WO2023131870A2 - Endonuclease variants and methods of use - Google Patents

Endonuclease variants and methods of use Download PDF

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Publication number
WO2023131870A2
WO2023131870A2 PCT/IB2023/000043 IB2023000043W WO2023131870A2 WO 2023131870 A2 WO2023131870 A2 WO 2023131870A2 IB 2023000043 W IB2023000043 W IB 2023000043W WO 2023131870 A2 WO2023131870 A2 WO 2023131870A2
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amino acid
endonuclease
seq
variant
lbcas12a
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French (fr)
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WO2023131870A8 (en
WO2023131870A3 (en
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Stefano STELLA
Nikolai WULFF
Faizaan MOHAMMAD
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Twelve Bio Aps
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Twelve Bio Aps
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Priority to CA3245296A priority Critical patent/CA3245296A1/en
Priority to EP23712320.3A priority patent/EP4460566A2/en
Priority to KR1020247026266A priority patent/KR20240145468A/ko
Priority to AU2023205461A priority patent/AU2023205461A1/en
Priority to IL314074A priority patent/IL314074A/en
Priority to JP2024541082A priority patent/JP2025502107A/ja
Priority to CN202380023337.1A priority patent/CN118742641A/zh
Priority to US18/726,872 priority patent/US20250388886A1/en
Application filed by Twelve Bio Aps filed Critical Twelve Bio Aps
Priority to MX2024008362A priority patent/MX2024008362A/es
Publication of WO2023131870A2 publication Critical patent/WO2023131870A2/en
Publication of WO2023131870A3 publication Critical patent/WO2023131870A3/en
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    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C12N9/222—Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes
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    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
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    • C12Y305/04—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amidines (3.5.4)
    • C12Y305/04005—Cytidine deaminase (3.5.4.5)
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]

Definitions

  • CRISPR-Cas systems Clustered regularly interspaced short palindromic repeats (CRISPR) that associate with Cas proteins to constitute an adaptive immune system that can combat attacks by foreign mobile genetic elements such as plasmids and phages.
  • CRISPR-Cas systems are classified into two classes (Classes 1 and 2) that are subdivided into six types (types I through VI).
  • Class 1 (types I, III and IV) systems use multiple Cas proteins in their CRISPR ribonucleoprotein effector nucleases and Class 2 systems (types II, V and VI) use a single Cas protein.
  • Class 2 type V is further classified into 4 subtypes (V-A, V–B, V–C, V–U).
  • V-A encodes the protein Cas12a (also known as Cpf1) and recently several high-resolution structures of Cas12a have provided an insight into its working mechanism.
  • Class 2 type V CRISPR-Cas12a is an RNA-guided endonuclease that has been harnessed as a genome editing tool. Broader use of these enzymes for gene and epigenetic editing requires improvement of certain properties.
  • SUMMARY The present disclosure provides, in some aspects, variant Cas12a endonucleases with improved properties, such as hyperactivity and low indiscriminate single strand DNA degradation activity.
  • Engineered variant endonucleases exhibit more efficient cleavage activity, relative to their wild-type reference Cas12a endonuclease.
  • engineered variant endonucleases of the present disclosure exhibit low to no indiscriminate single strand DNase activity.
  • variant Cas12a endonucleases that exhibit a preference for cleavage of one strand over the other strand of a double strand DNA. From structural studies of the LbCas12a ND2006 endonuclease, Applicants have identified a particular domain, referred to herein as the “LID-hub domain,” that is involved in a subset of catalytic events.
  • substitutions made at positions K932, N933, and V936 increase cleavage efficiency (“hyperactivity”) and certain substitutions made at positions K932, N933, V936, Q944, F983, and M986 reduce indiscriminate ssDNase activity.
  • Certain amino acid substitutions within the vicinity of the LID and LID-hub domains also impact activity (e.g., V938 or Q941).
  • variant Cas12a endonucleases comprise amino acid mutations at one or more amino acid positions within the lid region.
  • a variant Cas12a endonuclease comprises one or more mutations at an amino acid position corresponding to positions 925 to 937 of Lachnospiraceae bacterium ND2006 (e.g., SEQ ID NO: 1).
  • a variant Cas12a endonuclease comprises one or more mutations at an amino acid position corresponding to positions 936 to 948 of Lachnospiraceae bacterium COE1 (e.g., SEQ ID NO: 47).
  • variant Cas12a endonuclease(s) is interchangeable with the term “Cas12a variant.”
  • Some aspects relate to an engineered variant 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 amino acid position numbering of LbCas12a ND2006.
  • any one or more of the foregoing variant Cas12a endonucleases exhibits hyperactivity.
  • an engineered variant Cas12a endonuclease 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 amino acid position numbering of LbCas12a ND2006.
  • any one or more of the foregoing variant Cas12a endonucleases exhibits hyperactivity.
  • Some aspects relate to an engineered variant Cas12a endonuclease 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 amino acid position numbering of LbCas12a ND2006.
  • an engineered variant Cas12a endonuclease 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 amino acid position numbering of LbCas12a ND2006.
  • any one or more of the foregoing variant Cas12a endonucleases exhibits hypoactivity.
  • an engineered variant Cas12a endonuclease 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 amino acid position numbering of LbCas12a ND2006.
  • any one or more of the foregoing variant Cas12a endonucleases exhibits hypoactivity.
  • Yet other aspects relate to an engineered variant Cas12a endonuclease comprising a polypeptide sequence comprising mutations at an amino acid positions corresponding to 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 F983L; Q944D and T988F; S982W and F983L; S982W and T988F; or F983G and M986G with reference
  • any one or more of the foregoing variant Cas12a endonucleases exhibits hypoactivity.
  • Some aspects relate to an engineered variant Cas12a endonuclease 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 amino acid position numbering of LbCas12a ND2006.
  • as any one or more of the foregoing variant Cas12a endonucleases exhibits low (or no) ssDNase activity, such as low (or no) indiscriminate ssDNase activity.
  • an engineered variant Cas12a endonuclease 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 amino acid position numbering of LbCas12a ND2006.
  • any one or more of the foregoing variant Cas12a endonucleases exhibits low (or no) ssDNase activity, such as low (or no) indiscriminate ssDNase activity.
  • Yet other aspects relate to an engineered variant Cas12a endonuclease comprising a polypeptide sequence comprising mutations at an amino acid positions corresponding to positions: N933L and Q944M; or F983G and M986G with reference to amino acid position numbering of LbCas12a ND2006.
  • an engineered variant Cas12a endonuclease exhibits low (or no) ssDNase activity, such as low (or no) indiscriminate ssDNase activity.
  • an engineered variant Cas12a endonuclease is fused to an effector protein.
  • an engineered variant Cas12a endonuclease provided herein comprises an amino acid sequence having at least 85%, at least 90%, or least 95%, but less than 100% identity with the amino acid sequence of a wild-type Cas12a endonuclease selected from Acidaminococcus sp., Lachnospiraceae sp., and Francisella sp.
  • an engineered variant 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, a variant Cas12a endonuclease further comprises no more than 5 additional amino acid substitutions relative to a wild-type reference Cas12a endonuclease.
  • the present disclosure also provides an engineered variant 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.
  • polynucleotides encoding an engineered variant Cas12a endonuclease of the present disclosure.
  • cells comprising (a) an engineered variant Cas12a endonuclease of the present disclosure or a polynucleotide endonuclease of the present disclosure and (b) a guide RNA or a polynucleotide encoding a guide RNA.
  • Some aspects herein relate to a method comprising introducing into a cell (a) an engineered variant Cas12a endonuclease of the present disclosure or a polynucleotide of the present disclosure and optionally (b) a guide RNA or a polynucleotide encoding a guide RNA.
  • the present disclosure also provides uses of an engineered variant Cas12a endonuclease of the present disclosure for cleaving a nucleic acid.
  • a method for introducing a double strand break in a target nucleic acid comprises introducing into a cell comprising a target nucleic acid (a) an engineered variant Cas12a endonuclease of the present disclosure and (b) a guide RNA and incubating the cell to produce a double strand break in the target nucleic acid.
  • a method for introducing a double strand break in a target nucleic acid comprises introducing into a cell comprising a target nucleic acid (a) an engineered variant Cas12a endonuclease of the present disclosure and (b) a guide RNA and incubating the cell to produce a double strand break in the target nucleic acid.
  • the off-target single strand nucleic acid cleavage in the cell is reduced relative to off-target single strand nucleic acid cleavage in a control cell comprising a wild-type Cas12a endonuclease and a guide RNA.
  • a method for introducing a single strand break in a target nucleic acid comprises introducing into a cell comprising a target nucleic acid (a) an engineered variant Cas12a endonuclease of the present disclosure (b) a guide RNA, and incubating the cell to produce a single strand break in the target nucleic acid.
  • 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, optionally wherein the engineered polypeptide is an endonuclease that exhibits hyperactive, hypoactivity, and/or low ssDNase activity (e.g., low indiscriminate ssDNase activity) relative to a naturally-occurring Cas12a endonuclease (e.g., SEQ ID NO: 1).
  • the engineered polypeptide is an endonuclease that exhibits hyperactive, hypoactivity, and/or low ssDNase activity (e.g., low indiscriminate ssDNase activity) relative to a naturally-occurring
  • Some aspects relate to a fusion protein comprising an engineered variant Cas12a endonuclease of any one of the preceding aspects or embodiments and a base editing enzyme. Some aspects relate to an engineered polypeptide 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.
  • fusion protein comprising an engineered variant Cas12a endonuclease 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.
  • the base editing enzyme is capable of converting a purine into a different purine or a pyrimidine into a different pyrimidine.
  • the base editing enzyme comprises a deaminase, a guanine oxidase, or a guanine methyltransferase.
  • the deaminase is a cytidine deaminase or an adenosine deaminase.
  • the deaminase comprises a rAPOBEC1 polypeptide, an evoAPOBEC1 polypeptide, a hAPOBEC3A polypeptide, an evoCDA polypeptide, an evoFERNY polypeptide, or a TadA polypeptide.
  • fusion protein further comprises a uracil glycosylase inhibitor (UGI).
  • fusion protein further comprises one or more nuclear localization signal (NLS), optionally selected from an SV40 NLS, a nucleoprotein (NP) NLS, and a bipartite (BP) NLS.
  • NLS nuclear localization signal
  • fusion protein further comprises a uracil DNA glycosylase (UNG), optionally a human UNG (hUNG) or an Escherichia coli UNG (eUNG).
  • UNG uracil DNA glycosylase
  • hUNG human UNG
  • Escherichia coli UNG Escherichia coli UNG
  • fusion protein further comprises a N-methyl purine glycosylase (MPG), optionally wherein the MPG is positioned at or near the N-terminal or C- terminal ends of the fusion protein.
  • fusion protein further comprises one or more linker.
  • the linker comprises the sequence of SGSETPGTSESATPES (SEQ ID NO: 203). In some embodiments, the linker comprises the sequence of SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 204) In some embodiments, fusion protein further comprises a DNA binding domain (DBD). In some embodiments, the DBD is a Rad51 DBD. Some aspects relate to a polynucleotide encoding the fusion protein of any one of the preceding aspects or embodiments.
  • a cell comprising: a target nucleic acid comprising a target strand and a non-target strand; a guide RNA (gRNA) or a nucleic acid encoding a gRNA that binds to the target strand; and the fusion protein of any one of the preceding aspects or embodiments or the polynucleotide of any one of the preceding aspects or embodiments.
  • the cell is a human cell.
  • the human cell is from a human subject, wherein the human subject has a disease, disorder or condition associated with the target nucleic acid.
  • the cell is a plant cell.
  • Some aspects relate to a method comprising introducing into a cell (a) the fusion protein of any one of the preceding aspects or embodiments or the polynucleotide of any one of the preceding aspects or embodiments and optionally (b) a guide RNA or a polynucleotide encoding a guide RNA.
  • Some aspects relate to a method of gene editing comprising (i) contacting a target nucleic acid sequence with the 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.
  • the target nucleic acid is a target double-stranded DNA nucleic acid.
  • the guide RNA directs the fusion protein to bind to a specific segment of the target nucleic acid and in proximity to the target nucleobase.
  • the fusion protein cleaves the target nucleic acid.
  • the fusion protein comprises a cytidine deaminase and the target nucleobase is a cytidine.
  • the fusion protein comprises a guanine oxidase and the target nucleobase is a guanosine.
  • the fusion protein comprises a guanine methyltransferase and the target nucleobase is a guanosine.
  • the method is performed in a cell, optionally a human cell or a plant cell. In some embodiments, the method is performed in vitro or ex vivo. In other embodiments, the method is performed in vivo.
  • the target nucleic acid is a gene comprising a nucleobase mutation relative to a wild-type gene. In some embodiments, the gene comprising a nucleobase mutation is associated with a disease or disorder.
  • FIGs.1A-1X show an alignment of various wild-type Cas12a endonuclease amino acid sequences using the Clustal Omega online multiple sequence alignment program (top to bottom SEQ ID NO: 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.
  • FIGs.2A-2D show experimental data for various examples of the hyperactive Cas12a endonuclease variants of the present disclosure.
  • FIGs.3A-3E show experimental data for various examples of the hypoactive Cas12a endonuclease variants of the present disclosure.
  • FIGs.4A-4C show experimental data for various examples of the hypoactive Cas12a endonuclease variants of the present disclosure.
  • FIG.5 shows experimental data for various examples of the Cas12a endonuclease variants of the present disclosure having low indiscriminate ssDNase activity.
  • FIGs.6A-6C provide graphs of data comparing percent (%) of total reads having a C- to-T nucleotide edit at genomic positions corresponding to positions C8, C9, C10, C11, and C13 of the guide RNA (gRNA) using the LbBEv2 base editor in U2OS cells.
  • gRNA guide RNA
  • FIGs.7A-7D provide graphs of data comparing percent (%) of total reads having a C- to-T nucleotide edit at genomic positions corresponding to positions C8 and C10 of the gRNA using the LbBEv2, LbBEv3, LbBEv4, or LbBEv5 base editor in U2OS cells.
  • FIGs.8A-8D provide graphs of data comparing percent (%) of total reads having a C- to-T nucleotide edit at genomic positions corresponding to positions C9, C10, and C15 of the gRNA using the LbBEv2, LbBEv3, LbBEv4, or LbBEv5 base editor in U2OS cells.
  • FIGs.9A-9F provide data showing increased efficiency and specificity of base editing with LbBEv5 C-to-T base editor containing TBN04 (LbCas12a) as compared to LbBEv5 base editor containing inactive LbCas12a in U2OS cells (top to bottom SEQ ID NOs: 214-263).
  • FIGs.10A-10F provide data showing increased efficiency and specificity of base editing with the LbBEv5 C-to-T base editor (top to bottom SEQ ID NOs: 264-315).
  • FIGs.11A-11F provide data showing increased efficiency and specificity of base editing with the LbABE8e A-to-G base editor (top to bottom SEQ ID NO: 316-365).
  • FIGs.12A-12C provide data showing increased efficiency and specificity of base editing with A-to-G base editors comprising mutant Cas12a.
  • FIGs.13A-13B provide data showing the ability of base editors comprising a N- methyl purine glycosylase (MPG) to perform A-to-C base editing (SEQ ID NO: 409 and 366).
  • MPG N- methyl purine glycosylase
  • FIGs.12a Endonuclease Provided herein are variants of the Class II type V CRISPR-Cas12a endonuclease.
  • An “endonuclease” is an enzyme capable of cleaving the phosphodiester bond within a polynucleotide chain.
  • Endonucleases are specific (i.e., they recognize a given nucleotide sequence which directs the site of cleavage), while some are non-specific.
  • the present disclosure provides specific variant Cas12a endonucleases.
  • An endonuclease may cleave both strands of a double strand polynucleotide, or an endonuclease may demonstrate a preference for cleave cleaving one strand over the other strand of a double strand polynucleotide.
  • Cas12a The recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)-Cpf1 system, now reclassified as Cas12a, is a DNA-editing platform analogous to the widely used CRISPR-Cas9 system.
  • the Cas12a system exhibits several distinct features over the CRISPR-Cas9 system, such as increased specificity and a smaller gene size to encode the nuclease and the matching CRISPR guide RNA (crRNA), which could mitigate off-target and delivery problems, respectively, described for the Cas9 system.
  • crRNA CRISPR guide RNA
  • the Cas12a system exhibits reduced gene editing efficiency compared to Cas9.
  • RNA sequencing of small RNA molecules extracted from Francisella novicida U112 culture containing Cas12a-based CRISPR loci showed that mature crRNAs for Cas12a are 42–44 nucleotides (nt) in length, with the first 19/20 nt corresponding to the repeat sequence and the remaining 23-25 nt to the spacer sequence.
  • Cas12a processes its own pre-crRNA into mature crRNAs, without the requirement of a tracrRNA, making it a unique effector protein with both endoribonuclease and endonuclease activities.
  • Cas12a cuts it 4 nt upstream of the hairpin structures formed by the CRISPR repeats, producing intermediate crRNA molecules that undergo further processing in vivo into mature crRNAs.
  • Type V (Cas12a) CRISPR-Cas systems possesses a characteristic Ruv-C like nuclease domain, which has been shown to be related to IS605 family transposon encoded TnpB proteins. Crystallographic and cryo-EM data reveal that Cas12a adopts a bilobed structure formed by the REC and Nuc lobes.
  • the REC lobe is comprised of REC1 and REC2 domains
  • the Nuc lobe is comprised of the RuvC, the PAM-interacting (PI) and the WED domains, and additionally, the bridge helix (BH).
  • the RuvC endonuclease domain of this effector protein is made up of three discontinuous parts (RuvC I-III).
  • the RNase site for processing its own crRNA is situated in the WED-III subdomain, and the DNase site is located in the interface between the RuvC and the Nuc domains.
  • the crRNA is stabilized through interactions with the WED, RuvC and REC2 domains of the endonuclease, as well as two hydrated Mg2+ ions. This binary interference complex is then responsible for recognizing and degrading foreign DNA. See Paul, B. & Montoya, G. et al. Biomedical Journal 2020; 43(1): 8-17.
  • Protospacer adjacent motif (PAM) recognition is a critical initial step in identifying a prospective DNA molecule for degradation because the PAM allows the CRISPR-Cas systems to distinguish their own genomic DNA from invading nucleic acids.
  • Cas12a employs a multistep quality control mechanism to ensure the accurate and precise recognition of target spacer sequences.
  • the WED II-III, REC1 and PAM-interacting domains are responsible for PAM recognition and for initiating the hybridization of the DNA target with the crRNA.
  • the conserved loop-lysine helix-loop (LKL) region in the PI domain containing three conserved lysines (K667, K671, K677 in FnCas12a), inserts the helix into the PAM duplex with assistance from two conserved prolines in the LKL region.
  • Structural studies show the helix is inserted at an angle of 45° with respect to the dsDNA longitudinal axis, promoting the unwinding of the helical dsDNA.
  • the critical positioning of the three conserved lysines on the dsDNA initiates the uncoupling of the Watson–Crick interaction between the base pairs of the dsDNA after the PAM.
  • the target dsDNA unzipping allows the hybridization of the crRNA with the strand containing the PAM, the target strand, while the uncoupled DNA strand, non-target strand (NTS), is conducted towards the DNase site by the PAM-interacting domain.
  • NTS non-target strand
  • the PAM for LbCas12a and AsCas12a has a sequence of 5′-TTTN-3' and for FnCas12a a sequence of 5′-TTN-3′ and is situated upstream of the 5'end of the non-target strand [26,31,34]. It has also been shown that in addition to the canonical 5′-TTTN-3′ PAM, Cas12a also exhibits relaxed PAM recognition for suboptimal C-containing PAM sequences by forming altered interactions with the targeted DNA duplex. See Paul, B. & Montoya, G. et al. Exemplary, non-limiting, wild-type Cas12a protein sequences are provided in Table 1. Table 1. Non-limiting Examples of Wild-type Cas12a Sequences
  • a guide RNA is an RNA that functions to guide an RNA- or DNA-targeting enzyme to a specific target. Targeting requires a gRNA complementary to the target site as well as a 5' protospacer adjacent motif (PAM) on the DNA strand opposite the target sequence.
  • the gRNA for a Cas12a endonuclease is 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, a gRNA is about 40-44 nucleotides long.
  • the portion of the gRNA that base pairs to 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 to the protospacer is about 20-24 nucleotides long, e.g., about 21 nucleotides long. There is also a constant portion that binds to Cas12a, which is about 15-25 nucleotides long. In some embodiments, the constant portion that binds to Cas12a is about 20 nucleotides long).
  • the target sequence to which a gRNA binds should be next to a PAM sequence – e.g., TTTV, where V can represent A, C, or G.
  • V can represent A, C, or G.
  • the “V” of the TTTV is typically immediately adjacent to the most 5’ base of the non-targeted strand side of the protospacer element.
  • the PAM sequence may vary, dependent on the variant Cas12a endonuclease.
  • Variant Cas12a Endonucleases Provided herein, in some aspects, are engineered variant Cas12a endonucleases that have altered activity relative to a wild-type Case12 endonuclease.
  • variant Cas12a endonuclease refers to a non-naturally occurring endonuclease obtained by mutation of a wild-type (e.g., naturally-occurring) Cas12a gene, for example, a Cas12a gene from Table 1 (e.g., any one of SEQ ID NOs: 1-47). Variants of other wild-type Cas12 genes are contemplated herein. Thus, the variant Cas12a endonucleases provided herein are “engineered.” Mutations contemplated herein, with respect to an amino acid sequence, include, without limitation, substitutions, additions, and deletions. An amino acid “substitution” is a change in a single amino acid relative to a reference amino acid sequence.
  • a substitution at position E95 would include any amino acid, other than E, at position 95 (counting from the methionine (M) start codon) – e.g., E95R (R substituted for E) and E95Y (Y substituted for E).
  • the variant Cas12a endonucleases provided herein exhibit hyperactivity or low indiscriminate single strand deoxyribonuclease (DNase) activity, described in more detail elsewhere herein.
  • the activity (e.g., hyperactivity and/or indiscriminate single strand DNase activity) of a variant Cas12a endonuclease may be assessed using any method known in the art.
  • the activity of a variant Cas12a endonuclease is determined with a gel-based assay.
  • the activity of a variant Cas12a endonuclease is determined using fluorophores and/or a fluorophore-quencher system.
  • the activity of a variant Cas12a endonuclease may be assessed using short, labelled oligonucleotides, which measure the activity of Cas9 and Cas ⁇ , respectively (see, e.g., Jinek et al., Science, 2012, 337(6096): 816-821 and Pausche et al., Science, 2020, 396(6501): 333-337).
  • fluorophore-labeled short oligonucleotides are used to assess cleavage on both strands (see, e.g., Stella et al., Cell, 2018, 175: 1856-1871).
  • nickase activity is determined using optical tweezers (see, e.g., Paul et al., bioRxiv, 2021, doi.org/10.1101/2021.06.09.447528).
  • longer fluorophore-labeled oligonucleotides 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).
  • quencher-fluorophore-labeled single-stranded DNA is used to assess ssDNase activity (see, e.g., Chen et al., Science, 2018, 360(6387):436-439).
  • variant Cas12a endonuclease activity is assessed using single molecule fluorescence resonance energy transfer (FRET) (see, e.g., Son et al., PNAS, 2021, 118(49): e2113747118).
  • FRET single molecule fluorescence resonance energy transfer
  • an amino acid substitution is exemplified (e.g., E95R)
  • the present disclosure contemplates alternative substitutions having an “equivalent” charge, polarity, and or chemical class (defined by the amino acid side chain).
  • Table 2 provides the 20 naturally-occurring amino acids with a description of corresponding charge, polarity, and chemical class.
  • arginine has an equivalent charge to histidine and lysine; an equivalent polarity to asparagine, glutamine, serine, threonine, tyrosine, aspartic acid, glutamic acid, arginine, histidine, and lysine; and an equivalent chemical class/side chain to histidine and lysine.
  • E95H and E95K are examples of amino acid substitutions having an equivalent charge
  • E95N, E95Q, E95S, E95T, E95Y, E95D, E95E, E95H, and E95K are examples of amino acid substitutions having an equivalent polarity
  • E95H and E95K are examples of amino acid substitutions having an equivalent chemical class.
  • a given amino acid substitution is equivalent in charge, polarity, and chemical class.
  • E95H and E95K are examples of amino acid substitutions having an equivalent charge (i.e., positive), an equivalent polarity (i.e., polar), and an equivalent chemical class (i.e., basic).
  • a variant Cas12a endonuclease comprises a polypeptide sequence that 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 amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1), optionally wherein the variant Cas12a endonuclease exhibits hyperactivity.
  • a variant Cas12a endonuclease comprises a polypeptide sequence that comprise 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 amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1), optionally wherein the variant Cas12a endonuclease exhibits hypoactivity.
  • a variant Cas12a endonuclease comprises a polypeptide sequence that comprise 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 amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1), optionally wherein the variant Cas12a endonuclease exhibits low (or no) indiscriminate ssDNase activity.
  • a variant Cas12a endonuclease comprising a mutation at an amino acid position corresponding to a specific position with reference to amino acid position numbering of LbCas12a ND2006 encompasses variants of LbCas12a ND2006 (e.g., SEQ ID NO: 1), as well as variants of Cas12a orthologs of LbCas12a ND2006, including without limitation, variants of any one of the Cas12a endonucleases in 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, and in particular, with an LbCas12a ND2006 sequence, such as the amino acid sequence of SEQ ID NO: 1, as shown in FIGs.1A- 1X.
  • FIG.1A shows an alignment of various Cas12a homologs, highlighting that a variant Cas12a endonuclease comprising a mutation at an amino acid position corresponding to E95 with reference to amino acid position numbering of LbCas12a ND2006 includes: variant Cas12a endonucleases comprising a mutation at position I96 with reference to amino acid position numbering of AsCas12a BV3L6, and variant Cas12a endonucleases comprising a mutation at position K99 with reference to amino acid position numbering of FnCas12a.
  • variant Cas12a endonucleases of the present disclosure may share a certain percent identity relative to a wild-type Cas12a endonuclease.
  • a variant Cas12a endonuclease may comprise an amino acid sequence that includes any one or more mutation(s) (e.g., amino acid substitution(s)) 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 Cas12a endonucleases in Table 1 (e.g., SEQ ID NOs: 1-47), an ortholog thereof, or other wild-type Cas12a protein sequence.
  • Table 1 e.g., SEQ ID NOs: 1-47
  • a variant Cas12a endonuclease comprises a polypeptide sequence that comprise 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 amino acid position numbering of LbCas12a 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 Cas12a endonucleases in Table 1 (e.g., SEQ ID NOs: 1-47), an ortholog thereof, or other wild-type Cas12a protein sequence.
  • Table 1 e.g., SEQ ID NOs: 1-47
  • any one or more of the foregoing variant Cas12a endonucleases exhibits hyperactivity.
  • a variant Cas12a endonuclease comprises a polypeptide sequence that comprise 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 amino acid position numbering of LbCas12a 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 Cas12a endonucleases in Table 1 (e.g., SEQ ID NOs: 1-47), an ortholog
  • any one or more of the foregoing variant Cas12a endonucleases exhibits hyperactivity.
  • a variant Cas12a endonuclease comprises a polypeptide sequence that comprise 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 amino acid position numbering of LbCas12a 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 Cas12a endonucleases in Table 1 (e.g., SEQ ID NOs: 1-47), an ortholog thereof, or other wild-type Cas12a protein sequence.
  • Table 1 e.g., SEQ ID NOs: 1-47
  • any one or more of the foregoing variant Cas12a endonucleases exhibits hypoactivity.
  • a variant Cas12a endonuclease comprises a polypeptide sequence that comprise 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 amino acid position numbering of LbCas12a and has at least 80% (e.g., at least 80%, at least 80%, at least
  • any one or more of the foregoing variant Cas12a endonucleases exhibits hypoactivity.
  • a variant Cas12a endonuclease comprises a polypeptide sequence that comprise mutations at amino acid positions corresponding to 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 F983L; Q944D and T988F; S982W and F983L; S982W and T988F; or F983G and M986G with reference to amino acid position number
  • any one or more of the foregoing variant Cas12a endonucleases exhibits hypoactivity.
  • a variant Cas12a endonuclease comprises a polypeptide sequence that comprise 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 amino acid position numbering of LbCas12a 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 Cas12a endonucleases in Table 1 (e.g., SEQ ID NOs: 1-47), an ortholog thereof, or other wild-type Cas12a protein sequence.
  • Table 1 e.g., SEQ ID NOs: 1-47
  • any one or more of the foregoing variant Cas12a endonucleases exhibits low (or no) ssDNase activity.
  • a variant Cas12a endonuclease comprises a polypeptide sequence that comprise 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 amino acid position numbering of LbCas12a
  • any one or more of the foregoing variant Cas12a endonucleases exhibits low (or no) ssDNase activity.
  • a variant Cas12a endonuclease comprises a polypeptide sequence that comprise mutations at amino acid positions corresponding to positions: N933L and Q944M; or F983G and M986G with reference to amino acid position numbering of LbCas12a 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 Cas12a endonucleases in Table 1 (e.g., SEQ ID NOs: 1-47), an ortholog thereof, or other wild-type Cas12a protein sequence.
  • Table 1 e.g., SEQ ID NOs: 1-47
  • any one or more of the foregoing variant Cas12a endonucleases exhibits low (or no) ssDNase activity.
  • Identity refers to a relationship between two or among three 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 or among sequences as determined by the number of matches between or among strings of amino acids or strings of nucleotides. Identity is a measure of the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (e.g., “algorithms”).
  • Percent (%) identity as it applies to proteins or genes, for example, such as the Cas12a endonucleases described herein, is defined as the percentage of residues (amino acid or nucleic acid residues) in a first protein or gene sequence that are identical with the residues in a second protein or gene sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Methods and computer programs for the alignment are well known in the art. It is understood that identity depends on a calculation of percent identity but may differ in value due to gaps and penalties introduced in the calculation.
  • variants of a particular protein or gene have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to that 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.
  • tools for alignment include but are not limited to those of the BLAST suite (Altschul, S.F., et al. Nucleic Acids Res.1997;25:3389-3402); and those based on the Smith-Waterman algorithm (Smith, T.F. & Waterman, M.S. J. Mol. Biol. 1981;147:195-197).
  • a general global alignment technique based on dynamic programming is the Needleman–Wunsch algorithm (Needleman, S.B. & Wunsch, C.D. J. Mol. Biol. 1920;48:443-453).
  • a Fast Optimal Global Sequence Alignment Algorithm also has been developed that purportedly produces global alignment of nucleotide and amino acid sequences faster than other optimal global alignment methods, including the Needleman– Wunsch algorithm.
  • An alignment of the non-limiting examples of wild-type Cas12a endonuclease sequences is provided in FIGs.1A-1X.
  • 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 a double strand or single strand polynucleotide, binding to a crRNA, etc.).
  • 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
  • the wild-type Cas12a endonuclease exhibits indiscriminate ssDNase activity, cuts ⁇ 14 bp away from the PAM, and possesses RNase activity to self-process pre-crRNA. Further, its cleavage activity results in 5’ staggered overhangs, and its PAM site is 3’ to the targeting binding site.
  • a wild-type Cas9 endonuclease does not exhibit indiscriminate ssDNase activity, cuts ⁇ 3-4 bp away from the PAM, and does not possess RNase activity to self-process pre- crRNA (it requires accessory proteins to mediate pre-crRNA processing).
  • Cas12a “ortholog” refers to Cas12a genes (and proteins encoded by the genes) inferred to be descended from the same ancestral sequence separated by a speciation event: when a species diverges into two separate species, the copies of a single gene in the two resulting species are said to be orthologous.
  • Orthologs, or orthologous genes are genes in different species that originated by vertical descent from a single gene of the last common ancestor. Cas12a ortholog can be identified and characterized based on sequence similarities to the present Cas12a system, as has been described with Type II systems, for example.
  • orthologs of Cas12a include the Cas12a endonucleases of Table 1.
  • Cas12a Endonuclease Hyperactive Variants Some aspects of the present disclosure relate to hyperactive variant Cas12a endonucleases, i.e., variant Cas12a endonucleases that exhibit hyperactivity. “Hyperactivity” herein refers to polynucleotide cleavage activity of a variant endonuclease that is at least 10% greater than polynucleotide cleavage activity of the wild-type or other reference endonuclease.
  • a hyperactive variant Cas12a endonuclease has a higher reaction speed or initiates a cleavage reaction faster than the corresponding wild-type Cas12a endonuclease.
  • a hyperactive variant Cas12a endonucleases exhibits 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% greater than polynucleotide cleavage activity of the wild-type or other reference endonuclease. See, e.g., Zhang, L. et al. Nat Commun.2021 Jun 23;12(1):3908.
  • a variant 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 amino acid position numbering of LbCas12a and (b) exhibits hyperactivity, optionally wherein the variant Cas12a endonuclease has at least 85%, at least 90%, at least 95%, or at least 98% identity with a wild-type reference Cas12a endonuclease.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises a mutation at an amino acid position corresponding to position E95 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutation is E95R or E95Y.
  • a variant LbCas12a endonuclease comprises a mutation at position E95 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises a mutation at position E95 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit hyperactivity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises a mutation at an amino acid position corresponding to position E125 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutation is E125A or E125W.
  • a variant LbCas12a endonuclease comprises a mutation at position E125 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises a mutation at position E125 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit hyperactivity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises a mutation at an amino acid position corresponding to position N256 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutation is N256A.
  • a variant LbCas12a endonuclease comprises a mutation at position N256 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises a mutation at position N256 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit hyperactivity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises a mutation at an amino acid position corresponding to position R747 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutation is R747Y.
  • a variant LbCas12a endonuclease comprises a mutation at position R747 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises a mutation at position R747 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit hyperactivity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises a mutation at an amino acid position corresponding to position H759 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutation is H759V or H759D.
  • a variant LbCas12a endonuclease comprises a mutation at position H759 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises a mutation at position H759 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit hyperactivity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises a mutation at an amino acid position corresponding to position N813 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutation is N813R or N813H.
  • a variant LbCas12a endonuclease comprises a mutation at position N813 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises a mutation at position N813 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit hyperactivity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises a mutation at an amino acid position corresponding to position K932 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutation is K932L.
  • a variant LbCas12a endonuclease comprises a mutation at position K932 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises a mutation at position K932 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit hyperactivity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises a mutation at an amino acid position corresponding to position N933 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutation is N933E or N933V.
  • a variant LbCas12a endonuclease comprises a mutation at position N933 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises a mutation at position N933 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit hyperactivity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises a mutation at an amino acid position corresponding to position S934 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutation is S934Q.
  • a variant LbCas12a endonuclease comprises a mutation at position S934 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises a mutation at position S934 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit hyperactivity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises a mutation at an amino acid position corresponding to position V936 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutation is V936E, V936M, or V936K.
  • a variant LbCas12a endonuclease comprises a mutation at position V936 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises a mutation at position V936 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit hyperactivity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises a mutation at an amino acid position corresponding to position S982 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutation is S982N.
  • a variant LbCas12a endonuclease comprises a mutation at position S982 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises a mutation at position S982 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit hyperactivity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises a mutation at an amino acid position corresponding to position K984 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutation is K984R.
  • a variant LbCas12a endonuclease comprises a mutation at position K984 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises a mutation at position K984 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit hyperactivity.
  • a variant LbCas12a endonuclease comprises an E95 (e.g., E95R or E95Y) substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an E95 (e.g., E95R or E95Y) substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an E95 (e.g., E95R or E95Y) substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an E95 (e.g., E95R or E95Y) substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an E95 (e.g., E95R or E95Y) substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an E125 (e.g., E125A or E125W) substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an E125 (e.g., E125A or E125W) substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an E125 (e.g., E125A or E125W) substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an E125 (e.g., E125A or E125W) substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an E125 (e.g., E125A or E125W) substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an N256 (e.g., N256A) substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an N256 (e.g., N256A) substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an N256 (e.g., N256A) substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an N256 (e.g., N256A) substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an N256 (e.g., N256A) substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing variant Cas12a endonucleases may exhibit hyperactivity.
  • a variant LbCas12a endonuclease comprises an R747 (e.g., R747Y) substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an R747 (e.g., R747Y) substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an R747 (e.g., R747Y) substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an R747 (e.g., R747Y) substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an R747 (e.g., R747Y) substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing variant Cas12a endonucleases may exhibit hyperactivity.
  • a variant LbCas12a endonuclease comprises an H759 (e.g., H759V or H759D) substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an H759 (e.g., H759V or H759D) substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an H759 (e.g., H759V or H759D) substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an H759 (e.g., H759V or H759D) substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an H759 (e.g., H759V or H759D) substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing variant Cas12a endonucleases may exhibit hyperactivity.
  • a variant LbCas12a endonuclease comprises an N813 (e.g., N813R or N813H) substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an N813 (e.g., N813R or N813H) substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an N813 (e.g., N813R or N813H) substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an N813 (e.g., N813R or N813H) substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an N813 (e.g., N813R or N813H) substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing variant Cas12a endonucleases may exhibit hyperactivity.
  • a variant LbCas12a endonuclease comprises an K932 (e.g., K932L) substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an K932 (e.g., K932L) substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an K932 (e.g., K932L) substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an K932 (e.g., K932L) substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an K932 (e.g., K932L) substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing variant Cas12a endonucleases may exhibit hyperactivity.
  • a variant LbCas12a endonuclease comprises an N933 (e.g., N933E or N933V) substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an N933 (e.g., N933E or N933V) substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an N933 (e.g., N933E or N933V) substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an N933 (e.g., N933E or N933V) substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an N933 (e.g., N933E or N933V) substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing variant Cas12a endonucleases may exhibit hyperactivity.
  • a variant LbCas12a endonuclease comprises an S934Q (e.g., S934Q) substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an S934Q (e.g., S934Q) substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an S934Q (e.g., S934Q) substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1.
  • S934Q e.g., S934Q
  • a variant LbCas12a endonuclease comprises an S934Q (e.g., S934Q) substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an S934Q (e.g., S934Q) substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing variant Cas12a endonucleases may exhibit hyperactivity.
  • a variant LbCas12a endonuclease comprises an V936 (e.g., V936E, V936M, or V936K) substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an V936 (e.g., V936E, V936M, or V936K) substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an V936 (e.g., V936E, V936M, or V936K) substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an V936 (e.g., V936E, V936M, or V936K) substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an V936 (e.g., V936E, V936M, or V936K) substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing variant Cas12a endonucleases may exhibit hyperactivity.
  • a variant LbCas12a endonuclease comprises an S982 (e.g., S982N) substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an S982 (e.g., S982N) substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an S982 (e.g., S982N) substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an S982 (e.g., S982N) substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an S982 (e.g., S982N) substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing variant Cas12a endonucleases may exhibit hyperactivity.
  • a variant LbCas12a endonuclease comprises an K984 (e.g., K984R) substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an K984 (e.g., K984R) substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an K984 (e.g., K984R) substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an K984 (e.g., K984R) substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an K984 (e.g., K984R) substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing variant Cas12a endonucleases may exhibit hyperactivity.
  • K984 e.g., K984R
  • Any one or more of the foregoing variant Cas12a endonucleases may exhibit hyperactivity.
  • K984 e.g., K984R
  • Any one or more of the foregoing variant Cas12a endonucleases may exhibit hyperactivity.
  • Other aspects of the present disclosure provide hypoactive variant Cas12a endonucleases, i.e., variant Cas12a endonucleases that exhibit hypoactivity.
  • “Hypoactivity” herein refers to polynucleotide cleavage activity of a variant endonuclease that is at least 10% lower than polynucleotide cleavage activity of the wild-type or other reference endonuclease.
  • a hypoactive variant Cas12a endonucleases exhibits 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 polynucleotide cleavage activity of the wild-type or other reference endonuclease.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises a mutation at an amino acid position corresponding to position N256 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutation is N256K.
  • a variant LbCas12a endonuclease comprises a mutation at position N256 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises a mutation at position N256 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises a mutation at an amino acid position corresponding to position I831 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutation is I831A or I831Y.
  • a variant LbCas12a endonuclease comprises a mutation at position I831 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises a mutation at position I831 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises a mutation at an amino acid position corresponding to position K932 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutation is K932A, K932F, K932H, K932M, K932N, K932Q, K932R, K932S, K932T, K932W, or K932Y.
  • a variant LbCas12a endonuclease comprises a mutation at position K932 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises a mutation at position K932 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises a mutation at an amino acid position corresponding to position N933 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutation is N933L.
  • a variant LbCas12a endonuclease comprises a mutation at position N933 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises a mutation at position N933 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises a mutation at an amino acid position corresponding to position S934 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutation is S934W.
  • a variant LbCas12a endonuclease comprises a mutation at position S934 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises a mutation at position S934 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises a mutation at an amino acid position corresponding to position V936 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutation is V936G.
  • a variant LbCas12a endonuclease comprises a mutation at position V936 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises a mutation at position V936 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises a mutation at an amino acid position corresponding to position Q944 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutation is Q944D, Q944E, Q944K, or Q944M.
  • a variant LbCas12a endonuclease comprises a mutation at position Q944 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises a mutation at position Q944 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises a mutation at an amino acid position corresponding to position S982 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutation is S982T or S982W.
  • a variant LbCas12a endonuclease comprises a mutation at position S982 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises a mutation at position S982 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises a mutation at an amino acid position corresponding to position F983 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutation is F983G or F983L.
  • a variant LbCas12a endonuclease comprises a mutation at position F983 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises a mutation at position F983 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises a mutation at an amino acid position corresponding to position K984 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutation is K984F.
  • a variant LbCas12a endonuclease comprises a mutation at position K984 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises a mutation at position K984 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises a mutation at an amino acid position corresponding to position M986 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutation is M986G, M986L, or M986S.
  • a variant LbCas12a endonuclease comprises a mutation at position M986 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises a mutation at position M986 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises a mutation at an amino acid position corresponding to position T988 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutation is T988F.
  • a variant LbCas12a endonuclease comprises a mutation at position T988 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises a mutation at position T988 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises mutations at amino acid positions corresponding to positions K932 and F983 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutations are K932F and F983L.
  • a variant LbCas12a endonuclease comprises mutations at positions K932 and F983 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises mutations at positions K932 and F983 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises mutations at amino acid positions corresponding to positions K932 and T988 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutations are K932F and T988F.
  • a variant LbCas12a endonuclease comprises mutations at positions K932 and T988 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises mutations at positions K932 and T988 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises mutations at amino acid positions corresponding to positions K932 and Q944 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutations are K932R and Q944D.
  • a variant LbCas12a endonuclease comprises mutations at positions K932 and Q944 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises mutations at positions K932 and Q944 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises mutations at amino acid positions corresponding to positions K932 and F983 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutations are K932R and F983L.
  • a variant LbCas12a endonuclease comprises mutations at positions K932 and F983 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises mutations at positions K932 and F983 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises mutations at amino acid positions corresponding to positions K932 and T988 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutations are K932R and T988F.
  • a variant LbCas12a endonuclease comprises mutations at positions K932 and T988 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises mutations at positions K932 and T988 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises mutations at amino acid positions corresponding to positions K932 and F983 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutations are K932Y and F983L.
  • a variant LbCas12a endonuclease comprises mutations at positions K932 and F983 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises mutations at positions K932 and F983 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises mutations at amino acid positions corresponding to positions K932 and T988 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutations are K932Y and T988F.
  • a variant LbCas12a endonuclease comprises mutations at positions K932 and T988 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises mutations at positions K932 and T988 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises mutations at amino acid positions corresponding to positions N933 and Q944 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutations are N933L and Q944M.
  • a variant LbCas12a endonuclease comprises mutations at positions N933 and Q944 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises mutations at positions N933 and Q944 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises mutations at amino acid positions corresponding to positions V936 and Q944 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutations are V936G and Q944D.
  • a variant LbCas12a endonuclease comprises mutations at positions V936 and Q944 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises mutations at positions V936 and Q944 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises mutations at amino acid positions corresponding to positions V936 and S982 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutations are V936G and S982W.
  • a variant LbCas12a endonuclease comprises mutations at positions V936 and S982 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises mutations at positions V936 and S982 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises mutations at amino acid positions corresponding to positions V936 and M986 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutations are V936G and M986G.
  • a variant LbCas12a endonuclease comprises mutations at positions V936 and M986 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises mutations at positions V936 and M986 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises mutations at amino acid positions corresponding to positions V936 and T988 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutations are V936G and T988F.
  • a variant LbCas12a endonuclease comprises mutations at positions V936 and T988 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises mutations at positions V936 and T988 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises mutations at amino acid positions corresponding to positions Q944 and S982 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutations are Q944D and S982W.
  • a variant LbCas12a endonuclease comprises mutations at positions Q944 and S982 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises mutations at positions Q944 and S982 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises mutations at amino acid positions corresponding to positions Q944 and F983 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutations are Q944D and F983L.
  • a variant LbCas12a endonuclease comprises mutations at positions Q944 and F983 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises mutations at positions Q944 and F983 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises mutations at amino acid positions corresponding to positions Q944 and T988 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutations are Q944D and T988F.
  • a variant LbCas12a endonuclease comprises mutations at positions Q944 and T988 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises mutations at positions Q944 and T988 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises mutations at amino acid positions corresponding to positions S982 and F983 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutations are S982W and F983L.
  • a variant LbCas12a endonuclease comprises mutations at positions S982 and F983 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises mutations at positions S982 and F983 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises mutations at amino acid positions corresponding to positions S982 and T988 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutations are S982W and T988F.
  • a variant LbCas12a endonuclease comprises mutations at positions S982 and T988 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises mutations at positions S982 and T988 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises mutations at amino acid positions corresponding to positions F983 and M986 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutations are F983G and M986G.
  • a variant LbCas12a endonuclease comprises mutations at positions F983 and M986 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises mutations at positions F983 and M986 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • a variant LbCas12a endonuclease comprises an N256 (e.g., N256K) substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an N256 (e.g., N256K) substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an N256 (e.g., N256K) substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an N256 (e.g., N256K) substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an N256 (e.g., N256K) substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • a variant LbCas12a endonuclease comprises an I831 (e.g., I831A or I831Y) substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an I831 (e.g., I831A or I831Y) substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an I831 (e.g., I831A or I831Y) substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an I831 (e.g., I831A or I831Y) substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an I831 (e.g., I831A or I831Y) substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • a variant LbCas12a endonuclease comprises an K932 (e.g., K932A, K932F, K932H, K932M, K932N, K932Q, K932R, K932S, K932T, K932W, or K932Y) substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1.
  • K932 e.g., K932A, K932F, K932H, K932M, K932N, K932Q, K932R, K932S, K932T, K932W, or K932Y
  • a variant LbCas12a endonuclease comprises an K932 (e.g., K932A, K932F, K932H, K932M, K932N, K932Q, K932R, K932S, K932T, K932W, or K932Y) substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1.
  • K932 e.g., K932A, K932F, K932H, K932M, K932N, K932Q, K932R, K932S, K932T, K932W, or K932Y
  • a variant LbCas12a endonuclease comprises an K932 (e.g., K932A, K932F, K932H, K932M, K932N, K932Q, K932R, K932S, K932T, K932W, or K932Y) substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1.
  • K932 e.g., K932A, K932F, K932H, K932M, K932N, K932Q, K932R, K932S, K932T, K932W, or K932Y
  • a variant LbCas12a endonuclease comprises an K932 (e.g., K932A, K932F, K932H, K932M, K932N, K932Q, K932R, K932S, K932T, K932W, or K932Y) substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1.
  • K932 e.g., K932A, K932F, K932H, K932M, K932N, K932Q, K932R, K932S, K932T, K932W, or K932Y
  • a variant LbCas12a endonuclease comprises an K932 (e.g., K932A, K932F, K932H, K932M, K932N, K932Q, K932R, K932S, K932T, K932W, or K932Y) substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • a variant LbCas12a endonuclease comprises an N933 (e.g., N933L) substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an N933 (e.g., N933L) substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an N933 (e.g., N933L) substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an N933 (e.g., N933L) substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an N933 (e.g., N933L) substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • a variant LbCas12a endonuclease comprises an S934 (e.g., S934W) substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an S934 (e.g., S934W) substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an S934 (e.g., S934W) substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an S934 (e.g., S934W) substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an S934 (e.g., S934W) substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • a variant LbCas12a endonuclease comprises an V936 (e.g., V936G) substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an V936 (e.g., V936G) substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an V936 (e.g., V936G) substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an V936 (e.g., V936G) substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an V936 (e.g., V936G) substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • a variant LbCas12a endonuclease comprises an Q944 (e.g., Q944D, Q944E, Q944K, or Q944M) substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an Q944 (e.g., Q944D, Q944E, Q944K, or Q944M) substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an Q944 (e.g., Q944D, Q944E, Q944K, or Q944M) substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an Q944 (e.g., Q944D, Q944E, Q944K, or Q944M) substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an Q944 (e.g., Q944D, Q944E, Q944K, or Q944M) substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • a variant LbCas12a endonuclease comprises an S982 (e.g., S982T or S982W) substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an S982 (e.g., S982T or S982W) substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an S982 (e.g., S982T or S982W) substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an S982 (e.g., S982T or S982W) substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an S982 (e.g., S982T or S982W) substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • a variant LbCas12a endonuclease comprises an F983 (e.g., F983G or F983L) substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an F983 (e.g., F983G or F983L) substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an F983 (e.g., F983G or F983L) substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an F983 (e.g., F983G or F983L) substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an F983 (e.g., F983G or F983L) substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • a variant LbCas12a endonuclease comprises an K984 (e.g., K984F) substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an K984 (e.g., K984F) substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an K984 (e.g., K984F) substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an K984 (e.g., K984F) substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an K984 (e.g., K984F) substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • a variant LbCas12a endonuclease comprises an M986 (e.g., M986G, M986L, or M986S) substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an M986 (e.g., M986G, M986L, or M986S) substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an M986 (e.g., M986G, M986L, or M986S) substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an M986 (e.g., M986G, M986L, or M986S) substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an M986 (e.g., M986G, M986L, or M986S) substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • a variant LbCas12a endonuclease comprises an T988 (e.g., T988F) substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an T988 (e.g., T988F) substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an T988 (e.g., T988F) substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an T988 (e.g., T988F) substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an T988 (e.g., T988F) substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • a variant LbCas12a endonuclease comprises an K932F and F983L substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an K932F and F983L substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an K932F and F983L substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an K932F and F983L substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an K932F and F983L substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • a variant LbCas12a endonuclease comprises an K932F and T988F substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an K932F and T988F substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an K932F and T988F substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an K932F and T988F substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an K932F and T988F substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity. In some embodiments, a variant LbCas12a endonuclease comprises an K932R and Q944D substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an K932R and Q944D substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an K932R and Q944D substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an K932R and Q944D substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an K932R and Q944D substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • a variant LbCas12a endonuclease comprises an K932R and F983L substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an K932R and F983L substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an K932R and F983L substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an K932R and F983L substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an K932R and F983L substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • a variant LbCas12a endonuclease comprises an K932R and T988F substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an K932R and T988F substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an K932R and T988F substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an K932R and T988F substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an K932R and T988F substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity. In some embodiments, a variant LbCas12a endonuclease comprises an K932Y and F983L substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an K932Y and F983L substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an K932Y and F983L substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an K932Y and F983L substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an K932Y and F983L substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • a variant LbCas12a endonuclease comprises an K932Y and T988F substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an K932Y and T988F substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an K932Y and T988F substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an K932Y and T988F substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an K932Y and T988F substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • a variant LbCas12a endonuclease comprises an N933L and Q944M substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an N933L and Q944M substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an N933L and Q944M substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an N933L and Q944M substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an N933L and Q944M substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity. In some embodiments, a variant LbCas12a endonuclease comprises an V936G and Q944D substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an V936G and Q944D substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an V936G and Q944D substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an V936G and Q944D substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an V936G and Q944D substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • a variant LbCas12a endonuclease comprises an V936G and S982W substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an V936G and S982W substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an V936G and S982W substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an V936G and S982W substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an 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 foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • a variant LbCas12a endonuclease comprises an V936G and M986G substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an V936G and M986G substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an V936G and M986G substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an V936G and M986G substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an V936G and M986G substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity. In some embodiments, a variant LbCas12a endonuclease comprises an V936G and T988F substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an V936G and T988F substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an V936G and T988F substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an V936G and T988F substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an V936G and T988F substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • a variant LbCas12a endonuclease comprises an Q944D and S982W substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an Q944D and S982W substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an Q944D and S982W substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an Q944D and S982W substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an Q944D 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 foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • a variant LbCas12a endonuclease comprises an Q944D and F983L substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an Q944D and F983L substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an Q944D and F983L substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an Q944D and F983L substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an Q944D and F983L substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity. In some embodiments, a variant LbCas12a endonuclease comprises an Q944D and T988F substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an Q944D and T988F substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an Q944D and T988F substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an Q944D and T988F substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an Q944D and T988F substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • a variant LbCas12a endonuclease comprises an S982W and F983L substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an S982W and F983L substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an S982W and F983L substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an S982W and F983L substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an S982W and F983L substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • a variant LbCas12a endonuclease comprises an S982W and T988F substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an S982W and T988F substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an S982W and T988F substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an S982W and T988F substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an S982W and T988F substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity. In some embodiments, a variant LbCas12a endonuclease comprises an F983G and M986G substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an F983G and M986G substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an F983G and M986G substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an F983G and M986G substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an F983G and M986G substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing variant Cas12a endonucleases may exhibit hypoactivity.
  • V. Cas12a Endonuclease Low Indiscriminate ssDNase Variants In addition to high-specific double strand DNA (dsDNA) cleavage, Cas12a has also been shown to exhibit indiscriminate single strand DNA (ssDNA) degradation activity upon activation with a ssDNA complementary to the crRNA guide as well as with dsDNA complementary to the crRNA guide.
  • This activity is displayed by all Cas12a orthologs and degrades any available ssDNA molecule into single/double nucleotides. Comparisons of the structures of Cas12a before, during and after cleavage reveal the structural changes that result in such an indiscriminate activity.
  • the lid region which is involved in the checkpoints for accurate target recognition is responsible for this action. Before the crRNA-DNA hybrid is formed, the lid occludes the cleft where the catalytic residues reside. Upon formation of the hybrid, the lid changes conformation to form an ⁇ helix, thus interacting with the crRNA of the hybrid assembly, thus dissociating the polar interactions and making available the catalytic pocket.
  • indiscriminate single strand deoxyribonuclease ssDNase
  • This activity was unexpected in part because the mutations made in the parent wild-type enzyme are outside of the lid region – the region thought to be responsible for indiscriminate ssDNase activity.
  • Low ssDNase activity herein refers to indiscriminate ssDNA degradation activity of a variant endonuclease that is at least 10% lower than indiscriminate ssDNA degradation activity of the wild-type or other reference endonuclease.
  • a variant Cas12a endonucleases exhibits indiscriminate 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% lower than indiscriminate ssDNA degradation activity of the wild-type or other reference endonuclease.
  • a variant Cas12a endonucleases exhibits no (no measurable) indiscriminate ssDNase activity.
  • a variant 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 amino acid position numbering of LbCas12a and (b) exhibits low (or no) single indiscriminate ssDNase, optionally wherein the variant Cas12a endonuclease has at least 85%, at least 90%, at least 95%, or at least 98% identity with a wild-type reference Cas12a endonuclease.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises a mutation at an amino acid position corresponding to position N813 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutation is N813H, N813R, or N813W.
  • a variant LbCas12a endonuclease comprises a mutation at position N813 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises a mutation at position N813 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit low (or no) indiscriminate ssDNase activity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises a mutation at an amino acid position corresponding to position I831 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutation is I831A or I831Y.
  • a variant LbCas12a endonuclease comprises a mutation at position I831 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises a mutation at position I831 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit low (or no) indiscriminate ssDNase activity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises a mutation at an amino acid position corresponding to position K932 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutation is K932A, K932F, K932H, K932M, K932N, K932Q, K932R, K932S, K932T, K932W, or K932Y.
  • a variant LbCas12a endonuclease comprises a mutation at position K932 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises a mutation at position K932 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit low (or no) indiscriminate ssDNase activity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises a mutation at an amino acid position corresponding to position N933 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutation is N933E or N933L.
  • a variant LbCas12a endonuclease comprises a mutation at position N933 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises a mutation at position N933 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit low (or no) indiscriminate ssDNase activity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises a mutation at an amino acid position corresponding to position S934 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutation is S934K or S934Q.
  • a variant LbCas12a endonuclease comprises a mutation at position S934 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises a mutation at position S934 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit low (or no) indiscriminate ssDNase activity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises a mutation at an amino acid position corresponding to position V936 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutation is V936E or V936G.
  • a variant LbCas12a endonuclease comprises a mutation at position V936 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises a mutation at position V936 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit low (or no) indiscriminate ssDNase activity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises a mutation at an amino acid position corresponding to position Q944 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutation is Q944D, Q944E, or Q944K.
  • a variant LbCas12a endonuclease comprises a mutation at position Q944 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises a mutation at position Q944 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit low (or no) indiscriminate ssDNase activity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises a mutation at an amino acid position corresponding to position S982 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutation is S982W.
  • a variant LbCas12a endonuclease comprises a mutation at position S982 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises a mutation at position S982 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit low (or no) indiscriminate ssDNase activity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises a mutation at an amino acid position corresponding to position F983 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutation is F983G or F983L.
  • a variant LbCas12a endonuclease comprises a mutation at position F983 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises a mutation at position F983 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit low (or no) indiscriminate ssDNase activity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises a mutation at an amino acid position corresponding to position K984 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutation is K984F.
  • a variant LbCas12a endonuclease comprises a mutation at position K984 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises a mutation at position K984 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit low (or no) indiscriminate ssDNase activity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises a mutation at an amino acid position corresponding to position M986 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutation is M986F or M986G.
  • a variant LbCas12a endonuclease comprises a mutation at position M986 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises a mutation at position M986 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit low (or no) indiscriminate ssDNase activity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises a mutation at an amino acid position corresponding to position T988 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutation is T988F.
  • a variant LbCas12a endonuclease comprises a mutation at position T988 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises a mutation at position T988 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit low (or no) indiscriminate ssDNase activity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises mutations at amino acid positions corresponding to positions N933 and Q944 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutations are N933L and Q944M.
  • a variant LbCas12a endonuclease comprises mutations at positions N933 and Q944 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises mutations at positions N933 and Q944 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit low (or no) indiscriminate ssDNase activity.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises mutations at amino acid positions corresponding to positions F983 and M986 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutations are F983G and M986G.
  • a variant LbCas12a endonuclease comprises mutations at positions F983 and M986 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises mutations at positions F983 and M986 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1). Any one or more of the foregoing variant Cas12a endonucleases may exhibit low (or no) indiscriminate ssDNase activity.
  • a variant LbCas12a endonuclease comprises an N813 (e.g., N813H, N813R, or N813W) substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an N813 (e.g., N813H, N813R, or N813W) substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an N813 (e.g., N813H, N813R, or N813W) substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an N813 (e.g., N813H, N813R, or N813W) substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an N813 (e.g., N813H, N813R, or N813W) substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing variant Cas12a endonucleases may exhibit low (or no) ssDNase activity.
  • a variant LbCas12a endonuclease comprises an I831 (e.g., I831A or I831Y) substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an I831 (e.g., I831A or I831Y) substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an I831 (e.g., I831A or I831Y) substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an I831 (e.g., I831A or I831Y) substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an I831 (e.g., I831A or I831Y) substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing variant Cas12a endonucleases may exhibit low (or no) ssDNase activity.
  • a variant LbCas12a endonuclease comprises an K932 (e.g., K932A, K932F, K932H, K932M, K932N, K932Q, K932R, K932S, K932T, K932W, or K932Y) substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1.
  • K932 e.g., K932A, K932F, K932H, K932M, K932N, K932Q, K932R, K932S, K932T, K932W, or K932Y
  • a variant LbCas12a endonuclease comprises an K932 (e.g., K932A, K932F, K932H, K932M, K932N, K932Q, K932R, K932S, K932T, K932W, or K932Y) substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1.
  • K932 e.g., K932A, K932F, K932H, K932M, K932N, K932Q, K932R, K932S, K932T, K932W, or K932Y
  • a variant LbCas12a endonuclease comprises an K932 (e.g., K932A, K932F, K932H, K932M, K932N, K932Q, K932R, K932S, K932T, K932W, or K932Y) substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1.
  • K932 e.g., K932A, K932F, K932H, K932M, K932N, K932Q, K932R, K932S, K932T, K932W, or K932Y
  • a variant LbCas12a endonuclease comprises an K932 (e.g., K932A, K932F, K932H, K932M, K932N, K932Q, K932R, K932S, K932T, K932W, or K932Y) substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1.
  • K932 e.g., K932A, K932F, K932H, K932M, K932N, K932Q, K932R, K932S, K932T, K932W, or K932Y
  • a variant LbCas12a endonuclease comprises an K932 (e.g., K932A, K932F, K932H, K932M, K932N, K932Q, K932R, K932S, K932T, K932W, or K932Y) substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1.
  • K932 e.g., K932A, K932F, K932H, K932M, K932N, K932Q, K932R, K932S, K932T, K932W, or K932Y
  • a variant LbCas12a endonuclease comprises an N933 (e.g., N933E or N933L) substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an N933 (e.g., N933E or N933L) substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an N933 (e.g., N933E or N933L) substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an N933 (e.g., N933E or N933L) substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an N933 (e.g., N933E or N933L) substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing variant Cas12a endonucleases may exhibit low (or no) ssDNase activity.
  • a variant LbCas12a endonuclease comprises an S934 (e.g., S934K or S934Q) substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an S934 (e.g., S934K or S934Q) substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an S934 (e.g., S934K or S934Q) substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an S934 (e.g., S934K or S934Q) substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an S934 (e.g., S934K or S934Q) substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing variant Cas12a endonucleases may exhibit low (or no) ssDNase activity.
  • a variant LbCas12a endonuclease comprises an V936 (e.g., V936E or V936G) substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an V936 (e.g., V936E or V936G) substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an V936 (e.g., V936E or V936G) substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an V936 (e.g., V936E or V936G) substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an V936 (e.g., V936E or V936G) substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing variant Cas12a endonucleases may exhibit low (or no) ssDNase activity.
  • a variant LbCas12a endonuclease comprises an Q944 (e.g., Q944D, Q944E, or Q944K) substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an Q944 (e.g., Q944D, Q944E, or Q944K) substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an Q944 (e.g., Q944D, Q944E, or Q944K) substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an Q944 (e.g., Q944D, Q944E, or Q944K) substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an Q944 (e.g., Q944D, Q944E, or Q944K) substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing variant Cas12a endonucleases may exhibit low (or no) ssDNase activity.
  • a variant LbCas12a endonuclease comprises an S982 (e.g., S982W) substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an S982 (e.g., S982W) substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an S982 (e.g., S982W) substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an S982 (e.g., S982W) substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an S982 (e.g., S982W) substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing variant Cas12a endonucleases may exhibit low (or no) ssDNase activity.
  • a variant LbCas12a endonuclease comprises an F983 (e.g., F983G or F983L) substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an F983 (e.g., F983G or F983L) substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an F983 (e.g., F983G or F983L) substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an F983 (e.g., F983G or F983L) substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an F983 (e.g., F983G or F983L) substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing variant Cas12a endonucleases may exhibit low (or no) ssDNase activity.
  • a variant LbCas12a endonuclease comprises an K984 (e.g., K984F) substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an K984 (e.g., K984F) substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an K984 (e.g., K984F) substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an K984 (e.g., K984F) substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an K984 (e.g., K984F) substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing variant Cas12a endonucleases may exhibit low (or no) ssDNase activity.
  • a variant LbCas12a endonuclease comprises an M986 (e.g., M986F or M986G) substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an M986 (e.g., M986F or M986G) substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an M986 (e.g., M986F or M986G) substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an M986 (e.g., M986F or M986G) substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an M986 (e.g., M986F or M986G) substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing variant Cas12a endonucleases may exhibit low (or no) ssDNase activity.
  • a variant LbCas12a endonuclease comprises an T988 (e.g., T988F) substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an T988 (e.g., T988F) substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an T988 (e.g., T988F) substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an T988 (e.g., T988F) substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an T988 (e.g., T988F) substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing variant Cas12a endonucleases may exhibit low (or no) ssDNase activity.
  • a variant LbCas12a endonuclease comprises an N933L and Q944M substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an N933L and Q944M substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an N933L and Q944M substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an N933L and Q944M substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an N933L and Q944M substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing variant Cas12a endonucleases may exhibit low (or no) ssDNase activity. In some embodiments, a variant LbCas12a endonuclease comprises an F983G and M986G substitution and has at least 80% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an F983G and M986G substitution and has at least 85% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an F983G and M986G substitution and has at least 90% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant LbCas12a endonuclease comprises an F983G and M986G substitution and has at least 95% identity to the amino acid sequence of SEQ ID NO: 1.
  • a variant LbCas12a endonuclease comprises an F983G and M986G substitution and has at least 98% identity to the amino acid sequence of SEQ ID NO: 1. Any one or more of the foregoing variant Cas12a endonucleases may exhibit low (or no) ssDNase activity. Table 3. Variant Cas12a Endonucleases
  • nucleic acids comprise a polymer of nucleotides (nucleotide monomers). Thus, nucleic acids are also referred to as polynucleotides.
  • Nucleic acids may be or may 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 having a ⁇ -D-ribo configuration, ⁇ -LNA having an ⁇ -L-ribo configuration (a diastereomer of LNA), 2′-amino-LNA having a 2′-amino functionalization, and 2′-amino- ⁇ -LNA having a 2′-amino functionalization), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA) and/or chimeras and/or combinations thereof.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • TAA glycol nucleic acid
  • PNA peptide nucleic acid
  • LNA locked nu
  • the polynucleotide encoding the variant Cas12a endonuclease is an RNA, such as an mRNA.
  • Messenger RNA is RNA that encodes a (at least one) protein (a 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.
  • An mRNA provided herein typically comprises an open reading frame (ORF) encoding a variant Cas12a endonuclease.
  • an mRNA also comprises an ORF encoding a crRNA or multiple crRNAs.
  • an mRNA further comprises a 5 ⁇ cap, a 5 ⁇ untranslated region (UTR), a 3 ⁇ UTR, and a poly(A) tail.
  • An ORF is a continuous stretch of DNA or RNA beginning with a start codon (e.g., methionine (ATG or AUG)) and ending with a stop codon (e.g., TAA, TAG or TGA, or UAA, UAG or UGA).
  • AUG methionine
  • An ORF typically encodes a protein.
  • sequences disclosed herein may further comprise additional elements, e.g., 5’ and/or 3’ UTRs, but that those elements, unlike the ORF, need not necessarily be present in an RNA (e.g., mRNA) of the present disclosure.
  • an ORF encoding a variant Cas12a endonuclease of the disclosure is codon optimized. Codon optimization methods are known in the art. An open reading frame of any one or more of the variant Cas12a endonucleases provided herein may be codon optimized.
  • Codon optimization may be used to match codon frequencies in target and host organisms to ensure proper folding; bias GC content to increase RNA (e.g., mRNA) stability or reduce secondary structures; minimize tandem repeat codons or base runs that may impair gene construction or expression; customize transcriptional and translational control regions; insert or remove protein trafficking sequences; remove/add post translation modification sites in encoded protein (e.g., glycosylation sites); add, remove or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and RNA (e.g., mRNA) degradation sites; adjust translational rates to allow the various domains of the protein to fold properly; or reduce or eliminate problem secondary structures within the polynucleotide.
  • RNA e.g., mRNA
  • 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.
  • the open reading frame sequence is optimized using optimization algorithms.
  • a “5′ untranslated region” refers to a region of an mRNA that is directly upstream (i.e., 5′) from the start codon (i.e., the first codon of an mRNA transcript translated by a ribosome) that does not encode a polypeptide.
  • a “3′ untranslated region” refers to a region of an mRNA that is directly downstream (i.e., 3′) from the stop codon (i.e., the codon of an mRNA transcript that signals a termination of translation) that does not encode a polypeptide.
  • the 5’ UTR may comprise a promoter sequence.
  • an RNA comprises a 5′ terminal cap.
  • 5′-capping of polynucleotides may be completed concomitantly during an in vitro transcription reaction using, for example, the following chemical RNA cap analogs to generate the 5′-guanosine cap structure according to manufacturer protocols: 3 ⁇ -O-Me-m7G(5')ppp(5') G [the 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 may be completed post- transcriptionally using, for example, a Vaccinia Virus Capping Enzyme to generate the “Cap 0” structure: m7G(5')ppp(5')G
  • Cap 1 structure may be generated using both Vaccinia Virus Capping Enzyme and a 2′-O methyl-transferase to generate: m7G(5')ppp(5')G-2′-O-methyl.
  • Cap 2 structure may be generated from the Cap 1 structure followed by the 2′-O-methylation of the 5′-antepenultimate nucleotide using a 2′-O methyl-transferase.
  • Cap 3 structure may be generated from the Cap 2 structure followed by the 2′-O-methylation of the 5′-preantepenultimate nucleotide using a 2′-O methyl-transferase.
  • Enzymes may be derived from a recombinant source. Other cap analogs may be used.
  • a “poly(A) tail” is a region of mRNA that is downstream, e.g., directly downstream (i.e., 3′), from the 3′ UTR that contains multiple, consecutive adenosine monophosphates.
  • a poly(A) tail may contain 10 to 300 adenosine monophosphates. It can, in some instances, comprise up to about 400 adenine nucleotides.
  • a poly(A) tail may 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.
  • a poly(A) tail contains 50 to 250 adenosine monophosphates.
  • the poly(A) tail functions to protect mRNA from enzymatic degradation, e.g., in the cytoplasm, and aids in transcription termination, and/or export of the mRNA from the nucleus and translation.
  • the length of the 3′-poly(A) tail may be an essential element with respect to the stability of the individual mRNA.
  • a 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.
  • a poly(A) tail has a length of 100 nucleotides.
  • 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, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity, or demyristoylation activity.
  • deaminase activity e.g., adenosine deaminase and/or cyt
  • base editing fusion proteins comprising one or more base editing enzyme(s).
  • a “base editing enzyme” is an enzyme that is capable of converting a target nucleobase or base pair into a different nucleobase or base pair (e.g. conversion from 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 double-stranded breaks in a polynucleotide chain.
  • Base editing enzymes can be specific for DNA bases or specific for RNA bases. Any base editing enzyme may be utilized in a fusion protein as described herein.
  • a base editing enzyme may be capable of converting 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; or cytosine to uracil.
  • a base editing enzyme is capable converting a standard nucleobase (e.g., A, C, G, T, U) into a modified nucleobase (e.g., hypoxanthine, xanthine 7- methylguanine, 5,6-dihydrouracil, 5-methylcytosine, 5-hydroxymethylcytosine).
  • a base editing enzyme is capable converting a modified nucleobase (e.g., hypoxanthine, xanthine 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, 5- hydroxymethylcytosine) into a standard nucleobase (e.g., A, C, G, T, U).
  • a base editing enzyme is capable of converting a modified nucleobase into a different modified nucleobase.
  • a base editing enzyme converts a target base pair.
  • a base editing enzyme converts C-G base pairs to T-A base pairs.
  • a base editing enzyme converts A-T base pairs to G-C base pairs.
  • a base editing enzyme is a deaminase (e.g., cytidine deaminase or adenosine deaminase) that is capable of removing an amino group from a molecule.
  • Cytidine deaminases are capable of removing an amino group from a cytidine; adenosine deaminases are capable of removing an amino group from an adenosine.
  • a cytidine deaminase may be an apolipoprotein B mRNA editing enzyme complex (APOBEC1) family protein.
  • 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).
  • an adenosine deaminase is a TadA polypeptide.
  • a base editing enzyme is an oxidase (e.g., a guanine oxidase) that is capable of oxidizing a certain nucleobase.
  • a guanine oxidase functions to oxidize a certain guanine nucleobase in a target gene to form 8-oxoguanine (8-oxo-G).8-oxo- G induces steric rotation of the nucleobase around the glycosidic bond, forcing base pairing in the Hoogsteen orientation of 8-oxo-G.
  • Cellular recognition of the mismatched 8-oxo- G/cytosine paring leads to natural repair of the cytosine to an adenine.
  • a 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, TET 1 -CD, TET2 or TET3), an alpha-ketoglutarate-dependent hydroxylase (e.g., AlkB), .
  • XHD xanthine dehydrogenase
  • a cytochrome P450 enzyme e.g., CYP1A2, CYP2A6 or CYP3A6
  • TET-oxidase e.g., TET1, TET 1 -CD, TET2 or TET3
  • AlkB alpha-ketoglutarate-dependent hydroxylase
  • a xanthine dehydrogenase is a 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 a S.
  • a base editing enzyme is a methyltransferase (e.g., a guanine methyltransferase) that is capable of methylating a certain nucleobase.
  • a guanine methyltransferase functions to methylate a certain guanine nucleobase in a target gene to form N2,N2-dimethyl-guanine or N-methyl-guanine.
  • a guanine methyltransferase is a wild-type RlmA, Escherichia coli RlmA, human TrmTIOA, Escherichia coli TrmD, M. Jannaschii Trm5b, P. Abyssi Trm5a, a Trm5c from an archaeon, or a Staphylococcus scirui Cfr.
  • a base editing enzyme (e.g., a deaminase) is a base editing enzyme derived from a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse deaminase.
  • a base editing enzyme is a human base editing enzyme (e.g., a human deaminase, e.g., hAPOBEC polypeptide).
  • a base editing enzyme is a rat base editing enzyme (e.g., a rat deaminase, e.g., rAPOBEC1 polypeptide).
  • a base editing enzyme (e.g., a deaminase) is an evolved variant of a wild-type base editing enzyme.
  • a base editing enzyme is an evolved APOBEC polypeptide (e.g., evoAPOBEC1 polypeptide), an evolved cytidine deaminase polypeptide (e.g., evoCDA polypeptide), or an evolved FERNY polypeptide (e.g., evoFERNY polypeptide).
  • a base editing enzyme is as described in Thuronyi, et. al. Nat Biotechnol.2019 Sep; 37(9): 1070–1079, the contents of which are incorporated herein by reference.
  • a base editing enzyme is as described in US20200172931, the contents of which are incorporated herein by reference. Exemplary, non-limiting, base editing enzyme sequences are provided in Table 4. In some embodiments, a base editing enzyme comprises the amino acid sequence of any one of SEQ ID NOs: 27-69.
  • a base editing enzyme comprises an amino acid sequence that includes one or more mutation(s) (e.g., amino acid substitution(s)) 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), an ortholog thereof, or other base editing enzyme sequence. Table 4.
  • Table 4 e.g., SEQ ID NOs: 120-162
  • the fusion protein comprises one or more variant Cas12a endonuclease, one or more base editing enzymes, and one or more additional proteins (e.g., a protein, such as an enzyme, that regulates a biological activity).
  • additional proteins e.g., a protein, such as an enzyme, that regulates 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., a Rad51 DNA binding domain), reverse transcriptase, 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 deubiquitinating activity, a polypeptide having adenylation activity, a polypeptide having deaden
  • a fusion protein may comprise any one of the variant 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).
  • a fusion protein comprises any one of the base editing enzymes described herein.
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises a mutation at an amino acid position corresponding to position R833 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutation is R833L, R833K, or R833M.
  • a variant LbCas12a endonuclease comprises a mutation at position R833 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises a mutation at position R833 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises a mutation at an amino acid position corresponding to position K932 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutation is K932E or K932G.
  • a variant LbCas12a endonuclease comprises a mutation at position K932 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises a mutation at position K932 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises a mutation at an amino acid position corresponding to position Q944 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutation is Q944K.
  • a variant LbCas12a endonuclease comprises a mutation at position Q944 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises a mutation at position Q944 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises a mutation at an amino acid position corresponding to position K940 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutation is K940G.
  • a variant LbCas12a endonuclease comprises a mutation at position K940 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises a mutation at position K940 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises mutations at amino acid positions corresponding to positions K932, N933, V936, and S929 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutations are K932G, N933G, V936G, and S929G.
  • a variant LbCas12a endonuclease comprises mutations at positions K932, N933, V936, and S929 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises mutations at positions K932, N933, V936, and S929 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises mutations at amino acid positions corresponding to positions K940 and Q944 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutations are K940G and Q944K.
  • a variant LbCas12a endonuclease comprises mutations at positions K940 and Q944 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises mutations at positions K940 and Q944 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises mutations at amino acid positions corresponding to positions R836 and Q944 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutations are R836G and Q944K.
  • a variant LbCas12a endonuclease comprises mutations at positions R836 and Q944 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises mutations at positions R836 and Q944 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises mutations at amino acid positions corresponding to positions R833, E835, and Y943 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutations are R833M, E835D, and Y943T.
  • a variant LbCas12a endonuclease comprises mutations at positions R833, E835, and Y943 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises mutations at positions R833, E835, and Y943 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises mutations at amino acid positions corresponding to positions R836, Q944, and R935 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutations are R836G, Q944K, and R935G.
  • a variant LbCas12a endonuclease comprises mutations at positions R836, Q944, and R935 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises mutations at positions R836, Q944, and R935 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises mutations at amino acid positions corresponding to positions R833, E835, Y943, and R935 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutations are R833M, E835D, Y943T, and R935G.
  • a variant LbCas12a endonuclease comprises mutations at positions R833, E835, Y943, and R935 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises mutations at positions R833, E835, Y943, and R935 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises mutations at amino acid positions corresponding to positions R833, E835, Y943, and Q941 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutations are R833M, E835D, Y943T, and Q941K.
  • a variant LbCas12a endonuclease comprises mutations at positions R833, E835, Y943, and Q941 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises mutations at positions R833, E835, Y943, and Q941 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises mutations at amino acid positions corresponding to positions R833, E835, and E125 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutations are R833M, E835D, and E125A.
  • a variant LbCas12a endonuclease comprises mutations at positions R833, E835, and E125 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises mutations at positions R833, E835, and E125 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises mutations at amino acid positions corresponding to positions Y943, Q944, K932, N933, and E125 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutations are Y943F, Q944K, K932G, N933G, and E125A.
  • a variant LbCas12a endonuclease comprises mutations at positions Y943, Q944, K932, N933, and E125 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises mutations at positions Y943, Q944, K932, N933, and E125 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises mutations at amino acid positions corresponding to positions R836, Q944, R935, and E125 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutations are R836G, Q944K, R935G, and E125A.
  • a variant LbCas12a endonuclease comprises mutations at positions R836, Q944, R935, and E125 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises mutations at positions R836, Q944, R935, and E125 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises mutations at amino acid positions corresponding to positions R833, E835, Y943, R935, and E125 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutations are R833M, E835D, Y943T, R935G, and E125A.
  • a variant LbCas12a endonuclease comprises mutations at positions R833, E835, Y943, R935, and E125 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises mutations at positions R833, E835, Y943, R935, and E125 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises mutations at amino acid positions corresponding to positions R833, E835, Y943, Q941, and E125 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutations are R833M, E835D, Y943T, Q941K, and E125A.
  • a variant LbCas12a endonuclease comprises mutations at positions R833, E835, Y943, Q941, and E125 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises mutations at positions R833, E835, Y943, Q941, and E125 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • the variant Cas12a endonuclease comprises a polypeptide sequence that comprises mutations at amino acid positions corresponding to positions D832, Y943, Q944, K932, N933, and E125 with reference to amino acid position numbering of LbCas12a ND2006 (e.g., SEQ ID NO: 1).
  • the mutations are D832A, Y943F, Q944K, K932G, N933G, and E125A.
  • a variant LbCas12a endonuclease comprises mutations at positions D832, Y943, Q944, K932, N933, and E125 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a variant LbCas12a endonuclease comprises mutations at positions D832, Y943, Q944, K932, N933, and E125 with reference to amino acid position numbering 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 a wild-type reference LbCas12a ND2006 endonuclease (e.g., SEQ ID NO: 1).
  • a fusion protein comprises a variant Cas12a endonuclease that is located at or near the N-terminal end of the protein.
  • a fusion protein comprises a variant Cas12a endonuclease that is located at or near the C-terminal end of the protein. In some embodiments, a fusion protein comprises a base editing enzyme that is located at or near the N-terminal end of the protein. In some embodiments, a fusion protein comprises a base editing enzyme that is located at or near the C-terminal end of the protein. In some embodiments, a fusion protein comprises an N-terminal variant Cas12a endonuclease and a C-terminal base editing enzyme (i.e., variant Cas12a endonuclease is located closer to the N-terminus of the protein than the base editing enzyme).
  • a fusion protein comprises an N-terminal base editing enzyme and a C-terminal variant Cas12a endonuclease (i.e., base editing enzyme is located closer to the N-terminus of the protein than the variant Cas12a endonuclease).
  • a fusion protein may comprise one or more nuclear localization signals (NLSs).
  • NLSs nuclear localization signals
  • a fusion protein comprises 1, 2, 3, 4, 5, or more NLSs.
  • An NLS is an amino acid sequence that directs the fusion protein for import into the cell nucleus by nuclear transport.
  • an NLS is a positively charged amino acid sequence that comprises several lysine and/or arginine amino acids.
  • a fusion protein comprises an NLS that is located at or near the N-terminal end of the protein. In some embodiments, a fusion protein comprises an NLS that is located at or near the C-terminal end of the protein. In some embodiments, a fusion protein comprises an NLS that is located at or near the N-terminal end of the protein and an NLS that is located at or near the C-terminal end of the protein.
  • NLS include an SV40 NLS, a nucleoprotein (NP) NLS, and a bipartite (BP) NLS.
  • an SV40 NLS comprises the amino acid sequence of PKKKRKV (SEQ ID NO: 193).
  • a nucleoprotein NLS comprises the amino acid sequence of KRPAATKKAGQAKKKK (SEQ ID NO: 194).
  • a bipartite NLS comprises the amino acid sequence of KRTADGSEFESPKKKRKV (SEQ ID NO: 195).
  • a fusion protein comprises an SV40 NLS that is located at or near the N-terminal end of the protein and/or an SV40 NLS that is located at or near the C-terminal end of the protein.
  • a fusion protein comprises an SV40 NLS that is located at or near the N-terminal end of the protein, an SV40 NLS that is located at or near the C-terminal end of the protein, and an NP NLS that is located at or near the C-terminal end of the protein.
  • a fusion protein comprises an NP NLS that is located at or near the N-terminal end of the protein, an NP NLS that is located at or near the C-terminal end of the protein, and an SV40 NLS that is located at or near the C-terminal end of the protein.
  • a fusion protein comprises a BP NLS that is located at or near the N-terminal end of the protein, an SV40 NLS that is located at or near the C-terminal end of the protein, and an NP NLS that is located at or near the C-terminal end of the protein.
  • a fusion protein comprises a BP NLS that is located at or near the N-terminal end of the protein and a BP NLS that is located at or near the C-terminal end of the protein.
  • a fusion protein comprises a BP NLS that is located at or near the N-terminal end of the protein and an NP NLS that is located at or near the C-terminal end of the protein.
  • a fusion protein may comprise one or more linkers.
  • a linker for use in a fusion protein of the disclosure is generally an amino acid linker.
  • a linker functions to provide separation between different protein elements of the fusion protein (e.g., variant Cas12a endonuclease and base editing enzyme).
  • 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., to allow for each protein to fold and perform its function, e.g., enzymatic function).
  • a fusion protein comprises a linker between a variant Cas12a endonuclease and a base editing enzyme.
  • a linker is a flexible linker. In some embodiments, a linker is a flexible linker comprising serine and/or glycine amino acids. In some embodiments, a linker is an amino acid sequence, wherein the majority of the amino acids of the linker are serine and/or glycine amino acids. In some embodiments, a linker comprises the 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) wherein n is 1-10.
  • a linker comprises the amino acid sequence of GSSGGSGGSGGSGS (SEQ ID NO: 202). In some embodiments, a linker comprises the amino acid sequence of SGSETPGTSESATPES (SEQ ID NO: 203). In other embodiments, a linker comprises the amino acid sequence of SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 204). In some embodiments, a linker comprises the amino acid sequence of SGGSGGSGGS (SEQ ID NO: 205). In some embodiments, a linker comprises the amino acid sequence of GGGGGGS (SEQ ID NO: 206); GSSGGSGGSGGS (SEQ ID NO: 207); or SGGS (SEQ ID NO: 208).
  • the base editor fusion protein further comprises an inhibitor of base excision repair (“iBER”) that covalently or non-covalently binds to a mutated nucleobase to prevent its excision during subsequent mismatch repair.
  • iBER base excision repair
  • 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, a Methyl-CpG Binding Domain 4 (MBD4) inhibitor.
  • the iBER comprises a catalytically inactive OGG that binds 8-oxo-inosine to prevent its excision during subsequent mismatch repair.
  • a fusion protein may comprise one or more uracil glycosylase inhibitors (UGI).
  • a fusion protein comprises 1, 2, 3, 4, or 5 UGI polypeptides.
  • a UGI is a polypeptide that is capable of inhibiting a uracil-DNA glycosylase base-excision repair enzyme (e.g., from the uracil base excision repair (UBER) pathway).
  • UBER uracil base excision repair
  • a UGI is capable of inhibiting repair machinery such that the UGI polypeptide increases the efficiency of a C to T conversion.
  • a UGI polypeptide reduces the rate of conversions that are not the C to T conversion.
  • a UGI polypeptide may be a wild-type UGI polypeptide or a variant UGI polypeptide.
  • a UGI is a UGI from Bacillus subtilis bacteriophage PBS1.
  • a UGI polypeptide comprises the amino acid sequence of TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVML LTSDAPEYKPWALVIQDSNGENKIKML (SEQ ID NO: 209).
  • a UGI polypeptide comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 97% identity to SEQ ID NO: 209.
  • a fusion protein may comprise one or more DNA glycosylases.
  • a fusion protein comprises 1, 2, 3, 4, or 5 DNA glycosylases.
  • a fusion protein comprises a uracil DNA glycosylase (UNG).
  • UNG uracil DNA glycosylase
  • a fusion protein comprises a N-methyl purine glycosylase (MPG).
  • MPG N-methyl purine glycosylase
  • an N-methyl purine glycosylase (MPG) functions in recognition and repair of base pairs comprising deoxyinosine.
  • MPG is useful, in some embodiments, for A-to-C or A-to-T conversions, particularly when in combination with an adenosine deaminase (e.g., TadA deaminase).
  • a fusion protein comprises an MPG and an adenosine deaminase (e.g., TadA deaminase).
  • adenosine deaminase converts adenine to inosine; and the MPG subsequently removes the inosine to produce an abasic site.
  • the abasic site may subsequently be processed (e.g., to place a cytosine at that position).
  • a fusion protein comprises an MPG and a cytidine deaminase (e.g., APOBEC1 deaminase).
  • an MPG polypeptide comprises the amino acid sequence of SEQ ID NO: 210.
  • an MPG polypeptide comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 97% identity to SEQ ID NO: 210.
  • a UNG is a polypeptide that is capable of recognition and excision of uracil from DNA strands.
  • a UNG polypeptide is able to remove unwanted uracil bases from DNA molecules by cleaving the N-glycosidic bond and initiating the base- excision repair (BER) pathway.
  • a UNG is capable of increasing the efficiency of a C to G conversion.
  • a UNG polypeptide may be a human UNG (hUNG) or an Escherichia coli UNG (eUNG).
  • eUNG Escherichia coli UNG
  • a hUNG polypeptide is a mitochondrial UNG1 or the nuclear UNG2A.
  • a UNG polypeptide may be a wild-type UNG polypeptide or a variant UNG polypeptide.
  • an UNG polypeptide comprises the amino acid sequence ofANELTWHDVLAEEKQQPYFLNTLQTVASERQS GVTIYPPQKDVFNAFRFTELGDVKVVILGQDPYHGPGQAHGLAFSVRPGIAIPPSLLNMYKE LENTIPGFTRPNHGYLESWARQGVLLLNTVLTVRAGQAHSHASLGWETFTDKVISLINQHRE GVVFLLWGSHAQKKGAIIDKQRHHVLKAPHPSPLSAHRGFFGCNHFVLANQWLEQRGETPID WMPVLPAESE (SEQ ID NO: 211).
  • a UNG polypeptide comprises the amino acid sequence of IGQKTLYSFFSPSPARKRHAPSPEPA VQGTGVAGVPEESGDAAAIPAKKAPAGQEEPGTPPSSPLSAEQLDRIQRNKAAALLRLAARN VPVGFGESWKKHLSGEFGKPYFIKLMGFVAEERKHYTVYPPPHQVFTWTQMCDIKDVKVVIL GQDPYHGPNQAHGLCFSVQRPVPPPPSLENIYKELSTDIEDFVHPGHGDLSGWAKQGVLLLN AVLTVRAHQANSHKERGWEQFTDAVVSWLNQNSNGLVFLLWGSYAQKKGSAIDRKRHHVLQT AHPSPLSVYRGFFGCRHFSKTNELLQKSGKKPIDWKEL (SEQ ID NO: 212).
  • an UNG polypeptide comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 97% identity to SEQ ID NO: 211 or 212.
  • a fusion protein may comprise one or more DNA binding domains (DBD).
  • a fusion protein comprises 1, 2, 3, 4, or 5 DBDs.
  • a DBD is a DBD that recognizes a sequence-specific single-stranded DNA molecule.
  • a DBD is a DBD that recognizes a non-sequence-specific single-stranded DNA molecule.
  • a DBD is a DBD that recognizes a sequence-specific double-stranded DNA molecule.
  • a DBD is a DBD that recognizes a non-sequence-specific double-stranded DNA molecule.
  • a DBD comprises a Rad51 DNA binding domain (DBD).
  • a DBD may be a wild-type DBD polypeptide or a variant DBD polypeptide.
  • a DBD polypeptide comprises the amino acid sequence ofMAMQMQLEANADTSVEEESFGPQPISRLEQ CGINANDVKKLEEAGFHTVEAVAYAPKKELINIKGISEAKADKILAEAAKLVPMGFTTATEF HQRRSEIIQITTGSKELDKLLQ (SEQ ID NO: 213).
  • a DBD polypeptide comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 97% identity to SEQ ID NO: 213.
  • a fusion protein can be designed to be specific for a certain type of enzymatic conversion. For example, certain fusion proteins are designed for C to T conversion, A to G conversion, or C to G conversion.
  • a fusion protein comprising a variant 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).
  • a fusion protein comprising a variant 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).
  • a fusion protein comprising a variant Cas12a endonuclease and a base editing enzyme e.g., TadA
  • TadA is an A to G base editor (i.e., enzymatically converts A to G).
  • a fusion protein comprising a variant 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).
  • a fusion protein comprising a variant Cas12a endonuclease and a cytidine deaminase (e.g., APOBEC1) is a C to G base editor (i.e., enzymatically converts C to G).
  • a fusion protein comprising a variant Cas12a endonuclease, a cytidine deaminase (e.g., APOBEC1), and a UNG is a C to G base editor (i.e., enzymatically converts C to G).
  • a fusion protein sequences are provided in Table 5. Table 5. Non-limiting Examples of Fusion Proteins Comprising Variant Cas12a Endonucleases and Base Editing Enzyme
  • a fusion protein comprises the amino acid sequence of any one of SEQ ID NO: 163-185.
  • a fusion protein comprises an amino acid sequence that includes any one or more mutation(s) (e.g., amino acid substitution(s)) 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 protein in Table 5 (e.g., SEQ ID NOs: 163-185).
  • the cells may be eukaryotic cells or prokaryotic cells.
  • eukaryotic cells include animal cells, plant cells, and fungal cells.
  • the cells are mammalian cells.
  • the cells are human cells (e.g., human primary cells or human immortalized cells).
  • the cells are stem cells, such as adult stem cells or induced pluripotent stem cells (iPSCs).
  • iPSCs induced pluripotent stem cells
  • the cells may be selected from non-human primate cells, porcine cells, bovine cells, canine cells, feline cells, or rodent cells (e.g., rat or mouse cells).
  • Various human cell types are contemplated herein, including without limitation, immune cells (e.g., T cells (e.g., NKT cells, CD4+ T cells, CD8+ T cells, regulatory T cells, engineered T cells, e.g., CAR-T, TCR)), B cells, NK cells, tumor-infiltrating lymphocytes, etc.), neural cells, cardiovascular cells, epidermal cells, and metabolic cells.
  • the cells may be cancerous or non-cancerous.
  • the cells are tumor cells.
  • Other cell types are contemplated herein. IX.
  • variant Cas12a endonucleases have numerous uses, many of which are known in the art.
  • the variant Cas12a endonucleases of the present disclosure maybe used, in some instances, to improve those uses.
  • Several non-limiting examples of such uses include genome editing, bioengineering, diagnostics, and agricultural advancement.
  • Genome Editing In some embodiments, the variant Cas12a endonuclease are used for genome editing. Genome editing is a type of genetic engineering where a DNA is inserted, deleted or replaced in the genome of a living organism.
  • CRISPR-Cas systems as molecular tools for genome editing exploits their ability to produce a double strand break (DSB) at a specific genomic locus and depends entirely on the host cell DNA repair machinery to fix the lesion produced by these systems.
  • the repair mechanisms can be either of the following processes: homology-directed repair (HDR) or non-homologous end joining (NHEJ).
  • HDR utilizes a template DNA that is homologous to the break site (an unbroken sister chromatid or a homologous chromosome) to repair the DSB, whereas NHEJ is based on direct joining of broken ends of the DSB, making NHEJ the more error prone mechanism of the two.
  • HDR can thus be used to supply exogenous template DNA to implement a user defined change in the host genome.
  • NHEJ can be applied for gene disruption whereas HDR allows for the scope of introducing new genetic information or direct correction of the sequence at a specific locus.
  • Cas9 At the center of CRISPR mediated genome engineering today is Cas9, with applications including, but not limited to, gene knockout and precise genome editing. Despite the rapid advances in genome editing by Cas9, it still presents challenges owing to the possibility of off-target effects and difficulty of delivering the ribonucleoprotein particle.
  • Cas12a owing to its substantial differences with Cas9, presents an alternate molecular genome editing tool. The use of Cas12a in genome editing for various cell types has been probed in several studies up to date.
  • This auto-processing of its own crRNA has been used to modify multiple genetic elements simultaneously generating constitutive, conditional, inducible, orthogonal and multiplexed genome engineering of endogenous targets using multiple CRISPR RNAs delivered on a single plasmid.
  • the viability of this approach has been further established by other studies, in which multiplex gene regulation by Cas12a was successfully observed in bacteria, plants, as well as in mammalian cells.
  • Cas12a can also serve as a solution in cell types where use of Cas9 is toxic, such as in some industrial strains of Streptomyces.
  • Targeted mutagenesis in plants can also be achieved through co-expression of Cas12a and its cognate crRNA in vivo, as was shown in rice.
  • mutagenesis was more efficient through the use of pre-crRNAs with full-length direct repeat sequences than with mature crRNAs. Efficient mutagenesis through delivery of the pre- assembled ribonucleoprotein (RNP) particle was also observed in soybean and wild tobacco.
  • the RNP was assembled from recombinantly expressed Cas12a and in vitro transcribed or chemically synthesized crRNAs.
  • Successful gene editing of mammalian cells using Cas12a include correction of mutations causing Duchenne muscular dystrophy (DMD) in patient derived induced pluripotent stem cells (iPSCs) and in mdx mice, a popular model for studying DMD.
  • DMD Duchenne muscular dystrophy
  • iPSCs patient derived induced pluripotent stem cells
  • mdx mice a popular model for studying DMD.
  • Dystrophin expression was reinstated in iPSCs after Cas12a-mediated gene editing, while in the mdx mice, corrections in the pathophysiological hallmarks of muscular dystrophy were observed.
  • Delivery of the adenovirus vector with an AsCas12a expression cassette yielded successful mutations in primary human hepatocytes from humanized mice with chimeric liver.
  • Cas12a-mediated genome editing was also used to engineer rat models that mimic human atherosclerosis and this system may have potential applications in understanding early stage atherosclerosis. See Paul, B. & Montoya, G. et al. Biomedical Journal 2020; 43(1): 8-17, incorporated herein in its entirety. B.
  • the variant Cas12a endonuclease are used for bioengineering.
  • CRISPR-Cas nucleases in bioengineering of smart materials, for example hydrogels. These water-filled polymers are encapsulated by DNA.
  • Cas12a has been used to specifically degrade the DNA scaffold of DNA hydrogels, thus opening the possibility that this smart cutter can be turned into a programmable device to deliver the cargo of DNA encaged hydrogels in a determined location at a certain time.
  • the cleavage properties of Cas12a make it an ideal candidate to promote controlled delivery of the cargo.
  • the variant Cas12a endonucleases are used for detecting and/or quantifying nucleic acids.
  • the variants may be used as in vitro diagnostic tools for pathogenic (e.g., bacterial or viral) nucleic acids, or for identification of biomarkers indicative of disease, such as cancer (e.g., for identification and quantification of single CpG methylation sites – see, for example, van Dongen, JE et al.
  • the variant Cas12a endonucleases are used with the Specific Enhancer for Detection of PCR-amplified Nucleic Acids (SENA) method, which combines the transcleavage activity of Cas12a with the sensitivity offered by real-time PCR.
  • SENA Specific Enhancer for Detection of PCR-amplified Nucleic Acids
  • the variant Cas12a endonucleases are used with the DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR) technology, which performs simultaneous reverse transcription and isothermal amplification using loop-mediated amplification (RT–LAMP) for RNA extracted from a biological sample.
  • DETECTR DNA Endonuclease-Targeted CRISPR Trans Reporter
  • RT–LAMP loop-mediated amplification
  • the variant Cas12a endonucleases are used with the one-hour low-cost multipurpose highly efficient system (HOLMES) technology. See, e.g., Li, L. et al. ACS Synth Biol.2019; 8(10): 2228-2237.
  • HOLMES one-hour low-cost multipurpose highly efficient system
  • the variant Cas12a endonuclease are used for applications relating to agricultural advancement.
  • Cas12a editing has been widely utilized in many crops including rice, wheat, maize, soybean, cotton, tomato, citrus, tobacco, and the model plant Arabidopsis.
  • three Cas12a genome editing systems AsCas12a, FnCas12a, and LbCas12a have been demonstrated in plants with varied efficiency.
  • Rice is one of the most well-studied crops due to its agricultural importance, small genome size, ease of transformation and available genetic resources making it an ideal yield for the grasses. These factors have also made 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 average targeted mutation frequencies of 47.2% and 28.2%, respectively.
  • An independent study that targeted the disruption of OsPDS by LbCas12a resulted in a similarly high editing frequency of 32.3%. It was also demonstrated that pre-crRNAs were more efficient in generating mutants than mature crRNAs in rice. However, the opposite was observed in HEK293T cells.
  • LbCas12a was also used to create loss-of-function alleles of OsEPFL9 which regulates stomatal density. These lines increased water use efficiency eight-fold in T2 generation plants. See, Bandyopadhyay, A. et al. Front. Plant Sci.2020. Additional Embodiments Additional embodiments are described in the following numbered paragraphs: 1.
  • An engineered variant 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 amino acid position numbering of LbCas12a ND2006. 2.
  • the engineered variant Cas12a endonuclease of paragraph 1 wherein the variant Cas12a endonuclease exhibits hyperactivity, low single indiscriminate strand deoxyribonuclease (DNase) activity, target nickase activity (or a preference for cleaving one strand over the other of a dsDNA), or protospacer adjacent motif (PAM) nickase activity.
  • DNase low single indiscriminate strand deoxyribonuclease
  • PAM protospacer adjacent motif
  • the engineered variant Cas12a endonuclease of paragraph 6 wherein the mutation is a substitution of a polar, positively charged, and/or basic amino acid at position E95 with reference to amino acid position numbering of LbCas12a ND2006, preferably E95R or E95H, more preferably E95R.
  • the engineered variant Cas12a endonuclease of paragraph 6 wherein the mutation is a substitution of a polar, uncharged, and/or aromatic amino acid at position E95 with reference to amino acid position numbering of LbCas12a ND2006, preferably E95Y.
  • the mutation is a substitution of a polar, negatively charged, and/or acidic amino acid at position H759 with reference to amino acid position numbering of LbCas12a ND2006, preferably H759D. 34.
  • ssDNase single strand deoxyribonuclease
  • the engineered variant Cas12a endonuclease of paragraph 42 wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position N813 with reference to amino acid position numbering of LbCas12a ND2006, optionally wherein the polypeptide sequence has at least 90% identity to a wild-type reference Cas12a endonuclease, optionally to a wild-type reference Cas12a endonuclease of Table 1. 44.
  • the engineered variant Cas12a endonuclease of paragraph 42 wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position I831 with reference to amino acid position numbering of LbCas12a ND2006, optionally wherein the polypeptide sequence has at least 90% identity to a wild-type reference Cas12a endonuclease, optionally to a wild-type reference Cas12a endonuclease of Table 1. 47.
  • the mutation is a substitution of a polar, uncharged, and/or aromatic amino acid at position I831 with reference to amino acid position numbering of LbCas12a ND2006, preferably I831Y. 49.
  • the engineered variant Cas12a endonuclease of paragraph 42 wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position T870 with reference to amino acid position numbering of LbCas12a ND2006, optionally wherein the polypeptide sequence has at least 90% identity to a wild-type reference Cas12a endonuclease, optionally to a wild-type reference Cas12a endonuclease of Table 1. 50.
  • the engineered variant Cas12a endonuclease of paragraph 47 wherein the mutation is a substitution of a polar, uncharged, and/or aromatic amino acid at position T870 with reference to amino acid position numbering of LbCas12a ND2006, preferably T870Y. 51.
  • the engineered variant Cas12a endonuclease of paragraph 42 wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position G902 with reference to amino acid position numbering of LbCas12a ND2006, optionally wherein the polypeptide sequence has at least 90% identity to a wild-type reference Cas12a endonuclease, optionally to a wild-type reference Cas12a endonuclease of Table 1. 53.
  • the engineered variant Cas12a endonuclease of paragraph 52 wherein the mutation is a substitution of a polar, positively charged, and/or basic amino acid at position G902 with reference to amino acid position numbering of LbCas12a ND2006, preferably G902R, G902H, or G902K, more preferably G902R. 54.
  • the engineered variant Cas12a endonuclease of paragraph 42 wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position S982 with reference to amino acid position numbering of LbCas12a ND2006, optionally wherein the polypeptide sequence has at least 90% identity to a wild-type reference Cas12a endonuclease, optionally to a wild-type reference Cas12a endonuclease of Table 1. 56.
  • the engineered variant Cas12a endonuclease of paragraph 42 wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position K984 with reference to amino acid position numbering of LbCas12a ND2006, optionally wherein the polypeptide sequence has at least 90% identity to a wild-type reference Cas12a endonuclease, optionally to a wild-type reference Cas12a endonuclease of Table 1. 58.
  • the engineered variant Cas12a endonuclease of paragraph 57 wherein the mutation is a substitution of a nonpolar, uncharged, and/or aromatic amino acid at position K984 with reference to amino acid position numbering of LbCas12a ND2006, preferably K984F or K984W, more preferably K984F.
  • the engineered variant Cas12a endonuclease of paragraph 42 wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position T988 with reference to amino acid position numbering of LbCas12a ND2006, optionally wherein the polypeptide sequence has at least 90% identity to a wild-type reference Cas12a endonuclease, optionally to a wild-type reference Cas12a endonuclease of Table 1. 61.
  • the engineered variant Cas12a endonuclease of paragraph 60 wherein the mutation is a substitution of a nonpolar, uncharged, and/or aromatic amino acid at position T988 with reference to amino acid position numbering of LbCas12a ND2006, preferably T988F or T988W, more preferably T988F. 62.
  • the engineered variant Cas12a endonuclease of paragraph 62 wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position N260 with reference to amino acid position numbering of LbCas12a, optionally wherein the polypeptide sequence has at least 90% identity to a wild-type reference Cas12a endonuclease, optionally to a wild-type reference Cas12a endonuclease of Table 1. 64.
  • the engineered variant Cas12a endonuclease of paragraph 62 wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position G902 with reference to amino acid position numbering of LbCas12a ND2006, optionally wherein the polypeptide sequence has at least 90% identity to a wild-type reference Cas12a endonuclease, optionally to a wild-type reference Cas12a endonuclease of Table 1. 66.
  • the engineered variant Cas12a endonuclease of paragraph 65 wherein the mutation is a substitution of a nonpolar, uncharged, and/or aromatic amino acid at position G902 with reference to amino acid position numbering of LbCas12a ND2006, preferably G902W or G902F, more preferably G902W. 67.
  • the engineered variant Cas12a endonuclease of paragraph 67 wherein the mutation is a substitution of a polar, negatively charged, and/or amide amino acid amino acid at position K960 with reference to amino acid position numbering of LbCas12a ND2006, preferably K960E, optionally wherein the polypeptide sequence has at least 90% identity to a wild-type reference Cas12a endonuclease, optionally to a wild-type reference Cas12a endonuclease of Table 1. 69.
  • the engineered variant Cas12a endonuclease of any one of the preceding paragraphs comprising an amino acid sequence having at least 85%, at least 90%, or least 95%, but less than 100% identity with the amino acid sequence of a wild-type Cas12a endonuclease selected from Acidaminococcus sp., Lachnospiraceae sp., and Francisella sp. 70.
  • the engineered variant 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. 71.
  • An engineered variant Cas12a endonuclease comprising a polypeptide sequence comprising the amino acid sequence of any one of SEQ ID NOs: 48-119.
  • 72. A polynucleotide encoding the variant Cas12a endonuclease of any one of the preceding paragraphs.
  • 73. A cell comprising (a) the variant Cas12a endonuclease of any one of the preceding paragraphs or the polynucleotide of paragraph 72 and (b) a guide RNA or a polynucleotide encoding a guide RNA.
  • a method comprising introducing into a cell (a) the variant Cas12a endonuclease of any one of the preceding paragraphs or the polynucleotide of paragraph 72 and optionally (b) a guide RNA or a polynucleotide encoding a guide RNA.
  • a method comprising introducing into a cell (a) the variant Cas12a endonuclease of any one of the preceding paragraphs or the polynucleotide of paragraph 72 and optionally (b) a guide RNA or a polynucleotide encoding a guide RNA.
  • a guide RNA or a polynucleotide encoding a guide RNA e.g., a guide RNA or a polynucleotide encoding a guide RNA.
  • a method for introducing a double strand break in a target nucleic acid comprising introducing into a cell comprising a target nucleic acid (a) the variant Cas12a endonuclease of any one of paragraphs 5-41 and (b) a guide RNA; and incubating the cell to produce a double strand break in the target nucleic acid.
  • a method for introducing a double strand break in a target nucleic acid comprising introducing into a cell comprising a target nucleic acid (a) the variant Cas12a endonuclease of any one of paragraphs 42-61 and (b) a guide RNA; and incubating the cell to produce a double strand break in the target nucleic acid.
  • An engineered variant 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 amino acid position numbering of LbCas12a ND2006. 2.
  • the engineered variant Cas12a endonuclease of paragraph 1 wherein the engineered variant Cas12a endonuclease exhibits hyperactivity, low indiscriminate single strand deoxyribonuclease (DNase) activity, or target nickase activity (or a preference for cleaving one strand over the other of a dsDNA).
  • DNase indiscriminate single strand deoxyribonuclease
  • target nickase activity or a preference for cleaving one strand over the other of a dsDNA.
  • ssDNase single strand deoxyribonuclease
  • the engineered variant Cas12a endonuclease of paragraph 12 wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position K932 with reference to amino acid position numbering of LbCas12a ND2006, optionally wherein the polypeptide sequence has at least 90% identity to a wild-type reference Cas12a endonuclease, optionally to a wild-type reference Cas12a endonuclease of Table 1. 16.
  • the polypeptide sequence comprises a mutation at an amino acid position corresponding to position N933 with reference to amino acid position numbering of LbCas12a ND2006, optionally wherein the polypeptide sequence has at least 90% identity to a wild-type reference Cas12a endonuclease, optionally to a wild-type reference Cas12a endonuclease of Table 1.
  • the engineered variant Cas12a endonuclease of paragraph 12 wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position S934 with reference to amino acid position numbering of LbCas12a ND2006, optionally wherein the polypeptide sequence has at least 90% identity to a wild-type reference Cas12a endonuclease, optionally to a wild-type reference Cas12a endonuclease of Table 1.
  • 24. The engineered variant Cas12a endonuclease of paragraph 23, wherein the mutation is a substitution of a polar, uncharged, and/or acidic amino acid at position S934 with reference to amino acid position numbering of LbCas12a, preferably S934Q.
  • the engineered variant Cas12a endonuclease of paragraph 12 wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position V936 with reference to amino acid position numbering of LbCas12a ND2006, optionally wherein the polypeptide sequence has at least 90% identity to a wild-type reference Cas12a endonuclease, optionally to a wild-type reference Cas12a endonuclease of Table 1.
  • the mutation is a substitution of a polar, negatively charged, and/or amide amino acid at position V936 with reference to amino acid position numbering of LbCas12a, preferably V936E.
  • the engineered variant Cas12a endonuclease of paragraph 12 wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position K937 with reference to amino acid position numbering of LbCas12a ND2006, optionally wherein the polypeptide sequence has at least 90% identity to a wild-type reference Cas12a endonuclease, optionally to a wild-type reference Cas12a endonuclease of Table 1. 29.
  • the engineered variant Cas12a endonuclease of paragraph 28 wherein the mutation is a substitution of a polar, uncharged, and/or aromatic amino acid at position K937 with reference to amino acid position numbering of LbCas12a, preferably K937Y.
  • the mutation is a substitution of a polar, negatively charged, and/or acidic amino acid at position Q944 with reference to amino acid position numbering of LbCas12a, preferably Q944D.
  • the engineered variant Cas12a endonuclease of paragraph 39 wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position R833 with reference to amino acid position numbering of LbCas12a ND2006, optionally wherein the polypeptide sequence has at least 90% identity to a wild-type reference Cas12a endonuclease, optionally to a wild-type reference Cas12a endonuclease of Table 1. 41.
  • the engineered variant Cas12a endonuclease of paragraph 40 wherein the mutation is a substitution of a polar, positively charged, and/or basic amino acid at position R833 with reference to amino acid position numbering of LbCas12a, preferably R833K or R833H, more preferably R833K optionally wherein the engineered variant Cas12a endonuclease also exhibits low indiscriminate ssDNase activity. 42.
  • the engineered variant Cas12a endonuclease of paragraph 40 wherein the mutation is a substitution of a nonpolar, uncharged, and/or aliphatic amino acid at position R833 with reference to amino acid position numbering of LbCas12a, preferably R833L, R833A, R833I, R833P, or R833V, more preferably R833L, optionally wherein the engineered variant Cas12a endonuclease also exhibits low indiscriminate ssDNase activity. 44.
  • the engineered variant Cas12a endonuclease of paragraph 39 wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position E835 with reference to amino acid position numbering of LbCas12a ND2006, optionally wherein the polypeptide sequence has at least 90% identity to a wild-type reference Cas12a endonuclease, optionally to a wild-type reference Cas12a endonuclease of Table 1. 45.
  • the engineered variant Cas12a endonuclease of paragraph 44 wherein the mutation is a substitution of a polar, negatively charged, and/or acidic amino acid at position E835 with reference to amino acid position numbering of LbCas12a, preferably E835D, optionally wherein the engineered variant Cas12a endonuclease exhibits hypoactivity. 46.
  • the engineered variant Cas12a endonuclease of paragraph 44 wherein the mutation is a substitution of a nonpolar, uncharged, and/or aliphatic amino acid at position E835 with reference to amino acid position numbering of LbCas12a, preferably E835G, E835A, E835I, E835L, E835P, or E835V, more preferably E835G or E835A. 47.
  • the engineered variant Cas12a endonuclease of paragraph 39 wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position R836 with reference to amino acid position numbering of LbCas12a ND2006, optionally wherein the polypeptide sequence has at least 90% identity to a wild-type reference Cas12a endonuclease, optionally to a wild-type reference Cas12a endonuclease of Table 1.
  • the engineered variant Cas12a endonuclease of paragraph 47 wherein the mutation is a substitution of a nonpolar, uncharged, and/or aliphatic amino acid at position R836 with reference to amino acid position numbering of LbCas12a, preferably R836G, R836A, R836I, R836L, R836P, or R836V, more preferably R836G or R836A. 49.
  • the engineered variant Cas12a endonuclease of paragraph 39 wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position F931 with reference to amino acid position numbering of LbCas12a ND2006, optionally wherein the polypeptide sequence has at least 90% identity to a wild-type reference Cas12a endonuclease, optionally to a wild-type reference Cas12a endonuclease of Table 1. 50.
  • the engineered variant Cas12a endonuclease of paragraph 49 wherein the mutation is a substitution of a polar, positively charged, and/or basic amino acid at position F931 with reference to amino acid position numbering of LbCas12a, preferably F931H or F931K, more preferably F931H, optionally wherein the engineered variant Cas12a endonuclease exhibits hypoactivity. 51.
  • the engineered variant Cas12a endonuclease of paragraph 39 wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position K932 with reference to amino acid position numbering of LbCas12a ND2006, optionally wherein the polypeptide sequence has at least 90% identity to a wild-type reference Cas12a endonuclease, optionally to a wild-type reference Cas12a endonuclease of Table 1. 53.
  • the engineered variant Cas12a endonuclease of paragraph 52 wherein the mutation is a substitution of a nonpolar, uncharged, and/or aliphatic amino acid at position K932 with reference to amino acid position numbering of LbCas12a, preferably K932G or K932P, more preferably K932G, optionally wherein the engineered variant Cas12a endonuclease exhibits hypoactivity. 54.
  • the engineered variant Cas12a endonuclease of paragraph 52 wherein the mutation is a substitution of a polar, negatively charged, and/or amide amino acid at position K932 with reference to amino acid position numbering of LbCas12a, preferably K932E, optionally wherein the engineered variant Cas12a endonuclease exhibits hypoactivity.
  • the mutation is a substitution of a polar, positively charged, and/or basic amino acid at position K932 with reference to amino acid position numbering of LbCas12a, preferably K932H or K932R, more preferably K932H.
  • the engineered variant Cas12a endonuclease of paragraph 52 wherein the mutation is a substitution of a nonpolar, uncharged, and/or sulfur amino acid at position K932 with reference to amino acid position numbering of LbCas12a, preferably K932M or K932C, more preferably K932M.
  • the engineered variant Cas12a endonuclease of paragraph 52 wherein the mutation is a substitution of a polar, uncharged, and/or acidic amino acid at position K932 with reference to amino acid position numbering of LbCas12a, preferably K932Q.
  • the engineered variant Cas12a endonuclease of paragraph 39 wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position R935 with reference to amino acid position numbering of LbCas12a ND2006, optionally wherein the polypeptide sequence has at least 90% identity to a wild-type reference Cas12a endonuclease, optionally to a wild-type reference Cas12a endonuclease of Table 1. 61.
  • the engineered variant Cas12a endonuclease of paragraph 60 wherein the mutation is a substitution of a nonpolar, uncharged, and/or aliphatic amino acid at position R935 with reference to amino acid position numbering of LbCas12a, preferably R935L, R935G, R935I, or R935P, more preferably R935L, R935G, or R935I, optionally wherein the engineered variant Cas12a endonuclease having the R935I substitution exhibits hypoactivity. 62.
  • the engineered variant Cas12a endonuclease of paragraph 60 wherein the mutation is a substitution of a polar, positively charged, and/or basic amino acid at position R935 with reference to amino acid position numbering of LbCas12a, preferably R935H or R935K.
  • the engineered variant Cas12a endonuclease of paragraph 60 wherein the mutation is a substitution of a nonpolar, uncharged, and/or aromatic amino acid at position R935 with reference to amino acid position numbering of LbCas12a, preferably R935F or R935W, optionally wherein the engineered variant Cas12a endonuclease having the R935W substitution exhibits hypoactivity.
  • the engineered variant Cas12a endonuclease of paragraph 60 wherein the mutation is a substitution of a nonpolar, uncharged, and/or sulfur amino acid at position R935 with reference to amino acid position numbering of LbCas12a, preferably R935M or R935C, more preferably R935M.
  • the engineered variant Cas12a endonuclease of paragraph 39 wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position V936 with reference to amino acid position numbering of LbCas12a ND2006, optionally wherein the polypeptide sequence has at least 90% identity to a wild-type reference Cas12a endonuclease, optionally to a wild-type reference Cas12a endonuclease of Table 1. 68.
  • the engineered variant Cas12a endonuclease of paragraph 67 wherein the mutation is a substitution of a nonpolar, uncharged, and/or aliphatic amino acid at position V936 with reference to amino acid position numbering of LbCas12a, preferably V936G, V936I, V936L, or V936P, more preferably V936G, optionally wherein the engineered variant Cas12a endonuclease also exhibits low indiscriminate ssDNase activity. 69.
  • the engineered variant Cas12a endonuclease of paragraph 39 wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position V938 with reference to amino acid position numbering of LbCas12a ND2006, optionally wherein the polypeptide sequence has at least 90% identity to a wild-type reference Cas12a endonuclease, optionally to a wild-type reference Cas12a endonuclease of Table 1. 70.
  • the engineered variant Cas12a endonuclease of paragraph 39 wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position K940 with reference to amino acid position numbering of LbCas12a ND2006, optionally wherein the polypeptide sequence has at least 90% identity to a wild-type reference Cas12a endonuclease, optionally to a wild-type reference Cas12a endonuclease of Table 1. 72.
  • the engineered variant Cas12a endonuclease of paragraph 39 wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position Q941 with reference to amino acid position numbering of LbCas12a ND2006, optionally wherein the polypeptide sequence has at least 90% identity to a wild-type reference Cas12a endonuclease, optionally to a wild-type reference Cas12a endonuclease of Table 1. 74.
  • the mutation is a substitution of a polar, uncharged, and/or aromatic amino acid at position Q941 with reference to amino acid position numbering of LbCas12a, preferably Q941Y.
  • the engineered variant Cas12a endonuclease of paragraph 39 wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position Y943 with reference to amino acid position numbering of LbCas12a ND2006, optionally wherein the polypeptide sequence has at least 90% identity to a wild-type reference Cas12a endonuclease, optionally to a wild-type reference Cas12a endonuclease of Table 1. 77.
  • the engineered variant Cas12a endonuclease of paragraph 76 wherein the mutation is a substitution of a polar, uncharged, and/or hydroxyl amino acid at position Y943 with reference to amino acid position numbering of LbCas12a, preferably Y943T or Y943S, more preferably Y943T.
  • the engineered variant Cas12a endonuclease of paragraph 39 wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position Q944 with reference to amino acid position numbering of LbCas12a ND2006, optionally wherein the polypeptide sequence has at least 90% identity to a wild-type reference Cas12a endonuclease, optionally to a wild-type reference Cas12a endonuclease of Table 1. 80.
  • the engineered variant Cas12a endonuclease of paragraph 39 wherein the polypeptide sequence comprises a mutation at an amino acid position corresponding to position M986 with reference to amino acid position numbering of LbCas12a ND2006, optionally wherein the polypeptide sequence has at least 90% identity to a wild-type reference Cas12a endonuclease, optionally to a wild-type reference Cas12a endonuclease of Table 1.
  • the engineered variant Cas12a endonuclease of paragraph 82 wherein the mutation is a substitution of a nonpolar, uncharged, and/or aliphatic amino acid at position M986 with reference to amino acid position numbering of LbCas12a, preferably M986G, M986A, M986I, M986P, or M986V, more preferably M986G, optionally wherein the engineered variant Cas12a endonuclease also exhibits low indiscriminate ssDNase activity. 84.
  • An engineered variant 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 amino acid position numbering of LbCas12a ND20006, preferably wherein the engineered variant Cas12a endonuclease exhibits low indiscriminate single strand DNase activity, optionally wherein the engineered variant Cas12a endonuclease also exhibits hypoactivity.
  • An engineered variant Cas12a endonuclease comprising a polypeptide sequence comprising amino acid substitutions corresponding to the following amino acid substitutions: N933L and Q944M, with reference to amino acid position numbering of LbCas12a ND20006, preferably wherein the engineered variant Cas12a endonuclease exhibits low indiscriminate single strand DNase activity, optionally wherein the engineered variant Cas12a endonuclease also exhibits hypoactivity.
  • An engineered variant Cas12a endonuclease comprising a polypeptide sequence comprising amino acid substitutions corresponding to the following amino acid substitutions: R833K and E947D, with reference to amino acid position numbering of LbCas12a ND20006, preferably wherein the engineered variant Cas12a endonuclease exhibits target nickase activity (or a preference for cleaving one strand over the other of a dsDNA).
  • An engineered variant Cas12a endonuclease comprising a polypeptide sequence comprising amino acid substitutions corresponding to the following amino acid substitutions: E835G and E880G, with reference to amino acid position numbering of LbCas12a ND20006, preferably wherein the engineered variant Cas12a endonuclease exhibits target nickase activity (or a preference for cleaving one strand over the other of a dsDNA).
  • An engineered variant Cas12a endonuclease comprising a polypeptide sequence comprising amino acid substitutions corresponding to the following amino acid substitutions: S929G, K932G, and N933G, with reference to amino acid position numbering of LbCas12a ND20006, preferably wherein the engineered variant Cas12a endonuclease exhibits target nickase activity (or a preference for cleaving one strand over the other of a dsDNA).
  • An engineered variant 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 amino acid position numbering of LbCas12a ND20006, preferably wherein the engineered variant Cas12a endonuclease exhibits target nickase activity (or a preference for cleaving one strand over the other of a dsDNA).
  • An engineered variant 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 amino acid position numbering of LbCas12a ND20006, preferably wherein the engineered variant Cas12a endonuclease exhibits target nickase activity (or a preference for cleaving one strand over the other of a dsDNA).
  • An engineered variant 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 amino acid position numbering of LbCas12a ND20006, preferably wherein the engineered variant Cas12a endonuclease exhibits target nickase activity (or a preference for cleaving one strand over the other of a dsDNA). 92.
  • An engineered variant Cas12a endonuclease comprising a polypeptide sequence comprising amino acid substitutions corresponding to the following amino acid substitutions: G930A and F931L, with reference to amino acid position numbering of LbCas12a ND20006, preferably wherein the engineered variant Cas12a endonuclease exhibits target nickase activity (or a preference for cleaving one strand over the other of a dsDNA)and low indiscriminate single strand DNase activity. 93.
  • An engineered variant Cas12a endonuclease comprising a polypeptide sequence comprising amino acid substitutions corresponding to the following amino acid substitutions: G930A, F931L, and S934Q with reference to amino acid position numbering of LbCas12a, preferably wherein the engineered variant Cas12a endonuclease exhibits target nickase activity (or a preference for cleaving one strand over the other of a dsDNA).
  • An engineered variant Cas12a endonuclease comprising a polypeptide sequence comprising amino acid substitutions corresponding to the following amino acid substitutions: K932G and N933G, with reference to amino acid position numbering of LbCas12a ND20006, preferably wherein the engineered variant Cas12a endonuclease exhibits target nickase activity (or a preference for cleaving one strand over the other of a dsDNA).
  • An engineered variant Cas12a endonuclease comprising a polypeptide sequence comprising amino acid substitutions corresponding to the following amino acid substitutions: K932G, N933G, and V936F, with reference to amino acid position numbering of LbCas12a ND20006, preferably wherein the engineered variant Cas12a endonuclease exhibits target nickase activity (or a preference for cleaving one strand over the other of a dsDNA).
  • An engineered variant Cas12a endonuclease comprising a polypeptide sequence comprising amino acid substitutions corresponding to the following amino acid substitutions: V936G, F983G, and M986G, with reference to amino acid position numbering of LbCas12a ND20006, preferably wherein the engineered variant Cas12a endonuclease exhibits target nickase activity (or a preference for cleaving one strand over the other of a dsDNA).
  • An engineered variant Cas12a endonuclease comprising a polypeptide sequence comprising amino acid substitutions corresponding to the following amino acid substitutions: F983G and M986G, with reference to amino acid position numbering of LbCas12a ND20006, preferably wherein the engineered variant Cas12a endonuclease exhibits target nickase activity (or a preference for cleaving one strand over the other of a dsDNA) and low indiscriminate single strand DNase activity.
  • F983G and M986G with reference to amino acid position numbering of LbCas12a ND20006, preferably wherein the engineered variant Cas12a endonuclease exhibits target nickase activity (or a preference for cleaving one strand over the other of a dsDNA) and low indiscriminate single strand DNase activity.
  • An engineered variant Cas12a endonuclease comprising a polypeptide sequence comprising a Lid-hub domain, wherein the polypeptide comprises a mutation at an amino acid position in the Lid-hub domain or in the vicinity of the Lid-Hub domain.
  • the engineered variant 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 amino acid position numbering of LbCas12a ND20006.
  • the engineered variant Cas12a endonuclease of paragraph 100 wherein the mutation is a substitution corresponding to any one of the following amino acid substitutions: K932I, K932L, K932V, N933L, or V936M.
  • DNase indiscriminate single strand deoxyribonuclease
  • the engineered variant 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. 104.
  • the engineered variant Cas12a endonuclease of paragraph 104 wherein the mutation is a substitution corresponding to any one 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.
  • the engineered variant Cas12a endonuclease of any one of the preceding paragraphs comprising an amino acid sequence having at least 85%, at least 90%, or least 95%, but less than 100% identity with the amino acid sequence of a wild-type Cas12a endonuclease selected from Acidaminococcus sp., Lachnospiraceae sp., and Francisella sp. 107.
  • the engineered variant 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. 108.
  • An engineered variant Cas12a endonuclease comprising a polypeptide sequence comprising the amino acid sequence of any one of SEQ ID NOs: 48-119.
  • a cell comprising (a) the variant Cas12a endonuclease of any one of the preceding paragraphs or the polynucleotide of paragraph 109 and (b) a guide RNA or a polynucleotide encoding a guide RNA.
  • a method comprising introducing into a cell (a) the variant Cas12a endonuclease of any one of the preceding paragraphs or the polynucleotide of paragraph 109 and optionally (b) a guide RNA or a polynucleotide encoding a guide RNA. 112. Use of the variant Cas12a endonuclease of any one of the preceding paragraphs for cleaving a nucleic acid. 113.
  • a method for introducing a double strand break in a target nucleic acid comprising introducing into a cell comprising a target nucleic acid (a) the variant Cas12a endonuclease of any one of paragraphs 5-11 and (b) a guide RNA; and incubating the cell to produce a double strand break in the target nucleic acid.
  • a method for introducing a double strand break in a target nucleic acid comprising introducing into a cell comprising a target nucleic acid (a) the variant Cas12a endonuclease of any one of paragraphs 12-38, 84, or 85 and (b) a guide RNA; and incubating the cell to produce a double strand break in the target nucleic acid.
  • a target nucleic acid a variant Cas12a endonuclease of any one of paragraphs 12-38, 84, or 85 and (b) a guide RNA
  • a guide RNA a guide RNA
  • a method for introducing a single strand break in a target nucleic acid comprising introducing into a cell comprising a target nucleic acid (a) the variant Cas12a endonuclease of any one of paragraphs 39-83 or 86-97 and (b) a guide RNA; and incubating the cell to produce a single strand break in the target nucleic acid.
  • Cas12a Endonuclease Variants Purified Cas12a variants of Table 3 in complex with crRNA were tested against (1) dsDNA containing a quencher on one site of the cleavage site and a fluorophore on the other site of the cleavage site, on separate DNA strands, and (2) a combination dsDNA containing no quencher or fluorophore but together with a ssDNA containing both a quencher and fluorophore. The retained activity on dsDNA was confirmed by (1) where the quencher and fluorophore were separated upon cleavage, and the emission signal increases over time.
  • Hyperactive Cas12a endonuclease variants were identified as having a higher reaction speed or by initiating the reaction faster than the naturally-occurring (i.e., wildtype (WT)) Cas12a. Within the subset of Cas12a endonuclease variants that had hyperactivity, several variants were also identified as having low ssDNase activity. Low ssDNase activity is identified by a lowered ability to cleave the ssDNA and thereby separate the quencher and fluorophore when activated by specific dsDNA, thus by lacking an emission signal in comparable magnitude to that of the wildtype Cas12a in (2). See FIGs.2A-2D and Table 6.
  • Hypoactive Cas12a endonuclease variants were identified as having a lower reaction speed or by initiating the reaction slower than the wildtype (WT) Cas12a.
  • WT wildtype
  • several variants were also identified as having low ssDNase activity.
  • Low ssDNase activity is identified by a lowered ability to cleave the ssDNA and thereby separate the quencher and fluorophore when activated by specific dsDNA, thus by lacking an emission signal in comparable magnitude to that of the wildtype Cas12a in (2). See FIGs.3A-3E, 4A-4C, and Table 6.
  • U2OS cells were transfected using a plasmid expressing the base editor together with a plasmid expressing a gRNA (AGCCTCAC 8 C 9 C 10 CTC 13 TAGCCCT (SEQ ID NO: 186)). Transfected cells were sorted after 3 days and re-cultured for another 3 days after which the cells were lysed and genomic DNA was extracted. Genomic region around the gRNA was PCR amplified and editing was analyzed by next generation sequencing. In these experiments, LbCas12a was either catalytic inactive (inactive Cas12a comprising a D832A mutation) LbCas12a or a LbCas12a variant (TBN04).
  • FIG.6A-6C showed efficient base editing (FIG.6A-6C).
  • FIG.6A % of total reads showing base editing at individual positions. This includes the reads that show base editing without indels plus the reads that harbor base editing as well as the indels.
  • FIG. 6B % of total reads showing only base editing at individual positions. This only includes the reads that show base editing but no indels.
  • FIG.6C % of total reads showing base editing at only one specific position. This excludes all the reads with base editing at multiple positions as well as the reads harboring indels.
  • Inactive Cas12a was used as the control.
  • the improvement in the base editing efficiency with LbBEv2 was more significant when using a base editor containing LbCas12a variant (TBN04).
  • TBN04 base editor containing LbCas12a variant
  • LbBEv2 Structure of LbBEv2 is as follows: Linker3-rAPOBEC1-Linker4-LbCas12a-NP NLS-Linker5-UGI-Linker2-SV40 NLS; wherein Linker3 sequence is the amino acid sequence of GS, Linker4 sequence is the amino acid sequence of SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 204) and Linker5 sequence is the amino acid sequence of GSSGGSGGSGGS (SEQ ID NO: 207).
  • Linker3 sequence is the amino acid sequence of GS
  • Linker4 sequence is the amino acid sequence of SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 204)
  • Linker5 sequence is the amino acid sequence of GSSGGSGGSGGS (SEQ ID NO: 207).
  • C-to-T base editors fusion proteins comprising a LbCas12a Cas12a variant (TBN04) and cytidine deaminase
  • NLSs at the N-terminus LbVEv3, LbBEv4 and LbBEv5
  • U2OS cells were transfected using a plasmid expressing the base editor together with a plasmid expressing the gRNA. Transfected cells were sorted after 3 days and re-cultured for another 3 days after which the cells were lysed, and genomic DNA was extracted.
  • FIG.7A % of total reads showing base editing at individual positions. This includes the reads that show base editing without indels plus the reads that harbor base editing as well as the indels.
  • FIG.7B % of total reads showing only base editing at individual positions. This only includes the reads that show base editing but no indels.
  • FIG.7C % of total reads showing base editing at only one specific position. This excludes all the reads with base editing at multiple positions as well as the reads harboring indels.
  • FIG.7D % of total reads showing indels.
  • Inactive Cas12a was used as the control.
  • LbCas12a can be catalytic inactive (inactive LbCas12a comprising a D832A mutation) or a variant LbCas12a (TBN04).
  • FIG.8A-8D % of total reads showing base editing at individual positions. This includes the reads that show base editing without indels plus the reads that harbor base editing as well as the indels.
  • FIG.8B % of total reads showing only base editing at individual positions. This only includes the reads that show base editing but no indels.
  • FIG. 8C % of total reads showing base editing at only one specific position.
  • FIG.8D % of total reads showing indels. Inactive Cas12a was used as the control.
  • LbBEv3 Structure of LbBEv3 is as follows: SV40 NLS-Linker3-rAPOBEC1-Linker4-LbCas12a-NP NLS-Linker5-UGI-Linker2-SV40 NLS Structure of LbBEv4 is as follows: NP NLS-Linker3-rAPOBEC1-Linker4-LbCas12a-NP NLS-Linker5-UGI-Linker2-SV40 NLS Structure of LbBEv5 is as follows: Linker3-BP NLS-Linker3-rAPOBEC1-Linker4-LbCas12a-NP NLS-Linker5-UGI-Linker2- SV40 NLS Example 4.
  • C-to-T base editors fusion proteins comprising a Cas12a variant and cytidine deaminase
  • U2OS cells were transfected using a plasmid expressing the base editor together with a plasmid expressing the gRNA. Transfected cells were sorted after 3 days and re- cultured for another 3 days after which the cells were lysed and genomic DNA was extracted. Genomic region around the gRNA was PCR amplified and editing was analyzed by next generation sequencing.
  • C-to-T base editor containing the TBN04 shows overall higher base editing efficiency than base editors containing inactive Cas12a. Moreover, TBN04 base editor shows remarkable base selectivity where the majority of edited alleles has editing only at one specific position whereas C-to-T base editor containing inactive LbCas12a lacks base selectivity and edits multiple Cs in the editing window.
  • FIGs.9A-9D provide graphs of data comparing percent (%) of total reads having a C- to-T nucleotide edit at genomic positions corresponding to positions C6, C8, C9, C10, C11, and C13 of the gRNA using the LbBEv5 base editor.
  • FIG.9A % of total reads showing base editing at individual positions.
  • FIG.9B % of total reads showing only base editing at individual positions. This only includes the reads that show base editing but no indels.
  • FIG.9C % of total reads showing base editing at only one specific position. This excludes all the reads with base editing at multiple positions as well as the reads harboring indels.
  • FIG.9D % of total reads showing indels.
  • FIGs.9E-9F show allele frequency tables around the gRNA.
  • Fig.9E frequency of alleles harboring different edits by LbBEv5 containing inactive LbCas12a and gRNA AGCCTC 6 AC 8 C 9 C 10 C 11 TC 13 TAGCCCT (SEQ ID NO: 192) in U2OS cells.
  • FIG.9F frequency of alleles harboring different edits by LbBEv5 containing TBN04 (LbCas12a) and gRNA AGCCTC 6 AC 8 C 9 C 10 C 11 TC 13 TAGCCCT (SEQ ID NO: 192) in U2OS cells.
  • FIGs.10A-10D provide graphs of data comparing percent (%) of total reads having a C-to-T nucleotide edit at genomic positions corresponding to positions C8 and C10 of the gRNA using the LbBEv5 base editor.
  • FIG.10A % of total reads showing base editing at individual positions. This includes the reads that show base editing without indels plus the reads that harbor base editing as well as the indels.
  • FIG.10B % of total reads showing only base editing at individual positions. This only includes the reads that show base editing but no indels.
  • FIG.10C % of total reads showing base editing at only one specific position. This excludes all the reads with base editing at multiple positions as well as the reads harboring indels.
  • FIG.10D % of total reads showing indels.
  • FIGs.10E-10F show allele frequency tables around the gRNA.
  • FIG.10E frequency of alleles harboring different edits by LbBEv5 containing inactive LbCas12a and gRNA TTCTCCCC 8 TC 10 TGCTGGATAC (SEQ ID NO: 187) in U2OS cells.
  • FIG.10F frequency of alleles harboring different edits by LbBEv5 containing TBN04 (LbCas12a) and gRNA TTCTCCCC 8 TC 10 TGCTGGATAC (SEQ ID NO: 187) in U2OS cells.
  • Example 5 Efficiency and Specificity of A-to-G Base Editors The base editing efficiency and specificity of A-to-G base editor (fusion protein comprising a Cas12a variant and adenosine deaminase) containing inactive LbCas12a (Cas12a comprising a D832A mutation) or TBN04 (LbCas12a variant) was determined.
  • U2OS cells were transfected using a plasmid expressing the base editor together with a plasmid expressing the gRNA. Transfected cells were sorted after 3 days and re-cultured for another 3 days after which the cells were lysed and genomic DNA was extracted. Genomic region around the gRNA was PCR amplified and editing was analyzed by next generation sequencing.
  • the A-to-G base editor containing the TBN04 shows overall higher base editing efficiency than base editors containing inactive Cas12a. Moreover, TBN04 base editor shows significant base selectivity where the majority of edited alleles has editing only at one specific position.
  • FIGs.11A-11D provide graphs of data comparing percent (%) of total reads having a C-to-T nucleotide edit at genomic positions corresponding to positions A7, A8, A11, A12, and A15 of the gRNA using the LbABE8e base editor.
  • FIG.11A % of total reads showing A-to-G base editing at individual positions. This includes the reads that show base editing without indels plus the reads that harbor base editing as well as the indels.
  • FIG.11B % of total reads showing only base editing at individual positions. This only includes the reads that show base editing but no indels.
  • FIG.11C % of total reads showing base editing at only one specific position.
  • FIG.11D % of total reads showing indels.
  • FIGs.11E-11F show allele frequency tables around the gRNA.
  • Fig.11E frequency of alleles harboring different edits by LbABE8e containing inactive LbCas12a and gRNA TGCTGCA 7 A 8 GTA 11 A 12 GCA 15 TGCATTTG (SEQ ID NO: 188) in U2OS cells.
  • FIG.11F frequency of alleles harboring different edits by LbABE8e containing TBN04 (LbCas12a) and gRNA TGCTGCA 7 A 8 GTA 11 A 12 GCA 15 TGCATTTG (SEQ ID NO: 188) in U2OS cells.
  • the structure of LbABE8e is as follows: BP NLS-TadA-Linker4-LbCas12a-Linker2-BP NLS; wherein LbCas12a can be inactive LbCas12a or TBN04 (LbCas12a variant).
  • a selection of fusion proteins comprising a Cas12a variants as provided in Table 7 were assayed for their ability to produce indels and perform an A-to-G base conversion.
  • the Cas12a variants were placed into the LbABE8e structural framework.
  • the data in Table 7 and FIGs.12A-12C demonstrate that the A-to-G base editors were selective for the A 11 in the gRNA TGCTGCA 7 A 8 GTA 11 A 12 GCA 15 TGCATTTG (SEQ ID NO: 188).
  • FIGs.12A-12C provide graphs of data comparing percent (%) of total reads having a A-to-G nucleotide edit at genomic positions corresponding to positions A11 and A12 of the gRNA using the base editors.
  • FIG.12A % of total reads showing A-to-G base editing at individual positions. This includes the reads that show base editing without indels plus the reads that harbor base editing as well as the indels.
  • FIG.12B % of total reads showing only base editing at individual positions. This only includes the reads that show base editing but no indels.
  • FIG.12C % of total reads showing base editing at only one specific position (left) and % of total reads showing indels (right). This excludes all the reads with base editing at multiple positions as well as the reads harboring indels.
  • inosine is instead removed by MPG as part of the base excision (BER) repair pathway to form an AP site, which is further processed to result in an A-to-C conversion.
  • the base editing efficiency and specificity of these A-to-C base editors were determined.
  • HEK 293T cells were transfected using a plasmid expressing the base editor together with a plasmid expressing the gRNA. Transfected cells were sorted after 3 days and re-cultured for another 3 days after which the cells were lysed and genomic DNA was extracted.
  • Genomic region around the gRNA was PCR amplified and editing was analyzed by next generation sequencing.
  • the A-to-C base editor containing the TBN04 and MPG (TBN04_nMPG and TBN04_cMPG) showed higher A-to-C base editing efficiency than base editors containing inactive Cas12a.
  • the A-to-C base editors demonstrated high selectivity for specific sites. For gRNA TGCTGCA 7 A 8 GTA 11 A 12 GCA 15 TGCATTTG (SEQ ID NO: 189), the base editors showed selective editing at A 11 (FIG.13A).
  • FIG.13A % of total reads showing A-to-C, A-to-G, or A-to-T base editing at position A 11 of TGCTGCA 7 A 8 GTA 11 A 12 GCA 15 TGCATTTG (SEQ ID NO: 189).
  • FIG.13B % of total reads showing A-to-C, A-to-G, or A-to-T base editing at position A 9 of GTTTA 5 A 6 A 7 CA 9 CA 11 CCGGGTTA 19 A 20 TA 22 A 23 (SEQ ID NO: 190).
  • LbABE8e_nMPG MPG-Linker5-BP NLS-TadA-Linker4-LbCas12a-Linker2-BP NLS; wherein LbCas12a can be inactive LbCas12a or TBN04 (LbCas12a variant).
  • LbABE8e_cMPG is as follows: BP NLS-TadA-Linker4-LbCas12a-Linker5-MPG-Linker2-BP NLS; wherein LbCas12a can be inactive LbCas12a or TBN04 (LbCas12a variant).

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