A CAS12A mutant of Lacnospirace SP. with enhanced cleavage activity via a non-canonical TTTT protospacer-adjacent motif.

Abstract

Described herein are CAS12A mutants from Lachnospiraceae bacterium and methods for their use. These mutants have enhanced DNA cleavage activity at the non-canonical TTTT protospacer adjacent motif (PAM) compared to the wild-type enzyme.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 018,592, filed May 1, 2020, and No. 63 / 090,912, filed October 13, 2020, the contents of which are hereby incorporated by reference in their entirety. This application is related to International Application PCT / US2020 / 019168, filed February 21, 2020, and published as International Patent Application WO2020 / 172502 A1 on August 27, 2020, the contents of which are hereby incorporated by reference in their entirety.

[0002] Reference to Sequence Listing This application is filed with a computer - readable form of the sequence listing in accordance with 37 C.F.R. §1.821(c). The text file, "013670 - 9067 - WO01_sequence_listing_27 - APR - 2021_ST25.txt", submitted via EFS, was created on April 27, 2021, contains 5320 sequences, and has a file size of 30.2 megabytes, the contents of which are hereby incorporated by reference in their entirety.

[0003] This specification describes Cas12a variants derived from Lachnospiraceae bacterium and methods of using them. These variants have enhanced DNA cleavage activity at non - canonical TTTT protospacer - adjacent motifs (PAMs) compared to the wild - type enzyme.

Background Art

[0004] LbCas12a is an RNA guide endonuclease derived from the clustered, regularly spaced, short, palindromic repeat (CRISPR) adaptive immune system of the Lachnospirace bacterium ND2006 (Lb). See Zetsche et al., Cell 163: pp. 759-771 (2015). The Cas12a nuclease is classified as a class 2 V CRISPR system and, when it forms a complex with CRISPR RNA (crRNA), it results in alternating DNA double-strand breaks with a 5' overhang of 5 nucleotides. The LbCas12a:crRNA complex is called the CRISPR ribonucleoprotein (RNP) complex.

[0005] Cas12a is induced at a 21-24 nucleotide DNA target sequence, commonly known as a protospacer, by a target-site-specific complementary guide RNA (gRNA) of 21-24 nucleotides. The Cas12a-gRNA RNP complex mediates double-strand DNA breaks (DSBs), which are then repaired by either non-homologous end joining (NHEJ, usually introducing mutations or indels at the break site) or, if a suitable template nucleic acid is present, by a homologous recombination repair (HDR) system for precise editing.

[0006] Precise recognition of DNA targets by LbCas12a requires both crRNA and a canonical "TTTV" protospacer adjacency motif (PAM), which is a 4bp sequence immediately upstream of the protospacer, in contrast to the 2bp NGG PAM of Cas9 from Streptococcus pyogenes. See Jinek et al., Science 337:816-821 (2012). Cas12a has expanded the targetable loci in genome editing, particularly over T-rich sites that are inaccessible to the Cas9 system. Although targetable sites for Cas12a on the genome appear less frequently than those for Cas9, LbCas12a has gained considerable popularity in plant genome engineering due to its editing activity at ambient temperatures (20-30°C). Improving the cleavage activity and associated genome editing efficiency of LbCas12a is expected to significantly accelerate the development of agricultural products with enhanced properties.

[0007] It is desirable to further improve the utility of LbCas12a by enhancing its on-target activity through protein mutagenesis. Previous studies have been conducted to improve the utility of Cas12a (also known as AsCas12a) derived from the species Acidaminococcus sp. See U.S. Patent Application Publication US2020 / 0109382A1, which is incorporated herein by reference. However, transferring beneficial mutations identified in AsCas12a into the LbCas12a polypeptide sequence has not been straightforward. Despite the significant structural and sequence similarities between the two homologous Cas12a enzymes, most point mutations that improved the activity of AsCas12a are detrimental to LbCas12a. See Schindele and Puchta, Plant Biotechnol. J. 18(5):1118-1120 (2020).

[0008] What is needed is a novel LbCas12a variant that enhances the activity of this enzyme in non-canonical TTTT protospacer-adjacent motifs, particularly in human cells. [Overview of the Initiative]

[0009] One embodiment described herein is an isolated mutant LbCas12a polypeptide comprising at least one amino acid substitution introduced into the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2, provided that the mutant LbCas12a polypeptide provides improved CRISPR / LbCas12a-related nuclease activity at a non-canonical TTTT PAM site compared to the wild-type LbCas12a polypeptide sequence. In one embodiment, the mutant LbCas12a polypeptide has 95% to 99% identity with a polypeptide sequence selected from even-numbered sequences of SEQ ID NOs: 4 to 4022. In another embodiment, the mutant LbCas12a polypeptide has a polypeptide sequence selected from even-numbered sequences of SEQ ID NOs: 4 to 4022. In another embodiment, the mutant LbCas12a polypeptide includes at least one amino acid substitution at the position of 81, 125, 145, 146, 152, 182, 396, 478, 491, 582, 595, 606, 646, 665, 795, 799, 814, 839, 841, 858, 860, 890, 898, 909, 913, 981, 1020, 1083, or 1121 of the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2. In another embodiment, the mutant LbCas12a polypeptide comprises at least one amino acid substitution selected from F81E, E125K, N145R, G146R, T152K, R182V, S396D, K478R, V491D, N582R, K595R, Y606F, Y646H, D665N, E795Q, P799V, T814G, L839F, I841A, E858V, I860R, W890A, E898N, H909K, E913R, E981V, S1020E, V1083W, or K1121D of the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2. In another embodiment, mutant LbCas12a polypeptides include: F81E, SEQ ID NO: 802; E125K, SEQ ID NO: 428; N145R, SEQ ID NO: 2902; G146R, SEQ ID NO: 156; T152K, SEQ ID NO: 694; R182V, SEQ ID NO: 98; S396D, SEQ ID NO: 108; K478R, SEQ ID NO: 134; V491D, SEQ ID NO: 954; N582R, SEQ ID NO: 1730; K595R, SEQ ID NO: 184; Y606F,Select from sequence numbers 642;Y646H, 720;D665N, 1014;E795Q, 930;P799V, 706;T814G, 162;L839F, 834;I841A, 988;E858V, 114;I860R, 250;W890A, 3958;E898N, 48;H909K, 70;E913R, 1960;E981V, 880;S1020E, 532;V1083W, 132; or K1121D, 598. In another embodiment, a single substitution mutation introduced into the wild-type LbCas12a polypeptide sequence of Sequence ID No. 2, wherein the mutant LbCas12a polypeptide is at the following positions: (a) F81, E125, N145, G146, T152, R182, S396, K478, V491, N582, K595, Y606, Y646, D665, E795, P799, T814, L839, I841, E858, I860, W890, E898, H909, E913, E981, S1020, V1083, or K1121; Or (b) the following positions: containing a substitution mutation selected from the polysubstitution mutations introduced into the wild-type LbCas12a polypeptide sequence of SEQ ID NO: F81, E125, N145, G146, T152, R182, S396, K478, V491, N582, K595, Y606, Y646, D665, E795, P799, T814, L839, I841, E858, I860, W890, E898, H909, E913, E981, S1020, V1083 or K1121. In another embodiment, polysubstitution mutations include: E88A / E795Q, E125K / E795Q, G146R / E795Q, R182V / E795Q, V491D / E795Q, Q529I / E795Q, Y646H / E795Q, D665N / E795Q, T814K / E795Q, L839F / E795Q, Q906F / E795Q, E795Q / Q1170D, G146R / R182V / E795Q, G146R / E795Q / D665N, G146R / E795Q / E981V, G146R / E795Q / T814K, G146R / R182V / D665N, E125K / R182V / E981V,R182V / P799V / E981V, R182V / T814K / E981V, G146R / P799V / E981V, G146R / R182V / E795Q / F81E, G146R / R18 2V / E795Q / E125K, G146R / R182V / E795Q / E125A, G146R / R182V / E795Q / P799V, G146R / R182V / E795Q / T814K, Includes G146R / R182V / E795Q / E981V, G146R / R182V / E795Q / D665N, G146R / R182V / P799V / E981V, E125K / G146R / R182V / E795Q / D665N, G146R / R182V / E795Q / P799V / T814K, or E125K / G146R / R182V / E795Q / D665N / E981V. In another embodiment, polysubstitution mutations include: E88A / E795Q, E125K / E795Q, G146R / E795Q, R182V / E795Q, V491D / E795Q, Q529I / E795Q, Y646H / E795Q, D665N / E795Q, T814K / E795Q, L839F / E795Q, Q906F / E795Q, E795Q / Q1170D, G146R / R182V / E795Q, G146R / E795Q / D665N, G146R / E795Q / E981V, G146R / E795Q / T 814K, G146R / R182V / D665N, E125K / R182V / E981V, R182V / P799V / E981V, R182V / T814K / E981V , G146R / P799V / E981V, G146R / R182V / E795Q / F81E, G146R / R182V / E795Q / E125K, G146R / R18 2V / E795Q / E125A, G146R / R182V / E795Q / P799V, G146R / R182V / E795Q / T814K, G146R / R182V / Including E795Q / E981V, G146R / R182V / E795Q / D665N, G146R / R182V / P799V / E981V, E125K / G146R / R182V / E795Q / D665N, or G146R / R182V / E795Q / P799V / T814K, compared to the wild-type LbCas12a polypeptide sequence,In another embodiment, the mutant LbCas12a polypeptide provides improved CRISPR / LbCas12a-related nuclease activity at the non-canonical TTTT PAM site. Select from 6, 3968, 3970, 3972, 3974, 3976, 3978, 3980, 3982, 3984, 3986, 3988, 3990, 3992, 3994, 3996, 3998, 4000, 4002, 4004, 4006, 4008, 4010, 4012, 4014, 4016, 4018, 4020, or 4022. In another embodiment, mutant LbCas12a polypeptides include: F81E (SEQ ID NO: 802), E125K (SEQ ID NO: 428), N145R (SEQ ID NO: 2902), G146R (SEQ ID NO: 156), T152K (SEQ ID NO: 694), R182V (SEQ ID NO: 98), V491D (SEQ ID NO: 954), K478R (SEQ ID NO: 134), N582R (SEQ ID NO: 1730), D665N (SEQ ID NO: 1014), E795Q (SEQ ID NO: 930), P799V (SEQ ID NO: 706), E858V (SEQ ID NO: 114), I860R (SEQ ID NO: 250), E913R (SEQ ID NO: 1960), E981V (SEQ ID NO: 880), E88A / E795Q (SEQ ID NO: 3960), E125K / E795Q (SEQ ID NO: 3962), G146R / E795Q (SEQ ID NO: 3964), R182V / E795Q (SEQ ID NO: 3966), V491D / E795Q (SEQ ID NO: 3968), Q529I / E795Q (SEQ ID NO: 3970), Y646H / E795Q (SEQ ID NO: 3972), D665N / E795Q (SEQ ID NO: 3974), T814K / E795Q (SEQ ID NO: 3976), L839F / E795Q (SEQ ID NO: 3964) 78), Q906F / E795Q (SEQ ID NO: 3980), E795Q / Q1170D (SEQ ID NO: 3982), G146R / R182V / E795Q (SEQ ID NO: 3984), G146R / E795Q / D665N (SEQ ID NO: 3986), G146R / E795Q / E981V (SEQ ID NO: 3988), G146R / E795Q / T814K (SEQ ID NO: 3990),G146R / R182V / D665N (Sequence ID 3992), E125K / R182V / E981V (Sequence ID 3994), R182V / P799V / E981V (Sequence ID 3996), R182V / T814K / E981V (Sequence ID 3998), G146R / P799V / E981V (Sequence ID 4000), G146R / R182V / E795Q / F81E (Sequence ID 4002), G146R / R182V / E795Q / E125K (Sequence ID 4004), G146R / R182V / E795Q / E125A (Sequence ID 4006), G146R / R18 Select from 2V / E795Q / P799V (sequence number 4008), G146R / R182V / E795Q / T814K (sequence number 4010), G146R / R182V / E795Q / E981V (sequence number 4012), G146R / R182V / E795Q / D665N (sequence number 4014), G146R / R182V / P799V / E981V (sequence number 4016), E125K / G146R / R182V / E795Q / D665N (sequence number 4018), or G146R / R182V / E795Q / P799V / T814K (sequence number 4020). In another embodiment, the mutant LbCas12a polypeptide is G146R / R182V / E795Q (SEQ ID NO: 3984).

[0010] Another embodiment described herein is an isolated polynucleotide sequence encoding a mutant LbCas12a polypeptide comprising at least one amino acid substitution introduced into the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2, provided that the mutant LbCas12a polypeptide provides improved CRISPR / LbCas12a-related nuclease activity at a non-canonical TTTT PAM site compared to the wild-type LbCas12a polypeptide sequence. In one embodiment, the encoded mutant LbCas12a polypeptide has 95% to 99% identity with a polypeptide sequence selected from even-numbered sequences of SEQ ID NOs: 4 to 4022. In another embodiment, the encoded mutant LbCas12a polypeptide has a polypeptide sequence selected from even-numbered sequences of SEQ ID NOs: 4 to 4022. In yet another embodiment, the mutant LbCas12a polynucleotide sequence has 95% to 99% identity with a nucleotide sequence selected from odd-numbered sequences of SEQ ID NOs: 3 to 4021. In another embodiment, the mutant LbCas12a polynucleotide has a nucleotide sequence selected from the odd-numbered sequences of SEQ ID NOs: 3 to 4021. In yet another embodiment, the encoded mutant LbCas12a polypeptide has at least one amino acid substitution at the position of 81, 125, 145, 146, 152, 182, 396, 478, 491, 582, 595, 606, 646, 665, 795, 799, 814, 839, 841, 858, 860, 890, 898, 909, 913, 981, 1020, 1083, or 1121 of the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2. In another embodiment, the encoded mutant LbCas12a polypeptide, compared to the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2, is F81E, E125K, N145R, G146R, T152K, R182V, S396D, K478R, V491D, N582R, K595R, Y606F, Y646H, D665N, E795Q, P799V, T814G, L839F, I841A, E858V, I860R, W890A, E898N, H909K, E913R, E981V, S1020E,The mutant LbCas12a polynucleotide comprises at least one amino acid substitution selected from V1083W or K1121D. In another embodiment, the mutant LbCas12a polynucleotide comprises: F81E, SEQ ID NO: 802; E125K, SEQ ID NO: 428; N145R, SEQ ID NO: 2902; G146R, SEQ ID NO: 156; T152K, SEQ ID NO: 694; R182V, SEQ ID NO: 98; S396D, SEQ ID NO: 108; K478R, SEQ ID NO: 134; V491D, SEQ ID NO: 954; N582R, SEQ ID NO: 1730; K595R, SEQ ID NO: 184; Y606F, SEQ ID NO: 642; Y646H, SEQ ID NO: 720; D665N, SEQ ID NO: 1014 Selected from ;E795Q, SEQ ID NO: 930;P799V, SEQ ID NO: 706;T814G, SEQ ID NO: 162;L839F, SEQ ID NO: 834;I841A, SEQ ID NO: 988;E858V, SEQ ID NO: 114;I860R, SEQ ID NO: 250;W890A, SEQ ID NO: 3958;E898N, SEQ ID NO: 48;H909K, SEQ ID NO: 70;E913R, SEQ ID NO: 1960;E981V, SEQ ID NO: 880;S1020E, SEQ ID NO: 532;V1083W, SEQ ID NO: 132;or K1121D, SEQ ID NO: 598. In another embodiment, the encoded mutant LbCas12a polypeptide is a single substitution introduced into the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2, at the following positions: (a) F81, E125, N145, G146, T152, R182, S396, K478, V491, N582, K595, Y606, Y646, D665, E795, P799, T814, L839, I841, E858, I860, W890, E898, H909, E913, E981, S1020, V1083, or K1121. Mutations; or (b) at the following positions: F81, E125, N145, G146, T152, R182, S396, K478, V491, N582, K595, Y606, Y646, D665, E795, P799, T814, L839, I841, E858, I860, W890, E898, H909, E913, E981, S1020, V1083 or K1121, comprising substitution mutations selected from polysubstitution mutations introduced into the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2. In another embodiment, the polysubstitution mutations include: E88A / E795Q, E125K / E795Q,G146R / E795Q, R182V / E795Q, V491D / E795Q, Q529I / E795Q, Y646H / E795Q, D665N / E795Q, T814K / E795Q, L839F / E795Q, Q906F / E795Q, E795Q / Q1170D, G146R / R182V / E795Q, G146R / E795Q / D665N, G146R / E795Q / E981V, G146R / E795Q / T814K, G146R / R182V / D665N, E125K / R182V / E981V, R182V / P799V / E981V, R182V / T814K / E981V, G146R / P799V / E981V, G146R / R182V / E795Q / F81E, G146R / R182V / E795Q / E125K, G146R / R182V / E795Q / E125A, G146R / R182V / E795Q / P799V, G146R / R182V / E795Q / T814K, G146R / R182V / E795Q / E981V, G146R / Includes R182V / E795Q / D665N, G146R / R182V / P799V / E981V, E125K / G146R / R182V / E795Q / D665N, G146R / R182V / E795Q / P799V / T814K, or E125K / G146R / R182V / E795Q / D665N / E981V. In another embodiment, polysubstitution mutations include: E88A / E795Q, E125K / E795Q, G146R / E795Q, R182V / E795Q, V491D / E795Q, Q529I / E795Q, Y646H / E795Q, D665N / E795Q, T814K / E795Q, L839F / E795Q, Q906F / E795Q, E795Q / Q1170D, G146R / R182V / E795Q, G146R / E795Q / D665N, G146R / E795Q / E 981V, G146R / E795Q / T814K, G146R / R182V / D665N, E125K / R182V / E981V, R182V / P799V / E981V, R182V / T814K / E981V, G146R / P79 9V / E981V, G146R / R182V / E795Q / F81E, G146R / R182V / E795Q / E125K, G146R / R182V / E795Q / E125A, G146R / R182V / E795Q / P799V,The following polypeptide sequences, comprising G146R / R182V / E795Q / T814K, G146R / R182V / E795Q / E981V, G146R / R182V / E795Q / D665N, G146R / R182V / P799V / E981V, E125K / G146R / R182V / E795Q / D665N, or G146R / R182V / E795Q / P799V / T814K, provide improved CRISPR / LbCas12a-related nuclease activity at non-canonical TTTT PAM sites compared to wild-type LbCas12a polypeptide sequences. In another embodiment, the encoded mutant LbCas12a polypeptides are: F81E (SEQ ID NO: 802), E125K (SEQ ID NO: 428), N145R (SEQ ID NO: 2902), G146R (SEQ ID NO: 156), T152K (SEQ ID NO: 694), R182V (SEQ ID NO: 98), V491D (SEQ ID NO: 954), K478R (SEQ ID NO: 134), N582R (SEQ ID NO: 1730), D665N (SEQ ID NO: 1014), E795Q (SEQ ID NO: 930), P799V (SEQ ID NO: 706), E858V (SEQ ID NO: 114), I860R (SEQ ID NO: 250), E913R (SEQ ID NO: 1960), E981V (SEQ ID NO: 880), E88A / E795Q (SEQ ID NO: 3960), E125K / E795Q (SEQ ID NO: 3962), G146R / E795Q (SEQ ID NO: 3964), R182V / E795Q (SEQ ID NO: 3966), V491D / E795Q (SEQ ID NO: 3968), Q529I / E795Q (SEQ ID NO: 3970), Y646H / E795Q (SEQ ID NO: 3972), D665N / E795Q (SEQ ID NO: 3974), T814K / E795Q (SEQ ID NO: 3976), L839F / E795Q (SEQ ID NO: 3978), Q906F / E795Q (SEQ ID NO: 3980), E795Q / Q1170D (SEQ ID NO: 3982), G146R / R182V / E795Q (SEQ ID NO: 3984), G146R / E795Q / D665N (SEQ ID NO: 3986), G146R / E795Q / E981V (SEQ ID NO: 3988), G146R / E795Q / T814K (SEQ ID NO: 3990), G146R / R182V / D665N (SEQ ID NO: 3992), E125K / R182V / E981V (SEQ ID NO: 3994), R182V / P799V / E981V (SEQ ID NO: 3996),R182V / T814K / E981V (SEQ ID NO: 3998), G146R / P799V / E981V (SEQ ID NO: 4000), G146R / R182V / E795Q / F81E (SEQ ID NO: 4002), G146R / R182V / E795Q / E125K (SEQ ID NO: 4004), G146R / R182V / E795Q / E125A (SEQ ID NO: 4006), G146R / R182V / E795Q / P799V (SEQ ID NO: 4008), G146R / R182V / E795Q The following are selected from / T814K (SEQ ID NO: 4010), G146R / R182V / E795Q / E981V (SEQ ID NO: 4012), G146R / R182V / E795Q / D665N (SEQ ID NO: 4014), G146R / R182V / P799V / E981V (SEQ ID NO: 4016), E125K / G146R / R182V / E795Q / D665N (SEQ ID NO: 4018), or G146R / R182V / E795Q / P799V / T814K (SEQ ID NO: 4020). In another embodiment, the encoded mutant LbCas12a polypeptide is G146R / R182V / E795Q (SEQ ID NO: 3984). In another embodiment, mutant LbCas12a polynucleotides are: SEQ ID NOs: 801, 427, 2901, 155, 693, 97, 953, 133, 1729, 641, 719, 1013, 929, 705, 833, 987, 113, 249, 3957, 47, 69, 1959, 879, 531, 597, 3959, 3961, 3963, 396 Select from 5, 3967, 3969, 3971, 3973, 3975, 3977, 3979, 3981, 3983, 3985, 3987, 3989, 3991, 3993, 3995, 3997, 3999, 4001, 4003, 4005, 4007, 4009, 4011, 4013, 4015, 4017, 4019, or 4021. In another embodiment, the mutant LbCas12a polynucleotides are SEQ ID NOs: 801, 427, 2901, 155, 693, 97, 953, 133, 1729, 1013, 929, 705, 113, 249, 531, 597, 3959, 3961, 3963, 3965, 3967, 3969, 3971, 3973, 3975, 3977, 3979, 3981, 3983, 3985, 3987, 3989, 3991, 3993, 3995, 3997, 3999, 4001, 4003, 4005,Selected from 4007, 4009, 4011, 4013, 4015, 4017, or 4019, these sequences encode mutant LbCas12a polypeptides that provide improved CRISPR / LbCas12a-related nuclease activity at non-canonical TTTT PAM sites compared to wild-type LbCas12a polypeptide sequences. In another embodiment, the mutant LbCas12a polynucleotide is sequence number 3983.

[0011] Another embodiment described herein is a vector or plasmid comprising any of the polynucleotide sequences described herein.

[0012] Another embodiment described herein is a cell comprising either the polynucleotide sequence described herein or the vector or plasmid described herein.

[0013] Another embodiment described herein is an isolated ribonucleoprotein complex comprising a guide RNA and a mutant LbCas12a polypeptide having at least one amino acid substitution introduced into the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2, provided that the mutant LbCas12a polypeptide provides improved CRISPR / LbCas12a-related nuclease activity at a non-canonical TTTT PAM site compared to the wild-type LbCas12a polypeptide sequence. In one embodiment, the mutant LbCas12a polypeptide has a polypeptide sequence selected from even-numbered sequences of SEQ ID NOs: 4-4022. In another embodiment, the mutant LbCas12a polypeptide includes at least one amino acid substitution at the position of 81, 125, 145, 146, 152, 182, 396, 478, 491, 582, 595, 606, 646, 665, 795, 799, 814, 839, 841, 858, 860, 890, 898, 909, 913, 981, 1020, 1083, or 1121 of the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2. In another embodiment, the mutant LbCas12a polypeptide comprises at least one amino acid substitution selected from F81E, E125K, N145R, G146R, T152K, R182V, S396D, K478R, V491D, N582R, K595R, Y606F, Y646H, D665N, E795Q, P799V, T814G, L839F, I841A, E858V, I860R, W890A, E898N, H909K, E913R, E981V, S1020E, V1083W, or K1121D of the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2. In another embodiment, mutant LbCas12a polypeptides include: F81E, SEQ ID NO: 802; E125K, SEQ ID NO: 428; N145R, SEQ ID NO: 2902; G146R, SEQ ID NO: 156; T152K, SEQ ID NO: 694; R182V, SEQ ID NO: 98; S396D, SEQ ID NO: 108; K478R, SEQ ID NO: 134; V491D, SEQ ID NO: 954; N582R, SEQ ID NO: 1730; K595R, SEQ ID NO: 184; Y606F, SEQ ID NO: 642; Y646H, SEQ ID NO: 720; D665N, SEQ ID NO: 1014; E795Q, SEQ ID NO: 930; P799V,Select from sequence numbers 706;T814G, 162;L839F, 834;I841A, 988;E858V, 114;I860R, 250;W890A, 3958;E898N, 48;H909K, 70;E913R, 1960;E981V, 880;S1020E, 532;V1083W, 132; or K1121D, 598. In another embodiment, mutant LbCas12a polypeptides include: F81E (SEQ ID NO: 802), E125K (SEQ ID NO: 428), N145R (SEQ ID NO: 2902), G146R (SEQ ID NO: 156), T152K (SEQ ID NO: 694), R182V (SEQ ID NO: 98), V491D (SEQ ID NO: 954), K478R (SEQ ID NO: 134), N582R (SEQ ID NO: 1730), D665N (SEQ ID NO: 1014), E795Q (SEQ ID NO: 930), P799 V (sequence number 706), E858V (sequence number 114), I860R (sequence number 250), E913R (sequence number 1960), E981V (sequence number 880), E88A / E795Q (sequence number 3960), E125K / E795Q (sequence number 3962), G146R / E795Q (sequence number 3964), R182V / E795Q (sequence number 3966), V491D / E795Q (sequence number 3968), Q529I / E795Q (sequence number (No. 3970), Y646H / E795Q (Sequence ID 3972), D665N / E795Q (Sequence ID 3974), T814K / E795Q (Sequence ID 3976), L839F / E795Q (Sequence ID 3978), Q906F / E795Q (Sequence ID 3980), E795Q / Q1170D (Sequence ID 3982), G146R / R182V / E795Q (Sequence ID 3984), G146R / E795Q / D665N (Sequence ID 3986), G146R / E795Q / E981V (SEQ ID NO: 3988), G146R / E795Q / T814K (SEQ ID NO: 3990), G146R / R182V / D665N (SEQ ID NO: 3992), E125K / R182V / E981V (SEQ ID NO: 3994), R182V / P799V / E981V (SEQ ID NO: 3996), R182V / T814K / E981V (SEQ ID NO: 3998), G146R / P799V / E981V (SEQ ID NO: 4000),G146R / R182V / E795Q / F81E (Sequence ID 4002), G146R / R182V / E795Q / E125K (Sequence ID 4004), G146R / R182V / E795Q / E125A (Sequence ID 4006), G146R / R182V / E795Q / P799V (Sequence ID 4008), G146R / R182V / E795Q / T814K (Sequence ID 4010), G146R / R182V The mutant LbCas12a polypeptide is selected from / E795Q / E981V (SEQ ID NO: 4012), G146R / R182V / E795Q / D665N (SEQ ID NO: 4014), G146R / R182V / P799V / E981V (SEQ ID NO: 4016), E125K / G146R / R182V / E795Q / D665N (SEQ ID NO: 4018), or G146R / R182V / E795Q / P799V / T814K (SEQ ID NO: 4020). In another embodiment, the mutant LbCas12a polypeptide is G146R / R182V / E795Q (SEQ ID NO: 3984).

[0014] Another embodiment described herein is a method for enhancing the efficiency of gene editing at a non-canonical TTTT PAM site in a cell using a CRISPR ribonucleoprotein complex, comprising the step of contacting a cell with a CRISPR ribonucleoprotein complex comprising a guide RNA and a mutant LbCas12a polypeptide having at least one amino acid substitution introduced into the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2, provided that the mutant LbCas12a polypeptide provides improved CRISPR / LbCas12a-related nuclease activity at a non-canonical TTTT PAM site compared to the wild-type LbCas12a polypeptide sequence. In one embodiment, the mutant LbCas12a polypeptide has a polypeptide sequence selected from even-numbered sequences of SEQ ID NOs: 4 to 4022. In another embodiment, the mutant LbCas12a polypeptide includes at least one amino acid substitution at the position of 81, 125, 145, 146, 152, 182, 396, 478, 491, 582, 595, 606, 646, 665, 795, 799, 814, 839, 841, 858, 860, 890, 898, 909, 913, 981, 1020, 1083, or 1121 of the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2. In another embodiment, the mutant LbCas12a polypeptide comprises at least one amino acid substitution selected from F81E, E125K, N145R, G146R, T152K, R182V, S396D, K478R, V491D, N582R, K595R, Y606F, Y646H, D665N, E795Q, P799V, T814G, L839F, I841A, E858V, I860R, W890A, E898N, H909K, E913R, E981V, S1020E, V1083W, or K1121D of the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2. In another embodiment, mutant LbCas12a polypeptides include: F81E, SEQ ID NO: 802; E125K, SEQ ID NO: 428; N145R, SEQ ID NO: 2902; G146R, SEQ ID NO: 156; T152K, SEQ ID NO: 694; R182V, SEQ ID NO: 98; S396D, SEQ ID NO: 108; K478R, SEQ ID NO: 134; V491D, SEQ ID NO: 954; N582R, SEQ ID NO: 1730; K595R,Select from sequence numbers 184;Y606F, 642;Y646H, 720;D665N, 1014;E795Q, 930;P799V, 706;T814G, 162;L839F, 834;I841A, 988;E858V, 114;I860R, 250;W890A, 3958;E898N, 48;H909K, 70;E913R, 1960;E981V, 880;S1020E, 532;V1083W, 132; or K1121D, 598. In another embodiment, a single substitution mutation is introduced into the wild-type LbCas12a polypeptide sequence of Sequence ID No. 2, in which a mutant LbCas12a polypeptide is introduced at (a) a position selected from: F81, E125, N145, G146, T152, R182, S396, K478, V491, N582, K595, Y606, Y646, D665, E795, P799, T814, L839, I841, E858, I860, W890, E898, H909, E913, E981, S1020, V1083, or K1121; Or (b) the following positions: containing a substitution mutation selected from the polysubstitution mutations introduced into the wild-type LbCas12a polypeptide sequence of SEQ ID NO: F81, E125, N145, G146, T152, R182, S396, K478, V491, N582, K595, Y606, Y646, D665, E795, P799, T814, L839, I841, E858, I860, W890, E898, H909, E913, E981, S1020, V1083 or K1121. In another embodiment, polysubstitution mutations include: E88A / E795Q, E125K / E795Q, G146R / E795Q, R182V / E795Q, V491D / E795Q, Q529I / E795Q, Y646H / E795Q, D665N / E795Q, T814K / E795Q, L839F / E795Q, Q906F / E795Q, E795Q / Q1170D, G146R / R182V / E795Q, G146R / E795Q / D665N, G146R / E795Q / E981V, G146R / E795Q / T814K, G146R / R182V / D665N, E125K / R182V / E981V,R182V / P799V / E981V, R182V / T814K / E981V, G146R / P799V / E981V, G146R / R182V / E795Q / F81E, G146R / R182V / E795Q / E125K, G146R / R182V / E795Q / E125A, G146R / R182V / E795Q / P799V, G146R / Includes R182V / E795Q / T814K, G146R / R182V / E795Q / E981V, G146R / R182V / E795Q / D665N, G146R / R182V / P799V / E981V, E125K / G146R / R182V / E795Q / D665N, or G146R / R182V / E795Q / P799V / T814K. In another embodiment, mutant LbCas12a polypeptides include: F81E (SEQ ID NO: 802), E125K (SEQ ID NO: 428), N145R (SEQ ID NO: 2902), G146R (SEQ ID NO: 156), T152K (SEQ ID NO: 694), R182V (SEQ ID NO: 98), V491D (SEQ ID NO: 954), K478R (SEQ ID NO: 134), N582R (SEQ ID NO: 1730), D665N (variables). Column number 1014), E795Q (sequence number 930), P799V (sequence number 706), E858V (sequence number 114), I860R (sequence number 250), E913R (sequence number 1960), E981V (sequence number 880), E88A / E795Q (sequence number 3960), E125K / E795Q (sequence number 3962), G146R / E795Q (sequence number 3964), R18 2V / E795Q (SEQ ID NO: 3966), V491D / E795Q (SEQ ID NO: 3968), Q529I / E795Q (SEQ ID NO: 3970), Y646H / E795Q (SEQ ID NO: 3972), D665N / E795Q (SEQ ID NO: 3974), T814K / E795Q (SEQ ID NO: 3976), L839F / E795Q (SEQ ID NO: 3978), Q906F / E795Q (SEQ ID NO: 3976) 980), E795Q / Q1170D (Sequence ID 3982), G146R / R182V / E795Q (Sequence ID 3984), G146R / E795Q / D665N (Sequence ID 3986), G146R / E795Q / E981V (Sequence ID 3988), G146R / E795Q / T814K (Sequence ID 3990), G146R / R182V / D665N (Sequence ID 3992),E125K / R182V / E981V (SEQ ID NO: 3994), R182V / P799V / E981V (SEQ ID NO: 3996), R182V / T814K / E981V (SEQ ID NO: 3998), G146R / P799V / E981V (SEQ ID NO: 4000), G146R / R182V / E795Q / F81E (SEQ ID NO: 4002), G146R / R182V / E795Q / E125K (SEQ ID NO: 4004), G146R / R182V / E795Q / E125A (SEQ ID NO: 4006), G146R / R182V / E795Q / P799V Select from (Sequence ID 4008), G146R / R182V / E795Q / T814K (Sequence ID 4010), G146R / R182V / E795Q / E981V (Sequence ID 4012), G146R / R182V / E795Q / D665N (Sequence ID 4014), G146R / R182V / P799V / E981V (Sequence ID 4016), E125K / G146R / R182V / E795Q / D665N (Sequence ID 4018), or G146R / R182V / E795Q / P799V / T814K (Sequence ID 4020). In another embodiment, the mutant LbCas12a polypeptide is G146R / R182V / E795Q (SEQ ID NO: 3984).

[0015] Another embodiment described herein is a kit for enhancing the efficiency of gene editing at a non-canonical TTTT PAM site in a cell, comprising a CRISPR-ribonucleoprotein complex comprising a guide RNA and a mutant LbCas12a polypeptide comprising at least one amino acid substitution introduced into the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2, provided that the mutant LbCas12a polypeptide provides improved CRISPR / LbCas12a-related nuclease activity at a non-canonical TTTT PAM site compared to the wild-type LbCas12a polypeptide sequence. In one embodiment, the mutant LbCas12a polypeptide has a polypeptide sequence selected from even-numbered sequences of SEQ ID NOs: 4-4022. In another embodiment, the mutant LbCas12a polypeptide includes at least one amino acid substitution at the position of 81, 125, 145, 146, 152, 182, 396, 478, 491, 582, 595, 606, 646, 665, 795, 799, 814, 839, 841, 858, 860, 890, 898, 909, 913, 981, 1020, 1083, or 1121 of the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2. In another embodiment, the mutant LbCas12a polypeptide comprises at least one amino acid substitution selected from F81E, E125K, N145R, G146R, T152K, R182V, S396D, K478R, V491D, N582R, K595R, Y606F, Y646H, D665N, E795Q, P799V, T814G, L839F, I841A, E858V, I860R, W890A, E898N, H909K, E913R, E981V, S1020E, V1083W, or K1121D of the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2. In another embodiment, mutant LbCas12a polypeptides include: F81E, SEQ ID NO: 802; E125K, SEQ ID NO: 428; N145R, SEQ ID NO: 2902; G146R, SEQ ID NO: 156; T152K, SEQ ID NO: 694; R182V, SEQ ID NO: 98; S396D, SEQ ID NO: 108; K478R, SEQ ID NO: 134; V491D, SEQ ID NO: 954; N582R, SEQ ID NO: 1730; K595R, SEQ ID NO: 184; Y606F, SEQ ID NO: 642; Y646H,Select from sequence numbers 720;D665N, 1014;E795Q, 930;P799V, 706;T814G, 162;L839F, 834;I841A, 988;E858V, 114;I860R, 250;W890A, 3958;E898N, 48;H909K, 70;E913R, 1960;E981V, 880;S1020E, 532;V1083W, 132; or K1121D, 598. In another embodiment, a single substitution mutation is introduced into the wild-type LbCas12a polypeptide sequence of Sequence ID No. 2, in which a mutant LbCas12a polypeptide is introduced at (a) a position selected from: F81, E125, N145, G146, T152, R182, S396, K478, V491, N582, K595, Y606, Y646, D665, E795, P799, T814, L839, I841, E858, I860, W890, E898, H909, E913, E981, S1020, V1083, or K1121; Or (b) the following positions: containing a substitution mutation selected from the polysubstitution mutations introduced into the wild-type LbCas12a polypeptide sequence of SEQ ID NO: F81, E125, N145, G146, T152, R182, S396, K478, V491, N582, K595, Y606, Y646, D665, E795, P799, T814, L839, I841, E858, I860, W890, E898, H909, E913, E981, S1020, V1083 or K1121. In another embodiment, polysubstitution mutations include: E88A / E795Q, E125K / E795Q, G146R / E795Q, R182V / E795Q, V491D / E795Q, Q529I / E795Q, Y646H / E795Q, D665N / E795Q, T814K / E795Q, L839F / E795Q, Q906F / E79 5Q, E795Q / Q1170D, G146R / R182V / E795Q, G146R / E795Q / D665N, G146R / E795Q / E981V, G 146R / E795Q / T814K, G146R / R182V / D665N, E125K / R182V / E981V, R182V / P799V / E981V,R182V / T814K / E981V, G146R / P799V / E981V, G146R / R182V / E795Q / F81E, G146R / R182V / E7 95Q / E125K, G146R / R182V / E795Q / E125A, G146R / R182V / E795Q / P799V, G146R / R182V / E79 Includes 5Q / T814K, G146R / R182V / E795Q / E981V, G146R / R182V / E795Q / D665N, G146R / R182V / P799V / E981V, E125K / G146R / R182V / E795Q / D665N, or G146R / R182V / E795Q / P799V / T814K. In another embodiment, mutant LbCas12a polypeptides include: F81E (SEQ ID NO: 802), E125K (SEQ ID NO: 428), N145R (SEQ ID NO: 2902), G146R (SEQ ID NO: 156), T152K (SEQ ID NO: 694), R182V (SEQ ID NO: 98), V491D (SEQ ID NO: 954), K478R (SEQ ID NO: 134), N582R (SEQ ID NO: 1730), D665N (SEQ ID NO: 1014) ), E795Q (sequence number 930), P799V (sequence number 706), E858V (sequence number 114), I860R (sequence number 250), E913R (sequence number 1960), E981V (sequence number 880), E88A / E795Q (sequence number 3960), E125K / E795Q (sequence number 3962), G146R / E795Q (sequence number 3964), R182V / E795Q (sequence number 3960) 966), V491D / E795Q (sequence number 3968), Q529I / E795Q (sequence number 3970), Y646H / E795Q (sequence number 3972), D665N / E795Q (sequence number 3974), T814K / E795Q (sequence number 3976), L839F / E795Q (sequence number 3978), Q906F / E795Q (sequence number 3980), E795Q / Q1170D (sequence number (Sequence number 3982), G146R / R182V / E795Q (Sequence number 3984), G146R / E795Q / D665N (Sequence number 3986), G146R / E795Q / E981V (Sequence number 3988), G146R / E795Q / T814K (Sequence number 3990), G146R / R182V / D665N (Sequence number 3992), E125K / R182V / E981V (Sequence number 3994),R182V / P799V / E981V (SEQ ID NO: 3996), R182V / T814K / E981V (SEQ ID NO: 3998), G146R / P799V / E981V (SEQ ID NO: 4000), G146R / R182V / E795Q / F81E (SEQ ID NO: 4002), G146R / R182V / E795Q / E125K (SEQ ID NO: 4004), G146R / R182V / E795Q / E125A (SEQ ID NO: 4006), G146R / R182V / E795Q / P799V (SEQ ID NO: 4008), G14 The mutant LbCas12a polypeptide is selected from 6R / R182V / E795Q / T814K (SEQ ID NO: 4010), G146R / R182V / E795Q / E981V (SEQ ID NO: 4012), G146R / R182V / E795Q / D665N (SEQ ID NO: 4014), G146R / R182V / P799V / E981V (SEQ ID NO: 4016), E125K / G146R / R182V / E795Q / D665N (SEQ ID NO: 4018), or G146R / R182V / E795Q / P799V / T814K (SEQ ID NO: 4020). In another embodiment, the mutant LbCas12a polypeptide is G146R / R182V / E795Q (SEQ ID NO: 3984).

[0016] Another embodiment described herein is the use of a mutant LbCas12a polypeptide to improve CRISPR / LbCas12a-related nuclease activity at a non-canonical TTTT PAM site, wherein the mutant LbCas12a polypeptide comprises at least one amino acid substitution introduced into the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2, provided that the mutant LbCas12a polypeptide provides improved CRISPR / LbCas12a-related nuclease activity at a non-canonical TTTT PAM site compared to the wild-type LbCas12a polypeptide sequence. In one embodiment, the mutant LbCas12a polypeptide has a polypeptide sequence selected from the even-numbered sequences of SEQ ID NOs: 4 to 4022. In another embodiment, the mutant LbCas12a polypeptide includes at least one amino acid substitution at the position of 81, 125, 145, 146, 152, 182, 396, 478, 491, 582, 595, 606, 646, 665, 795, 799, 814, 839, 841, 858, 860, 890, 898, 909, 913, 981, 1020, 1083, or 1121 of the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2. In another embodiment, the mutant LbCas12a polypeptide comprises at least one amino acid substitution selected from F81E, E125K, N145R, G146R, T152K, R182V, S396D, K478R, V491D, N582R, K595R, Y606F, Y646H, D665N, E795Q, P799V, T814G, L839F, I841A, E858V, I860R, W890A, E898N, H909K, E913R, E981V, S1020E, V1083W, or K1121D of the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2. In another embodiment, mutant LbCas12a polypeptides include: F81E, SEQ ID NO: 802; E125K, SEQ ID NO: 428; N145R, SEQ ID NO: 2902; G146R, SEQ ID NO: 156; T152K, SEQ ID NO: 694; R182V, SEQ ID NO: 98; S396D, SEQ ID NO: 108; K478R, SEQ ID NO: 134; V491D, SEQ ID NO: 954; N582R, SEQ ID NO: 1730; K595R, SEQ ID NO: 184; Y606F, SEQ ID NO: 642; Y646H,Select from sequence numbers 720;D665N, 1014;E795Q, 930;P799V, 706;T814G, 162;L839F, 834;I841A, 988;E858V, 114;I860R, 250;W890A, 3958;E898N, 48;H909K, 70;E913R, 1960;E981V, 880;S1020E, 532;V1083W, 132; or K1121D, 598. In another embodiment, a single substitution mutation is introduced into the wild-type LbCas12a polypeptide sequence of Sequence ID No. 2, in which a mutant LbCas12a polypeptide is introduced at (a) a position selected from: F81, E125, N145, G146, T152, R182, S396, K478, V491, N582, K595, Y606, Y646, D665, E795, P799, T814, L839, I841, E858, I860, W890, E898, H909, E913, E981, S1020, V1083, or K1121; Or (b) the following positions: containing a substitution mutation selected from the polysubstitution mutations introduced into the wild-type LbCas12a polypeptide sequence of SEQ ID NO: F81, E125, N145, G146, T152, R182, S396, K478, V491, N582, K595, Y606, Y646, D665, E795, P799, T814, L839, I841, E858, I860, W890, E898, H909, E913, E981, S1020, V1083 or K1121. In another embodiment, polysubstitution mutations include: E88A / E795Q, E125K / E795Q, G146R / E795Q, R182V / E795Q, V491D / E795Q, Q529I / E795Q, Y646H / E795Q, D665N / E795Q, T814K / E795Q, L839F / E795Q, Q906F / E79 5Q, E795Q / Q1170D, G146R / R182V / E795Q, G146R / E795Q / D665N, G146R / E795Q / E981V, G 146R / E795Q / T814K, G146R / R182V / D665N, E125K / R182V / E981V, R182V / P799V / E981V,R182V / T814K / E981V, G146R / P799V / E981V, G146R / R182V / E795Q / F81E, G146R / R182V / E7 95Q / E125K, G146R / R182V / E795Q / E125A, G146R / R182V / E795Q / P799V, G146R / R182V / E79 Includes 5Q / T814K, G146R / R182V / E795Q / E981V, G146R / R182V / E795Q / D665N, G146R / R182V / P799V / E981V, E125K / G146R / R182V / E795Q / D665N, or G146R / R182V / E795Q / P799V / T814K. In another embodiment, mutant LbCas12a polypeptides include: F81E (SEQ ID NO: 802), E125K (SEQ ID NO: 428), N145R (SEQ ID NO: 2902), G146R (SEQ ID NO: 156), T152K (SEQ ID NO: 694), R182V (SEQ ID NO: 98), V491D (SEQ ID NO: 954), K478R (SEQ ID NO: 134), N582R (SEQ ID NO: 1730), D665N (SEQ ID NO: 1014) ), E795Q (sequence number 930), P799V (sequence number 706), E858V (sequence number 114), I860R (sequence number 250), E913R (sequence number 1960), E981V (sequence number 880), E88A / E795Q (sequence number 3960), E125K / E795Q (sequence number 3962), G146R / E795Q (sequence number 3964), R182V / E795Q (sequence number 3960) 966), V491D / E795Q (sequence number 3968), Q529I / E795Q (sequence number 3970), Y646H / E795Q (sequence number 3972), D665N / E795Q (sequence number 3974), T814K / E795Q (sequence number 3976), L839F / E795Q (sequence number 3978), Q906F / E795Q (sequence number 3980), E795Q / Q1170D (sequence number (Sequence number 3982), G146R / R182V / E795Q (Sequence number 3984), G146R / E795Q / D665N (Sequence number 3986), G146R / E795Q / E981V (Sequence number 3988), G146R / E795Q / T814K (Sequence number 3990), G146R / R182V / D665N (Sequence number 3992), E125K / R182V / E981V (Sequence number 3994),R182V / P799V / E981V (SEQ ID NO: 3996), R182V / T814K / E981V (SEQ ID NO: 3998), G146R / P799V / E981V (SEQ ID NO: 4000), G146R / R182V / E795Q / F81E (SEQ ID NO: 4002), G146R / R182V / E795Q / E125K (SEQ ID NO: 4004), G146R / R182V / E795Q / E125A (SEQ ID NO: 4006), G146R / R182V / E795Q / P799V (SEQ ID NO: 4008), G14 The mutant LbCas12a polypeptide is selected from 6R / R182V / E795Q / T814K (SEQ ID NO: 4010), G146R / R182V / E795Q / E981V (SEQ ID NO: 4012), G146R / R182V / E795Q / D665N (SEQ ID NO: 4014), G146R / R182V / P799V / E981V (SEQ ID NO: 4016), E125K / G146R / R182V / E795Q / D665N (SEQ ID NO: 4018), or G146R / R182V / E795Q / P799V / T814K (SEQ ID NO: 4020). In another embodiment, the mutant LbCas12a polypeptide is G146R / R182V / E795Q (SEQ ID NO: 3984).

[0017] Another embodiment described herein is a method for expressing and purifying a mutant LbCas13a protein, comprising: (a) inserting a nucleotide sequence encoding a mutant LbCas12a polypeptide having at least one amino acid substitution compared to the wild-type LbCas12a polypeptide sequence and having 95–99% identity with any of the even-numbered polypeptide sequences of SEQ ID NOs. 4–4022 into an expression plasmid; (b) transforming one or more cells with the expression plasmid; (c) inducing expression of the transformed plasmid; (d) isolating the cells; (e) extracting the mutant LbCas13a protein; and (f) purifying the mutant LbCas13a protein, wherein the mutant LbCas12a polypeptide provides improved CRISPR / LbCas12a-related nuclease activity at the non-canonical TTTT PAM site compared to the wild-type LbCas12a polypeptide sequence.

[0018] Another embodiment described herein is a mutant LbCas12a polypeptide produced by the method described herein. [Brief explanation of the drawing]

[0019] [Figure 1A] Figure 1A shows the exemplary phenotypic distribution of all LbCas12a variants covered by bacterial screening. For this analysis, the mean enrichment score of point mutations across two biological replications was used for selection. The enrichment scores of all synonymous variations (i.e., different codons encoding the same amino acid as the wild-type amino acid) clustered tightly around 0, which allowed for the quantification of the phenotype of WT-LbCas12a in selection. Figure 1B shows the distribution and enrichment of synonymous variations. Figure 1C shows the distribution and enrichment of non-synonymous variations. [Figure 1B] Figure 1A shows the exemplary phenotypic distribution of all LbCas12a variants covered by bacterial screening. For this analysis, the mean enrichment score of point mutations across two biological replications was used for selection. The enrichment scores of all synonymous variations (i.e., different codons encoding the same amino acid as the wild-type amino acid) clustered tightly around 0, which allowed for the quantification of the phenotype of WT-LbCas12a in selection. Figure 1B shows the distribution and enrichment of synonymous variations. Figure 1C shows the distribution and enrichment of non-synonymous variations. [Figure 1C]Figure 1A shows the exemplary phenotypic distribution of all LbCas12a variants covered by bacterial screening. For this analysis, the mean enrichment score of point mutations across two biological replications was used for selection. The enrichment scores of all synonymous variations (i.e., different codons encoding the same amino acid as the wild-type amino acid) clustered tightly around 0, which allowed for the quantification of the phenotype of WT-LbCas12a in selection. Figure 1B shows the distribution and enrichment of synonymous variations. Figure 1C shows the distribution and enrichment of non-synonymous variations. [Figure 2] Figure 2 shows the phenotypic (activity) scores of all mutations across the entire LbCas12a polypeptide sequence. Ten of the most active mutants are indicated. [Figure 3A] Figure 3A shows exemplary phenotypic scores for point mutations at position E795 of LbCas12a. [Figure 3B] Figure 3B shows exemplary phenotypic scores for N527N and N527K. Other point variants at this location were excluded from screening, which indicates that any variation at this location is harmful. [Figure 4A] Figure 4A shows exemplary phenotypic scores for point mutations at position D156. The majority of mutations are well tolerated at this position; leucine appears neutral; phenylalanine or histidine are harmful at this position. [Figure 4B] Figure 4B shows exemplary phenotypic scores for point mutations at position G532. Most mutations are harmful at this position. [Figure 5]Figures 5A–J show exemplary point mutations with enhanced DNA cleavage activity in wild-type LbCas12a (Figure 5A) and TTTT PAM (Figures 5B–J) compared to a negative control (without HPRT38346 gRNA; Figure 5B). The cleavage activity of the LbCas12a variant at the TTTT PAM site was measured by a bacterial-based activity assay. E. coli (E. coli) survival under arabinose selection depends on the successful cleavage of the HPRT38346 protospacer on the toxin expression plasmid using TTTT PAM. E. coli survival was dramatically reduced in the absence of HPRT38346 gRNA. See Figure 5A (wild-type) vs. Figure 5B (negative control). Compared to the wild type (Figure 5A), all exemplary mutants showed improved viability in the presence of gRNA (Figures 5C–5J, mutants: K478R; E898N; S396D; G146R; K595R; E795Q; T814G; and V1083W, respectively), which reflects enhanced cleavage activity of LbCas12a variants in non-canonical TTTT PAM. [Figure 6A] Figures 6A and 6B show the performance evaluation of LbCas12a variants as RNP complexes in human cells. The LbCas12a variants shown in Table 7 were purified and assembled as RNPs using four different crRNAs (Table 9). Figure 6A shows the editing efficiency of the LbCas12a variants using the T7 endonuclease I assay 48 hours after delivery to human HEK293 cells. Three replicates were performed. Averaged data and standard deviations are shown in Table 10. Figure 6B shows the normalized editing efficiency for each variant (normalized relative to wild-type LbCas12a) and the normalized editing efficiency presented as a multiplier of improvement compared to wild-type. For each variant, the first quartile (Q1), median, and third quartile (Q3) are shown. The dashed line represents the baseline activity of the wild-type LbCas12a protein. Two replicates were performed. Normalized data for each crRNA are shown in Table 11. [Figure 6B]Figures 6A-B show the performance evaluation of LbCas12a variants in human cells as RNP complexes. The LbCas12a variants shown in Table 7 were purified and assembled as RNPs using four different crRNAs (Table 9). Figure 6A shows the editing efficiency of the LbCas12a variants using the T7 endonuclease I assay 48 hours after delivery into human HEK293 cells. Three replicates were performed. The averaged data and standard deviations are shown in Table 10. Figure 6B shows the normalized editing efficiency of each variant (normalized to wild-type LbCas12a) and the normalized editing efficiency presented as the fold improvement compared to wild-type. For each variant, the first quartile (Q1), median, and third quartile (Q3) are shown. The dashed line represents the baseline activity of the wild-type LbCas12a protein. Two replicates were performed. The normalized data for each crRNA are shown in Table 11.

Mode for Carrying Out the Invention

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For example, cell and tissue culture, molecular biology, immunology, microbiology, genetics, and any nomenclature and techniques used in connection with protein and nucleic acid chemistry and hybridization described herein are well known and commonly used in the art. In case of conflict, including definitions, the present disclosure will control. Exemplary methods and materials are described below, but methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention.

[0021] As used herein, the terms "amino acid", "nucleotide", "polypeptide", "polynucleotide", and "vector" have their common meanings as understood by a person skilled in the art, a biochemist. 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 R) are used herein.

[0022] As used herein, terms such as "include", "including", "contain", "containing", "having", etc. mean "comprising". The present disclosure also contemplates other embodiments "comprising", "consisting of", and "consisting essentially of" the embodiments, aspects, or elements presented herein, whether or not explicitly recited.

[0023] As used herein, the terms "a", "an", "the", and similar terms used in the context of the present disclosure (particularly in the context of the claims) are to be construed to cover both the singular and the plural unless otherwise indicated herein or clearly contradicted by the context. In addition, "a", "an", or "the" means "one or more" unless otherwise specified.

[0024] As used herein, the term "or" may be conjunctive or disjunctive.

[0025] As used herein, the term "substantially" means mostly or to a considerable degree but not completely. [[ID=A]]

[0026] [[ID=B]] Where used herein, the terms “about” or “approximately” applied to one or more values ​​of interest refer to a value similar to the reference value described, or a value within an acceptable margin of error with respect to a particular value determined by those skilled in the art, but which will depend in part to the way in which the value is measured or determined, for example, to the limits of the measurement system. In one embodiment, the term “about” refers to any value, including both integer and fractional components, within a maximum variation of ±10% of the value modified by the term “about.” Alternatively, “about” may mean within a standard deviation of 3 or greater, according to convention in the art. Or, in relation to biological systems or biological 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. Where used herein, the symbol “~” means “about” or “approximately.”

[0027] All ranges disclosed herein include both endpoints as discrete values, as well as all integers and fractions specified within the 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 qualified with the term "approximately", the specified range is expanded, including the endpoint, by a maximum variation of ±10% of any value within the range or within a standard deviation of 3 or greater.

[0028] Where used herein, the terms “control” and “reference” are used interchangeably. A “reference” level or “control” level may be a predetermined value or range, used as a baseline or benchmark for evaluating measurement results. “Control” also refers to a control experiment or control cells.

[0029] As used herein, the term “effective amount” of a compound described herein means the amount of the compound described herein that induces a biological response, such as reducing or inhibiting enzyme or protein activity, improving symptoms, alleviating a condition, slowing or delaying the progression of a disease, or preventing a disease.

[0030] As used herein, the terms “inhibit,” “inhibit,” or “the act of inhibiting” refer to the reduction or suppression of a given condition, symptom, disorder, or disease, or a significant reduction in the baseline activity of a biological activity or process.

[0031] As used herein, the terms "Lachnospirace bacterium Cas12a" or "LbCas12a" are used interchangeably to refer to the Lachnospirace bacterium ND2006(Lb)Cas12a protein (formerly named Cpf1), a class 2 / V CRISPR RNA guided endonuclease. The terms "LbCas12a," "wild-type LbCas12a," "wild-type Lb enzyme," or "WT LbCas12a" refer to a protein having the amino acid sequence of naturally occurring Lachnospirace bacterium ND2006 Cas12a (e.g., encoded by the nucleotide sequences of SEQ ID NO: 2 and SEQ ID NO: 1), which possesses biochemical activity when combined with a suitable crRNA to form an active CRISPR / Cas12a endonuclease system.

[0032] The terms “mutant LbCas12a protein” or “variant LbCas12a protein” are used interchangeably and refer to LbCas12a protein forms having a different amino acid sequence from wild-type Lachnospirace bacterium ND2006 Cas12a, which possess biochemical activity when combined with appropriate crRNA to form an active CRISPR-Cas12a endonuclease system. This includes orthologs and Cas12a variants having a different amino acid sequence from wild-type Lachnospirace bacterium ND2006 Cas12a. The mutant LbCas12a proteins described herein have at least one amino acid substitution relative to the wild-type LbCas12a polypeptide sequence. In one embodiment, the mutant LbCas12a polypeptide has at least one amino acid substitution relative to the wild-type LbCas12a polypeptide sequence and exhibits improved CRISPR / LbCas12a-related nuclease activity at the non-canonical TTTT PAM site compared to the wild-type LbCas12a enzyme.

[0033] As used herein, the phrase "sequence of odd-numbered sequences of sequence numbers 3-4021" means sequence numbers 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 13 1, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193 ,195,197,199,201,203,205,207,209,211,213,215,217,219,221,223,225,227,229,231,233,235,237,239,241,243,245,247,249,251,253,255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 3 19, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 38 1, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443 ,445,447,449,451,453,455,457,459,461,463,465,467,469,471,473,475,477,479,481,483,485,487,489,491,493,495,497,499,501,503,505,507、509、511、513、515、517、519、521、523、525、527、529、531、533、535、537、539、541、543、545、547、549、551、553、555、557、559、561、563、565、567、569、571、573、575、577、579、581、583、585、587、589、591、593、595、597、599、601、603、605、607、609、611、613、615、617、619、621、623、625、627、629、631、633、635、637、639、641、643、645、647、649、651、653、655、657、659、661、663、665、667、669、671、673、675、677、679、681、683、685、687、689、691、693、695、697、699、701、703、705、707、709、711、713、715、717、719、721、723、725、727、729、731、733、735、737、739、741、743、745、747、749、751、753、755、757、759、761、763、765、767、769、771、773、775、777、779、781、783、785、787、789、791、793、795、797、799、801、803、805、807、809、811、813、815、817、819、821、823、825、827、829、831、833、835、837、839、841、843、845、847、849、851、853、855、857、859、861、863、865、867、869、871、873、875、877、879、881、 883、885、887、889、891、893、895、897、899、901、903、905、907、909、911、913、915、917、919、921、923、925、927、929、931、933、935、937、939、941、943、945、947、949、951、953、955、957、959、961、963、965、967、969、971、973、975、977、979、981、983、985、987、989、991、993、995、997、999、1001、1003、1005、1007、1009、1011、1013、1015、1017、1019、1021、1023、1025、1027、1029、1031、1033、1035、1037、1039、1041、1043、1045、1047、1049、1051、1053、1055、1057、1059、1061、1063、1065、1067、1069、1071、1073、1075、1077、1079、1081、1083、1085、1087、1089、1091、1093、1095、1097、1099、1101、1103、1105、1107、1109、1111、1113、1115、1117、1119、1121、1123、1125、1127、1129、1131、1133、1135、1137、1139、1141、1143、1145、1147、1149、1151、1153、1155、1157、1159、1161、1163、1165、1167、1169、1171、1173、1175、1177、1179、1181、1183、1185、1187、1189、1191、1193、1195、1197、1199、1201、1203、1205、1207、1209、1211、1213、1215、1217、1219、1221、1223、1225、1227、1229、1231、1233、1235、1237、1239、1241、1243、1245、1247、1249、1251、1253、1255、1257、1259、1261、1263、1265、1267、1269、1271、1273、1275、1277、1279、1281、1283、1285、1287、1289、1291、1293、1295、1297、1299、1301、1303、1305、1307、1309、1311、1313、1315、1317、1319、1321、1323、1325、1327、1329、1331、1333、1335、1337、1339、1341、1343、1345、1347、1349、1351、1353、1355、1357、1359、1361、1363、1365、1367、1369、1371、1373、1375、1377、1379、1381、1383、1385、1387、1389、1391、1393、1395、1397、1399、1401、1403、1405、1407、1409、1411、1413、1415、1417、1419、1421、1423、1425、1427、1429、1431、1433、1435、1437、1439、1441、1443、1445、1447、1449、1451、1453、1455、1457、1459、1461、1463、1465、1467、1469、1471、1473、1475、1477、1479、1481、1483、1485、1487、1489、1491、1493、1495、1497、1499、1501、1503、1505、1507、1509、1511、1513、1515、1517、1519、1521、1523、1525、1527、1529、1531、1533、1535、1537、1539、1541、1543、1545、1547、1549、1551、1553、1555、1557、1559、1561、1563、1565、1567、1569、1571、1573、1575、1577、1579、1581、1583、1585、1587、1589、1591、1593、1595、1597、1599、1601、1603、1605、1607、 1609、1611、1613、1615、1617、1619、1621、1623、1625、1627、1629、1631、1633、1635、1637、1639、1641、1643、1645、1647、1649、1651、1653、1655、1657、1659、1661、1663、1665、1667、1669、1671、1673、1675、1677、1679、1681、1683、1685、1687、1689、1691、1693、1695、1697、1699、1701、1703、1705、1707、1709、1711、1713、1715、1717、1719、1721、1723、1725、1727、1729、1731、1733、1735、1737、1739、1741、1743、1745、1747、1749、1751、1753、1755、1757、1759、1761、1763、1765、1767、1769、1771、1773、1775、1777、1779、1781、1783、1785、1787、1789、1791、1793、1795、1797、1799、1801、1803、1805、1807、1809、1811、1813、1815、1817、1819、1821、1823、1825、1827、1829、1831、1833、1835、1837、1839、1841、1843、1845、1847、1849、1851、1853、1855、1857、1859、1861、1863、1865、1867、1869、1871、1873、1875、1877、1879、1881、1883、1885、1887、1889、1891、1893、1895、1897、1899、1901、1903、1905、1907、1909、1911、1913、1915、1917、1919、1921、1923、1925、1927、1929、1931、1933、1935、1937、1939、1941、1943、1945、1947、1949、1951、1953、1955、1957、1959、1961、1963、1965、1967、1969、1971、1973、1975、1977、1979、1981、1983、1985、1987、1989、1991、1993、1995、1997、1999、2001、2003、2005、2007、2009、2011、2013、2015、2017、2019、2021、2023、2025、2027、2029、2031、2033、2035、2037、2039、2041、2043、2045、2047、2049、2051、2053、2055、2057、2059、2061、2063、2065、2067、2069、2071、2073、2075、2077、2079、2081、2083、2085、2087、2089、2091、2093、2095、2097、2099、2101、2103、2105、2107、2109、2111、2113、2115、2117、2119、2121、2123、2125、2127、2129、2131、2133、2135、2137、2139、2141、2143、2145、2147、2149、2151、2153、2155、2157、2159、2161、2163、2165、2167、2169、2171、2173、2175、2177、2179、2181、2183、2185、2187、2189、2191、2193、2195、2197、2199、2201、2203、2205、2207、2209、2211、2213、2215、2217、2219、2221、2223、2225、2227、2229、2231、2233、2235、2237、2239、2241、2243、2245、2247、 2249、2251、2253、2255、2257、2259、2261、2263、2265、2267、2269、2271、2273、2275、2277、2279、2281、2283、2285、2287、2289、2291、2293、2295、2297、2299、2301、2303、2305、2307、2309、2311、2313、2315、2317、2319、2321、2323、2325、2327、2329、2331、2333、2335、2337、2339、2341、2343、2345、2347、2349、2351、2353、2355、2357、2359、2361、2363、2365、2367、2369、2371、2373、2375、2377、2379、2381、2383、2385、2387、2389、2391、2393、2395、2397、2399、2401、2403、2405、2407、2409、2411、2413、2415、2417、2419、2421、2423、2425、2427、2429、2431、2433、2435、2437、2439、2441、2443、2445、2447、2449、2451、2453、2455、2457、2459、2461、2463、2465、2467、2469、2471、2473、2475、2477、2479、2481、2483、2485、2487、2489、2491、2493、2495、2497、2499、2501、2503、2505、2507、2509、2511、2513、2515、2517、2519、2521、2523、2525、2527、2529、2531、2533、2535、2537、2539、2541、2543、2545、2547、2549、2551、2553、2555、2557、2559、2561、2563、2565、2567、2569、2571、2573、2575、2577、2579、2581、2583、2585、2587、2589、2591、2593、2595、2597、2599、2601、2603、2605、2607、2609、2611、2613、2615、2617、2619、2621、2623、2625、2627、2629、2631、2633、2635、2637、2639、2641、2643、2645、2647、2649、2651、2653、2655、2657、2659、2661、2663、2665、2667、2669、2671、2673、2675、2677、2679、2681、2683、2685、2687、2689、2691、2693、2695、2697、2699、2701、2703、2705、2707、2709、2711、2713、2715、2717、2719、2721、2723、2725、2727、 2729、2731、2733、2735、2737、2739、2741、2743、2745、2747、2749、2751、2753、2755、2757、2759、2761、2763、2765、2767、2769、2771、2773、2775、2777、2779、2781、2783、2785、2787、2789、2791、2793、2795、2797、2799、2801、2803、2805、2807、2809、2811、2813、2815、2817、2819、2821、2823、2825、2827、2829、2831、2833、2835、2837、2839、2841、2843、2845、2847、2849、2851、2853、2855、2857、2859、2861、2863、2865、2867、2869、2871、2873、2875、2877、2879、2881、2883、2885、2887、2889、2891、2893、2895、2897、2899、2901、2903、2905、2907、2909、2911、2913、2915、2917、2919、2921、2923、2925、2927、2929、2931、2933、2935、2937、2939、2941、2943、2945、2947、2949、2951、2953、2955、2957、2959、2961、2963、2965、2967、2969、2971、2973、2975、2977、2979、2981、2983、2985、2987、2989、2991、2993、2995、2997、2999、3001、3003、3005、3007、3009、3011、3013、3015、3017、3019、3021、3023、3025、3027、3029、3031、3033、3035、3037、3039、3041、3043、3045、3047、3049、3051、3053、3055、3057、3059、3061、3063、3065、3067、3069、3071、3073、3075、3077、3079、3081、3083、3085、3087、3089、3091、3093、3095、3097、3099、3101、3103、3105、3107、3109、3111、3113、3115、3117、3119、3121、3123、3125、3127、3129、3131、3133、3135、3137、3139、3141、3143、3145、3147、3149、3151、3153、3155、3157、3159、3161、3163、3165、3167、3169、3171、3173、3175、3177、3179、3181、3183、3185、3187、3189、3191、3193、3195、3197、3199、3201、3203、3205、3207、3209、3211、3213、3215、3217、3219、3221、3223、3225、3227、3229、3231、3233、3235、3237、3239、3241、3243、3245、3247、3249、3251、3253、3255、3257、3259、3261、3263、3265、3267、3269、3271、3273、3275、3277、3279、3281、3283、3285、3287、3289、3291、3293、3295、3297、3299、3301、3303、3305、3307、3309、3311、3313、3315、3317、3319、3321、3323、3325、3327、3329、3331、3333、3335、3337、3339、3341、3343、3345、3347、3349、3351、3353、3355、3357、3359、3361、3363、3365、3367、 3369、3371、3373、3375、3377、3379、3381、3383、3385、3387、3389、3391、3393、3395、3397、3399、3401、3403、3405、3407、3409、3411、3413、3415、3417、3419、3421、3423、3425、3427、3429、3431、3433、3435、3437、3439、3441、3443、3445、3447、3449、3451、3453、3455、3457、3459、3461、3463、3465、3467、3469、3471、3473、3475、3477、3479、3481、3483、3485、3487、3489、3491、3493、3495、3497、3499、3501、3503、3505、3507、3509、3511、3513、3515、3517、3519、3521、3523、3525、3527、3529、3531、3533、3535、3537、3539、3541、3543、3545、3547、3549、3551、3553、3555、3557、3559、3561、3563、3565、3567、3569、3571、3573、3575、3577、3579、3581、3583、3585、3587、3589、3591、3593、3595、3597、3599、3601、3603、3605、3607、3609、3611、3613、3615、3617、3619、3621、3623、3625、3627、3629、3631、3633、3635、3637、3639、3641、3643、3645、3647、3649、3651、3653、3655、3657、3659、3661、3663、3665、3667、3669、3671、3673、3675、3677、3679、3681、3683、3685、3687、3689、3691、3693、3695、3697、3699、3701、3703、3705、3707、3709、3711、3713、3715、3717、3719、3721、3723、3725、3727、3729、3731、3733、3735、3737、3739、3741、3743、3745、3747、3749、3751、3753、3755、3757、3759、3761、3763、3765、3767、3769, 3771, 3773, 3775, 3777, 3779, 3781, 3783, 3785, 3787, 3789, 3791, 3793, 3795, 3797, 3799, 3801, 3803, 3805, 3807, 3809, 3811, 3813, 3815, 3817, 3819, 3821, 3823, 3825, 3827, 3829, 3831, 38 33, 3835, 3837, 3839, 3841, 3843, 3845, 3847, 3849, 3851, 3853, 3855, 3857, 3859, 3861, 3863, 3865, 3867, 3869, 3871, 3873, 3875, 3877, 3879, 3881, 3883, 3885, 3887, 3889, 3891, 3893, 3895, 3897, 3899, 3901, 3903, 3905, 3907, 3909, 3911, 3913, 3915, 3917, 3919, 3921, 3923, 3925, 3927, 3929, 3931, 3933, 3935, 3937, 3939, 3941, 3943, 3945, 3947, 3949, 3951, 3953, 3955, 3957, 3959, 3961, 39 This refers to the nucleotide sequences 63, 3965, 3967, 3969, 3971, 3973, 3975, 3977, 3979, 3981, 3983, 3985, 3987, 3989, 3991, 3993, 3995, 3997, 3999, 4001, 4003, 4005, 4007, 4009, 4011, 4013, 4015, 4017, 4019, or 4021.

[0034] As used herein, the phrase "the even-numbered sequence of sequence numbers 4-4022" means sequence numbers 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 1 32, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 19 4, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256 ,258,260,262,264,266,268,270,272,274,276,278,280,282,284,286,288,290,292,294,296,298,300,302,304,306,308,310,312,314,316,318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 3 82, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 44 4, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506,508、510、512、514、516、518、520、522、524、526、528、530、532、534、536、538、540、542、544、546、548、550、552、554、556、558、560、562、564、566、568、570、572、574、576、578、580、582、584、586、588、590、592、594、596、598、600、602、604、606、608、610、612、614、616、618、620、622、624、626、628、630、632、634、636、638、640、642、644、646、648、650、652、654、656、658、660、662、664、666、668、670、672、674、676、678、680、682、684、686、688、690、692、694、696、698、700、702、704、706、708、710、712、714、716、718、720、722、724、726、728、730、732、734、736、738、740、742、744、746、748、750、752、754、756、758、760、762、764、766、768、770、772、774、776、778、780、782、784、786、788、790、792、794、796、798、800、802、804、806、808、810、812、814、816、818、820、822、824、826、828、830、832、834、836、838、840、842、844、846、848、850、852、854、856、858、860、862、864、866、868、870、872、874、876、878、880、882、884、886、888、890、892、894、896、898、900、902、904、906、908、910、912、914、916、918、920、922、924、926、928、930、932、934、936、938、940、942、944、946、948、950、952、954、956、958、960、962、 964、966、968、970、972、974、976、978、980、982、984、986、988、990、992、994、996、998、1000、1002、1004、1006、1008、1010、1012、1014、1016、1018、1020、1022、1024、1026、1028、1030、1032、1034、1036、1038、1040、1042、1044、1046、1048、1050、1052、1054、1056、1058、1060、1062、1064、1066、1068、1070、1072、1074、1076、1078、1080、1082、1084、1086、1088、1090、1092、1094、1096、1098、1100、1102、1104、1106、1108、1110、1112、1114、1116、1118、1120、1122、1124、1126、1128、1130、1132、1134、1136、1138、1140、1142、1144、1146、1148、1150、1152、1154、1156、1158、1160、1162、1164、1166、1168、1170、1172、1174、1176、1178、1180、1182、1184、1186、1188、1190、1192、1194、1196、1198、1200、1202、1204、1206、1208、1210、1212、1214、1216、1218、1220、1222、1224、1226、1228、1230、1232、1234、1236、1238、1240、1242、1244、1246、1248、1250、1252、1254、1256、1258、1260、1262、1264、1266、1268、1270、1272、1274、1276、1278、1280、1282、1284、1286、1288、1290、1292、1294、1296、1298、1300、1302、1304、1306、1308、1310、1312、1314、1316、1318、1320、1322、1324、1326、1328、1330、1332、1334、1336、1338、1340、1342、1344、1346、1348、1350、1352、1354、1356、1358、1360、1362、1364、1366、1368、1370、1372、1374、1376、1378、1380、1382、1384、1386、1388、1390、1392、1394、1396、1398、1400、1402、1404、1406、1408、1410、1412、1414、1416、1418、1420、1422、1424、1426、1428、1430、1432、1434、1436、1438、1440、1442、1444、1446、1448、1450、1452、1454、1456、1458、1460、1462、1464、1466、1468、1470、1472、1474、1476、1478、1480、1482、1484、1486、1488、1490、1492、1494、1496、1498、1500、1502、1504、1506、1508、1510、1512、1514、1516、1518、1520、1522、1524、1526、1528、1530、1532、1534、1536、1538、1540、1542、1544、1546、1548、1550、1552、1554、1556、1558、1560、1562、1564、1566、1568、1570、1572、1574、1576、1578、1580、1582、1584、1586、1588、1590、1592、1594、1596、1598、1600、1602、1604、1606、1608、 1610、1612、1614、1616、1618、1620、1622、1624、1626、1628、1630、1632、1634、1636、1638、1640、1642、1644、1646、1648、1650、1652、1654、1656、1658、1660、1662、1664、1666、1668、1670、1672、1674、1676、1678、1680、1682、1684、1686、1688、1690、1692、1694、1696、1698、1700、1702、1704、1706、1708、1710、1712、1714、1716、1718、1720、1722、1724、1726、1728、1730、1732、1734、1736、1738、1740、1742、1744、1746、1748、1750、1752、1754、1756、1758、1760、1762、1764、1766、1768、1770、1772、1774、1776、1778、1780、1782、1784、1786、1788、1790、1792、1794、1796、1798、1800、1802、1804、1806、1808、1810、1812、1814、1816、1818、1820、1822、1824、1826、1828、1830、1832、1834、1836、1838、1840、1842、1844、1846、1848、1850、1852、1854、1856、1858、1860、1862、1864、1866、1868、1870、1872、1874、1876、1878、1880、1882、1884、1886、1888、1890、1892、1894、1896、1898、1900、1902、1904、1906、1908、1910、1912、1914、1916、1918、1920、1922、1924、1926、1928、1930、1932、1934、1936、1938、1940、1942、1944、1946、1948、1950、1952、1954、1956、1958、1960、1962、1964、1966、1968、1970、1972、1974、1976、1978、1980、1982、1984、1986、1988、1990、1992、1994、1996、1998、2000、2002、2004、2006、2008、2010、2012、2014、2016、2018、2020、2022、2024、2026、2028、2030、2032、2034、2036、2038、2040、2042、2044、2046、2048、2050、2052、2054、2056、2058、2060、2062、2064、2066、2068、2070、2072、2074、2076、2078、2080、2082、2084、2086、2088、2090、2092、2094、2096、2098、2100、2102、2104、2106、2108、2110、2112、2114、2116、2118、2120、2122、2124、2126、2128、2130、2132、2134、2136、2138、2140、2142、2144、2146、2148、2150、2152、2154、2156、2158、2160、2162、2164、2166、2168、2170、2172、2174、2176、2178、2180、2182、2184、2186、2188、2190、2192、2194、2196、2198、2200、2202、2204、2206、2208、2210、2212、2214、2216、2218、2220、2222、2224、2226、2228、2230、2232、2234、2236、2238、2240、2242、2244、2246、2248、 2250、2252、2254、2256、2258、2260、2262、2264、2266、2268、2270、2272、2274、2276、2278、2280、2282、2284、2286、2288、2290、2292、2294、2296、2298、2300、2302、2304、2306、2308、2310、2312、2314、2316、2318、2320、2322、2324、2326、2328、2330、2332、2334、2336、2338、2340、2342、2344、2346、2348、2350、2352、2354、2356、2358、2360、2362、2364、2366、2368、2370、2372、2374、2376、2378、2380、2382、2384、2386、2388、2390、2392、2394、2396、2398、2400、2402、2404、2406、2408、2410、2412、2414、2416、2418、2420、2422、2424、2426、2428、2430、2432、2434、2436、2438、2440、2442、2444、2446、2448、2450、2452、2454、2456、2458、2460、2462、2464、2466、2468、2470、2472、2474、2476、2478、2480、2482、2484、2486、2488、2490、2492、2494、2496、2498、2500、2502、2504、2506、2508、2510、2512、2514、2516、2518、2520、2522、2524、2526、2528、2530、2532、2534、2536、2538、2540、2542、2544、2546、2548、2550、2552、2554、2556、2558、2560、2562、2564、2566、2568、2570、2572、2574、2576、2578、2580、2582、2584、2586、2588、2590、2592、2594、2596、2598、2600、2602、2604、2606、2608、2610、2612、2614、2616、2618、2620、2622、2624、2626、2628、2630、2632、2634、2636、2638、2640、2642、2644、2646、2648、2650、2652、2654、2656、2658、2660、2662、2664、2666、2668、2670、2672、2674、2676、2678、2680、2682、2684、2686、2688、2690、2692、2694、2696、2698、2700、2702、2704、2706、2708、2710、2712、2714、2716、2718、2720、2722、2724、2726、2728、 2730、2732、2734、2736、2738、2740、2742、2744、2746、2748、2750、2752、2754、2756、2758、2760、2762、2764、2766、2768、2770、2772、2774、2776、2778、2780、2782、2784、2786、2788、2790、2792、2794、2796、2798、2800、2802、2804、2806、2808、2810、2812、2814、2816、2818、2820、2822、2824、2826、2828、2830、2832、2834、2836、2838、2840、2842、2844、2846、2848、2850、2852、2854、2856、2858、2860、2862、2864、2866、2868、2870、2872、2874、2876、2878、2880、2882、2884、2886、2888、2890、2892、2894、2896、2898、2900、2902、2904、2906、2908、2910、2912、2914、2916、2918、2920、2922、2924、2926、2928、2930、2932、2934、2936、2938、2940、2942、2944、2946、2948、2950、2952、2954、2956、2958、2960、2962、2964、2966、2968、2970、2972、2974、2976、2978、2980、2982、2984、2986、2988、2990、2992、2994、2996、2998、3000、3002、3004、3006、3008、3010、3012、3014、3016、3018、3020、3022、3024、3026、3028、3030、3032、3034、3036、3038、3040、3042、3044、3046、3048、3050、3052、3054、3056、3058、3060、3062、3064、3066、3068、3070、3072、3074、3076、3078、3080、3082、3084、3086、3088、3090、3092、3094、3096、3098、3100、3102、3104、3106、3108、3110、3112、3114、3116、3118、3120、3122、3124、3126、3128、3130、3132、3134、3136、3138、3140、3142、3144、3146、3148、3150、3152、3154、3156、3158、3160、3162、3164、3166、3168、3170、3172、3174、3176、3178、3180、3182、3184、3186、3188、3190、3192、3194、3196、3198、3200、3202、3204、3206、3208、3210、3212、3214、3216、3218、3220、3222、3224、3226、3228、3230、3232、3234、3236、3238、3240、3242、3244、3246、3248、3250、3252、3254、3256、3258、3260、3262、3264、3266、3268、3270、3272、3274、3276、3278、3280、3282、3284、3286、3288、3290、3292、3294、3296、3298、3300、3302、3304、3306、3308、3310、3312、3314、3316、3318、3320、3322、3324、3326、3328、3330、3332、3334、3336、3338、3340、3342、3344、3346、3348、3350、3352、3354、3356、3358、3360、3362、3364、3366、3368、 3370、3372、3374、3376、3378、3380、3382、3384、3386、3388、3390、3392、3394、3396、3398、3400、3402、3404、3406、3408、3410、3412、3414、3416、3418、3420、3422、3424、3426、3428、3430、3432、3434、3436、3438、3440、3442、3444、3446、3448、3450、3452、3454、3456、3458、3460、3462、3464、3466、3468、3470、3472、3474、3476、3478、3480、3482、3484、3486、3488、3490、3492、3494、3496、3498、3500、3502、3504、3506、3508、3510、3512、3514、3516、3518、3520、3522、3524、3526、3528、3530、3532、3534、3536、3538、3540、3542、3544、3546、3548、3550、3552、3554、3556、3558、3560、3562、3564、3566、3568、3570、3572、3574、3576、3578、3580、3582、3584、3586、3588、3590、3592、3594、3596、3598、3600、3602、3604、3606、3608、3610、3612、3614、3616、3618、3620、3622、3624、3626、3628、3630、3632、3634、3636、3638、3640、3642、3644、3646、3648、3650、3652、3654、3656、3658、3660、3662、3664、3666、3668、3670、3672、3674、3676、3678、3680、3682、3684、3686、3688、3690、3692、3694、3696、3698、3700、3702、3704、3706、3708、3710、3712、3714、3716、3718、3720、3722、3724、3726、3728、3730、3732、3734、3736、3738、3740、3742、3744、3746、3748、3750、3752、3754、3756、3758、3760、3762、3764、3766、3768、3770, 3772, 3774, 3776, 3778, 3780, 3782, 3784, 3786, 3788, 3790, 3792, 3794, 3796, 3798, 3800, 3802, 3804, 3806, 3808, 3810, 3812, 3814, 3816, 3818, 3820, 3822, 3824, 3826, 3828, 3830, 3832, 38 34, 3836, 3838, 3840, 3842, 3844, 3846, 3848, 3850, 3852, 3854, 3856, 3858, 3860, 3862, 3864, 3866, 3868, 3870, 3872, 3874, 3876, 3878, 3880, 3882, 3884, 3886, 3888, 3890, 3892, 3894, 3896, 3898, 3900, 3902, 3904, 3906, 3908, 3910, 3912, 3914, 3916, 3918, 3920, 3922, 3924, 3926, 3928, 3930, 3932, 3934, 3936, 3938, 3940, 3942, 3944, 3946, 3948, 3950, 3952, 3954, 3956, 3958, 3960, 3962, 39 This refers to polypeptide sequences of 64, 3966, 3968, 3970, 3972, 3974, 3976, 3978, 3980, 3982, 3984, 3986, 3988, 3990, 3992, 3994, 3996, 3998, 4000, 4002, 4004, 4006, 4008, 4010, 4012, 4014, 4016, 4018, 4020, or 4022.

[0035] Described herein are nucleic acids encoding mutant LbCas12a polypeptides that exhibit enhanced editing activity at a non-canonical TTTT protospacer-adjacent motif (PAM) compared to the wild-type LbCas12a enzyme.

[0036] Cas12a provides useful complement to Cas9 by expanding the range of PAM sequences that can be targeted from GC-rich regions (Cas9) to AT-rich regions (Cas12a) of the genome, thereby expanding the range of sequences that can be modified using CRISPR genome engineering. In addition to having T-rich PAM sites, another advantage of the Cas12a system compared to Cas9 is the use of a single short RNA molecule.

[0037] Described herein are isolated mutant Cas12a proteins containing at least one point mutation from the wild-type Cas12a amino acid sequence. The isolated mutant Cas12a proteins are active in a clustered, regularly spaced, short, palindromic repeat (CRISPR) / CRISPR-related protein endonuclease system ("CRISPR / Cas12a endonuclease system"). The CRISPR / Cas12a endonuclease system exhibits maintained on-target editing activity compared to the wild-type CRISPR / Cas endonuclease system. In another embodiment, the Cas12a protein is derived from Lachnospirace bacterium ND2006 (Lb). The wild-type LbCas12a protein (SEQ ID NO: 2) is encoded by the nucleotide sequence shown below (SEQ ID NO: 1).

[0038] Wild-type LbCas12a DNA sequence (SEQ ID NO: 1; 3684nt) [ka]

[0039] WT LbCas12a amino acid sequence (SEQ ID NO: 2;1228 AA) [ka]

[0040] This specification describes the phenotypes of select point mutations in LbCas12a in bacterial screening for DNA cleavage activity in non-canonical TTTT PAMs. Bacteria-based directed evolution methods were performed on LbCas12a to identify mutations with enhanced cleavage activity. A deep-scan mutagenic library was constructed containing all possible amino acid point mutations across the entire LbCas12a coding sequence; in this case, most clones contained only a single mutation. (Wrenbeck et al., Nat. Methods 13:928-930 (2016)). This type of library allows for direct evaluation of the phenotype of each point mutation by measuring relative viability across wild-type LbCas12a protein in bacterial screening.

[0041] Screening is performed by transforming screening strains containing a toxin plasmid with a mutant LbCas12a library and a crRNA targeting the HPRT38346 site on the toxin plasmid. After recovery and IPTG induction, cells were seeded in LB-chloramphenicol medium containing arabinose and incubated overnight at 37°C. Functional LbCas12 enzymes are capable of CRISPR activity and inactivate the toxin plasmid. If the mutant LbCas12a enzyme is not functional, the transformed screening E. coli cells do not survive. LbCas12a expression plasmids supported by viable E. coli cells were extracted and purified. Both input and selected plasmid libraries were amplified by PCR, randomly fragmented using the Nextera Library Prep Kit, and sequenced on Illumina NextSeq® with approximately 40 million reads per library. The frequency of mutations at each position of LbCas12a in both libraries was measured and normalized to the total coverage of each codon. The relative survival rate of each point mutation was calculated as the ratio of normalized frequencies between the selected library and the input library. Since the degree of cell survival under arabinose selection indicates the cleavage activity of the LbCas12a variant in the HPRT38346 protospacer, all variants enriched during the selection process across the wild class are variants with enhanced activity using TTTT PAM.

[0042] The phenotypic analysis of 17,278 point mutations in LbCas12a during bacterial screening was used to assess the superiority of cleavage activity in non-canonical TTTT PAM. Two biological replications were performed using phenotypic assays, which allowed for the isolation of a large accumulation of novel LbCas12a variants with enhanced cleavage activity. Mutant residues in the library are shown in Table 1; amino acids of wild-type mutations are listed above the wild-type sequence (actual mutant residues are not shown, e.g., only S2 is shown instead of S2H). Specific mutations and their activity levels are shown in Table 2. Figure 2 shows the activity scores of mutations across the entire wild-type LbCas12a amino acid sequence, indicating the top 10 variants. The phenotypic score (or activity score) is calculated as the natural logarithm of the ratio of normalized frequencies between the selected library (Round 4) and the input library (Round 3), in which case the normalized frequency of each variant at each position is calculated as the ratio of the variant to the synonym change at each position.

[0043] The phenotype of specific mutations was evaluated in E. coli as colony-forming units (CFUs) for each mutant. These mutants, possessing enhanced CFUs at the time of selection, indicated improved editing efficiency in E. coli, regardless of the actual mechanism.

[0044] [Table 1-1]

[0045] [Table 1-2]

[0046] In the wild-type LbCas12a polypeptide sequence shown above, the amino acids in bold and italics indicate wild-type amino acids that are substituted individually or in combination as described herein. The LbCas12a variants described herein are useful as research tools or for therapeutic use in any CRISPR / Cas12a DNA cleavage or gene editing experiment or treatment. The superior activity of these variants may enhance the editing efficiency of LbCas12a and potentially directly replace WT-LbCas12a in genome editing applications.

[0047] One embodiment described herein is an isolated mutant LbCas12a polypeptide comprising at least one amino acid substitution introduced into the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2, provided that the mutant LbCas12a polypeptide provides improved CRISPR / LbCas12a-related nuclease activity at a non-canonical TTTT PAM site compared to the wild-type LbCas12a polypeptide sequence. In one embodiment, the mutant LbCas12a polypeptide has 95% to 99% identity with a polypeptide sequence selected from even-numbered sequences of SEQ ID NOs: 4 to 4022. In another embodiment, the mutant LbCas12a polypeptide has a polypeptide sequence selected from even-numbered sequences of SEQ ID NOs: 4 to 4022. In another embodiment, the mutant LbCas12a polypeptide includes at least one amino acid substitution at the position of 81, 125, 145, 146, 152, 182, 396, 478, 491, 582, 595, 606, 646, 665, 795, 799, 814, 839, 841, 858, 860, 890, 898, 909, 913, 981, 1020, 1083, or 1121 of the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2. In another embodiment, the mutant LbCas12a polypeptide comprises at least one amino acid substitution selected from F81E, E125K, N145R, G146R, T152K, R182V, S396D, K478R, V491D, N582R, K595R, Y606F, Y646H, D665N, E795Q, P799V, T814G, L839F, I841A, E858V, I860R, W890A, E898N, H909K, E913R, E981V, S1020E, V1083W, or K1121D of the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2. In another embodiment, mutant LbCas12a polypeptides include: F81E, SEQ ID NO: 802; E125K, SEQ ID NO: 428; N145R, SEQ ID NO: 2902; G146R, SEQ ID NO: 156; T152K, SEQ ID NO: 694; R182V, SEQ ID NO: 98; S396D, SEQ ID NO: 108; K478R, SEQ ID NO: 134; V491D, SEQ ID NO: 954; N582R, SEQ ID NO: 1730; K595R, SEQ ID NO: 184; Y606F,Select from sequence numbers 642;Y646H, 720;D665N, 1014;E795Q, 930;P799V, 706;T814G, 162;L839F, 834;I841A, 988;E858V, 114;I860R, 250;W890A, 3958;E898N, 48;H909K, 70;E913R, 1960;E981V, 880;S1020E, 532;V1083W, 132; or K1121D, 598. In another embodiment, a single substitution mutation introduced into the wild-type LbCas12a polypeptide sequence of Sequence ID No. 2, wherein the mutant LbCas12a polypeptide is at the following positions: (a) F81, E125, N145, G146, T152, R182, S396, K478, V491, N582, K595, Y606, Y646, D665, E795, P799, T814, L839, I841, E858, I860, W890, E898, H909, E913, E981, S1020, V1083, or K1121; Or (b) the following positions: containing a substitution mutation selected from the polysubstitution mutations introduced into the wild-type LbCas12a polypeptide sequence of SEQ ID NO: F81, E125, N145, G146, T152, R182, S396, K478, V491, N582, K595, Y606, Y646, D665, E795, P799, T814, L839, I841, E858, I860, W890, E898, H909, E913, E981, S1020, V1083 or K1121. In another embodiment, polysubstitution mutations include: E88A / E795Q, E125K / E795Q, G146R / E795Q, R182V / E795Q, V491D / E795Q, Q529I / E795Q, Y646H / E795Q, D665N / E795Q, T814K / E795Q, L839F / E795Q, Q906F / E795Q, E795Q / Q1170D, G146R / R182V / E795Q, G146R / E795Q / D665N, G146R / E795Q / E981V, G146R / E795Q / T814K, G146R / R182V / D665N, E125K / R182V / E981V,R182V / P799V / E981V, R182V / T814K / E981V, G146R / P799V / E981V, G146R / R182V / E795Q / F81E, G146R / R18 2V / E795Q / E125K, G146R / R182V / E795Q / E125A, G146R / R182V / E795Q / P799V, G146R / R182V / E795Q / T814K, Includes G146R / R182V / E795Q / E981V, G146R / R182V / E795Q / D665N, G146R / R182V / P799V / E981V, E125K / G146R / R182V / E795Q / D665N, G146R / R182V / E795Q / P799V / T814K, or E125K / G146R / R182V / E795Q / D665N / E981V. In another embodiment, polysubstitution mutations include: E88A / E795Q, E125K / E795Q, G146R / E795Q, R182V / E795Q, V491D / E795Q, Q529I / E795Q, Y646H / E795Q, D665N / E795Q, T814K / E795Q, L839F / E795Q, Q906F / E795Q, E795Q / Q1170D, G146R / R182V / E795Q, G146R / E795Q / D665N, G146R / E795Q / E981V, G146R / E795Q / T 814K, G146R / R182V / D665N, E125K / R182V / E981V, R182V / P799V / E981V, R182V / T814K / E981V , G146R / P799V / E981V, G146R / R182V / E795Q / F81E, G146R / R182V / E795Q / E125K, G146R / R18 2V / E795Q / E125A, G146R / R182V / E795Q / P799V, G146R / R182V / E795Q / T814K, G146R / R182V / Including E795Q / E981V, G146R / R182V / E795Q / D665N, G146R / R182V / P799V / E981V, E125K / G146R / R182V / E795Q / D665N, or G146R / R182V / E795Q / P799V / T814K, compared to the wild-type LbCas12a polypeptide sequence,In another embodiment, the mutant LbCas12a polypeptide provides improved CRISPR / LbCas12a-related nuclease activity at the non-canonical TTTT PAM site. Select from 6, 3968, 3970, 3972, 3974, 3976, 3978, 3980, 3982, 3984, 3986, 3988, 3990, 3992, 3994, 3996, 3998, 4000, 4002, 4004, 4006, 4008, 4010, 4012, 4014, 4016, 4018, 4020, or 4022. In another embodiment, mutant LbCas12a polypeptides include: F81E (SEQ ID NO: 802), E125K (SEQ ID NO: 428), N145R (SEQ ID NO: 2902), G146R (SEQ ID NO: 156), T152K (SEQ ID NO: 694), R182V (SEQ ID NO: 98), V491D (SEQ ID NO: 954), K478R (SEQ ID NO: 134), N582R (SEQ ID NO: 1730), D665N (SEQ ID NO: 1014), E795Q (SEQ ID NO: 930), P799V (SEQ ID NO: 706), E858V (SEQ ID NO: 114), I860R (SEQ ID NO: 250), E913R (SEQ ID NO: 1960), E981V (SEQ ID NO: 880), E88A / E795Q (SEQ ID NO: 3960), E125K / E795Q (SEQ ID NO: 3962), G146R / E795Q (SEQ ID NO: 3964), R182V / E795Q (SEQ ID NO: 3966), V491D / E795Q (SEQ ID NO: 3968), Q529I / E795Q (SEQ ID NO: 3970), Y646H / E795Q (SEQ ID NO: 3972), D665N / E795Q (SEQ ID NO: 3974), T814K / E795Q (SEQ ID NO: 3976), L839F / E795Q (SEQ ID NO: 3964) 78), Q906F / E795Q (SEQ ID NO: 3980), E795Q / Q1170D (SEQ ID NO: 3982), G146R / R182V / E795Q (SEQ ID NO: 3984), G146R / E795Q / D665N (SEQ ID NO: 3986), G146R / E795Q / E981V (SEQ ID NO: 3988), G146R / E795Q / T814K (SEQ ID NO: 3990),G146R / R182V / D665N (Sequence ID 3992), E125K / R182V / E981V (Sequence ID 3994), R182V / P799V / E981V (Sequence ID 3996), R182V / T814K / E981V (Sequence ID 3998), G146R / P799V / E981V (Sequence ID 4000), G146R / R182V / E795Q / F81E (Sequence ID 4002), G146R / R182V / E795Q / E125K (Sequence ID 4004), G146R / R182V / E795Q / E125A (Sequence ID 4006), G146R / R18 Select from 2V / E795Q / P799V (sequence number 4008), G146R / R182V / E795Q / T814K (sequence number 4010), G146R / R182V / E795Q / E981V (sequence number 4012), G146R / R182V / E795Q / D665N (sequence number 4014), G146R / R182V / P799V / E981V (sequence number 4016), E125K / G146R / R182V / E795Q / D665N (sequence number 4018), or G146R / R182V / E795Q / P799V / T814K (sequence number 4020). In another embodiment, the mutant LbCas12a polypeptide is G146R / R182V / E795Q (SEQ ID NO: 3984).

[0048] Another embodiment described herein is an isolated polynucleotide sequence encoding a mutant LbCas12a polypeptide comprising at least one amino acid substitution introduced into the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2, provided that the mutant LbCas12a polypeptide provides improved CRISPR / LbCas12a-related nuclease activity at a non-canonical TTTT PAM site compared to the wild-type LbCas12a polypeptide sequence. In one embodiment, the encoded mutant LbCas12a polypeptide has 95% to 99% identity with a polypeptide sequence selected from even-numbered sequences of SEQ ID NOs: 4 to 4022. In another embodiment, the encoded mutant LbCas12a polypeptide has a polypeptide sequence selected from even-numbered sequences of SEQ ID NOs: 4 to 4022. In yet another embodiment, the mutant LbCas12a polynucleotide sequence has 95% to 99% identity with a nucleotide sequence selected from odd-numbered sequences of SEQ ID NOs: 3 to 4021. In another embodiment, the mutant LbCas12a polynucleotide has a nucleotide sequence selected from the odd-numbered sequences of SEQ ID NOs: 3 to 4021. In yet another embodiment, the encoded mutant LbCas12a polypeptide has at least one amino acid substitution at the position of 81, 125, 145, 146, 152, 182, 396, 478, 491, 582, 595, 606, 646, 665, 795, 799, 814, 839, 841, 858, 860, 890, 898, 909, 913, 981, 1020, 1083, or 1121 of the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2. In another embodiment, the encoded mutant LbCas12a polypeptide is compared to the wild-type LbCas12a polypeptide sequence of SEQ ID NO: F81E, E125K, N145R, G146R, T152K, R182V, S396D, K478R, V491D, N582R, K595R, Y606F, Y646H, D665N, E795Q, P799V, T814G, L839F, I841A, E858V, I860R, W890A, E898N, H909K, E913R, E981V, S1020E,The mutant LbCas12a polynucleotide comprises at least one amino acid substitution selected from V1083W or K1121D. In another embodiment, the mutant LbCas12a polynucleotide comprises: F81E, SEQ ID NO: 802; E125K, SEQ ID NO: 428; N145R, SEQ ID NO: 2902; G146R, SEQ ID NO: 156; T152K, SEQ ID NO: 694; R182V, SEQ ID NO: 98; S396D, SEQ ID NO: 108; K478R, SEQ ID NO: 134; V491D, SEQ ID NO: 954; N582R, SEQ ID NO: 1730; K595R, SEQ ID NO: 184; Y606F, SEQ ID NO: 642; Y646H, SEQ ID NO: 720; D665N, SEQ ID NO: 1014 Selected from ;E795Q, SEQ ID NO: 930;P799V, SEQ ID NO: 706;T814G, SEQ ID NO: 162;L839F, SEQ ID NO: 834;I841A, SEQ ID NO: 988;E858V, SEQ ID NO: 114;I860R, SEQ ID NO: 250;W890A, SEQ ID NO: 3958;E898N, SEQ ID NO: 48;H909K, SEQ ID NO: 70;E913R, SEQ ID NO: 1960;E981V, SEQ ID NO: 880;S1020E, SEQ ID NO: 532;V1083W, SEQ ID NO: 132;or K1121D, SEQ ID NO: 598. In another embodiment, the encoded mutant LbCas12a polypeptide is a single substitution introduced into the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2, at the following positions: (a) F81, E125, N145, G146, T152, R182, S396, K478, V491, N582, K595, Y606, Y646, D665, E795, P799, T814, L839, I841, E858, I860, W890, E898, H909, E913, E981, S1020, V1083, or K1121. Mutations; or (b) at the following positions: F81, E125, N145, G146, T152, R182, S396, K478, V491, N582, K595, Y606, Y646, D665, E795, P799, T814, L839, I841, E858, I860, W890, E898, H909, E913, E981, S1020, V1083 or K1121, comprising substitution mutations selected from polysubstitution mutations introduced into the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2. In another embodiment, the polysubstitution mutations include: E88A / E795Q, E125K / E795Q,G146R / E795Q, R182V / E795Q, V491D / E795Q, Q529I / E795Q, Y646H / E795Q, D665N / E795Q, T814K / E795Q, L839F / E795Q, Q906F / E795Q, E795Q / Q1170D, G146R / R182V / E795Q, G146R / E795Q / D665N, G146R / E795Q / E981V, G146R / E795Q / T814K, G146R / R182V / D665N, E125K / R182V / E981V, R182V / P799V / E981V, R182V / T814K / E981V, G146R / P799V / E981V, G146R / R182V / E795Q / F81E, G146R / R182V / E795Q / E125K, G146R / R182V / E795Q / E125A, G146R / R182V / E795Q / P799V, G146R / R182V / E795Q / T814K, G146R / R182V / E795Q / E981V, G146R / Includes R182V / E795Q / D665N, G146R / R182V / P799V / E981V, E125K / G146R / R182V / E795Q / D665N, G146R / R182V / E795Q / P799V / T814K, or E125K / G146R / R182V / E795Q / D665N / E981V. In another embodiment, polysubstitution mutations include: E88A / E795Q, E125K / E795Q, G146R / E795Q, R182V / E795Q, V491D / E795Q, Q529I / E795Q, Y646H / E795Q, D665N / E795Q, T814K / E795Q, L839F / E795Q, Q906F / E795Q, E795Q / Q1170D, G146R / R182V / E795Q, G146R / E795Q / D665N, G146R / E795Q / E 981V, G146R / E795Q / T814K, G146R / R182V / D665N, E125K / R182V / E981V, R182V / P799V / E981V, R182V / T814K / E981V, G146R / P79 9V / E981V, G146R / R182V / E795Q / F81E, G146R / R182V / E795Q / E125K, G146R / R182V / E795Q / E125A, G146R / R182V / E795Q / P799V,The following polypeptide sequences, comprising G146R / R182V / E795Q / T814K, G146R / R182V / E795Q / E981V, G146R / R182V / E795Q / D665N, G146R / R182V / P799V / E981V, E125K / G146R / R182V / E795Q / D665N, or G146R / R182V / E795Q / P799V / T814K, provide improved CRISPR / LbCas12a-related nuclease activity at non-canonical TTTT PAM sites compared to wild-type LbCas12a polypeptide sequences. In another embodiment, the encoded mutant LbCas12a polypeptides are: F81E (SEQ ID NO: 802), E125K (SEQ ID NO: 428), N145R (SEQ ID NO: 2902), G146R (SEQ ID NO: 156), T152K (SEQ ID NO: 694), R182V (SEQ ID NO: 98), V491D (SEQ ID NO: 954), K478R (SEQ ID NO: 134), N582R (SEQ ID NO: 1730), D665N (SEQ ID NO: 1014), E795Q (SEQ ID NO: 930), P799V (SEQ ID NO: 706), E858V (SEQ ID NO: 114), I860R (SEQ ID NO: 250), E913R (SEQ ID NO: 1960), E981V (SEQ ID NO: 880), E88A / E795Q (SEQ ID NO: 3960), E125K / E795Q (SEQ ID NO: 3962), G146R / E795Q (SEQ ID NO: 3964), R182V / E795Q (SEQ ID NO: 3966), V491D / E795Q (SEQ ID NO: 3968), Q529I / E795Q (SEQ ID NO: 3970), Y646H / E795Q (SEQ ID NO: 3972), D665N / E795Q (SEQ ID NO: 3974), T814K / E795Q (SEQ ID NO: 3976), L839F / E795Q (SEQ ID NO: 3978), Q906F / E795Q (SEQ ID NO: 3980), E795Q / Q1170D (SEQ ID NO: 3982), G146R / R182V / E795Q (SEQ ID NO: 3984), G146R / E795Q / D665N (SEQ ID NO: 3986), G146R / E795Q / E981V (SEQ ID NO: 3988), G146R / E795Q / T814K (SEQ ID NO: 3990), G146R / R182V / D665N (SEQ ID NO: 3992), E125K / R182V / E981V (SEQ ID NO: 3994), R182V / P799V / E981V (SEQ ID NO: 3996),R182V / T814K / E981V (SEQ ID NO: 3998), G146R / P799V / E981V (SEQ ID NO: 4000), G146R / R182V / E795Q / F81E (SEQ ID NO: 4002), G146R / R182V / E795Q / E125K (SEQ ID NO: 4004), G146R / R182V / E795Q / E125A (SEQ ID NO: 4006), G146R / R182V / E795Q / P799V (SEQ ID NO: 4008), G146R / R182V / E795Q The following are selected from / T814K (SEQ ID NO: 4010), G146R / R182V / E795Q / E981V (SEQ ID NO: 4012), G146R / R182V / E795Q / D665N (SEQ ID NO: 4014), G146R / R182V / P799V / E981V (SEQ ID NO: 4016), E125K / G146R / R182V / E795Q / D665N (SEQ ID NO: 4018), or G146R / R182V / E795Q / P799V / T814K (SEQ ID NO: 4020). In another embodiment, the encoded mutant LbCas12a polypeptide is G146R / R182V / E795Q (SEQ ID NO: 3984). In another embodiment, mutant LbCas12a polynucleotides are: SEQ ID NOs: 801, 427, 2901, 155, 693, 97, 953, 133, 1729, 641, 719, 1013, 929, 705, 833, 987, 113, 249, 3957, 47, 69, 1959, 879, 531, 597, 3959, 3961, 3963, 396 Select from 5, 3967, 3969, 3971, 3973, 3975, 3977, 3979, 3981, 3983, 3985, 3987, 3989, 3991, 3993, 3995, 3997, 3999, 4001, 4003, 4005, 4007, 4009, 4011, 4013, 4015, 4017, 4019, or 4021. In another embodiment, the mutant LbCas12a polynucleotides are SEQ ID NOs: 801, 427, 2901, 155, 693, 97, 953, 133, 1729, 1013, 929, 705, 113, 249, 531, 597, 3959, 3961, 3963, 3965, 3967, 3969, 3971, 3973, 3975, 3977, 3979, 3981, 3983, 3985, 3987, 3989, 3991, 3993, 3995, 3997, 3999, 4001, 4003, 4005,Selected from 4007, 4009, 4011, 4013, 4015, 4017, or 4019, these sequences encode mutant LbCas12a polypeptides that provide improved CRISPR / LbCas12a-related nuclease activity at non-canonical TTTT PAM sites compared to wild-type LbCas12a polypeptide sequences. In another embodiment, the mutant LbCas12a polynucleotide is sequence number 3983.

[0049] Another embodiment described herein is a vector or plasmid comprising any of the polynucleotide sequences described herein.

[0050] Another embodiment described herein is a cell comprising either the polynucleotide sequence described herein or the vector or plasmid described herein.

[0051] Another embodiment described herein is an isolated ribonucleoprotein complex comprising a guide RNA and a mutant LbCas12a polypeptide having at least one amino acid substitution introduced into the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2, provided that the mutant LbCas12a polypeptide provides improved CRISPR / LbCas12a-related nuclease activity at a non-canonical TTTT PAM site compared to the wild-type LbCas12a polypeptide sequence. In one embodiment, the mutant LbCas12a polypeptide has a polypeptide sequence selected from even-numbered sequences of SEQ ID NOs: 4-4022. In another embodiment, the mutant LbCas12a polypeptide includes at least one amino acid substitution at the position of 81, 125, 145, 146, 152, 182, 396, 478, 491, 582, 595, 606, 646, 665, 795, 799, 814, 839, 841, 858, 860, 890, 898, 909, 913, 981, 1020, 1083, or 1121 of the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2. In another embodiment, the mutant LbCas12a polypeptide comprises at least one amino acid substitution selected from F81E, E125K, N145R, G146R, T152K, R182V, S396D, K478R, V491D, N582R, K595R, Y606F, Y646H, D665N, E795Q, P799V, T814G, L839F, I841A, E858V, I860R, W890A, E898N, H909K, E913R, E981V, S1020E, V1083W, or K1121D of the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2. In another embodiment, mutant LbCas12a polypeptides include: F81E, SEQ ID NO: 802; E125K, SEQ ID NO: 428; N145R, SEQ ID NO: 2902; G146R, SEQ ID NO: 156; T152K, SEQ ID NO: 694; R182V, SEQ ID NO: 98; S396D, SEQ ID NO: 108; K478R, SEQ ID NO: 134; V491D, SEQ ID NO: 954; N582R, SEQ ID NO: 1730; K595R, SEQ ID NO: 184; Y606F, SEQ ID NO: 642; Y646H, SEQ ID NO: 720; D665N, SEQ ID NO: 1014; E795Q, SEQ ID NO: 930; P799V,Select from sequence numbers 706;T814G, 162;L839F, 834;I841A, 988;E858V, 114;I860R, 250;W890A, 3958;E898N, 48;H909K, 70;E913R, 1960;E981V, 880;S1020E, 532;V1083W, 132; or K1121D, 598. In another embodiment, mutant LbCas12a polypeptides include: F81E (SEQ ID NO: 802), E125K (SEQ ID NO: 428), N145R (SEQ ID NO: 2902), G146R (SEQ ID NO: 156), T152K (SEQ ID NO: 694), R182V (SEQ ID NO: 98), V491D (SEQ ID NO: 954), K478R (SEQ ID NO: 134), N582R (SEQ ID NO: 1730), D665N (SEQ ID NO: 1014), E795Q (SEQ ID NO: 930), P799 V (sequence number 706), E858V (sequence number 114), I860R (sequence number 250), E913R (sequence number 1960), E981V (sequence number 880), E88A / E795Q (sequence number 3960), E125K / E795Q (sequence number 3962), G146R / E795Q (sequence number 3964), R182V / E795Q (sequence number 3966), V491D / E795Q (sequence number 3968), Q529I / E795Q (sequence number (No. 3970), Y646H / E795Q (Sequence ID 3972), D665N / E795Q (Sequence ID 3974), T814K / E795Q (Sequence ID 3976), L839F / E795Q (Sequence ID 3978), Q906F / E795Q (Sequence ID 3980), E795Q / Q1170D (Sequence ID 3982), G146R / R182V / E795Q (Sequence ID 3984), G146R / E795Q / D665N (Sequence ID 3986), G146R / E795Q / E981V (SEQ ID NO: 3988), G146R / E795Q / T814K (SEQ ID NO: 3990), G146R / R182V / D665N (SEQ ID NO: 3992), E125K / R182V / E981V (SEQ ID NO: 3994), R182V / P799V / E981V (SEQ ID NO: 3996), R182V / T814K / E981V (SEQ ID NO: 3998), G146R / P799V / E981V (SEQ ID NO: 4000),G146R / R182V / E795Q / F81E (Sequence ID 4002), G146R / R182V / E795Q / E125K (Sequence ID 4004), G146R / R182V / E795Q / E125A (Sequence ID 4006), G146R / R182V / E795Q / P799V (Sequence ID 4008), G146R / R182V / E795Q / T814K (Sequence ID 4010), G146R / R182V The mutant LbCas12a polypeptide is selected from / E795Q / E981V (SEQ ID NO: 4012), G146R / R182V / E795Q / D665N (SEQ ID NO: 4014), G146R / R182V / P799V / E981V (SEQ ID NO: 4016), E125K / G146R / R182V / E795Q / D665N (SEQ ID NO: 4018), or G146R / R182V / E795Q / P799V / T814K (SEQ ID NO: 4020). In another embodiment, the mutant LbCas12a polypeptide is G146R / R182V / E795Q (SEQ ID NO: 3984).

[0052] Another embodiment described herein is a method for enhancing the efficiency of gene editing at a non-canonical TTTT PAM site in a cell using a CRISPR ribonucleoprotein complex, comprising the step of contacting a cell with a CRISPR ribonucleoprotein complex comprising a guide RNA and a mutant LbCas12a polypeptide having at least one amino acid substitution introduced into the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2, provided that the mutant LbCas12a polypeptide provides improved CRISPR / LbCas12a-related nuclease activity at a non-canonical TTTT PAM site compared to the wild-type LbCas12a polypeptide sequence. In one embodiment, the mutant LbCas12a polypeptide has a polypeptide sequence selected from even-numbered sequences of SEQ ID NOs: 4 to 4022. In another embodiment, the mutant LbCas12a polypeptide includes at least one amino acid substitution at the position of 81, 125, 145, 146, 152, 182, 396, 478, 491, 582, 595, 606, 646, 665, 795, 799, 814, 839, 841, 858, 860, 890, 898, 909, 913, 981, 1020, 1083, or 1121 of the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2. In another embodiment, the mutant LbCas12a polypeptide comprises at least one amino acid substitution selected from F81E, E125K, N145R, G146R, T152K, R182V, S396D, K478R, V491D, N582R, K595R, Y606F, Y646H, D665N, E795Q, P799V, T814G, L839F, I841A, E858V, I860R, W890A, E898N, H909K, E913R, E981V, S1020E, V1083W, or K1121D of the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2. In another embodiment, mutant LbCas12a polypeptides include: F81E, SEQ ID NO: 802; E125K, SEQ ID NO: 428; N145R, SEQ ID NO: 2902; G146R, SEQ ID NO: 156; T152K, SEQ ID NO: 694; R182V, SEQ ID NO: 98; S396D, SEQ ID NO: 108; K478R, SEQ ID NO: 134; V491D, SEQ ID NO: 954; N582R, SEQ ID NO: 1730; K595R,Select from sequence numbers 184;Y606F, 642;Y646H, 720;D665N, 1014;E795Q, 930;P799V, 706;T814G, 162;L839F, 834;I841A, 988;E858V, 114;I860R, 250;W890A, 3958;E898N, 48;H909K, 70;E913R, 1960;E981V, 880;S1020E, 532;V1083W, 132; or K1121D, 598. In another embodiment, a single substitution mutation is introduced into the wild-type LbCas12a polypeptide sequence of Sequence ID No. 2, in which a mutant LbCas12a polypeptide is introduced at (a) a position selected from: F81, E125, N145, G146, T152, R182, S396, K478, V491, N582, K595, Y606, Y646, D665, E795, P799, T814, L839, I841, E858, I860, W890, E898, H909, E913, E981, S1020, V1083, or K1121; Or (b) the following positions: containing a substitution mutation selected from the polysubstitution mutations introduced into the wild-type LbCas12a polypeptide sequence of SEQ ID NO: F81, E125, N145, G146, T152, R182, S396, K478, V491, N582, K595, Y606, Y646, D665, E795, P799, T814, L839, I841, E858, I860, W890, E898, H909, E913, E981, S1020, V1083 or K1121. In another embodiment, polysubstitution mutations include: E88A / E795Q, E125K / E795Q, G146R / E795Q, R182V / E795Q, V491D / E795Q, Q529I / E795Q, Y646H / E795Q, D665N / E795Q, T814K / E795Q, L839F / E795Q, Q906F / E795Q, E795Q / Q1170D, G146R / R182V / E795Q, G146R / E795Q / D665N, G146R / E795Q / E981V, G146R / E795Q / T814K, G146R / R182V / D665N, E125K / R182V / E981V,R182V / P799V / E981V, R182V / T814K / E981V, G146R / P799V / E981V, G146R / R182V / E795Q / F81E, G146R / R182V / E795Q / E125K, G146R / R182V / E795Q / E125A, G146R / R182V / E795Q / P799V, G146R / Includes R182V / E795Q / T814K, G146R / R182V / E795Q / E981V, G146R / R182V / E795Q / D665N, G146R / R182V / P799V / E981V, E125K / G146R / R182V / E795Q / D665N, or G146R / R182V / E795Q / P799V / T814K. In another embodiment, mutant LbCas12a polypeptides include: F81E (SEQ ID NO: 802), E125K (SEQ ID NO: 428), N145R (SEQ ID NO: 2902), G146R (SEQ ID NO: 156), T152K (SEQ ID NO: 694), R182V (SEQ ID NO: 98), V491D (SEQ ID NO: 954), K478R (SEQ ID NO: 134), N582R (SEQ ID NO: 1730), D665N (variables). Column number 1014), E795Q (sequence number 930), P799V (sequence number 706), E858V (sequence number 114), I860R (sequence number 250), E913R (sequence number 1960), E981V (sequence number 880), E88A / E795Q (sequence number 3960), E125K / E795Q (sequence number 3962), G146R / E795Q (sequence number 3964), R18 2V / E795Q (SEQ ID NO: 3966), V491D / E795Q (SEQ ID NO: 3968), Q529I / E795Q (SEQ ID NO: 3970), Y646H / E795Q (SEQ ID NO: 3972), D665N / E795Q (SEQ ID NO: 3974), T814K / E795Q (SEQ ID NO: 3976), L839F / E795Q (SEQ ID NO: 3978), Q906F / E795Q (SEQ ID NO: 3976) 980), E795Q / Q1170D (Sequence ID 3982), G146R / R182V / E795Q (Sequence ID 3984), G146R / E795Q / D665N (Sequence ID 3986), G146R / E795Q / E981V (Sequence ID 3988), G146R / E795Q / T814K (Sequence ID 3990), G146R / R182V / D665N (Sequence ID 3992),E125K / R182V / E981V (SEQ ID NO: 3994), R182V / P799V / E981V (SEQ ID NO: 3996), R182V / T814K / E981V (SEQ ID NO: 3998), G146R / P799V / E981V (SEQ ID NO: 4000), G146R / R182V / E795Q / F81E (SEQ ID NO: 4002), G146R / R182V / E795Q / E125K (SEQ ID NO: 4004), G146R / R182V / E795Q / E125A (SEQ ID NO: 4006), G146R / R182V / E795Q / P799V Select from (Sequence ID 4008), G146R / R182V / E795Q / T814K (Sequence ID 4010), G146R / R182V / E795Q / E981V (Sequence ID 4012), G146R / R182V / E795Q / D665N (Sequence ID 4014), G146R / R182V / P799V / E981V (Sequence ID 4016), E125K / G146R / R182V / E795Q / D665N (Sequence ID 4018), or G146R / R182V / E795Q / P799V / T814K (Sequence ID 4020). In another embodiment, the mutant LbCas12a polypeptide is G146R / R182V / E795Q (SEQ ID NO: 3984).

[0053] Another embodiment described herein is a kit for enhancing the efficiency of gene editing at a non-canonical TTTT PAM site in a cell, comprising a CRISPR-ribonucleoprotein complex comprising a guide RNA and a mutant LbCas12a polypeptide comprising at least one amino acid substitution introduced into the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2, provided that the mutant LbCas12a polypeptide provides improved CRISPR / LbCas12a-related nuclease activity at a non-canonical TTTT PAM site compared to the wild-type LbCas12a polypeptide sequence. In one embodiment, the mutant LbCas12a polypeptide has a polypeptide sequence selected from even-numbered sequences of SEQ ID NOs: 4-4022. In another embodiment, the mutant LbCas12a polypeptide includes at least one amino acid substitution at the position of 81, 125, 145, 146, 152, 182, 396, 478, 491, 582, 595, 606, 646, 665, 795, 799, 814, 839, 841, 858, 860, 890, 898, 909, 913, 981, 1020, 1083, or 1121 of the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2. In another embodiment, the mutant LbCas12a polypeptide comprises at least one amino acid substitution selected from F81E, E125K, N145R, G146R, T152K, R182V, S396D, K478R, V491D, N582R, K595R, Y606F, Y646H, D665N, E795Q, P799V, T814G, L839F, I841A, E858V, I860R, W890A, E898N, H909K, E913R, E981V, S1020E, V1083W, or K1121D of the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2. In another embodiment, mutant LbCas12a polypeptides include: F81E, SEQ ID NO: 802; E125K, SEQ ID NO: 428; N145R, SEQ ID NO: 2902; G146R, SEQ ID NO: 156; T152K, SEQ ID NO: 694; R182V, SEQ ID NO: 98; S396D, SEQ ID NO: 108; K478R, SEQ ID NO: 134; V491D, SEQ ID NO: 954; N582R, SEQ ID NO: 1730; K595R, SEQ ID NO: 184; Y606F, SEQ ID NO: 642; Y646H,Select from sequence numbers 720;D665N, 1014;E795Q, 930;P799V, 706;T814G, 162;L839F, 834;I841A, 988;E858V, 114;I860R, 250;W890A, 3958;E898N, 48;H909K, 70;E913R, 1960;E981V, 880;S1020E, 532;V1083W, 132; or K1121D, 598. In another embodiment, a single substitution mutation is introduced into the wild-type LbCas12a polypeptide sequence of Sequence ID No. 2, in which a mutant LbCas12a polypeptide is introduced at (a) a position selected from: F81, E125, N145, G146, T152, R182, S396, K478, V491, N582, K595, Y606, Y646, D665, E795, P799, T814, L839, I841, E858, I860, W890, E898, H909, E913, E981, S1020, V1083, or K1121; Or (b) the following positions: containing a substitution mutation selected from the polysubstitution mutations introduced into the wild-type LbCas12a polypeptide sequence of SEQ ID NO: F81, E125, N145, G146, T152, R182, S396, K478, V491, N582, K595, Y606, Y646, D665, E795, P799, T814, L839, I841, E858, I860, W890, E898, H909, E913, E981, S1020, V1083 or K1121. In another embodiment, polysubstitution mutations include: E88A / E795Q, E125K / E795Q, G146R / E795Q, R182V / E795Q, V491D / E795Q, Q529I / E795Q, Y646H / E795Q, D665N / E795Q, T814K / E795Q, L839F / E795Q, Q906F / E79 5Q, E795Q / Q1170D, G146R / R182V / E795Q, G146R / E795Q / D665N, G146R / E795Q / E981V, G 146R / E795Q / T814K, G146R / R182V / D665N, E125K / R182V / E981V, R182V / P799V / E981V,R182V / T814K / E981V, G146R / P799V / E981V, G146R / R182V / E795Q / F81E, G146R / R182V / E7 95Q / E125K, G146R / R182V / E795Q / E125A, G146R / R182V / E795Q / P799V, G146R / R182V / E79 Includes 5Q / T814K, G146R / R182V / E795Q / E981V, G146R / R182V / E795Q / D665N, G146R / R182V / P799V / E981V, E125K / G146R / R182V / E795Q / D665N, or G146R / R182V / E795Q / P799V / T814K. In another embodiment, mutant LbCas12a polypeptides include: F81E (SEQ ID NO: 802), E125K (SEQ ID NO: 428), N145R (SEQ ID NO: 2902), G146R (SEQ ID NO: 156), T152K (SEQ ID NO: 694), R182V (SEQ ID NO: 98), V491D (SEQ ID NO: 954), K478R (SEQ ID NO: 134), N582R (SEQ ID NO: 1730), D665N (SEQ ID NO: 1014) ), E795Q (sequence number 930), P799V (sequence number 706), E858V (sequence number 114), I860R (sequence number 250), E913R (sequence number 1960), E981V (sequence number 880), E88A / E795Q (sequence number 3960), E125K / E795Q (sequence number 3962), G146R / E795Q (sequence number 3964), R182V / E795Q (sequence number 3960) 966), V491D / E795Q (sequence number 3968), Q529I / E795Q (sequence number 3970), Y646H / E795Q (sequence number 3972), D665N / E795Q (sequence number 3974), T814K / E795Q (sequence number 3976), L839F / E795Q (sequence number 3978), Q906F / E795Q (sequence number 3980), E795Q / Q1170D (sequence number (Sequence number 3982), G146R / R182V / E795Q (Sequence number 3984), G146R / E795Q / D665N (Sequence number 3986), G146R / E795Q / E981V (Sequence number 3988), G146R / E795Q / T814K (Sequence number 3990), G146R / R182V / D665N (Sequence number 3992), E125K / R182V / E981V (Sequence number 3994),R182V / P799V / E981V (SEQ ID NO: 3996), R182V / T814K / E981V (SEQ ID NO: 3998), G146R / P799V / E981V (SEQ ID NO: 4000), G146R / R182V / E795Q / F81E (SEQ ID NO: 4002), G146R / R182V / E795Q / E125K (SEQ ID NO: 4004), G146R / R182V / E795Q / E125A (SEQ ID NO: 4006), G146R / R182V / E795Q / P799V (SEQ ID NO: 4008), G14 The mutant LbCas12a polypeptide is selected from 6R / R182V / E795Q / T814K (SEQ ID NO: 4010), G146R / R182V / E795Q / E981V (SEQ ID NO: 4012), G146R / R182V / E795Q / D665N (SEQ ID NO: 4014), G146R / R182V / P799V / E981V (SEQ ID NO: 4016), E125K / G146R / R182V / E795Q / D665N (SEQ ID NO: 4018), or G146R / R182V / E795Q / P799V / T814K (SEQ ID NO: 4020). In another embodiment, the mutant LbCas12a polypeptide is G146R / R182V / E795Q (SEQ ID NO: 3984).

[0054] Another embodiment described herein is the use of a mutant LbCas12a polypeptide to improve CRISPR / LbCas12a-related nuclease activity at a non-canonical TTTT PAM site, wherein the mutant LbCas12a polypeptide comprises at least one amino acid substitution introduced into the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2, provided that the mutant LbCas12a polypeptide provides improved CRISPR / LbCas12a-related nuclease activity at a non-canonical TTTT PAM site compared to the wild-type LbCas12a polypeptide sequence. In one embodiment, the mutant LbCas12a polypeptide has a polypeptide sequence selected from the even-numbered sequences of SEQ ID NOs: 4 to 4022. In another embodiment, the mutant LbCas12a polypeptide includes at least one amino acid substitution at the position of 81, 125, 145, 146, 152, 182, 396, 478, 491, 582, 595, 606, 646, 665, 795, 799, 814, 839, 841, 858, 860, 890, 898, 909, 913, 981, 1020, 1083, or 1121 of the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2. In another embodiment, the mutant LbCas12a polypeptide comprises at least one amino acid substitution selected from F81E, E125K, N145R, G146R, T152K, R182V, S396D, K478R, V491D, N582R, K595R, Y606F, Y646H, D665N, E795Q, P799V, T814G, L839F, I841A, E858V, I860R, W890A, E898N, H909K, E913R, E981V, S1020E, V1083W, or K1121D of the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2. In another embodiment, mutant LbCas12a polypeptides include: F81E, SEQ ID NO: 802; E125K, SEQ ID NO: 428; N145R, SEQ ID NO: 2902; G146R, SEQ ID NO: 156; T152K, SEQ ID NO: 694; R182V, SEQ ID NO: 98; S396D, SEQ ID NO: 108; K478R, SEQ ID NO: 134; V491D, SEQ ID NO: 954; N582R, SEQ ID NO: 1730; K595R, SEQ ID NO: 184; Y606F, SEQ ID NO: 642; Y646H,Select from sequence numbers 720;D665N, 1014;E795Q, 930;P799V, 706;T814G, 162;L839F, 834;I841A, 988;E858V, 114;I860R, 250;W890A, 3958;E898N, 48;H909K, 70;E913R, 1960;E981V, 880;S1020E, 532;V1083W, 132; or K1121D, 598. In another embodiment, a single substitution mutation is introduced into the wild-type LbCas12a polypeptide sequence of Sequence ID No. 2, in which a mutant LbCas12a polypeptide is introduced at (a) a position selected from: F81, E125, N145, G146, T152, R182, S396, K478, V491, N582, K595, Y606, Y646, D665, E795, P799, T814, L839, I841, E858, I860, W890, E898, H909, E913, E981, S1020, V1083, or K1121; Or (b) the following positions: containing a substitution mutation selected from the polysubstitution mutations introduced into the wild-type LbCas12a polypeptide sequence of SEQ ID NO: F81, E125, N145, G146, T152, R182, S396, K478, V491, N582, K595, Y606, Y646, D665, E795, P799, T814, L839, I841, E858, I860, W890, E898, H909, E913, E981, S1020, V1083 or K1121. In another embodiment, polysubstitution mutations include: E88A / E795Q, E125K / E795Q, G146R / E795Q, R182V / E795Q, V491D / E795Q, Q529I / E795Q, Y646H / E795Q, D665N / E795Q, T814K / E795Q, L839F / E795Q, Q906F / E79 5Q, E795Q / Q1170D, G146R / R182V / E795Q, G146R / E795Q / D665N, G146R / E795Q / E981V, G 146R / E795Q / T814K, G146R / R182V / D665N, E125K / R182V / E981V, R182V / P799V / E981V,R182V / T814K / E981V, G146R / P799V / E981V, G146R / R182V / E795Q / F81E, G146R / R182V / E7 95Q / E125K, G146R / R182V / E795Q / E125A, G146R / R182V / E795Q / P799V, G146R / R182V / E79 Includes 5Q / T814K, G146R / R182V / E795Q / E981V, G146R / R182V / E795Q / D665N, G146R / R182V / P799V / E981V, E125K / G146R / R182V / E795Q / D665N, or G146R / R182V / E795Q / P799V / T814K. In another embodiment, mutant LbCas12a polypeptides include: F81E (SEQ ID NO: 802), E125K (SEQ ID NO: 428), N145R (SEQ ID NO: 2902), G146R (SEQ ID NO: 156), T152K (SEQ ID NO: 694), R182V (SEQ ID NO: 98), V491D (SEQ ID NO: 954), K478R (SEQ ID NO: 134), N582R (SEQ ID NO: 1730), D665N (SEQ ID NO: 1014) ), E795Q (sequence number 930), P799V (sequence number 706), E858V (sequence number 114), I860R (sequence number 250), E913R (sequence number 1960), E981V (sequence number 880), E88A / E795Q (sequence number 3960), E125K / E795Q (sequence number 3962), G146R / E795Q (sequence number 3964), R182V / E795Q (sequence number 3960) 966), V491D / E795Q (sequence number 3968), Q529I / E795Q (sequence number 3970), Y646H / E795Q (sequence number 3972), D665N / E795Q (sequence number 3974), T814K / E795Q (sequence number 3976), L839F / E795Q (sequence number 3978), Q906F / E795Q (sequence number 3980), E795Q / Q1170D (sequence number (Sequence number 3982), G146R / R182V / E795Q (Sequence number 3984), G146R / E795Q / D665N (Sequence number 3986), G146R / E795Q / E981V (Sequence number 3988), G146R / E795Q / T814K (Sequence number 3990), G146R / R182V / D665N (Sequence number 3992), E125K / R182V / E981V (Sequence number 3994),R182V / P799V / E981V (SEQ ID NO: 3996), R182V / T814K / E981V (SEQ ID NO: 3998), G146R / P799V / E981V (SEQ ID NO: 4000), G146R / R182V / E795Q / F81E (SEQ ID NO: 4002), G146R / R182V / E795Q / E125K (SEQ ID NO: 4004), G146R / R182V / E795Q / E125A (SEQ ID NO: 4006), G146R / R182V / E795Q / P799V (SEQ ID NO: 4008), G14 The mutant LbCas12a polypeptide is selected from 6R / R182V / E795Q / T814K (SEQ ID NO: 4010), G146R / R182V / E795Q / E981V (SEQ ID NO: 4012), G146R / R182V / E795Q / D665N (SEQ ID NO: 4014), G146R / R182V / P799V / E981V (SEQ ID NO: 4016), E125K / G146R / R182V / E795Q / D665N (SEQ ID NO: 4018), or G146R / R182V / E795Q / P799V / T814K (SEQ ID NO: 4020). In another embodiment, the mutant LbCas12a polypeptide is G146R / R182V / E795Q (SEQ ID NO: 3984).

[0055] Another embodiment described herein is a method for expressing and purifying a mutant LbCas13a protein, comprising: (a) inserting into an expression plasmid a nucleotide sequence encoding a mutant LbCas12a polypeptide having at least one amino acid substitution compared to the wild-type LbCas12a polypeptide sequence and having 95–99% identity with any of the even-numbered polypeptide sequences of SEQ ID NOs. 4–4022; (b) transforming one or more cells with the expression plasmid; (c) inducing expression of the transformed plasmid; (d) isolating the cells; (e) extracting the mutant LbCas13a protein; and (f) purifying the mutant LbCas13a protein, wherein the mutant LbCas12a polypeptide provides improved CRISPR / LbCas12a-related nuclease activity at the non-canonical TTTT PAM site compared to the wild-type LbCas12a polypeptide sequence.

[0056] Another embodiment described herein is a mutant LbCas12a polypeptide produced by the method described herein.

[0057] Another embodiment described herein is a method for producing one or more of the nucleotide sequences described herein, or a polypeptide encoded by the nucleotide sequences described herein, the method comprising: transforming or transfecting cells with nucleic acids comprising the nucleotide sequences described herein; growing the cells; isolating further amounts of the nucleotide sequences described herein, if necessary; inducing the expression of the polypeptide encoded by the nucleotide sequences described herein; and isolating the polypeptide encoded by the nucleotides described herein.

[0058] Another embodiment described herein is a means for producing one or more of the nucleotide sequences described herein, or a polypeptide encoded by the nucleotide sequences described herein, the method comprising: transforming or transfecting cells with nucleic acids comprising the nucleotide sequences described herein; growing the cells; isolating further amounts of the nucleotide sequences described herein, if necessary; inducing the expression of the polypeptide encoded by the nucleotide sequences described herein; and isolating the polypeptide encoded by the nucleotides described herein.

[0059] Another embodiment described herein is a nucleotide sequence or polypeptide encoded by a nucleotide sequence, which is produced by the method or means described herein.

[0060] Another embodiment described herein is the use of an effective amount of polypeptide encoded by one or more of the nucleotide sequences described herein in a CRISPR / Cas12a system.

[0061] Another embodiment described herein is a research tool comprising a polypeptide encoded by the nucleotide sequence described herein.

[0062] Another embodiment described herein is a biochemical reagent or therapeutic agent comprising a polypeptide encoded by the nucleotide sequence described herein.

[0063] The polynucleotides described herein include variants having substitutions, deletions, and / or additions that may involve one or more nucleotides. Variants may be modified in the coding region, non-coding region, or both. Modifications in the coding region can result in conserved or non-conserved amino acid substitutions, deletions, or additions. Of these, silent substitutions, additions, and deletions that do not alter activity are particularly preferred.

[0064] Further embodiments described herein include nucleic acid molecules comprising a polynucleotide having a nucleotide sequence that is about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, more preferably at least about 90-99%, identical to (a) a polypeptide having an even-numbered amino acid sequence of sequence 4-4022, or degenerate, homologous, or codon-optimized variants thereof; and (c) a nucleotide sequence that can hybridize to a complementary chain of the nucleotide sequence of (a) or (b) above and can express a functional polypeptide of an even-numbered amino acid sequence of sequence 4-4022.

[0065] A polynucleotide having a nucleotide sequence that is at least, for example, 90-99% "identical" to a reference nucleotide sequence encoding the mutant LbCas12a is intended to have a nucleotide sequence identical to the reference sequence, except that the polynucleotide sequence may contain up to approximately 10-1 point mutations, additions, or deletions per 100 nucleotides of the reference nucleotide sequence encoding the mutant LbCas12a.

[0066] In other words, to obtain a polynucleotide having a nucleotide sequence that is approximately 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. Such mutations in the reference sequence can occur at the 5' or 3' terminal position of the reference nucleotide sequence, or anywhere between these terminal positions, and can be scattered individually among the nucleotides in the reference sequence or in one or more consecutive groups within the reference sequence. The same applies to polypeptide sequences that are approximately 90–99% identical to a reference polypeptide sequence.

[0067] 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. Whether nucleic acid sequences or peptide sequences, the percentage of identity between two sequences is generally described as the number of exact matches between the two aligned sequences divided by the length of the shorter sequence, multiplied by 100. Approximate alignment of nucleic acid sequences is possible using the local homology algorithm described by Smith and Waterman, Advances in Applied Mathematics 2: pp. 482-489 (1981).

[0068] Due to the degeneracy of the genetic code, numerous nucleic acid molecules, or their degenerate, homologous, or codon-optimized variants, will encode the mutant LbCas12a. These molecules will have sequences that are at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequences shown in the odd-numbered sequences of SEQ ID NOs: 3 to 4021.

[0069] The polynucleotides described herein include those encoding mutations, variations, substitutions, additions, deletions, and specific examples of the polypeptides described herein. For example, guidance on how to make 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: pp. 1306-1310 (1990), where the authors note the surprising tolerance of proteins to amino acid substitutions.

[0070] Therefore, polypeptide fragments, derivatives, or analogs of even-numbered sequences in Sequence ID No. 4 to 4022 may be (i) in which one or more amino acid residues (e.g., 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 residues, or more) are substituted with conserved or non-conserved amino acid residues (preferably conserved amino acid residues). Such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) one or more amino acid residues contain substituents (e.g., 1, 2, 3, 4, 5, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 residues, or more), or (iii) a mature polypeptide fused with another polypeptide or compound, for example, a compound that extends the half-life of the polypeptide (e.g., polyethylene glycol), or (iv) a mature polypeptide fused with further amino acids, such as an IgG Fc fusion region peptide or a leader sequence or secretion sequence or a sequence or precursor protein sequence used for the purification of the mature polypeptide, may or may not be present. Such fragments, derivatives, and analogs are considered to be within the scope of the art based on the teachings herein.

[0071] In addition, polypeptide fragments, derivatives, or analogs of the even-numbered sequences of SEQ ID NOs: 4-4022 may be substituted with one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues). In some examples, these polypeptides, their fragments, derivatives, or analogs will have polypeptide sequences that are at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polypeptide sequences shown in the even-numbered sequences of SEQ ID NOs: 4-4022, and will include functional or non-functional proteins or enzymes. Similarly, additions or deletions to polypeptides may occur either at the N-terminus or C-terminus, or within a non-conserved region of the polypeptide (which is assumed to be unimportant as it is not photogenically conserved).

[0072] In many cases as described herein, amino acid substitutions, mutations, additions, or deletions are of a minor nature, such as conservative amino acid substitutions that do not significantly affect protein folding or activity or addition or deletion to the N-terminus or C-terminus. It is preferable that this is the case. Naturally, the number of amino acid substitutions, additions, or deletions that a person skilled in the art would perform depends on many factors, including those described herein. Generally, the number of substitutions, additions, or deletions of any given polypeptide is 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 or less.

[0073] The applications of Cas12a and LbCas12a-based tools are numerous and diverse. These applications, though not limited to those listed above, include: gene editing in plants, yeast, and mammals; cell editing of organs in living animals; embryo editing; rapid creation of knockout / knock-in animal lines; creation of animal models of disease states; correction of disease states; insertion of reporter genes; and whole-genome functional screening.

[0074] It will be apparent to those skilled in the art that appropriate modifications and applications of the compositions, formulations, methods, processes, and applications described herein can be made without departing from the scope of any of their embodiments or aspects. The compositions and methods provided are illustrative and are not intended to limit the scope of any particular embodiment. 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, and processes described herein includes all actual or potential combinations of the embodiments, aspects, options, examples, and priorities described herein. The exemplary compositions and formulations described herein may omit any component, replace any component disclosed herein, or include any component otherwise disclosed 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 to the total mass of the other components in the formulation, is disclosed herein as if expressly disclosed. If the meaning of any term in any patent or publication incorporated by reference conflicts with the meaning of a term used herein, the meaning of the term or phrase herein shall prevail. Furthermore, the foregoing discussions merely disclose and describe exemplary embodiments. All patents and publications cited herein are incorporated herein by reference with respect to their specific teachings.

[0075] Various embodiments and aspects of the present invention described herein are summarized in the following sections: Section 1. Isolated mutant LbCas12a polypeptide comprising at least one amino acid substitution introduced into the wild-type LbCas12a polypeptide sequence of Sequence ID No. 2, provided that, compared to the wild-type LbCas12a polypeptide sequence, the mutant LbCas12a polypeptide provides improved CRISPR / LbCas12a-related nuclease activity at the non-canonical TTTT PAM site. Section 2. Isolated mutant LbCas12a polypeptide from Section 1, having 95% to 99% identity with polypeptide sequences selected from even-numbered sequences 4 to 4022. Section 3. Isolated mutant LbCas12a polypeptides from Section 1 or 2, having polypeptide sequences selected from even-numbered sequences 4-4022. Section 4. Isolated mutant LbCas12a polypeptides from any one of Sections 1-3, containing at least one amino acid substitution at the position of 81, 125, 145, 146, 152, 182, 396, 478, 491, 582, 595, 606, 646, 665, 795, 799, 814, 839, 841, 858, 860, 890, 898, 909, 913, 981, 1020, 1083, or 1121 of the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2. Section 5. Isolated mutant LbCas12a polypeptides from any one of Sections 1-4, comprising at least one amino acid substitution selected from F81E, E125K, N145R, G146R, T152K, R182V, S396D, K478R, V491D, N582R, K595R, Y606F, Y646H, D665N, E795Q, P799V, T814G, L839F, I841A, E858V, I860R, W890A, E898N, H909K, E913R, E981V, S1020E, V1083W, or K1121D of the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2. Section 6. F81E, SEQ ID NO: 802; E125K, SEQ ID NO: 428; N145R, SEQ ID NO: 2902; G146R, SEQ ID NO: 156; T152K, SEQ ID NO: 694; R182V, SEQ ID NO: 98; S396D, SEQ ID NO: 108; K478R, SEQ ID NO: 134; V491D, SEQ ID NO: 954; N582R, SEQ ID NO: 1730; K595R, SEQ ID NO: 184; Y606F, SEQ ID NO: 642; Y646H, SEQ ID NO: 720; D665N, SEQ ID NO: 1014; E795Q, SEQ ID NO: 930; P799V, SEQ ID NO: 706; T Isolated mutant LbCas12a polypeptide from any one of sections 1-5, selected from 814G, SEQ ID NO: 162; L839F, SEQ ID NO: 834; I841A, SEQ ID NO: 988; E858V, SEQ ID NO: 114; I860R, SEQ ID NO: 250; W890A, SEQ ID NO: 3958; E898N, SEQ ID NO: 48; H909K, SEQ ID NO: 70; E913R, SEQ ID NO: 1960; E981V, SEQ ID NO: 880; S1020E, SEQ ID NO: 532; V1083W, SEQ ID NO: 132; or K1121D, SEQ ID NO: 598. A single substitution mutation introduced into the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2, selected from the following positions in Section 7.(a): F81, E125, N145, G146, T152, R182, S396, K478, V491, N582, K595, Y606, Y646, D665, E795, P799, T814, L839, I841, E858, I860, W890, E898, H909, E913, E981, S1020, V1083 or K1121; or (b) The following locations: A polysubstitution mutation introduced into the wild-type LbCas12a polypeptide sequence of SEQ ID NO: F81, E125, N145, G146, T152, R182, S396, K478, V491, N582, K595, Y606, Y646, D665, E795, P799, T814, L839, I841, E858, I860, W890, E898, H909, E913, E981, S1020, V1083 or K1121. An isolated mutant LbCas12a polypeptide from any one of sections 1-6, containing a substitution mutation selected from the above. Section 8. Polysubstitution mutations include: E88A / E795Q, E125K / E795Q, G146R / E795Q, R182V / E795Q, V491D / E795Q, Q529I / E795Q, Y646H / E795Q, D665N / E795Q, T814K / E795Q, L839F / E795Q, Q906F / E795Q, E795Q / Q1170D, G146R / R 182V / E795Q, G146R / E795Q / D665N, G146R / E795Q / E981V, G146R / E795Q / T814K, G146R / R182V / D66 5N, E125K / R182V / E981V, R182V / P799V / E981V, R182V / T814K / E981V, G146R / P799V / E981V, G146R / R182V / E795Q / F81E, G146R / R182V / E795Q / E125K, G146R / R182V / E795Q / E125A, G146R / R182V / E79 5Q / P799V, G146R / R182V / E795Q / T814K, G146R / R182V / E795Q / E981V, G146R / R182V / E795Q / D665N, Isolated mutant LbCas12a polypeptides from any one of sections 1-7, including G146R / R182V / P799V / E981V, E125K / G146R / R182V / E795Q / D665N, G146R / R182V / E795Q / P799V / T814K, or E125K / G146R / R182V / E795Q / D665N / E981V. Section 9. Polysubstitution mutations: E88A / E795Q, E125K / E795Q, G146R / E795Q, R182V / E795Q, V491D / E795Q, Q529I / E795Q, Y646H / E795Q, D665N / E795Q, T814K / E795Q, L839F / E795Q, Q906F / E795Q, E795Q / Q1170 D, G146R / R182V / E795Q, G146R / E795Q / D665N, G146R / E795Q / E981V, G146R / E795Q / T814K, G1 46R / R182V / D665N, E125K / R182V / E981V, R182V / P799V / E981V, R182V / T814K / E981V, G146R / P799V / E981V, G146R / R182V / E795Q / F81E, G146R / R182V / E795Q / E125K, G146R / R182V / E795Q / E125A, G146R / R182V / E795Q / P799V, G146R / R182V / E795Q / T814K, G146R / R182V / E795Q / E98 Isolated mutant LbCas12a polypeptides from any one of sections 1-8, comprising 1V, G146R / R182V / E795Q / D665N, G146R / R182V / P799V / E981V, E125K / G146R / R182V / E795Q / D665N, or G146R / R182V / E795Q / P799V / T814K, providing improved CRISPR / LbCas12a-related nuclease activity at non-canonical TTTT PAM sites compared to wild-type LbCas12a polypeptide sequences. Section 10. The mutant LbCas12a polypeptide, Sequence numbers 802, 428, 2902, 156, 694, 98, 954, 134, 1730, 642, 720, 1014, 930, 706, 834, 988, 114, 250, 3958, 48, 70, 1960, 880, 532, 598, 3960, 3962, 3964, 3966, 3968, 3970, 3972, 3974, 3976, 3978 Isolated mutant LbCas12a polypeptide from any one of sections 1-9, selected from 3980, 3982, 3984, 3986, 3988, 3990, 3992, 3994, 3996, 3998, 4000, 4002, 4004, 4006, 4008, 4010, 4012, 4014, 4016, 4018, 4020, or 4022. Section 11. Mutant LbCas12a polypeptides include: F81E (SEQ ID NO: 802), E125K (SEQ ID NO: 428), N145R (SEQ ID NO: 2902), G146R (SEQ ID NO: 156), T152K (SEQ ID NO: 694), R182V (SEQ ID NO: 98), V491D (SEQ ID NO: 954), K478R (SEQ ID NO: 134), N582R (SEQ ID NO: 1730), D665N (SEQ ID NO: 1014), E795Q (SEQ ID NO: 930), P799V (SEQ ID NO: 706), E858V (SEQ ID NO: 114), I860R (SEQ ID NO: 250), E913R (SEQ ID NO: 19 60), E981V (sequence number 880), E88A / E795Q (sequence number 3960), E125K / E795Q (sequence number 3962), G146R / E795Q (sequence number 3964), R182V / E795Q (sequence number 3966), V491D / E795Q (sequence number 3968), Q529I / E795Q (sequence number 3970), Y646H / E795Q (sequence number 3972), D665N / E795Q (sequence number 3974), T814K / E795Q (sequence number 3976), L839F / E795Q (sequence number 3978), Q906F / E7 95Q (SEQ ID NO: 3980), E795Q / Q1170D (SEQ ID NO: 3982), G146R / R182V / E795Q (SEQ ID NO: 3984), G146R / E795Q / D665N (SEQ ID NO: 3986), G146R / E795Q / E981V (SEQ ID NO: 3988), G146R / E795Q / T814K (SEQ ID NO: 3990), G146R / R182V / D665N (SEQ ID NO: 3992), E125K / R182V / E981V (SEQ ID NO: 3994), R182V / P799V / E981V (SEQ ID NO: 3996), R182V / T814K / E 981V (SEQ ID NO: 3998), G146R / P799V / E981V (SEQ ID NO: 4000), G146R / R182V / E795Q / F81E (SEQ ID NO: 4002), G146R / R182V / E795Q / E125K (SEQ ID NO: 4004), G146R / R182V / E795Q / E125A (SEQ ID NO: 4006), G146R / R182V / E795Q / P799V (SEQ ID NO: 4008), G146R / R182V / E795Q / T814K (SEQ ID NO: 4010), G146R / R182V / E795Q / E981V (SEQ ID NO: 4012),An isolated mutant LbCas12a polypeptide from any one of sections 1-10, selected from G146R / R182V / E795Q / D665N (SEQ ID NO: 4014), G146R / R182V / P799V / E981V (SEQ ID NO: 4016), E125K / G146R / R182V / E795Q / D665N (SEQ ID NO: 4018), or G146R / R182V / E795Q / P799V / T814K (SEQ ID NO: 4020). Section 12.G146R / R182V / E795Q (SEQ ID NO: 3984) is an isolated mutant LbCas12a polypeptide from any one of sections 1 to 11.

[0076] Section 13. An isolated polynucleotide sequence encoding a mutant LbCas12a polypeptide comprising at least one amino acid substitution introduced into the wild-type LbCas12a polypeptide sequence of Sequence ID No. 2, provided that, compared to the wild-type LbCas12a polypeptide sequence, the mutant LbCas12a polypeptide provides improved CRISPR / LbCas12a-related nuclease activity at a non-canonical TTTT PAM site. Section 14. The isolated polynucleotide sequences from Section 13, wherein the encoded mutant LbCas12a polypeptide has 95%–99% identity with polypeptide sequences selected from even-numbered sequences of SEQ ID NOs. 4–4022. Section 15. The isolated polynucleotide sequence from Section 13 or 14, wherein the encoded mutant LbCas12a polypeptide has a polypeptide sequence selected from even-numbered sequences of SEQ ID NOs. 4–4022. Section 16. An isolated polynucleotide sequence from any one of Sections 13-15, wherein the mutant LbCas12a polynucleotide sequence has 95%-99% identity with a nucleotide sequence selected from odd-numbered sequences of SEQ ID NOs. 3-4021. Section 17. An isolated polynucleotide sequence from any one of Sections 13-16, wherein the mutant LbCas12a polynucleotide has a nucleotide sequence selected from odd-numbered sequences of SEQ ID NOs. 3-4021. Section 18. An isolated polynucleotide sequence from any one of Sections 13-17, wherein the encoded mutant LbCas12a polypeptide contains at least one amino acid substitution at the position of 81, 125, 145, 146, 152, 182, 396, 478, 491, 582, 595, 606, 646, 665, 795, 799, 814, 839, 841, 858, 860, 890, 898, 909, 913, 981, 1020, 1083, or 1121 of the wild-type LbCas12a polypeptide sequence of Sequence ID No. 2. Section 19. An isolated polynucleotide sequence from any one of Sections 13-18, wherein the encoded mutant LbCas12a polypeptide contains at least one amino acid substitution selected from F81E, E125K, N145R, G146R, T152K, R182V, S396D, K478R, V491D, N582R, K595R, Y606F, Y646H, D665N, E795Q, P799V, T814G, L839F, I841A, E858V, I860R, W890A, E898N, H909K, E913R, E981V, S1020E, V1083W, or K1121D, compared to the wild-type LbCas12a polypeptide sequence of Sequence ID No. 2.

[0077] Section 20. Mutant LbCas12a polynucleotides include F81E, SEQ ID NO: 802; E125K, SEQ ID NO: 428; N145R, SEQ ID NO: 2902; G146R, SEQ ID NO: 156; T152K, SEQ ID NO: 694; R182V, SEQ ID NO: 98; S396D, SEQ ID NO: 108; K478R, SEQ ID NO: 134; V491D, SEQ ID NO: 954; N582R, SEQ ID NO: 1730; K595R, SEQ ID NO: 184; Y606F, SEQ ID NO: 642; Y646H, SEQ ID NO: 720; D665N, SEQ ID NO: 1014; E795Q, SEQ ID NO: 930; P799V, Isolated mutant LbCas12a polynucleotides from any one of sections 13-19, selected from SEQ ID NOs: 706;T814G, SEQ ID NOs: 162;L839F, SEQ ID NOs: 834;I841A, SEQ ID NOs: 988;E858V, SEQ ID NOs: 114;I860R, SEQ ID NOs: 250;W890A, SEQ ID NOs: 3958;E898N, SEQ ID NOs: 48;H909K, SEQ ID NOs: 70;E913R, SEQ ID NOs: 1960;E981V, SEQ ID NOs: 880;S1020E, SEQ ID NOs: 532;V1083W, SEQ ID NOs: 132; or K1121D, SEQ ID NOs: 598. Section 21. The encoded mutant LbCas12a polypeptide, (a) A single substitution mutation introduced into the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2, selected from the following locations: F81, E125, N145, G146, T152, R182, S396, K478, V491, N582, K595, Y606, Y646, D665, E795, P799, T814, L839, I841, E858, I860, W890, E898, H909, E913, E981, S1020, V1083 or K1121; or (b) The following locations: A polysubstitution mutation introduced into the wild-type LbCas12a polypeptide sequence of SEQ ID NO: F81, E125, N145, G146, T152, R182, S396, K478, V491, N582, K595, Y606, Y646, D665, E795, P799, T814, L839, I841, E858, I860, W890, E898, H909, E913, E981, S1020, V1083 or K1121. An isolated mutant LbCas12a polynucleotide from any one of sections 13-20, containing a substitution mutation selected from the above. Section 22. Polysubstitution mutations include: E88A / E795Q, E125K / E795Q, G146R / E795Q, R182V / E795Q, V491D / E795Q, Q529I / E795Q, Y646H / E795Q, D665N / E795Q, T814K / E795Q, L839F / E795Q, Q906F / E795Q, E795Q / Q1170D, G146R / R 182V / E795Q, G146R / E795Q / D665N, G146R / E795Q / E981V, G146R / E795Q / T814K, G146R / R182V / D665 N, E125K / R182V / E981V, R182V / P799V / E981V, R182V / T814K / E981V, G146R / P799V / E981V, G146R / R 182V / E795Q / F81E, G146R / R182V / E795Q / E125K, G146R / R182V / E795Q / E125A, G146R / R182V / E795Q / P799V, G146R / R182V / E795Q / T814K, G146R / R182V / E795Q / E981V, G146R / R182V / E795Q / D665N, G1 Isolated mutant LbCas12a polynucleotides from any one of sections 13-21, containing 46R / R182V / P799V / E981V, E125K / G146R / R182V / E795Q / D665N, G146R / R182V / E795Q / P799V / T814K, or E125K / G146R / R182V / E795Q / D665N / E981V. Section 23. Polysubstitution mutations include: E88A / E795Q, E125K / E795Q, G146R / E795Q, R182V / E795Q, V491D / E795Q, Q529I / E795Q, Y646H / E795Q, D665N / E795Q, T814K / E795Q, L839F / E795Q, Q906F / E795Q, E795Q / Q117 0D, G146R / R182V / E795Q, G146R / E795Q / D665N, G146R / E795Q / E981V, G146R / E795Q / T814K, G 146R / R182V / D665N, E125K / R182V / E981V, R182V / P799V / E981V, R182V / T814K / E981V, G146R / P799V / E981V, G146R / R182V / E795Q / F81E, G146R / R182V / E795Q / E125K, G146R / R182V / E795 Q / E125A, G146R / R182V / E795Q / P799V, G146R / R182V / E795Q / T814K, G146R / R182V / E795Q / E9 Isolated mutant LbCas12a polynucleotides from any one of sections 13-22, comprising 81V, G146R / R182V / E795Q / D665N, G146R / R182V / P799V / E981V, E125K / G146R / R182V / E795Q / D665N, or G146R / R182V / E795Q / P799V / T814K, providing improved CRISPR / LbCas12a-related nuclease activity at non-canonical TTTT PAM sites compared to wild-type LbCas12a polypeptide sequences. Section 24. Encoded variant LbCas12a polypeptides include: F81E (SEQ ID NO: 802), E125K (SEQ ID NO: 428), N145R (SEQ ID NO: 2902), G146R (SEQ ID NO: 156), T152K (SEQ ID NO: 694), R182V (SEQ ID NO: 98), V491D (SEQ ID NO: 954), K478R (SEQ ID NO: 134), N582R (SEQ ID NO: 1730), D665N (SEQ ID NO: 1014), E795Q (SEQ ID NO: 930), P799V (SEQ ID NO: 706), E858V (SEQ ID NO: 114), I860R (SEQ ID NO: 250), E913R (SEQ ID NO: Column number 1960), E981V (sequence number 880), E88A / E795Q (sequence number 3960), E125K / E795Q (sequence number 3962), G146R / E795Q (sequence number 3964), R182V / E795Q (sequence number 3966), V491D / E795Q (sequence number 3968), Q529I / E795Q (sequence number 3970), Y646H / E795Q (sequence number 3972), D665N / E795Q (sequence number 3974), T814K / E795Q (sequence number 3976), L839F / E795Q (sequence number 3978), Q906F / E795Q (SEQ ID NO: 3980), E795Q / Q1170D (SEQ ID NO: 3982), G146R / R182V / E795Q (SEQ ID NO: 3984), G146R / E795Q / D665N (SEQ ID NO: 3986), G146R / E795Q / E981V (SEQ ID NO: 3988), G146R / E795Q / T814K (SEQ ID NO: 3990), G146R / R182V / D665N (SEQ ID NO: 3992), E125K / R182V / E981V (SEQ ID NO: 3994), R182V / P799V / E981V (SEQ ID NO: 3996), R182V / T814K / E981V (SEQ ID NO: 3998), G146R / P799V / E981V (SEQ ID NO: 4000), G146R / R182V / E795Q / F81E (SEQ ID NO: 4002), G146R / R182V / E795Q / E125K (SEQ ID NO: 4004), G146R / R182V / E795Q / E125A (SEQ ID NO: 4006), G146R / R182V / E795Q / P799V (SEQ ID NO: 4008), G146R / R182V / E795Q / T814K (SEQ ID NO: 4010), G146R / R182V / E795Q / E981V (SEQ ID NO: 4012),An isolated mutant LbCas12a polynucleotide from any one of sections 13-23, selected from G146R / R182V / E795Q / D665N (SEQ ID NO: 4014), G146R / R182V / P799V / E981V (SEQ ID NO: 4016), E125K / G146R / R182V / E795Q / D665N (SEQ ID NO: 4018), or G146R / R182V / E795Q / P799V / T814K (SEQ ID NO: 4020). Section 25. An isolated mutant LbCas12a polynucleotide from any one of sections 13-24, wherein the encoded mutant LbCas12a polypeptide is G146R / R182V / E795Q (SEQ ID NO: 3984). Section 26. Sequence IDs 801, 427, 2901, 155, 693, 97, 953, 133, 1729, 641, 719, 1013, 929, 705, 833, 987, 113, 249, 3957, 47, 69, 1959, 879, 531, 597, 3959, 3961, 3963, 3965, 3967, 3969, 3971, 3973, 3975, 3977 Isolated mutant LbCas12a polynucleotides from any one of sections 13-25, selected from 3979, 3981, 3983, 3985, 3987, 3989, 3991, 3993, 3995, 3997, 3999, 4001, 4003, 4005, 4007, 4009, 4011, 4013, 4015, 4017, 4019, or 4021. Section 27. Sequence IDs 801, 427, 2901, 155, 693, 97, 953, 133, 1729, 1013, 929, 705, 113, 249, 531, 597, 3959, 3961, 3963, 3965, 3967, 3969, 3971, 3973, 3975, 3977, 3979, 3981, 39 Isolated mutant LbCas12a polynucleotides from any one of sections 13–26, selected from 83, 3985, 3987, 3989, 3991, 3993, 3995, 3997, 3999, 4001, 4003, 4005, 4007, 4009, 4011, 4013, 4015, 4017, or 4019, encoding a mutant LbCas12a polypeptide that provides improved CRISPR / LbCas12a-related nuclease activity at non-canonical TTTT PAM sites compared to wild-type LbCas12a polypeptide sequences. Section 28. Sequence ID 3983, an isolated mutant LbCas12a polynucleotide from any one of sections 13-27. Section 29. A vector or plasmid containing a polynucleotide sequence from any one of sections 13-28. Cells containing a polynucleotide sequence from any one of sections 13-28 or a vector or plasmid from section 29.

[0078] Section 31. An isolated ribonucleoprotein complex comprising a guide RNA and a mutant LbCas12a polypeptide having at least one amino acid substitution introduced into the wild-type LbCas12a polypeptide sequence of Sequence ID No. 2, provided that, compared to the wild-type LbCas12a polypeptide sequence, the mutant LbCas12a polypeptide provides improved CRISPR / LbCas12a-related nuclease activity at a non-canonical TTTT PAM site. Section 32. The isolated ribonucleoprotein complex from Section 31, wherein the mutant LbCas12a polypeptide has a polypeptide sequence selected from even-numbered sequences of SEQ ID NOs: 4-4022. Section 33. Isolated ribonucleoprotein complexes from Section 31 or 32, wherein the mutant LbCas12a polypeptide contains at least one amino acid substitution at the position of 81, 125, 145, 146, 152, 182, 396, 478, 491, 582, 595, 606, 646, 665, 795, 799, 814, 839, 841, 858, 860, 890, 898, 909, 913, 981, 1020, 1083, or 1121 of the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2. Section 34. An isolated ribonucleoprotein complex from any one of sections 31-33, wherein the mutant LbCas12a polypeptide contains at least one amino acid substitution selected from F81E, E125K, N145R, G146R, T152K, R182V, S396D, K478R, V491D, N582R, K595R, Y606F, Y646H, D665N, E795Q, P799V, T814G, L839F, I841A, E858V, I860R, W890A, E898N, H909K, E913R, E981V, S1020E, V1083W, or K1121D of the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2. Section 35. Mutant LbCas12a polypeptides include: F81E, SEQ ID NO: 802; E125K, SEQ ID NO: 428; N145R, SEQ ID NO: 2902; G146R, SEQ ID NO: 156; T152K, SEQ ID NO: 694; R182V, SEQ ID NO: 98; S396D, SEQ ID NO: 108; K478R, SEQ ID NO: 134; V491D, SEQ ID NO: 954; N582R, SEQ ID NO: 1730; K595R, SEQ ID NO: 184; Y606F, SEQ ID NO: 642; Y646H, SEQ ID NO: 720; D665N, SEQ ID NO: 1014; E795Q, SEQ ID NO: 930; P79 An isolated ribonucleoprotein complex from any one of sections 31-34, selected from 9V, SEQ ID NO: 706; T814G, SEQ ID NO: 162; L839F, SEQ ID NO: 834; I841A, SEQ ID NO: 988; E858V, SEQ ID NO: 114; I860R, SEQ ID NO: 250; W890A, SEQ ID NO: 3958; E898N, SEQ ID NO: 48; H909K, SEQ ID NO: 70; E913R, SEQ ID NO: 1960; E981V, SEQ ID NO: 880; S1020E, SEQ ID NO: 532; V1083W, SEQ ID NO: 132; or K1121D, SEQ ID NO: 598.Section 36. Mutant LbCas12a polypeptides include: F81E (SEQ ID NO: 802), E125K (SEQ ID NO: 428), N145R (SEQ ID NO: 2902), G146R (SEQ ID NO: 156), T152K (SEQ ID NO: 694), R182V (SEQ ID NO: 98), V491D (SEQ ID NO: 954), K478R (SEQ ID NO: 134), N582R (SEQ ID NO: 1730), D665N (SEQ ID NO: 1014), E795Q (SEQ ID NO: 930), P799V (SEQ ID NO: 706), E858V (SEQ ID NO: 114), I860R (SEQ ID NO: 250), E913R (SEQ ID NO: 19 60), E981V (sequence number 880), E88A / E795Q (sequence number 3960), E125K / E795Q (sequence number 3962), G146R / E795Q (sequence number 3964), R182V / E795Q (sequence number 3966), V491D / E795Q (sequence number 3968), Q529I / E795Q (sequence number 3970), Y646H / E795Q (sequence number 3972), D665N / E795Q (sequence number 3974), T814K / E795Q (sequence number 3976), L839F / E795Q (sequence number 3978), Q906F / E7 95Q (SEQ ID NO: 3980), E795Q / Q1170D (SEQ ID NO: 3982), G146R / R182V / E795Q (SEQ ID NO: 3984), G146R / E795Q / D665N (SEQ ID NO: 3986), G146R / E795Q / E981V (SEQ ID NO: 3988), G146R / E795Q / T814K (SEQ ID NO: 3990), G146R / R182V / D665N (SEQ ID NO: 3992), E125K / R182V / E981V (SEQ ID NO: 3994), R182V / P799V / E981V (SEQ ID NO: 3996), R182V / T814K / E 981V (SEQ ID NO: 3998), G146R / P799V / E981V (SEQ ID NO: 4000), G146R / R182V / E795Q / F81E (SEQ ID NO: 4002), G146R / R182V / E795Q / E125K (SEQ ID NO: 4004), G146R / R182V / E795Q / E125A (SEQ ID NO: 4006), G146R / R182V / E795Q / P799V (SEQ ID NO: 4008), G146R / R182V / E795Q / T814K (SEQ ID NO: 4010), G146R / R182V / E795Q / E981V (SEQ ID NO: 4012),An isolated ribonucleoprotein complex from any one of sections 31-35, selected from G146R / R182V / E795Q / D665N (SEQ ID NO: 4014), G146R / R182V / P799V / E981V (SEQ ID NO: 4016), E125K / G146R / R182V / E795Q / D665N (SEQ ID NO: 4018), or G146R / R182V / E795Q / P799V / T814K (SEQ ID NO: 4020). Section 37. An isolated ribonucleoprotein complex from any one of sections 31-36, in which the mutant LbCas12a polypeptide is G146R / R182V / E795Q (SEQ ID NO: 3984).

[0079] Section 38. A method for enhancing the efficiency of gene editing at non-canonical TTTT PAM sites in cells using the CRISPR ribonucleoprotein complex, wherein: A method comprising the step of contacting a cell with a CRISPR-ribonucleoprotein complex comprising a guide RNA and a mutant LbCas12a polypeptide having at least one amino acid substitution introduced into the wild-type LbCas12a polypeptide sequence of Sequence ID No. 2, provided that the mutant LbCas12a polypeptide provides improved CRISPR / LbCas12a-related nuclease activity at a non-canonical TTTT PAM site compared to the wild-type LbCas12a polypeptide sequence. Section 39. The method of Section 38, wherein the mutant LbCas12a polypeptide has a polypeptide sequence selected from even-numbered sequences of SEQ ID NOs: 4 to 4022. Section 40. The method of Section 38 or 39, wherein the mutant LbCas12a polypeptide comprises at least one amino acid substitution at the position of 81, 125, 145, 146, 152, 182, 396, 478, 491, 582, 595, 606, 646, 665, 795, 799, 814, 839, 841, 858, 860, 890, 898, 909, 913, 981, 1020, 1083, or 1121 of the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2. Section 41. The method of any one of Sections 38-40, wherein the mutant LbCas12a polypeptide comprises at least one amino acid substitution selected from F81E, E125K, N145R, G146R, T152K, R182V, S396D, K478R, V491D, N582R, K595R, Y606F, Y646H, D665N, E795Q, P799V, T814G, L839F, I841A, E858V, I860R, W890A, E898N, H909K, E913R, E981V, S1020E, V1083W, or K1121D of the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2. Section 42. Mutant LbCas12a polypeptides include: F81E, SEQ ID NO: 802; E125K, SEQ ID NO: 428; N145R, SEQ ID NO: 2902; G146R, SEQ ID NO: 156; T152K, SEQ ID NO: 694; R182V, SEQ ID NO: 98; S396D, SEQ ID NO: 108; K478R, SEQ ID NO: 134; V491D, SEQ ID NO: 954; N582R, SEQ ID NO: 1730; K595R, SEQ ID NO: 184; Y606F, SEQ ID NO: 642; Y646H, SEQ ID NO: 720; D665N, SEQ ID NO: 1014; E795Q, SEQ ID NO: A method from any one of sections 38 to 41, selected from 930;P799V, SEQ ID NO: 706;T814G, SEQ ID NO: 162;L839F, SEQ ID NO: 834;I841A, SEQ ID NO: 988;E858V, SEQ ID NO: 114;I860R, SEQ ID NO: 250;W890A, SEQ ID NO: 3958;E898N, SEQ ID NO: 48;H909K, SEQ ID NO: 70;E913R, SEQ ID NO: 1960;E981V, SEQ ID NO: 880;S1020E, SEQ ID NO: 532;V1083W, SEQ ID NO: 132; or K1121D, SEQ ID NO: 598. Section 43. The mutant LbCas12a polypeptide: (a) A single substitution mutation introduced into the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2, selected from the following locations: F81, E125, N145, G146, T152, R182, S396, K478, V491, N582, K595, Y606, Y646, D665, E795, P799, T814, L839, I841, E858, I860, W890, E898, H909, E913, E981, S1020, V1083 or K1121; or (b) The following locations: A polysubstitution mutation introduced into the wild-type LbCas12a polypeptide sequence of SEQ ID NO: F81, E125, N145, G146, T152, R182, S396, K478, V491, N582, K595, Y606, Y646, D665, E795, P799, T814, L839, I841, E858, I860, W890, E898, H909, E913, E981, S1020, V1083 or K1121. A method from any one of sections 38-42, including a substitution mutation selected from the above. Section 44. Polysubstitution mutations: E88A / E795Q, E125K / E795Q, G146R / E795Q, R182V / E795Q, V491D / E795Q, Q529I / E795Q, Y646H / E795Q, D665N / E795Q, T814K / E795Q, L839F / E795Q, Q906F / E795Q, E795Q / Q1170D, G146R / R182V / E795Q, G146R / E795Q / D665N, G146R / E795Q / E981V, G146R / E795Q / T814K, G146R / R182V / D665N, E125K / R182V / E981V, R182V / P799V / E981V, R182V / T814K / E9 81V, G146R / P799V / E981V, G146R / R182V / E795Q / F81E, G146R / R182V / E795Q / E125K, G146R / R182V / E795Q / E125A, G146R / R182V / E795Q / P799V, G146R / R182V / E795Q / T814K, G146R / R The method of any one of sections 38 to 43, including 182V / E795Q / E981V, G146R / R182V / E795Q / D665N, G146R / R182V / P799V / E981V, E125K / G146R / R182V / E795Q / D665N, or G146R / R182V / E795Q / P799V / T814K. Section 45. Mutant LbCas12a polypeptides include: F81E (SEQ ID NO: 802), E125K (SEQ ID NO: 428), N145R (SEQ ID NO: 2902), G146R (SEQ ID NO: 156), T152K (SEQ ID NO: 694), R182V (SEQ ID NO: 98), V491D (SEQ ID NO: 954), K478R (SEQ ID NO: 134), N582R (SEQ ID NO: 1730), D665N (SEQ ID NO: 1014), E795Q (SEQ ID NO: 930), P799V (SEQ ID NO: 706), E858V (SEQ ID NO: 114), I860R (SEQ ID NO: 250), E913R (SEQ ID NO: 19 60), E981V (sequence number 880), E88A / E795Q (sequence number 3960), E125K / E795Q (sequence number 3962), G146R / E795Q (sequence number 3964), R182V / E795Q (sequence number 3966), V491D / E795Q (sequence number 3968), Q529I / E795Q (sequence number 3970), Y646H / E795Q (sequence number 3972), D665N / E795Q (sequence number 3974), T814K / E795Q (sequence number 3976), L839F / E795Q (sequence number 3978), Q906F / E7 95Q (SEQ ID NO: 3980), E795Q / Q1170D (SEQ ID NO: 3982), G146R / R182V / E795Q (SEQ ID NO: 3984), G146R / E795Q / D665N (SEQ ID NO: 3986), G146R / E795Q / E981V (SEQ ID NO: 3988), G146R / E795Q / T814K (SEQ ID NO: 3990), G146R / R182V / D665N (SEQ ID NO: 3992), E125K / R182V / E981V (SEQ ID NO: 3994), R182V / P799V / E981V (SEQ ID NO: 3996), R182V / T814K / E 981V (SEQ ID NO: 3998), G146R / P799V / E981V (SEQ ID NO: 4000), G146R / R182V / E795Q / F81E (SEQ ID NO: 4002), G146R / R182V / E795Q / E125K (SEQ ID NO: 4004), G146R / R182V / E795Q / E125A (SEQ ID NO: 4006), G146R / R182V / E795Q / P799V (SEQ ID NO: 4008), G146R / R182V / E795Q / T814K (SEQ ID NO: 4010), G146R / R182V / E795Q / E981V (SEQ ID NO: 4012),A method from any one of sections 38-44, selected from G146R / R182V / E795Q / D665N (sequence number 4014), G146R / R182V / P799V / E981V (sequence number 4016), E125K / G146R / R182V / E795Q / D665N (sequence number 4018), or G146R / R182V / E795Q / P799V / T814K (sequence number 4020). Section 46. The method described in any one of sections 38-45, where the mutant LbCas12a polypeptide is G146R / R182V / E795Q (SEQ ID NO: 3984).

[0080] Section 47. A kit for improving the efficiency of gene editing at non-canonical TTTT PAM sites in cells: A kit comprising a CRISPR-ribonucleoprotein complex containing a guide RNA and a mutant LbCas12a polypeptide having at least one amino acid substitution introduced into the wild-type LbCas12a polypeptide sequence of Sequence ID No. 2, provided that, compared to the wild-type LbCas12a polypeptide sequence, the mutant LbCas12a polypeptide provides improved CRISPR / LbCas12a-related nuclease activity at a non-canonical TTTT PAM site. Section 48. The kit of Section 47, wherein the mutant LbCas12a polypeptide has a polypeptide sequence selected from even-numbered sequences of SEQ ID NOs: 4-4022. Section 49. Kits of Section 47 or 48, wherein the mutant LbCas12a polypeptide contains at least one amino acid substitution at the position of 81, 125, 145, 146, 152, 182, 396, 478, 491, 582, 595, 606, 646, 665, 795, 799, 814, 839, 841, 858, 860, 890, 898, 909, 913, 981, 1020, 1083, or 1121 of the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2. Section 50. A kit from any one of sections 47-49, wherein the mutant LbCas12a polypeptide contains at least one amino acid substitution selected from F81E, E125K, N145R, G146R, T152K, R182V, S396D, K478R, V491D, N582R, K595R, Y606F, Y646H, D665N, E795Q, P799V, T814G, L839F, I841A, E858V, I860R, W890A, E898N, H909K, E913R, E981V, S1020E, V1083W, or K1121D of the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2. Section 51. Mutant LbCas12a polypeptides include: F81E, SEQ ID NO: 802; E125K, SEQ ID NO: 428; N145R, SEQ ID NO: 2902; G146R, SEQ ID NO: 156; T152K, SEQ ID NO: 694; R182V, SEQ ID NO: 98; S396D, SEQ ID NO: 108; K478R, SEQ ID NO: 134; V491D, SEQ ID NO: 954; N582R, SEQ ID NO: 1730; K595R, SEQ ID NO: 184; Y606F, SEQ ID NO: 642; Y646H, SEQ ID NO: 720; D665N, SEQ ID NO: 1014; E795Q, SEQ ID NO: A kit of any one of sections 47-50, selected from 930;P799V, SEQ ID NO: 706;T814G, SEQ ID NO: 162;L839F, SEQ ID NO: 834;I841A, SEQ ID NO: 988;E858V, SEQ ID NO: 114;I860R, SEQ ID NO: 250;W890A, SEQ ID NO: 3958;E898N, SEQ ID NO: 48;H909K, SEQ ID NO: 70;E913R, SEQ ID NO: 1960;E981V, SEQ ID NO: 880;S1020E, SEQ ID NO: 532;V1083W, SEQ ID NO: 132; or K1121D, SEQ ID NO: 598. Section 52. The mutant LbCas12a polypeptide: (a) A single substitution mutation introduced into the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2, selected from the following locations: F81, E125, N145, G146, T152, R182, S396, K478, V491, N582, K595, Y606, Y646, D665, E795, P799, T814, L839, I841, E858, I860, W890, E898, H909, E913, E981, S1020, V1083 or K1121; or (b) The following locations: A polysubstitution mutation introduced into the wild-type LbCas12a polypeptide sequence of SEQ ID NO: F81, E125, N145, G146, T152, R182, S396, K478, V491, N582, K595, Y606, Y646, D665, E795, P799, T814, L839, I841, E858, I860, W890, E898, H909, E913, E981, S1020, V1083 or K1121. A kit containing a substitution mutation selected from any one of sections 47-51. Section 53. Multiple substitution mutations include E88A / E795Q, E125K / E795Q, G146R / E795Q, R182V / E795Q, V491D / E795Q, Q529I / E795Q, Y646H / E795Q, D665N / E795Q, T814K / E795Q, L839F / E795Q, Q906F / E795Q, E795Q / Q1170D, G146R / R182V / E795Q, G146R / E795Q / D665N, G146R / E795Q / E981V, G146R / E795Q / T 814K, G146R / R182V / D665N, E125K / R182V / E981V, R182V / P799V / E981V, R182V / T814K / E98 1V, G146R / P799V / E981V, G146R / R182V / E795Q / F81E, G146R / R182V / E795Q / E125K, G146R / R182V / E795Q / E125A, G146R / R182V / E795Q / P799V, G146R / R182V / E795Q / T814K, G146R / R1 A kit containing any one of sections 47-52, including 82V / E795Q / E981V, G146R / R182V / E795Q / D665N, G146R / R182V / P799V / E981V, E125K / G146R / R182V / E795Q / D665N, or G146R / R182V / E795Q / P799V / T814K. Section 54. Mutant LbCas12a polypeptides include: F81E (SEQ ID NO: 802), E125K (SEQ ID NO: 428), N145R (SEQ ID NO: 2902), G146R (SEQ ID NO: 156), T152K (SEQ ID NO: 694), R182V (SEQ ID NO: 98), V491D (SEQ ID NO: 954), K478R (SEQ ID NO: 134), N582R (SEQ ID NO: 1730), D665N (SEQ ID NO: 1014), E795Q (SEQ ID NO: 930), P799V (SEQ ID NO: 706), E858V (SEQ ID NO: 114), I860R (SEQ ID NO: 250), E913R (SEQ ID NO: 19 60), E981V (sequence number 880), E88A / E795Q (sequence number 3960), E125K / E795Q (sequence number 3962), G146R / E795Q (sequence number 3964), R182V / E795Q (sequence number 3966), V491D / E795Q (sequence number 3968), Q529I / E795Q (sequence number 3970), Y646H / E795Q (sequence number 3972), D665N / E795Q (sequence number 3974), T814K / E795Q (sequence number 3976), L839F / E795Q (sequence number 3978), Q906F / E7 95Q (SEQ ID NO: 3980), E795Q / Q1170D (SEQ ID NO: 3982), G146R / R182V / E795Q (SEQ ID NO: 3984), G146R / E795Q / D665N (SEQ ID NO: 3986), G146R / E795Q / E981V (SEQ ID NO: 3988), G146R / E795Q / T814K (SEQ ID NO: 3990), G146R / R182V / D665N (SEQ ID NO: 3992), E125K / R182V / E981V (SEQ ID NO: 3994), R182V / P799V / E981V (SEQ ID NO: 3996), R182V / T814K / E 981V (SEQ ID NO: 3998), G146R / P799V / E981V (SEQ ID NO: 4000), G146R / R182V / E795Q / F81E (SEQ ID NO: 4002), G146R / R182V / E795Q / E125K (SEQ ID NO: 4004), G146R / R182V / E795Q / E125A (SEQ ID NO: 4006), G146R / R182V / E795Q / P799V (SEQ ID NO: 4008), G146R / R182V / E795Q / T814K (SEQ ID NO: 4010), G146R / R182V / E795Q / E981V (SEQ ID NO: 4012),A kit from any one of sections 47-53, selected from G146R / R182V / E795Q / D665N (sequence number 4014), G146R / R182V / P799V / E981V (sequence number 4016), E125K / G146R / R182V / E795Q / D665N (sequence number 4018), or G146R / R182V / E795Q / P799V / T814K (sequence number 4020). Section 55. A kit from any one of sections 47-54, in which the mutant LbCas12a polypeptide is G146R / R182V / E795Q (SEQ ID NO: 3984).

[0081] Section 56. Use of a mutant LbCas12a polypeptide to improve CRISPR / LbCas12a-related nuclease activity at a non-canonical TTTT PAM site, wherein the mutant LbCas12a polypeptide comprises at least one amino acid substitution introduced into the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2, provided that the mutant LbCas12a polypeptide provides improved CRISPR / LbCas12a-related nuclease activity at a non-canonical TTTT PAM site compared to the wild-type LbCas12a polypeptide sequence. Section 57. Use of Section 56, wherein the mutant LbCas12a polypeptide has a polypeptide sequence selected from even-numbered sequences of SEQ ID NOs. 4-4022. Section 58. Use of Section 56 or 57, wherein the mutant LbCas12a polypeptide contains at least one amino acid substitution at the position of 81, 125, 145, 146, 152, 182, 396, 478, 491, 582, 595, 606, 646, 665, 795, 799, 814, 839, 841, 858, 860, 890, 898, 909, 913, 981, 1020, 1083, or 1121 of the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2. Section 59. Use of any one of Sections 56-58, wherein the mutant LbCas12a polypeptide contains at least one amino acid substitution selected from F81E, E125K, N145R, G146R, T152K, R182V, S396D, K478R, V491D, N582R, K595R, Y606F, Y646H, D665N, E795Q, P799V, T814G, L839F, I841A, E858V, I860R, W890A, E898N, H909K, E913R, E981V, S1020E, V1083W, or K1121D of the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2. Section 60. Mutant LbCas12a polypeptides include: F81E, SEQ ID NO: 802; E125K, SEQ ID NO: 428; N145R, SEQ ID NO: 2902; G146R, SEQ ID NO: 156; T152K, SEQ ID NO: 694; R182V, SEQ ID NO: 98; S396D, SEQ ID NO: 108; K478R, SEQ ID NO: 134; V491D, SEQ ID NO: 954; N582R, SEQ ID NO: 1730; K595R, SEQ ID NO: 184; Y606F, SEQ ID NO: 642; Y646H, SEQ ID NO: 720; D665N, SEQ ID NO: 1014; E795Q, SEQ ID NO: Use of any one of sections 56-59, selected from 930;P799V, SEQ ID NO: 706;T814G, SEQ ID NO: 162;L839F, SEQ ID NO: 834;I841A, SEQ ID NO: 988;E858V, SEQ ID NO: 114;I860R, SEQ ID NO: 250;W890A, SEQ ID NO: 3958;E898N, SEQ ID NO: 48;H909K, SEQ ID NO: 70;E913R, SEQ ID NO: 1960;E981V, SEQ ID NO: 880;S1020E, SEQ ID NO: 532;V1083W, SEQ ID NO: 132;or K1121D, SEQ ID NO: 598. Section 61. The mutant LbCas12a polypeptide: (a) A single substitution mutation introduced into the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2, selected from the following locations: F81, E125, N145, G146, T152, R182, S396, K478, V491, N582, K595, Y606, Y646, D665, E795, P799, T814, L839, I841, E858, I860, W890, E898, H909, E913, E981, S1020, V1083 or K1121; or (b) The following locations: A polysubstitution mutation introduced into the wild-type LbCas12a polypeptide sequence of SEQ ID NO: F81, E125, N145, G146, T152, R182, S396, K478, V491, N582, K595, Y606, Y646, D665, E795, P799, T814, L839, I841, E858, I860, W890, E898, H909, E913, E981, S1020, V1083 or K1121. Use of any one of sections 56-60, including a substitution mutation selected from the above. Section 62. Polysubstitution mutations include: E88A / E795Q, E125K / E795Q, G146R / E795Q, R182V / E795Q, V491D / E795Q, Q529I / E795Q, Y646H / E795Q, D665N / E795Q, T814K / E795Q, L839F / E795Q, Q906F / E795Q, E795Q / Q1170D, G146R / R182V / E795Q, G146R / E795Q / D665N, G146R / E795Q / E981V, G146R / E795Q / T814K, G146R / R182V / D665N, E125K / R182V / E981V, R182V / P799V / E981V, R182V / T814K / E9 81V, G146R / P799V / E981V, G146R / R182V / E795Q / F81E, G146R / R182V / E795Q / E125K, G146R / R182V / E795Q / E125A, G146R / R182V / E795Q / P799V, G146R / R182V / E795Q / T814K, G146R / R Use of any one of sections 56-61, including 182V / E795Q / E981V, G146R / R182V / E795Q / D665N, G146R / R182V / P799V / E981V, E125K / G146R / R182V / E795Q / D665N, or G146R / R182V / E795Q / P799V / T814K. Section 63. Mutant LbCas12a polypeptides include: F81E (SEQ ID NO: 802), E125K (SEQ ID NO: 428), N145R (SEQ ID NO: 2902), G146R (SEQ ID NO: 156), T152K (SEQ ID NO: 694), R182V (SEQ ID NO: 98), V491D (SEQ ID NO: 954), K478R (SEQ ID NO: 134), N582R (SEQ ID NO: 1730), D665N (SEQ ID NO: 1014), E795Q (SEQ ID NO: 930), P799V (SEQ ID NO: 706), E858V (SEQ ID NO: 114), I860R (SEQ ID NO: 250), E913R (SEQ ID NO: 19 60), E981V (sequence number 880), E88A / E795Q (sequence number 3960), E125K / E795Q (sequence number 3962), G146R / E795Q (sequence number 3964), R182V / E795Q (sequence number 3966), V491D / E795Q (sequence number 3968), Q529I / E795Q (sequence number 3970), Y646H / E795Q (sequence number 3972), D665N / E795Q (sequence number 3974), T814K / E795Q (sequence number 3976), L839F / E795Q (sequence number 3978), Q906F / E7 95Q (SEQ ID NO: 3980), E795Q / Q1170D (SEQ ID NO: 3982), G146R / R182V / E795Q (SEQ ID NO: 3984), G146R / E795Q / D665N (SEQ ID NO: 3986), G146R / E795Q / E981V (SEQ ID NO: 3988), G146R / E795Q / T814K (SEQ ID NO: 3990), G146R / R182V / D665N (SEQ ID NO: 3992), E125K / R182V / E981V (SEQ ID NO: 3994), R182V / P799V / E981V (SEQ ID NO: 3996), R182V / T814K / E 981V (SEQ ID NO: 3998), G146R / P799V / E981V (SEQ ID NO: 4000), G146R / R182V / E795Q / F81E (SEQ ID NO: 4002), G146R / R182V / E795Q / E125K (SEQ ID NO: 4004), G146R / R182V / E795Q / E125A (SEQ ID NO: 4006), G146R / R182V / E795Q / P799V (SEQ ID NO: 4008), G146R / R182V / E795Q / T814K (SEQ ID NO: 4010), G146R / R182V / E795Q / E981V (SEQ ID NO: 4012),Use of any one of sections 56-62, selected from G146R / R182V / E795Q / D665N (sequence number 4014), G146R / R182V / P799V / E981V (sequence number 4016), E125K / G146R / R182V / E795Q / D665N (sequence number 4018), or G146R / R182V / E795Q / P799V / T814K (sequence number 4020). Section 64. Use of any one of sections 56-63, where the mutant LbCas12a polypeptide is G146R / R182V / E795Q (SEQ ID NO: 3984).

[0082] Section 65. A method for expressing and purifying a mutant LbCas13a protein, wherein: (a) Inserting into an expression plasmid a nucleotide sequence encoding a mutant LbCas12a polypeptide that contains at least one amino acid substitution compared to the wild-type LbCas12a polypeptide sequence and has 95-99% identity with any of the even-numbered polypeptide sequences from SEQ ID NOs. 4 to 4022; (b) the step of transforming one or more cells with an expression plasmid; (c) A step of inducing the expression of the transformed plasmid; (d) the step of separating the cells; (e) the step of extracting the mutant LbCas13a protein; (f) A step to purify the mutant LbCas13a protein, Includes, Here, the mutant LbCas12a polypeptide provides improved CRISPR / LbCas12a-related nuclease activity at the non-canonical TTTT PAM site compared to the wild-type LbCas12a polypeptide sequence. method. Section 66. Mutant LbCas12a polypeptide generated by the method in Section 65.

[0083] References Zetsche et al. "Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System," Cell 163: 759-771 (2015). Jinek et al., "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity," Science 337: 816-821 (2012). Schindele and Puchta, "Engineering CRISPR / LbCas12a for highly efficient, temperature-tolerant plant gene editing," Plant Biotechnol. J.18(5):1118-1120 (2020). Wrenbeck et al., "Plasmid-based one-pot saturation mutagenesis," Nat. Methods 13: 928-930 (2016). [Examples]

[0084] Example 1 High-throughput measurement of DNA cleavage activity of LbCas12a variants at the TTTT PAM site in Escherichia coli. A bacterial-based directed evolution method for LbCas12a was performed to identify mutations with enhanced cleavage activity. A deep-scan mutagenic library was constructed containing all possible point mutations at the amino acid level across the entire LbCas12a coding sequence, in which case most clones contained only one mutation. Wrenbeck et al., Nat. Methods 13:928-930 (2016), incorporated herein by reference. This type of library allowed for direct phenotypic evaluation of each point mutation by measuring its relative viability against the LbCas12a wild-type (WT) protein in bacterial screening.

[0085] Screening strains containing toxin plasmids were transformed with an LbCas12a library and crRNA targeting the HPRT38346 site on the toxin plasmid. After recovery and IPTG induction, cells were seeded in LB-chloramphenicol medium containing arabinose and incubated overnight at 37°C. LbCas12a expression plasmids supported by viable E. coli cells were extracted and purified. Both the input and selected plasmid libraries were amplified by PCR, randomly fragmented using the Nextera Library Prep Kit, and sequenced using Illumina NextSeq® with approximately 40 million reads per library. The frequency of mutations at each position of LbCas12a in both libraries was measured and normalized against 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. The phenotypic score (or activity score) is calculated as the natural logarithm of the ratio of normalization frequencies between the selected library (round 4) and the input library (round 3), where the normalization frequency of each variant at each position is calculated as the ratio of the specific variant to the synonym change at each position. Since the degree of cell viability under arabinose selection indicates the cleavage activity of the LbCas12a variant in the HPRT38346 protospacer, any variant enriched during the selection process across the WT is a variant with enhanced activity in TTTT PAM.

[0086] Table 2 lists the phenotypic scores (or activity scores) of 17,278 LbCas12a variants with single point remnants. The scores for synonymous changes (n=849, 69% of all locations) clustered tightly around 0 and were therefore used as baseline activity for wild-type LbCas12a (Figure 1A-C). Overall, 1,977 of the 17,278 point mutations (approximately 11.4%) showed some advantage with scores above the 99th percentile (0.248) of all synonymous changes (Figure 2; Table 2).

[0087] Table 2 shows the point mutations enriched across the WT protein. The phenotypic score is the natural logarithm of the relative enrichment of point mutations across two biological copies that exhibit a beneficial phenotype. The primers used to construct the saturated mutagenesis library are shown in Table 3.

[0088] [Table 2-1]

[0089] [Table 2-2]

[0090] [Table 2-3]

[0091] [Table 2-4]

[0092] [Table 2-5]

[0093] [Table 2-6]

[0094] [Table 2-7]

[0095] [Table 2-8]

[0096] [Table 2-9]

[0097] Table 2-10

[0098] Table 2-11

[0099] Table 2-12

[0100] Table 2-13

[0101] Table 2-14

[0102] Table 2-15

[0103] Table 2-16

[0104] Table 2-17

[0105] Table 2-18

[0106] Table 2-19

[0107] Table 2-20

[0108] Table 2-21

[0109] Table 2-22

[0110] Table 2-23

[0111] Table 2-24

[0112] Table 2-25

[0113] Table 2-26

[0114] Table 2-27

[0115] Table 2-28

[0116] Table 2-29

[0117] Table 2-30

[0118] Table 2-31

[0119] Table 2-32

[0120] Table 2-33

[0121] Table 2-34

[0122] Table 2-35

[0123] Table 3-1

[0124] Table 3-2

[0125] Table 3-3

[0126] Table 3-4

[0127] Table 3-5

[0128] Table 3-6

[0129] Table 3-7

[0130] Table 3-8

[0131] Table 3-9

[0132] Table 3-10

[0133] Table 3-11

[0134] Table 3-12

[0135] Table 3-13

[0136] Table 3-14

[0137] [Table 3-15]

[0138] [Table 3-16]

[0139] [Table 3-17]

[0140] [Table 3-18]

[0141] [Table 3-19]

[0142] [Table 3-20]

[0143] [Table 3-21]

[0144] [Table 3-22]

[0145] To demonstrate the usefulness of the results, we first evaluated the phenotypes of LbCas12a mutations at known locations, including N527, E795, D156, G532, and K538, which correspond to the M537, F870, E174, S542, and K548 mutations in AsCas12a, respectively.

[0146] Previous studies selected M537R and F870L, resulting in the creation of AsCas12a-Ultra with enhanced cleavage activity. See U.S. Patent Application Publication US2020 / 0109382A1, which is incorporated herein by reference. As second choices at each position, M537K and F870I were beneficial, but to a lesser extent (Table 4). However, only E795L was successfully translocated to LbCas12a, in which case N527R negatively affected enzyme activity. These discrepancies between AsCas12a and LbCas12a were accurately reflected throughout the entire screening process.

[0147] [Table 4]

[0148] Testing of phenotypic scores for mutations at E795 revealed a beneficial phenotype for E795L but not for E795I. Furthermore, E795Q was superior to the others as the optimal choice for this position (Figure 3A). In contrast, most mutations at position N527 were excluded from screening, with the exception of N527N and N527K. Moreover, N527K presented a strongly negative phenotype compared to synonymous changes (N527N) (Figure 3B). These data suggest that mutations at position N527 of LbCas12a are generally harmful, which is consistent with previous findings regarding the loss of activity in N527R mutants.

[0149] Since it has been previously shown that corresponding mutants in AsCas12a (E174R / S542R / K548R) improve activity, we further evaluated the effects of mutations at D156, G532, and K538 in LbCas12a (Table 5). Firstly, most mutations at D156, including D156R, had higher phenotypic scores than the wild type, suggesting that a similar mutant found in AsCas12a (E174R) could be transferred into LbCas12a with similar outcomes (Figure 4A). This screening further identified D156Q as the optimal mutant. Mutations at G532, including G532R, reduced activity (Figure 4B). Almost all mutants at K538 were excluded from the screening, suggesting that mutations at this location are generally unacceptable (data not shown).

[0150] [Table 5]

[0151] These results were consistent with previous studies showing that D156R is a beneficial mutation for LbCas12a (Figure 4A). See Schindele and Puchta, Plant Biotechnol J. 18(5):1118–1120 (2010), which is incorporated herein by reference. In summary, high-throughput data accurately reproduced and identified mutations with known outcomes. In addition, these experiments suggest that point mutations found in one Cas12a family member are unlikely to be reliably translocated to the other, as most of the activity-enhancing mutants identified in AsCas12a were detrimental in LbCas12a.

[0152] Driven by high-throughput results, a series of LbCas12a variants with novel point mutations (G146R, S396D, K478R, K595R, E795Q, T814G, E898N, and V1083W) were cloned and tested for their activity in the context of bacterial selection assays. As shown in Figures 5C–J, these variants improved bacterial viability by outperforming WT-LbCas12a during selection, suggesting that the variants can be added to WT-LbCas12a, thereby enhancing its intrinsic cleavage activity in TTTT PAM.

[0153] [Table 6]

[0154] Table 6 shows that the 10 mutants with the highest activity are not necessarily the best mutants for enhanced editing in TTTT-PAM, because the phenotype is measured in E. coli cells. Besides enhancing DNA cleavage, some mutants enriched the genome because they reduced toxicity to host cells or improved the solubility or expression levels of LbCas12a in E. coli. These variants are useful for genome editing in E. coli or other bacteria where the toxicity of the CRISPR enzyme (Cas12a or Cas9) is a known issue. WT enzymes are toxic, and this is thought to cause inferior transformation and editing efficiencies. The literature has shown that reducing the toxicity, activity, or expression levels of the CRISPR enzyme improves editing efficiency. However, this type of toxicity is not usually a problem in mammalian cells. Therefore, to obtain mutants that function better in human cells, a selected set of mutants were tested in HEK293 cells for further confirmation.

[0155] Example 2 LbCas12a mutant with enhanced activity Table 7 lists the LbCas12a variants evaluated in this study. Site-directed mutagenesis was performed to introduce specific point mutations into expression vectors using the primers listed in Table 8. The phenotypes of specific single, double, triple, quadruple, quintuple, and hexavalent mutations were evaluated in E. coli as colony-forming units (CFUs) for each mutant. These mutants, exhibiting enhanced CFUs at the time of selection, indicated improved editing efficiency in E. coli, regardless of the underlying mechanism.

[0156] [Table 7-1]

[0157] [Table 7-2]

[0158] [Table 8-1]

[0159] [Table 8-2]

[0160] LbCas12a mutants were transformed into E. coli BL21(DE3) cells. Colonies from a suitable strain were inoculated into TB medium containing kanamycin (0.05 mg / mL) and grown at 37°C until the OD reached approximately 0.9. The flask was then cooled to 18°C ​​for 30 minutes. Protein expression was induced by adding 500 μL of 1 M IPTG, followed by growth at 18°C ​​for 19 hours. The cells were harvested, the cell pellet was resuspended, and lysed in three passes through Avestin Emulsiflex C3 pre-cooled to 4°C at 15-20 kpsi. The lysate was centrifuged at 16,000 × g at 4°C for 20 minutes to remove cell debris.

[0161] The clarified lysate was applied to a HisTrap™ HP column (Cytiva). The procedure consisted of equilibrating the resin with His Bind® buffer (20 mM NaPO4 pH 6.8, 0.5 M NaCl, 10 mM imidazole, 5% glycerol), followed by loading the sample. The sample was washed with His-Bind buffer, followed by a 10% "B" wash consisting of additional standard washes and 10% His-Elution buffer (10 mM NaPO4 pH 6.8, 250 mM NaCl, 150 mM imidazole, 5% glycerol). Finally, the sample was eluted using His-Elution buffer.

[0162] The partially purified Cas13a variant was then applied to a HiTrap® Heparin HP column. The procedure consisted of equilibrating the resin with Heparin-Bind buffer (20 mM NaPO4 pH 6.8, 250 mM NaCl, 10% glycerol), followed by loading the sample. The sample was then washed with Heparin-Bind buffer, followed by a 5% "B" wash consisting of 5% Heparin Elution buffer (10 mM NaPO4 pH 6.8, 1 M NaCl, 10% glycerol). Finally, the purified protein was eluted using Heparin Elution buffer (10 mM NaPO4, pH 6.8, 1 M NaCl, 10% glycerol). Finally, the purified protein was eluted using heparin.

[0163] The purified LbCas12a variant was concentrated to approximately 10 mg / mL and stored at -20 °C in a storage buffer containing 25 mM Tris-HCl pH 7.4, 0.3 M NaCl, 1 mM EDTA, 1 mM DTT, and 50% glycerol.

[0164] Example 3 Editing efficiency of LbCas12a variants in human cells To evaluate the genome editing efficiency of each variant, LbCas12a-RNP (1 μM) was assembled using four crRNAs targeting the human HPRT gene (Table 9). The assembled RNP (50 nM) was delivered to HEK293 cells by Lonza nucleofection (SF buffer, CM-130), and the editing efficiency was measured by the T7 endonuclease I assay 48 hours after delivery (Table 10; Figure 6A).

[0165] [Table 9]

[0166] To facilitate comparison, the efficiency of each variant at each target was normalized to WT LbCas12a and is presented as a multiplier of improvement compared to WT in Table 11 and Figure 6B. Overall, 49 of the 57 purified variants containing single or multiple point mutations improved activity compared to WT at one or more sites (Tables 10-11). Triple mutant No. 38 (SEQ ID NO: 3984), G146R / R182V / E795Q ("RVQ") performed best, showing approximately a 3-fold improvement over WT in this assay. Stacking other beneficial mutations with RVQ gradually decreased its activity (see variant numbers 47-57, Tables 10-11, Figures 6A-B). Therefore, the triple mutant RVQ (No. 38, SEQ ID NO: 3984) appears to be the optimal LbCas12a variant for genome editing.

[0167] [Table 10-1]

[0168] [Table 10-2]

[0169] [Table 11-1]

[0170] Table 11-2

Claims

1. An isolated mutant LbCas12a polypeptide comprising at least three amino acid substitutions, G146R, R182V, and E795Q, relative to the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2, wherein the mutant LbCas12a polypeptide exhibits at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 3984, and provides improved CRISPR / LbCas12a-related nuclease activity at the non-canonical TTTT PAM site compared to the wild-type LbCas12a polypeptide sequence.

2. The isolated mutant LbCas12a polypeptide according to claim 1, wherein the mutant LbCas12a polypeptide comprises the amino acid sequence of SEQ ID NO: 3984.

3. An isolated nucleic acid encoding a mutant LbCas12a polypeptide comprising at least three amino acid substitutions G146R, R182V and E795Q relative to the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2, wherein the mutant LbCas12a polypeptide has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 3984, and provides improved CRISPR / LbCas12a-related nuclease activity at the non-canonical TTTT PAM site compared to the wild-type LbCas12a polypeptide sequence.

4. The isolated nucleic acid according to claim 3, wherein the mutant LbCas12a polypeptide comprises the amino acid sequence of SEQ ID NO: 3984.

5. A vector or plasmid comprising the isolated nucleic acid described in claim 3.

6. A cell comprising the isolated nucleic acid described in claim 3, or the vector or plasmid described in claim 5.

7. An isolated ribonucleoprotein complex comprising a guide RNA and a mutant LbCas12a polypeptide having at least three amino acid substitutions of G146R, R182V and E795Q relative to the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2, wherein the mutant LbCas12a polypeptide has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 3984, and the mutant LbCas12a polypeptide provides improved CRISPR / LbCas12a-related nuclease activity at the non-canonical TTTT PAM site compared to the wild-type LbCas12a polypeptide sequence.

8. The isolated ribonucleoprotein complex according to claim 7, wherein the mutant LbCas12a polypeptide comprises the amino acid sequence of SEQ ID NO: 3984.

9. A CRISPR ribonucleoprotein complex for use in a method to enhance the efficiency of gene editing at non-canonical TTTT PAM sites in cells, The CRISPR ribonucleoprotein complex comprises a guide RNA and a mutant LbCas12a polypeptide having at least three amino acid substitutions of G146R, R182V, and E795Q relative to the wild-type LbCas12a polypeptide sequence of SEQ ID NO:

2. The method includes the step of bringing the CRISPR ribonucleoprotein complex into contact with cells, Herein, the CRISPR ribonucleoprotein complex wherein the mutant LbCas12a polypeptide contains at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 3984, and the mutant LbCas12a polypeptide provides improved CRISPR / LbCas12a-related nuclease activity at the non-canonical TTTT PAM site compared to the wild-type LbCas12a polypeptide sequence.

10. The CRISPR ribonucleoprotein complex according to claim 9, wherein the mutant LbCas12a polypeptide comprises the amino acid sequence of SEQ ID NO: 3984.

11. A kit for enhancing the efficiency of gene editing at non-canonical TTTT PAM sites in cells: The kit comprises a CRISPR ribonucleoprotein complex comprising a guide RNA and a mutant LbCas12a polypeptide having at least three amino acid substitutions of G146R, R182V, and E795Q relative to the wild-type LbCas12a polypeptide sequence of SEQ ID NO: 2, wherein the mutant LbCas12a polypeptide has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 3984, and compared to the wild-type LbCas12a polypeptide sequence, the mutant LbCas12a polypeptide provides improved CRISPR / LbCas12a-related nuclease activity at the non-canonical TTTT PAM site.

12. The kit according to claim 11, wherein the mutant LbCas12a polypeptide comprises the amino acid sequence of SEQ ID NO: 3984.

13. A method for expressing and purifying a mutant LbCas12a protein, comprising: (a) Inserting a nucleotide sequence encoding a mutant LbCas12a polypeptide containing at least three amino acid substitutions, G146R, R182V, and E795Q, into an expression plasmid relative to the wild-type LbCas12a polypeptide sequence of Sequence ID No. 2; (b) the step of transforming one or more non-human cells with an expression plasmid; (c) A step of inducing the expression of the transformed plasmid; (d) the step of separating the non-human cells; (e) the step of extracting the mutant LbCas12a protein; (f) A step of purifying the mutant LbCas12a protein, Includes, Here, the mutant LbCas12a polypeptide contains at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 3984 and provides improved CRISPR / LbCas12a-related nuclease activity at the non-canonical TTTT PAM site compared to the wild-type LbCas12a polypeptide sequence. The aforementioned method.

Images