endonuclease exhibits high activity.

[0359] 6. The engineered mutant Cas12a endonuclease of paragraph 5, wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position K932, with reference to the numbering of the amino acid positions of LbCas12a ND2006, and wherein the polypeptide sequence may have at least 90% identity to a wild-type reference Cas12a endonuclease, optionally a wild-type reference Cas12a endonuclease of Table 1.

[0360] 7. The engineered mutant Cas12a endonuclease of paragraph 6, wherein the mutation is a non-polar, uncharged, and / or aliphatic amino acid substitution at position K932, preferably K932I, K932L, K932V, K932A, or K932P, more preferably K932I, K932L, or K932V, with reference to the numbering of the amino acid positions of LbCas12a ND2006.

[0361] 8. The engineered mutant Cas12a endonuclease of paragraph 5, wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position N933, with reference to the numbering of the amino acid positions of LbCas12a ND2006, and wherein the polypeptide sequence may have at least 90% identity to a wild-type reference Cas12a endonuclease, optionally a wild-type reference Cas12a endonuclease of Table 1.

[0362] 9. The engineered mutant Cas12a endonuclease of paragraph 8, wherein the mutation is a substitution of a non-polar, uncharged, and / or aliphatic amino acid at position N933, preferably N933L, N933A, N933G, N933I, or N933P, more preferably N933L, with reference to the numbering of the amino acid positions of LbCas12a.

[0363] 10. The engineered mutant Cas12a endonuclease of paragraph 5, wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position V936, with reference to the numbering of the amino acid positions of LbCas12a ND2006, and wherein the polypeptide sequence may have at least 90% identity to a wild-type reference Cas12a endonuclease, optionally a wild-type reference Cas12a endonuclease of Table 1.

[0364] 11. The engineered mutant Cas12a endonuclease of paragraph 10, wherein the mutation is a substitution of a non-polar, uncharged, and / or sulfate amino acid at position V936, preferably V936M or V936C, more preferably V936M, with reference to the numbering of the amino acid positions of LbCas12a.

[0365] 12. The engineered mutant Cas12a endonuclease of any one of paragraphs 1 to 4, wherein (a) the polypeptide sequence comprises a mutation at an amino acid position corresponding to position S929, K932, N933, S934, V936, K937, Q944, F983, or M986, with reference to the numbering of the amino acid positions of LbCas12a ND2006; and (b) the mutant Cas12a endonuclease exhibits reduced promiscuous single-stranded deoxyribonuclease (ssDNase) activity.

[0366] 13. The engineered mutant Cas12a endonuclease of paragraph 12, wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position S929, with reference to the numbering of the amino acid positions of LbCas12a, and wherein the polypeptide sequence may have at least 90% identity to a wild-type reference Cas12a endonuclease, optionally a wild-type reference Cas12a endonuclease of Table 1.

[0367] 14. The engineered mutant Cas12a endonuclease of paragraph 13, wherein the mutation is a substitution of a non-polar, uncharged and / or aliphatic amino acid at position S929, preferably S929L, S929A, S929I, S929P or S929V, more preferably S929L, with reference to the numbering of the amino acid positions of LbCas12a.

[0368] 15. The engineered mutant Cas12a endonuclease of paragraph 12, wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position K932, with reference to the numbering of the amino acid positions of LbCas12a ND2006, and wherein the polypeptide sequence may have at least 90% identity to a wild-type reference Cas12a endonuclease, optionally a wild-type reference Cas12a endonuclease of Table 1.

[0369] 16. The engineered mutant Cas12a endonuclease of paragraph 15, wherein the mutation is a substitution of a polar, positively charged, and / or basic amino acid at position K932, preferably K932R, with reference to the numbering of the amino acid positions of LbCas12a.

[0370] 17. The engineered mutant Cas12a endonuclease of paragraph 15, wherein the mutation is a polar, uncharged and / or hydroxy amino acid substitution at position K932, preferably K932T, with reference to the numbering of the amino acid positions of LbCas12a.

[0371] 18. The engineered mutant Cas12a endonuclease of paragraph 15, wherein the mutation is a substitution of a non-polar, uncharged, and / or aromatic amino acid at position K932, preferably K932F or K932W, with reference to the numbering of the amino acid positions of LbCas12a.

[0372] 19. The engineered mutant Cas12a endonuclease of paragraph 15, wherein the mutation is a polar, uncharged and / or aromatic amino acid substitution at position K932, preferably K932Y, with reference to the numbering of the amino acid positions of LbCas12a.

[0373] 20. The engineered mutant Cas12a endonuclease of paragraph 12, wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position N933, with reference to the numbering of the amino acid positions of LbCas12a ND2006, and wherein the polypeptide sequence may have at least 90% identity to a wild-type reference Cas12a endonuclease, optionally a wild-type reference Cas12a endonuclease of Table 1.

[0374] 21. The engineered mutant Cas12a endonuclease of paragraph 20, wherein the mutation is a substitution of a polar, negatively charged and / or amide amino acid at position N933, preferably N933E, with reference to the numbering of the amino acid positions of LbCas12a.

[0375] 22. The engineered mutant Cas12a endonuclease of paragraph 20, wherein the mutation is a substitution of a non-polar, uncharged and / or aliphatic amino acid at position N933, preferably N933V, N933A, N933G, N933I or N933P, more preferably N933V, with reference to the numbering of the amino acid positions of LbCas12a.

[0376] 23. The engineered mutant Cas12a endonuclease of paragraph 12, wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position S934, with reference to the numbering of the amino acid positions of LbCas12a ND2006, and wherein the polypeptide sequence may have at least 90% identity to a wild-type reference Cas12a endonuclease, optionally a wild-type reference Cas12a endonuclease of Table 1.

[0377] 24. The engineered mutant Cas12a endonuclease of paragraph 23, wherein the mutation is a substitution of a polar, uncharged and / or acidic amino acid at position S934, preferably S934Q, with reference to the numbering of the amino acid positions of LbCas12a.

[0378] 25. The engineered mutant Cas12a endonuclease of paragraph 12, wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position V936, with reference to the numbering of the amino acid positions of LbCas12a ND2006, and wherein the polypeptide sequence may have at least 90% identity to a wild-type reference Cas12a endonuclease, optionally a wild-type reference Cas12a endonuclease of Table 1.

[0379] 26. The engineered mutant Cas12a endonuclease of paragraph 25, wherein the mutation is a substitution of a polar, negatively charged and / or amide amino acid at position V936, preferably V936E, with reference to the numbering of the amino acid positions of LbCas12a.

[0380] 27. The engineered mutant Cas12a endonuclease of paragraph 25, wherein the mutation is a substitution of a polar, positively charged and / or basic amino acid at position V936, preferably V936K, V936R, or V936H, more preferably V936K, with reference to the numbering of the amino acid positions of LbCas12a.

[0381] 28. The engineered mutant Cas12a endonuclease of paragraph 12, wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position K937, with reference to the numbering of the amino acid positions of LbCas12a ND2006, and wherein the polypeptide sequence may have at least 90% identity to a wild-type reference Cas12a endonuclease, optionally a wild-type reference Cas12a endonuclease of Table 1.

[0382] 26. The engineered mutant Cas12a endonuclease of paragraph 28, wherein the mutation is a polar, uncharged and / or aromatic amino acid substitution at position K937, preferably K937Y, with reference to the numbering of the amino acid positions of LbCas12a.

[0383] 30. The engineered mutant Cas12a endonuclease of paragraph 12, wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position Q944, with reference to the numbering of the amino acid positions of LbCas12a ND2006, and wherein the polypeptide sequence may have at least 90% identity to a wild-type reference Cas12a endonuclease, optionally a wild-type reference Cas12a endonuclease of Table 1.

[0384] 31. The engineered mutant Cas12a endonuclease of paragraph 30, wherein the mutation is a polar, negatively charged and / or acidic amino acid substitution at position Q944, preferably Q944D, with reference to the numbering of the amino acid positions of LbCas12a.

[0385] 32. The engineered mutant Cas12a endonuclease of paragraph 30, wherein the mutation is a substitution of a non-polar, uncharged and / or sulfated amino acid at position Q944, preferably Q944M or Q944C, more preferably Q944M, with reference to the numbering of the amino acid positions of LbCas12a.

[0386] 33. The engineered mutant Cas12a endonuclease of paragraph 30, wherein the mutation is a substitution of a non-polar, uncharged and / or aliphatic amino acid at position Q944, preferably Q944G, Q944I, Q944A, Q944L, Q944P or Q944V, more preferably Q944G, with reference to the numbering of the amino acid positions of LbCas12a.

[0387] 34. The engineered mutant Cas12a endonuclease of paragraph 12, wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position F983, with reference to the numbering of the amino acid positions of LbCas12a ND2006, and wherein the polypeptide sequence may have at least 90% identity to a wild-type reference Cas12a endonuclease, optionally a wild-type reference Cas12a endonuclease in Table 1.

[0388] 35. The engineered mutant Cas12a endonuclease of paragraph 34, wherein the mutation is a substitution of a non-polar, uncharged and / or aliphatic amino acid at position F983, preferably F983L, F983A, F983G, F983I, F983P or F983V, more preferably F983L, with reference to the numbering of the amino acid positions of LbCas12a.

[0389] 36. The engineered mutant Cas12a endonuclease of paragraph 12, wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position M986, with reference to the numbering of the amino acid positions of LbCas12a ND2006, and wherein the polypeptide sequence may have at least 90% identity to a wild-type reference Cas12a endonuclease, optionally a wild-type reference Cas12a endonuclease in Table 1.

[0390] 37. The engineered mutant Cas12a endonuclease of paragraph 36, wherein the mutation is a substitution of a non-polar, uncharged and / or aromatic amino acid at position M986, preferably M986F or M986W, more preferably M986F, with reference to the numbering of the amino acid positions of LbCas12a.

[0391] 38. The engineered mutant Cas12a endonuclease of paragraph 36, wherein the mutation is a polar, uncharged and / or hydroxy amino acid substitution at position M986, preferably M986S or M986T, more preferably M986S, with reference to the numbering of the amino acid positions of LbCas12.

[0392] 39. The engineered mutant Cas12a endonuclease of any one of paragraphs 1 to 4, wherein (a) the polypeptide sequence comprises a mutation at an amino acid position corresponding to position R833, E835, R836, F931, K932, R935, V936, V938, K940, Q941, Y943, Q944, or M986, with reference to the numbering of the amino acid positions of LbCas12a ND2006; and (b) the mutant Cas12a endonuclease exhibits targeted nickase activity (or a preference for cleaving one strand of dsDNA over the other strand).

[0393] 40. The engineered mutant Cas12a endonuclease of paragraph 39, wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position R833, with reference to the numbering of the amino acid positions of LbCas12a ND2006, and wherein the polypeptide sequence may have at least 90% identity to a wild-type reference Cas12a endonuclease, optionally a wild-type reference Cas12a endonuclease of Table 1.

[0394] 41. The engineered mutant Cas12a endonuclease of paragraph 40, wherein the mutation is a substitution of a polar, positively charged and / or basic amino acid at position R833, preferably R833K or R833H, more preferably R833K, with reference to the numbering of the amino acid positions of LbCas12a, and may also exhibit reduced promiscuous ssDNase activity.

[0395] 42. The engineered mutant Cas12a endonuclease of paragraph 40, wherein the mutation is a substitution of a non-polar, uncharged and / or sulfated amino acid at position R833, preferably R833M or R833C, more preferably R833M, with reference to the numbering of the amino acid positions of LbCas12a, and may also exhibit reduced promiscuous ssDNase activity.

[0396] 43. The engineered mutant Cas12a endonuclease of paragraph 40, wherein the mutation is a substitution of a non-polar, uncharged and / or aliphatic amino acid at position R833, preferably R833L, R833A, R833I, R833P or R833V, more preferably R833L, with reference to the numbering of the amino acid positions of LbCas12a, and may also exhibit reduced promiscuous ssDNase activity.

[0397] 44. The engineered mutant Cas12a endonuclease of paragraph 39, wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position E835, with reference to the numbering of the amino acid positions of LbCas12a ND2006, and wherein the polypeptide sequence may have at least 90% identity to a wild-type reference Cas12a endonuclease, optionally a wild-type reference Cas12a endonuclease in Table 1.

[0398] 45. The engineered mutant Cas12a endonuclease of paragraph 44, wherein the mutation is a substitution of a polar, negatively charged and / or acidic amino acid at position E835, preferably E835D, with reference to the numbering of the amino acid positions of LbCas12a, and may exhibit reduced activity.

[0399] 46. ​​The engineered mutant Cas12a endonuclease of paragraph 44, wherein the mutation is a substitution of a non-polar, uncharged and / or aliphatic amino acid at position E835, preferably E835G, E835A, E835I, E835L, E835P, or E835V, more preferably E835G or E835A, with reference to the numbering of the amino acid positions of LbCas12a.

[0400] 47. The engineered mutant Cas12a endonuclease of paragraph 39, wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position R836, with reference to the numbering of the amino acid positions of LbCas12a ND2006, and wherein the polypeptide sequence may have at least 90% identity to a wild-type reference Cas12a endonuclease, optionally a wild-type reference Cas12a endonuclease of Table 1.

[0401] 48. The engineered mutant Cas12a endonuclease of paragraph 47, wherein the mutation is a substitution of a non-polar, uncharged and / or aliphatic amino acid at position R836, preferably R836G, R836A, R836I, R836L, R836P, or R836V, more preferably R836G or R836A, with reference to the numbering of the amino acid positions of LbCas12a.

[0402] 49. The engineered mutant Cas12a endonuclease of paragraph 39, wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position F931, with reference to the numbering of the amino acid positions of LbCas12a ND2006, and wherein the polypeptide sequence may have at least 90% identity to a wild-type reference Cas12a endonuclease, optionally a wild-type reference Cas12a endonuclease in Table 1.

[0403] 50. The engineered mutant Cas12a endonuclease of paragraph 49, wherein the mutation is a substitution of a polar, positively charged, and / or basic amino acid at position F931, preferably F931H or F931K, more preferably F931H, with reference to the numbering of the amino acid positions of LbCas12a, and may exhibit reduced activity.

[0404] 51. The engineered mutant Cas12a endonuclease of paragraph 49, wherein the mutation is a substitution of a non-polar, uncharged and / or aliphatic amino acid at position F931, preferably F931L, F931A, F931G, F931I, F931P or F931V, more preferably F931L, with reference to the numbering of the amino acid positions of LbCas12a.

[0405] 52. The engineered mutant Cas12a endonuclease of paragraph 39, wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position K932, with reference to the numbering of the amino acid positions of LbCas12a ND2006, and wherein the polypeptide sequence may have at least 90% identity to a wild-type reference Cas12a endonuclease, optionally a wild-type reference Cas12a endonuclease of Table 1.

[0406] 53. The engineered mutant Cas12a endonuclease of paragraph 52, wherein the mutation is a substitution of a non-polar, uncharged, and / or aliphatic amino acid at position K932, preferably K932G or K932P, more preferably K932G, with reference to the numbering of the amino acid positions of LbCas12a, and may exhibit reduced activity.

[0407] 54. The engineered mutant Cas12a endonuclease of paragraph 52, wherein the mutation is a substitution of a polar, negatively charged and / or amide amino acid at position K932, preferably K932E, with reference to the numbering of the amino acid positions of LbCas12a, and may exhibit reduced activity.

[0408] 55. The engineered mutant Cas12a endonuclease of paragraph 52, wherein the mutation is a substitution of a polar, positively charged and / or basic amino acid at position K932, preferably K932H or K932R, more preferably K932H, with reference to the numbering of the amino acid positions of LbCas12a.

[0409] 56. The engineered mutant Cas12a endonuclease of paragraph 52, wherein the mutation is a substitution of a non-polar, uncharged, and / or sulfate amino acid at position K932, preferably K932M or K932C, more preferably K932M, with reference to the numbering of the amino acid positions of LbCas12a.

[0410] 57. The engineered mutant Cas12a endonuclease of paragraph 52, wherein the mutation is a substitution of a polar, uncharged, and / or amide amino acid at position K932, preferably K932N, with reference to the numbering of the amino acid positions of LbCas12a.

[0411] 58. The engineered mutant Cas12a endonuclease of paragraph 52, wherein the mutation is a polar, uncharged and / or acidic amino acid substitution at position K932, preferably K932Q, with reference to the numbering of the amino acid positions of LbCas12a.

[0412] 59. The engineered mutant Cas12a endonuclease of paragraph 52, wherein the mutation is a polar, uncharged, and / or hydroxy amino acid substitution at position K932, preferably K932S, with reference to the numbering of the amino acid positions of LbCas12a.

[0413] 60. The engineered mutant Cas12a endonuclease of paragraph 39, wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position R935, with reference to the numbering of the amino acid positions of LbCas12a ND2006, and wherein the polypeptide sequence may have at least 90% identity to a wild-type reference Cas12a endonuclease, optionally a wild-type reference Cas12a endonuclease of Table 1.

[0414] 61. The engineered mutant Cas12a endonuclease of paragraph 60, wherein the mutation is a substitution of a non-polar, uncharged and / or aliphatic amino acid at position R935, with reference to the numbering of the amino acid positions of LbCas12a, preferably R935L, R935G, R935I or R935P, more preferably R935L, R935G or R935I, and wherein the engineered mutant Cas12a endonuclease having an R935I substitution may exhibit reduced activity.

[0415] 62. The engineered mutant Cas12a endonuclease of paragraph 60, wherein the mutation is a substitution of a polar, positively charged, and / or basic amino acid at position R935, preferably R935H or R935K, with reference to the numbering of the amino acid positions of LbCas12a.

[0416] 63. The engineered mutant Cas12a endonuclease of paragraph 60, wherein the mutation is a substitution of a non-polar, uncharged, and / or aromatic amino acid at position R935, preferably R935F or R935W, with reference to the numbering of the amino acid positions of LbCas12a, and wherein the engineered mutant Cas12a endonuclease having an R935W substitution may exhibit reduced activity.

[0417] 64. The engineered mutant Cas12a endonuclease of paragraph 60, wherein the mutation is a substitution of a non-polar, uncharged, and / or sulfate amino acid at position R935, preferably R935M or R935C, more preferably R935M, with reference to the numbering of the amino acid positions of LbCas12a.

[0418] 65. The engineered mutant Cas12a endonuclease of paragraph 60, wherein the mutation is a substitution of a polar, uncharged, and / or amide amino acid at position R935, preferably R935N, with reference to the numbering of the amino acid positions of LbCas12a.

[0419] 66. The engineered mutant Cas12a endonuclease of paragraph 60, wherein the mutation is a polar, uncharged, and / or hydroxy amino acid substitution at position R935, preferably R935S or R935T, with reference to the numbering of the amino acid positions of LbCas12a.

[0420] 67. The engineered mutant Cas12a endonuclease of paragraph 39, wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position V936, with reference to the numbering of the amino acid positions of LbCas12a ND2006, and wherein the polypeptide sequence may have at least 90% identity to a wild-type reference Cas12a endonuclease, optionally a wild-type reference Cas12a endonuclease in Table 1.

[0421] 68. The engineered mutant Cas12a endonuclease of paragraph 67, wherein the mutation is a substitution of a non-polar, uncharged and / or aliphatic amino acid at position V936, preferably V936G, V936I, V936L, or V936P, more preferably V936G, with reference to the numbering of the amino acid positions of LbCas12a, and may also exhibit reduced promiscuous ssDNase activity.

[0422] 69. The engineered mutant Cas12a endonuclease of paragraph 39, wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position V938, with reference to the numbering of the amino acid positions of LbCas12a ND2006, and wherein the polypeptide sequence may have at least 90% identity to a wild-type reference Cas12a endonuclease, optionally a wild-type reference Cas12a endonuclease in Table 1.

[0423] 70. The engineered mutant Cas12a endonuclease of paragraph 69, wherein the mutation is a substitution of a polar, negatively charged and / or amide amino acid at position V938, preferably V938E, with reference to the numbering of the amino acid positions of LbCas12a, and may also exhibit reduced promiscuous ssDNase activity.

[0424] 71. The engineered mutant Cas12a endonuclease of paragraph 39, wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position K940, with reference to the numbering of the amino acid positions of LbCas12a ND2006, and wherein the polypeptide sequence may have at least 90% identity to a wild-type reference Cas12a endonuclease, optionally a wild-type reference Cas12a endonuclease of Table 1.

[0425] 72. The engineered mutant Cas12a endonuclease of paragraph 71, wherein the mutation is a substitution of a non-polar, uncharged, and / or aliphatic amino acid at position K940, preferably K940G, K940A, K940I, K940L, K940P, or K940V, more preferably K940G, with reference to the numbering of the amino acid positions of LbCas12a.

[0426] 73. The engineered mutant Cas12a endonuclease of paragraph 39, wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position Q941, with reference to the numbering of the amino acid positions of LbCas12a ND2006, and wherein the polypeptide sequence may have at least 90% identity to a wild-type reference Cas12a endonuclease, optionally a wild-type reference Cas12a endonuclease of Table 1.

[0427] 74. The engineered mutant Cas12a endonuclease of paragraph 73, wherein the mutation is a substitution of a polar, positively charged, and / or basic amino acid at position Q941, preferably Q941K, Q941R or Q941H, with reference to the numbering of the amino acid positions of LbCas12a.

[0428] 75. The engineered mutant Cas12a endonuclease of paragraph 73, wherein the mutation is a substitution of a polar, uncharged and / or aromatic amino acid at position Q941, preferably Q941Y, with reference to the numbering of the amino acid positions of LbCas12a.

[0429] 76. The engineered mutant Cas12a endonuclease of paragraph 39, wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position Y943, with reference to the numbering of the amino acid positions of LbCas12a ND2006, and wherein the polypeptide sequence may have at least 90% identity to a wild-type reference Cas12a endonuclease, optionally a wild-type reference Cas12a endonuclease in Table 1.

[0430] 77. The engineered mutant Cas12a endonuclease of paragraph 76, wherein the mutation is a polar, uncharged and / or hydroxy amino acid substitution at position Y943, preferably Y943T or Y943S, more preferably Y943T, with reference to the numbering of the amino acid positions of LbCas12.

[0431] 78. The engineered mutant Cas12a endonuclease of paragraph 76, wherein the mutation is a substitution of a non-polar, uncharged and / or aromatic amino acid at position Y943, preferably Y943F or Y943W, more preferably Y943F, with reference to the numbering of the amino acid positions of LbCas12a.

[0432] 79. The engineered mutant Cas12a endonuclease of paragraph 39, wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position Q944, with reference to the numbering of the amino acid positions of LbCas12a ND2006, and wherein the polypeptide sequence may have at least 90% identity to a wild-type reference Cas12a endonuclease, optionally a wild-type reference Cas12a endonuclease of Table 1.

[0433] 80. The engineered mutant Cas12a endonuclease of paragraph 79, wherein the mutation is a substitution of a polar, positively charged, and / or basic amino acid at position Q944, preferably Q944K, Q944R, or Q944H, more preferably Q944K, with reference to the numbering of the amino acid positions of LbCas12a.

[0434] 81. The engineered mutant Cas12a endonuclease of paragraph 79, wherein the mutation is a substitution of a polar, negatively charged and / or amide amino acid at position Q944, preferably Q944E, with reference to the numbering of the amino acid positions of LbCas12a.

[0435] 82. The engineered mutant Cas12a endonuclease of paragraph 39, wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position M986, with reference to the numbering of the amino acid positions of LbCas12a ND2006, and wherein the polypeptide sequence may have at least 90% identity to a wild-type reference Cas12a endonuclease, optionally a wild-type reference Cas12a endonuclease of Table 1.

[0436] 83. The engineered mutant Cas12a endonuclease of paragraph 82, wherein the mutation is a substitution of a non-polar, uncharged, and / or aliphatic amino acid at position M986, preferably M986G, M986A, M986I, M986P, or M986V, more preferably M986G, with reference to the numbering of the amino acid positions of LbCas12a, and may also exhibit reduced promiscuous ssDNase activity.

[0437] 84. An engineered mutant Cas12a endonuclease comprising a polypeptide sequence comprising amino acid substitutions corresponding to the following amino acid substitutions: R833L, S929L, K932M, Q944F and E947M, with reference to the numbering of the amino acid positions of LbCas12a ND20006, preferably exhibiting low promiscuous single-stranded DNase activity, and optionally exhibiting low activity.

[0438] 85. An engineered mutant Cas12a endonuclease comprising a polypeptide sequence comprising amino acid substitutions corresponding to the following amino acid substitutions: N933L and Q944M, with reference to the numbering of the amino acid positions of LbCas12a ND20006, and preferably exhibiting low promiscuous single-stranded DNase activity, and optionally low activity.

[0439] 86. An engineered mutant Cas12a endonuclease comprising a polypeptide sequence comprising amino acid substitutions corresponding to the following amino acid substitutions: R833K and E947D, with reference to the numbering of the amino acid positions of LbCas12a ND20006, preferably wherein the engineered mutant Cas12a endonuclease exhibits targeted nickase activity (or preference for cleaving one strand of dsDNA over the other).

[0440] 87. An engineered mutant Cas12a endonuclease comprising a polypeptide sequence comprising amino acid substitutions corresponding to the following amino acid substitutions: E835G and E880G, with reference to the numbering of the amino acid positions of LbCas12a ND20006, preferably wherein the engineered mutant Cas12a endonuclease exhibits targeted nickase activity (or preference for cleaving one strand of dsDNA over the other).

[0441] 88. An engineered mutant Cas12a endonuclease comprising a polypeptide sequence comprising amino acid substitutions corresponding to the following amino acid substitutions: S929G, K932G and N933G, with reference to the numbering of the amino acid positions of LbCas12a ND20006, preferably exhibiting targeted nickase activity (or preference for cleaving one strand of dsDNA over the other).

[0442] 89. An engineered mutant Cas12a endonuclease comprising a polypeptide sequence comprising amino acid substitutions corresponding to the following amino acid substitutions: S929G, K932G, N933G and V936G, with reference to the numbering of the amino acid positions of LbCas12a ND20006, preferably wherein the engineered mutant Cas12a endonuclease exhibits targeted nickase activity (or preference for cleaving one strand of dsDNA over the other).

[0443] 90. An engineered mutant Cas12a endonuclease comprising a polypeptide sequence comprising amino acid substitutions corresponding to the following amino acid substitutions: S929G, K932G, N933G and V936F, with reference to the numbering of the amino acid positions of LbCas12a ND20006, preferably exhibiting targeted nickase activity (or preference for cleaving one strand of dsDNA over the other).

[0444] 91. An engineered mutant Cas12a endonuclease comprising a polypeptide sequence comprising amino acid substitutions corresponding to the following amino acid substitutions: S929G, V936G, F983G and M986G, with reference to the numbering of the amino acid positions of LbCas12a ND20006, preferably wherein the engineered mutant Cas12a endonuclease exhibits targeted nickase activity (or preference for cleaving one strand of dsDNA over the other).

[0445] 92. An engineered mutant Cas12a endonuclease comprising a polypeptide sequence comprising amino acid substitutions corresponding to the following amino acid substitutions: G930A and F931L, with reference to the numbering of the amino acid positions of LbCas12a ND20006, preferably exhibiting targeted nickase activity (or preference for cleaving one strand of dsDNA over the other) and reduced promiscuous single-stranded DNase activity.

[0446] 93. An engineered mutant Cas12a endonuclease comprising a polypeptide sequence containing amino acid substitutions corresponding to the following amino acid substitutions: G930A, F931L, and S934Q, with reference to the numbering of the amino acid positions of LbCas12a, preferably exhibiting targeted nickase activity (or preference for cleaving one strand of dsDNA over the other).

[0447] 94. An engineered mutant Cas12a endonuclease comprising a polypeptide sequence comprising amino acid substitutions corresponding to the following amino acid substitutions: K932G and N933G, with reference to the numbering of the amino acid positions of LbCas12a ND20006, preferably exhibiting targeted nickase activity (or preference for cleaving one strand of dsDNA over the other).

[0448] 95. An engineered mutant Cas12a endonuclease comprising a polypeptide sequence comprising amino acid substitutions corresponding to the following amino acid substitutions: K932G, N933G and V936F, with reference to the numbering of the amino acid positions of LbCas12a ND20006, preferably exhibiting targeted nickase activity (or preference for cleaving one strand of dsDNA over the other).

[0449] 96. An engineered mutant Cas12a endonuclease comprising a polypeptide sequence comprising amino acid substitutions corresponding to the following amino acid substitutions: V936G, F983G and M986G, with reference to the numbering of the amino acid positions of LbCas12a ND20006, preferably wherein the engineered mutant Cas12a endonuclease exhibits targeted nickase activity (or preference for cleaving one strand of dsDNA over the other).

[0450] 97. An engineered mutant Cas12a endonuclease comprising a polypeptide sequence comprising amino acid substitutions corresponding to the following amino acid substitutions: F983G and M986G, with reference to the numbering of the amino acid positions of LbCas12a ND20006, preferably exhibiting targeted nickase activity (or preference for cleaving one strand of dsDNA over the other) and reduced promiscuous single-stranded DNase activity.

[0451] 98. An engineered mutant Cas12a endonuclease comprising a polypeptide sequence that includes a lid-hub domain, wherein the polypeptide comprises a mutation in the lid-hub domain or at an amino acid position adjacent to the lid-hub domain.

[0452] 99. The engineered mutant Cas12a endonuclease of paragraph 98, wherein the mutation is a substitution at a position corresponding to the following amino acid positions: R833, E835, E880, R836, S929, G930, F931, K932, N933, S934, R935, V936, K937, V938, K940, Q941, Y943, Q944, E947, F983, or M986, with reference to the numbering of the amino acid positions of LbCas12a ND20006.

[0453] 100. An engineered mutant Cas12a endonuclease according to paragraph 98 or 99, exhibiting high activity.

[0454] 101. The engineered mutant Cas12a endonuclease of paragraph 100, wherein the mutation corresponds to any one of the following amino acid substitutions: K932I, K932L, K932V, N933L, or V936M.

[0455] 102. The engineered mutant Cas12a endonuclease of paragraph 98 or 99, which exhibits low promiscuous single-stranded deoxyribonuclease (DNase) activity.

[0456] 103. The engineered mutant Cas12a endonuclease of paragraph 102, wherein the mutation is a substitution corresponding to any one of the following amino acid substitutions: S929L, K932F, K932R, K932T, K932Y, K932W, N933E, N933V, S934Q, V936E, V936K, K937Y, Q944D, Q944G, Q944M, F983L, M986F, or M986S.

[0457] 104. An engineered mutant Cas12a endonuclease according to paragraph 98 or 99, which exhibits targeted nickase activity (or a preference for cleaving one strand of dsDNA over the other).

[0458] 105. The mutation is any of the following amino acid substitutions: R833K, R833L, R833M, E835A, E835D, E835G, R836A, R836G, F931H, F931L, K932E, K932G, K932H, K932M, K932N, K932Q, K932S, R935F, R935G, R935H, R935I, R935K, R935L , R935M, R935N, R935S, R935T, R935W, V936G, V938E, K940G, Q941H, Q941K, Q941R, Q941Y, Y943F, Y943T, Q944E, Q944K, or M986G.

[0459] 106. The engineered mutant Cas12a endonuclease of any one of the preceding paragraphs, comprising an amino acid sequence having at least 85%, at least 90%, or at least 95% but less than 100% identity to the amino acid sequence of a wild-type Cas12a endonuclease selected from Acidaminococcus species, Lachnospira species, and Francisella species.

[0460] 107. The engineered mutant Cas12a endonuclease of any one of the preceding paragraphs, further comprising no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acid substitutions relative to the wild-type reference Cas12a endonuclease.

[0461] 108. An engineered mutant Cas12a endonuclease comprising a polypeptide sequence comprising the amino acid sequence of any one of SEQ ID NOs: 48 to 119.

[0462] 109. A polynucleotide encoding a mutant Cas12a endonuclease according to any one of the preceding paragraphs.

[0463] 110. A cell comprising (a) a mutant Cas12a endonuclease according to any one of the preceding paragraphs or a polynucleotide according to paragraph 109 and (b) a guide RNA or a polynucleotide encoding a guide RNA.

[0464] 111. A method comprising introducing into a cell (a) a mutant Cas12a endonuclease according to any one of the preceding paragraphs or a polynucleotide according to paragraph 109, and, optionally, (b) a guide RNA or a polynucleotide encoding the guide RNA.

[0465] 112. Use of a mutant Cas12a endonuclease according to any one of the preceding paragraphs for cleaving a nucleic acid.

[0466] 113. A method for introducing a double-strand break into a target nucleic acid, comprising: Introducing into a cell containing a target nucleic acid (a) a mutant Cas12a endonuclease according to any one of paragraphs 5 to 11 and (b) a guide RNA; incubating the cells to cause a double-stranded break in the target nucleic acid; A method comprising:

[0467] 114. A method for introducing a double-strand break into a target nucleic acid, comprising: Introducing into a cell containing a target nucleic acid (a) a mutant Cas12a endonuclease according to any one of paragraphs 12 to 38, 84, or 85, and (b) a guide RNA; incubating the cells to cause a double-stranded break in the target nucleic acid; A method comprising:

[0468] 115. The method of paragraph 114, wherein off-target single-stranded nucleic acid cleavage in the cell is reduced compared to off-target single-stranded nucleic acid cleavage in a control cell comprising a wild-type Cas12a endonuclease and a guide RNA.

[0469] 116. A method for introducing a single-strand break into a target nucleic acid, comprising: Introducing into a cell containing a target nucleic acid (a) a mutant Cas12a endonuclease according to any one of paragraphs 39 to 83 or 86 to 97 and (b) a guide RNA; incubating the cells to produce a single-stranded break in the target nucleic acid; A method comprising: EXAMPLES

[0470] [Example 1] Cas12a endonuclease mutants The purified Cas12a mutants in Table 3 complexed with crRNA were tested on (1) dsDNA containing a quencher at one site of the cleavage site and a fluorophore at the other site of the cleavage site on separate DNA strands, and (2) combined dsDNA with ssDNA containing neither a quencher nor a fluorophore, but both a quencher and a fluorophore. Retention of activity on dsDNA was confirmed by separation of the quencher and fluorophore upon cleavage, resulting in an increase in the luminescence signal over time (1).

[0471] High activity Cas12a endonuclease mutants were identified by having a higher reaction rate or a faster reaction initiation than naturally occurring (i.e., wild type (WT)) Cas12a. Among the subset of Cas12a endonuclease mutants with high activity, some mutants were also identified to have low ssDNase activity. Low ssDNase activity is identified by a reduced ability to cleave ssDNA and thereby separate the quencher and fluorophore when activated by a specific dsDNA, and thus a lack of a luminescence signal of comparable magnitude to wild type Cas12a (2). See Figures 2A-2D and Table 6.

[0472] Low activity Cas12a endonuclease mutants were identified by having a lower reaction rate or a slower reaction initiation than wild-type (WT) Cas12a. Among the subset of Cas12a endonuclease mutants with low activity, some mutants were also identified to have low ssDNase activity. Low ssDNase activity is identified by a reduced ability to cleave ssDNA and thereby separate the quencher and fluorophore when activated by a specific dsDNA, and thus by a lack of a luminescence signal of a magnitude comparable to that of wild-type Cas12a (2). See Figures 3A-E, 4A-C and Table 6.

[0473] Three Cas12a endonuclease mutants were identified that exhibited reduced ssDNase activity while possessing an activity profile similar to wild-type Cas12a (see Figure 5 and Table 6).

[0474] [Table 6-1] [Table 6-2]

[0475] [Example 2] Evaluation of interenzyme linkers The activity of different C to T base editors (Cas12a mutants and fusion proteins containing cytidine deaminase) containing linkers of various sizes and sequences between the different domains was tested in U2OS cells. 10 CTC 13 U2OS cells were transfected with a plasmid expressing the base editor together with a plasmid expressing TAGCCCT (SEQ ID NO: 186). Transfected cells were selected after 3 days and recultured for an additional 3 days, after which the cells were lysed and genomic DNA was extracted. Genomic regions around the gRNA were amplified by PCR and editing was analyzed by next generation sequencing. In these experiments, LbCas12a was either catalytically inactive (inactive Cas12a containing the D832A mutation), LbCas12a, or an LbCas12a mutant (TBN04).

[0476] LbBEv2 (containing linker 4 between rAPOBEC1 and LbCas12a and linker 5 between LbCas12a and UGI) showed efficient base editing (Figure 6A-6C). Figure 6A: % of total reads showing base editing at individual positions. This includes reads showing base editing without indels and reads carrying base editing and indels. Figure 6B: % of total reads showing only base editing at individual positions. This includes only reads showing base editing but not indels. Figure 6C: % of total reads showing base editing only at one specific position. This excludes total reads with base editing at multiple positions as well as reads carrying indels. Inactive Cas12a was used as a control.

[0477] The improvement in base editing efficiency by LbBEv2 was more pronounced when using a base editor containing the LbCas12a mutant (...

Claims

1. 1. An engineered mutant Cas12a endonuclease comprising a polypeptide sequence comprising a mutation at an amino acid position corresponding to position E95, E125, N256, R747, H759, N813, K932, N933, S934, V936, S982, or K984, with reference to the numbering of the amino acid positions of LbCasl2a ND2006, wherein optionally the endonuclease exhibits enhanced activity.

2. The engineered mutant Cas12a endonuclease of claim 1, wherein the mutation is E95R, E95Y, E125A, E125W, N256A, R747Y, H759V, H759D, N813R, N813H, K932L, N933E, N933V, S934Q, V936E, V936M, V936K, S982N, or K984R.

3. 1. An engineered mutant Cas12a endonuclease comprising a polypeptide sequence comprising a mutation at an amino acid position corresponding to position N256, 1831, K932, N933, S934, V936, Q944, S982, F983, K984, M986, or T988, with reference to the numbering of the amino acid positions of LbCasl2a ND2006, wherein optionally the endonuclease exhibits enhanced activity.

4. The engineered mutant Cas12a endonuclease of claim 3, wherein the mutation is N256K, I831A, I831Y, K932A, K932F, K932H, K932M, K932N, K932Q, K932R, K932S, K932T, K932W, K932Y, N933L, S934W, V936G, Q944D, Q944E, Q944K, Q944M, S982T, S982W, F983G, F983L, K984F, M986G, M986L, M986S, or T988F.

5. 4. The engineered mutant Cas12a endonuclease of claim 3, comprising a mutation at an amino acid position corresponding to position Q944, wherein the mutation may be Q944D, Q944E, Q944K, or Q944M, and further wherein the polypeptide sequence has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to the amino acid sequence of SEQ ID NO: 86, 87, 88, or 89.

6. 5. The engineered mutant Casl2a endonuclease of claim 4, wherein the polypeptide sequence comprises mutations selected from K932F and F983L, K932F and T988F, K932R and Q944D, K932R and F983L, K932R and T988F, K932Y and F983L, K932Y and T988F, N933L and Q944M, V936G and Q944D, V936G and S982W, V936G and M986G, V936G and T988F, Q944D and S982W, Q944D and F983L, Q944D and T988F, S982W and F983L, S982W and T988F, or F983G and M986G.

7. The following: (i) comprising the mutations K932R and Q944D, wherein the polypeptide sequence may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to the amino acid sequence of SEQ ID NO: 104; (ii) comprises the mutations N933L and Q944M, and the polypeptide sequence may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to the amino acid sequence of SEQ ID NO: 109; (iii) the mutations V936G and Q944D, and the polypeptide sequence may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to the amino acid sequence of SEQ ID NO: 110; (iv) mutations Q944D and S982W, and the polypeptide sequence may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to the amino acid sequence of SEQ ID NO: 114; (v) comprising the mutations Q944D and F983L, wherein the polypeptide sequence may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to the amino acid sequence of SEQ ID NO: 115; or (vi) mutations Q944D and T988F, and the polypeptide sequence may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to the amino acid sequence of SEQ ID NO: 116; 7. The engineered mutant Cas12a endonuclease of claim 6, wherein:

8. 1. An engineered mutant Cas12a endonuclease comprising a polypeptide sequence comprising a mutation at an amino acid position corresponding to position N813, 1831, K932, N933, S934, V936, Q944, S982, F983, K984, M986, or T988, with reference to the numbering of the amino acid positions of LbCasl2a ND2006, optionally wherein the endonuclease exhibits reduced promiscuous ssDNase activity.

9. The engineered mutant Cas12a endonuclease of claim 8, wherein the mutation is N813H, N813R, N813W, I831A, I831Y, K932A, K932F, K932H, K932M, K932N, K932Q, K932R, K932S, K932T, K932W, K932Y, N933E, N933L, S934K, S934Q, V936E, V936G, Q944D, Q944E, Q944K, S982W, F983G, F983L, K984F, M986F, M986G, or T988F.

10. 9. The engineered mutant Cas12a endonuclease of claim 8, comprising a mutation at an amino acid position corresponding to position Q944, wherein the mutation may be Q944D, Q944E, or Q944K, and further wherein the polypeptide sequence has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to the amino acid sequence of SEQ ID NO: 86, 87, or 88.

11. The engineered mutant Cas12a endonuclease of claim 9, wherein the mutations are (i) N933L and Q944M, or (ii) F983G and M986G.

12. The engineered mutant Cas12a endonuclease of claim 11, comprising mutations N933L and Q944M, and wherein the polypeptide sequence may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity to the amino acid sequence of SEQ ID NO:

109.

13. An engineered mutant Cas12a endonuclease comprising a polypeptide sequence containing one or more mutations at amino acid positions corresponding to positions R833, E835, R836, F931, R935, K940, Q941, Y943, and / or Q944, with reference to the amino acid position numbering of LbCas12a ND2006.

14. The engineered mutant Cas12a endonuclease of claim 13, wherein one or more mutations are selected from R833L, R833K, R833M, E835D, R836G, R935G, K940G, Q941K, Y943T, Y943F, and Q944K.

15. The engineered mutant Cas12a endonuclease of claim 14, wherein the mutations are K940G and Q944K; R836G and Q944K; R833M, E835D and Y943T; R836G, Q944K and R935G; R833M, E835D, Y943T and R935G; or R833M, E835D, Y943T and Q941K.

16. A fusion protein comprising an engineered mutant Cas12a endonuclease according to claims 1 to 15, and a base editing enzyme, wherein, optionally, the base editing enzyme comprises a deaminase, a guanine oxidase, or a guanine methyltransferase.

17. 17. The fusion protein of claim 16, wherein the deaminase is a cytidine deaminase or an adenosine deaminase, and optionally the deaminase comprises a rAPOBEC1 polypeptide, an evoAPOBEC1 polypeptide, a hAPOBEC3A polypeptide, an evoCDA polypeptide, an evoFERNY polypeptide, or a TadA polypeptide.

18. (i) further comprising a uracil glycosylase inhibitor (UGI); (ii) further comprising one or more nuclear localization signals (NLS), which may be selected from an SV40 NLS, a nucleoprotein (NP) NLS, and a bipartite (BP) NLS; (iii) uracil DNA glycosylase (UNG), optionally further comprising human UNG (hUNG) or Escherichia coli UNG (eUNG); (iv) further comprising an N-methylpurine glycosylase (MPG), optionally located at or near the N-terminus or C-terminus of the fusion protein; (v) further comprising one or more linkers, optionally wherein the linker comprises the sequence SGSETPGTSESATPES (SEQ ID NO: 203) or SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 204); and / or (vi) further comprising a DNA binding domain (DBD), wherein the DBD is a Rad51 DBD; 18. The fusion protein of claim 17.

19. A polynucleotide encoding an engineered mutant Cas12a endonuclease described in any one of claims 1 to 15 or a fusion protein comprising an engineered mutant Cas12a endonuclease described in claims 1 to 15 and a base editing enzyme.

20. 20. A cell comprising (a) a polynucleotide according to claim 19, and (b) a guide RNA or a polynucleotide encoding the guide RNA, optionally wherein the cell is a human cell.

21. 1. A method of gene editing, comprising: (i) contacting a target nucleic acid sequence with the fusion protein of claim 16 and a guide RNA, wherein the target nucleic acid comprises a target nucleobase; (ii) modifying the target nucleobase; A method comprising:

22. 22. The method of claim 21, which is carried out in vitro, ex vivo or in vivo.