Eubacterium rectum CAS12A mutant

Abstract

A series of recombinant Cas12a proteins from Eubacterium rectum are described in the invention. In one aspect, there are provided various mutations to the wild-type Eubacterium rectum Cas12a polypeptide (ERCAS12A) that have enhanced activity.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 462,772, filed April 28, 2023, which is incorporated herein by reference in its entirety.

[0003] References to sequence lists

[0004] This application is filed together with a sequence list XML in ST.26 XML format, pursuant to 37 CFR § 1.831 and PCT Rule 13 iii. The sequence list XML file “013670-0028-WO01_sequence_listing_xml_25-APR-2024.xml”, filed with the USPTO Patent Centre, was created on April 25, 2024, contains 1667 sequences, has a file size of 1.94 megabytes (2,043,904 bytes), and is incorporated herein by reference in its entirety. Background Technology

[0005] Eubacterium rectalum Cas12a (ERCAS12A) is a modified RNA-guided endonuclease derived from the clustered regularly interspaced short palindromic repeat (CRISPR) adaptive immune system of Eubacterium rectalum (now known as Agathobacter rectalis). Zetsche et al., Cell 163: 759-771 (2015), which is incorporated herein by reference for such teaching. Cas12a is guided by a target RNA of approximately 21 to 24 nt specific to the target DNA sequence of approximately 21 to 24 nt, or commonly referred to as the protospacer. The Cas12a-gRNA ribonucleoprotein (RNP) complex mediates double-stranded DNA break (DSB), which is then repaired by non-homologous end joining (NHEJ), which typically introduces mutations or indels at the cleavage site, or by homology-directed repair (HDR) systems for precise editing (if a suitable template nucleic acid is available).

[0006] For ERCAS12A variants to recognize the correct DNA targets, both crRNA and the classic “TTTV” protospacer adjacent motif (PAM) (a 4-bp sequence immediately upstream of the protospacer) are crucial. Compared to the 2-bp NGG PAM of Cas9 from Streptococcus pyogenes, Cas12a expands the targetable loci in genome editing, particularly at T-rich sites inaccessible to the Cas9 system. Jinek et al., Science 337: 816-821 (2012), which is incorporated herein by reference for such teaching. Although the targetable sites of Cas12a are less frequent on the genome than those of Cas9, ERCAS12A remains an alternative enzyme for genome editing applications. Improving the cleavage activity and associated genome editing efficiency of ERCAS12A will greatly facilitate the development of human cell therapies with enhanced properties.

[0007] Previous studies have improved the utility of Cas12a (LbCas12a) from Lachnospiraceae bacterium ND2006 and Cas12a (AsCas12a) from Acidaminococcus sp. See, for example, Gao et al., Nat. Biotechnol. 35(8): 789-792 (2017); Kleinstiver et al., Nat. Biotechnol. 37(3): 276-282 (2019); Zhang et al., Nat. Communications 12: 3908 (2021); U.S. Patent Application Publication Nos. US 2021 / 0348144 A1 and US 2023 / 0040148 A1, each incorporated herein by reference for such teaching. However, transferring beneficial mutations from AsCas12a and LbCas12a to ERCAS12A is neither straightforward nor predictable. Despite the significant structural similarity among these homologous proteins, most point mutations that improve AsCas12a and LbCas12a activity are detrimental to ERCAS12A.

[0008] What is needed is a mutation that enhances ERCAS12A activity. Summary of the Invention

[0009] One embodiment described herein is an isolated polypeptide comprising an E. rectum Cas12a (ERCAS12A) mutant having one or more amino acid substitutions introduced into the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 2, wherein the ERCAS12A mutant polypeptide provides an improvement in nuclease activity compared to the wild-type ERCAS12A polypeptide. In one aspect, the ERCAS12A mutant has 98% to 99% identity with a polypeptide sequence selected from even-numbered sequences of SEQ ID NO: 4 to 210. In another aspect, the ERCAS12A mutant has a polypeptide sequence selected from even-numbered sequences of SEQ ID NO: 4 to 210. In yet another aspect, the ERCAS12A mutant comprises a polypeptide sequence located at SEQ ID NO: 2. The wild-type ERCAS12A polypeptide sequence of 2 may contain one or more amino acid substitutions at the following positions: position 18, position 35, position 39, position 39, position 40, position 56, position 56, position 59, position 59, position 158, position 165, position 181, position 290, position 291, position 296, position 297, position 297, position 303. 303rd, 305th, 307th, 353rd, 444th, 465th, 469th, 470th, 498th, 507th, 507th, 507th, 510th, 510th, 510th, 512th, 512th, 513th, 513th, 513th, 514th, 514th 515th, 516th, 516th, 517th, 518th, 520th, 522nd, 541st, 553rd, 556th, 556th, 562nd, 562nd, 562nd, 583rd, 585th, 653rd, 712th, 716th, 731st, 732nd, 744th, 744th Positions 744, 748, 751, 754, 757, 757, 771, 787, 825, 826, 826, 832, 836, 836, 839, 840, 907, 907, 916, 935, 936, 974, or 1019. In another aspect, the ERCAS12A mutant comprises one or more amino acid substitutions selected from the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 2:

[0010] I936R, K974G, K1019V, or combinations thereof.

[0011] In another aspect, the ERCAS12A mutant was selected from:

[0012]

[0013] In another aspect, the ERCAS12A mutant was selected from:

[0014]

[0015]

[0016] Another embodiment described herein is an isolated polynucleotide sequence encoding an *E. rectum* Cas12a (ERCAS12A) mutant having one or more amino acid substitutions introduced into the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 2, wherein the ERCAS12A mutant provides improved gene editing compared to the wild-type ERCAS12A polypeptide. In one aspect, the encoded ERCAS12A mutant has 98% to 99% identity with a polypeptide sequence selected from even-numbered sequences of SEQ ID NO: 4 to 210. In another aspect, the encoded ERCAS12A mutant has a polypeptide sequence selected from even-numbered sequences of SEQ ID NO: 4 to 210. In yet another aspect, the ERCAS12A mutant polynucleotide sequence has 95% to 99% identity with a nucleotide sequence selected from odd-numbered sequences of SEQ ID NO: 3 to 209. In another aspect, the ERCAS12A mutant polynucleotide has a nucleotide sequence selected from the odd-numbered sequences of SEQ ID NO: 3 to 209. In another aspect, the encoded ERCAS12A mutant comprises one or more amino acid substitutions at the following positions in the wild-type ERCAS12A polypeptide sequence located at SEQ ID NO: 2: positions 18, 35, 39, 39, 40, 56, 56, 59, 59, 158, 165, 181, 290, 291, 296, 297, 297, 297, 303. 303rd, 305th, 307th, 353rd, 444th, 465th, 469th, 470th, 498th, 507th, 507th, 507th, 510th, 510th, 510th, 512th, 512th, 513th, 513th, 513th, 514th, 514th 515th, 516th, 516th, 517th, 518th, 520th, 522nd, 541st, 553rd, 556th, 556th, 562nd, 562nd, 562nd, 583rd, 585th, 653rd, 712th, 716th, 731st, 732nd, 744th, 744th The 744th, 748th, 751st, 754th, 757th, 757th, 771st, 787th, 825th, 826th, 826th, 832nd, 836th, 836th, 839th, 840th, 907th, 907th, 916th, 935th, 936th, 974th, or 1019th position.In another aspect, the encoded ERCAS12A mutant contains one or more amino acid substitutions selected from the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 2:

[0017] , or a combination thereof.

[0018] In another aspect, the ERCAS12A mutant polynucleotide encoding the polynucleotide is selected from:

[0019]

[0020]

[0021] In another aspect, the ERCAS12A mutant polynucleotide is selected from:

[0022]

[0023]

[0024] In another aspect, the ERCAS12A mutant polynucleotide is selected from:

[0025]

[0026] Another embodiment described herein is a vector or plasmid containing the polynucleotide sequence described herein.

[0027] Another embodiment described herein is a cell containing nucleic acids having one or more of the polynucleotide sequences described herein.

[0028] Another embodiment described herein is an isolated CRISPR ribonucleic acid protein complex comprising a guide RNA and an ERCAS12A mutant polypeptide having one or more amino acid substitutions introduced into the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 2, wherein the ERCAS12A mutant provides improved gene editing compared to the wild-type ERCAS12A polypeptide. In one aspect, the ERCAS12A mutant has a polypeptide sequence selected from even-numbered sequences of SEQ ID NO: 4 to 210. In another aspect, the ERCAS12A mutant comprises a sequence located at SEQ ID NO: The wild-type ERCAS12A polypeptide sequence of 2 may contain one or more amino acid substitutions at the following positions: position 18, position 35, position 39, position 39, position 40, position 56, position 56, position 59, position 59, position 158, position 165, position 181, position 290, position 291, position 296, position 297, position 297, position 303. 303rd, 305th, 307th, 353rd, 444th, 465th, 469th, 470th, 498th, 507th, 507th, 507th, 510th, 510th, 510th, 512th, 512th, 513th, 513th, 513th, 514th, 514th 515th, 516th, 516th, 517th, 518th, 520th, 522nd, 541st, 553rd, 556th, 556th, 562nd, 562nd, 562nd, 583rd, 585th, 653rd, 712th, 716th, 731st, 732nd, 744th, 744th Positions 744, 748, 751, 754, 757, 757, 771, 787, 825, 826, 826, 832, 836, 836, 839, 840, 907, 907, 916, 935, 936, 974, or 1019. In another aspect, the ERCAS12A mutant comprises one or more amino acid substitutions selected from the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 2:

[0029]

[0030] , or a combination thereof.

[0031] In another aspect, the ERCAS12A mutant was selected from:

[0032]

[0033]

[0034] In another aspect, the ERCAS12A mutant was selected from:

[0035]

[0036] Another embodiment described herein is an isolated CRISPR / Cpf1 endonuclease system comprising crRNA and an ERCAS12A mutant polypeptide having one or more amino acid substitutions introduced into the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 2, wherein the ERCAS12A mutant polypeptide provides improved genome editing compared to the wild-type ERCAS12A polypeptide. In one aspect, the ERCAS12A mutant has a polypeptide sequence selected from even-numbered sequences of SEQ ID NO: 4 to 210. In another aspect, the ERCAS12A mutant comprises a sequence located at SEQ ID NO: The wild-type ERCAS12A polypeptide sequence of 2 may contain one or more amino acid substitutions at the following positions: position 18, position 35, position 39, position 39, position 40, position 56, position 56, position 59, position 59, position 158, position 165, position 181, position 290, position 291, position 296, position 297, position 297, position 303. 303rd, 305th, 307th, 353rd, 444th, 465th, 469th, 470th, 498th, 507th, 507th, 507th, 510th, 510th, 510th, 512th, 512th, 513th, 513th, 513th, 514th, 514th 515th, 516th, 516th, 517th, 518th, 520th, 522nd, 541st, 553rd, 556th, 556th, 562nd, 562nd, 562nd, 583rd, 585th, 653rd, 712th, 716th, 731st, 732nd, 744th, 744th The 744th, 748th, 751st, 754th, 757th, 757th, 771st, 787th, 825th, 826th, 826th, 832nd, 836th, 836th, 839th, 840th, 907th, 907th, 916th, 935th, 936th, 974th, or 1019th position.

[0037] In another aspect, the ERCAS12A mutant comprises one or more amino acid substitutions selected from the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 2:

[0038]

[0039] , or a combination thereof.

[0040] In another aspect, the ERCAS12A mutant was selected from:

[0041]

[0042]

[0043] In another aspect, the ERCAS12A mutant was selected from:

[0044]

[0045] Another embodiment described herein is a method for performing gene editing, comprising: contacting a candidate editing target locus with a CRISPR ribonucleic acid protein complex comprising crRNA and an ERCAS12A mutant polypeptide, the ERCAS12A mutant polypeptide comprising one or more amino acid substitutions introduced into the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 2, wherein the ERCAS12A mutant provides an improvement in gene editing compared to the wild-type ERCAS12A polypeptide. In one aspect, the ERCAS12A mutant has a polypeptide sequence selected from even-numbered sequences of SEQ ID NO: 4 to 210. In another aspect, the ERCAS12A mutant comprises a sequence located at SEQ ID NO: 210. The wild-type ERCAS12A polypeptide sequence of 2 may contain one or more amino acid substitutions at the following positions: position 18, position 35, position 39, position 39, position 40, position 56, position 56, position 59, position 59, position 158, position 165, position 181, position 290, position 291, position 296, position 297, position 297, position 303. 303rd, 305th, 307th, 353rd, 444th, 465th, 469th, 470th, 498th, 507th, 507th, 507th, 510th, 510th, 510th, 512th, 512th, 513th, 513th, 513th, 514th, 514th 515th, 516th, 516th, 517th, 518th, 520th, 522nd, 541st, 553rd, 556th, 556th, 562nd, 562nd, 562nd, 583rd, 585th, 653rd, 712th, 716th, 731st, 732nd, 744th, 744th Positions 744, 748, 751, 754, 757, 757, 771, 787, 825, 826, 826, 832, 836, 836, 839, 840, 907, 907, 916, 935, 936, 974, or 1019. In another aspect, the ERCAS12A mutant comprises one or more amino acid substitutions selected from the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 2:

[0046]

[0047] , or a combination thereof.

[0048] In another aspect, the ERCAS12A mutant was selected from:

[0049]

[0050] In another aspect, the ERCAS12A mutant was selected from:

[0051]

[0052]

[0053] Another embodiment described herein is a kit comprising a CRISPR ribonucleic acid-protein complex containing a guide RNA and an ERCAS12A mutant peptide comprising one or more amino acid substitutions introduced into the wild-type ERCAS12A peptide sequence of SEQ ID NO: 2, wherein the ERCAS12A mutant peptide provides improved gene editing compared to the wild-type ERCAS12A peptide. In one aspect, the ERCAS12A mutant has a peptide sequence selected from even-numbered sequences of SEQ ID NO: 4 to 210. In another aspect, the ERCAS12A mutant comprises one or more amino acid substitutions selected from the wild-type ERCAS12A peptide sequence of SEQ ID NO: 2:

[0054]

[0055] , or a combination thereof.

[0056] In another aspect, the ERCAS12A mutant was selected from:

[0057]

[0058]

[0059] In another aspect, the ERCAS12A mutant was selected from:

[0060]

[0061] Another embodiment described herein is the use of the ERCAS12A mutant peptide for improving the activity of CRISPR / LbCas12a-related nucleases at non-canonical TTTT PAM sites, wherein the ERCAS12A mutant introduces one or more amino acid substitutions into the wild-type ERCAS12A peptide sequence of SEQ ID NO: 2, wherein the ERCAS12A mutant provides improved gene editing compared to the wild-type ERCAS12A peptide. In one aspect, the ERCAS12A mutant has a peptide sequence selected from even-numbered sequences of SEQ ID NO: 4 to 210. In another aspect, the ERCAS12A mutant is selected from:

[0062]

[0063] Attached Figure Description

[0064] This patent or application document contains at least one color drawing. Upon request and payment of the necessary fees, the official authority will provide a copy of this patent or application publication with the color drawing.

[0065] Figure 1A Figure B shows a high-throughput characterization of ERCAS12A cleavage activity in E. coli. Three biological replicates were selected and deep sequencing was performed to determine the enrichment of each ERCAS12A variant. Figure 1A Phenotypic scores for synonymous (n=834) and non-synonymous variations (n=22409) in this selection are shown. Notably, most synonymous variations did not lead to significant enrichment and were used as baseline activity for wild-type ERCAS12A. Figure 1B Enrichment scores for 23,243 ERCAS12A point mutations are shown in two biological replicates. The results of the high-throughput screening are highly reproducible (R... 2 =0.66).

[0066] Figure 2 DNA cleavage activity of the ERCAS12A variants in *E. coli* is shown. Each ERCAS12A mutant was cloned and evaluated against the background of *E. coli*. *E. coli* survival was determined by obtaining the ratio of colony counts under conditions of selection pressure with and without it. Most mutations resulted in higher survival rates than wild-type (WT) ERCAS12A (approximately 10%). Original data are available at [link to original data]. Figures 3 to 5 As shown in the image.

[0067] Figure 3Some examples of point mutations in ERCAS12A with enhanced DNA cleavage activity are shown. Clearly, the mutations improve survival at the TTTC and TTTT PAM sites, indicating improved cleavage activity relative to WT-ERCAS12A.

[0068] Figure 4 Some examples of point mutations in ERCAS12A with enhanced DNA cleavage activity are shown.

[0069] Figure 5 Some examples of point mutations in ERCAS12A with enhanced DNA cleavage activity are shown.

[0070] Figure 6 The cleavage activity of the ERCAS12A variants in *E. coli* is shown. Each ErCas12a mutant was cloned and evaluated against the background of *E. coli*. *E. coli* survival was determined by obtaining the ratio of colony counts with and without selection pressure. Most mutations resulted in higher survival rates than WT ERCAS12A (approximately 18%).

[0071] Figure 7A Figure 1 shows the performance of the ERCAS12A variant as an RNP in human cells. The ERCAS12A variant and WT protein were purified and assembled into an RNP using six different crRNAs. Figure 7A To B shows the electroporation reaction at 200 nM ( Figure 7A ) or 50 nM RNP ( Figure 7B Editing efficiency of each variant was measured by T7EI 48 hours after delivery. NC: Negative control in the absence of ERCAS12A nuclease. Figure 7C D shows the results at 200 nM RNP ( Figure 7C ) and 50 nM RNP ( Figure 7D Normalized activity of each variant relative to WT ERCAS12A. The dashed line represents the baseline activity of the WT protein.

[0072] Figure 8A Figure C shows the on-target editing efficiency of the ERCAS12A point mutation in human cells. Figure 8A The relative editing efficiencies of the ERCAS12A variant at two target sites in human cells are shown. Plasmids encoding each variant were delivered via liposome transfection, and editing efficiencies were measured using the T7 endonuclease I assay (T7EI) 2 days after delivery. Figure 8B The editing efficiency of positive hits from liposome transfection experiments is shown when delivered as RNP. Figure 8CThe normalized editing efficiency of the ERCAS12A point mutation relative to WT is shown.

[0073] Figure 9A Figure B illustrates the performance of a stacked ERCAS12A variant with multiple point mutations as an RNP in human cells. The ERCAS12A variant and WT protein were purified and assembled into an RNP using six different crRNAs. Figure 9A Editing efficiency for each variant, measured by T7EI, is shown 48 hours after delivery at 50 nM in the electroporation reaction. NC: Negative control in the absence of ERCAS12A nuclease. Figure 9B The normalized activity of each variant relative to WT ERCAS12A at 50 nM RNP is shown.

[0074] Figure 10 shows the relative editing efficiency of the ERCAS12A variant at nine target sites in human cells. Plasmids encoding each variant were delivered via liposome transfection, and editing efficiency was measured by NGS 3 days after delivery.

[0075] Figure 11 The on-target editing efficiency of ERCAS12A and Y513G in human cells was demonstrated. ERCAS12A, Y513G, and WT proteins were purified and assembled into RNPs using seven different crRNAs, and the editing efficiency was measured by NGS two days after delivery.

[0076] Figure 12 illustrates the on-target editing efficiency of the ERCAS12A mutant in human cells. WT ERCAS12A, Y513G, S514P, Y513G / F840L, and AsCas12a-Ultra (e.g., AsCas12a M537R / F870L) proteins were purified and assembled into RNPs using different crRNAs, and editing efficiency was measured by NGS 2 days after delivery.

[0077] Figure 13 The performance of stacked ERCAS12A variants with multiple point mutations as RNPs in human cells is demonstrated. ERCAS12A variants and WT proteins were purified and assembled into RNPs using different crRNAs. The following RNPs (D303R / S469K / T826F / F840L; F465R / S469K / T826F / F840L; D303R / S469K / K516P / T826F / F840L) showed improved activity compared to ERCAS12A WT.

[0078] Figure 14 A summary of the on-target editing efficiencies of various ERCAS12A point mutations in human cells is presented. Detailed Implementation

[0079] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For example, any terms and techniques used herein in conjunction with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization are well known and commonly used in the art. In case of conflict, this disclosure (including definitions) shall prevail. Exemplary methods and materials are described below, but similar or equivalent methods and materials may be used in the practice or testing of the embodiments and aspects described herein.

[0080] Terms such as “comprising,” “including,” “containing,” “with,” “having,” etc., used herein mean “comprising / including.” Whether expressly stated or not, this disclosure also includes other embodiments that “include the embodiments or elements presented herein,” “comprise the embodiments or elements presented herein,” and “comprise substantially the embodiments or elements presented herein.”

[0081] As used herein, unless otherwise specified herein or clearly contradicted by the context, nouns and similar terms without quantifiers used in the context of this disclosure (especially in the context of the claims) shall be construed as encompassing both the singular and plural. Furthermore, unless otherwise stated, nouns without quantifiers mean “one / type or more / types”.

[0082] The term "or" as used in this article can be either combined or separated.

[0083] As used herein, the terms “amino acid,” “nucleotide,” “polynucleotide,” “carrier,” “peptide,” and “protein” have their common meanings as understood by one of ordinary biochemists in the art. Standard single-letter nucleotides (A, C, G, T, U) and standard single-letter amino acids (A, C, G, T, U) are used herein. ).

[0084] The terms “nucleic acid” and “polynucleotide” used in this article have the same meaning and are interchangeable.

[0085] The terms “amino acid sequence,” “polypeptide,” and “protein” used in this article have the same meaning and are interchangeable.

[0086] The term “substantially” as used in this article means to a great or significant degree, but not entirely.

[0087] As used herein, the term "about" or "approximately" when applied to one or more target values ​​means a value similar to the reference value or within an acceptable margin of error for a particular value as determined by one of ordinary skill in the art, depending in part on how the value was measured or determined, such as limitations of the measurement system. In one aspect, the term "about" means any value, including integer and decimal parts, within a variation of up to ±10% of the value modified by the term "about". Alternatively, "about" may mean within three or more standard deviations, as practiced in the art. Or, for example, with respect to biological systems or processes, the term "about" may mean within an order of magnitude of a value, in some embodiments within five times a value, and in some embodiments within two times a value. The symbol "~" used herein means "about" or "approximately".

[0088] All ranges disclosed herein include the two endpoints of a discrete value and all integers and decimals specified within that range. For example, a range of 0.1 to 2.0 includes 0.1, 0.2, 0.3, 0.4... 2.0. If the endpoints are modified by the term "about," the specified range extends to at most ±10% variation of any value within that range, or extends to within 3 or more standard deviations, including the endpoints.

[0089] The terms “control” or “reference” as used herein are used interchangeably. A “reference” or “control” level can be a predetermined value or range that serves as a baseline or benchmark for evaluating measurement results. “Control” also refers to a control experiment or control cells.

[0090] The phrase “effective amount” used in this article refers to an amount of a compound described herein that will elicit a biological response, such as a reduction or inhibition of enzyme or protein activity, or to improve symptoms, alleviate symptoms, slow or delay disease progression, or prevent disease.

[0091] As used in this article, the term “inhibition” and its variations refer to the reduction or suppression of a given condition, symptom, disorder, or disease, or a significant reduction in the baseline activity of a biological activity or process.

[0092] The terms “Er. rectum Cas12a” or “ERCAS12A” used in this article are used interchangeably and refer to the Er. rectum (Er) Cas12a protein (formerly known as Cpf1), a class 2 / V CRISPR RNA-guided endonuclease.

[0093] The terms “ERCAS12A”, “wild-type ERCAS12A”, or “WT ERCAS12A” refer to a protein that has the amino acid sequence of naturally occurring Escherichia coli Cas12a (e.g., SEQ ID NO: 2, encoded by the nucleotide sequence of SEQ ID NO: 1) and is biochemically active when bound to a suitable crRNA to form an active CRISPR / Cas12a endonuclease system.

[0094] The terms "mutant ERCAS12A protein" or "variant ERCAS12A protein" are used interchangeably and refer to a form of *E. rectum* Cas12a protein that has an amino acid sequence different from that of wild-type *E. rectum* Cas12a protein and is biochemically active when bound to a suitable crRNA to form an active CRISPR-Cas12a endonuclease system. This includes orthologs and Cas12a variants that have an amino acid sequence different from that of wild-type *E. rectum* Cas12a. The mutant ERCAS12A protein described herein has at least one amino acid substitution relative to the wild-type ERCAS12A polypeptide sequence. In one aspect, the mutant ERCAS12A polypeptide has at least one amino acid substitution relative to the wild-type ERCAS12A polypeptide sequence and exhibits improved CRISPR / ERCAS12A-associated nuclease activity at non-classical TTTT PAM sites compared to the wild-type ERCAS12A enzyme.

[0095] As used herein, the term “substantially purified” when applied to a composition means a composition having a purity of at least 90% or greater, including 90%, 95%, 99%, and greater than 99%.

[0096] As used herein, the term "isolated" refers to a substantially purified composition when used to describe a composition (e.g., a polynucleotide, polypeptide, or ribonucleic acid protein complex), or, in the case of a ribonucleic acid protein complex, to a composition in which at least one component is substantially purified. Further regarding isolated ribonucleic acid protein complexes, preferably, all components are substantially purified.

[0097] As used herein, the term "affinity tag" refers to a ligand that allows detection and / or selection of an oligonucleotide sequence to which the ligand is linked. For the purposes of this disclosure, a bait may include an affinity tag. In particular, affinity tags are typically located at one or both of the N-terminus and / or C-terminus of a polypeptide, using conventional chemical conjugation techniques or recombinant DNA techniques. Exemplary affinity tags include biotin, digoxigenin, streptavidin, multiple histidines (e.g., (His6)), glutathione-S-transferase (GST), HaloTag®, AviTag, calmodulin-tag, polyglutamate tag, FLAG-tag, HA-tag, Myc-tag, S-tag, SBP-tag, Softag3, V5 tag, Xpress tag, hapten, etc.

[0098] As used herein, the term "eukaryotic cell" includes cells of a particular organism or cells derived from a particular organism, such as a plant or mammal, including but not limited to humans, or non-human eukaryotic or animal or mammalian, as discussed herein, such as mice, rats, rabbits, dogs, livestock, or non-human mammals or primates. In some embodiments, the following may be excluded: processes for modifying human germline genetic identity, and / or processes for modifying animal genetic identity that may cause suffering to animals but have no substantial medical benefit to humans or animals, and animals resulting from such processes. Preferred human cells include cells derived from somatic cells and germline cells. Exemplary somatic cells include cells derived from major organ and tissue systems, including the immune system and hematopoietic system.

[0099] The phrase “odd-numbered sequences of SEQ ID NO: 3 to 209” as used in this article refers to the nucleotide sequences of SEQ ID NO: 3, 5, 7... 207 or 209. See Table 14, where all odd-numbered sequences are nucleotide sequences.

[0100] The phrase “even-numbered sequences of SEQ ID NO: 4 to 210” as used in this article refers to the polypeptide sequences of SEQ ID NO: 4, 6, 8... 206 or 210. See Table 14, where all even-numbered sequences are polypeptide sequences.

[0101] This article describes the E. rectum Cas12a (ERCAS12A) variant and methods for enhancing the efficacy of ERCAS12A and its variants.

[0102] Bacterial-based directed evolution of ERCAS12A was used to identify mutations with enhanced cleavage activity. First, a deep-scan mutagenesis library was created containing all possible point mutations at the amino acid level across the entire coding sequence of ERCAS12A. Most clones contained only one mutation. This type of library allows for direct evaluation of the phenotype by measuring the relative survival of each point mutation relative to the wild-type (WT) protein in bacterial selection. Briefly, selection strains carrying the viral plasmid were transformed with the ERCAS12A library and crRNA targeting the HPRT38346 site on the viral plasmid. After resuscitation and IPTG induction, cells were seeded on LB-chloramphenicol medium containing arabinose and incubated overnight at 37°C. ERCAS12A expression plasmids carried by surviving *E. coli* cells were extracted and purified. Input plasmid libraries and selected plasmid libraries were amplified by PCR, randomly fragmented using the Nextera library prep kit, and sequenced on Illumina NextSeq with approximately 40 million reads per library. The frequency of mutations at each position of ERCAS12A in both libraries was measured and normalized against the total coverage of each codon. The relative survival of each point mutation was calculated as the ratio of the normalized frequencies between the selected and input libraries. Since the degree of cell survival under arabinose selection indicates the cleavage activity of ERCAS12A variants at the protospacer region of HPRT38346, any variant enriched relative to WT during selection was a variant with enhanced activity at TTTT PAM.

[0103] A large set of putative mutations with enhanced activity in the E. coli background was described. In bacterial screening measuring cleavage activity at non-classical TTTTPAM sites, 22,049 point mutations of ERCAS12A were identified. Two biological replicates of the phenotypic measurements enabled the isolation of a large set of ERCAS12A variants with enhanced cleavage activity.

[0104] The genome editing efficiency of ERCAS12A variants with single or multiple point mutations in human cells was evaluated. ERCAS12A variants were column-purified and delivered as ribonucleic acid proteins (RNPs) to human HEK293 cells for editing six genomic targets with classic TTTV PAM. The editing efficiency of each mutant was quantified by a T7EI assay and compared with that of WTERCAS12A. This allowed for rigorous validation of the activity of putative candidates in the absence of protein overexpression in the human cell context. Editing efficiency was measured by a T7 endonuclease I assay (T7E1) performed 72 hours after delivery.

[0105] Genome editing efficiencies of 10 ERCAS12A variants with single or multiple point mutations in human cells were evaluated. ERCAS12A variants were column-purified and delivered as ribonucleic acid proteins (RNPs) to human HEK293 cells for editing six genomic targets with classic TTTV PAM. The editing efficiency of each mutant was quantified by a T7EI assay and compared with WTERCAS12A. This allowed for rigorous validation of the activity of putative candidates in the absence of protein overexpression in the human cell context. Editing efficiency was measured by a T7 endonuclease I assay performed 72 hours after delivery.

[0106] One embodiment described herein is a CRISPR-associated protein comprising a polypeptide encoding an ERCAS12A variant. The isolated polypeptide comprising the ERCAS12A variant is selected from polypeptides shown in Table 2 that have one or more amino acid substitutions relative to wild-type ERCAS12A (SEQ ID NO: 2).

[0107] Another embodiment described herein is an ErCas12a variant selected from the peptides shown in Table 2 that have one or more amino acid substitutions relative to wild-type ERCAS12A (SEQ ID NO: 2), wherein the selected ERCAS12A variant exhibits improved genome editing relative to wild-type ERCAS12A (SEQ ID NO: 2).

[0108] Another embodiment described herein provides a CRISPR ribonucleic acid protein complex. The CRISPR ribonucleic acid protein contains a polypeptide encoding an ERCAS12A variant. The ERCAS12A variant is selected from polypeptides shown in Table 2 that have one or more amino acid substitutions relative to wild-type ERCAS12A (SEQ ID NO: 2).

[0109] Another embodiment described herein provides a method for improving gene editing efficiency at the TTTN PAM site in cells containing a CRISPR ribonucleoprotein complex. The method includes the step of contacting the cell with the CRISPR ribonucleoprotein complex. The CRISPR ribonucleoprotein complex contains guide RNA and a CRISPR-associated protein. The CRISPR-associated protein contains a polypeptide encoding an ERCAS12A variant. The ERCAS12A variant is selected from polypeptides shown in Table 2 that have one or more amino acid substitutions relative to wild-type ERCAS12A (SEQ ID NO: 2).

[0110] Another implementation described herein provides a kit containing guide RNA and CRISPR-related proteins. The CRISPR-related proteins contain a polypeptide encoding the ERCAS12A variant.

[0111] Another embodiment described herein is a CRISPR-associated protein comprising a polypeptide encoding an ERCAS12A variant, containing at least one variant amino acid selected from the following amino acid positions:

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120] Or a combination thereof, provided that the variant ERCAS12A provides an improvement in the activity of CRISPR / ERCAS12A-related nucleases.

[0121] In one aspect, the ERCAS12A variant contains at least one variant amino acid selected from the following amino acid positions:

[0122]

[0123] Or a combination thereof.

[0124] Another embodiment described herein provides a CRISPR ribonucleic acid-protein complex. The CRISPR ribonucleic acid-protein complex comprises a guide RNA and a CRISPR-associated protein. The CRISPR-associated protein comprises a polypeptide encoding an ERCAS12A variant, which contains at least one variant amino acid selected from the following amino acid positions:

[0125]

[0126] Or a combination thereof, provided that the variant ERCAS12A provides an improvement in the activity of CRISPR / ERCAS12A-related nucleases.

[0127] Another embodiment described herein provides a method for improving gene editing efficiency in cells containing a CRISPR ribonucleic acid protein complex. The method includes contacting cells with a CRISPR ribonucleic acid protein complex comprising guide RNA and a CRISPR-associated protein. The CRISPR-associated protein comprises a polypeptide encoding an ERCAS12A variant, which contains at least one variant amino acid selected from the following amino acid positions:

[0128]

[0129] Or a combination thereof, provided that the variant ERCAS12A provides an improvement in the activity of CRISPR / ERCAS12A-related nucleases.

[0130] Another embodiment described herein provides a kit comprising guide RNA and a CRISPR-associated protein, the CRISPR-associated protein containing a polypeptide encoding an ERCAS12A variant. The ERCAS12A variant contains at least one variant amino acid selected from the following amino acid positions:

[0131]

[0132] Or a combination thereof, provided that the variant ERCAS12A provides an improvement in the activity of CRISPR / ERCAS12A-related nucleases.

[0133] Another embodiment described herein provides a nucleic acid encoding a CRISPR-related protein comprising a polypeptide encoding an ERCAS12A variant. The ERCAS12A variant comprises at least one variant amino acid selected from the following amino acid positions:

[0134]

[0135] Or a combination thereof, provided that the variant ERCAS12A provides an improvement in the activity of CRISPR / ERCAS12A-related nucleases.

[0136] Another embodiment described herein is a polynucleotide sequence encoding an ERCAS12A variant. The polynucleotide sequence comprises a nucleotide sequence having at least 90% to 99% identity with any odd-numbered sequence of SEQ ID NO: 3 to 209 (e.g., SEQ ID NO: 3, 5, 7... 207 or 209). In one aspect, the polynucleotide sequence comprises any odd-numbered sequence of SEQ ID NO: 3 to 209 (e.g., SEQ ID NO: 3, 5, 7... 207 or 209).

[0137] Another embodiment described herein provides a CAS endonuclease system comprising an expression cassette encoding a polynucleotide sequence encoding the ERCAS12A polypeptide. The polynucleotide sequence comprises a nucleotide sequence having at least 90% to 99% identity with any odd-numbered sequence of SEQ ID NO: 3 to 209 (e.g., 3, 5, 7... 207 or 209). In one aspect, the polynucleotide sequence comprises any odd-numbered sequence of SEQ ID NO: 3 to 209 (e.g., 3, 5, 7... 207 or 209).

[0138] Another embodiment described herein is a CAS endonuclease system comprising an amino acid sequence encoding an ERCAS12A polypeptide. The amino acid sequence comprises a polypeptide containing an ERCAS12A variant selected from those shown in Table 1 that have one or more amino acid substitutions relative to wild-type ERCAS12A (SEQ ID NO: 2), wherein the ERCAS12A variant exhibits improved genome editing relative to wild-type ERCAS12A (SEQ ID NO: 2).

[0139] Another embodiment described herein is a method for genome editing in eukaryotic cells. This method includes the step of introducing a CAS endonuclease system into eukaryotic cells, the CAS endonuclease system comprising an expression cassette encoding a polynucleotide sequence encoding an ERCAS12A polypeptide comprising an ERCAS12A variant selected from those shown in Table 1 having one or more amino acid substitutions relative to wild-type ERCAS12A (SEQ ID NO: 2), wherein the ERCAS12A variant exhibits improved genome editing relative to wild-type ERCAS12A (SEQ ID NO: 2).

[0140] Another embodiment described herein is a method for performing genome editing in eukaryotic cells. This method includes the step of introducing a CAS endonuclease system into eukaryotic cells, the CAS endonuclease system comprising an amino acid sequence encoding an ERCAS12A polypeptide comprising an ERCAS12A variant selected from polypeptides shown in Table 1 that have one or more amino acid substitutions relative to wild-type ERCAS12A (SEQ ID NO: 2), wherein the ERCAS12A variant exhibits improved genome editing relative to wild-type ERCAS12A (SEQ ID NO: 2).

[0141] Another embodiment described herein is a CRISPR-related protein comprising a fusion peptide. This fusion peptide comprises an ERCAS12A open reading frame, a nuclear localization signal, an amino acid linker (optionally), and an affinity tag (optionally).

[0142] Another embodiment described herein is a method for genome editing in eukaryotic cells. This method includes the step of introducing a CAS endonuclease system into eukaryotic cells, said CAS endonuclease system containing CRISPR-related proteins.

[0143] Another embodiment is a polypeptide encoded by the nucleotide sequence described herein. In one aspect, the polypeptide has at least 85% to 99% identity with the even-numbered sequences of SEQ ID NO: 4 to 210 (e.g., SEQ ID NO: 4, 6, 8... 206 or 210). In another aspect, the polypeptide is selected from the even-numbered sequences of SEQ ID NO: 4 to 210 (e.g., SEQ ID NO: 4, 6, 8... 206 or 210).

[0144] Another embodiment described herein is a method for preparing one or more nucleotide sequences described herein or polypeptides encoded by nucleotide sequences described herein, the method comprising: transforming or transfecting cells with a nucleic acid containing nucleotide sequences described herein; culturing cells; optionally, isolating an additional amount of nucleotide sequences described herein; inducing expression of polypeptides encoded by nucleotide sequences described herein; and isolating polypeptides encoded by nucleotide sequences described herein.

[0145] Another embodiment described herein is a means for preparing one or more nucleotide sequences described herein or polypeptides encoded by nucleotide sequences described herein, the method comprising: transforming or transfecting cells with a nucleic acid containing nucleotide sequences described herein; culturing cells; optionally, isolating an additional amount of nucleotide sequences described herein; inducing expression of polypeptides encoded by nucleotide sequences described herein; and isolating polypeptides encoded by nucleotide sequences described herein.

[0146] Another embodiment described herein is a nucleotide sequence or a polypeptide encoded by the method or means described herein.

[0147] Another embodiment described herein uses an effective amount of a polypeptide encoded by one or more of the odd-numbered sequences of SEQ ID NO: 3 to 209 (e.g., 3, 5, 7... 207 or 209).

[0148] Another implementation described herein is a research tool comprising a polypeptide encoded by the nucleotide sequence described herein.

[0149] Another embodiment described herein is a reagent comprising a polypeptide encoded by the nucleotide sequence described herein.

[0150] The polynucleotides described herein include variants with substitutions, deletions, and / or additions, which may involve one or more nucleotides. These variants may occur in coding regions, non-coding regions, or both. Changes in coding regions may result in conserved or non-conserved amino acid substitutions, deletions, or additions. Silent substitutions, additions, and deletions are particularly preferred among these, as they do not alter the binding properties and activity.

[0151] Other embodiments described herein include nucleic acid molecules comprising polynucleotides having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the following, and more preferably, at least about 90% to 99% or 100% identity: (a) a nucleotide sequence encoding a polypeptide having an amino acid sequence in an even-numbered sequence of SEQ ID NO: 4 to 210 (e.g., SEQ ID NO: 4, 6, 8... 206 or 210), or a degenerate, homologous, or codon-optimized variant thereof; (b) a nucleotide sequence encoding an even-numbered sequence of SEQ ID NO: 4 to 210 (e.g., SEQ ID NO: 4, 6, 8...). (a) The nucleotide sequence of a polypeptide having an amino acid sequence in (b) 206 or 210, or a degenerate, homologous, or codon-optimized variant thereof; and (c) The nucleotide sequence of a functional polypeptide capable of hybridizing with a complementary sequence to any nucleotide sequence in (a) or (b) above and capable of expressing an amino acid sequence in an even-numbered sequence of SEQ ID NO: 4 to 210 (e.g., SEQ ID NO: 4, 6, 8 ... 206 or 210).

[0152] A polynucleotide having at least, for example, 90% to 99% “identity” with a reference nucleotide sequence encoding odd-numbered sequences (e.g., 3, 5, 7 ... 207 or 209) means that for every 100 nucleotides of a reference nucleotide sequence encoding odd-numbered sequences (e.g., 3, 5, 7 ... 207 or 209), the polynucleotide sequence may contain up to about 10 point mutations, additions, or deletions, and otherwise the nucleotide sequence of the polynucleotide is identical to the reference sequence.

[0153] In other words, to obtain a polynucleotide sequence having at least 90% to 99% identity with a reference nucleotide sequence, up to 10% of the nucleotides in the reference sequence may be deleted, added, or replaced with other nucleotides, or the number of nucleotides that may be inserted into the reference sequence may be up to 10% of the total nucleotides in the reference sequence. These mutations in the reference sequence may occur at the 5' or 3' ends of the reference nucleotide sequence or at any position between these ends, either individually among nucleotides in the reference sequence or within one or more consecutive groups of nucleotides in the reference sequence. The same applies to polypeptide sequences having at least about 90% to 99% identity with a reference polypeptide sequence.

[0154] As mentioned above, two or more polynucleotide sequences can be compared by determining their percentage of identity. Similarly, two or more amino acid sequences can be compared by determining their percentage of identity. The percentage of identity between two sequences (whether nucleic acid or peptide) is typically described as the number of exact matches between the two aligned sequences divided by the length of the shorter sequence and multiplied by 100. Approximate alignment of nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2: 4 82-489 (1981). This algorithm is extended to peptide sequences, using a scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, MO Dayhoff ed., 5 Supplement 3: 353-358, National Biomedical Research Foundation, Washington, DC, USA, and normalized by Gribskov, Nucl. Acids Res. 14(6): 6745-6763 (1986).

[0155] For example, due to the degeneracy of the genetic code, those skilled in the art will recognize that a large number of nucleic acid molecules, or their degenerate, homologous, or codon-optimized variants, having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the nucleic acid sequences shown in SEQ ID NO: 3 to 209 (e.g., SEQ ID NO: 4, 6, 8... 206, or 210), will encode the even-numbered sequences of SEQ ID NO: 4 to 210 (e.g., SEQ ID NO: 4, 6, 8... 206, or 210).

[0156] The polynucleotides described in this article include those that encode the polypeptides described herein, including mutations, alterations, substitutions, additions, deletions, and specific instances thereof. For example, Bowie, JU et al., “Deciphering the Messagein Protein Sequences: Tolerance to Amino Acid Substitutions,” Science 247:1306-1310 (1990) provides guidance on how to perform phenotypic silencing of amino acid substitutions, in which the authors note the remarkable tolerance of proteins to amino acid substitutions.

[0157] Therefore, fragments, derivatives, or analogs of polypeptides with even-numbered sequences of SEQ ID NO: 4 to 210 (e.g., SEQ ID NO: 4, 6, 8... 206 or 210) can be (i) those in which one or more amino acid residues (e.g., 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 residues, or even more) are replaced by conserved or non-conserved amino acid residues (preferably conserved amino acid residues), such replaced amino acid residues may or may not be These are: (i) those encoded by the genetic code; or (ii) those in which one or more amino acid residues contain substituents (e.g., 1, 2, 3, 4, 5, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 residues, or even more); or (iii) those in which the mature polypeptide is fused with another polypeptide or compound (e.g., a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol); or (iv) those in which the mature polypeptide is fused with another amino acid (e.g., an IgG Fc fusion region peptide or a leader sequence or secreted sequence or a sequence used to purify the mature polypeptide or a proteogenic sequence). Based on the teachings herein, those skilled in the art will consider such fragments, derivatives, and analogues to be within the scope of this invention.

[0158] Additionally, fragments, derivatives, or analogs of the polypeptides with even-numbered sequences of SEQ ID NO: 4 to 210 (e.g., SEQ ID NO: 4, 6, 8... 206, or 210) may be substituted with one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues). In some cases, these polypeptides, their fragments, derivatives, or analogs will have a polypeptide sequence with at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the polypeptide sequences shown in SEQ ID NO: 4 to 210 (e.g., SEQ ID NO: 4, 6, 8... 206, or 210), and will contain functional or non-functional proteins or enzymes. Similarly, the addition or deletion of peptides can be made at the N-terminus or C-terminus of the peptide or in non-conserved regions (which are considered non-critical because they are not phylogenetically conserved).

[0159] As described herein, in many cases, amino acid substitutions, mutations, additions, or deletions are preferably minor in nature, such as conserved amino acid substitutions or additions or deletions at the N-terminus or C-terminus that do not significantly affect protein folding or activity. Of course, the number of amino acid substitutions, additions, or deletions performed by a person skilled in the art depends on many factors, including those described herein. Typically, the number of substitutions, additions, or deletions for any given polypeptide will not exceed about 100, 90, 80, 70, 60, 50, 40, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 5, 6, 4, 3, 2, or 1.

[0160] Another embodiment described herein is a multinucleotide vector comprising one or more nucleotide sequences described herein.

[0161] Another embodiment described herein is a cell comprising one or more of the nucleotide sequences or polynucleotide vectors described herein.

[0162] It will be apparent to those skilled in the art that suitable modifications and adaptations can be made to the compositions, formulations, methods, processes, apparatuses, components, and applications described herein without departing from the scope of any embodiments or aspects thereof. The compositions, apparatuses, components, and methods provided are exemplary and not intended to limit the scope of any disclosed embodiments. All embodiments, aspects, and options disclosed herein can be combined in any variation or iteration. The scope of the compositions, formulations, methods, apparatuses, components, and processes described herein includes all actual or potential combinations of the embodiments, aspects, options, examples, and preferences described herein. Any component or step may be omitted, substituted for, or included in any composition, formulation, apparatus, component, or method described herein, or any component or step disclosed elsewhere herein. The mass ratio of any component of any composition or formulation disclosed herein to the mass of any other component in the formulation or to the total mass of the other components in the formulation is disclosed herein as if explicitly disclosed. In the event of any conflict between the meaning of any term in any patent or publication incorporated by reference and the meaning of the term used in this disclosure, the meaning of the term or phrase in this disclosure shall prevail. All patents and publications cited in this article are incorporated herein by reference for their specific teaching purposes.

[0163] The various embodiments and aspects of the invention described herein are summarized by the following provisions:

[0164] Clause 1. An isolated polypeptide comprising a *E. rectum* Cas12a (ERCAS12A) mutant having one or more amino acid substitutions introduced into the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 2, wherein the ERCAS12A mutant polypeptide provides an improvement in nuclease activity compared to the wild-type ERCAS12A polypeptide.

[0165] Clause 2. The isolated polypeptide described in Clause 1, wherein the ERCAS12A mutant has 98% to 99% identity with the polypeptide sequence selected from the even-numbered sequences of SEQ ID NO: 4 to 210.

[0166] Clause 3. The isolated polypeptide described in Clause 1 or 2, wherein the ERCAS12A mutant has a polypeptide sequence selected from the even-numbered sequences of SEQ ID NO: 4 to 210.

[0167] Clause 4. The isolated polypeptide of any one of Clauses 1 to 3, wherein the ERCAS12A mutant comprises one or more amino acid substitutions at the following positions of the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 2: position 18, position 35, position 39, position 39, position 40, position 56, position 56, position 59, position 59, position 158, position 165, position 181, position 290, position 291, position 296, position 297, position 297, position 297, position 303 303rd, 305th, 307th, 353rd, 444th, 465th, 469th, 470th, 498th, 507th, 507th, 507th, 510th, 510th, 510th, 512th, 512th, 513th, 513th, 513th, 514th, 514th 515th, 516th, 516th, 517th, 518th, 520th, 522nd, 541st, 553rd, 556th, 556th, 562nd, 562nd, 562nd, 583rd, 585th, 653rd, 712th, 716th, 731st, 732nd, 744th, 744th The 744th, 748th, 751st, 754th, 757th, 757th, 771st, 787th, 825th, 826th, 826th, 832nd, 836th, 836th, 839th, 840th, 907th, 907th, 916th, 935th, 936th, 974th, or 1019th position.

[0168] Clause 5. The isolated polypeptide of any one of Clauses 1 to 4, wherein the ERCAS12A mutant comprises one or more amino acid substitutions selected from the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 2:

[0169]

[0170] , or a combination thereof.

[0171] Clause 6. The isolated polypeptide of any one of Clauses 1 to 5, wherein the ERCAS12A mutant is selected from:

[0172]

[0173] .

[0174] Clause 7. The isolated polypeptide of any one of Clauses 1 to 6, wherein the ERCAS12A mutant is selected from:

[0175] .

[0176] Clause 8. An isolated polynucleotide sequence encoding a *E. rectum* Cas12a (ERCAS12A) mutant having one or more amino acid substitutions introduced into the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 2, wherein the ERCAS12A mutant provides improved gene editing compared to the wild-type ERCAS12A polypeptide.

[0177] Clause 9. The isolated polynucleotide sequence described in Clause 8, wherein the encoded ERCAS12A mutant has 98% to 99% identity with the polypeptide sequence selected from the even-numbered sequences of SEQ ID NO: 4 to 210.

[0178] Clause 10. The isolated polynucleotide sequence described in Clause 8 or 9, wherein the encoded ERCAS12A mutant has a polypeptide sequence selected from even-numbered sequences of SEQ ID NO: 4 to 210.

[0179] Clause 11. The isolated polynucleotide sequence of any one of Clauses 8 to 10, wherein the ERCAS12A mutant polynucleotide sequence has 95% to 99% identity with the nucleotide sequence of the odd-numbered sequences selected from SEQ ID NO: 3 to 209.

[0180] Clause 12. The isolated polynucleotide sequence of any one of Clauses 8 to 11, wherein the ERCAS12A mutant polynucleotide has a nucleotide sequence selected from odd-numbered sequences of SEQ ID NO: 3 to 209.

[0181] Clause 13. The isolated polynucleotide sequence of any one of Clauses 8 to 12, wherein the encoded ERCAS12A mutant comprises one or more amino acid substitutions at the following positions of the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 2: position 18, position 35, position 39, position 39, position 40, position 56, position 56, position 59, position 59, position 158, position 165, position 181, position 290, position 291, position 296, position 297, position 297, position 297, position 303 303rd, 305th, 307th, 353rd, 444th, 465th, 469th, 470th, 498th, 507th, 507th, 507th, 510th, 510th, 510th, 512th, 512th, 513th, 513th, 513th, 514th, 514th 515th, 516th, 516th, 517th, 518th, 520th, 522nd, 541st, 553rd, 556th, 556th, 562nd, 562nd, 562nd, 583rd, 585th, 653rd, 712th, 716th, 731st, 732nd, 744th, 744th The 744th, 748th, 751st, 754th, 757th, 757th, 771st, 787th, 825th, 826th, 826th, 832nd, 836th, 836th, 839th, 840th, 907th, 907th, 916th, 935th, 936th, 974th, or 1019th position.

[0182] Clause 14. The isolated polynucleotide sequence of any one of Clauses 8 to 13, wherein the encoded ERCAS12A mutant comprises one or more amino acid substitutions selected from the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 2:

[0183]

[0184] , or a combination thereof.

[0185] Clause 15. The isolated polynucleotide sequence of any one of Clauses 8 to 14, wherein the ERCAS12A mutant polynucleotide encoding is selected from: Q18V (SEQ ID NO: 4);

[0186] .

[0187] Clause 16. The isolated polynucleotide sequence of any one of Clauses 8 to 15, wherein the ERCAS12A mutant polynucleotide is selected from:

[0188]

[0189] .

[0190] Clause 17. The isolated polynucleotide sequence of any one of Clauses 8 to 16, wherein the ERCAS12A mutant polynucleotide is selected from:

[0191] .

[0192] Clause 18. A vector or plasmid comprising the polynucleotide sequence of any one of Clauses 8 to 17.

[0193] Clause 19. A cell comprising a nucleic acid containing one or more of the polynucleotide sequences described in any one of Clauses 8 to 18.

[0194] Clause 20. An isolated CRISPR ribonucleic acid protein complex comprising a guide RNA and an ERCAS12A mutant polypeptide having one or more amino acid substitutions introduced into the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 2, wherein the ERCAS12A mutant provides improved gene editing compared to the wild-type ERCAS12A polypeptide.

[0195] Clause 21. The isolated CRISPR ribonucleic acid protein complex as described in Clause 20, wherein the ERCAS12A mutant has a polypeptide sequence selected from even-numbered sequences of SEQ ID NO: 4 to 210.

[0196] Clause 22. The isolated CRISPR ribonucleic acid protein complex as described in Clause 20 or 21, wherein the ERCAS12A mutant comprises one or more amino acid substitutions at the following positions in the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 2: position 18, position 35, position 39, position 39, position 40, position 56, position 56, position 59, position 59, position 158, position 165, position 181, position 290, position 291, position 296, position 297, position 297, position 297, position 303 303rd, 305th, 307th, 353rd, 444th, 465th, 469th, 470th, 498th, 507th, 507th, 507th, 510th, 510th, 510th, 512th, 512th, 513th, 513th, 513th, 514th, 514th 515th, 516th, 516th, 517th, 518th, 520th, 522nd, 541st, 553rd, 556th, 556th, 562nd, 562nd, 562nd, 583rd, 585th, 653rd, 712th, 716th, 731st, 732nd, 744th, 744th The 744th, 748th, 751st, 754th, 757th, 757th, 771st, 787th, 825th, 826th, 826th, 832nd, 836th, 836th, 839th, 840th, 907th, 907th, 916th, 935th, 936th, 974th, or 1019th position.

[0197] Clause 23. The isolated CRISPR ribonucleic acid protein complex of any one of Clauses 20 to 22, wherein the ERCAS12A mutant comprises one or more amino acid substitutions selected from the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 2:

[0198]

[0199]

[0200] , or a combination thereof.

[0201] Clause 24. The isolated CRISPR ribonucleic acid-protein complex of any one of Clauses 20 to 23, wherein the ERCAS12A mutant is selected from:

[0202]

[0203] .

[0204] Clause 25. The isolated CRISPR ribonucleic acid-protein complex of any one of Clauses 20 to 24, wherein the ERCAS12A mutant is selected from:

[0205] .

[0206] Clause 26. An isolated CRISPR / Cpf1 endonuclease system comprising crRNA and an ERCAS12A mutant polypeptide having one or more amino acid substitutions introduced into the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 2, wherein the ERCAS12A mutant polypeptide provides improved genome editing compared to the wild-type ERCAS12A polypeptide.

[0207] Clause 27. The isolated CRISPR / Cpf1 endonuclease system described in Clause 26, wherein the ERCAS12A mutant has a polypeptide sequence selected from even-numbered sequences of SEQ ID NO: 4 to 210.

[0208] Clause 28. The isolated CRISPR / Cpf1 endonuclease system described in Clause 26 or 27, wherein the ERCAS12A mutant comprises one or more amino acid substitutions at the following positions in the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 2: position 18, position 35, position 39, position 39, position 40, position 56, position 56, position 59, position 59, position 158, position 165, position 181, position 290, position 291, position 296, position 297, position 297, position 297, position 303 303rd, 305th, 307th, 353rd, 444th, 465th, 469th, 470th, 498th, 507th, 507th, 507th, 510th, 510th, 510th, 512th, 512th, 513th, 513th, 513th, 514th, 514th 515th, 516th, 516th, 517th, 518th, 520th, 522nd, 541st, 553rd, 556th, 556th, 562nd, 562nd, 562nd, 583rd, 585th, 653rd, 712th, 716th, 731st, 732nd, 744th, 744th The 744th, 748th, 751st, 754th, 757th, 757th, 771st, 787th, 825th, 826th, 826th, 832nd, 836th, 836th, 839th, 840th, 907th, 907th, 916th, 935th, 936th, 974th, or 1019th position.

[0209] Clause 29. The isolated CRISPR / Cpf1 endonuclease system of any one of Clauses 26 to 28, wherein the ERCAS12A mutant comprises one or more amino acid substitutions selected from the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 2:

[0210]

[0211]

[0212] , or a combination thereof.

[0213] Clause 30. The isolated CRISPR / Cpf1 endonuclease system as described in any one of Clauses 26 to 29, wherein the ERCAS12A mutant is selected from:

[0214]

[0215] .

[0216] Clause 31. The isolated CRISPR / Cpf1 endonuclease system as described in any one of Clauses 26 to 30, wherein the ERCAS12A mutant is selected from:

[0217] .

[0218] Article 32. Methods for performing gene editing, comprising:

[0219] The candidate editing target locus is contacted with a CRISPR ribonucleic acid protein complex comprising crRNA and an ERCAS12A mutant peptide, the ERCAS12A mutant peptide comprising one or more amino acid substitutions introduced into the wild-type ERCAS12A peptide sequence of SEQ ID NO: 2, wherein the ERCAS12A mutant provides improved gene editing compared to the wild-type ERCAS12A peptide.

[0220] Clause 33. The method of Clause 32, wherein the ERCAS12A mutant has a polypeptide sequence selected from even-numbered sequences of SEQ ID NO: 4 to 210.

[0221] Clause 34. The method of Clause 32 or 33, wherein the ERCAS12A mutant comprises one or more amino acid substitutions at the following positions of the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 2: position 18, position 35, position 39, position 39, position 40, position 56, position 56, position 59, position 59, position 158, position 165, position 181, position 290, position 291, position 296, position 297, position 297, position 297, position 303 303rd, 305th, 307th, 353rd, 444th, 465th, 469th, 470th, 498th, 507th, 507th, 507th, 510th, 510th, 510th, 512th, 512th, 513th, 513th, 513th, 514th, 514th 515th, 516th, 516th, 517th, 518th, 520th, 522nd, 541st, 553rd, 556th, 556th, 562nd, 562nd, 562nd, 583rd, 585th, 653rd, 712th, 716th, 731st, 732nd, 744th, 744th The 744th, 748th, 751st, 754th, 757th, 757th, 771st, 787th, 825th, 826th, 826th, 832nd, 836th, 836th, 839th, 840th, 907th, 907th, 916th, 935th, 936th, 974th, or 1019th position.

[0222] Clause 35. The method of any one of Clauses 32 to 34, wherein the ERCAS12A mutant comprises one or more amino acid substitutions selected from the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 2:

[0223]

[0224]

[0225] , or a combination thereof.

[0226] Clause 36. The method of any one of Clauses 32 to 35, wherein the ERCAS12A mutant is selected from:

[0227]

[0228] .

[0229] Clause 37. The method of any one of Clauses 32 to 36, wherein the ERCAS12A mutant is selected from:

[0230] .

[0231] Clause 38. A kit comprising a CRISPR ribonucleic acid protein complex containing a guide RNA and an ERCAS12A mutant peptide comprising one or more amino acid substitutions introduced into the wild-type ERCAS12A peptide sequence of SEQ ID NO: 2, wherein the ERCAS12A mutant provides improved gene editing compared to the wild-type ERCAS12A peptide.

[0232] Clause 39. The kit described in Clause 38, wherein the ERCAS12A mutant has a polypeptide sequence selected from even-numbered sequences of SEQ ID NO: 4 to 210.

[0233] Clause 40. The kit described in Clause 38 or 39, wherein the ERCAS12A mutant comprises one or more amino acid substitutions selected from the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 2: Q18V,

[0234]

[0235] , or a combination thereof.

[0236] Clause 41. The kit described in any one of Clauses 38 to 40, wherein the ERCAS12A mutant is selected from:

[0237]

[0238] .

[0239] Clause 42. The kit described in any one of Clauses 38 to 41, wherein the ERCAS12A mutant is selected from:

[0240] .

[0241] Clause 43. Use of the ERCAS12A mutant polypeptide for improved gene editing, wherein the ERCAS12A mutant is introduced into one or more amino acid substitutions in the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 2, wherein the ERCAS12A mutant provides improved gene editing compared to the wild-type ERCAS12A polypeptide.

[0242] Clause 44. The use described in Clause 43, wherein the ERCAS12A mutant has a polypeptide sequence selected from even-numbered sequences of SEQ ID NO: 4 to 210.

[0243] Clause 45. The use described in Clause 43 or 44, wherein the ERCAS12A mutant is selected from: Q18V (SEQ ID NO: 4);

[0244]

[0245] .

[0246] Example

[0247] Example 1

[0248] High-throughput measurement of DNA cleavage activity of the ERCAS12A variant at the TTTT PAM site in Escherichia coli.

[0249] The following examples demonstrate the ability of the present invention to improve gene editing efficiency compared to ERCAS12A (a variant of E. rectalis Cas12a).

[0250] The wild-type nucleotide sequence (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO: 2) of ERCAS12A are shown in Table 1.

[0251]

[0252]

[0253] Phenotypic scores for 22,409 ERCAS12A variants with single-point mutations are listed in Table 2. Scores for synonymous changes (n = 849, 69% of all positions) clustered closely around 0 and were therefore used as baseline activity for ERCAS12A (SEQ ID NO: 2). Figure 1A Enrichment measurements for each ERCAS12A point mutation were highly consistent across biological replicates. Figure 1B Overall, 2,564 of the 22,409 point mutations (approximately 11.4%) showed some benefit, with scores greater than the highest score of all synonymous variations (0.41) (Table 2).

[0254] Specific mutated residues that confer certain benefits are shown in the sequence of the wild-type ERCAS12A polypeptide (SEQ ID NO: 2). Mutated residues are bolded; non-bold residues are not mutated. There are 2564 individual mutations at 763 specific residues (approximately 60%) out of 1263 amino acid residues.

[0255]

[0256]

[0257] Table 2 shows the ERCAS12A mutations with improved DNA cleavage activity in *E. coli*. Enrichment scores for the three biological replicates were measured by deep sequencing. Mutants with a mean enrichment score greater than the highest score (0.41) of all synonyms were considered positive hits.

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[0316] The polynucleotide and polypeptide sequences of the 2564 mutants shown in Table 2 are not provided. Those skilled in the art will understand that mutations can be generated from wild-type ERCAS12A nucleotide or polypeptide sequences by changing the codons of wild-type amino acids and replacing them with codons of mutant amino acids. For example, in ERCAS12A mutant 1 shown in Table 2, "M1G" indicates that methionine 1 in the polypeptide sequence has been replaced with glycine. Similarly, the wild-type codon "ATG" for methionine 1 can be replaced with any of the E. coli glycine codons "GGT", "GGC", "GGA", or "GGG" to produce a mutant polynucleotide coding sequence encoding glycine at position 1. The codons of ERCAS12A amino acids can be optimized for specific organisms, such as E. coli, Homo sapiens, or other organisms, by changing the coding strand codons to the most commonly used codons in the corresponding organism.

[0317] To demonstrate the utility of these results, selective cloning was performed using cells with point mutations (

[0318]

[0319] A group of ERCAS12A variants were analyzed, and their activity was tested in the context of bacterial selection assays. (e.g.) Figures 2 to 5As shown, most of the characterized mutants exhibited improved bacterial survival relative to WT-ERCAS12A (SEQ ID NO: 2) during selection, thus validating the results of high-throughput screening. Since bacterial survival in assays is correlated with the intrinsic cleavage activity of the ERCAS12A nuclease, these mutants with improved performance relative to the WT enzyme hold great promise for human genome engineering.

[0320] Table 3 shows the primer sequences used to generate the ERCAS12A saturated mutagenic library. Standard recombination methods and techniques were used. Screening libraries were constructed using the methods described in Wrenbeck et al., Nat. Methods 13(11):928-930 (2016).

[0321] The selection was performed by transforming selectable strains carrying the viral plasmid with a mutant ERCAS12A library and crRNA targeting the HPRT38346 site on the viral plasmid. After resuscitation and IPTG induction, cells were seeded on LB-chloramphenicol medium containing arabinose and incubated overnight at 37°C. The functional ERCAS12A enzyme was capable of CRISPR activity and inactivating the viral plasmid. If the mutant LbCas12a enzyme was not functional, the transformed selectable *E. coli* cells did not survive. The ERCAS12A expression plasmid carried by the surviving *E. coli* cells was extracted and purified. Both the input plasmid library and the selected plasmid library were amplified by PCR, randomly fragmented, and sequenced. The frequency of mutations at each position of ERCAS12A in both libraries was measured and normalized to the total coverage per codon. The relative survival rate of each point mutation was calculated as the ratio of the normalized frequencies between the selected and input libraries. Since the degree of cell survival under arabinose selection indicates the cleavage activity of the ERCAS12A variant at the protospacer of HPRT38346, any variant that is enriched relative to WT during selection is a variant with enhanced activity at TTTT PAM.

[0322]

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[0364] Example 3

[0365] Editing efficiency of ErCas12a variant in human cells

[0366] This example evaluated the DNA cleavage activity of 20 ErCas12a point mutations in the E. coli background (Figure 7). The cleavage activity of each mutant was quantified by calculating the survival rate of E. coli with or without inducing the virulence reporter gene. Compared with WT-ERCAS12A, 31 of the 40 point mutations showed improved DNA cleavage, further validating the results of the sequencing-based high-throughput phenotypic measurements in Example 1.

[0367] Eight representative ERCAS12A point mutations were selected for further characterization in human cells. These point mutations include... Y907V. Site-directed mutagenesis was performed using primers listed in Table 4 to introduce specific point mutations into the expression vector. Each protein was expressed in *E. coli* BL21DE3 cells and purified sequentially using nickel-affinity chromatography and heparin chromatography. To evaluate the genome editing efficiency of each variant, ERCAS12A-RNPs were assembled using six crRNAs targeting the human HPRT gene (Table 5). The assembled RNPs (50 nM or 200 nM) were delivered to HEK293 cells via Lonza nuclear transfection (SF buffer, DS-150 procedure), and editing efficiency was measured by a T7 endonuclease I (T7EI) assay performed 48 hours after delivery. Figure 8ATo B). For ease of comparison, the efficiency of the variant at each target was normalized relative to WT-ERCAS12A and expressed as a factor of improvement relative to WT ( Figure 8C To D). Single mutant variants showed improved activity relative to WT at one or more sites, especially at low RNP concentrations (50 nM, Figure 8B D).

[0368]

[0369] Example 4

[0370] Editing efficiency of ErCas12 variants in human cells

[0371] This example evaluated the editing efficiency of 42 additional ErCas12a point mutations selected based on previous E. coli screening data. Site-directed mutagenesis was performed using primers listed in Table 6 to introduce specific point mutations into expression vectors. Purified plasmid liposomes were transfected into human HEK293 cells using two crRNAs targeting the human HPRT gene (Table 7). HPRT editing efficiency was measured by a T7EI assay performed 2 days after delivery. For ease of comparison, the efficiency of each variant at the target site was normalized relative to WT ERCAS12A and expressed as a fold improvement relative to WT (Figure 9). When delivered as plasmids, 21 of the 42 mutants improved the on-target efficiency of ERCAS12A (Figure 8, Table 7). Four point mutations, including S158G, D303R, F465R, and F840L, were selected for further characterization in the case of RNP. Of the four mutations, three (D303R, F465R, and F840L) improved on-target editing efficiency at almost all target sites. Figure 8B To C).

[0372]

[0373]

[0374]

[0375]

[0376] Given the availability of a large set of single-point mutations with enhanced activity, mutations are combined or "stacked" to further improve ERCAS12A activity. The point mutations described in the previous examples were stacked in nine combinations, and the editing efficiency of these ERCAS12A variants was evaluated in human cells using RNP. Figure 9A(To B). Notably, all superimposed variants enhanced the on-target editing efficiency of ERCAS12A at one or more target sites. Among these, the variant with double mutations (S469K / T826F) exhibited superior performance.

[0377] Example 5

[0378] Editing efficiency of ErCas12 variants in human cell lines

[0379] The editing efficiency of an additional 20 ERCAS12A point mutations was evaluated. Twenty additional ERCAS12A point mutations were selected based on E. coli screening data. Site-directed mutagenesis was performed using primers listed in Table 8 to introduce specific point mutations into the expression vector. Purified plasmid liposomes were transfected into human HEK293 cells using crRNAs targeting eight human therapeutically relevant genes (Table 9). The editing efficiency of each gene was measured by NGS performed 3 days after delivery. Figure 10A To H).

[0380]

[0381]

[0382]

[0383]

[0384] Example 6

[0385] Editing efficiency of ERCAS12A variant in human cells

[0386] The editing efficiency of the ERCAS12A Y513G point mutation selected from plasmid screening was evaluated. Site-directed mutagenesis was performed using primers listed in Table 10 to introduce point mutations into protein expression vectors. Purified proteins were tested in human HEK293 cells using RNPs targeting seven human therapy-related genes (Table 11). The editing efficiency of each gene was measured by NGS performed 2 days after delivery. Figure 11 ).

[0387] Then, site-directed mutagenesis was used to add additional mutations to ERCAS12A Y513G, Y832R, and F840L, and S514P was achieved on the protein expression vector using the primers listed in Table 10. Using four crRNAs targeting human therapy-related genes (Table 11), the purified proteins were tested by RNP in human HEK293 cells. The editing efficiency of each gene was measured by NGS performed 2 days after delivery. Figure 12A(To D). The experiment also included Acidaminococcus sp. Cas12a M537R / F870L (AsCas12a-Ultra) as a positive control, as well as cases with and without electroporation enhancers.

[0388]

[0389]

[0390]

[0391] Example 7

[0392] Another mutation, K516P, overlaps with the best ERCAS12A superimposed mutant identified in the previous examples (e.g., The proteins were stacked together. Site-directed mutagenesis was used to add the point mutation, and the primers are listed in Table 12. The protein was expressed in E. coli BL21DE3 cells and purified sequentially using nickel-affinity chromatography, heparin chromatography, and hydroxyapatite chromatography. To evaluate the genome editing efficiency of each variant, the ERCAS12A mutant-RNP was assembled using the crRNAs shown in Table 13. The assembled RNP (2 µM) was delivered to HEK293 cells via nuclear transfection, and the percentage of insertions and deletions was measured by NGS performed 48 hours after delivery. Figure 13 Superimposed mutants Each of these mutations improved activity relative to ERCAS12A WT at these sites. The superposition of these beneficial mutations produced ERCAS12A variants that were more effective than any single point mutation.

[0393]

[0394] The nucleotide and polypeptide sequences of the selected ERCAS12A variants are provided in Table 14.

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[0502]

Claims

1. An isolated polypeptide comprising an Eubacterium rectale Cas12a (ERCAS12A) mutant having one or more amino acid substitutions introduced into the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 2, wherein the ERCAS12A mutant polypeptide provides an improvement in nuclease activity compared to the wild-type ERCAS12A polypeptide.

2. The isolated polypeptide of claim 1, wherein the ERCAS12A mutant has 98% to 99% identity with the polypeptide sequence selected from even-numbered sequences of SEQ ID NO: 4 to 210.

3. The isolated polypeptide of claim 1, wherein the ERCAS12A mutant has a polypeptide sequence selected from even-numbered sequences of SEQ ID NO: 4 to 210.

4. The isolated polypeptide of claim 1, wherein the ERCAS12A mutant comprises one or more amino acid substitutions at the following positions in the wild-type ERCAS12A polypeptide sequence of SEQ ID NO:2: position 18, position 35, position 39, position 39, position 40, position 56, position 56, position 59, position 59, position 158, position 165, position 181, position 290, position 291, position 296, position 297, position 297, position 297, position 3 03rd, 303rd, 305th, 307th, 353rd, 444th, 465th, 469th, 470th, 498th, 507th, 507th, 507th, 510th, 510th, 510th, 512th, 512th, 513th, 513th, 513th, 514th, 514th 515th, 516th, 516th, 517th, 518th, 520th, 522nd, 541st, 553rd, 556th, 556th, 562nd, 562nd, 562nd, 583rd, 585th, 653rd, 712th, 716th, 731st, 732nd, 744th, 744th The 744th, 748th, 751st, 754th, 757th, 757th, 771st, 787th, 825th, 826th, 826th, 832nd, 836th, 836th, 839th, 840th, 907th, 907th, 916th, 935th, 936th, 974th, or 1019th positions.

5. The isolated polypeptide of claim 1, wherein the ERCAS12A mutant comprises one or more amino acid substitutions selected from the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 2: , or a combination thereof.

6. The isolated polypeptide of claim 1, wherein the ERCAS12A mutant is selected from: 。 7. The isolated polypeptide of claim 1, wherein the ERCAS12A mutant is selected from: 。 8. An isolated polynucleotide sequence encoding a mutant of Escherichia coli Cas12a (ERCAS12A) having one or more amino acid substitutions introduced into the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 2, wherein the ERCAS12A variant polypeptide provides an improvement in nuclease activity compared to the wild-type ERCAS12A polypeptide.

9. The isolated polynucleotide sequence of claim 8, wherein the encoded ERCAS12A mutant has 98% to 99% identity with the polypeptide sequence selected from the even-numbered sequences of SEQ ID NO: 4 to 210.

10. The isolated polynucleotide sequence of claim 8, wherein the encoded ERCAS12A mutant has a polypeptide sequence selected from even-numbered sequences of SEQ ID NO: 4 to 210.

11. The isolated polynucleotide sequence of claim 8, wherein the ERCAS12A mutant polynucleotide sequence has 95% to 99% identity with the nucleotide sequences selected from odd-numbered sequences of SEQ ID NO: 3 to 209.

12. The isolated polynucleotide sequence of claim 8, wherein the ERCAS12A mutant polynucleotide has a nucleotide sequence selected from odd-numbered sequences of SEQ ID NO: 3 to 209.

13. The isolated polynucleotide sequence of claim 8, wherein the encoded ERCAS12A mutant comprises one or more amino acid substitutions at the following positions of the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 2: positions 18, 35, 39, 39, 40, 56, 56, 59, 59, 158, 165, 181, 290, 291, 296, 297, 297, 297, 303 303rd, 305th, 307th, 353rd, 444th, 465th, 469th, 470th, 498th, 507th, 507th, 507th, 510th, 510th, 510th, 512th, 512th, 513th, 513th, 513th, 514th, 514th 515th, 516th, 516th, 517th, 518th, 520th, 522nd, 541st, 553rd, 556th, 556th, 562nd, 562nd, 562nd, 583rd, 585th, 653rd, 712th, 716th, 731st, 732nd, 744th, 744th The 744th, 748th, 751st, 754th, 757th, 757th, 771st, 787th, 825th, 826th, 826th, 832nd, 836th, 836th, 839th, 840th, 907th, 907th, 916th, 935th, 936th, 974th, or 1019th position.

14. The isolated polynucleotide sequence of claim 8, wherein the encoded ERCAS12A mutant comprises one or more amino acid substitutions selected from the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 2: Or a combination thereof.

15. The isolated polynucleotide sequence of claim 8, wherein the encoding ERCAS12A mutant polynucleotide is selected from: 。 16. The isolated polynucleotide sequence of claim 8, wherein the ERCAS12A mutant polynucleotide is selected from: 。 17. The isolated polynucleotide sequence of claim 8, wherein the ERCAS12A mutant polynucleotide is selected from: 。 18. A vector or plasmid comprising the polynucleotide sequence of claim 8.

19. A cell comprising a nucleic acid containing one or more of the polynucleotide sequences of claim 8.

20. An isolated CRISPR ribonucleic acid protein complex comprising a guide RNA and an ERCAS12A mutant polypeptide having one or more amino acid substitutions introduced into the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 2, wherein the ERCAS12A mutant polypeptide provides improved gene editing compared to the wild-type ERCAS12A polypeptide.

21. The isolated CRISPR ribonucleic acid protein complex of claim 20, wherein the ERCAS12A mutant has a polypeptide sequence selected from even-numbered sequences of SEQ ID NO: 4 to 210.

22. The isolated CRISPR ribonucleic acid protein complex of claim 20, wherein the ERCAS12A mutant comprises one or more amino acid substitutions at the following positions of the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 2: positions 18, 35, 39, 39, 40, 56, 56, 59, 59, 158, 165, 181, 290, 291, 296, 297, 297, 297, 303 303rd, 305th, 307th, 353rd, 444th, 465th, 469th, 470th, 498th, 507th, 507th, 507th, 510th, 510th, 510th, 512th, 512th, 513th, 513th, 513th, 514th, 514th 515th, 516th, 516th, 517th, 518th, 520th, 522nd, 541st, 553rd, 556th, 556th, 562nd, 562nd, 562nd, 583rd, 585th, 653rd, 712th, 716th, 731st, 732nd, 744th, 744th The 744th, 748th, 751st, 754th, 757th, 757th, 771st, 787th, 825th, 826th, 826th, 832nd, 836th, 836th, 839th, 840th, 907th, 907th, 916th, 935th, 936th, 974th, or 1019th position.

23. The isolated CRISPR ribonucleic acid protein complex of claim 20, wherein the ERCAS12A mutant comprises one or more amino acid substitutions selected from the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 2: Or a combination thereof.

24. The isolated CRISPR ribonucleic acid protein complex of claim 20, wherein the ERCAS12A mutant is selected from: 。 25. The isolated CRISPR ribonucleic acid protein complex of claim 20, wherein the ERCAS12A mutant is selected from: 。 26. An isolated CRISPR / Cpf1 endonuclease system comprising crRNA and an ERCAS12A mutant polypeptide having one or more amino acid substitutions introduced into the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 2, wherein the ERCAS12A mutant polypeptide provides improved genome editing compared to the wild-type ERCAS12A polypeptide.

27. The isolated CRISPR / Cpf1 endonuclease system of claim 26, wherein the ERCAS12A mutant has a polypeptide sequence selected from even-numbered sequences of SEQ ID NO: 4 to 210.

28. The isolated CRISPR / Cpf1 endonuclease system of claim 26, wherein the ERCAS12A mutant comprises one or more amino acid substitutions at the following positions of the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 2: positions 18, 35, 39, 39, 40, 56, 56, 59, 59, 158, 165, 181, 290, 291, 296, 297, 297, 297, 303 303rd, 305th, 307th, 353rd, 444th, 465th, 469th, 470th, 498th, 507th, 507th, 507th, 510th, 510th, 510th, 512th, 512th, 513th, 513th, 513th, 514th, 514th 515th, 516th, 516th, 517th, 518th, 520th, 522nd, 541st, 553rd, 556th, 556th, 562nd, 562nd, 562nd, 583rd, 585th, 653rd, 712th, 716th, 731st, 732nd, 744th, 744th The 744th, 748th, 751st, 754th, 757th, 757th, 771st, 787th, 825th, 826th, 826th, 832nd, 836th, 836th, 839th, 840th, 907th, 907th, 916th, 935th, 936th, 974th, or 1019th position.

29. The isolated CRISPR / Cpf1 endonuclease system of claim 26, wherein the ERCAS12A mutant comprises one or more amino acid substitutions selected from the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 2: , or a combination thereof.

30. The isolated CRISPR / Cpf1 endonuclease system of claim 26, wherein the ERCAS12A mutant is selected from: 。 31. The isolated CRISPR / Cpf1 endonuclease system of claim 26, wherein the ERCAS12A mutant is selected from: 。 32. Methods for performing gene editing, including: The candidate editing target locus is contacted with a CRISPR ribonucleic acid protein complex comprising crRNA and an ERCAS12A mutant peptide, the ERCAS12A mutant peptide comprising one or more amino acid substitutions introduced into the wild-type ERCAS12A peptide sequence of SEQ ID NO: 2, wherein the ERCAS12A mutant provides improved gene editing compared to the wild-type ERCAS12A peptide.

33. The method of claim 32, wherein the ERCAS12A mutant has a polypeptide sequence selected from even-numbered sequences of SEQ ID NO: 4 to 210.

34. The method of claim 32, wherein the ERCAS12A mutant comprises one or more amino acid substitutions at the following positions in the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 2: positions 18, 35, 39, 39, 40, 56, 56, 59, 59, 158, 165, 181, 290, 291, 296, 297, 297, 297, 303. 303rd, 305th, 307th, 353rd, 444th, 465th, 469th, 470th, 498th, 507th, 507th, 507th, 510th, 510th, 510th, 512th, 512th, 513th, 513th, 513th, 514th, 514th 515th, 516th, 516th, 517th, 518th, 520th, 522nd, 541st, 553rd, 556th, 556th, 562nd, 562nd, 562nd, 583rd, 585th, 653rd, 712th, 716th, 731st, 732nd, 744th, 744th The 744th, 748th, 751st, 754th, 757th, 757th, 771st, 787th, 825th, 826th, 826th, 832nd, 836th, 836th, 839th, 840th, 907th, 907th, 916th, 935th, 936th, 974th, or 1019th position.

35. The method of claim 32, wherein the ERCAS12A mutant comprises one or more amino acid substitutions selected from the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 2: Or a combination thereof.

36. The method of claim 32, wherein the ERCAS12A mutant is selected from: 。 37. The method of claim 32, wherein the ERCAS12A mutant is selected from: 。 38. A kit comprising a CRISPR ribonucleic acid protein complex containing a guide RNA and an ERCAS12A mutant peptide comprising one or more amino acid substitutions introduced into the wild-type ERCAS12A peptide sequence of SEQ ID NO: 2, wherein the ERCAS12A mutant provides improved gene editing compared to the wild-type ERCAS12A peptide.

39. The kit of claim 38, wherein the ERCAS12A mutant has a polypeptide sequence selected from even-numbered sequences of SEQ ID NO: 4 to 210.

40. The kit of claim 38, wherein the ERCAS12A mutant comprises one or more amino acid substitutions selected from the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 2: Or a combination thereof.

41. The kit of claim 38, wherein the ERCAS12A mutant is selected from: 。 42. The kit of claim 38, wherein the ERCAS12A mutant is selected from: 。 43. Use of the ERCAS12A mutant polypeptide for improved purposes, wherein the ERCAS12A mutant is introduced into one or more amino acid substitutions in the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 2, wherein the ERCAS12A mutant provides improved gene editing compared to the wild-type ERCAS12A polypeptide.

44. The use of claim 43, wherein the ERCAS12A mutant has a polypeptide sequence selected from even-numbered sequences of SEQ ID NO: 4 to 210.

45. The use according to claim 43, wherein the ERCAS12A mutant is selected from: 。

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