Proctobacteria CAS12A mutant

Abstract

A series of recombinant Cas12a proteins derived from Eubacterium rectale are described herein. In one embodiment, various mutations having enhanced activity against wild-type Eubacterium rectale Cas12a polypeptide (ERCAS12A) are provided.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 462,772, filed on April 28, 2023, which is hereby incorporated by reference in its entirety.

[0002] Reference to Sequence Listing This application was filed with a sequence listing XML in ST.26 XML format in accordance with 37 C.F.R.§1.831 and PCT Rule 13ter. The sequence listing XML file “01367,0 - 0028 - WO01_sequence_listing_xml_25 - APR - 2024.xml,” filed with the USPTO Patent Center, was created on April 25, 2024, contains 1,667 sequences, has a file size of 1.94 M bytes (2,043,904 bytes), and is hereby incorporated by reference in its entirety.

Background Art

[0003] Eubacterium rectale Cas12a (ERCAS12A) is an engineered RNA guide endonuclease derived from the clustered regularly interspaced short palindromic repeat (CRISPR) adaptive immune system of Eubacterium rectalis (now known as Agatobacter rectalis). For these instructions, see Zetsche et al., Cell 163: 759-771 (2015), incorporated herein by reference. Cas12a is guided by a target-site-specific complementary RNA of approximately 21-24 nt to a DNA target sequence of approximately 21-24 nt, commonly referred to as a protospacer. The Cas12a-gRNA ribonucleoprotein (RNP) complex mediates double-strand DNA breaks (DSBs), which are typically repaired by non-homologous end joining (NHEJ), which introduces mutations or indels at the break site, or by homologous recombination repair (HDR) for precise editing when a suitable template nucleic acid is present.

[0004] Key to recognizing the correct DNA target for the ERCAS12A variant are the crRNA and the canonical "TTTV" protospacer adjacency motif (PAM), which is a 4-bp sequence located immediately upstream of the protospacer. Compared to the 2-bp NGG PAM of Cas9 from Streptococcus pyogenes, Cas12a amplified targetable loci in genome editing, particularly across T-rich sites inaccessible with the Cas9 system. For these teachings, see Jinek et al., Science 337: 816-821 (2012), incorporated herein by reference. Although the frequency of Cas12a targetable sites in the genome is lower than that of Cas9, ERCAS12A is an alternative enzyme for use in genome engineering applications. The improved cleavage activity and associated genome editing efficiency of ERCAS12A would greatly facilitate the development of human cell therapies with enhanced properties.

[0005] Previous studies have improved the usefulness of Cas12a derived from the Lachnospiraceae bacterium ND2006 (LbCas12a) and the Acidaminococcus sp. (AsCas12a). For these teachings, 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 No. 2021 / 0348144A1 and U.S. Patent Application Publication No. 2023 / 0040148A1, respectively, which are incorporated herein by reference. However, the transfer of benign mutations from AsCas12a and LbCas12a to ERCAS12A was neither simple nor predictable. Despite the significant structural similarities between these homologous proteins, the majority of point mutations that improved the activity of AsCas12a and LbCas12a were detrimental to ERCAS12A.

[0006] What is needed is a mutation that enhances the activity of ERCAS12A. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] U.S. Patent Application Publication No. 2021 / 0348144A1 [Patent Document 2] U.S. Patent No. 2023 / 0040148A1 [Non-patent literature]

[0008] [Non-Patent Document 1] Zetsche et al., Cell 163: 759-771 (2015) [Non-Patent Document 2] Jinek et al., Science 337: 816-821 (2012) [Non-Patent Document 3] Gao et al., Nat. Biotechnol. 35(8): 789-792 (2017) [Non-Patent Document 4] Kleinstiver et al., Nat. Biotechnol. 37(3): 276-282 (2019) [Non-Patent Document 5] Zhang et al., Nat. Communications 12: 3908 (2021) [Non-Patent Document 6] Smith and Waterman, Advances in Applied Mathematics 2: 4 82-489 (1981) [Non-Patent Document 7] Dayhoff, Atlas of Protein Sequences and Structure, edited by MO Dayhoff, 5 suppl. 3: 353-358, National Biomedical Research Foundation, Washington, DC, USA. [Non-Patent Document 8] Gribskov, Nucl. Acids Res. 14(6): 6745-6763, (1986) [Non-Patent Document 9] Bowie, JU et al., “Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions,” Science 247: 1306-1310 (1990) [Non-Patent Document 10] Wrenbeck et al., Nat. Methods 13(11):928-930 (2016) [Overview of the project] [Means for solving the problem]

[0009]

[0010]

[0011] Another embodiment described herein is a vector or plasmid containing the polynucleotide sequence described herein.

[0012] Another embodiment described herein is a cell comprising a nucleic acid containing one or more of the polynucleotide sequences described herein.

[0013]

[0014]

[0015]

[0016] Another embodiment described herein is a kit comprising a CRISPR ribonucleoprotein 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. In one embodiment, the ERCAS12A mutant has a polypeptide sequence selected from even-numbered sequences of SEQ ID NOs: 4-210. In another embodiment, the ERCAS12A mutant has Q18V, I35L, G39K, G39Q, I40Q, D56P, D56R, D59A, D59R, S158G, K165Y, S181V, A290K, D291N, V296R, P297F, P297R, P297V, D303 C, D303R, E305K, Y307K, Q353R, L444W, F465R, S469K, V470K, I498K, Y507R, Y507S, Y507W, Q 510K, Q510V, Q510Y, P512G, P512V, Y513G, Y513L, Y513R, Y513S, S514P, S514R, T515P, K516 P, K516R, K517P, I518P, L520M, F522A, A541V, G553M, N556E, N556R, D562I, D562K, D562V, N 583V, L585K, F653R, K712R, N716R, E731P, N732T, A744N, A744P, A744T, F748A, S751A, K754 In another embodiment, the ERCAS12A mutant includes one or more amino acid substitutions selected from G, I757K, I757W, E771R, N787A, A825R, T826F, T826K, Y832R, Y836K, Y836Q, Y839L, F840L, Y907I, Y907V, Q916Y, E935R, I936R, K974G, K1019V, or combinations thereof. In another embodiment, the ERCAS12A mutant includes Q18V (SEQ ID NO: 4); I35L (SEQ ID NO: 6); G39Q (SEQ ID NO: 8); G39K (SEQ ID NO: 10); I40Q (SEQ ID NO: 12); D56R (SEQ ID NO: 14); D56P (SEQ ID NO: 16); D59R (SEQ ID NO: 18); D59A (SEQ ID NO: 20); S158G (SEQ ID NO: 22); K165Y (SEQ ID NO: 24);S181V (sequence number 26); A290K (sequence number 28); D291N (sequence number 30); V296R (sequence number 32); P297V (sequence number 34); P297R (sequence number 36); P297F (sequence number 38); D303R (sequence number 40); D303C (sequence number 42); E305K (sequence number 44); Y307K (sequence number 46); Q353R (sequence number 48); L444W (sequence number 50); F465R (sequence number 52); S469K (sequence number 54); V470K (sequence number 56); I498K (sequence number 58); Y507W (sequence number 6 0);Y507S(Sequence ID 62);Y507R(Sequence ID 64);Q510Y(Sequence ID 66);Q510V(Sequence ID 68);Q510K(Sequence ID 70);P512V(Sequence ID 72);P512G(Sequence ID 74);Y513S(Sequence ID 76);Y513R(Sequence ID 78);Y513L(Sequence ID 80);Y513G(Sequence ID 82);S514R(Sequence ID 84);S514P(Sequence ID 86);T515P(Sequence ID 88);K516R(Sequence ID 90);K516P(Sequence ID 92);K517P(Sequence ID 94);I518P(Sequence ID number 96); L520M (Sequence ID 98); F522A (Sequence ID 100); A541V (Sequence ID 102); G553M (Sequence ID 104); N556R (Sequence ID 106); N556E (Sequence ID 108); D562V (Sequence ID 110); D562K (Sequence ID 112); D562I (Sequence ID 114); N583V (Sequence ID 116); L585K (Sequence ID 118); F653R (Sequence ID 120); K712R (Sequence ID 122); N716R (Sequence ID 124); E731P (Sequence ID 126); N732T (Sequence ID 128); A744T (Sequence ID 130); A744P (Sequence ID 132); A744N (Sequence ID 134); F748A (Sequence ID 136); S751A (Sequence ID 138); K754G (Sequence ID 140); I757W (Sequence ID 142); I757K (Sequence ID 144); E771R (Sequence ID 146); N787A (Sequence ID 148); A825R (Sequence ID 150); T826K (Sequence ID 152); T826F (Sequence ID 154); Y832R (Sequence ID 156); Y836Q (Sequence ID 158); Y836K (Sequence ID 160); Y839L (Sequence ID 162);F840L (Accession No. 164); Y907V (Accession No. 166); Y907I (Accession No. 168); Q916Y (Accession No. 170); E935R (Accession No. 172); I936R (Accession No. 174); K974G (Accession No. 176); K1019V (Accession No. 178); I35L / S469K (Accession No. 180); I35L / T826F (Accession No. 182); I40Q / S469K (Accession No. 184); I40Q / T826F (Accession No. 186); S469K / K516P (Accession No. 188); S469K / T826F (Accession No. 190); Y513G / Y832R (Accession No. 192); Y513G / F840L (Accession No. 194); I35L / S469K / T826F (Accession No. 196); I40Q / S469K / T826F (Accession No. 198); S469K / K516P / T826F (Accession No. 200); S469K / T826F / F840L (Accession No. 202); D303R / S469K / T826F / F840L (Accession No. 204); F465R / S469K / T826F / F840L (Accession No. 206); D303R / F465R / S469K / T826F / F840L (Accession No. 208); or D303R / S469K / K516P / T826F / F840L (Accession No. 210). In another aspect, the ERCAS12A variant is selected from D303R / S469K / T826F / F840L (Accession No. 204); F465R / S469K / T826F / F840L (Accession No. 206); D303R / F465R / S469K / T826F / F840L (Accession No. 208); or D303R / S469K / K516P / T826F / F840L (Accession No. 210).;

[0017] Another embodiment described herein involves the use of an ERCAS12A mutant polypeptide to improve CRISPR / LbCas12a-related nuclease activity at a non-canonical TTTT PAM site, wherein the ERCAS12A mutant has one or more amino acid substitutions introduced into the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 2, and the ERCAS12A mutant provides improved gene editing compared to the wild-type ERCAS12A polypeptide. In one embodiment, the ERCAS12A mutant has a polypeptide sequence selected from even-numbered sequences of SEQ ID NOs: 4 to 210. In another embodiment, the ERCAS12A mutants include Q18V (sequence number 4); I35L (sequence number 6); G39Q (sequence number 8); G39K (sequence number 10); I40Q (sequence number 12); D56R (sequence number 14); D56P (sequence number 16); D59R (sequence number 18); D59A (sequence number 20); S158G (sequence number 22); K165Y (sequence number 24); S181V (sequence number 26); A290K (Sequence ID 28); D291N (Sequence ID 30); V296R (Sequence ID 32); P297V (Sequence ID 34); P297R (Sequence ID 36); P297F (Sequence ID 38); D303R (Sequence ID 40); D303C (Sequence ID 42); E305K (Sequence ID 44); Y307K (Sequence ID 46); Q353R (Sequence ID 48); L444W (Sequence ID 50); F465R (Sequence ID 52); S469 K(sequence number 54);V470K(sequence number 56);I498K(sequence number 58);Y507W(sequence number 60);Y507S(sequence number 62);Y507R(sequence number 64);Q510Y(sequence number 66);Q510V(sequence number 68);Q510K(sequence number 70);P512V(sequence number 72);P512G(sequence number 74);Y513S(sequence number 76);Y513R(sequence number 78);Y51 3L(SEQ ID NO: 80);Y513G(SEQ ID NO: 82);S514R(SEQ ID NO: 84);S514P(SEQ ID NO: 86);T515P(SEQ ID NO: 88);K516R(SEQ ID NO: 90);K516P(SEQ ID NO: 92);K517P(SEQ ID NO: 94);I518P(SEQ ID NO: 96);L520M(SEQ ID NO: 98);F522A(SEQ ID NO: 100);A541V(SEQ ID NO: 102);G553M(SEQ ID NO: 104);N556R(sequence code 106);N556E(sequence code 108);D562V(sequence code 110);D562K(sequence code 112);D562I(sequence code 114);N583V(sequence code 116);L585K(sequence code 118);F653R(sequence code 120);K712R(sequence code 122);N716R(sequence code 124);E731P(sequence code 126);N732T(sequence code 128);A744T(sequence code 130);A744P(sequence code 132);A744N(sequence code 134);F748A(sequence code 136);S751 A (sequence number 138); K754G (sequence number 140); I757W (sequence number 142); I757K (sequence number 144); E771R (sequence number 146); N787A (sequence number 148); A825R (sequence number 150); T826K (sequence number 152); T826F (sequence number 154); Y832R (sequence number 156); Y836Q (sequence number 158); Y836K (sequence number 160); Y839L (sequence number 162); F840L (sequence number 164); Y907V (sequence number 166); Y907I (sequence number 168); Q916Y (sequence number Code 170); E935R (Sequence ID 172); I936R (Sequence ID 174); K974G (Sequence ID 176); K1019V (Sequence ID 178); I35L / S469K (Sequence ID 180); I35L / T826F (Sequence ID 182); I40Q / S469K (Sequence ID 184); I40Q / T826F (Sequence ID 186); S469K / K516P (Sequence ID 188); S469K / T826F (Sequence ID 190); Y513G / Y832R (Sequence ID 192); Y513G / F840L (Sequence ID 194); I35L / S469K / T82 6F (sequence number 196); I40Q / S469K / T826F (sequence number 198); S469K / K516P / T826F (sequence number 200); S469K / T826F / F840L (sequence number 202); D303R / S469K / T826F / F840L (sequence number 204); F465R / S469K / T826F / F840L (sequence number 206); D303R / F465R / S469K / T826F / F840L (sequence number 208); or D303R / S469K / K516P / T826F / F840L (sequence number 210) are selected.

[0018] The patent and application files include at least one drawing executed in color. A copy of this patent or patent application publication containing color drawings will be provided by the authority upon request and payment of the required fees.

Brief Description of the Drawings

[0019] [Figure 1A] Shows high-throughput characterization of the cleavage activity of ERCAS12A in Escherichia coli (E. coli). Three biological replicates of the selection were performed and deep sequencing was carried out to determine the enrichment of each ERCAS12A variant. Figure 1A shows the phenotypic scores of synonymous changes (n = 834) and non-synonymous changes (n = 22409) during the selection process. Notably, most of the synonymous changes did not result in obvious enrichment and were used as the baseline activity of wild-type ERCAS12A. [Figure 1B] Shows high-throughput characterization of the cleavage activity of ERCAS12A in Escherichia coli. Three biological replicates of the selection were performed and deep sequencing was carried out to determine the enrichment of each ERCAS12A variant. Figure 1B shows the enrichment scores of 23,243 point mutations of ERCAS12A over two biological replicates. The results of the high-throughput screening are highly reproducible (R2 = 0.66). [Figure 2] Shows the DNA cleavage activity of ERCAS12A variants in Escherichia coli. Each ERCAS12A mutant was cloned and evaluated for Escherichia coli. The survival rate of Escherichia coli was determined by taking the ratio of the number of colonies with or without selection pressure. Most mutations resulted in a higher survival rate than wild-type (WT) ERCAS12A (about 10%). The raw data are shown in Figures 3 - 5. [Figure 3] Shows examples of point mutations in ERCAS12A with enhanced DNA cleavage activity. Clearly, the mutations increased the survival rate at TTTC and TTTT PAM sites and showed improved cleavage activity compared to WT-ERCAS12A. [Figure 4]An example of a point mutation in ERCAS12A with enhanced DNA cleavage activity is shown. [Figure 5] An example of a point mutation in ERCAS12A with enhanced DNA cleavage activity is shown. [Figure 6] This study demonstrates the cleavage activity of ERCAS12A variants in Escherichia coli. Each ErCas12a variant was cloned and evaluated in E. coli. E. coli viability was determined by the ratio of colony numbers with and without selective pressure. Most mutations resulted in higher viability than WT-ERCAS12A (approximately 18%). [Figure 7A] This paper demonstrates the performance of ERCAS12A variants as RNPs in human cells. ERCAS12A variants and WT proteins were purified and assembled as RNPs using six different crRNAs. Figure 7A shows the editing efficiency of each variant, measured by T7EI (T7 endonuclease I) 48 hours after delivery with 200 nM RNP in electroporation. NC: Negative control lacking ERCAS12A nuclease. [Figure 7B] This paper demonstrates the performance of ERCAS12A variants as RNPs in human cells. ERCAS12A variants and WT proteins were purified and assembled as RNPs using six different crRNAs. Figure 7B shows the editing efficiency of each variant, measured by T7EI 48 hours after delivery with 50 nM RNP in electroporation. NC: Negative control lacking ERCAS12A nuclease. [Figure 7C] This study demonstrates the performance of ERCAS12A variants as RNPs in human cells. ERCAS12A variants and WT proteins were purified and assembled as RNPs using six different crRNAs. Figure 7C shows the normalized activity of each variant against WT ERCAS12A at 200 nM RNP. The dashed line represents the baseline activity of the WT protein. [Figure 7D]This paper demonstrates the performance of ERCAS12A variants as RNPs in human cells. ERCAS12A variants and WT proteins were purified and assembled as RNPs using six different crRNAs. Figure 7D shows the normalized activity of each variant against WT ERCAS12A at 50 nM RNP. The dashed line represents the baseline activity of the WT protein. [Figure 8A] This shows the on-target editing efficiency of ERCAS12A point mutations in human cells. Figure 8A shows the relative editing efficiency of ERCAS12A variants in human cells across two target sites. Plasmids encoding each variant were delivered by lipofection, and editing efficiency was measured using the T7EI assay two days after delivery. [Figure 8B] This shows the on-target editing efficiency of ERCAS12A point mutations in human cells. Figure 8B shows the editing efficiency of positive hits from lipofection experiments when delivered as RNP. [Figure 8C] This shows the on-target editing efficiency of ERCAS12A point mutations in human cells. Figure 8C shows the normalized editing efficiency of ERCAS12A point mutations compared to WT cells. [Figure 9A] This report demonstrates the performance of stacked ERCAS12A variants with numerous point mutations in human cells as RNPs. ERCAS12A variants and WT proteins were purified and assembled as RNPs using six different crRNAs. Figure 9A shows the editing efficiency of each variant, measured by T7EI 48 hours after delivery at 50 nM in electroporation. NC: Negative control lacking ERCAS12A nuclease. [Figure 9B] This paper demonstrates the performance of stacked ERCAS12A variants with multiple point mutations in human cells as RNPs. ERCAS12A variants and WT proteins were purified and assembled as RNPs using six different crRNAs. Figure 9B shows the normalized activity of each variant against WT ERCAS12A in 50 nM RNPs. [Figure 10]This study shows the relative editing efficiency of ERCAS12A variants in human cells across nine target sites. Plasmids encoding each variant were delivered by lipofection. Editing efficiency was measured by NGS three days after delivery. [Figure 11] This study demonstrates the on-target editing efficiency of ERCAS12A Y513G in human cells. ERCAS12A Y513G and WT proteins were purified and assembled as RNPs using seven different crRNAs. Editing efficiency was measured by NGS two days after delivery. [Figure 12] This study demonstrates the on-target editing efficiency of ERCAS12A mutants in human cells. WT ERCAS12A, Y513G, S514P, Y513G / F840L, and AsCas12a-Ultra (e.g., AsCas12a M537R / F870L) proteins were purified and assembled as RNPs using various crRNAs. Editing efficiency was measured by NGS two days after delivery. [Figure 13] This study demonstrates the performance of stacked ERCAS12A variants with multiple point mutations as RNPs in human cells. ERCAS12A variants and WT proteins were purified and assembled as RNPs using various crRNAs. D303R / S469K / T826F / F840L, F465R / S469K / T826F / F840L, and D303R / S469K / K516P / T826F / F840L improved activity compared to ERCAS12A WT. [Figure 14] This paper summarizes the on-target editing efficiencies of various ERCAS12A point mutations in human cells. [Modes for carrying out the invention]

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. For example, all terms and techniques used relating to cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry, and hybridization described herein are well known and commonly used in the art. In case of any conflict, the disclosure, including definitions, shall prevail. Exemplary methods and materials are described below, but similar or equivalent methods and materials may be used when carrying out or testing the embodiments and aspects described herein.

[0021] As used herein, the term “include,” “include,” “contain,” “containing,” “having,” etc., means “comprising.” This disclosure also intends other embodiments that “include,” “consist of,” and “essentially consist of,” the embodiments or elements presented herein, whether expressly described or not.

[0022] As used herein, the terms “a, an, the” and similar terms used in relation to this disclosure (particularly in relation to the claims) should be construed to include both singular and plural unless otherwise indicated herein or explicitly denied by the context. Furthermore, “a, an, or the” means “one or more” unless otherwise specified.

[0023] As used herein, the term "or" may be a conjunction or a separator.

[0024] As used herein, the terms “amino acid,” “nucleotide,” “polynucleotide,” “vector,” “polypeptide,” and “protein” have the common meanings understood by a biochemist with the usual skills in this art. In this specification, standard single-letter nucleotides (A, C, G, T, U) and standard single-letter amino acids (A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y) are used.

[0025] As used herein, the terms "nucleic acid" and "polynucleotide" are interchangeable and have the same meaning.

[0026] As used herein, the terms "amino acid sequence," "polypeptide," and "protein" are interchangeable and have the same meaning.

[0027] As used herein, the term “substantially” means “to a great or significant degree,” but not “completely.”

[0028] As used herein, the terms “about” or “approximately” applied to one or more values ​​of interest refer to a value similar to the described reference value or a value within an acceptable margin of error for a particular value determined by those skilled in the art, which will in part depend on how the value was measured or determined, for example, the limitations of the measuring system. In one embodiment, the term “about” refers to any value containing both integer and fractional components that is within ±10% of the value modified by the term “about.” Alternatively, “about” may mean within three or more standard deviations for the implementation in the art. Or, for example with respect to biological systems or processes, the term “about” may mean within one order of magnitude of a value, within five times in some embodiments, and within two times in some embodiments. As used herein, the symbol “~” may mean “about” or “approximately.”

[0029] All ranges disclosed herein include both endpoints as discrete values ​​and all integers and fractions specified within that range. For example, the range 0.1 to 2.0 includes 0.1, 0.2, 0.3, 0.4...2.0. Where an endpoint is modified by the term "approximately", the specified range is extended, including the endpoint, by a variation of up to ±10% of any value within the range, or a variation within a range of three or more standard deviations.

[0030] As used herein, the terms “control” and “reference” are interchangeable. The “reference” or “control” level may be a predetermined value or range, which is used as a baseline or benchmark for evaluating measured results. “Control” also refers to a control experiment or control cells.

[0031] As used herein, the term "effective amount" of a compound described herein refers to the amount of the compound described herein that induces a biological response, for example, by reducing or inhibiting the activity of an enzyme or protein, or by improving symptoms, alleviating a condition, slowing or delaying the progression of a disease, or preventing a disease.

[0032] As used herein, the term “inhibit” (“inhibition” or “inhibiting”) means the reduction or suppression of a given condition, symptom, or disorder, or disease, or a significant decrease in the baseline activity of a biological activity or process.

[0033] As used herein, the terms “Erectobacter rectum Cas12a” or “ERCAS12A” are interchangeable and refer to the Erectobacter rectum (Er) Cas12a protein (formerly named Cpf1), i.e., a class 2 / type V CRISPR RNA guided endonuclease.

[0034] The terms "ERCAS12A," "wild-type ERCAS12A," or "WT ERCAS12A" refer to a biochemically active protein that possesses the amino acid sequence of naturally occurring *Cas12A* from *Bacillus oryzae* (e.g., sequence number 2 encoded by the nucleotide sequence of sequence number 1) and forms an active CRISPR / Cas12a endonuclease system when combined with a suitable crRNA.

[0035] The terms "mutant ERCAS12A protein" or "variant ERCAS12A protein" are interchangeable and refer to a form of *Proteus rectus* Cas12a protein that has a different amino acid sequence from the wild-type *Proteus rectus* Cas12a protein and possesses biochemical activity to form an active CRISPR / Cas12a endonuclease system when combined with a suitable crRNA. This includes orthologs and Cas12a variants that have a different amino acid sequence from the wild-type *Proteus rectus* Cas12a. The mutant ERCAS12A proteins described herein have at least one amino acid substitution from the wild-type ERCAS12A polypeptide sequence. In one embodiment, the mutant ERCAS12A polypeptide has at least one amino acid substitution from the wild-type ERCAS12A polypeptide sequence and exhibits improved CRISPR / ERCAS12A-related nuclease activity at the non-canonical TTTT PAM site compared to the wild-type ERCAS12A enzyme.

[0036] As used herein, the term “substantially purified” as applied to a composition means having a purity of at least 90%, and including compositions with a purity of 90%, 95%, 99%, and greater than 99%.

[0037] As used herein, the term “isolated” refers, when used to describe compositions such as polynucleotides, polypeptides, or ribonucleoprotein complexes, to a substantially purified composition, or, in the case of ribonucleoprotein complexes, to at least one component that is substantially purified. More specifically with respect to isolated ribonucleoprotein complexes, preferably all components are substantially purified.

[0038] As used herein, the term “affinity tag” refers to a ligand that enables the detection and / or selection of an oligonucleotide sequence to which the ligand is bound. For the purposes of this disclosure, decoys may include affinity tags. In particular, affinity tags are typically located at one or both of the N-terminus and / or C-terminus of a polypeptide by conventional chemical coupling techniques or recombinant DNA techniques. Exemplary affinity tags include, among others, biotin, digoxigenin, streptavidin, polyhistines, e.g., (His6), glutathione-S-transferase (GST), HaloTag®, AviTag, calmodulin tag, polyglutamine tag, FLAG tag, HA tag, Myc tag, S tag, SBP tag, Softag 3, V5 tag, Xpress tag, and haptens.

[0039] As used herein, the term “eukaryotic cell” includes cells of certain living organisms, such as humans, or non-human eukaryotes or animals or mammals as discussed herein, including but not limited to mice, rats, rabbits, dogs, livestock, or non-human mammals or primates, or plants or mammals, or cells derived therefrom. In some embodiments, processes that alter the genetic identity of the human germline and / or animals, and animals resulting from such processes, which may cause suffering without substantial medical benefit to humans or animals, may be excluded. Preferred human cells include somatic cells and cells derived from germline cells. Exemplary somatic cells include cells from any major organ and tissue system, including the immune system and hematopoietic system.

[0040] In this specification, the phrase "odd-numbered sequences of SEQ ID NOs. 3-209" refers to the nucleotide sequences of SEQ ID NOs. 3, 5, 7...207, or 209. See Table 14, where all odd-numbered sequences are nucleotide sequences.

[0041] In this specification, the phrase "even-numbered sequences of sequence numbers 4-210" refers to polypeptide sequences of sequence numbers 4, 6, 8...206, or 210. See Table 14, where all even-numbered sequences are polypeptide sequences.

[0042] This specification describes a variant of *Erythrocyte rectum* Cas12a (ERCAS12A) and a method for enhancing the usefulness of ERCAS12A and its variants.

[0043] We identified mutations with enhanced cleavage activity using bacterial-based, targeted ERCAS12A evolution. First, we created a deep-scan mutagenesis library 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 allowed for direct evaluation of the phenotype of each point mutation by measuring its relative viability against wild-type (WT) protein in bacterial screening. Briefly, screening strains carrying the toxin plasmid were transformed with the ERCAS12A library and crRNA targeting the HPRT38346 site on the toxin plasmid. After harvesting and IPTG induction, cells were seeded in LB-chloramphenicol medium containing arabinose and incubated overnight at 37°C. The ERCAS12A expression plasmid held by the viable E. coli cells was extracted and purified. Both the input plasmid library and the selected plasmid library 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 was measured in both libraries and normalized to the total coverage of each codon. The relative viability of each point mutation was calculated as the ratio of the normalized frequencies between the selected library and the input library. Since the degree of cell viability under arabinose selection is an indicator of the cleavage activity of ERCAS12A variants in the HPRT38346 protospacer, any variant enriched against WT during selection is considered to be a variant with enhanced activity in TTTT PAM.

[0044] This report describes a large accumulation of mutations associated with *E. coli* that are thought to have enhanced activity. It details the phenotyping of 22,049 point mutations in ERCAS12A during bacterial screening to measure cleavage activity in non-canonical TTTT PAM. Two biological replicates were performed for phenotypic measurement. This enabled the isolation of a large accumulation of ERCAS12A variants with enhanced cleavage activity.

[0045] 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 to human HEK293 cells as ribonucleoproteins (RNPs) for editing six genomic targets using canonical TTTV PAM. The editing efficiency of each variant was quantified by the T7EI assay and compared to WT ERCAS12A. This allowed for rigorous validation of the activity of hypothetical candidates in human cells without protein overexpression. Editing efficiency was measured 72 hours after delivery by the T7 endonuclease I assay (T7E1).

[0046] Genome editing efficiency of 10 ERCAS12A variants with single or multiple point mutations in human cells. ERCAS12A variants were column-purified and delivered to human HEK293 cells as ribonucleoproteins (RNPs) for editing six genomic targets using canonical TTTV PAM. The editing efficiency of each variant was quantified by the T7EI assay and compared to WT ERCAS12A. This allowed for rigorous validation of the activity of hypothetical candidates in human cells without protein overexpression. Editing efficiency was measured 72 hours after delivery by the T7 endonuclease I assay (T7E1).

[0047] One embodiment described herein is a CRISPR-related protein containing a polypeptide encoding a variant of ERCAS12A. The isolated polypeptide containing the ERCAS12A variant is selected from polypeptides having one or more amino acid substitutions relative to wild-type ERCAS12A (SEQ ID NO: 2) as shown in Table 2.

[0048] Another embodiment described herein is an ErCas12A variant selected from polypeptides having one or more amino acid substitutions shown in Table 2 relative to wild-type ERCAS12A (SEQ ID NO: 2), wherein the selected ERCAS12A variant exhibits improved genome editing compared to wild-type ERCAS12A (SEQ ID NO: 2).

[0049] Another embodiment described herein provides a CRISPR ribonucleoprotein complex comprising a polypeptide encoding a variant of ERCAS12A. The variant of ERCAS12A is selected from polypeptides having one or more amino acid substitutions relative to wild-type ERCAS12A (SEQ ID NO: 2) as presented in Table 2.

[0050] Another embodiment described herein provides a method for increasing the efficiency of gene editing at TTTN PAM sites in cells using a CRISPR ribonucleoprotein complex. This method includes contacting cells with a CRISPR ribonucleoprotein complex. The CRISPR ribonucleoprotein complex comprises a guide RNA and a CRISPR-related protein. The CRISPR-related protein includes a polypeptide encoding a variant of ERCAS12A. The variant of ERCAS12A is selected from polypeptides having one or more amino acid substitutions compared to wild-type ERCAS12A (SEQ ID NO: 2) as presented in Table 2.

[0051] Another embodiment described herein provides a kit comprising a guide RNA and a CRISPR-related protein. The CRISPR-related protein comprises a polypeptide encoding a variant of ERCAS12A.

[0052] Another embodiment described herein is a CRISPR-related protein comprising a polypeptide encoding a variant of ERCAS12A, provided that the variant improves CRISPR / ERCAS12A-related nuclease activity, including M1G, N2C, N3I, T5D, T5F, T5V, N6P, N7A, N7D, N7E, N7I, N7Q, N7Y, F8H, F8K, Q9D, N10A, N10E, F11E, F11Q, I12L, I12Q, I14E, I14H, I14L, I14N, I14V, S15K, S15M, S15T, S16H, S1 6M, S16N, Q18I, Q18M, Q18N, Q18R, Q18T, Q18V, Q18W, A24E, I26P, P27C, P27F , P27G, P27H, P27K, P27L, P27M, P27R, P27Y, T28G, T30D, T30F, T30H, T30Q, T 30V, T30W, T31A, T31C, T31D, T31G, T31H, T31L, T31M, T31N, T31P, T31S, Q32 A, Q32C, Q32F, Q32G, Q32I, Q32K, Q32L, Q32M, Q32N, Q32P, Q32S, Q32T, Q32V, Q 32W, Q32Y, Q33F, Q33I, Q33P, Q33V, Q33W, Q33Y, F34A, F34L, F34P, F34R, F34 W, I35A, I35C, I35E, I35F, I35G, I35K, I35L, I35M, I35N, I35P, I35Q, I35S, I35T, I35V, I35W, I35Y, V36K, V36M, V36P, K37P, N38A, N38D, N38E, N38G, N3 8H, N38I, N38L, N38M, N38P, N38Q, N38R, N38S, N38T, N38V, G39A, G39C, G39D, G39E, G39F, G39H, G39I, G39K, G39L, G39M, G39N, G39P, G39Q, G39R, G39S, G3 9T, G39V, G39W, G39Y, I40C, I40D, I40E, I40K, I40L, I40N, I40P, I40Q, I40R, I40T, I40V, I40W, I40Y, E43C, E43I, E43L, E43Q, E43S, E43T, E43V, E45F, E4 5R, L46C, L46D, L46E, L46G, L46H, L46K, L46N, L46S, L46T, E49C, E49F, 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93E、L693G、L693M、L693Q、L693T、L693W、Q694C、Q694G、Q694I、Q694K、Q694 R、Q694S、Q694W、Q694Y、E695S、G697I、G697N、G697P、Q698A、Q698F、L699C、 L699F、L699Y、Y700A、Y700G、Y700I、Y700T、Y700V、L701P、L701W、F702A、F70 2C、F702G、F702H、F702L、F702M、F702N、F702Q、F702S、F702T、F702V、Q703C 、K711L、K711P、K712A、K712C、K712F、K712G、K712H、K712Q、K712R、K712S、K 712W, K712Y, S713R, T714N, T714W, T714Y, N716A, N716C, N716I, N716K, N716M, N716Q, N716R, N716T, N716V, D717A, D717P, L719K, K725Y, L727H, L727I L727T, F728T, S729H, S729K, E730G, E730I, E730L, E730M, E730V, E731A, E731L, E731P, N732D, N732T, L733I, L733K, D735K, D735R, V737Q, L738E, N741 C、N741G、E743N、A744C、A744N、A744P、A744S、A744T、E745P、F748A、F748C、 F748D、F748E、F748G、F748H、F748I、F748M、F748N、F748Q、F748R、F748S、F74 8T、F748V、F748W、K750A、K750E、K750F、K750G、K750H、K750L、K750N、K750P 、K750S、K750T、K750W、K750Y、S751A、S751C、S751D、S751E、S751F、S751G、S 751H、S751L、S751M、S751N、S751P、S751Q、S751R、S751T、S751V、S752C、S75 2F、S752G、S752H、S752I、S752L、S752M、S752Q、S752V、S752W、S752Y、I753E、K754A, K754C, K754D, K754E, K754F, K754G, K754H, K754I, K754L, K754N, K754P, K754Q, K754S, K754T, K754V, K754W, K754Y, P756H, P756I, P756K, P756 L, P756M, P756N, P756S, P756T, I757A, I757C, I757F, I757G, I757K, I757L, I757M, I757P, I757Q, I757R, I757S, I757W, I757Y, I758A, I758C, I758E, I75 8G, I758K, I758Q, I758S, I758T, I758V, G762K, I764Q, V766Y, N767R, R768K, T769L, E771A, E771C, E771F, E771H, E771I, E771K, E771M, E771Q, E771R, E 771S, E771T, E771V, E771W, E771Y, A772W, E773I, E773P, E774H, E774I, E774K, E774L, E774M, E774Q, E774R, E774S, E774T, E774V, E774W, K775C, Q777R Q777V, F778G, F778K, F778Q, F778R, I783Q, V784F, V784H, V784I, V784L, V784R, V784W, V784Y, N787A, N787C, N787F, N787G, N787H, N787M, N787P, N787 Q、N787R、N787S、N787W、I788F、I788Y、P789C、E790A、E790C、E790F、E790G、 E790H、E790I、E790K、E790M、E790N、E790P、E790Q、E790R、E790S、E790T、E79 0V、E790W、E790Y、N791E、N791F、N791G、Y793Q、Y793R、Y793V、Y793W、Q794L 、Q794P、Y797W、K798L、F800W、N801A、N801S、K803A、S804F、S804I、K806C、S 809G、A813H、E823A、E823P、E823S、A825H、A825K、A825R、T826C、T826F、T82 6H、T826I、T826K、T826L、T826M、T826N、T826Q、T826R、T826W、T826Y、N827D、N827E、V829T、D831L、D831M、D831N、D831Q、D831R、Y832A、Y832E、Y832G、Y832K、Y832M、Y832N、Y832Q、Y832R、Y836A、Y836C、Y836D、Y836E、Y836G、Y836H、Y836I、Y836K、Y836L、Y836M、Y836N、Y836Q、Y836R、Y836S、Y836T、Y836V、D837P、Y839A、Y839C、Y839H、Y839I、Y839K、Y839L、Y839M、Y839N、Y839Q、Y839R、Y839S、Y839T、Y839V、L841C、L841G、L841I、L841V、L841Y、M843F、M843Q、P844C、I845Q、T846G、T846L、T846Q、T846W、T846Y、N848D、F849R、A851N、A851T、I857A、I857N、D859P、R860E、R860K、I861A、L862D、L862H、L862Y、A866D、A866Y、K869C、H872M、H872S、I876L、V886A、V886G、V886S、T891I、C892D、C892H、C892I、C892T、G893K、N894V、E897Y、Q898G、F901G、F901L、F901M、F901Q、I903V、Y907I、Y907M、Y907T、Y907V、Y909F、Y909W、Q910A、Q910N、Q910S、Q910Y、I911A、I911D、I911E、I911G、I911H、I911Q、I911W、I911Y、K912C、K912H、K912Y、K914A、K914C、K914F、K914M、K914Q、K914S、K914T、Q916A、Q916D、Q916L、Q916M、Q916N、Q916V、Q916W、Q916Y、G918D、G918E、G918F、G918H、G918I、G918K、G918L、G918M、G918Q、G918T、G918Y、A919E、A919F、A919I、A919L、A919M、A919Y、Q921A、Q921T、I922A、I922C、I922H、I922R、I922W、I922Y、K925C、E926N、E926S、E926T、K928W、E929C、E929G、E929N、E929P、E929S、G931H, G931Q, K934S, E935A, E935C, E935F, E935G, E935H, E935K, E935L, E935M, E935N, E935Q, E935R, E935S, E935T, E935W, E935Y, I936A, I936C, I936F I936G, I936K, I936L, I936Q, I936R, I936S, I936T, I936Y, L941C, L941N, S942A, L943I, V944C, I945M, H946W, E947F, E947M, E947P, I948L, S949I, S949 K, S949M, S949Q, S949T, S949V, M951G, V952E, V952W, I953A, I953P, K954A, K954P, Y955C, Y955W, N956C, N956F, N956W, N956Y, A957F, A957P, A957T, I95 8V, I959F, D963C, F968W, K970C, K974A, K974C, K974E, K974G, K974M, K974P, K974Q, K974S, K974T, K974W, K974Y, V975I, R977H, K982S, K982Y, F983A, M9 86G、I988E、I988K、L991C、L991W、N992A、N992L、L994A、V995P、F996T、F996 V、D998T、D998W、I999H、I999P、I1001W、T1002C、T1002L、T1002S、T1002V、E1 003M、E1003S、N1004A、N1004G、N1004P、N1004Q、N1004R、L1007A、L1007C、L 1007V、L1008F、Y1011I、Y1011L、Y1011V、T1014C、Y1015A、Y1015E、Y1015G、Y 1015M、Y1015Q、Y1015T、I1016A、I1016E、I1016N、I1016P、P1017D、P1017E、 P1017G、P1017N、P1017Q、P1017W、K1019A、K1019C、K1019G、K1019S、K1019V、 K1021A、K1021F、K1021V、K1021W、V1023C、V1023N、G1024Y、Q1026M、Q1026N、 C1027N、C1029N、Y1032K、Y1032Q、Y1032R、Y1032W、A1036F、A1036H、K1040A、K1040I、K1040T、I1050D、I1050H、I1050W、K1052C、K1052G、K1052I、K1052W、F1053Y、K1054F、K1054I、K1054L、K1054M、K1054V、K1054W、D1055C、D1055F、D1055I、D1055L、D1055M、D1055P、D1055Q、D1055T、D1055V、L1056C、L1056E、L1056I、T1057Y、V1058H、V1058W、A1060N、A1060Q、K1061C、K1061D、K1061E、K1061F、K1061I、K1061L、K1061Q、K1061R、K1061S、K1061T、K1061W、R1062A、R1062D、R1062H、R1062M、R1062V、R1062Y、E1063F、E1063G、E1063W、I1065W、F1068N、D1069M、S1070E、R1072D、R1072E、D1074R、D1074S、S1075V、E1076T、K1077R、C1081A、C1081I、T1083E、T1083K、T1083M、T1083Y、N1087A、N1087W、N1088A、N1088P、I1090G、T1091C、T1091W、Q1092A、Q1092C、Q1092F、Q1092I、Q1092Y、N1093C、T1094I、T1094P、T1094Y、V1095A、V1095D、V1095R、M1096C、S1097D、K1098C、K1098V、K1098W、S1099P、S1099Q、S1100D、S1100G、S1100H、S1100M、S1100Q、Y1106I、K1111R、K1111V、R1113E、R1113I、R1113K、R1113N、R1113T、R1118F、R1118M、R1118Q、F1119A、F1119N、F1119W、S1120D、D1124T、T1125D、T1125E、T1125H、T1125M、T1125W、I1126A、I1126C、I1126E、T1129H、T1129I、T1129V、D1131L、D1131S、E1133T、K1134I、T1135Q、T1135R、T1135V、E1137L、E1137N、E1137Q、M1138D、M1138G、T1139Q、T1139V、T1139Y、I1141C, I1141P, N1142A, W1143F, D1145F, G1146K, R1150N, Q1151C, D1155M, I1158A, V1159D, F1163C, F1163N, E1164P, I1165A, L 1179D, L1179E, L1179N, R1182K, R1182N, R1182Q, R1182Y, D1183Y, Y1184W, R1186M, L1187M, L1192K, L1192Y, E1194C, E1194F, E1 194S, N1195S, N1195Y, N1196A, I1197G, I1197W, A1204C, A1204D, A1204F, A1204M, A1204N, A1204Q, D1206C, D1206F, D1206I, A12 07Q, L1208A, L1208Q, D1211C, D1211S, D1211T, A1212C, A1212M, A1212S, A1212T, A1212V, N1215G, E1227T, I1228H, I1228Y, K1229 T, Q1230C, Q1230G, Q1230T, E1233W, E1233Y, N1234A, N1234E, N1234F, N1234I, W1235E, W1235Y, K1236F, K1236P, E1237A, E1237F , E1237G, E1237I, G1239H, G1239K, G1239L, G1239Q, G1239T, K1240Y, F1241P, S1242E, S1242Q, D1244I, K1245G, K1245V, S1249E, It contains at least one variant amino acid selected from the amino acid positions of S1249Q, S1249W, N1250A, N1250G, N1250L, N1250P, D1252L, F1254T, D1255M, F1256R, F1256W, K1260F, K1260G, K1260H, R1261M, R1261N, R1261V, R1261W, R1261Y, Y1262M, L1263A, L1263E, L1263N, L1263Q, or combinations thereof.

[0053] In one embodiment, the ERCAS12A variants are Q18V, I35L, G39K, G39Q, I40Q, D56P, D56R, D59A, D59R, S158G, K165Y, S181V, A290K, D291N, V296R, P297F, P297R, P297V, D303C, D303R, E305K, Y307K, Q353R, L444W, F465R, S469K, V470K, I498K, Y507R, Y507S, Y507W, Q510K, Q510V, Q510 Y, P512G, P512V, Y513G, Y513L, Y513R, Y513S, S514P, S514R, T515P, K516P, K516R, K517P, I518P, L520M, F522A, A541V, G553M, N55 6E, N556R, D562I, D562K, D562V, N583V, L585K, F653R, K712R, N716R, E731P, N732T, A744N, A744P, A744T, F748A, S751A, K754G, I7 57K, I757W, E771R, N787A, A825R, T826F, T826K, Y832R, Y836K, Y836Q, Y839L, F840L, Y907I, Y907V, Q916Y, E935R, I936R, K974G, K 1019V, I35L / S469K, I35L / T826F, I40Q / S469K, I40Q / T826F, S469K / T826F, S469K / K516P, Y513G / F840L, Y513G / Y832R, I35L / S469 It contains at least one variant amino acid selected from the amino acid positions of K / T826F, I40Q / S469K / T826F, S469K / K516P / T826F, S469K / T826F / F840L, D303R / S469K / T826F / F840L, F465R / S469K / T826F / F840L, D303R / F465R / S469K / T826F / F840L, D303R / S469K / K516P / T826F / F840L, or combinations thereof.

[0054] Another embodiment described herein provides a CRISPR ribonucleoprotein complex comprising a guide RNA and a CRISPR-related protein.CRISPR-related proteins, assuming that the variant ERCAS12A improves CRISPR / ERCAS12A-related nuclease activity, include Q18V, I35L, G39K, G39Q, I40Q, D56P, D56R, D59A, D59R, S158G, K165Y, S181V, A290K, D291N, V296R, P297F, P297R, P297V, D303C, D303R, E305K, Y307K, Q353R, L444W, F465R, S469K, V470K, I498K, Y507R, Y507S, Y507W, Q510K, Q510V, Q510Y, P512G, P512V, Y513G, Y513L, Y513R, Y513S, S514P, S514R, T515P, K516P, K516R, K517P, I518P, L520M, F5 22A, A541V, G553M, N556E, N556R, D562I, D562K, D562V, N583V, L585K, F653R, K712R, N716R, E731P, N732T, A744N, A744P, A744T, F748A, S751A , K754G, I757K, I757W, E771R, N787A, A825R, T826F, T826K, Y832R, Y83 6K, Y836Q, Y839L, F840L, Y907I, Y907V, Q916Y, E935R, I936R, K974G, K 1019V, I35L / S469K, I35L / T826F, I40Q / S469K, I40Q / T826F, S469K / T8 26F, S469K / K516P, Y513G / F840L, Y513G / Y832R, I35L / S469K / T826F, I4 The polypeptide comprises a variant of ERCAS12A containing at least one variant amino acid selected from the amino acid positions of 0Q / S469K / T826F, S469K / K516P / T826F, S469K / T826F / F840L, D303R / S469K / T826F / F840L, F465R / S469K / T826F / F840L, D303R / F465R / S469K / T826F / F840L, D303R / S469K / K516P / T826F / F840L, or combinations thereof.

[0055] Another embodiment described herein provides a method for increasing the efficiency of gene editing in cells using a CRISPR ribonucleoprotein complex. This method includes contacting cells with a CRISPR ribonucleoprotein complex comprising a guide RNA and a CRISPR-related protein.CRISPR-related proteins, assuming that the variant ERCAS12A improves CRISPR / ERCAS12A-related nuclease activity, include Q18V, I35L, G39K, G39Q, I40Q, D56P, D56R, D59A, D59R, S158G, K165Y, S181V, A290K, D291N, V296R, P297F, P297R, P297V, D303C, D303R, E305K, Y307K, Q353R, L444W, F465R, S469K, V470K, I498K, Y507R, Y507S, Y507W, Q510K, Q510V, Q510Y, P512G, P512V, Y513G, Y513L, Y513R, Y513S, S514P, S514R, T515P, K516P, K516R, K517P, I518P, L520M, F5 22A, A541V, G553M, N556E, N556R, D562I, D562K, D562V, N583V, L585K, F653R, K712R, N716R, E731P, N732T, A744N, A744P, A744T, F748A, S751A , K754G, I757K, I757W, E771R, N787A, A825R, T826F, T826K, Y832R, Y83 6K, Y836Q, Y839L, F840L, Y907I, Y907V, Q916Y, E935R, I936R, K974G, K 1019V, I35L / S469K, I35L / T826F, I40Q / S469K, I40Q / T826F, S469K / T8 26F, S469K / K516P, Y513G / F840L, Y513G / Y832R, I35L / S469K / T826F, I4 The polypeptide comprises a variant of ERCAS12A containing at least one variant amino acid selected from the amino acid positions of 0Q / S469K / T826F, S469K / K516P / T826F, S469K / T826F / F840L, D303R / S469K / T826F / F840L, F465R / S469K / T826F / F840L, D303R / F465R / S469K / T826F / F840L, D303R / S469K / K516P / T826F / F840L, or combinations thereof.

[0056] In another embodiment described herein, a kit is provided comprising a guide RNA and a CRISPR-related protein comprising a polypeptide encoding a variant of ERCAS12A. The variants of ERCAS12A are Q18V, I35L, G39K, G39Q, I40Q, D56P, D56R, D59A, D59R, S158G, K165Y, S181V, A290K, D291N, V296R, P297F, P297R, P297V, D303C, D303R, E305K, Y307K, Q353R, L444W, F465R, S469K, V470K, I498 K, Y507R, Y507S, Y507W, Q510K, Q510V, Q510Y, P512G, P512V, Y513G, Y513L, Y513R, Y513S, S514P, S514R, T515P, K516P, K516R, K517P, I51 8P, L520M, F522A, A541V, G553M, N556E, N556R, D562I, D562K, D562V, N583V, L585K, F653R, K712R, N716R, E731P, N732T, A744N, A744P, A7 44T, F748A, S751A, K754G, I757K, I757W, E771R, N787A, A825R, T826F, T826K, Y832R, Y836K, Y836Q, Y839L, F840L, Y907I, Y907V, Q916Y, E 935R, I936R, K974G, K1019V, I35L / S469K, I35L / T826F, I40Q / S469K, I40Q / T826F, S469K / T826F, S469K / K516P, Y513G / F840L, Y513G / Y83 It contains at least one variant amino acid selected from the amino acid positions of 2R, I35L / S469K / T826F, I40Q / S469K / T826F, S469K / K516P / T826F, S469K / T826F / F840L, D303R / S469K / T826F / F840L, F465R / S469K / T826F / F840L, D303R / F465R / S469K / T826F / F840L, D303R / S469K / K516P / T826F / F840L, or combinations thereof.

[0057] Another embodiment described herein provides nucleic acids encoding CRISPR-related proteins, including polypeptides encoding variants of ERCAS12A. The variants of ERCAS12A are, on the premise that the variant ERCAS12A improves CRISPR / ERCAS12A-related nuclease activity, Q18V, I35L, G39K, G39Q, I40Q, D56P, D56R, D59A, D59R, S158G, K165Y, S181V, A290K, D291N, V296R, P297F, P297R, P297V, D303C, D303R, E305K, Y307K, Q353R, L444W, F465R, S469K, V470K, I498 K, Y507R, Y507S, Y507W, Q510K, Q510V, Q510Y, P512G, P512V, Y513G, Y513L, Y513R, Y513S, S514P, S514R, T515P, K516P, K516R, K517P, I51 8P, L520M, F522A, A541V, G553M, N556E, N556R, D562I, D562K, D562V, N583V, L585K, F653R, K712R, N716R, E731P, N732T, A744N, A744P, A7 44T, F748A, S751A, K754G, I757K, I757W, E771R, N787A, A825R, T826F, T826K, Y832R, Y836K, Y836Q, Y839L, F840L, Y907I, Y907V, Q916Y, E 935R, I936R, K974G, K1019V, I35L / S469K, I35L / T826F, I40Q / S469K, I40Q / T826F, S469K / T826F, S469K / K516P, Y513G / F840L, Y513G / Y83 It contains at least one variant amino acid selected from the amino acid positions of 2R, I35L / S469K / T826F, I40Q / S469K / T826F, S469K / K516P / T826F, S469K / T826F / F840L, D303R / S469K / T826F / F840L, F465R / S469K / T826F / F840L, D303R / F465R / S469K / T826F / F840L, D303R / S469K / K516P / T826F / F840L, or combinations thereof.

[0058] Another embodiment described herein is a polynucleotide sequence encoding an ERCAS12A variant. The polynucleotide sequence comprises a nucleotide sequence having at least 90–99% identity with any one of the odd-numbered sequences of SEQ ID NOs: 3–209 (e.g., SEQ ID NOs: 3, 5, 7…207, or 209). In one embodiment, the polynucleotide sequence comprises any one of the odd-numbered sequences of SEQ ID NOs: 3–209 (e.g., SEQ ID NOs: 3, 5, 7…207, or 209).

[0059] 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 90–99% identity with any one of the odd-numbered sequences of SEQ ID NOs: 3–209 (e.g., SEQ ID NOs: 3, 5, 7…207, or 209). In one embodiment, the polynucleotide sequence comprises any one of the odd-numbered sequences of SEQ ID NOs: 3–209 (e.g., SEQ ID NOs: 3, 5, 7…207, or 209).

[0060] Another embodiment described herein is a CAS endonuclease system comprising an amino acid sequence encoding the ERCAS12A polypeptide. The amino acid sequence comprises a polypeptide containing an ERCAS12A variant selected from polypeptides having one or more amino acid substitutions shown in Table 1 relative to wild-type ERCAS12A (SEQ ID NO: 2), the ERCAS12A variant exhibiting improved genome editing compared to wild-type ERCAS12A (SEQ ID NO: 2).

[0061] Another embodiment described herein is a method for performing genome editing in eukaryotic cells. This method includes introducing a CAS endonuclease system into eukaryotic cells, the CAS endonuclease system comprising an expression cassette encoding a polynucleotide sequence encoding an ERCAS12A polypeptide containing an ERCAS12A variant selected from polypeptides having one or more amino acid substitutions shown in Table 1 relative to wild-type ERCAS12A (SEQ ID NO: 2), the ERCAS12A variant exhibiting improved genome editing relative to wild-type ERCAS12A (SEQ ID NO: 2).

[0062] 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, wherein the CAS endonuclease system comprises an amino acid sequence encoding an ERCAS12A polypeptide containing an ERCAS12A variant selected from polypeptides having one or more amino acid substitutions shown in Table 1 relative to wild-type ERCAS12A (SEQ ID NO: 2), the ERCAS12A variant exhibits improved genome editing relative to wild-type ERCAS12A (SEQ ID NO: 2).

[0063] Another embodiment described herein is a CRISPR-related protein comprising a fusion polypeptide. The fusion polypeptide comprises an ERCAS12A open reading frame, a nuclear localization signal, an optional amino acid linker, and an optional affinity tag.

[0064] 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 a CRISPR-related protein.

[0065] Another embodiment is a polypeptide encoded by the nucleotide sequences described herein. In one embodiment, the polypeptide has at least 85% to 99% identity with the even-numbered sequences of SEQ ID NOs: 4-210 (e.g., SEQ ID NOs: 4, 6, 8...206, or 210). In another embodiment, the polypeptide is selected from the even-numbered sequences of SEQ ID NOs: 4-210 (e.g., SEQ ID NOs: 4, 6, 8...206, or 210).

[0066] Another embodiment described herein is a process for producing one or more nucleotide sequences described herein or polypeptides encoded by such nucleotide sequences, the process comprising the steps of transforming or transfecting cells with nucleic acids containing the nucleotide sequences described herein, growing the cells, optionally isolating further amounts of the nucleotide sequences described herein, inducing the expression of polypeptides encoded by the nucleotide sequences described herein, and isolating polypeptides encoded by the nucleotides described herein.

[0067] Another embodiment described herein is a means for producing one or more nucleotide sequences described herein or polypeptides encoded by such nucleotide sequences, the process comprising the steps of transforming or transfecting cells with nucleic acids comprising the nucleotide sequences described herein, growing the cells, optionally isolating further amounts of the nucleotide sequences described herein, inducing the expression of polypeptides encoded by such nucleotide sequences, and isolating polypeptides encoded by such nucleotide sequences.

[0068] Another embodiment described herein is a nucleotide sequence or polypeptide encoded by a nucleotide sequence produced by the method or means described herein.

[0069] Another embodiment described herein is the use of an effective amount of polypeptide encoded by one or more odd-numbered sequences of SEQ ID NOs: 3-209 (e.g., 3, 5, 7...207, or 209).

[0070] Another embodiment described herein is a research tool comprising a polypeptide encoded by the nucleotide sequence described herein.

[0071] Another embodiment described herein is a drug comprising a polypeptide encoded by the nucleotide sequence described herein.

[0072] The polynucleotides described herein include variants having substitutions, deletions, and / or additions that may involve one or more nucleotides. Variants can be modified in the coding region, the non-coding region, or both. Modifications in the coding region may result in conserved or non-conserved amino acid substitutions, deletions, or additions. Of these, silent substitutions, additions, and deletions that do not alter the binding properties and activity are particularly preferred.

[0073] Further embodiments described herein include (a) nucleotide sequences encoding polypeptides having amino acid sequences in even-numbered sequences of SEQ ID NOs: 4-210 (e.g., SEQ ID NOs: 4, 6, 8...206, or 210) or degenerate, homologous, or codon-optimized variants thereof, (b) nucleotide sequences encoding polypeptides having amino acid sequences in even-numbered sequences of SEQ ID NOs: 4-210 (e.g., SEQ ID NOs: 4, 6, 8...206, or 210) or degenerate, homologous, or codon-optimized variants thereof, and (c) The present invention comprises a nucleic acid molecule containing a polynucleotide having a nucleotide sequence that can hybridize with a complement of either the nucleotide sequence of (a) or (b) above, and which can express a functional polypeptide of an even-numbered amino acid sequence of sequence numbers 4-210 (e.g., sequence numbers 4, 6, 8...206, or 210), and which is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, more preferably at least about 90-99%, or 100% identical to a nucleotide sequence.

[0074] A polynucleotide having a nucleotide sequence that is at least, for example, 90-99% "identical" to a reference nucleotide sequence encoding an odd-numbered sequence of sequence numbers 3-209 (e.g., 3, 5, 7...207, or 209) is intended to have a nucleotide sequence identical to the reference sequence, except that the polynucleotide sequence may contain approximately 10-1 point mutations, additions, or deletions per 100 nucleotides of the reference nucleotide sequence encoding an odd-numbered sequence of sequence numbers 3-209 (e.g., 3, 5, 7...207, or 209).

[0075] In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 90-99% identical to a reference nucleotide sequence, up to 10% of the nucleotides in the reference sequence may be deleted, added, or substituted with other nucleotides, or up to 10% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations in the reference sequence may occur at the 5'- or 3'-terminal positions of the reference nucleotide sequence, or individually between nucleotides in the reference sequence, or scattered within one or more consecutive groups in the reference sequence, at any position between these terminal positions. This also applies to polypeptide sequences that are at least approximately 90-99% identical to a reference polypeptide sequence.

[0076] As described 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 of two sequences is generally described by dividing the number of exact matches between the two aligned sequences by the length of the shorter sequence and multiplying by 100, whether they are nucleic acid or peptide sequences. 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 was developed by Dayhoff, Atlas of Protein Sequences and Structure, MO Dayhoff ed., 5 suppl. 3: 353-358, National Biomedical Research Foundation, Washington, DC, USA, and can be extended to peptide sequences by using a scoring matrix normalized by Gribskov, Nucl. Acids Res. 14(6): 6745-6763, (1986).

[0077] For example, due to the degeneracy of genetic coding, a person skilled in the art will recognize that a number of nucleic acid molecules having sequences that are at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the odd-numbered sequences of SEQ ID NOs. 3-209 (e.g., SEQ ID NOs. 4, 6, 8-206, or 210) or their degenerate, homologous, or codon-optimized variants, can code for even-numbered sequences of SEQ ID NOs.

[0078] The polynucleotides described herein include polynucleotides encoding mutations, alterations, substitutions, additions, deletions, and specific examples of the polypeptides described herein. For example, guidance on methods for producing phenotypic silent amino acid substitutions is provided in Bowie, JU et al., "Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions," Science 247: 1306-1310 (1990), where the authors demonstrate the surprising tolerance of proteins to amino acid substitutions.

[0079] That is, polypeptide fragments, derivatives, or analogs of even-numbered sequences of SEQ ID NOs: 4-210 (e.g., SEQ ID NOs: 4, 6, 8...206, or 210) may (i) have 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 or more residues) substituted with conserved or unconserved amino acid residues (preferably conserved amino acid residues). Such substituted amino acid residues may be those encoded by a gene code, or (ii) one or more amino acid residues containing substituents (e.g., 1, 2, 3, 4, 5, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 or more residues), or (iii) a mature polypeptide fused with another polypeptide or compound, such as a compound that extends the half-life of a polypeptide (e.g., polyethylene glycol), or (iv) an additional amino acid fused with a mature polypeptide, such as an IgG Fc fusion region polypeptide or a sequence employed for the purification of a leader or secretion sequence or a mature polypeptide or proprotein sequence, or otherwise. Such fragments, derivatives, and analogs are considered to be within the scope of the art based on the teachings herein.

[0080] Furthermore, polypeptide fragments, derivatives, or analogs of even-numbered sequences of SEQ ID NOs. 4-210 (e.g., SEQ ID NOs. 4, 6, 8...206, or 210) may be substituted with one or more conserved or unconserved amino acid residues (preferably conserved amino acid residues). In some cases, these polypeptides, their fragments, derivatives, or analogs will have polypeptide sequences that are at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the polypeptide sequences represented by even-numbered sequences of SEQ ID NOs. 4-210 (e.g., SEQ ID NOs. 4, 6, 8...206, or 210), and will include functional or non-functional proteins or enzymes. Similarly, additions or deletions to polypeptides may occur at the N- or C-terminus of the polypeptide or within non-conserved regions (these regions are not conserved as pictured and are therefore considered unimportant).

[0081] As described herein, in many cases amino acid substitutions, mutations, additions, or deletions are minor, preferably such as conservative amino acid substitutions that do not significantly affect protein folding or activity or addition or deletion to the N- or C-terminus. Of course, the number of amino acid substitutions, additions, or deletions performed by those skilled in the art depends on many factors, including those described herein. Generally, the number of substitutions, additions, or deletions for any given polypeptide does 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.

[0082] Another embodiment described herein is a polynucleotide vector comprising one or more nucleotide sequences described herein.

[0083] Another embodiment described herein is a cell comprising one or more nucleotide sequences or polynucleotide vectors described herein.

[0084] It will be apparent to those skilled in the art that suitable modifications and adaptations can be made to the compositions, formulations, methods, processes, apparatus, assemblies, and applications described herein without departing from the scope of any embodiment or aspect thereof. The compositions, apparatus, assemblies, and methods provided are illustrative and are not intended to limit the scope of any of the disclosed embodiments. All of the various embodiments, aspects, and options disclosed herein can be combined in any variation or iteration. The scope of the compositions, formulations, methods, apparatus, assemblies, and processes described herein includes all actual or possible combinations of the embodiments, aspects, options, examples, and preferred examples described herein. The compositions, formulations, apparatus, assemblies, or methods described herein may exclude any component or step, substitute any component or step disclosed herein, or include any component or step disclosed elsewhere herein. The ratio of the mass of any component of any of the compositions or formulations disclosed herein to the mass of any other component in the formulation or the total mass of the other components in the formulation is thus disclosed as if they were expressly disclosed. If the meaning of any term in any patent or publication incorporated by reference conflicts with the meaning of any term used in this disclosure, the meaning of the term or phrase in this disclosure shall prevail. All patents and publications referenced herein are incorporated herein by reference for their specific teachings.

[0085] The various embodiments and aspects of the present invention described herein are summarized in the following sections. Section 1. Isolated polypeptide comprising a Proctobacter rectum Cas12a (ERCAS12A) mutant having one or more amino acid substitutions introduced into the wild-type ERCAS12A polypeptide sequence of Sequence ID No. 2, wherein the ERCAS12A mutant polypeptide provides improved nuclease activity compared to the wild-type ERCAS12A polypeptide. Section 2. The isolated polypeptide described in Section 1, wherein the ERCAS12A mutant has 98% to 99% identity with polypeptide sequences selected from even-numbered sequences of SEQ ID NOs. 4 to 210. Section 3. The isolated polypeptide described in Section 1 or 2, wherein the ERCAS12A mutant has a polypeptide sequence selected from even-numbered sequences of SEQ ID NOs. 4 to 210. Section 4. The ERCAS12A mutant has the following characteristics in the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 18, 35, 39, 39, 40, 56, 56, 59, 59, 158, 165, 181, 290, 291, 296, 297, 297, 297, 303, 303, 305, 307, 353, 444, 465, 469, 470, 498, 507, 507, 507, 510, 510, 510, 512, 512, 513, 513, 513, 513, 514, 514, 515, 516, 516, 517, 5 An isolated polypeptide as described in any one of sections 1 to 3, comprising one or more amino acid substitutions at positions 18, 520, 522, 541, 553, 556, 556, 562, 562, 583, 585, 653, 712, 716, 731, 732, 744, 744, 744, 748, 751, 754, 757, 757, 771, 787, 825, 826, 826, 832, 836, 836, 839, 840, 907, 907, 916, 935, 936, 974, or 1019. Section 5. ERCAS12A mutants have the following variants of the wild-type ERCAS12A polypeptide sequence in SEQ ID NO: Q18V, I35L, G39K, G39Q, I40Q, D56P, D56R, D59A, D59R, S158G, K165Y, S181V, A290K, D291N, V296R, P297F, P297R, P297V, D303C, D303R, E305K , Y307K, Q353R, L444W, F465R, S469K, V470K, I498K, Y507R, Y507S, Y507W, Q510K, Q510V, Q510 Y, P512G, P512V, Y513G, Y513L, Y513R, Y513S, S514P, S514R, T515P, K516P, K516R, K517P, I518 P, L520M, F522A, A541V, G553M, N556E, N556R, D562I, D562K, D562V, N583V, L585K, F653R, K71 2R, N716R, E731P, N732T, A744N, A744P, A744T, F748A, S751A, K754G, I757K, I757W, E771R, N78 An isolated polypeptide as described in any one of sections 1 to 4, comprising one or more amino acid substitutions selected from 7A, A825R, T826F, T826K, Y832R, Y836K, Y836Q, Y839L, F840L, Y907I, Y907V, Q916Y, E935R, I936R, K974G, K1019V, or any combination thereof. Section 6. ERCAS12A mutants include Q18V (sequence number 4); I35L (sequence number 6); G39Q (sequence number 8); G39K (sequence number 10); I40Q (sequence number 12); D56R (sequence number 14); D56P (sequence number 16); D59R (sequence number 18); D59A (sequence number 20); S158G (sequence number 22); K165Y (sequence number 24); S181V (sequence number 26); A290K (sequence number 28); D291N (sequence number 30); V296R (sequence number 32); P297V (sequence number 34); P297R (sequence number 36); P297F (sequence number 32) Number 38); D303R (Sequence ID 40); D303C (Sequence ID 42); E305K (Sequence ID 44); Y307K (Sequence ID 46); Q353R (Sequence ID 48); L444W (Sequence ID 50); F465R (Sequence ID 52); S469K (Sequence ID 54); V470K (Sequence ID 56); I498K (Sequence ID 58); Y507W (Sequence ID 60); Y507S (Sequence ID 62); Y507R (Sequence ID 64); Q510Y (Sequence ID 66); Q510V (Sequence ID 68); Q510K (Sequence ID 70); P512V (Sequence ID 72); P51 2G(Sequence ID 74);Y513S(Sequence ID 76);Y513R(Sequence ID 78);Y513L(Sequence ID 80);Y513G(Sequence ID 82);S514R(Sequence ID 84);S514P(Sequence ID 86);T515P(Sequence ID 88);K516R(Sequence ID 90);K516P(Sequence ID 92);K517P(Sequence ID 94);I518P(Sequence ID 96);L520M(Sequence ID 98);F522A(Sequence ID 100);A541V(Sequence ID 102);G553M(Sequence ID 104);N556R(Sequence ID 106);N556E(Array Number 108); D562V (Sequence ID 110); D562K (Sequence ID 112); D562I (Sequence ID 114); N583V (Sequence ID 116); L585K (Sequence ID 118); F653R (Sequence ID 120); K712R (Sequence ID 122); N716R (Sequence ID 124); E731P (Sequence ID 126); N732T (Sequence ID 128); A744T (Sequence ID 130); A744P (Sequence ID 132); A744N (Sequence ID 134); F748A (Sequence ID 136); S751A (Sequence ID 138); K754G (Sequence ID 140);I757W (sequence number 142); I757K (sequence number 144); E771R (sequence number 146); N787A (sequence number 148); A825R (sequence number 150); T826K (sequence number 152); T826F (sequence number 154); Y832R (sequence number 156); Y836Q (sequence number 158); Y836K (sequence number 160); Y839L (sequence number 162); F840L (sequence number 164); Y907 V(sequence code 166);Y907I(sequence code 168);Q916Y(sequence code 170);E935R(sequence code 172);I936R(sequence code 174);K974G(sequence code 176);K1019V(sequence code 178);I35L / S469K(sequence code 180);I35L / T826F(sequence code 182);I40Q / S469K(sequence code 184);I40Q / T826F(sequence code 186);S4 69K / K516P (Sequence ID 188); S469K / T826F (Sequence ID 190); Y513G / Y832R (Sequence ID 192); Y513G / F840L (Sequence ID 194); I35L / S469K / T826F (Sequence ID 196); I40Q / S469K / T826F (Sequence ID 198); S469K / K516P / T826F (Sequence ID 200); S469K / T826F / F840L (Sequence ID 2 An isolated polypeptide as described in any one of sections 1 to 5, selected from: 02); D303R / S469K / T826F / F840L (SEQ ID NO: 204); F465R / S469K / T826F / F840L (SEQ ID NO: 206); D303R / F465R / S469K / T826F / F840L (SEQ ID NO: 208); or D303R / S469K / K516P / T826F / F840L (SEQ ID NO: 210). Section 7. An isolated polypeptide as described in any one of Sections 1 to 6, wherein the ERCAS12A mutant is selected from D303R / S469K / T826F / F840L (SEQ ID NO: 204); F465R / S469K / T826F / F840L (SEQ ID NO: 206); D303R / F465R / S469K / T826F / F840L (SEQ ID NO: 208); or xD303R / S469K / K516P / T826F / F840L (SEQ ID NO: 210). Section 8. Isolated polynucleotide sequences encoding a *Cas12a* (ERCAS12A) mutant of *Erythritis rectum* 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. Section 9. The isolated polynucleotide sequence described in Section 8, wherein the encoded ERCAS12A variant has 98%–99% identity with the polypeptide sequence selected from the even-numbered sequences of SEQ ID NOs. 4–210. Section 10. An isolated polynucleotide sequence as described in Section 8 or 9, wherein the encoded ERCAS12A variant has a polypeptide sequence selected from even-numbered sequences of SEQ ID NOs. 4 to 210. Section 11. An isolated polynucleotide sequence described in any one of Sections 8 to 10, wherein the ERCAS12A mutant polynucleotide sequence has 95% to 99% identity with a nucleotide sequence selected from odd-numbered sequences of SEQ ID NOs. 3 to 209. Section 12. An isolated polynucleotide sequence described in any one of Sections 8 to 11, wherein the ERCAS12A mutant polynucleotide has a nucleotide sequence selected from the odd-numbered sequences of SEQ ID NOs. 3 to 209. Section 13. The encoded ERCAS12A mutant has the following characteristics in the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 18, 35, 39, 39, 40, 56, 56, 59, 59, 158, 165, 181, 290, 291, 296, 297, 297, 297, 303, 303, 305, 307, 353, 444, 465, 469, 470, 498, 507, 507, 507, 510, 510, 510, 512, 512, 513, 513, 513, 513, 514, 514, 515, 516, 516, 517, 5 An isolated polynucleotide sequence as described in any one of sections 8 to 12, comprising one or more amino acid substitutions at positions 18, 520, 522, 541, 553, 556, 556, 562, 562, 583, 585, 653, 712, 716, 731, 732, 744, 744, 744, 748, 751, 754, 757, 757, 771, 787, 825, 826, 826, 832, 836, 836, 839, 840, 907, 907, 916, 935, 936, 974, or 1019. Section 14. The encoded ERCAS12A mutants are Q18V, I35L, G39K, G39Q, I40Q, D56P, D56R, D59A, D59R, S158G, K165Y, S181V, A290K, D291N, V296R, P297F, P297R, P297V, D303C, D303R, E 305K, Y307K, Q353R, L444W, F465R, S469K, V470K, I498K, Y507R, Y507S, Y507W, Q510K, Q510V, Q5 10Y, P512G, P512V, Y513G, Y513L, Y513R, Y513S, S514P, S514R, T515P, K516P, K516R, K517P, I51 8P, L520M, F522A, A541V, G553M, N556E, N556R, D562I, D562K, D562V, N583V, L585K, F653R, K712 R, N716R, E731P, N732T, A744N, A744P, A744T, F748A, S751A, K754G, I757K, I757W, E771R, N787A Isolated polynucleotide sequences as described in any one of sections 8 to 13, comprising one or more amino acid substitutions selected from A825R, T826F, T826K, Y832R, Y836K, Y836Q, Y839L, F840L, Y907I, Y907V, Q916Y, E935R, I936R, K974G, K1019V, or combinations thereof. Section 15. The encoded ERCAS12A mutant polynucleotides are Q18V (sequence number 4); I35L (sequence number 6); G39Q (sequence number 8); G39K (sequence number 10); I40Q (sequence number 12); D56R (sequence number 14); D56P (sequence number 16); D59R (sequence number 18); D59A (sequence number 20); S158G (sequence number 22); K165Y (sequence number 24); S181V (sequence number 26); A290K (sequence number 28); D291N (sequence number 30); V296R (sequence number 32); P297V (sequence number 34); P297R (distributed Column number 36); P297F (Sequence number 38); D303R (Sequence number 40); D303C (Sequence number 42); E305K (Sequence number 44); Y307K (Sequence number 46); Q353R (Sequence number 48); L444W (Sequence number 50); F465R (Sequence number 52); S469K (Sequence number 54); V470K (Sequence number 56); I498K (Sequence number 58); Y507W (Sequence number 60); Y507S (Sequence number 62); Y507R (Sequence number 64); Q510Y (Sequence number 66); Q510V (Sequence number 68); Q510K (Sequence number 70); P5 12V (Sequence ID 72); P512G (Sequence ID 74); Y513S (Sequence ID 76); Y513R (Sequence ID 78); Y513L (Sequence ID 80); Y513G (Sequence ID 82); S514R (Sequence ID 84); S514P (Sequence ID 86); T515P (Sequence ID 88); K516R (Sequence ID 90); K516P (Sequence ID 92); K517P (Sequence ID 94); I518P (Sequence ID 96); L520M (Sequence ID 98); F522A (Sequence ID 100); A541V (Sequence ID 102); G553M (Sequence ID 104); N556R (Array Number 106); N556E (Sequence ID 108); D562V (Sequence ID 110); D562K (Sequence ID 112); D562I (Sequence ID 114); N583V (Sequence ID 116); L585K (Sequence ID 118); F653R (Sequence ID 120); K712R (Sequence ID 122); N716R (Sequence ID 124); E731P (Sequence ID 126); N732T (Sequence ID 128); A744T (Sequence ID 130); A744P (Sequence ID 132); A744N (Sequence ID 134); F748A (Sequence ID 136); S751A (Sequence ID 138);K754G (Sequence No. 140); I757W (Sequence No. 142); I757K (Sequence No. 144); E771R (Sequence No. 146); N787A (Sequence No. 148); A825R (Sequence No. 150); T826K (Sequence No. 152); T826F (Sequence No. 154); Y832R (Sequence No. 156); Y836Q (Sequence No. 158); Y836K (Sequence No. 160); Y839L (Sequence No. 162); F840L (Sequence No. (Sequence number 164); Y907V (Sequence number 166); Y907I (Sequence number 168); Q916Y (Sequence number 170); E935R (Sequence number 172); I936R (Sequence number 174); K974G (Sequence number 176); K1019V (Sequence number 178); I35L / S469K (Sequence number 180); I35L / T826F (Sequence number 182); I40Q / S469K (Sequence number 184); I40Q / T826F (Sequence number 18 6);S469K / K516P(Sequence ID 188);S469K / T826F(Sequence ID 190);Y513G / Y832R(Sequence ID 192);Y513G / F840L(Sequence ID 194);I35L / S469K / T826F(Sequence ID 196);I40Q / S469K / T826F(Sequence ID 198);S469K / K516P / T826F(Sequence ID 200);S469K / T826F / F840L(Sequence ID 2 An isolated polynucleotide sequence as described in any one of sections 8 to 14, selected from: 02); D303R / S469K / T826F / F840L (SEQ ID NO: 204); F465R / S469K / T826F / F840L (SEQ ID NO: 206); D303R / F465R / S469K / T826F / F840L (SEQ ID NO: 208); or D303R / S469K / K516P / T826F / F840L (SEQ ID NO: 210). Section 16. ERCAS12A mutant polynucleotides include Q18V (sequence number 3); I35L (sequence number 5); G39Q (sequence number 7); G39K (sequence number 9); I40Q (sequence number 11); D56R (sequence number 13); D56P (sequence number 15); D59R (sequence number 17); D59A (sequence number 19); S158G (sequence number 21); K165Y (sequence number 23); S181V (sequence number 25); A290K (sequence number 27); D291N (sequence number 29); V296R (sequence number 31); P297V (sequence number 33); P297R (sequence number 35); P2 97F(sequence number 37);D303R(sequence number 39);D303C(sequence number 41);E305K(sequence number 43);Y307K(sequence number 45);Q353R(sequence number 47);L444W(sequence number 49);F465R(sequence number 51);S469K(sequence number 53);V470K(sequence number 55);I498K(sequence number 57);Y507W(sequence number 59);Y507S(sequence number 61);Y507R(sequence number 63);Q510Y(sequence number 65);Q510V(sequence number 67);Q510K(sequence number 69);P512V(sequence number 71) ;P512G(sequence number 73);Y513S(sequence number 75);Y513R(sequence number 77);Y513L(sequence number 79);Y513G(sequence number 81);S514R(sequence number 83);S514P(sequence number 85);T515P(sequence number 87);K516R(sequence number 89);K516P(sequence number 91);K517P(sequence number 93);I518P(sequence number 95);L520M(sequence number 97);F522A(sequence number 99);A541V(sequence number 101);G553M(sequence number 103);N556R(sequence number 105);N556E( SEQ ID NO: 107); D562V (SEQ ID NO: 109); D562K (SEQ ID NO: 111); D562I (SEQ ID NO: 113); N583V (SEQ ID NO: 115); L585K (SEQ ID NO: 117); F653R (SEQ ID NO: 119); K712R (SEQ ID NO: 121); N716R (SEQ ID NO: 123); E731P (SEQ ID NO: 125); N732T (SEQ ID NO: 127); A744T (SEQ ID NO: 129); A744P (SEQ ID NO: 131); A744N (SEQ ID NO: 132); F748A (SEQ ID NO: 135); S751A (SEQ ID NO: 137); K754G (SEQ ID NO: 139);I757W (sequence number 141); I757K (sequence number 143); E771R (sequence number 145); N787A (sequence number 147); A825R (sequence number 149); T826K (sequence number 151); T826F (sequence number 153); Y832R (sequence number 155); Y836Q (sequence number 157); Y836K (sequence number 159); Y839L (sequence number 161); F840L (sequence number 163); Y907V (Sequence code 165);Y907I(Sequence code 167);Q916Y(Sequence code 169);E935R(Sequence code 171);I936R(Sequence code 173);K974G(Sequence code 175);K1019V(Sequence code 177);I35L / S469K(Sequence code 179);I35L / T826F(Sequence code 181);I40Q / S469K(Sequence code 183);I40Q / T826F(Sequence code 185);S469K / K516P(sequence number 187);S469K / T826F(sequence number 189);Y513G / Y832R(sequence number 191);Y513G / F840L(sequence number 193);I35L / S469K / T826F(sequence number 195);I40Q / S469K / T826F(sequence number 197);S469K / K516P / T826F(sequence number 199);S469K / T826F / F840L(sequence number 201); An isolated polynucleotide sequence as described in any one of sections 8 to 15, selected from D303R / S469K / T826F / F840L (SEQ ID NO: 203); F465R / S469K / T826F / F840L (SEQ ID NO: 205); D303R / F465R / S469K / T826F / F840L (SEQ ID NO: 207); or D303R / S469K / K516P / T826F / F840L (SEQ ID NO: 209). Section 17. An isolated polynucleotide sequence described in any one of Sections 8 to 16, wherein the ERCAS12A mutant polynucleotide is selected from D303R / S469K / T826F / F840L (SEQ ID NO: 203); F465R / S469K / T826F / F840L (SEQ ID NO: 205); D303R / F465R / S469K / T826F / F840L (SEQ ID NO: 207); or D303R / S469K / K516P / T826F / F840L (SEQ ID NO: 209). Section 18. A vector or plasmid containing a polynucleotide sequence as described in any one of Sections 8 to 17. Section 19. A cell containing nucleic acid comprising one or more polynucleotide sequences described in any one of Sections 8 to 18. Section 20. An isolated CRISPR ribonucleoprotein 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. Section 21. The isolated CRISPR ribonucleoprotein complex described in Section 20, wherein the ERCAS12A mutant has a polypeptide sequence selected from even-numbered sequences of SEQ ID NOs. 4–210. Section 22. The ERCAS12A mutant has the following characteristics in the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 18, 35, 39, 39, 40, 56, 56, 59, 59, 158, 165, 181, 290, 291, 296, 297, 297, 297, 303, 303, 305, 307, 353, 444, 465, 469, 470, 498, 507, 507, 507, 510, 510, 510, 512, 512, 513, 513, 513, 513, 514, 514, 515, 516, 516, 517, 518, 52 An isolated CRISPR ribonucleoprotein complex as described in Section 20 or 21, comprising one or more amino acid substitutions at positions 0, 522, 541, 553, 556, 556, 562, 562, 583, 585, 653, 712, 716, 731, 732, 744, 744, 744, 748, 751, 754, 757, 757, 771, 787, 825, 826, 826, 832, 836, 836, 839, 840, 907, 907, 916, 935, 936, 974, or 1019. Section 23. ERCAS12A mutants have the following variants of the wild-type ERCAS12A polypeptide sequence in SEQ ID NO: Q18V, I35L, G39K, G39Q, I40Q, D56P, D56R, D59A, D59R, S158G, K165Y, S181V, A290K, D291N, V296R, P297F, P297R, P297V, D303C, D303R, E305K, Y30 7K, Q353R, L444W, F465R, S469K, V470K, I498K, Y507R, Y507S, Y507W, Q510K, Q510V, Q510Y, P512 G, P512V, Y513G, Y513L, Y513R, Y513S, S514P, S514R, T515P, K516P, K516R, K517P, I518P, L520M, F522A, A541V, G553M, N556E, N556R, D562I, D562K, D562V, N583V, L585K, F653R, K712R, N716R, E 731P, N732T, A744N, A744P, A744T, F748A, S751A, K754G, I757K, I757W, E771R, N787A, A825R, T82 An isolated CRISPR ribonucleoprotein complex as described in any one of sections 20 to 22, comprising one or more amino acid substitutions selected from 6F, T826K, Y832R, Y836K, Y836Q, Y839L, F840L, Y907I, Y907V, Q916Y, E935R, I936R, K974G, K1019V, or combinations thereof. Section 24. ERCAS12A mutants include Q18V (sequence number 4); I35L (sequence number 6); G39Q (sequence number 8); G39K (sequence number 10); I40Q (sequence number 12); D56R (sequence number 14); D56P (sequence number 16); D59R (sequence number 18); D59A (sequence number 20); S158G (sequence number 22); K165Y (sequence number 24); S181V (sequence number 26); A290K (sequence number 28); D291N (sequence number 30); V296R (sequence number 32); P297V (sequence number 34); P297R (sequence number 36); P297F (sequence number 32) Number 38); D303R (Sequence ID 40); D303C (Sequence ID 42); E305K (Sequence ID 44); Y307K (Sequence ID 46); Q353R (Sequence ID 48); L444W (Sequence ID 50); F465R (Sequence ID 52); S469K (Sequence ID 54); V470K (Sequence ID 56); I498K (Sequence ID 58); Y507W (Sequence ID 60); Y507S (Sequence ID 62); Y507R (Sequence ID 64); Q510Y (Sequence ID 66); Q510V (Sequence ID 68); Q510K (Sequence ID 70); P512V (Sequence ID 72); P51 2G(Sequence ID 74);Y513S(Sequence ID 76);Y513R(Sequence ID 78);Y513L(Sequence ID 80);Y513G(Sequence ID 82);S514R(Sequence ID 84);S514P(Sequence ID 86);T515P(Sequence ID 88);K516R(Sequence ID 90);K516P(Sequence ID 92);K517P(Sequence ID 94);I518P(Sequence ID 96);L520M(Sequence ID 98);F522A(Sequence ID 100);A541V(Sequence ID 102);G553M(Sequence ID 104);N556R(Sequence ID 106);N556E(Array Number 108); D562V (Sequence ID 110); D562K (Sequence ID 112); D562I (Sequence ID 114); N583V (Sequence ID 116); L585K (Sequence ID 118); F653R (Sequence ID 120); K712R (Sequence ID 122); N716R (Sequence ID 124); E731P (Sequence ID 126); N732T (Sequence ID 128); A744T (Sequence ID 130); A744P (Sequence ID 132); A744N (Sequence ID 134); F748A (Sequence ID 136); S751A (Sequence ID 138); K754G (Sequence ID 140);I757W (sequence number 142); I757K (sequence number 144); E771R (sequence number 146); N787A (sequence number 148); A825R (sequence number 150); T826K (sequence number 152); T826F (sequence number 154); Y832R (sequence number 156); Y836Q (sequence number 158); Y836K (sequence number 160); Y839L (sequence number 162); F840L (sequence number 164); Y907V (array Number 166); Y907I (Sequence ID 168); Q916Y (Sequence ID 170); E935R (Sequence ID 172); I936R (Sequence ID 174); K974G (Sequence ID 176); K1019V (Sequence ID 178); I35L / S469K (Sequence ID 180); I35L / T826F (Sequence ID 182); I40Q / S469K (Sequence ID 184); I40Q / T826F (Sequence ID 186); S469K / K516 P(sequence code 188);S469K / T826F(sequence code 190);Y513G / Y832R(sequence code 192);Y513G / F840L(sequence code 194);I35L / S469K / T826F(sequence code 196);I40Q / S469K / T826F(sequence code 198);S469K / K516P / T826F(sequence code 200);S469K / T826F / F840L(sequence code 202);D303R / S4 An isolated CRISPR ribonucleoprotein complex as described in any one of sections 20 to 23, selected from 69K / T826F / F840L (SEQ ID NO: 204); F465R / S469K / T826F / F840L (SEQ ID NO: 206); D303R / F465R / S469K / T826F / F840L (SEQ ID NO: 208); or D303R / S469K / K516P / T826F / F840L (SEQ ID NO: 210). Section 25. An isolated CRISPR ribonucleoprotein complex as described in any one of sections 20 to 24, wherein the ERCAS12A mutant is selected from D303R / S469K / T826F / F840L (SEQ ID NO: 204); F465R / S469K / T826F / F840L (SEQ ID NO: 206); D303R / F465R / S469K / T826F / F840L (SEQ ID NO: 208); or D303R / S469K / K516P / T826F / F840L (SEQ ID NO: 210). Section 26. An isolated CRISPR / Cpf1 endonuclease system comprising an ERCAS12A mutant polypeptide having one or more amino acid substitutions introduced into the wild-type ERCAS12A polypeptide sequence of crRNA and SEQ ID NO: 2, wherein the ERCAS12A mutant polypeptide provides improved genome editing compared to the wild-type ERCAS12A polypeptide. Section 27. The isolated CRISPR / Cpf1 endonuclease system described in Section 26, wherein the ERCAS12A mutant has a polypeptide sequence selected from even-numbered sequences of SEQ ID NOs. 4-210. Section 28. The ERCAS12A mutant has the following sequences in the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 18, 35, 39, 39, 40, 56, 56, 59, 59, 158, 165, 181, 290, 291, 296, 297, 297, 297, 303, 303, 305, 307, 353, 444, 465, 469, 470, 498, 507, 507, 507, 510, 510, 510, 512, 512, 513, 513, 513, 513, 514, 514, 515, 516, 516, 517, 518, 520, An isolated CRISPR / Cpf1 endonuclease system as described in Section 26 or 27, comprising one or more amino acid substitutions at positions 522, 541, 553, 556, 556, 562, 562, 583, 585, 653, 712, 716, 731, 732, 744, 744, 744, 748, 751, 754, 757, 757, 771, 787, 825, 826, 826, 832, 836, 836, 839, 840, 907, 907, 916, 935, 936, 974, or 1019. Section 29. The ERCAS12A mutants have the following variants of the wild-type ERCAS12A polypeptide sequence in SEQ ID NO: Q18V, I35L, G39K, G39Q, I40Q, D56P, D56R, D59A, D59R, S158G, K165Y, S181V, A290K, D291N, V296R, P297F, P297R, P297V, D303C, D303R, E305K, Y307 K, Q353R, L444W, F465R, S469K, V470K, I498K, Y507R, Y507S, Y507W, Q510K, Q510V, Q510Y, P512G, P512V, Y513G, Y513L, Y513R, Y513S, S514P, S514R, T515P, K516P, K516R, K517P, I518P, L520M, F5 22A, A541V, G553M, N556E, N556R, D562I, D562K, D562V, N583V, L585K, F653R, K712R, N716R, E731 P, N732T, A744N, A744P, A744T, F748A, S751A, K754G, I757K, I757W, E771R, N787A, A825R, T826F, An isolated CRISPR / Cpf1 endonuclease system as described in any one of sections 26 to 28, comprising one or more amino acid substitutions selected from T826K, Y832R, Y836K, Y836Q, Y839L, F840L, Y907I, Y907V, Q916Y, E935R, I936R, K974G, K1019V, or combinations thereof. Section 30. ERCAS12A mutants include Q18V (sequence number 4); I35L (sequence number 6); G39Q (sequence number 8); G39K (sequence number 10); I40Q (sequence number 12); D56R (sequence number 14); D56P (sequence number 16); D59R (sequence number 18); D59A (sequence number 20); S158G (sequence number 22); K165Y (sequence number 24); S181V (sequence number 26); A290K (sequence number 28); D291N (sequence number 30); V296R (sequence number 32); P297V (sequence number 34); P297R (sequence number 36); P297F (sequence number 32) Number 38); D303R (Sequence ID 40); D303C (Sequence ID 42); E305K (Sequence ID 44); Y307K (Sequence ID 46); Q353R (Sequence ID 48); L444W (Sequence ID 50); F465R (Sequence ID 52); S469K (Sequence ID 54); V470K (Sequence ID 56); I498K (Sequence ID 58); Y507W (Sequence ID 60); Y507S (Sequence ID 62); Y507R (Sequence ID 64); Q510Y (Sequence ID 66); Q510V (Sequence ID 68); Q510K (Sequence ID 70); P512V (Sequence ID 72); P51 2G(Sequence ID 74);Y513S(Sequence ID 76);Y513R(Sequence ID 78);Y513L(Sequence ID 80);Y513G(Sequence ID 82);S514R(Sequence ID 84);S514P(Sequence ID 86);T515P(Sequence ID 88);K516R(Sequence ID 90);K516P(Sequence ID 92);K517P(Sequence ID 94);I518P(Sequence ID 96);L520M(Sequence ID 98);F522A(Sequence ID 100);A541V(Sequence ID 102);G553M(Sequence ID 104);N556R(Sequence ID 106);N556E(Array Number 108); D562V (Sequence ID 110); D562K (Sequence ID 112); D562I (Sequence ID 114); N583V (Sequence ID 116); L585K (Sequence ID 118); F653R (Sequence ID 120); K712R (Sequence ID 122); N716R (Sequence ID 124); E731P (Sequence ID 126); N732T (Sequence ID 128); A744T (Sequence ID 130); A744P (Sequence ID 132); A744N (Sequence ID 134); F748A (Sequence ID 136); S751A (Sequence ID 138); K754G (Sequence ID 140);I757W (Sequence number 142); I757K (Sequence number 144); E771R (Sequence number 146); N787A (Sequence number 148); A825R (Sequence number 150); T826K (Sequence number 152); T826F (Sequence number 154); Y832R (Sequence number 156); Y836Q (Sequence number 158); Y836K (Sequence number 160); Y839L (Sequence number 162); F840L (Sequence number 164); Y907V (Sequence number No. 166); Y907I (Sequence ID 168); Q916Y (Sequence ID 170); E935R (Sequence ID 172); I936R (Sequence ID 174); K974G (Sequence ID 176); K1019V (Sequence ID 178); I35L / S469K (Sequence ID 180); I35L / T826F (Sequence ID 182); I40Q / S469K (Sequence ID 184); I40Q / T826F (Sequence ID 186); S469K / K516P ( S469K / T826F(Sequence ID 190);Y513G / Y832R(Sequence ID 192);Y513G / F840L(Sequence ID 194);I35L / S469K / T826F(Sequence ID 196);I40Q / S469K / T826F(Sequence ID 198);S469K / K516P / T826F(Sequence ID 200);S469K / T826F / F840L(Sequence ID 202);D303R / S469K An isolated CRISPR / Cpf1 endonuclease system as described in any one of sections 26 to 29, selected from / T826F / F840L (SEQ ID NO: 204); F465R / S469K / T826F / F840L (SEQ ID NO: 206); D303R / F465R / S469K / T826F / F840L (SEQ ID NO: 208); or D303R / S469K / K516P / T826F / F840L (SEQ ID NO: 210). Section 31. An isolated CRISPR / Cpf1 endonuclease system as described in any one of sections 26 to 30, wherein the ERCAS12A mutant is selected from D303R / S469K / T826F / F840L (SEQ ID NO: 204); F465R / S469K / T826F / F840L (SEQ ID NO: 206); D303R / F465R / S469K / T826F / F840L (SEQ ID NO: 208); or D303R / S469K / K516P / T826F / F840L (SEQ ID NO: 210). Section 32. A method for performing gene editing, comprising the step of contacting a candidate editing target site locus with a CRISPR ribonucleoprotein complex 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 provides improved gene editing compared to the wild-type ERCAS12A polypeptide. Section 33. The method according to Section 32, wherein the ERCAS12A mutant has a polypeptide sequence selected from even-numbered sequences of SEQ ID NOs. 4 to 210. Section 34. The ERCAS12A mutant has the following characteristics in the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 18, 35, 39, 39, 40, 56, 56, 59, 59, 158, 165, 181, 290, 291, 296, 297, 297, 297, 303, 303, 305, 307, 353, 444, 465, 469, 470, 498, 507, 507, 507, 510, 510, 510, 512, 512, 513, 513, 513, 513, 514, 514, 515, 516, 51 The method according to Section 32 or 33, comprising one or more amino acid substitutions at positions 6, 517, 518, 520, 522, 541, 553, 556, 556, 562, 562, 562, 583, 585, 653, 712, 716, 731, 732, 744, 744, 744, 748, 751, 754, 757, 757, 771, 787, 825, 826, 826, 832, 836, 836, 839, 840, 907, 907, 916, 935, 936, 974, or 1019. Section 35. ERCAS12A mutants have the following variants of the wild-type ERCAS12A polypeptide sequence in SEQ ID NO: Q18V, I35L, G39K, G39Q, I40Q, D56P, D56R, D59A, D59R, S158G, K165Y, S181V, A290K, D291N, V296R, P297F, P297R, P297V, D303C, D303R, E30 5K, Y307K, Q353R, L444W, F465R, S469K, V470K, I498K, Y507R, Y507S, Y507W, Q510K, Q510V, Q 510Y, P512G, P512V, Y513G, Y513L, Y513R, Y513S, S514P, S514R, T515P, K516P, K516R, K517P, I518P, L520M, F522A, A541V, G553M, N556E, N556R, D562I, D562K, D562V, N583V, L585K, F653 R, K712R, N716R, E731P, N732T, A744N, A744P, A744T, F748A, S751A, K754G, I757K, I757W, E77 The method according to any one of sections 32 to 34, comprising one or more amino acid substitutions selected from 1R, N787A, A825R, T826F, T826K, Y832R, Y836K, Y836Q, Y839L, F840L, Y907I, Y907V, Q916Y, E935R, I936R, K974G, K1019V, or combinations thereof. Section 36. ERCAS12A mutants include Q18V (sequence number 4); I35L (sequence number 6); G39Q (sequence number 8); G39K (sequence number 10); I40Q (sequence number 12); D56R (sequence number 14); D56P (sequence number 16); D59R (sequence number 18); D59A (sequence number 20); S158G (sequence number 22); K165Y (sequence number 24); S181V (sequence number 26); A290K (sequence number 28); D291N (sequence number 30); V296R (sequence number 32); P297V (sequence number 34); P297R (sequence number 36); P297F (sequence number 32) Number 38); D303R (Sequence ID 40); D303C (Sequence ID 42); E305K (Sequence ID 44); Y307K (Sequence ID 46); Q353R (Sequence ID 48); L444W (Sequence ID 50); F465R (Sequence ID 52); S469K (Sequence ID 54); V470K (Sequence ID 56); I498K (Sequence ID 58); Y507W (Sequence ID 60); Y507S (Sequence ID 62); Y507R (Sequence ID 64); Q510Y (Sequence ID 66); Q510V (Sequence ID 68); Q510K (Sequence ID 70); P512V (Sequence ID 72); P51 2G(Sequence ID 74);Y513S(Sequence ID 76);Y513R(Sequence ID 78);Y513L(Sequence ID 80);Y513G(Sequence ID 82);S514R(Sequence ID 84);S514P(Sequence ID 86);T515P(Sequence ID 88);K516R(Sequence ID 90);K516P(Sequence ID 92);K517P(Sequence ID 94);I518P(Sequence ID 96);L520M(Sequence ID 98);F522A(Sequence ID 100);A541V(Sequence ID 102);G553M(Sequence ID 104);N556R(Sequence ID 106);N556E(Array Number 108); D562V (Sequence ID 110); D562K (Sequence ID 112); D562I (Sequence ID 114); N583V (Sequence ID 116); L585K (Sequence ID 118); F653R (Sequence ID 120); K712R (Sequence ID 122); N716R (Sequence ID 124); E731P (Sequence ID 126); N732T (Sequence ID 128); A744T (Sequence ID 130); A744P (Sequence ID 132); A744N (Sequence ID 134); F748A (Sequence ID 136); S751A (Sequence ID 138); K754G (Sequence ID 140);I757W (sequence number 142); I757K (sequence number 144); E771R (sequence number 146); N787A (sequence number 148); A825R (sequence number 150); T826K (sequence number 152); T826F (sequence number 154); Y832R (sequence number 156); Y836Q (sequence number 158); Y836K (sequence number 160); Y839L (sequence number 162); F840L (sequence number 164); Y9 07V (sequence number 166); Y907I (sequence number 168); Q916Y (sequence number 170); E935R (sequence number 172); I936R (sequence number 174); K974G (sequence number 176); K1019V (sequence number 178); I35L / S469K (sequence number 180); I35L / T826F (sequence number 182); I40Q / S469K (sequence number 184); I40Q / T826F (sequence number 186) ;S469K / K516P(Sequence ID 188);S469K / T826F(Sequence ID 190);Y513G / Y832R(Sequence ID 192);Y513G / F840L(Sequence ID 194);I35L / S469K / T826F(Sequence ID 196);I40Q / S469K / T826F(Sequence ID 198);S469K / K516P / T826F(Sequence ID 200);S469K / T826F / F840L( The method described in any one of sections 32 to 35, selected from: Sequence ID 202); D303R / S469K / T826F / F840L (Sequence ID 204); F465R / S469K / T826F / F840L (Sequence ID 206); D303R / F465R / S469K / T826F / F840L (Sequence ID 208); or D303R / S469K / K516P / T826F / F840L (Sequence ID 210). Section 37. The method according to any one of Sections 32 to 36, wherein the ERCAS12A mutant is selected from D303R / S469K / T826F / F840L (SEQ ID NO: 204); F465R / S469K / T826F / F840L (SEQ ID NO: 206); D303R / F465R / S469K / T826F / F840L (SEQ ID NO: 208); or D303R / S469K / K516P / T826F / F840L (SEQ ID NO: 210). Section 38. A kit comprising a CRISPR ribonucleoprotein complex containing 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. Section 39. The kit described in Section 38, wherein the ERCAS12A mutant has a polypeptide sequence selected from even-numbered sequences of sequence numbers 4 to 210. Section 40. ERCAS12A mutants have the following variants of the wild-type ERCAS12A polypeptide sequence in SEQ ID NO: Q18V, I35L, G39K, G39Q, I40Q, D56P, D56R, D59A, D59R, S158G, K165Y, S181V, A290K, D291N, V296R, P297F, P297R, P297V, D303C, D303R, E 305K, Y307K, Q353R, L444W, F465R, S469K, V470K, I498K, Y507R, Y507S, Y507W, Q510K, Q510V , Q510Y, P512G, P512V, Y513G, Y513L, Y513R, Y513S, S514P, S514R, T515P, K516P, K516R, K51 7P, I518P, L520M, F522A, A541V, G553M, N556E, N556R, D562I, D562K, D562V, N583V, L585K, F 653R, K712R, N716R, E731P, N732T, A744N, A744P, A744T, F748A, S751A, K754G, I757K, I757W The kit described in Section 38 or 39, comprising one or more amino acid substitutions selected from E771R, N787A, A825R, T826F, T826K, Y832R, Y836K, Y836Q, Y839L, F840L, Y907I, Y907V, Q916Y, E935R, I936R, K974G, K1019V, or combinations thereof. Section 41. ERCAS12A mutants include Q18V (sequence number 4); I35L (sequence number 6); G39Q (sequence number 8); G39K (sequence number 10); I40Q (sequence number 12); D56R (sequence number 14); D56P (sequence number 16); D59R (sequence number 18); D59A (sequence number 20); S158G (sequence number 22); K165Y (sequence number 24); S181V (sequence number 26); A290K (sequence number 28); D291N (sequence number 30); V296R (sequence number 32); P297V (sequence number 34); P297R (sequence number 36); P297F (sequence number 32) Number 38); D303R (Sequence ID 40); D303C (Sequence ID 42); E305K (Sequence ID 44); Y307K (Sequence ID 46); Q353R (Sequence ID 48); L444W (Sequence ID 50); F465R (Sequence ID 52); S469K (Sequence ID 54); V470K (Sequence ID 56); I498K (Sequence ID 58); Y507W (Sequence ID 60); Y507S (Sequence ID 62); Y507R (Sequence ID 64); Q510Y (Sequence ID 66); Q510V (Sequence ID 68); Q510K (Sequence ID 70); P512V (Sequence ID 72); P51 2G(Sequence ID 74);Y513S(Sequence ID 76);Y513R(Sequence ID 78);Y513L(Sequence ID 80);Y513G(Sequence ID 82);S514R(Sequence ID 84);S514P(Sequence ID 86);T515P(Sequence ID 88);K516R(Sequence ID 90);K516P(Sequence ID 92);K517P(Sequence ID 94);I518P(Sequence ID 96);L520M(Sequence ID 98);F522A(Sequence ID 100);A541V(Sequence ID 102);G553M(Sequence ID 104);N556R(Sequence ID 106);N556E(Array Number 108); D562V (Sequence ID 110); D562K (Sequence ID 112); D562I (Sequence ID 114); N583V (Sequence ID 116); L585K (Sequence ID 118); F653R (Sequence ID 120); K712R (Sequence ID 122); N716R (Sequence ID 124); E731P (Sequence ID 126); N732T (Sequence ID 128); A744T (Sequence ID 130); A744P (Sequence ID 132); A744N (Sequence ID 134); F748A (Sequence ID 136); S751A (Sequence ID 138); K754G (Sequence ID 140);I757W (sequence number 142); I757K (sequence number 144); E771R (sequence number 146); N787A (sequence number 148); A825R (sequence number 150); T826K (sequence number 152); T826F (sequence number 154); Y832R (sequence number 156); Y836Q (sequence number 158); Y836K (sequence number 160); Y839L (sequence number 162); F840L (sequence number 164); Y9 07V (sequence number 166); Y907I (sequence number 168); Q916Y (sequence number 170); E935R (sequence number 172); I936R (sequence number 174); K974G (sequence number 176); K1019V (sequence number 178); I35L / S469K (sequence number 180); I35L / T826F (sequence number 182); I40Q / S469K (sequence number 184); I40Q / T826F (sequence number 186) ;S469K / K516P(Sequence ID 188);S469K / T826F(Sequence ID 190);Y513G / Y832R(Sequence ID 192);Y513G / F840L(Sequence ID 194);I35L / S469K / T826F(Sequence ID 196);I40Q / S469K / T826F(Sequence ID 198);S469K / K516P / T826F(Sequence ID 200);S469K / T826F / F840L(Distribution ID) A kit as described in any one of sections 38 to 40, selected from column number 202); D303R / S469K / T826F / F840L (sequence number 204); F465R / S469K / T826F / F840L (sequence number 206); D303R / F465R / S469K / T826F / F840L (sequence number 208); or D303R / S469K / K516P / T826F / F840L (sequence number 210). Section 42. A kit as described in any one of sections 38 to 41, wherein the ERCAS12A mutant is selected from D303R / S469K / T826F / F840L (SEQ ID NO: 204); F465R / S469K / T826F / F840L (SEQ ID NO: 206); D303R / F465R / S469K / T826F / F840L (SEQ ID NO: 208); or D303R / S469K / K516P / T826F / F840L (SEQ ID NO: 210). Section 43. Use of ERCAS12A mutant polypeptide to improve gene editing, wherein the ERCAS12A mutant has one or more amino acid substitutions introduced into the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 2, and the ERCAS12A mutant provides improved gene editing compared to the wild-type ERCAS12A polypeptide. Section 44. The use described in Section 43, wherein the ERCAS12A mutant has a polypeptide sequence selected from even-numbered sequences of SEQ ID NOs. 4 to 210. Section 45. ERCAS12A mutants include Q18V (sequence number 4); I35L (sequence number 6); G39Q (sequence number 8); G39K (sequence number 10); I40Q (sequence number 12); D56R (sequence number 14); D56P (sequence number 16); D59R (sequence number 18); D59A (sequence number 20); S158G (sequence number 22); K165Y (sequence number 24); S181V (sequence number 26); A290K (sequence number 28); D291N (sequence number 30); V296R (sequence number 32); P297V (sequence number 34); P297R (sequence number 36); P297F (sequence number 32) Number 38); D303R (Sequence ID 40); D303C (Sequence ID 42); E305K (Sequence ID 44); Y307K (Sequence ID 46); Q353R (Sequence ID 48); L444W (Sequence ID 50); F465R (Sequence ID 52); S469K (Sequence ID 54); V470K (Sequence ID 56); I498K (Sequence ID 58); Y507W (Sequence ID 60); Y507S (Sequence ID 62); Y507R (Sequence ID 64); Q510Y (Sequence ID 66); Q510V (Sequence ID 68); Q510K (Sequence ID 70); P512V (Sequence ID 72); P51 2G(Sequence ID 74);Y513S(Sequence ID 76);Y513R(Sequence ID 78);Y513L(Sequence ID 80);Y513G(Sequence ID 82);S514R(Sequence ID 84);S514P(Sequence ID 86);T515P(Sequence ID 88);K516R(Sequence ID 90);K516P(Sequence ID 92);K517P(Sequence ID 94);I518P(Sequence ID 96);L520M(Sequence ID 98);F522A(Sequence ID 100);A541V(Sequence ID 102);G553M(Sequence ID 104);N556R(Sequence ID 106);N556E(Array Number 108); D562V (Sequence ID 110); D562K (Sequence ID 112); D562I (Sequence ID 114); N583V (Sequence ID 116); L585K (Sequence ID 118); F653R (Sequence ID 120); K712R (Sequence ID 122); N716R (Sequence ID 124); E731P (Sequence ID 126); N732T (Sequence ID 128); A744T (Sequence ID 130); A744P (Sequence ID 132); A744N (Sequence ID 134); F748A (Sequence ID 136); S751A (Sequence ID 138); K754G (Sequence ID 140);I757W (sequence number 142); I757K (sequence number 144); E771R (sequence number 146); N787A (sequence number 148); A825R (sequence number 150); T826K (sequence number 152); T826F (sequence number 154); Y832R (sequence number 156); Y836Q (sequence number 158); Y836K (sequence number 160); Y839L (sequence number 162); F840L (sequence number 164); Y907V (sequence number 166); Y907I (sequence number 168); Q916Y (sequence number 170); E935R (sequence number 172); I936R (sequence number 174); K974G (sequence number 176); K1019V (sequence number 178); I35L / S469K (sequence number 180); I35L / T826F (sequence number 182); I40Q / S469K (sequence number 184); I40Q / T826F (sequence number 186);S469K / K516P(Sequence ID 188);S469K / T826F(Sequence ID 190);Y513G / Y832R(Sequence ID 192);Y513G / F840L(Sequence ID 194);I35L / S469K / T826F(Sequence ID 196);I40Q / S469K / T826F(Sequence ID 198);S469K / K516P / T826F(Sequence ID 200);S469K / T826F / F Use as described in Section 43 or 44, selected from 840L (SEQ ID NO: 202); D303R / S469K / T826F / F840L (SEQ ID NO: 204); F465R / S469K / T826F / F840L (SEQ ID NO: 206); D303R / F465R / S469K / T826F / F840L (SEQ ID NO: 208); or D303R / S469K / K516P / T826F / F840L (SEQ ID NO: 210). [Examples]

[0086] (Example 1) High-throughput measurement of DNA cleavage activity of ERCAS12A variants in the TTTT PAM site of E. coli. The following examples demonstrate the ability of the present invention to increase gene editing efficiency compared to ERCAS12A (a variant of *Cas12a* from *Acute Bacteria*).

[0087] The nucleotide sequence (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO: 2) of wild-type ERCAS12A are shown in Table 1.

[0088] [Table 1A]

[0089] [Table 1B]

[0090] Table 2 lists the phenotypic scores of 22,409 ERCAS12A variants with a single point mutation. The scores for synonymous mutations (n=849, 69% of all locations) were tightly clustered around 0 and therefore functioned as baseline activity for ERCAS12A (SEQ ID NO: 2) (Figure 1A). The measure of enrichment for each ERCAS12A point mutation was highly consistent across biological replicates (Figure 1B). Overall, 2,564 of the 22,409 point mutations (approximately 11.4%) showed some degree of advantage with scores higher than the maximum score for all synonymous mutations (0.41) (Table 2).

[0091] The specific mutated residues that provided some degree of advantage are shown in the wild-type ERCAS12A polypeptide sequence (SEQ ID NO: 2). Mutant residues are shown in bold, while residues not shown in bold are not mutated. Of the 1263 amino acid residues, 763 (approximately 60%) had 2564 individual mutations.

[0092] [ka]

[0093] Table 2 shows ERCAS12A mutations exhibiting improved DNA cleavage activity in E. coli. Enrichment scores across three biological replicates were measured by deep sequencing. Mutants with an average enrichment score greater than the highest score (0.41) among all synonymous variants were considered positive hits.

[0094] [Table 2-1]

[0095] [Table 2-2]

[0096] [Table 2-3]

[0097] [Table 2-4]

[0098] [Table 2-5]

[0099] [Table 2-6]

[0100] [Table 2-7]

[0101] [Table 2-8]

[0102] [Table 2-9]

[0103] Table 2-10

[0104] Table 2-11

[0105] Table 2-12

[0106] Table 2-13

[0107] Table 2-14

[0108] Table 2-15

[0109] Table 2-16

[0110] Table 2-17

[0111] Table 2-18

[0112] Table 2-19

[0113] Table 2-20

[0114] Table 2-21

[0115] Table 2-22

[0116] Table 2-23

[0117] Table 2-24

[0118] Table 2-25

[0119] Table 2-26

[0120] Table 2-27

[0121] Table 2-28

[0122] Table 2-29

[0123] Table 2-30

[0124] Table 2-31

[0125] Table 2-32

[0126] Table 2-33

[0127] Table 2-34

[0128] Table 2-35

[0129] Table 2-36

[0130] Table 2-37

[0131] Table 2-38

[0132] Table 2-39

[0133] Table 2-40

[0134] Table 2-41

[0135] Table 2-42

[0136] Table 2-43

[0137] Table 2-44

[0138] Table 2-45

[0139] Table 2-46

[0140] Table 2-47

[0141] Table 2-48

[0142] Table 2-49

[0143] Table 2-50

[0144] Table 2-51

[0145] Table 2-52

[0146] Table 2-53

[0147] Table 2-54

[0148] Table 2-55

[0149] The sequences of the 2564 mutant polynucleotides and polypeptides shown in Table 2 are not provided. Those skilled in the art will understand that mutations can be created from the wild-type ERCAS12A nucleotide or polypeptide sequence by altering the codons of the wild-type amino acids and replacing them with the codons of the mutant amino acids. For example, ERCAS12A mutant 1, "M1G," shown in Table 2, indicates that methionine 1 in the polypeptide sequence has been replaced with glycine. Similarly, the wild-type codon of methionine 1, "ATG," can be replaced with one of the E. coli glycine codons "GGT," "GGC," "GGA," or "GGG" to create a mutant polynucleotide coding sequence encoding glycine at position 1. The codons of the ERCAS12A amino acids can be optimized for specific organisms, such as E. coli, humans (Homo sapiens), or other organisms, by changing the codons in the coding chain to the codons most commonly used in each organism.

[0150] To demonstrate the usefulness of these results, sets of ERCAS12A variants with point mutations (I35L, G39K, G39Q, I40Q, D56P, D56R, D291N, V296R, P297F, P297V, F465R, S469K, I498K, Y507S, Y507W, Q510V, and Q510Y) were selectively cloned and their activity in relation to bacterial selection assays was tested. As shown in Figures 2–5, most of the characterized mutants improved bacterial viability compared to WT-ERCAS12A (SEQ ID NO: 2) during selection, thus validating the results of high-throughput screening. Since viability in bacterial assays is related to the intrinsic cleavage activity of the ERCAS12A nuclease, these mutants exhibit improved performance compared to the WT enzyme and are extremely promising for human genome engineering.

[0151] Table 3 shows the primer sequences used to generate the ERCAS12A saturated mutagenesis library. Standard recombination methods and techniques were used. The screening library was constructed using the method described by Wrenbeck et al., Nat. Methods 13(11):928-930 (2016).

[0152] This screening function involves transforming screening strains carrying the toxin plasmid with a mutant ERCAS12A library and crRNA targeting the HPRT38346 site on the toxin plasmid. After harvesting and IPTG induction, cells were seeded in LB-chloramphenicol medium containing arabinose and incubated overnight at 37°C. Functional ERCAS12A enzymes are capable of CRISPR activity and inactivating the toxin plasmid. If the mutant LbCas12a enzyme is not functional, the transformed screening E. coli cells will not survive. The ERCAS12A expression plasmid held 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 was measured in both libraries and normalized to the total coverage of each codon. The relative viability of each point mutation was calculated as the ratio of the normalized frequencies between the selected library and the input library. Since the degree of cell viability under arabinose selection is an indicator of the cleavage activity of the ERCAS12A variant in the HPRT38346 protospacer, any variant enriched in WT during selection is a variant with enhanced activity in TTTT PAM.

[0153] [Table 3-1]

[0154] [Table 3-2]

[0155] Table 3-3

[0156] Table 3-4

[0157] Table 3-5

[0158] Table 3-6

[0159] Table 3-7

[0160] Table 3-8

[0161] Table 3-9

[0162] Table 3-10

[0163] Table 3-11

[0164] Table 3-12

[0165] Table 3-13

[0166] Table 3-14

[0167] Table 3-15

[0168] Table 3-16

[0169] Table 3-17

[0170] Table 3-18

[0171] Table 3-19

[0172] Table 3-20

[0173] Table 3-21

[0174] Table 3-22

[0175] Table 3-23

[0176] Table 3-24

[0177] Table 3-25

[0178] Table 3-26

[0179] Table 3-27

[0180] Table 3-28

[0181] Table 3-29

[0182] Table 3-30

[0183] Table 3-31

[0184] Table 3-32

[0185] Table 3-33

[0186] Table 3-34

[0187] Table 3-35

[0188] Table 3-36

[0189] Table 3-37

[0190] Table 3-38

[0191] Table 3-39

[0192] Table 3-40

[0193] Table 3-41

[0194] Table 3-42

[0195] Table 3-43

[0196] Table 3-44

[0197] Table 3-45

[0198] Table 3-46

[0199] Table 3-47

[0200] Table 3-48

[0201] Table 3-49

[0202] Table 3-50

[0203] Table 3-51

[0204] Table 3-52

[0205] Table 3-53

[0206] [Table 3-54]

[0207] [Table 3-55]

[0208] (Example 3) Editing efficiency of ErCas12a variants in human cells In this example, the DNA cleavage activity of 20 ErCas12a point mutations associated with Escherichia coli was evaluated (Figure 7). The cleavage activity of each mutant was quantified by calculating the viability of Escherichia coli with and without induction of a toxicity reporter gene. Compared to WT-ERCAS12A, 31 of the 40 point mutations showed improved DNA cleavage, thus further validating the results of the sequencing-based high-throughput phenotypic measurement in Example 1.

[0209] Eight representative ERCAS12A point mutations were selected for further characterization in human cells. These point mutations included I35L, G39Q, I40Q, S469K, Y507S, K516P, T826F, and Y907V. Site-directed mutagenesis was performed to introduce specific point mutations into expression vectors using the primers listed in Table 4. Each protein was expressed in E. coli BL21DE3 cells and purified sequentially using nickel affinity and heparin chromatography. To evaluate the genome editing efficiency of each variant, ERCAS12A-RNP was assembled using six crRNAs targeting the human HPRT gene (Table 5). Assembled RNP (50 nM or 200 nM) was delivered to HEK293 cells by Lonza nucleofection (SF buffer, DS-150 program), and editing efficiency was measured 48 hours after delivery by T7 endonuclease I assay (T7EI) (Figures 8A and 8B). To facilitate comparison, the efficiency of each variant at each target was normalized to WT-ERCAS12A and presented as a multiple improvement compared to WT (Figures 8C and 8D). Single mutant variants showed improved activity compared to WT at one or more sites, particularly at low RNP concentrations (50 nM, Figures 8B and 8D).

[0210] [Table 4]

[0211] [Table 5]

[0212] (Example 4) Editing efficiency of ErCas12 variants in human cells In this example, we evaluate the editing efficiency of 42 additional ErCas12a point mutations selected based on previous E. coli screening data. Site-directed mutagenesis was performed to introduce specific point mutations into expression vectors using the primers listed in Table 6. Purified plasmids were lipofected into human HEK293 cells using two crRNAs targeting the human HPRT gene (Table 7). HPRT editing efficiency was measured by the T7EI assay two days after delivery. To facilitate comparison, the efficiency of each variant at each target was normalized to WT ERCAS12A and presented as a multiplier improvement over WT (Figure 9). Of the 42 variants, 21 improved the on-target efficiency of ERCAS12A when delivered as plasmids (Figure 8, Table 7). Four point mutations, including S158G, D303R, F465R, and F840L, were selected for further characterization as RNPs. Three of the four mutations (D303R, F465R, and F840L) improved on-target editing efficiency at almost all target sites (Figures 8B and 8C).

[0213] [Table 6A]

[0214] [Table 6B]

[0215] [Table 7]

[0216] To further improve ERCAS12A activity, mutations were combined or "stacked" using large accumulations of single point mutations that had available enhanced activity. Nine combinations of point mutations described in previous examples were stacked, and the editing efficiency of these ERCAS12A variants was evaluated as RNP in human cells (Figures 9A and 9B). Notably, all stacked variants enhanced the on-target editing efficiency of ERCAS12A across one or more target sites. Among these, the variant with a double mutation (S469K / T826F) showed superior performance.

[0217] (Example 5) Editing efficiency of ErCas12 variants in human cell lines The editing efficiency of another 20 ERCAS12A point mutations was evaluated. A further 20 ERCAS12A point mutations were selected based on E. coli screening data. Site-directed mutagenesis was performed to introduce specific point mutations into expression vectors using the primers listed in Table 8. Purified plasmids were lipofected into human HEK293 cells using eight crRNAs targeting therapeutically relevant human genes (Table 9). The editing efficiency of each gene was measured by NGS three days after delivery (Figures 10A-10H).

[0218] [Table 8A]

[0219] [Table 8B]

[0220] [Table 9]

[0221] (Example 6) Editing efficiency of ERCAS12A variants in human cells The editing efficiency of ERCAS12A Y513G point mutations selected from plasmid screening was evaluated. Site-directed mutagenesis was performed to introduce point mutations into protein expression vectors using the primers listed in Table 10. Purified proteins targeting seven therapeutically relevant human crRNAs were tested as RNPs in human HEK293 cells (Table 11). The editing efficiency of each gene was measured by NGS two days after delivery (Figure 11).

[0222] Next, using site-directed mutagenesis, S514P was generated by adding further mutations, Y513G, Y832R, and F840L, to ERCAS12A on a protein expression vector using the primers listed in Table 10. The purified proteins were tested as RNPs in human HEK293 cells using four crRNAs targeting therapeutically relevant human genes (Table 11). The editing efficiency of each gene was measured by NGS two days after delivery (Figures 12A-12D). This experiment also included Acidaminococcus sp. Cas12a M537R / F870L (AsCas12a-Ultra) as a positive control, with and without electroporation enhancers.

[0223] [Table 10]

[0224] [Table 11]

[0225] (Example 7) The best ERCAS12A stacking mutant identified in previous examples was stacked with another mutation, namely K516P (e.g., D303R / S469K / K516P / T826F / F840L). This point mutation was added using site-directed mutagenesis. The primers are listed in Table 12. The proteins were expressed in E. coli BL21DE3 cells and purified sequentially using nickel affinity, heparin chromatography, and hydroxyapatite chromatography. To evaluate the genome editing efficiency of each variant, ERCAS12A mutant-RNP was assembled using the crRNAs found in Table 13. The assembled RNP (2 μM) was delivered to HEK293 cells by nucleofection, and the percentage of indels was measured by NGS 48 hours after delivery (Figure 13). Each of the stacked mutants (D303R / S469K / T826F / F840L; F465R / S469K / T826F / F840L; D303R / S469K / K516P / T826F / F840L) improved activity against ERCAS12A WT at these sites. The stacking of these beneficial mutants resulted in a more potent ERCAS12A variant than any of the individual point mutations.

[0226] [Table 12]

[0227] [Table 13]

[0228] The nucleotide and polypeptide sequences of the selected ERCAS12A variant are provided in Table 14.

[0229] [Table 14-1]

[0230] [Table 14-2]

[0231] Table 14-3

[0232] Table 14-4

[0233] Table 14-5

[0234] Table 14-6

[0235] Table 14-7

[0236] Table 14-8

[0237] Table 14-9

[0238] Table 14-10

[0239] Table 14-11

[0240] Table 14-12

[0241] Table 14-13

[0242] Table 14-14

[0243] Table 14-15

[0244] Table 14-16

[0245] Table 14-17

[0246] Table 14-18

[0247] Table 14-19

[0248] Table 14-20

[0249] Table 14-21

[0250] Table 14-22

[0251] Table 14-23

[0252] Table 14-24

[0253] Table 14-25

[0254] Table 14-26

[0255] Table 14-27

[0256] Table 14-28

[0257] Table 14-29

[0258] Table 14-30

[0259] Table 14-31

[0260] Table 14-32

[0261] Table 14-33

[0262] Table 14-34

[0263] Table 14-35

[0264] Table 14-36

[0265] Table 14-37

[0266] Table 14-38

[0267] Table 14-39

[0268] Table 14-40

[0269] Table 14-41

[0270] Table 14-42

[0271] Table 14-43

[0272] Table 14-44

[0273] Table 14-45

[0274] Table 14-46

[0275] Table 14-47

[0276] Table 14-48

[0277] Table 14-49

[0278] Table 14-50

[0279] Table 14-51

[0280] Table 14-52

[0281] Table 14-53

[0282] Table 14-54

[0283] Table 14-55

[0284] Table 14-56

[0285] Table 14-57

[0286] Table 14-58

[0287] Table 14-59

[0288] Table 14-60

[0289] Table 14-61

[0290] Table 14-62

[0291] Table 14-63

[0292] Table 14-64

[0293] Table 14-65

[0294] Table 14-66

[0295] Table 14-67

[0296] Table 14-68

[0297] Table 14-69

[0298] Table 14-70

[0299] Table 14-71

[0300] Table 14-72

[0301] Table 14-73

[0302] Table 14-74

[0303] Table 14-75

[0304] Table 14-76

[0305] Table 14-77

[0306] Table 14-78

[0307] Table 14-79

[0308] Table 14-80

[0309] Table 14-81

[0310] Table 14-82

[0311] Table 14-83

[0312] Table 14-84

[0313] Table 14-85

[0314] Table 14-86

[0315] Table 14-87

[0316] Table 14-88

[0317] Table 14-89

[0318]

Table 14-90

[0319] Table 14-91

[0320] Table 14-92

[0321] Table 14-93

[0322] Table 14-94

[0323] Table 14-95

[0324] Table 14-96

[0325] Table 14-97

[0326] Table 14-98

[0327] Table 14-99

[0328]

Table 14-100

[0329] Table 14-101

[0330] Table 14-102

[0331] Table 14-103

[0332] Table 14-104

[0333]

Table 14-105

[0334] Table 14-106

[0335] Table 14-107

[0336]

Table 14-108

[0337] Table 14-109

[0338]

Table 14-110

[0339] Table 14-111

[0340] Table 14-112

[0341] Table 14-113

Claims

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

2. The isolated polypeptide according to claim 1, wherein the ERCAS12A mutant has 98% to 99% identity with polypeptide sequences selected from even-numbered sequences of SEQ ID NOs. 4 to 210.

3. The isolated polypeptide according to claim 1, wherein the ERCAS12A mutant has a polypeptide sequence selected from even-numbered sequences of sequence numbers 4 to 210.

4. The ERCAS12A mutant has the following characteristics in the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 18, 35, 39, 39, 40, 56, 56, 59, 59, 158, 165, 181, 290, 291, 296, 297, 297, 297, 303, 303, 305, 307, 353, 444, 465, 469, 470, 498, 507, 507, 507, 510, 510, 510, 512, 512, 513, 513, 513, 513, 514, 514, 515, 516, 516, 5 An isolated polypeptide according to claim 1, comprising one or more amino acid substitutions at positions 17, 518, 520, 522, 541, 553, 556, 556, 562, 562, 562, 583, 585, 653, 712, 716, 731, 732, 744, 744, 744, 748, 751, 754, 757, 757, 771, 787, 825, 826, 826, 832, 836, 836, 839, 840, 907, 907, 916, 935, 936, 974, or 1019.

5. The ERCAS12A mutants are Q18V, I35L, G39K, G39Q, I40Q, D56P, D56R, D59A, D59R, S158G, K165Y, S181V, A290K, D291N, V296R, P297F, P297R, P297V, D303C, D303R, E30 5K, Y307K, Q353R, L444W, F465R, S469K, V470K, I498K, Y507R, Y507S, Y507W, Q510K, Q510V, Q 510Y, P512G, P512V, Y513G, Y513L, Y513R, Y513S, S514P, S514R, T515P, K516P, K516R, K517P, I518P, L520M, F522A, A541V, G553M, N556E, N556R, D562I, D562K, D562V, N583V, L585K, F653 R, K712R, N716R, E731P, N732T, A744N, A744P, A744T, F748A, S751A, K754G, I757K, I757W, E77 An isolated polypeptide according to claim 1, comprising one or more amino acid substitutions selected from 1R, N787A, A825R, T826F, T826K, Y832R, Y836K, Y836Q, Y839L, F840L, Y907I, Y907V, Q916Y, E935R, I936R, K974G, K1019V, or combinations thereof.

6. ERCAS12A mutants include Q18V (sequence number 4); I35L (sequence number 6); G39Q (sequence number 8); G39K (sequence number 10); I40Q (sequence number 12); D56R (sequence number 14); D56P (sequence number 16); D59R (sequence number 18); D59A (sequence number 20); S158G (sequence number 22); K165Y (sequence number 24); S181V (sequence number 26); A290K (sequence number 28); D291N (sequence number 30); V296R (sequence number 32); P297V (sequence number 34); P297R (sequence number 36); P297F (sequence number 32) Number 38); D303R (Sequence ID 40); D303C (Sequence ID 42); E305K (Sequence ID 44); Y307K (Sequence ID 46); Q353R (Sequence ID 48); L444W (Sequence ID 50); F465R (Sequence ID 52); S469K (Sequence ID 54); V470K (Sequence ID 56); I498K (Sequence ID 58); Y507W (Sequence ID 60); Y507S (Sequence ID 62); Y507R (Sequence ID 64); Q510Y (Sequence ID 66); Q510V (Sequence ID 68); Q510K (Sequence ID 70); P512V (Sequence ID 72); P51 2G(Sequence ID 74);Y513S(Sequence ID 76);Y513R(Sequence ID 78);Y513L(Sequence ID 80);Y513G(Sequence ID 82);S514R(Sequence ID 84);S514P(Sequence ID 86);T515P(Sequence ID 88);K516R(Sequence ID 90);K516P(Sequence ID 92);K517P(Sequence ID 94);I518P(Sequence ID 96);L520M(Sequence ID 98);F522A(Sequence ID 100);A541V(Sequence ID 102);G553M(Sequence ID 104);N556R(Sequence ID 106);N556E(Array Number 108); D562V (Sequence ID 110); D562K (Sequence ID 112); D562I (Sequence ID 114); N583V (Sequence ID 116); L585K (Sequence ID 118); F653R (Sequence ID 120); K712R (Sequence ID 122); N716R (Sequence ID 124); E731P (Sequence ID 126); N732T (Sequence ID 128); A744T (Sequence ID 130); A744P (Sequence ID 132); A744N (Sequence ID 134); F748A (Sequence ID 136); S751A (Sequence ID 138); K754G (Sequence ID 140);I757W (sequence number 142); I757K (sequence number 144); E771R (sequence number 146); N787A (sequence number 148); A825R (sequence number 150); T826K (sequence number 152); T826F (sequence number 154); Y832R (sequence number 156); Y836Q (sequence number 158); Y836K (sequence number 160); Y839L (sequence number 162); F840L (sequence number 164); Y9 07V (sequence number 166); Y907I (sequence number 168); Q916Y (sequence number 170); E935R (sequence number 172); I936R (sequence number 174); K974G (sequence number 176); K1019V (sequence number 178); I35L / S469K (sequence number 180); I35L / T826F (sequence number 182); I40Q / S469K (sequence number 184); I40Q / T826F (sequence number 186 );S469K / K516P(Sequence ID 188);S469K / T826F(Sequence ID 190);Y513G / Y832R(Sequence ID 192);Y513G / F840L(Sequence ID 194);I35L / S469K / T826F(Sequence ID 196);I40Q / S469K / T826F(Sequence ID 198);S469K / K516P / T826F(Sequence ID 200);S469K / T826F / F840L An isolated polypeptide according to claim 1, selected from (SEQ ID NO: 202); D303R / S469K / T826F / F840L (SEQ ID NO: 204); F465R / S469K / T826F / F840L (SEQ ID NO: 206); D303R / F465R / S469K / T826F / F840L (SEQ ID NO: 208); or D303R / S469K / K516P / T826F / F840L (SEQ ID NO: 210).

7. The isolated polypeptide according to claim 1, wherein the ERCAS12A mutant is selected from D303R / S469K / T826F / F840L (SEQ ID NO: 204); F465R / S469K / T826F / F840L (SEQ ID NO: 206); D303R / F465R / S469K / T826F / F840L (SEQ ID NO: 208); or D303R / S469K / K516P / T826F / F840L (SEQ ID NO: 210).

8. An isolated polynucleotide sequence encoding a rectal bacterium Cas12a (ERCAS12A) variant having one or more amino acid substitutions introduced into the wild-type ERCAS12A polypeptide sequence of Sequence ID No. 2, wherein the ERCAS12A variant polypeptide provides improved nuclease activity compared to the wild-type ERCAS12A polypeptide.

9. The isolated polynucleotide sequence according to claim 8, wherein the encoded ERCAS12A variant has 98% to 99% identity with polypeptide sequences selected from even-numbered sequences of sequence numbers 4 to 210.

10. The isolated polynucleotide sequence according to claim 8, wherein the encoded ERCAS12A variant has a polypeptide sequence selected from even-numbered sequences of sequence numbers 4 to 210.

11. The isolated polynucleotide sequence according to claim 8, wherein the ERCAS12A mutant polynucleotide sequence has 95% to 99% identity with a nucleotide sequence selected from the odd-numbered sequences of SEQ ID NOs. 3 to 209.

12. The isolated polynucleotide sequence according to claim 8, wherein the ERCAS12A mutant polynucleotide has a nucleotide sequence selected from the odd-numbered sequences of SEQ ID NOs. 3 to 209.

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

14. The encoded ERCAS12A variants are Q18V, I35L, G39K, G39Q, I40Q, D56P, D56R, D59A, D59R, S158G, K165Y, S181V, A290K, D291N, V296R, P297F, P297R, P297V, D303C, D303R of the wild-type ERCAS12A polypeptide sequence in SEQ ID NO: 2 , E305K, Y307K, Q353R, L444W, F465R, S469K, V470K, I498K, Y507R, Y507S, Y507W, Q510K, Q510V , Q510Y, P512G, P512V, Y513G, Y513L, Y513R, Y513S, S514P, S514R, T515P, K516P, K516R, K517P , I518P, L520M, F522A, A541V, G553M, N556E, N556R, D562I, D562K, D562V, N583V, L585K, F653 R, K712R, N716R, E731P, N732T, A744N, A744P, A744T, F748A, S751A, K754G, I757K, I757W, E771 The isolated polynucleotide sequence according to claim 8, comprising one or more amino acid substitutions selected from R, N787A, A825R, T826F, T826K, Y832R, Y836K, Y836Q, Y839L, F840L, Y907I, Y907V, Q916Y, E935R, I936R, K974G, K1019V, or combinations thereof.

15. The encoded ERCAS12A mutant polynucleotides are Q18V (sequence number 4); I35L (sequence number 6); G39Q (sequence number 8); G39K (sequence number 10); I40Q (sequence number 12); D56R (sequence number 14); D56P (sequence number 16); D59R (sequence number 18); D59A (sequence number 20); S158G (sequence number 22); K165Y (sequence number 24); S181V (sequence number 26); A290K (sequence number 28); D291N (sequence number 30); V296R (sequence number 32); P297V (sequence number 34); P297R (sequence number 36) );P297F(sequence number 38);D303R(sequence number 40);D303C(sequence number 42);E305K(sequence number 44);Y307K(sequence number 46);Q353R(sequence number 48);L444W(sequence number 50);F465R(sequence number 52);S469K(sequence number 54);V470K(sequence number 56);I498K(sequence number 58);Y507W(sequence number 60);Y507S(sequence number 62);Y507R(sequence number 64);Q510Y(sequence number 66);Q510V(sequence number 68);Q510K(sequence number 70);P512V(sequence number 72);P512G(Sequence ID 74);Y513S(Sequence ID 76);Y513R(Sequence ID 78);Y513L(Sequence ID 80);Y513G(Sequence ID 82);S514R(Sequence ID 84);S514P(Sequence ID 86);T515P(Sequence ID 88);K516R(Sequence ID 90);K516P(Sequence ID 92);K517P(Sequence ID 94);I518P(Sequence ID 96);L520M(Sequence ID 98);F522A(Sequence ID 100);A541V(Sequence ID 102);G553M(Sequence ID 104);N556R(Sequence ID 106);N556 E(sequence code 108); D562V(sequence code 110); D562K(sequence code 112); D562I(sequence code 114); N583V(sequence code 116); L585K(sequence code 118); F653R(sequence code 120); K712R(sequence code 122); N716R(sequence code 124); E731P(sequence code 126); N732T(sequence code 128); A744T(sequence code 130); A744P(sequence code 132); A744N(sequence code 134); F748A(sequence code 136); S751A(sequence code 138); K754G(sequence code 140);I757W (sequence number 142); I757K (sequence number 144); E771R (sequence number 146); N787A (sequence number 148); A825R (sequence number 150); T826K (sequence number 152); T826F (sequence number 154); Y832R (sequence number 156); Y836Q (sequence number 158); Y836K (sequence number 160); Y839L (sequence number 162); F840L (sequence number 164); Y90 7V (sequence number 166); Y907I (sequence number 168); Q916Y (sequence number 170); E935R (sequence number 172); I936R (sequence number 174); K974G (sequence number 176); K1019V (sequence number 178); I35L / S469K (sequence number 180); I35L / T826F (sequence number 182); I40Q / S469K (sequence number 184); I40Q / T826F (sequence number 186); S469K / K516P (Sequence ID 188); S469K / T826F (Sequence ID 190); Y513G / Y832R (Sequence ID 192); Y513G / F840L (Sequence ID 194); I35L / S469K / T826F (Sequence ID 196); I40Q / S469K / T826F (Sequence ID 198); S469K / K516P / T826F (Sequence ID 200); S469K / T826F / F840L (Array An isolated polynucleotide sequence according to claim 8, selected from: (Sequence ID 202); D303R / S469K / T826F / F840L (Sequence ID 204); F465R / S469K / T826F / F840L (Sequence ID 206); D303R / F465R / S469K / T826F / F840L (Sequence ID 208); or D303R / S469K / K516P / T826F / F840L (Sequence ID 210).

16. ERCAS12A mutant polynucleotides include Q18V (sequence number 3); I35L (sequence number 5); G39Q (sequence number 7); G39K (sequence number 9); I40Q (sequence number 11); D56R (sequence number 13); D56P (sequence number 15); D59R (sequence number 17); D59A (sequence number 19); S158G (sequence number 21); K165Y (sequence number 23); S181V (sequence number 25); A290K (sequence number 27); D291N (sequence number 29); V296R (sequence number 31); P297V (sequence number 33); P297R (sequence number 35); P2 97F(sequence number 37);D303R(sequence number 39);D303C(sequence number 41);E305K(sequence number 43);Y307K(sequence number 45);Q353R(sequence number 47);L444W(sequence number 49);F465R(sequence number 51);S469K(sequence number 53);V470K(sequence number 55);I498K(sequence number 57);Y507W(sequence number 59);Y507S(sequence number 61);Y507R(sequence number 63);Q510Y(sequence number 65);Q510V(sequence number 67);Q510K(sequence number 69);P512V(sequence number 71) ;P512G(sequence number 73);Y513S(sequence number 75);Y513R(sequence number 77);Y513L(sequence number 79);Y513G(sequence number 81);S514R(sequence number 83);S514P(sequence number 85);T515P(sequence number 87);K516R(sequence number 89);K516P(sequence number 91);K517P(sequence number 93);I518P(sequence number 95);L520M(sequence number 97);F522A(sequence number 99);A541V(sequence number 101);G553M(sequence number 103);N556R(sequence number 105);N556E( SEQ ID NO: 107); D562V (SEQ ID NO: 109); D562K (SEQ ID NO: 111); D562I (SEQ ID NO: 113); N583V (SEQ ID NO: 115); L585K (SEQ ID NO: 117); F653R (SEQ ID NO: 119); K712R (SEQ ID NO: 121); N716R (SEQ ID NO: 123); E731P (SEQ ID NO: 125); N732T (SEQ ID NO: 127); A744T (SEQ ID NO: 129); A744P (SEQ ID NO: 131); A744N (SEQ ID NO: 132); F748A (SEQ ID NO: 135); S751A (SEQ ID NO: 137); K754G (SEQ ID NO: 139);I757W (sequence number 141); I757K (sequence number 143); E771R (sequence number 145); N787A (sequence number 147); A825R (sequence number 149); T826K (sequence number 151); T826F (sequence number 153); Y832R (sequence number 155); Y836Q (sequence number 157); Y836K (sequence number 159); Y839L (sequence number 161); F840L (sequence number 163); Y90 7V (sequence number 165); Y907I (sequence number 167); Q916Y (sequence number 169); E935R (sequence number 171); I936R (sequence number 173); K974G (sequence number 175); K1019V (sequence number 177); I35L / S469K (sequence number 179); I35L / T826F (sequence number 181); I40Q / S469K (sequence number 183); I40Q / T826F (sequence number 185); S469K / K516P (sequence number 187); S469K / T826F (sequence number 189); Y513G / Y832R (sequence number 191); Y513G / F840L (sequence number 193); I35L / S469K / T826F (sequence number 195); I40Q / S469K / T826F (sequence number 197); S469K / K516P / T826F (sequence number 199); S469K / T826F / F840L (array An isolated polynucleotide sequence according to claim 8, selected from: (Sequence ID 201); D303R / S469K / T826F / F840L (Sequence ID 203); F465R / S469K / T826F / F840L (Sequence ID 205); D303R / F465R / S469K / T826F / F840L (Sequence ID 207); or D303R / S469K / K516P / T826F / F840L (Sequence ID 209).

17. The isolated polynucleotide sequence according to claim 8, wherein the ERCAS12A mutant polynucleotide is selected from D303R / S469K / T826F / F840L (SEQ ID NO: 203); F465R / S469K / T826F / F840L (SEQ ID NO: 205); D303R / F465R / S469K / T826F / F840L (SEQ ID NO: 207); or D303R / S469K / K516P / T826F / F840L (SEQ ID NO: 209).

18. A vector or plasmid comprising the polynucleotide sequence described in claim 8.

19. A cell comprising a nucleic acid containing one or more polynucleotide sequences as described in claim 8.

20. An isolated CRISPR ribonucleoprotein 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 ribonucleoprotein complex according to claim 20, wherein the ERCAS12A mutant has a polypeptide sequence selected from even-numbered sequences of SEQ ID NOs. 4 to 210.

22. The ERCAS12A mutant has the following characteristics in the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 18, 35, 39, 39, 40, 56, 56, 59, 59, 158, 165, 181, 290, 291, 296, 297, 297, 297, 303, 303, 305, 307, 353, 444, 465, 469, 470, 498, 507, 507, 507, 510, 510, 510, 512, 512, 513, 513, 513, 513, 514, 514, 515, 516, 516, 517, 518, 5 An isolated CRISPR ribonucleoprotein complex according to claim 20, comprising one or more amino acid substitutions at positions 20, 522, 541, 553, 556, 556, 562, 562, 583, 585, 653, 712, 716, 731, 732, 744, 744, 744, 748, 751, 754, 757, 757, 771, 787, 825, 826, 826, 832, 836, 836, 839, 840, 907, 907, 916, 935, 936, 974, or 1019.

23. The ERCAS12A mutants are Q18V, I35L, G39K, G39Q, I40Q, D56P, D56R, D59A, D59R, S158G, K165Y, S181V, A290K, D291N, V296R, P297F, P297R, P297V, D303C, D303R, E305K, Y307K, Q353R, L444W, F465R, S469K, V470K, I498K, Y507R, Y507S, Y507W, Q510K, Q510V, Q510Y, P512G, P512V, Y513G, Y513L, Y513R, Y513S, S514P, S514R, T515P, K516P, K516R, K517P, I518P, L 520M, F522A, A541V, G553M, N556E, N556R, D562I, D562K, D562V, N583V, L585K, F653R, K712R, N 716R, E731P, N732T, A744N, A744P, A744T, F748A, S751A, K754G, I757K, I757W, E771R, N787A, A8 An isolated CRISPR ribonucleoprotein complex according to claim 20, comprising one or more amino acid substitutions selected from 25R, T826F, T826K, Y832R, Y836K, Y836Q, Y839L, F840L, Y907I, Y907V, Q916Y, E935R, I936R, K974G, K1019V, or combinations thereof.

24. ERCAS12A mutants include Q18V (sequence number 4); I35L (sequence number 6); G39Q (sequence number 8); G39K (sequence number 10); I40Q (sequence number 12); D56R (sequence number 14); D56P (sequence number 16); D59R (sequence number 18); D59A (sequence number 20); S158G (sequence number 22); K165Y (sequence number 24); S181V (sequence number 26); A290K (sequence number 28); D291N (sequence number 30); V296R (sequence number 32); P297V (sequence number 34); P297R (sequence number 36); P297F (sequence number 32) Number 38); D303R (Sequence ID 40); D303C (Sequence ID 42); E305K (Sequence ID 44); Y307K (Sequence ID 46); Q353R (Sequence ID 48); L444W (Sequence ID 50); F465R (Sequence ID 52); S469K (Sequence ID 54); V470K (Sequence ID 56); I498K (Sequence ID 58); Y507W (Sequence ID 60); Y507S (Sequence ID 62); Y507R (Sequence ID 64); Q510Y (Sequence ID 66); Q510V (Sequence ID 68); Q510K (Sequence ID 70); P512V (Sequence ID 72); P51 2G(Sequence ID 74);Y513S(Sequence ID 76);Y513R(Sequence ID 78);Y513L(Sequence ID 80);Y513G(Sequence ID 82);S514R(Sequence ID 84);S514P(Sequence ID 86);T515P(Sequence ID 88);K516R(Sequence ID 90);K516P(Sequence ID 92);K517P(Sequence ID 94);I518P(Sequence ID 96);L520M(Sequence ID 98);F522A(Sequence ID 100);A541V(Sequence ID 102);G553M(Sequence ID 104);N556R(Sequence ID 106);N556E(Array Number 108); D562V (Sequence ID 110); D562K (Sequence ID 112); D562I (Sequence ID 114); N583V (Sequence ID 116); L585K (Sequence ID 118); F653R (Sequence ID 120); K712R (Sequence ID 122); N716R (Sequence ID 124); E731P (Sequence ID 126); N732T (Sequence ID 128); A744T (Sequence ID 130); A744P (Sequence ID 132); A744N (Sequence ID 134); F748A (Sequence ID 136); S751A (Sequence ID 138); K754G (Sequence ID 140);I757W (sequence number 142); I757K (sequence number 144); E771R (sequence number 146); N787A (sequence number 148); A825R (sequence number 150); T826K (sequence number 152); T826F (sequence number 154); Y832R (sequence number 156); Y836Q (sequence number 158); Y836K (sequence number 160); Y839L (sequence number 162); F840L (sequence number 164); Y907V ( SEQ ID NO: 166); Y907I (SEQ ID NO: 168); Q916Y (SEQ ID NO: 170); E935R (SEQ ID NO: 172); I936R (SEQ ID NO: 174); K974G (SEQ ID NO: 176); K1019V (SEQ ID NO: 178); I35L / S469K (SEQ ID NO: 180); I35L / T826F (SEQ ID NO: 182); I40Q / S469K (SEQ ID NO: 184); I40Q / T826F (SEQ ID NO: 186); S469K / K516P (Sequence ID 188); S469K / T826F (Sequence ID 190); Y513G / Y832R (Sequence ID 192); Y513G / F840L (Sequence ID 194); I35L / S469K / T826F (Sequence ID 196); I40Q / S469K / T826F (Sequence ID 198); S469K / K516P / T826F (Sequence ID 200); S469K / T826F / F840L (Sequence ID 202); D3 An isolated CRISPR ribonucleoprotein complex according to claim 20, selected from 03R / S469K / T826F / F840L (SEQ ID NO: 204); F465R / S469K / T826F / F840L (SEQ ID NO: 206); D303R / F465R / S469K / T826F / F840L (SEQ ID NO: 208); or D303R / S469K / K516P / T826F / F840L (SEQ ID NO: 210).

25. The isolated CRISPR ribonucleoprotein complex according to claim 20, wherein the ERCAS12A mutant is selected from D303R / S469K / T826F / F840L (SEQ ID NO: 204); F465R / S469K / T826F / F840L (SEQ ID NO: 206); D303R / F465R / S469K / T826F / F840L (SEQ ID NO: 208); or D303R / S469K / K516P / T826F / F840L (SEQ ID NO: 210).

26. An isolated CRISPR / Cpf1 endonuclease system comprising an ERCAS12A mutant polypeptide having one or more amino acid substitutions introduced into the crRNA and the wild-type ERCAS12A polypeptide sequence of Sequence 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 according to claim 26, wherein the ERCAS12A mutant has a polypeptide sequence selected from even-numbered sequences of SEQ ID NOs. 4 to 210.

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

29. The ERCAS12A mutants are Q18V, I35L, G39K, G39Q, I40Q, D56P, D56R, D59A, D59R, S158G, K165Y, S181V, A290K, D291N, V296R, P297F, P297R, P297V, D303C, D303R, E305K, Y in relation to the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 2 307K, Q353R, L444W, F465R, S469K, V470K, I498K, Y507R, Y507S, Y507W, Q510K, Q510V, Q510Y, P5 12G, P512V, Y513G, Y513L, Y513R, Y513S, S514P, S514R, T515P, K516P, K516R, K517P, I518P, L52 0M, F522A, A541V, G553M, N556E, N556R, D562I, D562K, D562V, N583V, L585K, F653R, K712R, N71 6R, E731P, N732T, A744N, A744P, A744T, F748A, S751A, K754G, I757K, I757W, E771R, N787A, A825 An isolated CRISPR / Cpf1 endonuclease system according to claim 26, comprising one or more amino acid substitutions selected from R, T826F, T826K, Y832R, Y836K, Y836Q, Y839L, F840L, Y907I, Y907V, Q916Y, E935R, I936R, K974G, K1019V, or combinations thereof.

30. ERCAS12A mutants include Q18V (sequence number 4); I35L (sequence number 6); G39Q (sequence number 8); G39K (sequence number 10); I40Q (sequence number 12); D56R (sequence number 14); D56P (sequence number 16); D59R (sequence number 18); D59A (sequence number 20); S158G (sequence number 22); K165Y (sequence number 24); S181V (sequence number 26); A290K (sequence number 28); D291N (sequence number 30); V296R (sequence number 32); P297V (sequence number 34); P297R (sequence number 36); P297F (sequence number 32) Number 38); D303R (Sequence ID 40); D303C (Sequence ID 42); E305K (Sequence ID 44); Y307K (Sequence ID 46); Q353R (Sequence ID 48); L444W (Sequence ID 50); F465R (Sequence ID 52); S469K (Sequence ID 54); V470K (Sequence ID 56); I498K (Sequence ID 58); Y507W (Sequence ID 60); Y507S (Sequence ID 62); Y507R (Sequence ID 64); Q510Y (Sequence ID 66); Q510V (Sequence ID 68); Q510K (Sequence ID 70); P512V (Sequence ID 72); P51 2G(Sequence ID 74);Y513S(Sequence ID 76);Y513R(Sequence ID 78);Y513L(Sequence ID 80);Y513G(Sequence ID 82);S514R(Sequence ID 84);S514P(Sequence ID 86);T515P(Sequence ID 88);K516R(Sequence ID 90);K516P(Sequence ID 92);K517P(Sequence ID 94);I518P(Sequence ID 96);L520M(Sequence ID 98);F522A(Sequence ID 100);A541V(Sequence ID 102);G553M(Sequence ID 104);N556R(Sequence ID 106);N556E(Array Number 108); D562V (Sequence ID 110); D562K (Sequence ID 112); D562I (Sequence ID 114); N583V (Sequence ID 116); L585K (Sequence ID 118); F653R (Sequence ID 120); K712R (Sequence ID 122); N716R (Sequence ID 124); E731P (Sequence ID 126); N732T (Sequence ID 128); A744T (Sequence ID 130); A744P (Sequence ID 132); A744N (Sequence ID 134); F748A (Sequence ID 136); S751A (Sequence ID 138); K754G (Sequence ID 140);I757W (sequence number 142); I757K (sequence number 144); E771R (sequence number 146); N787A (sequence number 148); A825R (sequence number 150); T826K (sequence number 152); T826F (sequence number 154); Y832R (sequence number 156); Y836Q (sequence number 158); Y836K (sequence number 160); Y839L (sequence number 162); F840L (sequence number 164); Y907V (distribution Column number 166); Y907I (Sequence number 168); Q916Y (Sequence number 170); E935R (Sequence number 172); I936R (Sequence number 174); K974G (Sequence number 176); K1019V (Sequence number 178); I35L / S469K (Sequence number 180); I35L / T826F (Sequence number 182); I40Q / S469K (Sequence number 184); I40Q / T826F (Sequence number 186); S469K / K5 16P (Sequence ID 188); S469K / T826F (Sequence ID 190); Y513G / Y832R (Sequence ID 192); Y513G / F840L (Sequence ID 194); I35L / S469K / T826F (Sequence ID 196); I40Q / S469K / T826F (Sequence ID 198); S469K / K516P / T826F (Sequence ID 200); S469K / T826F / F840L (Sequence ID 202); D303R An isolated CRISPR / Cpf1 endonuclease system according to claim 26, selected from / S469K / T826F / F840L (SEQ ID NO: 204); F465R / S469K / T826F / F840L (SEQ ID NO: 206); D303R / F465R / S469K / T826F / F840L (SEQ ID NO: 208); or D303R / S469K / K516P / T826F / F840L (SEQ ID NO: 210).

31. The isolated CRISPR / Cpf1 endonuclease system according to claim 26, wherein the ERCAS12A mutant is selected from D303R / S469K / T826F / F840L (SEQ ID NO: 204); F465R / S469K / T826F / F840L (SEQ ID NO: 206); D303R / F465R / S469K / T826F / F840L (SEQ ID NO: 208); or D303R / S469K / K516P / T826F / F840L (SEQ ID NO: 210).

32. A method for performing gene editing, comprising the step of contacting a candidate editing target site locus with a CRISPR ribonucleoprotein complex 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 provides improved gene editing compared to the wild-type ERCAS12A polypeptide.

33. The method according to claim 32, wherein the ERCAS12A mutant has a polypeptide sequence selected from even-numbered sequences of sequence numbers 4 to 210.

34. The ERCAS12A mutant has the following sequences in the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 18, 35, 39, 39, 40, 56, 56, 59, 59, 158, 165, 181, 290, 291, 296, 297, 297, 297, 303, 303, 305, 307, 353, 444, 465, 469, 470, 498, 507, 507, 507, 510, 510, 510, 512, 512, 513, 513, 513, 513, 514, 514, 515, 516, 5 The method according to claim 32, comprising one or more amino acid substitutions at positions 16, 517, 518, 520, 522, 541, 553, 556, 556, 562, 562, 562, 583, 585, 653, 712, 716, 731, 732, 744, 744, 744, 748, 751, 754, 757, 757, 771, 787, 825, 826, 826, 832, 836, 836, 839, 840, 907, 907, 916, 935, 936, 974, or 1019.

35. The ERCAS12A mutants are Q18V, I35L, G39K, G39Q, I40Q, D56P, D56R, D59A, D59R, S158G, K165Y, S181V, A290K, D291N, V296R, P297F, P297R, P297V, D303C, D303R, E 305K, Y307K, Q353R, L444W, F465R, S469K, V470K, I498K, Y507R, Y507S, Y507W, Q510K, Q510 V, Q510Y, P512G, P512V, Y513G, Y513L, Y513R, Y513S, S514P, S514R, T515P, K516P, K516R, K5 17P, I518P, L520M, F522A, A541V, G553M, N556E, N556R, D562I, D562K, D562V, N583V, L585K , F653R, K712R, N716R, E731P, N732T, A744N, A744P, A744T, F748A, S751A, K754G, I757K, I75 The method according to claim 32, comprising one or more amino acid substitutions selected from 7W, E771R, N787A, A825R, T826F, T826K, Y832R, Y836K, Y836Q, Y839L, F840L, Y907I, Y907V, Q916Y, E935R, I936R, K974G, K1019V, or combinations thereof.

36. ERCAS12A mutants include Q18V (sequence number 4); I35L (sequence number 6); G39Q (sequence number 8); G39K (sequence number 10); I40Q (sequence number 12); D56R (sequence number 14); D56P (sequence number 16); D59R (sequence number 18); D59A (sequence number 20); S158G (sequence number 22); K165Y (sequence number 24); S181V (sequence number 26); A290K (sequence number 28); D291N (sequence number 30); V296R (sequence number 32); P297V (sequence number 34); P297R (sequence number 36); P297F (sequence number 32) Number 38); D303R (Sequence ID 40); D303C (Sequence ID 42); E305K (Sequence ID 44); Y307K (Sequence ID 46); Q353R (Sequence ID 48); L444W (Sequence ID 50); F465R (Sequence ID 52); S469K (Sequence ID 54); V470K (Sequence ID 56); I498K (Sequence ID 58); Y507W (Sequence ID 60); Y507S (Sequence ID 62); Y507R (Sequence ID 64); Q510Y (Sequence ID 66); Q510V (Sequence ID 68); Q510K (Sequence ID 70); P512V (Sequence ID 72); P51 2G(Sequence ID 74);Y513S(Sequence ID 76);Y513R(Sequence ID 78);Y513L(Sequence ID 80);Y513G(Sequence ID 82);S514R(Sequence ID 84);S514P(Sequence ID 86);T515P(Sequence ID 88);K516R(Sequence ID 90);K516P(Sequence ID 92);K517P(Sequence ID 94);I518P(Sequence ID 96);L520M(Sequence ID 98);F522A(Sequence ID 100);A541V(Sequence ID 102);G553M(Sequence ID 104);N556R(Sequence ID 106);N556E(Array Number 108); D562V (Sequence ID 110); D562K (Sequence ID 112); D562I (Sequence ID 114); N583V (Sequence ID 116); L585K (Sequence ID 118); F653R (Sequence ID 120); K712R (Sequence ID 122); N716R (Sequence ID 124); E731P (Sequence ID 126); N732T (Sequence ID 128); A744T (Sequence ID 130); A744P (Sequence ID 132); A744N (Sequence ID 134); F748A (Sequence ID 136); S751A (Sequence ID 138); K754G (Sequence ID 140);I757W (sequence number 142); I757K (sequence number 144); E771R (sequence number 146); N787A (sequence number 148); A825R (sequence number 150); T826K (sequence number 152); T826F (sequence number 154); Y832R (sequence number 156); Y836Q (sequence number 158); Y836K (sequence number 160); Y839L (sequence number 162); F840L (sequence number 164); Y907V (sequence number 166); Y907I (sequence number 168); Q916Y (sequence number 170); E935R (sequence number 172); I936R (sequence number 174); K974G (sequence number 176); K1019V (sequence number 178); I35L / S469K (sequence number 180); I35L / T826F (sequence number 182); I40Q / S469K (sequence number 184); I40Q / T826F (sequence number Code 186); S469K / K516P (Sequence ID 188); S469K / T826F (Sequence ID 190); Y513G / Y832R (Sequence ID 192); Y513G / F840L (Sequence ID 194); I35L / S469K / T826F (Sequence ID 196); I40Q / S469K / T826F (Sequence ID 198); S469K / K516P / T826F (Sequence ID 200); S469K / T826F The method according to claim 32, selected from / F840L (sequence number 202); D303R / S469K / T826F / F840L (sequence number 204); F465R / S469K / T826F / F840L (sequence number 206); D303R / F465R / S469K / T826F / F840L (sequence number 208); or D303R / S469K / K516P / T826F / F840L (sequence number 210).

37. The method according to claim 32, wherein the ERCAS12A mutant is selected from D303R / S469K / T826F / F840L (SEQ ID NO: 204); F465R / S469K / T826F / F840L (SEQ ID NO: 206); D303R / F465R / S469K / T826F / F840L (SEQ ID NO: 208); or D303R / S469K / K516P / T826F / F840L (SEQ ID NO: 210).

38. A kit comprising a CRISPR ribonucleoprotein complex containing 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.

39. The kit according to claim 38, wherein the ERCAS12A mutant has a polypeptide sequence selected from even-numbered sequences of sequence numbers 4 to 210.

40. The ERCAS12A mutants are Q18V, I35L, G39K, G39Q, I40Q, D56P, D56R, D59A, D59R, S158G, K165Y, S181V, A290K, D291N, V296R, P297F, P297R, P297V, D303C, D303R, E 305K, Y307K, Q353R, L444W, F465R, S469K, V470K, I498K, Y507R, Y507S, Y507W, Q510K, Q510 V, Q510Y, P512G, P512V, Y513G, Y513L, Y513R, Y513S, S514P, S514R, T515P, K516P, K516R, K5 17P, I518P, L520M, F522A, A541V, G553M, N556E, N556R, D562I, D562K, D562V, N583V, L585K , F653R, K712R, N716R, E731P, N732T, A744N, A744P, A744T, F748A, S751A, K754G, I757K, I75 The kit according to claim 38, comprising one or more amino acid substitutions selected from 7W, E771R, N787A, A825R, T826F, T826K, Y832R, Y836K, Y836Q, Y839L, F840L, Y907I, Y907V, Q916Y, E935R, I936R, K974G, K1019V, or combinations thereof.

41. ERCAS12A mutants include Q18V (sequence number 4); I35L (sequence number 6); G39Q (sequence number 8); G39K (sequence number 10); I40Q (sequence number 12); D56R (sequence number 14); D56P (sequence number 16); D59R (sequence number 18); D59A (sequence number 20); S158G (sequence number 22); K165Y (sequence number 24); S181V (sequence number 26); A290K (sequence number 28); D291N (sequence number 30); V296R (sequence number 32); P297V (sequence number 34); P297R (sequence number 36); P297F (sequence number 32) Number 38); D303R (Sequence ID 40); D303C (Sequence ID 42); E305K (Sequence ID 44); Y307K (Sequence ID 46); Q353R (Sequence ID 48); L444W (Sequence ID 50); F465R (Sequence ID 52); S469K (Sequence ID 54); V470K (Sequence ID 56); I498K (Sequence ID 58); Y507W (Sequence ID 60); Y507S (Sequence ID 62); Y507R (Sequence ID 64); Q510Y (Sequence ID 66); Q510V (Sequence ID 68); Q510K (Sequence ID 70); P512V (Sequence ID 72); P51 2G(Sequence ID 74);Y513S(Sequence ID 76);Y513R(Sequence ID 78);Y513L(Sequence ID 80);Y513G(Sequence ID 82);S514R(Sequence ID 84);S514P(Sequence ID 86);T515P(Sequence ID 88);K516R(Sequence ID 90);K516P(Sequence ID 92);K517P(Sequence ID 94);I518P(Sequence ID 96);L520M(Sequence ID 98);F522A(Sequence ID 100);A541V(Sequence ID 102);G553M(Sequence ID 104);N556R(Sequence ID 106);N556E(Array Number 108); D562V (Sequence ID 110); D562K (Sequence ID 112); D562I (Sequence ID 114); N583V (Sequence ID 116); L585K (Sequence ID 118); F653R (Sequence ID 120); K712R (Sequence ID 122); N716R (Sequence ID 124); E731P (Sequence ID 126); N732T (Sequence ID 128); A744T (Sequence ID 130); A744P (Sequence ID 132); A744N (Sequence ID 134); F748A (Sequence ID 136); S751A (Sequence ID 138); K754G (Sequence ID 140);I757W (sequence number 142); I757K (sequence number 144); E771R (sequence number 146); N787A (sequence number 148); A825R (sequence number 150); T826K (sequence number 152); T826F (sequence number 154); Y832R (sequence number 156); Y836Q (sequence number 158); Y836K (sequence number 160); Y839L (sequence number 162); F840L (sequence number 164); Y907V (sequence number 166); Y907I (sequence number 168); Q916Y (sequence number 170); E935R (sequence number 172); I936R (sequence number 174); K974G (sequence number 176); K1019V (sequence number 178); I35L / S469K (sequence number 180); I35L / T826F (sequence number 182); I40Q / S469K (sequence number 184); I40Q / T826F (sequence number 186);S469K / K516P(Sequence ID 188);S469K / T826F(Sequence ID 190);Y513G / Y832R(Sequence ID 192);Y513G / F840L(Sequence ID 194);I35L / S469K / T826F(Sequence ID 196);I40Q / S469K / T826F(Sequence ID 198);S469K / K516P / T826F(Sequence ID 200);S469K / T826F / A kit according to claim 38, selected from F840L (sequence number 202); D303R / S469K / T826F / F840L (sequence number 204); F465R / S469K / T826F / F840L (sequence number 206); D303R / F465R / S469K / T826F / F840L (sequence number 208); or D303R / S469K / K516P / T826F / F840L (sequence number 210).

42. The kit according to claim 38, wherein the ERCAS12A mutant is selected from D303R / S469K / T826F / F840L (SEQ ID NO: 204); F465R / S469K / T826F / F840L (SEQ ID NO: 206); D303R / F465R / S469K / T826F / F840L (SEQ ID NO: 208); or D303R / S469K / K516P / T826F / F840L (SEQ ID NO: 210).

43. Use of ERCAS12A mutant polypeptide to improve gene editing, wherein the ERCAS12A mutant has one or more amino acid substitutions introduced into the wild-type ERCAS12A polypeptide sequence of SEQ ID NO: 2, and the ERCAS12A mutant provides improved gene editing compared to the wild-type ERCAS12A polypeptide.

44. The use according to claim 43, wherein the ERCAS12A mutant has a polypeptide sequence selected from even-numbered sequences of sequence numbers 4 to 210.

45. ERCAS12A mutants include Q18V (sequence number 4); I35L (sequence number 6); G39Q (sequence number 8); G39K (sequence number 10); I40Q (sequence number 12); D56R (sequence number 14); D56P (sequence number 16); D59R (sequence number 18); D59A (sequence number 20); S158G (sequence number 22); K165Y (sequence number 24); S181V (sequence number 26); A290K (sequence number 28); D291N (sequence number 30); V296R (sequence number 32); P297V (sequence number 34); P297R (sequence number 36); P297F (sequence number 32) Number 38); D303R (Sequence ID 40); D303C (Sequence ID 42); E305K (Sequence ID 44); Y307K (Sequence ID 46); Q353R (Sequence ID 48); L444W (Sequence ID 50); F465R (Sequence ID 52); S469K (Sequence ID 54); V470K (Sequence ID 56); I498K (Sequence ID 58); Y507W (Sequence ID 60); Y507S (Sequence ID 62); Y507R (Sequence ID 64); Q510Y (Sequence ID 66); Q510V (Sequence ID 68); Q510K (Sequence ID 70); P512V (Sequence ID 72); P51 2G(Sequence ID 74);Y513S(Sequence ID 76);Y513R(Sequence ID 78);Y513L(Sequence ID 80);Y513G(Sequence ID 82);S514R(Sequence ID 84);S514P(Sequence ID 86);T515P(Sequence ID 88);K516R(Sequence ID 90);K516P(Sequence ID 92);K517P(Sequence ID 94);I518P(Sequence ID 96);L520M(Sequence ID 98);F522A(Sequence ID 100);A541V(Sequence ID 102);G553M(Sequence ID 104);N556R(Sequence ID 106);N556E(Array Number 108); D562V (Sequence ID 110); D562K (Sequence ID 112); D562I (Sequence ID 114); N583V (Sequence ID 116); L585K (Sequence ID 118); F653R (Sequence ID 120); K712R (Sequence ID 122); N716R (Sequence ID 124); E731P (Sequence ID 126); N732T (Sequence ID 128); A744T (Sequence ID 130); A744P (Sequence ID 132); A744N (Sequence ID 134); F748A (Sequence ID 136); S751A (Sequence ID 138); K754G (Sequence ID 140);I757W (sequence number 142); I757K (sequence number 144); E771R (sequence number 146); N787A (sequence number 148); A825R (sequence number 150); T826K (sequence number 152); T826F (sequence number 154); Y832R (sequence number 156); Y836Q (sequence number 158); Y836K (sequence number 160); Y839L (sequence number 162); F840L (sequence number 164); Y907V (sequence number 166); Y907I (sequence number 168); Q916Y (sequence number 170); E935R (sequence number 172); I936R (sequence number 174); K974G (sequence number 176); K1019V (sequence number 178); I35L / S469K (sequence number 180); I35L / T826F (sequence number 182); I40Q / S469K (sequence number 184); I40Q / T826F (sequence number Code 186); S469K / K516P (Sequence ID 188); S469K / T826F (Sequence ID 190); Y513G / Y832R (Sequence ID 192); Y513G / F840L (Sequence ID 194); I35L / S469K / T826F (Sequence ID 196); I40Q / S469K / T826F (Sequence ID 198); S469K / K516P / T826F (Sequence ID 200); S469K / T826F The use described in claim 43, selected from / F840L (sequence number 202); D303R / S469K / T826F / F840L (sequence number 204); F465R / S469K / T826F / F840L (sequence number 206); D303R / F465R / S469K / T826F / F840L (sequence number 208); or D303R / S469K / K516P / T826F / F840L (sequence number 210).

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