Compositions containing Cas12i2 mutant polypeptides and their use

Abstract

The present invention relates to mutant Cas12i2 polypeptides, methods for making mutant Cas12i2 polypeptides, processes for characterizing mutant Cas12i2 polypeptides, cells comprising mutant Cas12i2 polypeptides, and methods for using mutant Cas12i2 polypeptides. The present invention further relates to complexes comprising mutant Cas12i2 polypeptides, methods for making the complexes, processes for characterizing the complexes, cells comprising the complexes, and methods for using the complexes.

Description

[Technical Field]

[0001] Sequence List This application includes a sequence listing submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy made on March 31, 2021, is named 51451-008WO5_Sequence_Listing_03_30_2021_ST25 and has a size of 1,589,401 bytes. [Background technology]

[0002] Clustered, regularly arranged short palindromic sequence repeats (CRISPR) and CRISPR-related (Cas) genes, collectively known as the CRISPR-Cas or CRISPR / Cas system, are adaptive immune systems in archaea and bacteria that protect specific species from exogenous genetic elements. [Overview of the project] [Problems that the invention aims to solve]

[0003] In view of the above background, the present invention offers specific advantages and advancements that surpass the prior art. [Means for solving the problem]

[0004] The present invention, as disclosed herein, is not limited to specific advantages or functions, but provides a variant Cas12i2 polypeptide comprising a sequence having at least 95% identity with the sequence shown in any one of SEQ ID NOs: 3-146 or any one of SEQ ID NOs: 495-512.

[0005] In one embodiment of the mutant Cas12i2 polypeptide, the mutant Cas12i2 polypeptide contains the sequence shown in Sequence ID No. 4.

[0006] In another embodiment of the mutant Cas12i2 polypeptide, the mutant Cas12i2 polypeptide contains the sequence shown in Sequence ID No. 5.

[0007] In another embodiment of the mutant Cas12i2 polypeptide, the mutant Cas12i2 polypeptide contains the sequence shown in SEQ ID NO: 495.

[0008] In another embodiment of the mutant Cas12i2 polypeptide, the mutant Cas12i2 polypeptide contains the sequence shown in SEQ ID NO: 496.

[0009] In another embodiment of the mutant Cas12i2 polypeptide, the mutant Cas12i2 polypeptide further comprises one or more substitutions from Table 2.

[0010] In another embodiment of the mutant Cas12i2 polypeptide, the mutant Cas12i2 polypeptide is a variant of the Cas12i2 polypeptide containing the sequence shown in Sequence ID No. 2.

[0011] The present invention further provides a composition comprising a variant Cas12i2 polypeptide as described herein, the composition further comprising an RNA guide or a nucleic acid encoding an RNA guide, the RNA guide comprising a direct repeat sequence and a spacer sequence.

[0012] In one embodiment of the composition, the direct repeat sequence comprises a nucleotide sequence having at least 95% sequence identity with any one of sequence numbers 492 to 494.

[0013] In another embodiment of the composition, the direct repeat sequence comprises a nucleotide sequence shown in any one of SEQ ID NOs: 492-494.

[0014] In another embodiment of the composition, the spacer sequence comprises about 11 to about 50 nucleotides.

[0015] In another embodiment of the composition, the spacer sequence comprises about 15 to about 35 nucleotides.

[0016] In another embodiment of the composition, the spacer sequence is bound to a target nucleic acid sequence adjacent to the 5'-NTTN-3' sequence.

[0017] In another embodiment of the mutant Cas12i2 polypeptide or composition, the mutant Cas12i2 polypeptide further comprises at least one nuclear localization signal (NLS), at least one nuclear export signal (NES), or at least one NLS and at least one NES.

[0018] In another embodiment of the mutant Cas12i2 polypeptide or composition, the mutant Cas12i2 polypeptide further comprises a peptide tag, a fluorescent protein, a base editing domain, a DNA methylation domain, a histone residue modification domain, a localization factor, a transcription modifier, a light-dependent regulatory factor, a chemically inducible factor, or a chromatin visualization factor.

[0019] In another embodiment of the composition, the composition is present in a delivery system comprising nanoparticles, liposomes, exosomes, microvesicles, or gene guns.

[0020] The present invention further provides nucleic acid molecules encoding the variant Cas12i2 polypeptide as described herein.

[0021] The present invention further provides cells comprising the composition or mutant Cas12i2 polypeptide as described herein.

[0022] In one aspect of a cell, it can be a eukaryotic cell or a prokaryotic cell.

[0023] In another embodiment of the cell, the cell is a mammalian cell or a plant cell.

[0024] In another embodiment of the cell, the cell is a human cell.

[0025] The present invention further provides compositions or formulations comprising the variant Cas12i2 polypeptide as described herein and optionally an RNA guide and / or cells.

[0026] The present invention further provides a composition comprising a mutant Cas12i2 polypeptide and an RNA guide, wherein the Cas12i2 mutant polypeptide and the RNA guide form a complex, and this mutant Cas12i2 polypeptide exhibits increased complex formation with the RNA guide compared to the parent polypeptide.

[0027] The present invention further provides a composition comprising a variant Cas12i2 polypeptide and an RNA guide, wherein the Cas12i2 variant polypeptide and the RNA guide form a complex, and this Cas12i2 variant polypeptide exhibits increased binding affinity to the RNA guide compared to the parent polypeptide.

[0028] The present invention further provides a composition comprising a variant Cas12i2 polypeptide and an RNA guide, wherein the Cas12i2 variant polypeptide and the RNA guide form a complex, and the Cas12i2 variant polypeptide and the RNA guide exhibit increased protein-RNA interaction compared to the parent polypeptide and RNA guide.

[0029] In one embodiment of the composition, the variant Cas12i2 polypeptide exhibits increased complex formation, increased binding affinity to RNA guides, and / or increased stability over a certain temperature range, for example, 20°C to 65°C.

[0030] In another embodiment of the composition, the variant Cas12i2 polypeptide exhibits increased complex formation, increased binding affinity to RNA guides, and / or increased stability over a range of incubation times.

[0031] In another embodiment of the composition, the variant Cas12i2 polypeptide exhibits increased complex formation, increased binding affinity to RNA guides, and / or increased stability in buffers having a pH in the range of approximately 7.3 to approximately 8.6.

[0032] In another embodiment of the composition, the mutant Cas12i2 polypeptide is the T of the mutant binary complex. m The value is T of the parent complex. m When the temperature is at least 8°C higher than the specified value, it exhibits increased complex formation, increased binding affinity to the RNA guide, and / or increased stability.

[0033] In another embodiment of the composition, the parent polypeptide comprises the amino acid sequence of SEQ ID NO: 2.

[0034] In another embodiment of the composition, the variant Cas12i2 polypeptide exhibits enzymatic activity equal to or greater than that of the parent polypeptide.

[0035] In another embodiment of the composition, equivalent or greater enzyme activity occurs in a temperature range of approximately 20°C to approximately 90°C.

[0036] In another embodiment of the composition, the variant Cas12i2 polypeptide exhibits increased stability and / or protein-RNA interactions.

[0037] In another embodiment of the composition, the variant Cas12i2 polypeptide further lacks enzymatic activity.

[0038] In another embodiment of the composition, the variant Cas12i2 polypeptide exhibits further increased enzyme activity.

[0039] In another embodiment of the composition, the variant Cas12i2 polypeptide exhibits increased RNA guide complex formation, RNA guide binding activity, and / or RNA guide binding specificity.

[0040] In another embodiment of the composition, the variant Cas12i2 polypeptide exhibits altered on-target binding.

[0041] In another embodiment of the composition, the variant Cas12i2 polypeptide exhibits altered off-target binding.

[0042] The present invention further provides a composition comprising a mutant Cas12i2 polypeptide and an RNA guide, wherein the Cas12i2 mutant polypeptide and the RNA guide form a mutant binary complex, and this mutant binary complex exhibits reduced complex dissociation compared to the complex formed by the parent polypeptide and the RNA guide.

[0043] The present invention further provides a composition comprising a mutant Cas12i2 polypeptide and an RNA guide, wherein the Cas12i2 mutant polypeptide and the RNA guide form a mutant binary complex, and this RNA guide exhibits reduced dissociation from the mutant Cas12i2 polypeptide compared to the RNA guide of the parent complex.

[0044] The present invention further provides a composition comprising a variant Cas12i2 polypeptide and an RNA guide, wherein the Cas12i2 variant polypeptide and the RNA guide form a variant binary complex, and this variant binary complex exhibits increased stability compared to the complex formed by the parent polypeptide and the RNA guide.

[0045] In one embodiment of the composition, the mutant binary complex exhibits increased ternary complex formation, increased binding affinity to the target nucleic acid, and / or increased stability over a certain temperature range, for example, 20°C to 65°C.

[0046] In another embodiment of the composition, the mutant binary complex exhibits increased stability over a range of incubation times.

[0047] In another embodiment of the composition, the mutant binary complex exhibits increased stability in a buffer having a pH in the range of approximately 7.3 to approximately 8.6.

[0048] In another embodiment of the composition, the mutant binary complex is the T of the mutant binary complex. m The value is T of the parent complex. m When the temperature is at least 8°C higher than the specified value, it exhibits increased ternary complex formation, increased binding affinity to target nucleic acids, and / or increased stability.

[0049] In another embodiment of the composition, the parent polypeptide comprises the amino acid sequence of SEQ ID NO: 2.

[0050] In another embodiment of the composition, the variant Cas12i2 polypeptide exhibits enzymatic activity equal to or greater than that of the parent polypeptide.

[0051] In another embodiment of the composition, equivalent or greater enzyme activity occurs in a temperature range of approximately 20°C to approximately 90°C.

[0052] In another embodiment of the composition, the variant Cas12i2 polypeptide exhibits increased stability and / or protein-RNA interactions.

[0053] In another embodiment of the composition, the variant Cas12i2 polypeptide further lacks enzymatic activity.

[0054] In another embodiment of the composition, the variant Cas12i2 polypeptide exhibits further increased enzyme activity.

[0055] In another embodiment of the composition, the variant Cas12i2 polypeptide exhibits increased RNA guide complex formation, RNA guide binding activity, and / or RNA guide binding specificity.

[0056] In another embodiment of the composition, the variant Cas12i2 polypeptide exhibits altered on-target binding.

[0057] In another embodiment of the composition, the variant Cas12i2 polypeptide exhibits altered off-target binding.

[0058] The present invention further provides a method for complexing the variant Cas12i2 polypeptide as described herein with an RNA guide as described herein, for example, an RNA guide.

[0059] The present invention further provides a composition comprising a mutant Cas12i2 polypeptide, an RNA guide, and a target nucleic acid, wherein the mutant Cas12i2 polypeptide and the RNA guide form a mutant binary complex, and this mutant binary complex exhibits increased ternary complex formation with the target nucleic acid compared to the parent binary complex.

[0060] The present invention further provides a composition comprising a variant Cas12i2 polypeptide, an RNA guide, and a target nucleic acid, wherein the Cas12i2 variant polypeptide and the RNA guide form a variant binary complex, and this variant binary complex exhibits increased binding affinity to the target nucleic acid compared to the parent binary complex.

[0061] The present invention further provides a composition comprising a mutant Cas12i2 polypeptide, an RNA guide, and a target nucleic acid, wherein the Cas12i2 mutant polypeptide and the RNA guide form a mutant binary complex, and this mutant binary complex exhibits increased protein-RNA interaction compared to the parent binary complex.

[0062] The present invention further provides a composition comprising a mutant Cas12i2 polypeptide, an RNA guide, and a target nucleic acid, wherein the Cas12i2 mutant polypeptide and the RNA guide form a mutant binary complex, and this mutant binary complex exhibits increased protein-DNA interaction compared to the parent binary complex.

[0063] In one embodiment of the composition, the mutant binary complex exhibits increased ternary complex formation, increased binding affinity to the target nucleic acid, and / or increased stability over a certain temperature range, for example, 20°C to 65°C.

[0064] In another embodiment of the composition, the mutant binary complex exhibits increased ternary complex formation, increased binding affinity to the target nucleic acid, and / or increased stability over a range of incubation times.

[0065] In another embodiment of the composition, the mutant binary complex exhibits increased ternary complex formation, increased binding affinity to target nucleic acids, and / or increased stability in a buffer having a pH in the range of about 7.3 to about 8.6.

[0066] In another embodiment of the composition, the mutant binary complex is the T of the binary complex. m The value is T of the parent binary complex. m When the temperature is at least 8°C higher than the specified value, it exhibits increased ternary complex formation, increased binding affinity to target nucleic acids, and / or increased stability.

[0067] In another embodiment of the composition, the parent polypeptide comprises the amino acid sequence of SEQ ID NO: 2.

[0068] In another embodiment of the composition, the mutant binary complex exhibits enzymatic activity equal to or greater than that of the parent binary complex.

[0069] In another embodiment of the composition, equivalent or greater enzyme activity occurs in a temperature range of approximately 20°C to approximately 90°C.

[0070] In another embodiment of the composition, the mutant binary complex exhibits increased stability and / or protein-RNA interaction.

[0071] In another embodiment of the composition, the mutant binary complex exhibits increased stability and / or protein-DNA interaction.

[0072] In another embodiment of the composition, the mutant binary complex further lacks enzymatic activity.

[0073] In another embodiment of the composition, the mutant binary complex exhibits further increased enzyme activity.

[0074] In another embodiment of the composition, the variant Cas12i2 polypeptide exhibits increased RNA guide complex formation, RNA guide binding activity, and / or RNA guide binding specificity.

[0075] In another embodiment of the composition, the mutant binary complex exhibits increased target nucleic acid complex formation, target nucleic acid activity, and / or target nucleic acid specificity.

[0076] In another embodiment of the composition, the mutant binary complex exhibits altered on-target binding.

[0077] In another embodiment of the composition, the mutant binary complex exhibits altered off-target binding.

[0078] The present invention further provides a composition comprising a variant Cas12i2 polypeptide, an RNA guide, and a target nucleic acid, wherein the Cas12i2 variant polypeptide and the RNA guide form a variant binary complex, and the variant binary complex and the target nucleic acid form a variant ternary complex, and this variant ternary complex exhibits reduced complex dissociation compared to the parent ternary complex.

[0079] The present invention further provides a composition comprising a mutant Cas12i2 polypeptide, an RNA guide, and a target nucleic acid, wherein the Cas12i2 mutant polypeptide and the RNA guide form a mutant binary complex, and the mutant binary complex and the target nucleic acid form a mutant ternary complex, and this target nucleic acid exhibits dissociation from the mutant ternary complex that is reduced compared to the parent ternary complex.

[0080] The present invention further provides a composition comprising a variant Cas12i2 polypeptide, an RNA guide, and a target nucleic acid, wherein the Cas12i2 variant polypeptide and the RNA guide form a variant binary complex, and the variant binary complex and the target nucleic acid form a variant ternary complex, the variant ternary complex exhibiting increased stability compared to the parent ternary complex.

[0081] In one embodiment of the composition, the mutant ternary complex exhibits increased stability over a certain temperature range, for example, 20°C to 65°C.

[0082] In another embodiment of the composition, the mutant ternary complex exhibits increased stability over a range of incubation times.

[0083] In another embodiment of the composition, the mutant ternary complex exhibits increased stability in a buffer having a pH in the range of approximately 7.3 to approximately 8.6.

[0084] In another embodiment of the composition, the mutant ternary complex is the T of the mutant ternary complex. m The value is T of the parent ternary complex. m Increased stability is observed when the temperature is at least 8°C higher than the specified value.

[0085] In another embodiment of the composition, the parent polypeptide comprises the amino acid sequence of SEQ ID NO: 2.

[0086] In another embodiment of the composition, the variant Cas12i2 polypeptide exhibits enzymatic activity equal to or greater than that of the parent polypeptide.

[0087] In another embodiment of the composition, equivalent or greater enzyme activity occurs in a temperature range of approximately 20°C to approximately 90°C.

[0088] In another embodiment of the composition, the variant Cas12i2 polypeptide exhibits increased stability and / or protein-RNA interactions.

[0089] In another embodiment of the composition, the mutant binary complex exhibits increased stability and / or protein-DNA interaction.

[0090] In another embodiment of the composition, the mutant ternary complex exhibits increased stability.

[0091] In another embodiment of the composition, the mutant binary complex further lacks enzymatic activity.

[0092] In another embodiment of the composition, the mutant binary complex exhibits further increased enzyme activity.

[0093] In another embodiment of the composition, the variant Cas12i2 polypeptide exhibits increased RNA guide complex formation, RNA guide binding activity, and / or RNA guide binding specificity.

[0094] In another embodiment of the composition, the mutant binary complex exhibits increased target nucleic acid complex formation, target nucleic acid binding activity, and / or target nucleic acid binding specificity.

[0095] In another embodiment of the composition, the mutant binary complex exhibits altered on-target binding.

[0096] In another embodiment of the composition, the mutant binary complex exhibits altered off-target binding.

[0097] The present invention further provides a method for complexing a mutant binary complex as described herein with a target nucleic acid, such as DNA, as described herein.

[0098] The present invention further provides a composition comprising a mutant Cas12i2 polypeptide and an RNA guide, wherein the mutant Cas12i2 polypeptide and the RNA guide form a mutant binary complex, and this mutant binary complex exhibits increased binding affinity to the target nucleic acid compared to the parent binary complex.

[0099] The present invention further provides a composition comprising a mutant Cas12i2 polypeptide and an RNA guide, wherein the Cas12i2 mutant polypeptide and the RNA guide form a mutant binary complex, and this mutant binary complex exhibits increased target binding affinity of the target nucleic acid to the target gene locus compared to the parent binary complex.

[0100] In one aspect of the composition, the mutant binary complex exhibits increased ternary complex formation and / or increased stability over a range of temperatures, such as 20°C to 65°C.

[0101] In another aspect of the composition, the mutant binary complex exhibits increased ternary complex formation and / or increased stability over a range of incubation times.

[0102] In another aspect of the composition, the mutant binary complex exhibits increased ternary complex formation and / or increased stability in a buffer having a pH in the range of about 7.3 to about 8.6.

[0103] In another aspect of the composition, the mutant binary complex has a T m value that is at least 8°C higher than the T m value of the parental binary complex and exhibits increased ternary complex formation and / or increased stability.

[0104] In another aspect of the composition, the parental polypeptide comprises the amino acid sequence of SEQ ID NO: 2.

[0105] In another aspect of the composition, the mutant binary complex exhibits enzymatic activity equal to or greater than that of the parental binary complex.

[0106] In another aspect of the composition, the enzymatic activity equal to or greater than that of the parental binary complex occurs in the temperature range of about 20°C to about 90°C.

[0107] In another aspect of the composition, the mutant binary complex exhibits increased stability and / or protein-RNA interaction.

[0108] In another aspect of the composition, the mutant binary complex exhibits increased stability and / or protein-DNA interaction.

[0109] In another aspect of the composition, the mutant binary complex further lacks enzymatic activity.

[0110] In another embodiment of the composition, the mutant binary complex exhibits further increased enzyme activity.

[0111] In another embodiment of the composition, the variant Cas12i2 polypeptide exhibits increased RNA guide complex formation, RNA guide binding activity, and / or RNA guide binding specificity.

[0112] In another embodiment of the composition, the mutant binary complex exhibits increased target nucleic acid complex formation, target nucleic acid activity, and / or target nucleic acid specificity.

[0113] In another embodiment of the composition, the mutant binary complex exhibits altered on-target binding.

[0114] In another embodiment of the composition, the mutant binary complex exhibits altered off-target binding.

[0115] The present invention further provides a composition comprising a plurality of mutant Cas12i2 polypeptides and two or more distinct RNA guides, wherein the distinct RNA guides individually form mutant binary complexes with separate mutant Cas12i2 polypeptides, and these mutant binary complexes specifically bind to two or more target gene loci of a target nucleic acid.

[0116] The present invention further provides a composition comprising a plurality of mutant Cas12i2 polypeptides and two or more distinct RNA guides, wherein the distinct RNA guides individually form mutant binary complexes with separate mutant Cas12i2 polypeptides, and these mutant binary complexes exhibit increased on-target binding to two or more target gene loci of the target nucleic acid compared to the parent binary complex.

[0117] The present invention further provides a composition comprising a plurality of mutant Cas12i2 polypeptides and two or more distinct RNA guides, wherein the distinct RNA guides individually form mutant binary complexes with separate mutant Cas12i2 polypeptides, and these mutant binary complexes exhibit increased on-target binding to two or more target loci of the target nucleic acid compared to the parent binary complex.

[0118] The present invention further provides a composition comprising a plurality of mutant Cas12i2 polypeptides and two or more distinct RNA guides, wherein the distinct RNA guides individually form mutant binary complexes with separate mutant Cas12i2 polypeptides, and these mutant binary complexes exhibit on-target ternary complex formation with two or more target gene loci of a target nucleic acid.

[0119] The present invention further provides a composition comprising a plurality of mutant Cas12i2 polypeptides and two or more distinct RNA guides, wherein the distinct RNA guides individually form mutant binary complexes with separate mutant Cas12i2 polypeptides, and these mutant binary complexes exhibit increased ternary complex formation with two or more target loci of the target nucleic acid compared to the parental binary complex.

[0120] In one embodiment of the composition, the mutant binary complex exhibits increased ternary complex formation and / or increased stability with the target nucleic acid over a certain temperature range, for example, 20°C to 65°C.

[0121] In another embodiment of the composition, the mutant binary complex exhibits increased ternary complex formation and / or increased stability with the target nucleic acid over a range of incubation times.

[0122] In another embodiment of the composition, the mutant binary complex exhibits increased ternary complex formation and / or increased stability with the target nucleic acid in a buffer having a pH in the range of about 7.3 to about 8.6.

[0123] In another embodiment of the composition, the mutant binary complex is the T of the binary complex.m The value is T of the parent binary complex. m When the temperature is at least 8°C higher than the specified value, it exhibits increased ternary complex formation with the target nucleic acid and / or increased stability.

[0124] In another embodiment of the composition, the parent polypeptide comprises the amino acid sequence of SEQ ID NO: 2.

[0125] In another embodiment of the composition, the variant Cas12i2 polypeptide exhibits enzymatic activity equal to or greater than that of the parent polypeptide.

[0126] In another embodiment of the composition, equivalent or greater enzyme activity occurs in a temperature range of approximately 20°C to approximately 90°C.

[0127] In another embodiment of the composition, the variant Cas12i2 polypeptide exhibits increased stability and / or protein-RNA interactions.

[0128] In another embodiment of the composition, the mutant binary complex exhibits increased stability and / or protein-DNA interaction.

[0129] In another embodiment of the composition, the mutant binary complex further lacks enzymatic activity.

[0130] In another embodiment of the composition, the mutant binary complex exhibits further increased enzyme activity.

[0131] In another embodiment of the composition, the variant Cas12i2 polypeptide exhibits increased RNA guide complex formation, RNA guide binding activity, and / or RNA guide binding specificity.

[0132] In another embodiment of the composition, the mutant binary complex exhibits increased target nucleic acid ternary complex formation, target nucleic acid binding affinity, and / or target nucleic acid binding specificity.

[0133] In another embodiment of the composition, the mutant binary complex exhibits altered on-target binding.

[0134] In another embodiment of the composition, the mutant binary complex exhibits altered off-target binding.

[0135] The present invention further provides a method for complexing the mutant binary complex described herein with a target nucleic acid, such as DNA, as described herein.

[0136] The present invention further provides a composition comprising a variant Cas12i2 polypeptide and an RNA guide, wherein the Cas12i2 variant polypeptide and the RNA guide form a variant binary complex, and this variant binary complex exhibits increased on-target binding affinity of the target nucleic acid to the target gene locus compared to the parent binary complex.

[0137] The present invention further provides a composition comprising a mutant Cas12i2 polypeptide and an RNA guide, wherein the mutant Cas12i2 polypeptide and the RNA guide form a mutant binary complex, and this mutant binary complex exhibits reduced binding affinity of the target nucleic acid to the non-target gene locus compared to the parent binary complex.

[0138] The present invention further provides a composition comprising a mutant Cas12i2 polypeptide and an RNA guide, wherein the mutant Cas12i2 polypeptide and the RNA guide form a mutant binary complex, which exhibits increased activity at the on-target locus of the target nucleic acid compared to the parent binary complex.

[0139] The present invention further provides a composition comprising a mutant Cas12i2 polypeptide and an RNA guide, wherein the mutant Cas12i2 polypeptide and the RNA guide form a mutant binary complex, and this mutant binary complex exhibits reduced activity at the non-target locus of the target nucleic acid compared to the parent binary complex.

[0140] In one embodiment of the composition, the mutant binary complex exhibits increased ternary complex formation and / or increased stability at the target gene locus over a certain temperature range, for example, 20°C to 65°C.

[0141] In another embodiment of the composition, the mutant binary complex exhibits increased ternary complex formation and / or increased stability at the target locus over a range of incubation times.

[0142] In another embodiment of the composition, the mutant binary complex exhibits increased ternary complex formation and / or increased stability at the target gene locus in a buffer having a pH in the range of approximately 7.3 to approximately 8.6.

[0143] In another embodiment of the composition, the mutant binary complex is the T of the binary complex. m The value is T of the parent binary complex. m When the temperature is at least 8°C higher than the specified value, increased ternary complex formation and / or increased stability are observed at the target gene locus.

[0144] In another embodiment of the composition, the parent binary complex comprises a parent polypeptide having the amino acid sequence of SEQ ID NO: 2.

[0145] In another embodiment of the composition, the variant Cas12i2 polypeptide exhibits enzymatic activity equal to or greater than that of the parent polypeptide.

[0146] In another embodiment of the composition, equivalent or greater enzyme activity occurs in a temperature range of approximately 20°C to approximately 90°C.

[0147] In another embodiment of the composition, the mutant binary complex exhibits increased stability and / or protein-RNA interaction.

[0148] In another embodiment of the composition, the mutant binary complex exhibits increased stability and / or protein-DNA interaction.

[0149] In another embodiment of the composition, the variant Cas12i2 polypeptide exhibits increased binary complex formation, RNA guide binding activity, and / or RNA guide binding specificity.

[0150] The present invention further provides a composition comprising a plurality of mutant Cas12i2 polypeptides and two or more distinct RNA guides, wherein the distinct RNA guides individually form plurality of mutant binary complexes with separate mutant Cas12i2 polypeptides, and these plurality of mutant binary complexes exhibit increased on-target binding to two or more target gene loci of the target nucleic acid compared to plurality of parental binary complexes.

[0151] The present invention further provides a composition comprising a plurality of mutant Cas12i2 polypeptides and two or more distinct RNA guides, wherein the distinct RNA guides individually form plurality of mutant binary complexes with separate mutant Cas12i2 polypeptides, and these plurality of mutant binary complexes exhibit reduced off-target binding to two or more non-target gene loci of the target nucleic acid compared to plurality of parental binary complexes.

[0152] The present invention further provides a composition comprising a plurality of mutant Cas12i2 polypeptides and two or more distinct RNA guides, wherein the distinct RNA guides individually form plurality of mutant binary complexes with separate mutant Cas12i2 polypeptides, and these plurality of mutant binary complexes exhibit increased on-target activity at two or more target gene loci of the target nucleic acid compared to plurality of parental binary complexes.

[0153] The present invention further provides a composition comprising a plurality of mutant Cas12i2 polypeptides and two or more distinct RNA guides, wherein the distinct RNA guides individually form plurality of mutant binary complexes with separate mutant Cas12i2 polypeptides, and these plurality of mutant binary complexes exhibit reduced off-target activity at two or more non-target gene loci of the target nucleic acid compared to plurality of parental binary complexes.

[0154] In one embodiment of the composition, multiple mutant binary complexes exhibit increased ternary complex formation and / or increased stability at the target gene locus of the target nucleic acid over a certain temperature range, for example, 20°C to 65°C.

[0155] In another embodiment of the composition, multiple mutant binary complexes exhibit increased ternary complex formation and / or increased stability at the target locus of the target nucleic acid over a range of incubation times.

[0156] In another embodiment of the composition, multiple mutant binary complexes exhibit increased ternary complex formation and / or increased stability at the target gene locus of the target nucleic acid in a buffer having a pH in the range of approximately 7.3 to approximately 8.6.

[0157] In another embodiment of the composition, multiple variant binary complexes are the T of the binary complex. m The value is T of the parent binary complex. m When the temperature is at least 8°C higher than the specified value, the target nucleic acid exhibits increased ternary complex formation and / or increased stability at the target gene locus.

[0158] In another embodiment of the composition, the multiple parent binary complexes include a parent polypeptide having the amino acid sequence of SEQ ID NO: 2.

[0159] In another embodiment of the composition, the variant Cas12i2 polypeptide exhibits enzymatic activity equal to or greater than that of the parent polypeptide.

[0160] In another embodiment of the composition, equivalent or greater enzyme activity occurs in a temperature range of approximately 20°C to approximately 90°C.

[0161] In another embodiment of the composition, the variant Cas12i2 polypeptide exhibits increased stability and / or protein-RNA interactions.

[0162] In another embodiment of the composition, multiple mutant binary complexes exhibit increased stability and / or protein-DNA interactions.

[0163] In another embodiment of the composition, the variant Cas12i2 polypeptide exhibits increased binary complex formation, RNA guide binding activity, and / or RNA guide binding specificity.

[0164] The present invention further provides a method for complexing a mutant binary complex as described herein with a target nucleic acid, such as DNA, as described herein.

[0165] The present invention further provides a method for complexing a plurality of mutant binary complexes described herein with a target nucleic acid, such as DNA, as described herein.

[0166] In one embodiment of the compositions described herein or the Cas12i2 polypeptide, the variant Cas12i2 polypeptide comprises at least one of the D581, G624, F626, D835, L836, P868, S879, D911, I926, V1020, V1030, E1035, and S1046 substitutions of the amino acid sequence of SEQ ID NO: 2.

[0167] In another embodiment of the composition or Cas12i2 polypeptide, the variant Cas12i2 polypeptide includes at least one of the following substitutions in the amino acid sequence of SEQ ID NO: D581G, D581R, G624R, F626G, F626R, D835G, D835R, L836G, L836R, P868G, P868R, P868T, S879G, S879R, D911G, D911R, I926G, I926R, V1020G, V1020R, V1030G, V1030R, E1035G, E1035R, S1046G, and S1046R.

[0168] In another embodiment of the composition or Cas12i2 polypeptide, the variant Cas12i2 polypeptide comprises at least one of the D581R, G624R, F626R, P868T, D911R, I926R, V1030G, E1035R, and S1046G substitutions in the amino acid sequence of SEQ ID NO: 2.

[0169] In another embodiment of the composition or the Cas12i2 polypeptide, the variant Cas12i2 polypeptide comprises at least one substitution listed in Table 1.

[0170] In another embodiment of the composition or Cas12i2 polypeptide, the variant Cas12i2 polypeptide comprises one amino acid sequence from SEQ ID NOs: 3 to 146 or one amino acid sequence from SEQ ID NOs: 495 to 512.

[0171] In another embodiment of the composition or the Cas12i2 polypeptide, the variant Cas12i2 polypeptide comprises at least one of an epitope peptide, a nuclear localization signal, and a nuclear export signal.

[0172] In another embodiment of the composition, the RNA guide comprises a DNA targeting sequence.

[0173] In another embodiment of the composition, the DNA targeting sequence is an RNA guide.

[0174] In another embodiment of the composition, the DNA targeting sequence is 13 to 30 nucleotides.

[0175] In another embodiment of the composition, the RNA guide comprises a direct repeat sequence ligated to a DNA targeting sequence.

[0176] In another embodiment of the composition, the composition further comprises a target nucleic acid.

[0177] In another embodiment of the composition, the target nucleic acid is present in cells.

[0178] In another embodiment of the composition, the variant Cas12i2 polypeptide and RNA guide are encoded by a vector, such as an expression vector.

[0179] The present invention further provides cells comprising the composition described herein.

[0180] The present invention further provides a method for expressing the vector as described herein.

[0181] The present invention further provides a method for preparing the compositions described herein.

[0182] The present invention further provides a method for delivering the compositions described herein.

[0183] The present invention further provides kits or systems comprising the compositions described herein or one or more components thereof.

[0184] In one embodiment of the compositions described herein, the RNA guide comprises or consists of 43 nucleotides or about 43 nucleotides.

[0185] In another embodiment of the composition, the RNA guide is a tracr-less RNA guide.

[0186] In another embodiment of the composition or the Cas12i2 polypeptide, the mutant Cas12i2 polypeptide exhibits approximately 40 times higher enzymatic activity than the parent polypeptide.

[0187] In another embodiment of the composition or the Cas12i2 polypeptide, the variant Cas12i2 polypeptide exhibits increased on-target specificity compared to the parent polypeptide.

[0188] In another embodiment of the composition or the Cas12i2 polypeptide, the variant Cas12i2 polypeptide exhibits reduced off-target specificity compared to the parent polypeptide.

[0189] In another embodiment of the composition, the mutant Cas12i2 polypeptide selectively induces a deletion adjacent to the 5'-NTTN-3' sequence (wherein N is any nucleotide).

[0190] In another embodiment of the composition, the deletion is located downstream of the 5'-NTTN-3' sequence.

[0191] In another embodiment of the composition, the parent polypeptide does not induce deletion.

[0192] In another embodiment of the composition, the deletion length is greater than the length of the Cas9 polypeptide-induced deletion.

[0193] In another embodiment of the composition, the deletion is located in the genes of a cell.

[0194] In another embodiment of the composition, the deletion is up to about 40 nucleotides long.

[0195] In another embodiment of the composition, the deletion is approximately 4 to 40 nucleotides in length.

[0196] In another embodiment of the composition, the deletion is approximately 4 to 25 nucleotides in length.

[0197] In another embodiment of the composition, the deletion is approximately 10 to 25 nucleotides in length.

[0198] In another embodiment of the composition, the deletion is approximately 10 to 15 nucleotides long.

[0199] In another embodiment of the composition, the deletion begins within approximately 5 to 15 nucleotides of the 5'-NTTN-3' sequence.

[0200] In another embodiment of the composition, the deletion begins within approximately 5 to 10 nucleotides of the 5'-NTTN-3' sequence.

[0201] In another embodiment of the composition, the deletion begins within approximately 10 to 15 nucleotides of the 5'-NTTN-3' sequence.

[0202] In another embodiment of the composition, the deletion begins within approximately 5 to 15 nucleotides downstream of the 5'-NTTN-3' sequence.

[0203] In another embodiment of the composition, the deletion begins within approximately 5 to 10 nucleotides downstream of the 5'-NTTN-3' sequence.

[0204] In another embodiment of the composition, the deletion begins within approximately 10 to 15 nucleotides downstream of the 5'-NTTN-3' sequence.

[0205] In another embodiment of the composition, the deletion ends within approximately 20 to 30 nucleotides of the 5'-NTTN-3' sequence.

[0206] In another embodiment of the composition, the deletion ends within approximately 20 to 25 nucleotides of the 5'-NTTN-3' sequence.

[0207] In another embodiment of the composition, the deletion ends within approximately 25 to 30 nucleotides of the 5'-NTTN-3' sequence.

[0208] In another embodiment of the composition, the deletion ends within approximately 20 to 30 nucleotides downstream of the 5'-NTTN-3' sequence.

[0209] In another embodiment of the composition, the deletion ends within approximately 20 to 25 nucleotides downstream of the 5'-NTTN-3' sequence.

[0210] In another embodiment of the composition, the deletion ends within approximately 25 to 30 nucleotides downstream of the 5'-NTTN-3' sequence.

[0211] In another embodiment of the composition, the deletion begins within approximately 5 to 15 nucleotides downstream of the 5'-NTTN-3' sequence and ends within approximately 20 to 30 nucleotides downstream of the 5'-NTTN-3' sequence.

[0212] In another embodiment of the composition, the deletion begins within approximately 5 to 15 nucleotides downstream of the 5'-NTTN-3' sequence and ends within approximately 20 to 25 nucleotides downstream of the 5'-NTTN-3' sequence.

[0213] In another embodiment of the composition, the deletion begins within approximately 5 to 15 nucleotides downstream of the 5'-NTTN-3' sequence and ends within approximately 25 to 30 nucleotides downstream of the 5'-NTTN-3' sequence.

[0214] In another embodiment of the composition, the deletion begins within approximately 5 to 10 nucleotides downstream of the 5'-NTTN-3' sequence and ends within approximately 20 to 30 nucleotides downstream of the 5'-NTTN-3' sequence.

[0215] In another embodiment of the composition, the deletion begins within approximately 5 to 10 nucleotides downstream of the 5'-NTTN-3' sequence and ends within approximately 20 to 25 nucleotides downstream of the 5'-NTTN-3' sequence.

[0216] In another embodiment of the composition, the deletion begins within approximately 5 to 10 nucleotides downstream of the 5'-NTTN-3' sequence and ends within approximately 25 to 30 nucleotides downstream of the 5'-NTTN-3' sequence.

[0217] In another embodiment of the composition, the deletion begins within approximately 10 to 15 nucleotides downstream of the 5'-NTTN-3' sequence and ends within approximately 20 to 30 nucleotides downstream of the 5'-NTTN-3' sequence.

[0218] In another embodiment of the composition, the deletion begins within approximately 10 to 15 nucleotides downstream of the 5'-NTTN-3' sequence and ends within approximately 20 to 25 nucleotides downstream of the 5'-NTTN-3' sequence.

[0219] In another embodiment of the composition, the deletion begins within approximately 10 to 15 nucleotides downstream of the 5'-NTTN-3' sequence and ends within approximately 25 to 30 nucleotides downstream of the 5'-NTTN-3' sequence.

[0220] In another embodiment of the composition, the 5'-NTTN-3' sequence is 5'-NTTY-3', 5'-NTTC-3', 5'-NTTT-3', 5'-NTTA-3', 5'-NTTB-3', 5'-NTTG-3', 5'-CTTY-3', 5'-DTTR'3', 5'-CTTR-3', 5'-DTTT-3', 5'-ATTN-3', or 5'-GTTN-3' (wherein Y is C or T, B is any nucleotide except A, D is any nucleotide except C, and R is A or G).

[0221] In another embodiment of the composition, the 5'-NTTN-3' sequence is 5'-CTTT-3', 5'-CTTC-3', 5'-GTTT-3', 5'-GTTC-3', 5'-TTTC-3', 5'-GTTA-3', or 5'-GTTG-3'.

[0222] In another embodiment of the composition, the deletion is located in the exon of a gene, for example, B2M, TRAC, PDCD1.

[0223] In another embodiment of the composition, the deletion overlaps with a gene mutation.

[0224] In another embodiment of the composition, the deletion overlaps with a gene insertion.

[0225] In another embodiment of the composition, the deletion removes the repeat elongation of the gene.

[0226] In another embodiment of the composition, the deletion disrupts one or both alleles of the gene.

[0227] In another embodiment of the composition, the deletion is induced in eukaryotic or prokaryotic cells.

[0228] In another embodiment of the composition, the deletion is induced in animal cells, plant cells, or fungal cells, or the cells are derived from animal cells, plant cells, or fungal cells.

[0229] In another embodiment of the composition, the deletion is induced in or originates from mammalian cells.

[0230] In another embodiment of the composition, the deletion is induced in or derived from human cells.

[0231] In another embodiment of the composition, the deletion is induced in primary cells.

[0232] In another embodiment of the composition, the deletion is induced in the cell line.

[0233] In another embodiment of the composition, the deletion is induced in T cells.

[0234] In another embodiment of the composition, the deletion induces the formation of stem cells (e.g., totipotent / omnipotent stem cells, pluripotent stem cells, compound pluripotent stem cells, oligopluripotent or unipotent stem cells), differentiated cells, or terminally differentiated cells.

[0235] In another embodiment of the composition, two or more deletions (e.g., multiplexed targeted deletions) are induced.

[0236] The present invention further provides a method for achieving deletion in cells, the method comprising contacting the cell's DNA with a mutant Cas12i2 polypeptide or complex as described herein.

[0237] The present invention further provides compositions or formulations comprising a variant Cas12i2 polypeptide as described herein, an RNA guide, and cells.

[0238] The present invention further provides a method for preparing the compositions described herein.

[0239] The present invention further provides a method for complexing the variant Cas12i2 polypeptide as described herein with an RNA guide as described herein, for example, an RNA guide.

[0240] The present invention further provides a method for complexing a mutant binary complex with a target nucleic acid, as described herein.

[0241] The present invention further provides a method for delivering the compositions described herein.

[0242] The present invention further provides a composition comprising a mutant Cas12i2 polypeptide, wherein the mutant Cas12i2 polypeptide includes substitutions that increase the interaction between the mutant Cas12i2 polypeptide and the nucleic acid compared to the parent polypeptide.

[0243] In one embodiment of the composition, the variant Cas12i2 polypeptide comprises the sequence shown in SEQ ID NO: 4.

[0244] In another embodiment of the composition, the variant Cas12i2 polypeptide comprises the sequence shown in any one of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 495, and SEQ ID NO: 496.

[0245] In another embodiment of the composition, the interaction is an electrostatic interaction.

[0246] In another embodiment of the composition, the interaction is a nonspecific interaction.

[0247] In another embodiment of the composition, the interactions are aromatic, hydrophobic, van der Waals, and / or cation-π interactions.

[0248] In another embodiment of the composition, the substitution is located at or adjacent to the nucleic acid interface.

[0249] In another aspect of the composition, the nucleic acid is an RNA guide comprising a direct repeat sequence and a spacer sequence.

[0250] In another aspect of the composition, the direct repeat sequence comprises a nucleotide sequence having at least 95% sequence identity with any one of SEQ ID NOs: 492 to 494.

[0251] In another aspect of the composition, the direct repeat sequence comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 492 to 494.

[0252] In another aspect of the composition, the substitution increases the interaction between the mutant Cas12i2 polypeptide and the direct repeat sequence.

[0253] In another aspect of the composition, the substitution increases binary complex formation as compared to the parental polypeptide.

[0254] In another aspect of the composition, the binary complex comprising the mutant Cas12i2 polypeptide exhibits increased stability as compared to the parental binary complex.

[0255] In another aspect of the composition, the substitution is an arginine, lysine, glutamine, asparagine, histidine, tyrosine or serine substitution.

[0256] In another aspect of the composition, the substitution is at the RNA binding interface.

[0257] In another aspect of the composition, the substitution is a substitution in the Wedge domain or the Rec2 domain.

[0258] In another aspect of the composition, the substitution is the substitution listed in Table 4.

[0259] In another aspect of the composition, the variant Cas12i2 polypeptide comprises a sequence having at least 95% identity with a sequence shown in any one of SEQ ID NOs: 3 to 146 or any one of SEQ ID NOs: 495 to 512.

[0260] In another aspect of the composition, the variant Cas12i2 polypeptide comprises a sequence having at least 95% identity with a sequence shown in any one of SEQ ID NOs: 3 to 146 or any one of SEQ ID NOs: 495 to 512, and further has substitutions listed in Table 4.

[0261] In another aspect of the composition, the variant Cas12i2 polypeptide comprises a sequence shown in any one of SEQ ID NOs: 3 to 146 or any one of SEQ ID NOs: 495 to 512.

[0262] In another aspect of the composition, the variant Cas12i2 polypeptide comprises a sequence shown in any one of SEQ ID NOs: 3 to 146 or any one of SEQ ID NOs: 495 to 512, and further has substitutions listed in Table 4.

[0263] In another aspect of the composition, the direct repeat sequence is shown in SEQ ID NO: 492, and the variant Cas12i2 polypeptide comprises a sequence shown in SEQ ID NO:4.

[0264] In another aspect of the composition, the direct repeat sequence is shown in SEQ ID NO: 492, and the variant Cas12i2 polypeptide comprises a sequence shown in any one of SEQ ID NOs: 3, SEQ ID NO: 5, SEQ ID NO: 495 and SEQ ID NO: 496.

[0265] In another aspect of the composition, the nucleic acid is a target nucleic acid.

[0266] In another aspect of the composition, the nucleic acid is double-stranded DNA.

[0267] In another aspect of the composition, the substitution increases the interaction between the variant Cas12i2 polypeptide and the double-stranded DNA.

[0268] In another embodiment of the composition, the double-stranded DNA comprises a PAM sequence.

[0269] In another embodiment of the composition, the substitution increases ternary complex formation compared to the parent polypeptide.

[0270] In another embodiment of the composition, the ternary complex containing the mutant Cas12i2 polypeptide exhibits increased stability compared to the parent ternary complex.

[0271] In another embodiment of the composition, the substitution is arginine, lysine, glutamine, asparagine, histidine, or serine substitution.

[0272] In another embodiment of the composition, the substitution is located at the double-stranded DNA binding interface.

[0273] In another embodiment of the composition, the substitution is in the Rec1 domain, the PI domain, or the Wedge domain.

[0274] In another embodiment of the composition, the substitutions are those listed in Table 5.

[0275] In another embodiment of the composition, the variant Cas12i2 polypeptide comprises a sequence having at least 95% identity with the sequence shown in any one of SEQ ID NOs: 3-146 or any one of SEQ ID NOs: 495-512.

[0276] In another embodiment of the composition, the variant Cas12i2 polypeptide comprises a sequence having at least 95% identity with any one of SEQ ID NOs: 3-146 or any one of SEQ ID NOs: 495-512, and further having the substitutions listed in Table 5.

[0277] In another embodiment of the composition, the variant Cas12i2 polypeptide comprises the sequence shown in any one of SEQ ID NOs: 3-146 or any one of SEQ ID NOs: 495-512.

[0278] In another aspect of the composition, the variant Cas12i2 polypeptide comprises a sequence shown in any one of SEQ ID NOs: 3 to 146 or any one of SEQ ID NOs: 495 to 512, and further has the substitutions listed in Table 5.

[0279] In another aspect of the composition, the nucleic acid is single-stranded DNA.

[0280] In another aspect of the composition, the single-stranded DNA comprises a non-target strand.

[0281] In another aspect of the composition, the single-stranded DNA comprises a target strand.

[0282] In another aspect of the composition, the substitution increases ternary complex formation as compared to the parental polypeptide.

[0283] In another aspect of the composition, the ternary complex comprising the variant Cas12i2 polypeptide exhibits increased stability as compared to the parental ternary complex.

[0284] In another aspect of the composition, the substitution is an arginine, lysine, glutamine, asparagine, histidine or alanine substitution.

[0285] In another aspect of the composition, the substitution is at the single-stranded DNA binding interface.

[0286] In another aspect of the composition, the substitution is a substitution in the PI domain, Rec1 domain, Wedge domain, RuvC domain, Rec2 domain or Nuc domain.

[0287] In another aspect of the composition, the substitution is the substitution listed in Table 6.

[0288] In another aspect of the composition, the variant Cas12i2 polypeptide comprises a sequence having at least 95% identity with a sequence shown in any one of SEQ ID NOs: 3 to 146 or any one of SEQ ID NOs: 495 to 512.

[0289] In another embodiment of the composition, the variant Cas12i2 polypeptide comprises a sequence having at least 95% identity with any one of SEQ ID NOs: 3-146 or any one of SEQ ID NOs: 495-512, and further having the substitutions listed in Table 6.

[0290] In another embodiment of the composition, the variant Cas12i2 polypeptide comprises the sequence shown in any one of SEQ ID NOs: 3-146 or any one of SEQ ID NOs: 495-512.

[0291] In another embodiment of the composition, the variant Cas12i2 polypeptide comprises the sequence shown in any one of SEQ ID NOs: 3-146 or any one of SEQ ID NOs: 495-512, and further comprises the substitutions listed in Table 6.

[0292] In another embodiment of the composition, the substitution increases the interaction between the mutant Cas12i2 polypeptide and the DNA / RNA hybrid molecule.

[0293] In another embodiment of the composition, the DNA / RNA hybrid molecule is a heteroduplex comprising an RNA guide spacer sequence and a target strand.

[0294] In another embodiment of the composition, the substitution stabilizes the heteroduplex.

[0295] In another embodiment of the composition, the substitution increases ternary complex formation compared to the parent polypeptide.

[0296] In another embodiment of the composition, the ternary complex containing the mutant Cas12i2 polypeptide exhibits increased stability compared to the parent ternary complex.

[0297] In another embodiment of the composition, the substitution is arginine, lysine, glutamine, asparagine, histidine, or serine substitution.

[0298] In another embodiment of the composition, the substitution is in the Rec1 domain, PI domain, Rec2 domain, or RuvC2 motif.

[0299] In another embodiment of the composition, the substitutions are those listed in Table 7.

[0300] In another embodiment of the composition, the variant Cas12i2 polypeptide comprises a sequence having at least 95% identity with the sequence shown in any one of SEQ ID NOs: 3-146 or any one of SEQ ID NOs: 495-512.

[0301] In another embodiment of the composition, the variant Cas12i2 polypeptide comprises a sequence having at least 95% identity with any one of SEQ ID NOs: 3-146 or any one of SEQ ID NOs: 495-512, and further having the substitutions listed in Table 7.

[0302] In another embodiment of the composition, the variant Cas12i2 polypeptide comprises the sequence shown in any one of SEQ ID NOs: 3-146 or any one of SEQ ID NOs: 495-512.

[0303] In another embodiment of the composition, the variant Cas12i2 polypeptide comprises the sequence shown in any one of SEQ ID NOs: 3-146 or any one of SEQ ID NOs: 495-512, and further comprises the substitutions listed in Table 7.

[0304] In another embodiment of the composition, the substitution increases the interaction between the mutant Cas12i2 polypeptide and the bases of a heteroduplex comprising (a) a double-stranded DNA duplex and / or (b) an RNA guide spacer sequence and a target strand.

[0305] In another embodiment of the composition, the double-stranded DNA double helix comprises a PAM sequence.

[0306] In another embodiment of the composition, the interactions are aromatic, hydrophobic, van der Waals, and / or cation-π interactions.

[0307] In another embodiment of the composition, the substitution stabilizes the R-loop.

[0308] In another embodiment of the composition, the substitution increases ternary complex formation compared to the parent polypeptide.

[0309] In another embodiment of the composition, the ternary complex containing the mutant Cas12i2 polypeptide exhibits increased stability compared to the parent ternary complex.

[0310] In another embodiment of the composition, the substitution is an arginine, lysine, tryptophan, phenylalanine, tyrosine, methionine, histidine, glutamine, threonine, or valine substitution.

[0311] In another embodiment of the composition, the substitution is in the Wedge domain, the Rec1 domain, or the RuvC domain.

[0312] In another embodiment of the composition, the substitutions are those listed in Table 8.

[0313] In another embodiment of the composition, the variant Cas12i2 polypeptide comprises a sequence having at least 95% identity with the sequence shown in any one of SEQ ID NOs: 3-146 or any one of SEQ ID NOs: 495-512.

[0314] In another embodiment of the composition, the variant Cas12i2 polypeptide comprises a sequence having at least 95% identity with any one of SEQ ID NOs: 3-146 or any one of SEQ ID NOs: 495-512, and further having the substitutions listed in Table 8.

[0315] In another embodiment of the composition, the variant Cas12i2 polypeptide comprises the sequence shown in any one of SEQ ID NOs: 3-146 or any one of SEQ ID NOs: 495-512.

[0316] In another embodiment of the composition, the variant Cas12i2 polypeptide comprises the sequence shown in any one of SEQ ID NOs: 3-146 or any one of SEQ ID NOs: 495-512, and further comprises the substitutions listed in Table 8.

[0317] In another embodiment of the composition, the parent polypeptide comprises the amino acid sequence of SEQ ID NO: 2.

[0318] In another embodiment of the composition, the mutant Cas12i2 polypeptide exhibits increased enzymatic activity compared to the parent polypeptide.

[0319] In another embodiment of the composition, the composition further comprises an RNA guide comprising a direct repeat sequence and a spacer sequence.

[0320] In another embodiment of the composition, the direct repeat sequence comprises a nucleotide sequence having at least 95% sequence identity with any one of sequence numbers 492 to 494.

[0321] In another embodiment of the composition, the direct repeat sequence comprises a nucleotide sequence shown in any one of SEQ ID NOs: 492-494.

[0322] In another embodiment of the composition, the direct repeat sequence is shown in SEQ ID NO: 492, and the variant Cas12i2 polypeptide comprises the sequence shown in SEQ ID NO: 4.

[0323] 239. In another embodiment of the composition, the direct repeat sequence is shown in SEQ ID NO: 492, and the variant Cas12i2 polypeptide comprises the sequence shown in any one of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 495, and SEQ ID NO: 496.

[0324] The present invention further provides cells comprising the compositions described herein.

[0325] In one embodiment of the cell, the composition does not substantially affect the cell's viability.

[0326] The present invention further provides a composition comprising a mutant Cas12i2 polypeptide, wherein the mutant Cas12i2 polypeptide includes substitutions that increase the mobility of the mutant Cas12i2 polypeptide in DNA binding compared to the parent polypeptide.

[0327] In one embodiment of the composition, the variant Cas12i2 polypeptide comprises the sequence shown in Sequence ID No. 4.

[0328] In another embodiment of the composition, the Cas12i2 polypeptide comprises the sequence shown in any one of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 495, and SEQ ID NO: 496.

[0329] In another embodiment of the composition, the substitution increases the binding of the mutant Cas12i2 polypeptide to DNA.

[0330] In another embodiment of the composition, the substitution increases the binding of the mutant Cas12i2 polypeptide to double-stranded DNA.

[0331] In another embodiment of the composition, the substitution increases the binding of the mutant Cas12i2 polypeptide to single-stranded DNA.

[0332] In another embodiment of the composition, the substitution increases ternary complex formation compared to the parent polypeptide.

[0333] In another embodiment of the composition, the ternary complex containing the mutant Cas12i2 polypeptide exhibits increased stability compared to the parent ternary complex.

[0334] In another embodiment of the composition, the substitution is the substitution of a bulk amino acid with an amino acid having a smaller side chain.

[0335] In another embodiment of the composition, the substitution is an alanine, valine, glycine, or serine substitution.

[0336] In another embodiment of the composition, the substitution is located in the helix II domain of the mutant Cas12i2 polypeptide.

[0337] In another embodiment of the composition, the substitutions are those listed in Table 9.

[0338] In another embodiment of the composition, the variant Cas12i2 polypeptide comprises a sequence having at least 95% identity with the sequence shown in any one of SEQ ID NOs: 3-146 or any one of SEQ ID NOs: 495-512.

[0339] In another embodiment of the composition, the variant Cas12i2 polypeptide comprises a sequence having at least 95% identity with the sequence shown in any one of SEQ ID NOs: 3-146 or any one of SEQ ID NOs: 495-512, and further having the substitutions listed in Table 9.

[0340] In another embodiment of the composition, the variant Cas12i2 polypeptide comprises the sequence shown in any one of SEQ ID NOs: 3-146 or any one of SEQ ID NOs: 495-512.

[0341] In another embodiment of the composition, the variant Cas12i2 polypeptide comprises the sequence shown in any one of SEQ ID NOs: 3-146 or any one of SEQ ID NOs: 495-512, and further comprises the substitutions listed in Table 9.

[0342] The present invention further provides a composition comprising a mutant Cas12i2 polypeptide, the mutant Cas12i2 polypeptide comprising substitutions that stabilize the interdomain interface formed during ternary complex formation compared to the parent polypeptide.

[0343] In one embodiment of the composition, the variant Cas12i2 polypeptide comprises the sequence shown in Sequence ID No. 4.

[0344] In another embodiment of the composition, the variant Cas12i2 polypeptide comprises the sequence shown in any one of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 495, and SEQ ID NO: 496.

[0345] In another embodiment of the composition, the interdomain interface is formed when single-stranded DNA comes into contact with the active site of the mutant Cas12i2 polypeptide.

[0346] In another embodiment of the composition, the interdomain interface is a helix II domain-Nuc domain interface.

[0347] In another embodiment of the composition, the substitution increases ternary complex formation compared to the parent polypeptide.

[0348] In another embodiment of the composition, the ternary complex containing the mutant Cas12i2 polypeptide exhibits increased stability compared to the parent ternary complex.

[0349] In another embodiment of the composition, the substitution is an aspartic acid, glutamic acid, arginine, or lysine substitution.

[0350] In another embodiment of the composition, the substitutions are those listed in Table 10.

[0351] In another embodiment of the composition, the variant Cas12i2 polypeptide comprises a sequence having at least 95% identity with the sequence shown in any one of SEQ ID NOs: 3-146 or any one of SEQ ID NOs: 495-512.

[0352] In another embodiment of the composition, the variant Cas12i2 polypeptide comprises a sequence having at least 95% identity with any one of SEQ ID NOs: 3-146 or any one of SEQ ID NOs: 495-512, and further having the substitutions listed in Table 10.

[0353] In another embodiment of the composition, the variant Cas12i2 polypeptide comprises the sequence shown in any one of SEQ ID NOs: 3-146 or any one of SEQ ID NOs: 495-512.

[0354] In another embodiment of the composition, the variant Cas12i2 polypeptide comprises the sequence shown in any one of SEQ ID NOs: 3-146 or any one of SEQ ID NOs: 495-512, and further comprises the substitutions listed in Table 10.

[0355] In another embodiment of the composition, the parent polypeptide comprises the amino acid sequence of SEQ ID NO: 2.

[0356] In another embodiment of the composition, the mutant Cas12i2 polypeptide exhibits increased enzymatic activity compared to the parent polypeptide.

[0357] In another embodiment of the composition, the composition further comprises an RNA guide comprising a direct repeat sequence and a spacer sequence.

[0358] In another embodiment of the composition, the direct repeat sequence comprises a nucleotide sequence having at least 95% sequence identity with any one of sequence numbers 492 to 494.

[0359] In another embodiment of the composition, the direct repeat sequence comprises a nucleotide sequence shown in any one of SEQ ID NOs: 492-494.

[0360] In another embodiment of the composition, the direct repeat sequence is shown in SEQ ID NO: 492, and the variant Cas12i2 polypeptide comprises the sequence shown in SEQ ID NO: 4.

[0361] In another embodiment of the composition, the direct repeat sequence is shown in SEQ ID NO: 492, and the variant Cas12i2 polypeptide comprises the sequence shown in any one of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 495, and SEQ ID NO: 496.

[0362] The present invention further provides cells comprising the composition described herein.

[0363] In one embodiment of the cell, the composition does not substantially affect the cell's viability.

[0364] The present invention further provides a composition comprising a mutant Cas12i2 polypeptide, wherein the mutant Cas12i2 polypeptide comprises substitutions that increase the on-target specificity of the mutant Cas12i2 polypeptide compared to the parent polypeptide.

[0365] In one embodiment of the composition, the variant Cas12i2 polypeptide comprises the sequence shown in Sequence ID No. 4.

[0366] In another embodiment of the composition, the variant Cas12i2 polypeptide comprises the sequence shown in any one of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 495, and SEQ ID NO: 496.

[0367] In another embodiment of the composition, the substitution increases on-target DNA binding.

[0368] In another embodiment of the composition, the substitution reduces off-target DNA binding.

[0369] In another embodiment of the composition, the substitution increases on-target ternary complex formation compared to the parent polypeptide.

[0370] In another embodiment of the composition, the on-target ternary complex containing the mutant Cas12i2 polypeptide exhibits increased stability compared to the parent ternary complex.

[0371] In another embodiment of the composition, the substitution is the substitution of an amino acid that contacts the spacer sequence of the RNA guide.

[0372] In another embodiment of the composition, the substitution is the substitution of a bulk amino acid with an amino acid having a smaller side chain.

[0373] In another embodiment of the composition, the substitution is an alanine, serine, valine, glutamine, or asparagine substitution.

[0374] In another embodiment of the composition, the substitution is in the Wedge domain, Rec1 domain, Rec2 domain, or RuvC domain.

[0375] In another embodiment of the composition, the substitution is a substitution in the helix II domain.

[0376] In another embodiment of the composition, the substitutions are those listed in Table 11.

[0377] In another embodiment of the composition, the variant Cas12i2 polypeptide comprises a sequence having at least 95% identity with the sequence shown in any one of SEQ ID NOs: 3-146 or any one of SEQ ID NOs: 495-512.

[0378] In another embodiment of the composition, the variant Cas12i2 polypeptide comprises a sequence having at least 95% identity with any one of SEQ ID NOs: 3-146 or any one of SEQ ID NOs: 495-512, and further having the substitutions listed in Table 11.

[0379] In another embodiment of the composition, the variant Cas12i2 polypeptide comprises the sequence shown in any one of SEQ ID NOs: 3-146 or any one of SEQ ID NOs: 495-512.

[0380] In another embodiment of the composition, the variant Cas12i2 polypeptide comprises the sequence shown in any one of SEQ ID NOs: 3-146 or any one of SEQ ID NOs: 495-512, and further comprises the substitutions listed in Table 11.

[0381] In another embodiment of the composition, the substitution reduces the catalytic reaction rate (Kcat) of the mutant Cas12i2 polypeptide.

[0382] In another embodiment of the composition, the substitution is an alanine, serine, threonine, valine, leucine, methionine, asparagine, or isoleucine substitution.

[0383] In another embodiment of the composition, the substitution is in the Wedge domain, Rec1 domain, Rec2 domain, or RuvC domain.

[0384] In another embodiment of the composition, the substitution is a substitution in the RuvC domain.

[0385] In another embodiment of the composition, the substitutions are those listed in Table 12.

[0386] In another embodiment of the composition, the variant Cas12i2 polypeptide comprises a sequence having at least 95% identity with the sequence shown in any one of SEQ ID NOs: 3-146 or any one of SEQ ID NOs: 495-512.

[0387] In another embodiment of the composition, the variant Cas12i2 polypeptide comprises a sequence having at least 95% identity with any one of SEQ ID NOs: 3-146 or any one of SEQ ID NOs: 495-512, and further having the substitutions listed in Table 12.

[0388] In another embodiment of the composition, the variant Cas12i2 polypeptide comprises the sequence shown in any one of SEQ ID NOs: 3-146 or any one of SEQ ID NOs: 495-512.

[0389] In another embodiment of the composition, the variant Cas12i2 polypeptide comprises the sequence shown in any one of SEQ ID NOs: 3-146 or any one of SEQ ID NOs: 495-512, and further comprises the substitutions listed in Table 12.

[0390] In another embodiment of the composition, the mutant Cas12i2 polypeptide exhibits increased on-target enzyme activity compared to the parent polypeptide.

[0391] In another embodiment of the composition, the mutant Cas12i2 polypeptide exhibits reduced off-target enzyme activity compared to the parent polypeptide.

[0392] In another embodiment of the composition, the variant Cas12i2 polypeptide exhibits off-target editing, which is less than 10% of on-target editing.

[0393] In another embodiment of the composition, the variant Cas12i2 polypeptide exhibits off-target editing, which is less than 5% of on-target editing.

[0394] In another embodiment of the composition, the composition further comprises an RNA guide comprising a direct repeat sequence and a spacer sequence.

[0395] In another embodiment of the composition, the direct repeat sequence comprises a nucleotide sequence having at least 95% sequence identity with any one of sequence numbers 492 to 494.

[0396] In another embodiment of the composition, the direct repeat sequence comprises a nucleotide sequence shown in any one of SEQ ID NOs: 492-494.

[0397] In another embodiment of the composition, the direct repeat sequence is shown in SEQ ID NO: 492, and the variant Cas12i2 polypeptide comprises the sequence shown in SEQ ID NO: 4.

[0398] In another embodiment of the composition, the direct repeat sequence is shown in SEQ ID NO: 492, and the variant Cas12i2 polypeptide comprises the sequence shown in any one of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 495, and SEQ ID NO: 496.

[0399] The present invention further provides cells comprising the composition described herein.

[0400] In one embodiment of the cell, the composition does not substantially affect the cell's viability.

[0401] In another embodiment of the composition, the variant Cas12i2 polypeptide exhibits a higher ratio of on-target binding to off-target binding compared to the Cas9 polypeptide.

[0402] In another embodiment of the composition, the variant Cas12i2 polypeptide exhibits a higher ratio of on-target activity to off-target activity compared to the Cas9 polypeptide.

[0403] In another embodiment of the composition, the variant Cas12i2 polypeptide exhibits a higher ratio of on-target editing to off-target editing compared to the Cas9 polypeptide.

[0404] In another embodiment of the composition, the variant Cas12i2 polypeptide exhibits fewer off-target bindings compared to the Cas9 polypeptide.

[0405] In another embodiment of the composition, the variant Cas12i2 polypeptide exhibits less off-target activity compared to the Cas9 polypeptide.

[0406] In another embodiment of the composition, the variant Cas12i2 polypeptide exhibits less off-target editing compared to the Cas9 polypeptide.

[0407] In another embodiment of the composition, off-target binding, off-target activity, and / or off-target editing by the mutant Cas12i2 polypeptide is at least 10% less than that by the Cas9 polypeptide.

[0408] In another embodiment of the composition, off-target binding, off-target activity, and / or off-target editing by the mutant Cas12i2 polypeptide are at least 20% less than off-target binding, off-target activity, and / or off-target editing by the Cas9 polypeptide.

[0409] In another embodiment of the composition, off-target binding, off-target activity, and / or off-target editing by the mutant Cas12i2 polypeptide are at least 30% less than off-target binding, off-target activity, and / or off-target editing by the Cas9 polypeptide.

[0410] In another embodiment of the composition, off-target binding, off-target activity, and / or off-target editing by the mutant Cas12i2 polypeptide are at least 40% less than off-target binding, off-target activity, and / or off-target editing by the Cas9 polypeptide.

[0411] In another embodiment of the composition, off-target binding, off-target activity, and / or off-target editing by the mutant Cas12i2 polypeptide are at least 50% less than off-target binding, off-target activity, and / or off-target editing by the Cas9 polypeptide.

[0412] In yet another embodiment, the present invention provides cells comprising the composition described herein.

[0413] In one embodiment of the cell, the composition does not substantially affect the cell's viability.

[0414] definition The present invention will be described with respect to detailed embodiments and with reference to specific drawings, but the invention is not limited thereto and is limited only by the claims. The terms set forth below shall be understood in their general sense unless otherwise indicated.

[0415] As used herein, the term “activity” refers to biological activity. In some embodiments, activity refers to effector activity. In some embodiments, activity includes enzymatic activity, such as the catalytic ability of an effector. For example, activity may include nuclease activity. In some embodiments, activity includes binding activity, such as the ability of an effector to guide RNA and / or bind to a target nucleic acid.

[0416] As used herein, the term “adjacent to” means that a nucleotide or amino acid sequence is adjacent to another nucleotide or amino acid sequence. In some embodiments, a nucleotide sequence is adjacent to another nucleotide sequence if there are no nucleotides separating the two sequences. In some embodiments, a nucleotide sequence is adjacent to another nucleotide sequence if the two sequences are separated by a small number of nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides). In some embodiments, a first sequence is adjacent to a second sequence if the two sequences are separated by about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In some embodiments, the term “adjacent to” is used to mean that a protein residue interacts with another protein residue. In some embodiments, the term “adjacent to” is used to mean that a protein residue interacts with a nucleotide or nucleic acid. In some embodiments, the term “adjacent to” is used to mean that a protein domain or motif interacts with another protein domain or motif. In some embodiments, the term “adjacent to” is used to mean that a protein domain or motif interacts with a nucleotide or nucleic acid sequence. As used herein, the term “adjacent to” is used to mean the positioning of an indel (insertion / deletion) in the modified cell of this disclosure.

[0417] As used herein, the term “complex” refers to an assembly of two or more molecules. In some embodiments, the complex includes polypeptide and nucleic acid molecules that are interacting with each other (e.g., bound, in contact with, or adherent to each other).

[0418] As used herein, the term “binary complex” refers to an assembly of two molecules (e.g., a polypeptide and a nucleic acid molecule). In some embodiments, a binary complex refers to an assembly of a polypeptide and a targeting portion (e.g., an RNA guide). In some embodiments, a binary complex refers to a ribonucleoprotein (RNP). As used herein, the term “mutant binary complex” refers to an assembly of a mutant Cas12i2 polypeptide and an RNA guide. As used herein, the term “parent binary complex” refers to an assembly of a parent polypeptide and an RNA guide, or a reference polypeptide and an RNA guide.

[0419] As used herein, the term “ternary complex” refers to an assembly of three molecules (e.g., a polypeptide and two nucleic acid molecules). In some embodiments, “ternary complex” refers to an assembly of a polypeptide, an RNA molecule, and a DNA molecule. In some embodiments, a ternary complex refers to an assembly of a polypeptide, a targeting portion (e.g., an RNA guide), and a target nucleic acid (e.g., a target DNA molecule). In some embodiments, “ternary complex” refers to an assembly of a binary complex (e.g., a ribonucleoprotein) and a third molecule (e.g., a target nucleic acid). As used herein, the terms “mutant ternary complex,” “mutant Cas12i2 ternary complex,” and “Cas12i2 mutant ternary complex” refer to an assembly of a mutant Cas12i2 polypeptide, an RNA guide, and a target nucleic acid (e.g., a mutant Cas12i2 ribonucleoprotein and a target nucleic acid). As used herein, the terms “parental ternary complex,” “parental Cas12i2 ternary complex,” and “Cas12i2 parental ternary complex” refer to an assembly of a parental polypeptide, an RNA guide, and a target nucleic acid (e.g., a parental ribonucleoprotein and a target nucleic acid) or a reference polypeptide, an RNA guide, and a target nucleic acid (e.g., a reference ribonucleoprotein and a target nucleic acid).

[0420] As used herein, the term “deletion” refers to the loss or removal of a nucleotide in a nucleic acid sequence. A deletion may be a deletion in the genome of an organism. A deletion may be a deletion in a cell. A deletion may be a DNA sequence. A deletion may be an RNA sequence. A deletion may be a frameshift mutation or a non-frameshift mutation. Cas12i2-induced deletions as described herein may refer to deletions of up to approximately 100 nucleotides from a nucleic acid molecule, such as approximately 4 to 100 nucleotides, approximately 4 to 50 nucleotides, approximately 4 to 40 nucleotides, approximately 4 to 25 nucleotides, approximately 10 to 25 nucleotides, and approximately 10 to 15 nucleotides. In some embodiments, Cas12i2-induced deletions as described herein appear downstream of a 5'-NTTN-3' sequence. In some embodiments, the term “Cas12i2-induced” refers to a deletion resulting from the induction of DNA cleavage by the Cas12i2 polypeptide. In some embodiments, the term "Cas12i2-inducible" refers to a deletion resulting from DNA breakage induced by the Cas12i2 polypeptide and subsequently repaired by the cell's DNA repair mechanism.

[0421] As used herein, the term “domain” refers to a distinct functional and / or structural unit of a polypeptide. In some embodiments, a domain may comprise a conserved amino acid sequence.

[0422] As used herein, the terms “editing efficiency” and “indel activity” refer to the ability of an enzyme (e.g., a mutant Cas12i2 polypeptide) to introduce indels (insertions / deletions) into a sequence. For example, in some embodiments, an enzyme that introduces indels into each of 10 target loci exhibits 100% editing efficiency. An enzyme that introduces indels into 5 of the 10 target loci exhibits 50% editing efficiency. In another example, an enzyme that introduces indels into target loci in 50% of multiple cells exhibits 50% editing efficiency. As used herein, the terms “editing efficiency,” “indel activity,” and “on-target editing” refer to the ability of an enzyme (e.g., a mutant Cas12i2 polypeptide) to selectively introduce indels into target loci. In some embodiments, the editing efficiency at a target locus is compared to the editing efficiency at a non-target locus. In some embodiments, editing at a target locus is compared to editing at a non-target locus.

[0423] As used herein, the term “effector activity” refers to biological activity. In some embodiments, effector activity includes enzymatic activity, such as the catalytic ability of an effector. For example, effector activity may include nuclease activity.

[0424] As used herein, the term “interface” refers to one or more residues of a mutant Cas12i2 polypeptide (e.g., a domain / motif or a portion of a domain / motif) that are in contact with (e.g., interact with or adjacent to) a separate domain / motif or portion of a separate domain / motif of a nucleic acid molecule or mutant Cas12i2 polypeptide. In some embodiments, the interface is the surface area embedded between adjacent domains or motifs. In some embodiments, the interface is the surface area between the polypeptide and the ligand (e.g., DNA or RNA) where the polypeptide and the ligand are in contact. As used herein, the term “nucleic acid interface” refers to residues of a mutant Cas12i2 polypeptide that are in close proximity to (e.g., adjacent to) or interacting with a nucleic acid sequence (e.g., a DNA sequence or an RNA sequence). As used herein, the term “RNA binding interface” refers to residues of a mutant Cas12i2 polypeptide that are in close proximity to (e.g., adjacent to) or interacting with an RNA guide (e.g., a direct repeat of an RNA guide). As used herein, the term “double-stranded DNA binding interface” refers to residues of the variant Cas12i2 polypeptide that are in close proximity to (e.g., adjacent to) and / or interact with double-stranded DNA. As used herein, the term “single-stranded DNA binding interface” refers to residues of the variant Cas12i2 polypeptide that are in close proximity to (e.g., adjacent to) and / or interact with single-stranded DNA. As used herein, the term “inter-domain interface” refers to a interface where one domain is in close proximity to (e.g., adjacent to) another domain. In some embodiments, inter-domain interfaces (e.g., helix II domain-Nuc domain interface) are formed during complex formation (e.g., during ternary complex formation).

[0425] As used herein, the terms “parent,” “parent polypeptide,” and “parent sequence” refer to the original polypeptide (e.g., starting polypeptide) from which the changes are made to produce the variant Cas12i2 polypeptide of the present invention. In some embodiments, the parent is a polypeptide having the same amino acid sequence as the variant, with one or more mutations at one or more designated positions. In exemplary embodiments, a mutation refers to a change in an amino acid within the polypeptide sequence. The parent may be a naturally occurring (wild-type) polypeptide. In detailed embodiments, the parent is a polypeptide having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 70%, at least 72%, at least 73%, at least 74%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the polypeptide of SEQ ID NO: 2. In some embodiments, mutations may include structural changes that do not alter the original amino acid sequence of the parent, such as binding, fusion, or other modifications. In some embodiments, the parent polypeptide sequence includes amino acid mutations and structural modifications.

[0426] As used herein, “multiple variant binary complexes” refers to multiple binary complexes comprising multiple variant Cas12i2 polypeptides. Multiple binary complexes comprising multiple parent polypeptides (e.g., the wild-type Cas12i2 polypeptide parent of SEQ ID NO: 2) are referred to herein as “multiple parent binary complexes.” Multiple Cas12i2 polypeptides, e.g., multiple variant Cas12i2 polypeptides, and two or more distinct targeting moieties, e.g., two or more distinct RNA guides, individually form two or more binary complexes (e.g., multiple binary complexes). In such embodiments, complex formation can be achieved simultaneously in a single composition or independently in separate compositions.

[0427] As used herein, the term “protospacer adjacency motif” or “PAM” refers to a DNA sequence adjacent to a target sequence to which a binary complex comprising a polypeptide (e.g., an enzyme such as Cas12i2 or a variant thereof) and a targeting moiety (e.g., an RNA guide) binds. In some embodiments, the PAM is required for enzyme activity. For double-stranded targets, the targeting moiety (e.g., an RNA guide) binds to the first strand of the target, and the PAM sequence, as described herein, is present on the second complementary strand. For example, in some embodiments, the RNA guide binds to the target strand (e.g., a spacer complementary strand), and the PAM sequence, as described herein, is present on the non-target strand (i.e., a non-spacer complementary strand). In some embodiments, the target strand (i.e., the spacer complementary strand) contains a 5'-NAAN-3' sequence.

[0428] As used herein, the terms “reference composition,” “reference molecule,” “reference sequence,” “reference,” and “reference complex” refer to a control, such as a negative control or parent (e.g., a parent sequence, parent protein, wild-type protein, or a complex containing the parent sequence). For example, a reference molecule refers to a Cas12i2 polypeptide compared to a mutant Cas12i2 polypeptide. Similarly, a reference RNA guide refers to a targeting portion of a modified RNA guide compared to a reference molecule. A mutant or modified molecule may be compared to a reference molecule based on its sequence (e.g., a mutant or modified molecule may have X% sequence identity or homology with the reference molecule), thermal stability, or activity (e.g., a mutant or modified molecule may have X% activity of the reference molecule). For example, if the reference molecule is a Cas12i2 polypeptide, a mutant or modified molecule may be characterized as having less than 10% of the activity of the reference Cas12i2 polypeptide, or at least 10% higher activity than the reference Cas12i2 polypeptide. Examples of reference Cas12i2 polypeptides include naturally occurring, unmodified Cas12i2 polypeptides, such as those derived from archaea or other bacterial species. In certain embodiments, the reference Cas12i2 polypeptide is a naturally occurring Cas12i2 polypeptide that has the closest sequence identity or homology to the comparative mutant Cas12i2 polypeptide. In certain embodiments, the reference Cas12i2 polypeptide is a parent molecule having a naturally occurring or known sequence that was mutated to obtain the mutant Cas12i2 polypeptide.

[0429] As used herein, the terms “RNA guide” or “RNA guide sequence” refer to any RNA molecule that facilitates the targeting of polypeptides described herein to target nucleic acids. For example, an RNA guide may be a molecule that recognizes (e.g., binds to) a target nucleic acid. An RNA guide may be designed to be complementary to a specific nucleic acid sequence. RNA guides include DNA targeting sequences and direct repeat (DR) sequences. The terms CRISPR RNA (crRNA), pre-crRNA, and mature crRNA also refer to RNA guides as used herein.

[0430] As used herein, the term “substantially identical” refers to a sequence, polynucleotide, or polypeptide having a degree of identity with a reference sequence.

[0431] As used herein, the term “targeting moiety” refers to a molecule or component (e.g., a nucleic acid and / or RNA guide) that facilitates the targeting of another molecule or component to a target nucleic acid. In some embodiments, the targeting moiety specifically interacts with or associates with the target nucleic acid.

[0432] As used herein, the terms “target nucleic acid,” “target sequence,” “target substrate,” and “on-target locus” refer to the nucleic acid sequence to which the targeting portion (e.g., an RNA guide) specifically binds. In some embodiments, the DNA targeting sequence of the RNA guide binds to the target nucleic acid. The binding of the binary complex to the target locus is referred to herein as “on-target binding.”

[0433] As used herein, the terms “non-target” and “off-target” refer to nucleic acid sequences other than those to which the targeting moiety specifically binds or is intended to specifically bind. A non-target locus is an unintended target of the targeting moiety (e.g., an RNA guide). Binding of the binary complex to a non-target locus is referred to herein as “off-target binding.” In some embodiments, a non-target locus is a locus on a target nucleic acid. In some embodiments, a non-target locus is a locus on a nucleic acid other than the target nucleic acid (e.g., a non-target nucleic acid).

[0434] As used herein, the terms “upstream” and “downstream” refer to the relative positions within a single nucleic acid (e.g., DNA) sequence in a nucleic acid molecule. As used herein, the terms “upstream” and “downstream” refer to the relative positions of two sequences. “Upstream” and “downstream” each refer to the 5' to 3' orientation in which RNA transcription occurs. The first sequence is upstream of the second sequence when the 3' end of the first sequence appears before the 5' end of the second sequence. The first sequence is downstream of the second sequence when the 5' end of the first sequence appears after the 3' end of the second sequence. As used herein, the terms “upstream” and “downstream” are used to refer to the relative position of a deletion relative to a 5'-NTTN'-3' sequence. When used herein, the 5'-NTTN-3' sequence is upstream of the Cas12i2-induced deletion, and the Cas12i2-induced deletion is downstream of the 5'-NTTN-3' sequence.

[0435] As used herein, the terms “mutant Cas12i2 polypeptide” and “mutant effector polypeptide” refer to polypeptides that have alterations, e.g., substitutions, insertions, deletions, and / or fusions, at one or more residue positions compared to the parent polypeptide. As used herein, the terms “mutant Cas12i2 polypeptide” and “mutant effector polypeptide” refer to polypeptides that have alterations compared to the polypeptide of SEQ ID NO: 2. In certain embodiments, for example, the following items are provided: (Item 1) A variant Cas12i2 polypeptide containing a sequence that has at least 95% identity with the sequence shown in any one of sequence numbers 3-146 or any one of sequence numbers 495-512. (Item 2) A variant Cas12i2 polypeptide described in item 1, comprising the sequence shown in sequence number 4. (Item 3) A variant Cas12i2 polypeptide according to item 1 or 2, comprising the sequence shown in Sequence ID No. 5. (Item 4) The variant Cas12i2 polypeptide described in item 1, comprising the sequence shown in sequence number 495. (Item 5) The variant Cas12i2 polypeptide described in item 1, comprising the sequence shown in sequence number 496. (Item 6) A variant Cas12i2 polypeptide described in any one of items 1 to 5, further comprising the substitutions in Table 2. (Item 7) A variant of the Cas12i2 polypeptide containing the sequence shown in Sequence ID No. 2, as described in any one of items 1 to 6. (Item 8) A composition comprising a variant Cas12i2 polypeptide described in any one of items 1 to 7, further comprising an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide comprises a direct repeat sequence and a spacer sequence. (Item 9) The composition according to item 8, wherein the direct repeat sequence comprises a nucleotide sequence having at least 95% sequence identity with any one of sequence numbers 492 to 494. (Item 10) The composition according to item 9, wherein the direct repeat sequence comprises a nucleotide sequence shown in any one of sequence numbers 492 to 494. (Item 11) The spacer sequence comprises about 11 to about 50 nucleotides, according to any one of items 8 to 10. (Item 12) The spacer sequence comprises about 15 to about 35 nucleotides, according to the composition described in item 11. (Item 13) The composition according to any one of items 8 to 12, wherein the spacer sequence is bound to a target nucleic acid sequence, and the target nucleic acid sequence is adjacent to a 5'-NTTN-3' sequence. (Item 14) The mutant Cas12i2 polypeptide further comprises at least one nuclear localization signal (NLS), at least one nuclear export signal (NES), or at least one NLS and at least one NES, as described in any one of items 1 to 7 or the composition described in any one of items 8 to 13. (Item 15) The mutant Cas12i2 polypeptide further comprises a peptide tag, a fluorescent protein, a base editing domain, a DNA methylation domain, a histone residue modification domain, a localization factor, a transcription modifier, a light-dependent regulatory factor, a chemically inducible factor, or a chromatin visualization factor, as described in any one of items 1 to 7 or 14, or as described in any one of items 8 to 14. (Item 16) A composition according to any one of items 8 to 15, which is present in a delivery system comprising nanoparticles, liposomes, exosomes, microvesicles, or gene guns. (Item 17) A nucleic acid molecule encoding a variant Cas12i2 polypeptide described in any one of items 1-7, 14, or 15. (Item 18) Cells containing the composition described in any one of items 1 to 17 or the mutant Cas12i2 polypeptide. (Item 19) A cell, either a eukaryotic or prokaryotic cell, as described in item 18. (Item 20) A cell, which is a mammalian cell or a plant cell, as described in item 18 or 19. (Item 21) Human cells, as described in any one of items 18-20. (Item 22) A composition or formulation comprising a variant Cas12i2 polypeptide described in any one of items 1-7, 14, or 15, and optionally an RNA guide and / or cells. (Item 23) A composition comprising a variant Cas12i2 polypeptide and an RNA guide, wherein the Cas12i2 variant polypeptide and the RNA guide form a complex, and the variant Cas12i2 polypeptide exhibits increased complex formation with the RNA guide compared to the parent polypeptide. (Item 24) A composition comprising a variant Cas12i2 polypeptide and an RNA guide, wherein the Cas12i2 variant polypeptide and the RNA guide form a complex, and the Cas12i2 variant polypeptide exhibits increased binding affinity to the RNA guide compared to the parent polypeptide. (Item 25) A composition comprising a variant Cas12i2 polypeptide and an RNA guide, wherein the Cas12i2 variant polypeptide and the RNA guide form a complex, and the Cas12i2 variant polypeptide and the RNA guide exhibit increased protein-RNA interaction compared to the parent polypeptide and the RNA guide. (Item 26) The composition according to any one of items 23 to 25, wherein the mutant Cas12i2 polypeptide exhibits increased complex formation, increased binding affinity to the RNA guide, and / or increased stability over a certain temperature range, for example, 20°C to 65°C. (Item 27) The composition according to any one of items 23 to 26, wherein the mutant Cas12i2 polypeptide exhibits increased complex formation, increased binding affinity to the RNA guide, and / or increased stability over a certain range of incubation times. (Item 28) The composition according to any one of items 23 to 27, wherein the mutant Cas12i2 polypeptide exhibits increased complex formation, increased binding affinity to the RNA guide, and / or increased stability in a buffer having a pH in the range of about 7.3 to about 8.6. (Item 29) The aforementioned mutant Cas12i2 polypeptide is the T of the mutant binary complex. m The value is T of the parent complex. m The composition according to any one of items 23 to 28, which exhibits increased complex formation, increased binding affinity to the RNA guide, and / or increased stability when the temperature is at least 8°C higher than the value. (Item 30) The parent polypeptide comprises the amino acid sequence of SEQ ID NO: 2, according to any one of items 23 to 29. (Item 31) The composition according to any one of items 23 to 30, wherein the mutant Cas12i2 polypeptide exhibits enzymatic activity equal to or greater than that of the parent polypeptide. (Item 32) The composition described in item 31 exhibits enzyme activity equivalent to or greater than that described above in a temperature range of approximately 20°C to approximately 90°C. (Item 33) The aforementioned mutant Cas12i2 polypeptide exhibits increased stability and / or protein-RNA interactions, as described in any one of items 23 to 32. (Item 34) The composition according to any one of items 23 to 33, wherein the aforementioned mutant Cas12i2 polypeptide further lacks enzymatic activity. (Item 35) The composition according to item 34, wherein the mutant Cas12i2 polypeptide further exhibits increased enzyme activity. (Item 36) The composition according to any one of items 23 to 35, wherein the mutant Cas12i2 polypeptide exhibits increased RNA guide complex formation, RNA guide binding activity, and / or RNA guide binding specificity. (Item 37) The aforementioned mutant Cas12i2 polypeptide exhibits altered on-target binding, as described in any one of items 23 to 36. (Item 38) The aforementioned mutant Cas12i2 polypeptide exhibits altered off-target binding, as described in any one of items 23 to 37. (Item 39) A composition comprising a variant Cas12i2 polypeptide and an RNA guide, wherein the Cas12i2 variant polypeptide and the RNA guide form a variant binary complex, and the variant binary complex exhibits reduced complex dissociation compared to the complex formed by the parent polypeptide and the RNA guide. (Item 40) A composition comprising a mutant Cas12i2 polypeptide and an RNA guide, wherein the Cas12i2 mutant polypeptide and the RNA guide form a mutant binary complex, and the RNA guide exhibits dissociation from the mutant Cas12i2 polypeptide, with a reduced amount compared to the RNA guide of the parent complex. (Item 41) A composition comprising a variant Cas12i2 polypeptide and an RNA guide, wherein the Cas12i2 variant polypeptide and the RNA guide form a variant binary complex, and the variant binary complex exhibits increased stability compared to the complex formed by the parent polypeptide and the RNA guide. (Item 42) The mutant binary complex exhibits increased ternary complex formation, increased binding affinity to target nucleic acids, and / or increased stability over a certain temperature range, for example, 20°C to 65°C, according to any one of items 39 to 41. (Item 43) The mutant binary complex exhibits increased stability over a certain range of incubation times, according to any one of items 39 to 42. (Item 44) The mutant binary complex exhibits increased stability in a buffer having a pH in the range of about 7.3 to about 8.6, according to any one of items 39 to 43. (Item 45) The mutant binary complex is the T of the mutant binary complex. m The value is T of the parent composite. m The composition according to any one of items 39 to 44, which exhibits increased ternary complex formation, increased binding affinity to the target nucleic acid, and / or increased stability when the temperature is at least 8°C higher than the value. (Item 46) The parent polypeptide comprises the amino acid sequence of SEQ ID NO: 2, according to any one of items 39 to 45. (Item 47) The composition according to any one of items 39 to 46, wherein the mutant Cas12i2 polypeptide exhibits enzymatic activity equal to or greater than that of the parent polypeptide. (Item 48) The composition described in item 47 exhibits enzyme activity equivalent to or greater than that described above in a temperature range of approximately 20°C to approximately 90°C. (Item 49) The aforementioned variant Cas12i2 polypeptide exhibits increased stability and / or protein-RNA interactions, as described in any one of items 39 to 48. (Item 50) The composition according to any one of items 39 to 49, wherein the aforementioned mutant Cas12i2 polypeptide further lacks enzymatic activity. (Item 51) The composition according to any one of items 39 to 49, wherein the mutant Cas12i2 polypeptide further exhibits increased enzyme activity. (Item 52) The composition according to any one of items 39 to 51, wherein the mutant Cas12i2 polypeptide exhibits increased RNA guide complex formation, RNA guide binding activity, and / or RNA guide binding specificity. (Item 53) The composition according to any one of items 39 to 52, wherein the mutant Cas12i2 polypeptide exhibits altered on-target binding. (Item 54) The aforementioned mutant Cas12i2 polypeptide exhibits altered off-target binding, as described in any one of items 39 to 53. (Item 55) A method for complexing a mutant Cas12i2 polypeptide described in any one of items 39 to 54 with an RNA guide described in any one of items 39 to 54, for example, an RNA guide. (Item 56) A composition comprising a mutant Cas12i2 polypeptide, an RNA guide, and a target nucleic acid, wherein the mutant Cas12i2 polypeptide and the RNA guide form a mutant binary complex, and the mutant binary complex exhibits increased ternary complex formation with the target nucleic acid compared to the parent binary complex. (Item 57) A composition comprising a variant Cas12i2 polypeptide, an RNA guide, and a target nucleic acid, wherein the Cas12i2 variant polypeptide and the RNA guide form a variant binary complex, and the variant binary complex exhibits increased binding affinity to the target nucleic acid compared to the parent binary complex. (Item 58) A composition comprising a variant Cas12i2 polypeptide, an RNA guide, and a target nucleic acid, wherein the Cas12i2 variant polypeptide and the RNA guide form a variant binary complex, and the variant binary complex exhibits increased protein-RNA interaction compared to the parent binary complex. (Item 59) A composition comprising a variant Cas12i2 polypeptide, an RNA guide, and a target nucleic acid, wherein the Cas12i2 variant polypeptide and the RNA guide form a variant binary complex, and the variant binary complex exhibits increased protein-DNA interaction compared to the parent binary complex. (Item 60) The composition according to any one of items 56 to 59, wherein the mutant binary complex exhibits increased ternary complex formation, increased binding affinity to the target nucleic acid, and / or increased stability over a certain temperature range, for example, 20°C to 65°C. (Item 61) The composition according to any one of items 56 to 60, wherein the mutant binary complex exhibits increased ternary complex formation, increased binding affinity to the target nucleic acid, and / or increased stability over a range of incubation times. (Item 62) The composition according to any one of items 56 to 61, wherein the mutant binary complex exhibits increased ternary complex formation, increased binding affinity to the target nucleic acid, and / or increased stability in a buffer having a pH in the range of about 7.3 to about 8.6. (Item 63) The mutant binary complex is the T of the binary complex. m The value is T of the parent binary complex. m The composition according to any one of items 56 to 62, which exhibits increased ternary complex formation, increased binding affinity to the target nucleic acid, and / or increased stability when the temperature is at least 8°C higher than the value. (Item 64) The parent polypeptide is a composition according to any one of items 56 to 63, comprising the amino acid sequence of SEQ ID NO: 2. (Item 65) The mutant binary complex exhibits enzymatic activity equal to or greater than that of the parent binary complex, according to any one of items 56 to 64. (Item 66) The composition described in item 65 exhibits enzyme activity equivalent to or greater than that described above in a temperature range of approximately 20°C to approximately 90°C. (Item 67) The mutant binary complex exhibits increased stability and / or protein-RNA interactions, according to any one of items 56 to 66. (Item 68) The mutant binary complex exhibits increased stability and / or protein-DNA interaction, according to any one of items 56 to 67. (Item 69) The composition according to any one of items 56 to 68, wherein the mutant binary complex further lacks enzymatic activity. (Item 70) The aforementioned mutant binary complex exhibits increased enzyme activity, as described in any one of items 56 to 68. (Item 71) The composition according to any one of items 56 to 70, wherein the mutant Cas12i2 polypeptide exhibits increased RNA guide complex formation, RNA guide binding activity, and / or RNA guide binding specificity. (Item 72) The mutant binary complex exhibits increased target nucleic acid complex formation, target nucleic acid activity, and / or target nucleic acid specificity, according to any one of items 56 to 71. (Item 73) The mutant binary complex exhibits altered on-target binding, according to any one of items 56 to 72. (Item 74) The mutant binary complex exhibits altered off-target binding, according to any one of items 56 to 73. (Item 75) A composition comprising a variant Cas12i2 polypeptide, an RNA guide, and a target nucleic acid, wherein the Cas12i2 variant polypeptide and the RNA guide form a variant binary complex, the variant binary complex and the target nucleic acid form a variant ternary complex, and the variant ternary complex exhibits reduced complex dissociation compared to the parent ternary complex. (Item 76) A composition comprising a variant Cas12i2 polypeptide, an RNA guide, and a target nucleic acid, wherein the Cas12i2 variant polypeptide and the RNA guide form a variant binary complex, the variant binary complex and the target nucleic acid form a variant ternary complex, and the target nucleic acid exhibits dissociation from the variant ternary complex to a degree less than that of the parent ternary complex. (Item 77) A composition comprising a variant Cas12i2 polypeptide, an RNA guide, and a target nucleic acid, wherein the Cas12i2 variant polypeptide and the RNA guide form a variant binary complex, the variant binary complex and the target nucleic acid form a variant ternary complex, and the variant ternary complex exhibits increased stability compared to the parent ternary complex. (Item 78) The mutant ternary complex exhibits increased stability over a certain temperature range, for example, 20°C to 65°C, according to any one of items 75 to 77. (Item 79) The mutant ternary complex exhibits increased stability over a certain range of incubation times, according to any one of items 75 to 78. (Item 80) The mutant ternary complex exhibits increased stability in a buffer having a pH in the range of approximately 7.3 to approximately 8.6, according to the composition according to any one of items 75 to 79. (Item 81) The mutant ternary complex is the T of the mutant ternary complex. m The value is T of the parent ternary complex. m A composition according to any one of items 75 to 80, which exhibits increased stability when the temperature is at least 8°C higher than the value. (Item 82) The parent polypeptide is a composition according to any one of items 75 to 81, comprising the amino acid sequence of SEQ ID NO: 2. (Item 83) The composition according to any one of items 75 to 82, wherein the mutant Cas12i2 polypeptide exhibits enzymatic activity equal to or greater than that of the parent polypeptide. (Item 84) The composition described in item 83 exhibits enzyme activity equivalent to or greater than that described above in a temperature range of approximately 20°C to approximately 90°C. (Item 85) The aforementioned variant Cas12i2 polypeptide exhibits increased stability and / or protein-RNA interactions, as described in any one of items 75 to 84. (Item 86) The mutant binary complex exhibits increased stability and / or protein-DNA interaction, according to any one of items 75 to 85. (Item 87) The mutant ternary complex exhibits increased stability, as described in any one of items 75 to 86. (Item 88) The aforementioned mutant binary complex further lacks enzymatic activity, according to any one of items 75 to 87. (Item 89) The aforementioned mutant binary complex exhibits increased enzyme activity, as described in any one of items 75 to 87. (Item 90) The composition according to any one of items 75 to 89, wherein the mutant Cas12i2 polypeptide exhibits increased RNA guide complex formation, RNA guide binding activity, and / or RNA guide binding specificity. (Item 91) The mutant binary complex exhibits increased target nucleic acid complex formation, target nucleic acid binding activity, and / or target nucleic acid binding specificity, according to any one of items 75 to 90. (Item 92) The mutant binary complex exhibits altered on-target binding, according to any one of items 75 to 91. (Item 93) The mutant binary complex exhibits altered off-target binding, according to any one of items 75 to 92. (Item 94) A method for complexing a mutant binary complex described in any one of items 75 to 93 with a target nucleic acid, such as DNA, described in any one of items 75 to 93. (Item 95) A composition comprising a mutant Cas12i2 polypeptide and an RNA guide, wherein the mutant Cas12i2 polypeptide and the RNA guide form a mutant binary complex, and the mutant binary complex exhibits increased binding affinity to a target nucleic acid compared to the parent binary complex. (Item 96) A composition comprising a variant Cas12i2 polypeptide and an RNA guide, wherein the Cas12i2 variant polypeptide and the RNA guide form a variant binary complex, and the variant binary complex exhibits increased target binding affinity of the target nucleic acid to the target gene locus compared to the parent binary complex. (Item 97) The mutant binary complex exhibits increased ternary complex formation and / or increased stability over a certain temperature range, for example, 20°C to 65°C, according to the composition of item 95 or 96. (Item 98) The composition according to any one of items 95 to 97, wherein the mutant binary complex exhibits increased ternary complex formation and / or increased stability over a certain range of incubation times. (Item 99) The mutant binary complex exhibits increased ternary complex formation and / or increased stability in a buffer having a pH in the range of about 7.3 to about 8.6, according to the composition according to any one of items 95 to 98. (Item 100) The mutant binary complex is the T of the binary complex. m The value is T of the parent binary complex. m The composition according to any one of items 95 to 99, which exhibits increased ternary complex formation and / or increased stability when the temperature is at least 8°C higher than the value. (Item 101) The parent polypeptide is a composition according to any one of items 95 to 100, comprising the amino acid sequence of SEQ ID NO: 2. (Item 102) The mutant binary complex exhibits enzymatic activity equal to or greater than that of the parent binary complex, according to any one of items 95 to 101. (Item 103) The composition described in item 102 exhibits enzyme activity equivalent to or greater than that described above in a temperature range of approximately 20°C to approximately 90°C. (Item 104) The mutant binary complex exhibits increased stability and / or protein-RNA interaction, according to any one of items 95 to 103. (Item 105) The mutant binary complex exhibits increased stability and / or protein-DNA interaction, according to any one of items 95 to 104. (Item 106) The aforementioned mutant binary complex further lacks enzymatic activity, the composition according to any one of items 95 to 105. (Item 107) The aforementioned mutant binary complex exhibits increased enzyme activity, as described in any one of items 95 to 105. (Item 108) The composition according to any one of items 95 to 107, wherein the mutant Cas12i2 polypeptide exhibits increased RNA guide complex formation, RNA guide binding activity, and / or RNA guide binding specificity. (Item 109) The mutant binary complex exhibits increased target nucleic acid complex formation, target nucleic acid activity, and / or target nucleic acid specificity, according to any one of items 95 to 108. (Item 110) The mutant binary complex exhibits altered on-target binding, according to any one of items 95 to 109. (Item 111) The mutant binary complex exhibits altered off-target binding, according to any one of items 95 to 110. (Item 112) A composition comprising multiple variant Cas12i2 polypeptides and two or more distinct RNA guides, wherein the distinct RNA guides individually form variant binary complexes with separate variant Cas12i2 polypeptides, and the variant binary complexes specifically bind to two or more target gene loci of a target nucleic acid. (Item 113) A composition comprising multiple mutant Cas12i2 polypeptides and two or more distinct RNA guides, wherein the distinct RNA guides individually form mutant binary complexes with separate mutant Cas12i2 polypeptides, and the mutant binary complexes exhibit increased on-target binding to two or more target gene loci of a target nucleic acid compared to the parent binary complex. (Item 114) A composition comprising multiple mutant Cas12i2 polypeptides and two or more distinct RNA guides, wherein the distinct RNA guides individually form mutant binary complexes with separate mutant Cas12i2 polypeptides, and the mutant binary complexes exhibit increased on-target binding to two or more target gene loci of a target nucleic acid compared to the parent binary complex. (Item 115) A composition comprising multiple mutant Cas12i2 polypeptides and two or more distinct RNA guides, wherein the distinct RNA guides individually form mutant binary complexes with separate mutant Cas12i2 polypeptides, and the mutant binary complexes exhibit on-target ternary complex formation with two or more target gene loci of a target nucleic acid. (Item 116) A composition comprising multiple mutant Cas12i2 polypeptides and two or more distinct RNA guides, wherein the distinct RNA guides individually form mutant binary complexes with separate mutant Cas12i2 polypeptides, and the mutant binary complexes exhibit increased ternary complex formation with two or more target loci of the target nucleic acid compared to the parent binary complex. (Item 117) The composition according to any one of items 112 to 116, wherein the mutant binary complex exhibits increased ternary complex formation and / or increased stability with the target nucleic acid over a certain temperature range, for example, 20°C to 65°C. (Item 118) The composition according to any one of items 112 to 117, wherein the mutant binary complex exhibits increased ternary complex formation and / or increased stability with the target nucleic acid over a range of incubation times. (Item 119) The composition according to any one of items 112 to 118, wherein the mutant binary complex exhibits increased ternary complex formation and / or increased stability with the target nucleic acid in a buffer having a pH in the range of about 7.3 to about 8.6. (Item 120) The mutant binary complex is the T of the binary complex. m The value is T of the parent binary complex. m The composition according to any one of items 112 to 119, which exhibits increased ternary complex formation with the target nucleic acid and / or increased stability when the temperature is at least 8°C higher than the value. (Item 121) The parent polypeptide is a composition according to any one of items 112 to 120, comprising the amino acid sequence of SEQ ID NO: 2. (Item 122) The mutant Cas12i2 polypeptide exhibits enzymatic activity equal to or greater than that of the parent polypeptide, as described in any one of items 112 to 121. (Item 123) The composition described in item 122 exhibits enzyme activity equivalent to or greater than that described above in a temperature range of approximately 20°C to approximately 90°C. (Item 124) The aforementioned variant Cas12i2 polypeptide exhibits increased stability and / or protein-RNA interactions, as described in any one of items 112 to 123. (Item 125) The mutant binary complex exhibits increased stability and / or protein-DNA interactions, according to any one of items 112 to 124. (Item 126) The aforementioned mutant binary complex further lacks enzymatic activity, the composition according to any one of items 112 to 125. (Item 127) The aforementioned mutant binary complex exhibits increased enzyme activity, as described in any one of items 112 to 125. (Item 128) The composition according to any one of items 112 to 127, wherein the mutant Cas12i2 polypeptide exhibits increased RNA guide complex formation, RNA guide binding activity, and / or RNA guide binding specificity. (Item 129) The mutant binary complex exhibits increased target nucleic acid ternary complex formation, target nucleic acid binding affinity, and / or target nucleic acid binding specificity, according to any one of items 112 to 128. (Item 130) The mutant binary complex exhibits altered on-target binding, as described in any one of items 112 to 129. (Item 131) The mutant binary complex exhibits altered off-target binding, as described in any one of items 112 to 130. (Item 132) A method for complexing a mutant binary complex described in any one of items 112 to 131 with a target nucleic acid, such as DNA, described in any one of items 112 to 131. (Item 133) A composition comprising a variant Cas12i2 polypeptide and an RNA guide, wherein the Cas12i2 variant polypeptide and the RNA guide form a variant binary complex, and the variant binary complex exhibits increased on-target binding affinity of the target nucleic acid to the target gene locus compared to the parent binary complex. (Item 134) A composition comprising a mutant Cas12i2 polypeptide and an RNA guide, wherein the mutant Cas12i2 polypeptide and the RNA guide form a mutant binary complex, and the mutant binary complex exhibits a reduced binding affinity of the target nucleic acid to a non-target gene locus compared to the parent binary complex. (Item 135) A composition comprising a mutant Cas12i2 polypeptide and an RNA guide, wherein the mutant Cas12i2 polypeptide and the RNA guide form a mutant binary complex, and the mutant binary complex exhibits increased activity at the on-target locus of the target nucleic acid compared to the parent binary complex. (Item 136) A composition comprising a mutant Cas12i2 polypeptide and an RNA guide, wherein the mutant Cas12i2 polypeptide and the RNA guide form a mutant binary complex, and the mutant binary complex exhibits reduced activity at the non-target locus of the target nucleic acid compared to the parent binary complex. (Item 137) The composition according to any one of items 133 to 136, wherein the mutant binary complex exhibits increased ternary complex formation and / or increased stability at the target gene locus over a certain temperature range, for example, 20°C to 65°C. (Item 138) The composition according to any one of items 133 to 137, wherein the mutant binary complex exhibits increased ternary complex formation and / or increased stability at the target gene locus over a certain range of incubation times. (Item 139) The composition according to any one of items 133 to 138, wherein the mutant binary complex exhibits increased ternary complex formation and / or increased stability at the target gene locus in a buffer having a pH in the range of about 7.3 to about 8.6. (Item 140) The mutant binary complex is the T of the binary complex. m The value is T of the parent binary complex. m The composition according to any one of items 133 to 139, which exhibits increased ternary complex formation and / or increased stability at the target gene locus when the temperature is at least 8°C higher than the value. (Item 141) The parent binary complex comprises a parent polypeptide having the amino acid sequence of SEQ ID NO: 2, according to any one of items 133 to 140. (Item 142) The composition according to item 141, wherein the mutant Cas12i2 polypeptide exhibits enzymatic activity equal to or greater than that of the parent polypeptide. (Item 143) The composition described in item 142 exhibits enzyme activity equivalent to or greater than that described above in a temperature range of approximately 20°C to approximately 90°C. (Item 144) The mutant binary complex exhibits increased stability and / or protein-RNA interactions, as described in any one of items 133 to 143. (Item 145) The mutant binary complex exhibits increased stability and / or protein-DNA interaction, as described in any one of items 133 to 144. (Item 146) The composition according to any one of items 133 to 145, wherein the mutant Cas12i2 polypeptide exhibits increased binary complex formation, RNA guide binding activity and / or RNA guide binding specificity. (Item 147) A composition comprising multiple mutant Cas12i2 polypeptides and two or more distinct RNA guides, wherein the distinct RNA guides individually form multiple mutant binary complexes with separate mutant Cas12i2 polypeptides, and the multiple mutant binary complexes exhibit increased on-target binding of target nucleic acids to two or more target gene loci compared to multiple parent binary complexes. (Item 148) A composition comprising multiple mutant Cas12i2 polypeptides and two or more distinct RNA guides, wherein the distinct RNA guides individually form multiple mutant binary complexes with separate mutant Cas12i2 polypeptides, and the multiple mutant binary complexes exhibit reduced off-target binding to two or more non-target gene loci of the target nucleic acid compared to multiple parent binary complexes. (Item 149) A composition comprising multiple mutant Cas12i2 polypeptides and two or more distinct RNA guides, wherein the distinct RNA guides individually form multiple mutant binary complexes with separate mutant Cas12i2 polypeptides, and the multiple mutant binary complexes exhibit increased on-target activity at two or more target gene loci of a target nucleic acid compared to multiple parent binary complexes. (Item 150) A composition comprising multiple mutant Cas12i2 polypeptides and two or more distinct RNA guides, wherein the distinct RNA guides individually form multiple mutant binary complexes with separate mutant Cas12i2 polypeptides, and the multiple mutant binary complexes exhibit reduced off-target activity at two or more non-target gene loci of a target nucleic acid compared to multiple parent binary complexes. (Item 151) The composition according to any one of items 147 to 150, wherein the plurality of mutant binary complexes exhibit increased ternary complex formation and / or increased stability at the target gene locus of the target nucleic acid over a certain temperature range, for example, 20°C to 65°C. (Item 152) The composition according to any one of items 147 to 151, wherein the plurality of mutant binary complexes exhibit increased ternary complex formation and / or increased stability at the target gene locus of the target nucleic acid over a range of incubation times. (Item 153) The composition according to any one of items 147 to 152, wherein the plurality of mutant binary complexes exhibit increased ternary complex formation and / or increased stability at the target gene locus of the target nucleic acid in a buffer having a pH in the range of about 7.3 to about 8.6. (Item 154) The plurality of mutant binary complexes are T m The value is T of the parent binary complex. m The composition according to any one of items 147 to 153, which exhibits increased ternary complex formation and / or increased stability at the target gene locus of the target nucleic acid when the temperature is at least 8°C higher than the value. (Item 155) The composition according to any one of items 147 to 154, wherein the plurality of parent binary complexes comprises a parent polypeptide having the amino acid sequence of SEQ ID NO: 2. (Item 156) The mutant Cas12i2 polypeptide exhibits enzymatic activity equal to or greater than that of the parent polypeptide, as described in any one of items 147 to 155. (Item 157) The composition described in item 156 exhibits enzyme activity equivalent to or greater than that described above in a temperature range of approximately 20°C to approximately 90°C. (Item 158) The aforementioned variant Cas12i2 polypeptide exhibits increased stability and / or protein-RNA interactions, as described in any one of items 147 to 157. (Item 159) The composition according to any one of items 147 to 158, wherein the plurality of mutant binary complexes exhibit increased stability and / or protein-DNA interactions. (Item 160) The aforementioned variant Cas12i2 polypeptide exhibits increased binary complex formation, RNA guide binding activity, and / or RNA guide binding specificity, as described in any one of items 147 to 159. (Item 161) A method for complexing a mutant binary complex described in any one of items 147 to 160 with a target nucleic acid, such as DNA, described in any one of items 147 to 160. (Item 162) A method for complexing multiple mutant binary complexes described in any one of items 147 to 161 with a target nucleic acid, such as DNA, described in any one of items 147 to 161. (Item 163) The mutant Cas12i2 polypeptide comprises at least one of the D581, G624, F626, D835, L836, P868, S879, D911, I926, V1020, V1030, E1035, and S1046 substitutions in the amino acid sequence of SEQ ID NO: 2, as described in any one of items 23-54, 56-93, 95-131, or 133-160. (Item 164) The mutant Cas12i2 polypeptide comprises at least one of the following substitutions in the amino acid sequence of SEQ ID NO: D581G, D581R, G624R, F626G, F626R, D835G, D835R, L836G, L836R, P868G, P868R, P868T, S879G, S879R, D911G, D911R, I926G, I926R, V1020G, V1020R, V1030G, V1030R, E1035G, E1035R, S1046G, and S1046R, as described in any one of items 23-54, 56-93, 95-131, 133-160, or 163. (Item 165) The mutant Cas12i2 polypeptide comprises at least one of the D581R, G624R, F626R, P868T, D911R, I926R, V1030G, E1035R, and S1046G substitutions in the amino acid sequence of SEQ ID NO: 2, as described in any one of items 23-54, 56-93, 95-131, 133-160, 163, or 164. (Item 166) The mutant Cas12i2 polypeptide comprises at least one substitution listed in Table 1, as described in any one of items 23-54, 56-93, 95-131, 133-160, or 163-165. (Item 167) The mutant Cas12i2 polypeptide comprises one amino acid sequence from any one of SEQ ID NOs. 3 to 146 or one amino acid sequence from any one of SEQ ID NOs. 495 to 512, as described in any one of the compositions described in items 23 to 54, 56 to 93, 95 to 131, 133 to 160, or 163 to 166. (Item 168) The mutant Cas12i2 polypeptide comprises at least one of an epitope peptide, a nuclear localization signal, and a nuclear export signal, as described in any one of items 23-54, 56-93, 95-131, 133-160, or 163-167. (Item 169) The RNA guide is a composition according to any one of items 23-54, 56-93, 95-131, 133-160, or 163-168, comprising a DNA targeting sequence. (Item 170) The DNA targeting sequence is an RNA guide, as described in item 169. (Item 171) The composition according to item 169 or 170, wherein the DNA targeting sequence is 13 to 30 nucleotides. (Item 172) The RNA guide comprises a direct repeat sequence linked to a DNA targeting sequence, according to any one of items 23-54, 56-93, 95-131, 133-160, or 163-171. (Item 173) A composition according to any one of items 23-54, 56-93, 95-131, 133-160, or 163-172, further comprising a target nucleic acid. (Item 174) The target nucleic acid is the composition described in item 173, which is present in cells. (Item 175) The composition according to any one of items 23-54, 56-93, 95-131, 133-160, or 163-174, which encodes the mutant Cas12i2 polypeptide and RNA guide in a vector, for example, an expression vector. (Item 176) Cells comprising a composition described in any one of items 23-54, 56-93, 95-131, 133-160, or 163-175. (Item 177) A method for expressing the vector described in item 175. (Item 178) A method for preparing any one of the compositions described in items 23-54, 56-93, 95-131, 133-160, or 163-175. (Item 179) A method for delivering a composition as described in any one of items 23-54, 56-93, 95-131, 133-160, or 163-175. (Item 180) A kit or system comprising a composition or one or more components thereof as described in any one of items 23-54, 56-93, 95-131, 133-160, or 163-175. (Item 181) The RNA guide is a composition according to any one of items 23-54, 56-93, 95-131, 133-160, or 163-175, comprising or consisting of 43 nucleotides or about 43 nucleotides. (Item 182) The RNA guide is a tracr-less RNA guide, as described in any one of items 23-54, 56-93, 95-131, 133-160, 163-175, or 181. (Item 183) The composition according to any one of items 23-54, 56-93, 95-131, 133-160, 163-175, 181, or 182, wherein the mutant Cas12i2 polypeptide further exhibits enzymatic activity approximately 40 times higher than that of the parent polypeptide. (Item 184) The composition according to any one of items 23-54, 56-93, 95-131, 133-160, 163-175, or 181-183, wherein the mutant Cas12i2 polypeptide exhibits increased on-target specificity compared to the parent polypeptide. (Item 185) The composition according to any one of items 23-54, 56-93, 95-131, 133-160, 163-175, or 181-184, wherein the mutant Cas12i2 polypeptide exhibits reduced off-target specificity compared to the parent polypeptide. (Item 186) The mutant Cas12i2 polypeptide selectively induces a deletion adjacent to the 5'-NTTN-3' sequence (wherein N is any nucleotide), as described in any one of items 23-54, 56-93, 95-131, 133-160, 163-175, or 181-185. (Item 187) The deletion is located downstream of the 5'-NTTN-3' sequence, as described in item 186. (Item 188) The parent polypeptide is a composition according to item 186 or 187, which does not induce the deletion. (Item 189) The composition according to any one of items 186 to 188, wherein the length of the deletion is greater than the length of the Cas9 polypeptide-induced deletion. (Item 190) The deletion is in the genes of a cell, as described in any one of items 186 to 189. (Item 191) The composition according to any one of items 186 to 190, wherein the deletion is at most about 40 nucleotides long. (Item 192) The composition according to any one of items 186 to 191, wherein the deletion is approximately 4 to 40 nucleotides in length. (Item 193) The composition according to any one of items 186 to 192, wherein the deletion is approximately 4 to 25 nucleotides in length. (Item 194) The composition according to any one of items 186 to 193, wherein the deletion is approximately 10 to 25 nucleotides in length. (Item 195) The composition according to any one of items 186 to 194, wherein the deletion is approximately 10 to 15 nucleotides in length. (Item 196) The composition according to any one of items 186 to 195, wherein the deletion begins within approximately 5 to 15 nucleotides of the 5'-NTTN-3' sequence. (Item 197) The deletion is located within approximately 5 to 10 nucleotides of the 5'-NTTN-3' sequence, as described in any one of items 186 to 196. (Item 198) The deletion is located within approximately 10 to 15 nucleotides of the 5'-NTTN-3' sequence, as described in any one of items 186 to 197. (Item 199) The composition according to any one of items 186 to 198, wherein the deletion begins within approximately 5 to 15 nucleotides downstream of the 5'-NTTN-3' sequence. (Item 200) The composition according to any one of items 186 to 199, wherein the deletion begins within approximately 5 to 10 nucleotides downstream of the 5'-NTTN-3' sequence. (Item 201) The composition according to any one of items 186 to 200, wherein the deletion begins within approximately 10 to 15 nucleotides downstream of the 5'-NTTN-3' sequence. (Item 202) The composition according to any one of items 186 to 201, wherein the deletion ends within approximately 20 to 30 nucleotides of the 5'-NTTN-3' sequence. (Item 203) The composition according to any one of items 186 to 202, wherein the deletion ends within approximately 20 to 25 nucleotides of the 5'-NTTN-3' sequence. (Item 204) The composition according to any one of items 186 to 203, wherein the deletion ends within approximately 25 to 30 nucleotides of the 5'-NTTN-3' sequence. (Item 205) The composition according to any one of items 186 to 204, wherein the deletion ends within about 20 to about 30 nucleotides downstream of the 5'-NTTN-3' sequence. (Item 206) The composition according to any one of items 186 to 205, wherein the deletion ends within approximately 20 to 25 nucleotides downstream of the 5'-NTTN-3' sequence. (Item 207) The composition according to any one of items 186 to 206, wherein the deletion ends within approximately 25 to 30 nucleotides downstream of the 5'-NTTN-3' sequence. (Item 208) The composition according to any one of items 186 to 207, wherein the deletion begins within approximately 5 to 15 nucleotides downstream of the 5'-NTTN-3' sequence and ends within approximately 20 to 30 nucleotides downstream of the 5'-NTTN-3' sequence. (Item 209) The composition according to any one of items 186 to 208, wherein the deletion begins within approximately 5 to 15 nucleotides downstream of the 5'-NTTN-3' sequence and ends within approximately 20 to 25 nucleotides downstream of the 5'-NTTN-3' sequence. (Item 210) The composition according to any one of items 186 to 209, wherein the deletion begins within approximately 5 to 15 nucleotides downstream of the 5'-NTTN-3' sequence and ends within approximately 25 to 30 nucleotides downstream of the 5'-NTTN-3' sequence. (Item 211) The composition according to any one of items 186 to 210, wherein the deletion begins within approximately 5 to 10 nucleotides downstream of the 5'-NTTN-3' sequence and ends within approximately 20 to 30 nucleotides downstream of the 5'-NTTN-3' sequence. (Item 212) The composition according to any one of items 186 to 211, wherein the deletion begins within approximately 5 to 10 nucleotides downstream of the 5'-NTTN-3' sequence and ends within approximately 20 to 25 nucleotides downstream of the 5'-NTTN-3' sequence. (Item 213) The composition according to any one of items 186 to 212, wherein the deletion begins within approximately 5 to 10 nucleotides downstream of the 5'-NTTN-3' sequence and ends within approximately 25 to 30 nucleotides downstream of the 5'-NTTN-3' sequence. (Item 214) The composition according to any one of items 186 to 213, wherein the deletion begins within approximately 10 to 15 nucleotides downstream of the 5'-NTTN-3' sequence and ends within approximately 20 to 30 nucleotides downstream of the 5'-NTTN-3' sequence. (Item 215) The composition according to any one of items 186 to 214, wherein the deletion begins within approximately 10 to 15 nucleotides downstream of the 5'-NTTN-3' sequence and ends within approximately 20 to 25 nucleotides downstream of the 5'-NTTN-3' sequence. (Item 216) The composition according to any one of items 186 to 215, wherein the deletion begins within about 10 to about 15 nucleotides downstream of the 5'-NTTN-3' sequence and ends within about 25 to about 30 nucleotides downstream of the 5'-NTTN-3' sequence. (Item 217) The composition according to any one of items 186 to 216, wherein the 5'-NTTN-3' sequence is 5'-NTTY-3', 5'-NTTC-3', 5'-NTTT-3', 5'-NTTA-3', 5'-NTTB-3', 5'-NTTG-3', 5'-CTTY-3', 5'-DTTR'3', 5'-CTTR-3', 5'-DTTT-3', 5'-ATTN-3', or 5'-GTTN-3' (wherein Y is C or T, B is any nucleotide other than A, D is any nucleotide other than C, and R is A or G). (Item 218) The composition according to any one of items 186 to 217, wherein the 5'-NTTN-3' sequence is 5'-CTTT-3', 5'-CTTC-3', 5'-GTTT-3', 5'-GTTC-3', 5'-TTTC-3', 5'-GTTA-3', or 5'-GTTG-3'. (Item 219) The deletion is located in an exon of the gene, for example, B2M, TRAC, or PDCD1, as described in any one of items 186 to 218. (Item 220) The composition according to any one of items 186 to 219, wherein the deletion overlaps with the mutation in the gene. (Item 221) The composition according to any one of items 186 to 220, wherein the deletion overlaps with the insertion of the gene. (Item 222) The deletion removes the repeat extension of the gene, as described in any one of items 186 to 221. (Item 223) The composition according to any one of items 186 to 222, wherein the deletion disrupts one or both alleles of the gene. (Item 224) The deletion is induced in eukaryotic or prokaryotic cells, as described in any one of items 186 to 223. (Item 225) The deletion is induced in animal cells, plant cells or fungal cells, or the cells are derived from animal cells, plant cells or fungal cells, according to any one of items 186 to 224. (Item 226) The deletion is induced in or derived from mammalian cells, as described in any one of items 186 to 225. (Item 227) The deletion is induced in or derived from human cells, as described in any one of items 186 to 226. (Item 228) The aforementioned deletion is induced in primary cells, as described in any one of items 186 to 227. (Item 229) The aforementioned deletion is induced in a cell line, as described in any one of items 186 to 228. (Item 230) The deletion is induced in T cells, as described in any one of items 186 to 229. (Item 231) The deletion is induced in stem cells (e.g., totipotent / totipotent stem cells, pluripotent stem cells, compound pluripotent stem cells, oligopotent stem cells, or unipotent stem cells), differentiated cells, or terminally differentiated cells, according to any one of items 186 to 230. (Item 232) Two or more deletions (e.g., multiplexed targeted deletions) are induced in the compositions described in any one of items 186 to 231. (Item 233) A method for achieving a deletion in a cell, comprising contacting the DNA of the cell with a mutant Cas12i2 polypeptide or complex described in any one of items 1-16, 22-54, 56-93, 95-131, 133-160, 163-175 or 181-232. (Item 234) A composition or formulation comprising a mutant Cas12i2 polypeptide described in any one of items 1-16, 22-54, 56-93, 95-131, 133-160, 163-175, or 181-232, an RNA guide, and cells. (Item 235) A method for preparing any one of the compositions described in items 1 to 234. (Item 236) A method for complexing a mutant Cas12i2 polypeptide described in any one of items 1 to 235 with an RNA guide described in any one of items 1 to 235, for example, an RNA guide. (Item 237) A method for complexing a mutant binary complex described in any one of items 1 to 236 with a target nucleic acid. (Item 238) A method for delivering a composition described in any one of items 1 to 237. (Item 239) A composition comprising a mutant Cas12i2 polypeptide, wherein the mutant Cas12i2 polypeptide includes substitutions that increase the interaction between the mutant Cas12i2 polypeptide and the nucleic acid compared to the parent polypeptide. (Item 240) The aforementioned variant Cas12i2 polypeptide comprises the sequence shown in Sequence ID No. 4, as described in item 239. (Item 241) The composition according to item 239, wherein the variant Cas12i2 polypeptide comprises the sequence shown in any one of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 495, and SEQ ID NO: 496. (Item 242) The composition according to item 239, wherein the interaction is an electrostatic interaction. (Item 243) The composition according to item 239, wherein the interaction is a nonspecific interaction. (Item 244) The composition according to item 239, wherein the interaction is aromatic, hydrophobic, van der Waals, and / or cation-π interaction. (Item 245) The composition according to item 239, wherein the substitution is located at or adjacent to the nucleic acid interface. (Item 246) The composition according to any one of items 239 to 245, wherein the nucleic acid is an RNA guide comprising a direct repeat sequence and a spacer sequence. (Item 247) The composition according to item 246, wherein the direct repeat sequence comprises a nucleotide sequence having at least 95% sequence identity with any one of sequence numbers 492 to 494. (Item 248) The composition according to item 246, wherein the direct repeat sequence comprises a nucleotide sequence shown in any one of sequence numbers 492 to 494. (Item 249) The composition according to any one of items 246 to 248, wherein the substitution increases the interaction between the mutant Cas12i2 polypeptide and the direct repeat sequence. (Item 250) The composition according to any one of items 239 to 249, wherein the substitution increases binary complex formation compared to the parent polypeptide. (Item 251) The binary complex comprising the mutant Cas12i2 polypeptide exhibits increased stability compared to the parent binary complex, according to any one of items 239 to 250. (Item 252) The composition according to any one of items 239 to 251, wherein the substitution is an arginine, lysine, glutamine, asparagine, histidine, tyrosine, or serine substitution. (Item 253) The substitution is located at the RNA binding interface, and the composition is as described in any one of items 239 to 252. (Item 254) The composition according to any one of items 239 to 253, wherein the substitution is a substitution in the Wedge domain or the Rec2 domain. (Item 255) The substitution is one of the substitutions listed in Table 4, and the composition is as described in any one of items 239 to 254. (Item 256) The composition according to any one of items 239 to 254, wherein the mutant Cas12i2 polypeptide comprises a sequence having at least 95% identity with the sequence shown in any one of sequence numbers 3 to 146 or any one of sequence numbers 495 to 512. (Item 257) The composition according to any one of items 239 to 254, wherein the mutant Cas12i2 polypeptide has at least 95% identity with the sequence shown in any one of sequence numbers 3 to 146 or any one of sequence numbers 495 to 512, and further comprises a sequence having the substitutions listed in Table 4. (Item 258) The composition according to any one of items 239 to 254, wherein the mutant Cas12i2 polypeptide comprises the sequence shown in any one of sequence numbers 3 to 146 or any one of sequence numbers 495 to 512. (Item 259) The composition according to any one of items 239 to 254, wherein the mutant Cas12i2 polypeptide comprises the sequence shown in any one of sequence numbers 3 to 146 or any one of sequence numbers 495 to 512, and further comprises the substitutions listed in Table 4. (Item 260) The composition according to any one of items 246 or 249-254, wherein the direct repeat sequence is shown in SEQ ID NO: 492, and the variant Cas12i2 polypeptide comprises the sequence shown in SEQ ID NO: 4. (Item 261) The composition according to any one of items 246 or 249-254, wherein the direct repeat sequence is shown in SEQ ID NO: 492, and the variant Cas12i2 polypeptide comprises the sequence shown in any one of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 495, and SEQ ID NO: 496. (Item 262) The nucleic acid is a target nucleic acid, and the composition is as described in any one of items 239 to 245. (Item 263) The composition according to any one of items 239-245 or 262, wherein the nucleic acid is double-stranded DNA. (Item 264) The composition according to item 263, wherein the substitution increases the interaction between the mutant Cas12i2 polypeptide and the double-stranded DNA. (Item 265) The composition according to item 263 or 264, wherein the double-stranded DNA comprises a PAM sequence. (Item 266) The composition according to any one of items 239-245 or 262-265, wherein the substitution increases ternary complex formation compared to the parent polypeptide. (Item 267) The ternary complex containing the mutant Cas12i2 polypeptide exhibits increased stability compared to the parent ternary complex, according to any one of items 239-245 or 262-266. (Item 268) The composition according to any one of items 239-245 or 262-267, wherein the substitution is an arginine, lysine, glutamine, asparagine, histidine, or serine substitution. (Item 269) The substitution is located at the double-stranded DNA binding interface, as described in any one of items 239-245 or 262-268. (Item 270) The composition according to any one of items 239-245 or 262-269, wherein the substitution is in the Rec1 domain, the PI domain, or the Wedge domain. (Item 271) The substitution is one of the substitutions listed in Table 5, and the composition is as described in any one of items 239-245 or 262-270. (Item 272) The composition according to any one of items 239-245 or 262-270, wherein the mutant Cas12i2 polypeptide comprises a sequence having at least 95% identity with the sequence shown in any one of sequence numbers 3-146 or any one of sequence numbers 495-512. (Item 273) The composition according to any one of items 239-245 or 262-270, wherein the mutant Cas12i2 polypeptide has at least 95% identity with the sequence shown in any one of sequence numbers 3-146 or any one of sequence numbers 495-512, and further comprises a sequence having the substitutions listed in Table 5. (Item 274) The composition according to any one of items 239-245 or 262-270, wherein the mutant Cas12i2 polypeptide comprises the sequence shown in any one of sequence numbers 3-146 or any one of sequence numbers 495-512. (Item 275) The composition according to any one of items 239-245 or 262-270, wherein the mutant Cas12i2 polypeptide comprises the sequence shown in any one of sequence numbers 3-146 or any one of sequence numbers 495-512, and further comprises the substitutions listed in Table 5. (Item 276) The composition according to any one of items 239 to 245, wherein the nucleic acid is single-stranded DNA. (Item 277) The single-stranded DNA comprises the non-target strand, as described in item 276. (Item 278) The single-stranded DNA is the composition according to item 276, comprising the target strand. (Item 279) The composition according to any one of items 239-245 or 276-278, wherein the substitution increases ternary complex formation compared to the parent polypeptide. (Item 280) The ternary complex containing the mutant Cas12i2 polypeptide exhibits increased stability compared to the parent ternary complex, according to any one of items 239-245 or 276-279. (Item 281) The composition according to any one of items 239-245 or 276-280, wherein the substitution is an arginine, lysine, glutamine, asparagine, histidine, or alanine substitution. (Item 282) The substitution is located at the single-stranded DNA binding interface in the composition according to any one of items 239-245 or 276-281. (Item 283) The composition according to any one of items 239-245 or 276-282, wherein the substitution is in the PI domain, Rec1 domain, Wedge domain, RuvC domain, Rec2 domain, or Nuc domain. (Item 284) The substitution is one of the substitutions listed in Table 6, and the composition is as described in any one of items 239-245 or 276-283. (Item 285) The composition according to any one of items 239-245 or 276-283, wherein the mutant Cas12i2 polypeptide comprises a sequence having at least 95% identity with the sequence shown in any one of sequence numbers 3-146 or any one of sequence numbers 495-512. (Item 286) The composition according to any one of items 239-245 or 276-283, wherein the mutant Cas12i2 polypeptide has at least 95% identity with the sequence shown in any one of sequence numbers 3-146 or any one of sequence numbers 495-512, and further comprises a sequence having the substitutions listed in Table 6. (Item 287) The composition according to any one of items 239-245 or 276-283, wherein the mutant Cas12i2 polypeptide comprises the sequence shown in any one of sequence numbers 3-146 or any one of sequence numbers 495-512. (Item 288) The composition according to any one of items 239-245 or 276-283, wherein the mutant Cas12i2 polypeptide comprises the sequence shown in any one of sequence numbers 3-146 or any one of sequence numbers 495-512, and further comprises the substitutions listed in Table 6. (Item 289) The composition according to any one of items 239 to 245, wherein the substitution increases the interaction between the mutant Cas12i2 polypeptide and the DNA / RNA hybrid molecule. (Item 290) The composition according to item 289, wherein the DNA / RNA hybrid molecule is a heteroduplex comprising an RNA guide spacer sequence and a target strand. (Item 291) The substitution stabilizes the heterodouble hemisphere, as described in any one of items 239-245, 289, or 290. (Item 292) The composition according to any one of items 239-245 or 289-291, wherein the substitution increases ternary complex formation compared to the parent polypeptide. (Item 293) The ternary complex containing the mutant Cas12i2 polypeptide exhibits increased stability compared to the parent ternary complex, according to any one of items 239-245 or 289-292. (Item 294) The composition according to any one of items 239-245 or 289-293, wherein the substitution is an arginine, lysine, glutamine, asparagine, histidine, or serine substitution. (Item 295) The composition according to any one of items 239-245 or 289-294, wherein the substitution is in the Rec1 domain, PI domain, Rec2 domain, or RuvC2 motif. (Item 296) The substitution is one of the substitutions listed in Table 7, and the composition is as described in any one of items 239-245 or 289-295. (Item 297) The composition according to any one of items 239-245 or 289-295, wherein the mutant Cas12i2 polypeptide comprises a sequence having at least 95% identity with the sequence shown in any one of sequence numbers 3-146 or any one of sequence numbers 495-512. (Item 298) The composition according to any one of items 239-245 or 289-295, wherein the mutant Cas12i2 polypeptide has at least 95% identity with the sequence shown in any one of sequence numbers 3-146 or any one of sequence numbers 495-512, and further comprises a sequence having the substitutions listed in Table 7. (Item 299) The composition according to any one of items 239-245 or 289-295, wherein the mutant Cas12i2 polypeptide comprises the sequence shown in any one of sequence numbers 3-146 or any one of sequence numbers 495-512. (Item 300) The composition according to any one of items 239-245 or 289-295, wherein the mutant Cas12i2 polypeptide comprises the sequence shown in any one of sequence numbers 3-146 or any one of sequence numbers 495-512, and further comprises the substitutions listed in Table 7. (Item 301) The composition according to any one of items 239 to 245, wherein the substitution increases the interaction between the mutant Cas12i2 polypeptide and the bases of a heteroduplex comprising (a) a double-stranded DNA double helix and / or (b) an RNA guide spacer sequence and a target strand. (Item 302) The double-stranded DNA duplex is the composition described in item 301, comprising a PAM sequence. (Item 303) The composition according to any one of items 239-245 or 301 or 302, wherein the interaction is aromatic, hydrophobic, van der Waals, and / or cation-π interaction. (Item 304) The substitution stabilizes the R loop, as described in any one of items 239-245 or 301-303. (Item 305) The composition according to any one of items 239-245 or 301-304, wherein the substitution increases ternary complex formation compared to the parent polypeptide. (Item 306) The ternary complex containing the mutant Cas12i2 polypeptide exhibits increased stability compared to the parent ternary complex, according to any one of items 239-245 or 301-305. (Item 307) The composition according to any one of items 239-245 or 301-306, wherein the substitution is an arginine, lysine, tryptophan, phenylalanine, tyrosine, methionine, histidine, glutamine, threonine, or valine substitution. (Item 308) The composition according to any one of items 239-245 or 301-307, wherein the substitution is in the Wedge domain, the Rec1 domain, or the RuvC domain. (Item 309) The substitution is one of the substitutions listed in Table 8, and the composition is as described in any one of items 239-245 or 301-308. (Item 310) The composition according to any one of items 239-245 or 301-308, wherein the mutant Cas12i2 polypeptide comprises a sequence having at least 95% identity with the sequence shown in any one of sequence numbers 3-146 or any one of sequence numbers 495-512. (Item 311) The composition according to any one of items 239-245 or 301-308, wherein the mutant Cas12i2 polypeptide has at least 95% identity with the sequence shown in any one of sequence numbers 3-146 or any one of sequence numbers 495-512, and further comprises a sequence having the substitutions listed in Table 8. (Item 312) The composition according to any one of items 239-245 or 301-308, wherein the mutant Cas12i2 polypeptide comprises the sequence shown in any one of sequence numbers 3-146 or any one of sequence numbers 495-512. (Item 313) The composition according to any one of items 239-245 or 301-308, wherein the mutant Cas12i2 polypeptide comprises the sequence shown in any one of sequence numbers 3-146 or any one of sequence numbers 495-512, and further comprises the substitutions listed in Table 8. (Item 314) The parent polypeptide comprises the amino acid sequence of SEQ ID NO: 2, according to any one of items 239 to 313. (Item 315) The composition according to any one of items 239 to 314, wherein the mutant Cas12i2 polypeptide exhibits increased enzymatic activity compared to the parent polypeptide. (Item 316) A composition according to any one of items 239 to 315, further comprising an RNA guide containing a direct repeat sequence and a spacer sequence. (Item 317) The composition according to item 316, wherein the direct repeat sequence comprises a nucleotide sequence having at least 95% sequence identity with any one of sequence numbers 492 to 494. (Item 318) The composition according to item 316, wherein the direct repeat sequence comprises a nucleotide sequence shown in any one of sequence numbers 492 to 494. (Item 319) The composition according to item 316, wherein the direct repeat sequence is shown in SEQ ID NO: 492, and the variant Cas12i2 polypeptide comprises the sequence shown in SEQ ID NO: 4. (Item 320) The composition according to item 316, wherein the direct repeat sequence is shown in SEQ ID NO: 492, and the variant Cas12i2 polypeptide comprises the sequence shown in any one of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 495, and SEQ ID NO: 496. (Item 321) Cells containing the composition described in any one of items 239 to 320. (Item 322) The cell according to item 321, wherein the composition does not substantially affect the viability of the cell. (Item 323) A composition comprising a mutant Cas12i2 polypeptide, wherein the mutant Cas12i2 polypeptide includes substitutions that increase the mobility of the mutant Cas12i2 polypeptide in DNA binding compared to the parent polypeptide. (Item 324) The aforementioned variant Cas12i2 polypeptide comprises the sequence shown in Sequence ID No. 4, as described in item 323. (Item 325) The composition according to item 323, wherein the Cas12i2 polypeptide comprises the sequence shown in any one of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 495, and SEQ ID NO: 496. (Item 326) The composition according to any one of items 323 to 325, wherein the substitution increases the binding of the mutant Cas12i2 polypeptide to DNA. (Item 327) The composition according to any one of items 323 to 326, wherein the substitution increases the binding of the mutant Cas12i2 polypeptide to double-stranded DNA. (Item 328) The composition according to any one of items 323 to 327, wherein the substitution increases the binding of the mutant Cas12i2 polypeptide to single-stranded DNA. (Item 329) The composition according to any one of items 323 to 328, wherein the substitution increases ternary complex formation compared to the parent polypeptide. (Item 330) The ternary complex containing the mutant Cas12i2 polypeptide exhibits increased stability compared to the parent ternary complex, according to any one of items 323 to 329. (Item 331) The composition according to any one of items 323 to 330, wherein the substitution is a substitution from a bulk amino acid to an amino acid having a smaller side chain. (Item 332) The composition according to any one of items 323 to 331, wherein the substitution is an alanine, valine, glycine, or serine substitution. (Item 333) The substitution is in the helix II domain of the variant Cas12i2 polypeptide, as described in any one of items 323 to 332. (Item 334) The substitution is one of the substitutions listed in Table 9, and the composition is one of the items 323 to 333. (Item 335) The composition according to any one of items 323 to 332, wherein the mutant Cas12i2 polypeptide comprises a sequence having at least 95% identity with the sequence shown in any one of sequence numbers 3 to 146 or any one of sequence numbers 495 to 512. (Item 336) The composition according to any one of items 323 to 332, wherein the mutant Cas12i2 polypeptide has at least 95% identity with the sequence shown in any one of sequence numbers 3 to 146 or any one of sequence numbers 495 to 512, and further comprises a sequence having the substitutions listed in Table 9. (Item 337) The composition according to any one of items 323 to 332, wherein the mutant Cas12i2 polypeptide comprises the sequence shown in any one of sequence numbers 3 to 146 or any one of sequence numbers 495 to 512. (Item 338) The composition according to any one of items 323 to 332, wherein the mutant Cas12i2 polypeptide comprises the sequence shown in any one of sequence numbers 3 to 146 or any one of sequence numbers 495 to 512, and further comprises the substitutions listed in Table 9. (Item 339) A composition comprising a mutant Cas12i2 polypeptide, wherein the mutant Cas12i2 polypeptide includes substitutions that stabilize the interdomain interface formed during ternary complex formation, compared to the parent polypeptide. (Item 340) The aforementioned variant Cas12i2 polypeptide comprises the sequence shown in Sequence ID No. 4, as described in item 339. (Item 341) The composition according to item 339, wherein the variant Cas12i2 polypeptide comprises the sequence shown in any one of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 495, and SEQ ID NO: 496. (Item 342) The composition according to any one of items 339 to 341, wherein the interdomain interface is formed when single-stranded DNA comes into contact with the active site of the mutant Cas12i2 polypeptide. (Item 343) The composition according to any one of items 339 to 342, wherein the interdomain interface is a helix II domain-Nuc domain interface. (Item 344) The composition according to any one of items 339 to 343, wherein the substitution increases ternary complex formation compared to the parent polypeptide. (Item 345) The ternary complex containing the mutant Cas12i2 polypeptide exhibits increased stability compared to the parent ternary complex, according to any one of items 339 to 344. (Item 346) The composition according to any one of items 339 to 345, wherein the substitution is an aspartic acid, glutamic acid, arginine, or lysine substitution. (Item 347) The substitution is one of the substitutions listed in Table 10, and the composition is as described in any one of items 339 to 346. (Item 348) The composition according to any one of items 339 to 346, wherein the mutant Cas12i2 polypeptide comprises a sequence having at least 95% identity with the sequence shown in any one of sequence numbers 3 to 146 or any one of sequence numbers 495 to 512. (Item 349) The composition according to any one of items 339 to 346, wherein the mutant Cas12i2 polypeptide has at least 95% identity with the sequence shown in any one of sequence numbers 3 to 146 or any one of sequence numbers 495 to 512, and further comprises a sequence having the substitutions listed in Table 10. (Item 350) The composition according to any one of items 339 to 346, wherein the mutant Cas12i2 polypeptide comprises the sequence shown in any one of sequence numbers 3 to 146 or any one of sequence numbers 495 to 512. (Item 351) The composition according to any one of items 339 to 346, wherein the mutant Cas12i2 polypeptide comprises the sequence shown in any one of sequence numbers 3 to 146 or any one of sequence numbers 495 to 512, and further comprises the substitutions listed in Table 10. (Item 352) The parent polypeptide comprises the amino acid sequence of SEQ ID NO: 2, according to any one of items 323 to 351. (Item 353) The composition according to any one of items 323 to 352, wherein the mutant Cas12i2 polypeptide exhibits increased enzymatic activity compared to the parent polypeptide. (Item 354) A composition according to any one of items 323 to 353, further comprising an RNA guide containing a direct repeat sequence and a spacer sequence. (Item 355) The composition according to item 354, wherein the direct repeat sequence comprises a nucleotide sequence having at least 95% sequence identity with any one of sequence numbers 492 to 494. (Item 356) The composition according to item 354, wherein the direct repeat sequence comprises a nucleotide sequence shown in any one of sequence numbers 492 to 494. (Item 357) The composition according to item 354, wherein the direct repeat sequence is shown in SEQ ID NO: 492, and the variant Cas12i2 polypeptide comprises the sequence shown in SEQ ID NO: 4. (Item 358) The composition according to item 354, wherein the direct repeat sequence is shown in SEQ ID NO: 492, and the variant Cas12i2 polypeptide comprises the sequence shown in any one of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 495, and SEQ ID NO: 496. (Item 359) Cells containing the composition described in any one of items 323 to 358. (Item 360) The cell according to item 359, wherein the composition does not substantially affect the viability of the cell. (Item 361) A composition comprising a mutant Cas12i2 polypeptide, wherein the mutant Cas12i2 polypeptide comprises a substitution that increases the on-target specificity of the mutant Cas12i2 polypeptide compared to the parent polypeptide. (Item 362) The aforementioned variant Cas12i2 polypeptide comprises the sequence shown in Sequence ID No. 4, as described in item 361. (Item 363) The composition according to item 361, wherein the variant Cas12i2 polypeptide comprises the sequence shown in any one of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 495, and SEQ ID NO: 496. (Item 364) The composition according to any one of items 361 to 363, wherein the substitution increases on-target DNA binding. (Item 365) The composition according to any one of items 361 to 364, wherein the substitution reduces off-target DNA binding. (Item 366) The composition according to any one of items 361 to 365, wherein the substitution increases on-target ternary complex formation compared to the parent polypeptide. (Item 367) The on-target ternary complex containing the mutant Cas12i2 polypeptide exhibits increased stability compared to the parent ternary complex, according to any one of items 361 to 366. (Item 368) The composition according to any one of items 361 to 367, wherein the substitution is an amino acid substitution that contacts the spacer sequence of the RNA guide. (Item 369) The composition according to any one of items 361 to 368, wherein the substitution is a substitution from a bulk amino acid to an amino acid having a smaller side chain. (Item 370) The composition according to any one of items 361 to 369, wherein the substitution is an alanine, serine, valine, glutamine, or asparagine substitution. (Item 371) The composition according to any one of items 361 to 370, wherein the substitution is in the Wedge domain, Rec1 domain, Rec2 domain, or RuvC domain. (Item 372) The composition according to any one of items 361 to 371, wherein the substitution is a substitution in the helix II domain. (Item 373) The aforementioned substitution is one of the substitutions listed in Table 11, and the composition is as described in any one of items 361 to 372. (Item 374) The composition according to any one of items 361 to 372, wherein the mutant Cas12i2 polypeptide comprises a sequence having at least 95% identity with the sequence shown in any one of sequence numbers 3 to 146 or any one of sequence numbers 495 to 512. (Item 375) The composition according to any one of items 361 to 372, wherein the mutant Cas12i2 polypeptide has at least 95% identity with the sequence shown in any one of sequence numbers 3 to 146 or any one of sequence numbers 495 to 512, and further comprises a sequence having the substitutions listed in Table 11. (Item 376) The composition according to any one of items 361 to 372, wherein the mutant Cas12i2 polypeptide comprises the sequence shown in any one of sequence numbers 3 to 146 or any one of sequence numbers 495 to 512. (Item 377) The composition according to any one of items 361 to 372, wherein the mutant Cas12i2 polypeptide comprises the sequence shown in any one of sequence numbers 3 to 146 or any one of sequence numbers 495 to 512, and further comprises the substitutions listed in Table 11. (Item 378) The composition according to any one of items 361 to 367, wherein the substitution reduces the catalytic reaction rate (Kcat) of the mutant Cas12i2 polypeptide. (Item 379) The composition according to any one of items 361-367 or 378, wherein the substitution is an alanine, serine, threonine, valine, leucine, methionine, asparagine, or isoleucine substitution. (Item 380) The composition according to any one of items 361-367 or 378 or 379, wherein the substitution is in the Wedge domain, Rec1 domain, Rec2 domain, or RuvC domain. (Item 381) The composition according to any one of items 361-367 or 378-380, wherein the substitution is a substitution in the RuvC domain. (Item 382) The substitution is one of the substitutions listed in Table 12, and the composition is as described in any one of items 361-367 or 378-381. (Item 383) The composition according to any one of items 361-367 or 378-381, wherein the mutant Cas12i2 polypeptide comprises a sequence having at least 95% identity with the sequence shown in any one of sequence numbers 3-146 or any one of sequence numbers 495-512. (Item 384) The composition according to any one of items 361-367 or 378-381, wherein the mutant Cas12i2 polypeptide has at least 95% identity with the sequence shown in any one of sequence numbers 3-146 or any one of sequence numbers 495-512, and further comprises a sequence having the substitutions listed in Table 12. (Item 385) The composition according to any one of items 361-367 or 378-381, wherein the mutant Cas12i2 polypeptide comprises the sequence shown in any one of SEQ ID NOs. 3-146 or any one of SEQ ID NOs. 495-512. (Item 386) The composition according to any one of items 361-367 or 378-381, wherein the mutant Cas12i2 polypeptide comprises the sequence shown in any one of sequence numbers 3-146 or any one of sequence numbers 495-512, and further comprises the substitutions listed in Table 12. (Item 387) The composition according to any one of items 361 to 386, wherein the mutant Cas12i2 polypeptide exhibits increased on-target enzyme activity compared to the parent polypeptide. (Item 388) The composition according to any one of items 361 to 387, wherein the mutant Cas12i2 polypeptide exhibits reduced off-target enzyme activity compared to the parent polypeptide. (Item 389) The composition according to any one of items 361 to 388, wherein the mutant Cas12i2 polypeptide exhibits off-target editing that is 10% or less of on-target editing. (Item 390) The composition according to any one of items 361 to 388, wherein the mutant Cas12i2 polypeptide exhibits off-target editing that is 5% or less of on-target editing. (Item 391) A composition according to any one of items 361 to 390, further comprising an RNA guide containing a direct repeat sequence and a spacer sequence. (Item 392) The composition according to item 391, wherein the direct repeat sequence comprises a nucleotide sequence having at least 95% sequence identity with any one of sequence numbers 492 to 494. (Item 393) The composition according to item 391, wherein the direct repeat sequence comprises a nucleotide sequence shown in any one of sequence numbers 492 to 494. (Item 394) The composition according to item 391, wherein the direct repeat sequence is shown in SEQ ID NO: 492, and the variant Cas12i2 polypeptide comprises the sequence shown in SEQ ID NO: 4. (Item 395) The composition according to item 391, wherein the direct repeat sequence is shown in SEQ ID NO: 492, and the variant Cas12i2 polypeptide comprises the sequence shown in any one of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 495, and SEQ ID NO: 496. (Item 396) The composition according to any one of items 365 to 395, wherein the mutant Cas12i2 polypeptide exhibits a higher ratio of on-target binding to off-target binding compared to the Cas9 polypeptide. (Item 397) The composition according to any one of items 365 to 396, wherein the mutant Cas12i2 polypeptide exhibits a higher ratio of on-target activity to off-target activity compared to the Cas9 polypeptide. (Item 398) The composition according to any one of items 365 to 397, wherein the variant Cas12i2 polypeptide exhibits a higher ratio of on-target editing to off-target editing compared to the Cas9 polypeptide. (Item 399) The composition according to any one of items 365 to 395, wherein the mutant Cas12i2 polypeptide exhibits fewer off-target bindings compared to the Cas9 polypeptide. (Item 400) The composition according to any one of items 365 to 396, wherein the mutant Cas12i2 polypeptide exhibits less off-target activity compared to the Cas9 polypeptide. (Item 401) The composition according to any one of items 365 to 397, wherein the mutant Cas12i2 polypeptide exhibits less off-target editing compared to the Cas9 polypeptide. (Item 402) The composition according to any one of items 365 to 401, wherein the off-target binding, off-target activity, and / or off-target editing by the mutant Cas12i2 polypeptide is at least 10% less than that by the Cas9 polypeptide. (Item 403) The composition according to any one of items 365 to 402, wherein the off-target binding, off-target activity, and / or off-target editing by the mutant Cas12i2 polypeptide is at least 20% less than that by the Cas9 polypeptide. (Item 404) The composition according to any one of items 365 to 403, wherein the off-target binding, off-target activity, and / or off-target editing by the mutant Cas12i2 polypeptide is at least 30% less than that by the Cas9 polypeptide. (Item 405) The composition according to any one of items 365 to 404, wherein the off-target binding, off-target activity, and / or off-target editing by the mutant Cas12i2 polypeptide is at least 40% less than that by the Cas9 polypeptide. (Item 406) The composition according to any one of items 365 to 405, wherein the off-target binding, off-target activity, and / or off-target editing by the mutant Cas12i2 polypeptide is at least 50% less than that by the Cas9 polypeptide. (Item 407) Cells containing the composition described in any one of items 365 to 406. (Item 408) The cell according to item 407, wherein the composition does not substantially affect the viability of the cell. [Brief explanation of the drawing]

[0436] [Figure 1] This is a schematic diagram showing the wild-type Cas12i2 (SEQ ID NO: 2) and Cas12i2 mutants shown in SEQ ID NOs: 3-5, 495, or 496. The RuvC motif and mutated residues are illustrated. [Figure 2] This paper demonstrates the introduction of a single mutation using overlap PCR and the creation of a linear DNA template expressing the mutant Cas12i2 sequence. [Figure 3A]a) A DNA EMSA gel showing the ability of RNPs prepared with wild-type Cas12i2 (SEQ ID NO: 2), the mutant Cas12i2 of SEQ ID NO: 3, or the mutant Cas12i2 of SEQ ID NO: 4, and b) crRNA1 (SEQ ID NO: 147) to bind to the AAVS1 dsDNA target (SEQ ID NO: 150). Bounded and unbound dsDNA bands are indicated. [Figure 3B] a) A DNA EMSA gel showing the ability of RNPs prepared with wild-type Cas12i2 (SEQ ID NO: 2), the mutant Cas12i2 of SEQ ID NO: 3, or the mutant Cas12i2 of SEQ ID NO: 4, and b) crRNA2 (SEQ ID NO: 148) to bind to the VEGFA dsDNA target (SEQ ID NO: 151). Bounded and unbound dsDNA bands are indicated. [Figure 3C] a) A DNA EMSA gel showing the ability of RNPs prepared with wild-type Cas12i2 (SEQ ID NO: 2), the mutant Cas12i2 of SEQ ID NO: 3, or the mutant Cas12i2 of SEQ ID NO: 4, and b) crRNA3 (SEQ ID NO: 149) to bind to the EMX1 dsDNA target (SEQ ID NO: 152). Bounded and unbound dsDNA bands are indicated. [Figure 3D] a) A DNA EMSA gel showing the ability of RNP prepared with wild-type Cas12i2 (SEQ ID NO: 2), the mutant Cas12i2 of SEQ ID NO: 3, or the mutant Cas12i2 of SEQ ID NO: 4, and b) crRNA1 (SEQ ID NO: 147) to bind to the EMX1 dsDNA target (SEQ ID NO: 152). An unbound dsDNA band is indicated. [Figure 3E] This gel shows the movement of samples containing a) crRNA1 (sequence number 147) and DNA target 1 (sequence number 150), b) crRNA2 (sequence number 148) and DNA target 2 (sequence number 151), c) crRNA3 (sequence number 149) and DNA target 3 (sequence number 152), and d) crRNA1 (sequence number 147) and DNA target 3 (sequence number 152). [Figure 4] This is a schematic diagram of the fluorescence depletion assay described in Example 10 for measuring the activity of the Cas12i2 mutant. [Figure 5A-5T]This graph shows the GFP depletion ratio (non-target / target) for wild-type Cas12i2 (solid line), the mutant Cas12i2 of sequence number 3 (dotted line), and the mutant Cas12i2 of sequence number 4 (dashed line). The depletion ratio was calculated from measurements taken over 12 hours. Twenty GFP targets are shown: top1 (Figure 5A), top2 (Figure 5B), top3 (Figure 5C), top4 (Figure 5D), top5 (Figure 5E), top6 (Figure 5F), top7 (Figure 5G), top8 (Figure 5H), top9 (Figure 5I), top10 (Figure 5J), bot1 (Figure 5K), bot2 (Figure 5L), bot3 (Figure 5M), bot4 (Figure 5N), bot5 (Figure 5O), bot6 (Figure 5P), bot7 (Figure 5Q), bot8 (Figure 5R), bot9 (Figure 5S), and bot10 (Figure 5T). [Figure 6A] The indel measurements for 15 targeted gene regions in wild-type Cas12i2, the mutant Cas12i2 of SEQ ID NO: 3, or the mutant Cas12i2 of SEQ ID NO: 4, as evaluated by next-generation sequencing, are shown. [Figure 6B] Using the data in Figure 6A, we show the proportion of 15 gene regions that were found to be targetable by wild-type Cas12i2, the sequence number 3 mutant Cas12i2, or the sequence number 4 mutant Cas12i2. [Figure 7A] We will compare the indel rates on the AAVS1 target using wild-type Cas12i2 (SEQ ID NO: 2) or Cas12i2 mutants SEQ ID NOs: 3-5, 495, or 496, 46, 47, 50-63, 65-68, 79, 84, 87-90, 95-97, 99, 101, 103, 104, 112, 114-118, 123, 130, and 131. [Figure 7B] We will compare the indel rates on the EMX1 target using wild-type Cas12i2 or Cas12i2 mutants of sequence numbers 3-5, 495 or 496, 46, 47, 50-63, 65-68, 76, 79, 84, 86-90, 95-97, 99, 101, 103, 104, 112 and 114-124. [Figure 7C]We will compare the indel rates on the VEGFA1 target using wild-type Cas12i2 or Cas12i2 mutants of sequence numbers 3-5, 495 or 496, 46, 47, 50-63, 65-68, 76, 79, 84, 86-90, 95-97, 99, 101, 103, 104, 112 and 114-124. [Figure 8] This shows the indel activity in primary T cells of a mutant binary complex containing the variant Cas12i2 (SEQ ID NO: 4) and several individual crRNAs that target B2M at various concentrations. Error bars represent the standard deviation of the mean of 4 technical replicas from a representative donor. [Figure 9] This shows the reduction in B2M expression in primary T cells by a mutant binary complex containing the variant Cas12i2 (SEQ ID NO: 4) and several individual crRNAs that target B2M at various concentrations. Error bars represent the standard deviation of the mean of 4 technical replicas from a representative donor. [Figure 10] This graph shows the viability of primary T cells (by DAPI staining) 7 days after introduction of a variant Cas12i2 RNP that targets various concentrations of B2M. Error bars represent the standard deviation of the mean of 4 technical replicas from a representative donor. [Figure 11A] Indel activity in primary T cells by a mutant binary complex containing the variant Cas12i2 (SEQ ID NO: 4) and several individual crRNAs targeting TRAC at various concentrations. Error bars represent the standard deviation of the mean of 4 technical replicas from a representative donor. [Figure 11B] This graph shows the viability of primary T cells (by DAPI staining) 7 days after introduction of a variant Cas12i2 RNP that targets various concentrations of TRAC. Error bars represent the standard deviation of the mean of 4 technical replicas from a representative donor. [Figure 12A]This shows the indel activity in primary T cells of a mutant binary complex containing the variant Cas12i2 (sequence number 4) and several individual crRNAs that target PDCD1 at various concentrations. Error bars represent the standard deviation of the mean of four technical replicas from one representative donor. [Figure 12B] This graph shows the viability of primary T cells (by DAPI staining) 7 days after introduction of the Cas12i2 RNP mutant, which targets various concentrations of PDCD1. Error bars represent the standard deviation of the mean of 4 technical replicas from a representative donor. [Figure 13] This is a schematic diagram illustrating how to calculate the Levenshtein distance (edit distance) using exemplary on-target and non-target sequences. The on-target sequence and four non-target sequences are shown, each with an edit distance of 1, 2, 3, or 4. Each substitution, insertion, or deletion residue is indicated in bold. The PAM sequences for the target sequence and each non-target sequence are indicated to the left of the dotted line. [Figure 14A] This document shows the on-target indel ratios at eight AAVS1 loci, eight EMX1 loci, and eight VEGFA loci using the Cas12i2 mutant of Sequence ID No. 3, and further shows the off-target indel ratios at loci with edit distances of 1, 2, 3, or 4 compared to the target loci. [Figure 14B] This document shows the on-target indel ratios at eight AAVS1 loci, eight EMX1 loci, and eight VEGFA loci using the Cas12i2 mutant of Sequence ID No. 4, and further shows the off-target indel ratios at loci with edit distances of 1, 2, 3, or 4 compared to the target loci. [Figure 14C] This study shows the on-target indel rates at eight AAVS1 loci, eight EMX1 loci, and eight VEGFA loci using the Cas12i2 mutant of Sequence ID No. 5, and further shows the off-target indel rates at loci with edit distances of 1, 2, 3, or 4 compared to the target loci. [Figure 15]This is a schematic diagram showing the steps of the tagmentation-based tag integration site sequencing (TTISS) method used to analyze the specificity and activity of the Cas12i2 mutant in Example 16. [Figure 16A] On-target and off-target reads for the Cas12i2 and SpCas9 variants of sequence number 4 in the target AAVS1_T5 are shown. [Figure 16B] On-target and off-target reads for the variants Cas12i2 and SpCas9 of sequence number 4 in the target EMX1_T2 are shown. [Figure 16C] On-target and off-target reads for the variants Cas12i2 and SpCas9 of sequence number 4 in the target EMX1_T4 are shown. [Figure 16D] On-target and off-target reads for the sequence number 4 variants Cas12i2 and SpCas9 in the target VEGFA_T6 are shown. [Figure 17A] This graph shows indels induced by several modified Cas12i2 mutants in EMX1_T6 and VEGFA_T7. [Figure 17B] This graph shows indels induced by Cas12i2 mutants of sequence numbers 3-5 and 495 in EMX1_T6 and VEGFA_T7. [Figure 18] This graph shows indels induced by Cas12i2 mutants of sequence numbers 4, 495, and 496 in AAVS1_T6, AAVS1_T7, EMX1_T2, EMX1_T6, and VEGFA_T5. [Figure 19] This is a schematic diagram showing the domain structure of the Cas12i2 polypeptide. [Figure 20A] The location of the D581R substitution in the Cas12i2 structure is illustrated. The D581R substitution can form electrostatic contact with the PAM sequence DNA. D581R can interact with the non-target strand. [Figure 20B]The positions of the I926R and V1030G substitutions near the active site in the Cas12i2 structure are illustrated. I926R can interact with single-stranded DNA near the active site and stabilizes the interface with Rec1. V1030G is located in the C-terminal region of the structure. [Figure 20C] The stabilization of unpaired, non-stacking bases by direct repeats is illustrated. Direct repeat sequences interact with Cas12i2 over a wide range. In addition, some RNA guide bases pair with each other. However, some bases remain exposed (indicated by asterisks), and these exposed bases, for example, do not pair with other bases and therefore have limited interaction with Cas12i2. Substitutions that increase the interaction between Cas12i2 and the RNA guide, particularly in the exposed regions, can stabilize the binary complex and increase enzyme activity. [Figure 21A] This is a schematic diagram illustrating the formation of a ternary complex. The double-stranded DNA downstream of the PAM thaws, and the RNA guide spacer binds to the target strand, forming a heteroduplex. The PAM sequence remains as intact double-stranded DNA, so the terminal PAM double-stranded DNA base pairs are partially exposed to the environment and proteins. The terminal base pairs of the heteroduplex are also exposed. The exposed bases are designated as the "heteroduplex end" and the "dsDNA double-strand end." [Figure 21B] This shows the exposed bases at the DNA fusion and heterodouble-strand annealing sites in the Cas12i2 structure. This specification describes substitutions that stabilize the ends of double-stranded and heterodouble-stranded DNA. Such substitutions can lower the energy barrier to the unwinding of the initial target. [Figure 22A] This shows the conformational changes required for the transition from a binary to a ternary complex. Most of the changes at the Cα position between the binary and ternary structures are observed in the helix II domain. The vectors indicate a Cα displacement of ≥3.0 Å and approximately 25° rotation of helix II around the axis. This specification describes substitutions that enhance ternary complex formation. [Figure 22B]Figure 22B shows regions within the helix II domain where substitution can increase the mobility of the helix II domain. Further, Figure 22B shows regions within the Cas12i2 structure where substitution can stabilize the interface between the helix II domain and the Nuc domain. [Modes for carrying out the invention]

[0437] This disclosure relates to novel variants of the effector of SEQ ID NO: 2, as well as methods for producing and using them. This disclosure further relates to complexes containing variants of the effector of SEQ ID NO: 2, as well as methods for producing and using them. In some embodiments, this specification describes compositions containing complexes having one or more properties. In some embodiments, methods for delivering compositions containing complexes are described.

[0438] composition In some embodiments, the present invention as described herein includes a composition comprising a complex (e.g., a binary complex). In some embodiments, the present invention as described herein includes a composition comprising a complex comprising a Cas12i2 polypeptide and a targeting moiety. In some embodiments, the composition of the present invention comprises a mutant Cas12i2 polypeptide and an RNA guide, the mutant Cas12i2 polypeptide exhibiting increased complex formation with the RNA guide compared to the parent polypeptide.

[0439] In some embodiments, the composition of the present invention comprises a complex comprising a variant Cas12i2 polypeptide and an RNA guide, wherein the Cas12i2 variant polypeptide and RNA guide have higher binding affinity compared to the parent polypeptide and RNA guide.

[0440] In some embodiments, the composition of the present invention comprises a complex comprising a variant Cas12i2 polypeptide and an RNA guide, wherein the Cas12i2 variant polypeptide and RNA guide have a stronger protein-RNA interaction compared to the parent polypeptide and RNA guide. In some embodiments, the protein-RNA interaction is an ionic interaction.

[0441] In some embodiments, the composition of the present invention comprises a complex comprising a mutant Cas12i2 polypeptide and an RNA guide, which is more stable than the complex formed by the parent polypeptide and the RNA guide.

[0442] In some embodiments, the present invention as described herein comprises a composition comprising a complex (e.g., a ternary complex). In some embodiments, the composition of the present invention comprises a mutant Cas12i2 polypeptide, an RNA guide, and a target nucleic acid, wherein the mutant Cas12i2 polypeptide exhibits increased complex formation (e.g., ternary complex formation) with the RNA guide and the target nucleic acid compared to the parent polypeptide.

[0443] In some embodiments, the composition of the present invention comprises a binary complex comprising a mutant Cas12i2 polypeptide and an RNA guide, the composition further comprising a target nucleic acid. In some embodiments, the binary complex exhibits increased ternary complex formation with the target nucleic acid compared to the parent binary complex.

[0444] In some embodiments, the composition of the present invention comprises a variant Cas12i2 polypeptide, an RNA guide, and a target nucleic acid, wherein the Cas12i2 variant polypeptide and RNA guide have a higher binding affinity to the target nucleic acid compared to the parent polypeptide and RNA guide.

[0445] In some embodiments, the composition of the present invention comprises a binary complex comprising a variant Cas12i2 polypeptide and an RNA guide, the composition further comprising a target nucleic acid. In some embodiments, the binary complex has a higher binding affinity to the target nucleic acid compared to the parent binary complex.

[0446] In some embodiments, the composition of the present invention comprises a ternary complex comprising a variant Cas12i2 polypeptide, an RNA guide, and a target nucleic acid, wherein the ternary complex is more stable than the complex formed by the parent polypeptide, the RNA guide, and the target nucleic acid.

[0447] In some embodiments, the composition of the present invention comprises a binary complex comprising a mutant Cas12i2 polypeptide and an RNA guide, the composition further comprising a target nucleic acid. In some embodiments, the binary complex forms a ternary complex with the target nucleic acid that is more stable than the ternary complex formed by the parent binary complex and the target nucleic acid.

[0448] In some embodiments, the present invention as described herein includes a composition comprising a complex (e.g., a ternary complex). In some embodiments, the composition of the present invention comprises a mutant Cas12i2 polypeptide, an RNA guide, and a target nucleic acid, wherein the mutant Cas12i2 polypeptide and RNA guide form a mutant binary complex having a higher binding affinity to the target nucleic acid compared to the parent binary complex.

[0449] In some embodiments, the composition of the present invention comprises a binary complex comprising a mutant Cas12i2 polypeptide and an RNA guide, the composition further comprising a target nucleic acid. In some embodiments, the binary complex has a higher target binding affinity to the target locus of the target nucleic acid compared to the parent binary complex.

[0450] In some embodiments, the composition of the present invention comprises a plurality of mutant Cas12i2 polypeptides and two or more distinct RNA guides. In some embodiments, the distinct RNA guides individually form mutant binary complexes with separate mutant Cas12i2 polypeptides, and these mutant binary complexes exhibit higher on-target binding to two or more target loci of the target nucleic acid compared to the parent binary complex.

[0451] In some embodiments, the composition of the present invention comprises a plurality of mutant Cas12i2 polypeptides and two or more distinct RNA guides. In some embodiments, the distinct RNA guides individually form mutant binary complexes with separate mutant Cas12i2 polypeptides, and these mutant binary complexes exhibit higher on-target ternary complex formation with two or more target loci of the target nucleic acid compared to the parental binary complex.

[0452] In some embodiments, the composition of the present invention comprises a plurality of mutant Cas12i2 polypeptides and two or more distinct RNA guides. In some embodiments, the distinct RNA guides individually form mutant binary complexes with separate mutant Cas12i2 polypeptides, and these mutant binary complexes form more stable ternary complexes with two or more target loci of the target nucleic acid compared to the parental binary complex.

[0453] In some embodiments, the present invention as described herein includes a composition comprising a complex. In some embodiments, the present invention includes a binary complex comprising a variant Cas12i2 polypeptide and an RNA guide.

[0454] In some embodiments, the present invention comprises a ternary complex comprising a mutant Cas12i2 polypeptide, an RNA guide, and a target gene locus of a target nucleic acid. In some embodiments, the composition of the present invention comprises a plurality of mutant Cas12i2 polypeptides and two or more distinct RNA guides, each of which individually forms a mutant binary complex with a separate mutant Cas12i2 polypeptide.

[0455] In some embodiments, the composition of the present invention comprises a mutant Cas12i2 polypeptide and an RNA guide, the mutant Cas12i2 polypeptide and RNA guide forming a mutant binary complex having a higher on-target binding affinity to the target locus of the target nucleic acid compared to the parent binary complex. In some embodiments, the composition of the present invention comprises multiple mutant binary complexes having higher on-target binding to two or more target loci of the target nucleic acid compared to multiple parent binary complexes.

[0456] In some embodiments, the composition of the present invention comprises a binary complex comprising a mutant Cas12i2 polypeptide and an RNA guide, the binary complex having a lower binding affinity to non-target loci of the target nucleic acid compared to the parent binary complex. In some embodiments, the composition of the present invention comprises multiple mutant binary complexes exhibiting lower off-target binding to two or more non-target loci of the target nucleic acid compared to multiple parent binary complexes.

[0457] In some embodiments, the composition of the present invention comprises a binary complex comprising a mutant Cas12i2 polypeptide and an RNA guide, the binary complex having higher activity at the on-target locus of the target nucleic acid compared to the parent binary complex. In some embodiments, the composition of the present invention comprises multiple mutant binary complexes having higher on-target activity at two or more target loci of the target nucleic acid compared to multiple parent binary complexes.

[0458] In some embodiments, the composition of the present invention comprises a binary complex comprising a mutant Cas12i2 polypeptide and an RNA guide, the binary complex having lower activity at non-target loci of the target nucleic acid compared to the parent binary complex. In some embodiments, the composition of the present invention comprises multiple mutant binary complexes having lower off-target activity at two or more non-target loci of the target nucleic acid compared to multiple parent binary complexes.

[0459] Cas12i2 polypeptide variant In some embodiments, the compositions of the present invention include the variant Cas12i2 polypeptide described herein.

[0460] In some embodiments, the polypeptide of the present invention is a variant of a parent polypeptide, where the parent is encoded by a polynucleotide comprising a nucleotide sequence such as SEQ ID NO: 1 or an amino acid sequence such as SEQ ID NO: 2.

[0461] [Table 1]

[0462] [Table 2]

[0463] [Table 3]

[0464] In one embodiment of the present invention, the mutant polypeptide sequence comprises one or more mutations.

[0465] The nucleic acid sequences encoding the parent polypeptide described herein may be substantially identical to a reference nucleic acid sequence, e.g., Sequence ID No. 1. In some embodiments, the variant Cas12i2 polypeptide is encoded by a nucleic acid containing a sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 99.5% sequence identity with a reference nucleic acid sequence, e.g., the nucleic acid sequence encoding the parent polypeptide, e.g., Sequence ID No. 1. The percentage identity between two such nucleic acids may be determined by manually examining two optimally aligned nucleic acid sequences or by using a software program or algorithm with standard parameters (e.g., BLAST, ALIGN, CLUSTAL). One indicator that two nucleic acid sequences are substantially identical is that, under stringent conditions (e.g., within a moderate to high stringency range), the nucleic acid molecule hybridizes to the complementary sequence of the other.

[0466] In some embodiments, the mutant Cas12i2 polypeptide is encoded by a nucleic acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or higher sequence identity, but not 100%, with a reference nucleic acid sequence, e.g., the nucleic acid sequence encoding the parent polypeptide, e.g., SEQ ID NO: 1.

[0467] In some embodiments, the variant Cas12i2 polypeptide of the present invention comprises a polypeptide sequence having 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% but not 100% identity with SEQ ID NO: 2. In some embodiments, the variant Cas12i2 polypeptide of the present invention comprises a polypeptide sequence having 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher but not 100% identity with SEQ ID NO: 2.

[0468] In some embodiments, the present invention describes a variant Cas12i2 polypeptide having a specified degree of amino acid sequence identity with respect to one or more reference polypeptides, such as a parent polypeptide, for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99%, but not 100%, of the amino acid sequence identity of SEQ ID NO: 2. Homology or identity can be determined by amino acid sequence alignment using a program such as BLAST, ALIGN, or CLUSTAL, as described herein.

[0469] In some embodiments, the mutant Cas12i2 polypeptide comprises changes in one or more (e.g., several) amino acids of the parent polypeptide, where at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 ,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,10 7, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 15 6, 157, 158, 159, 160, 161, 162, 162, 164, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 193, 194, 195, 196, 197, 198, 199, 200 or more have changed.

[0470] In some embodiments, the mutant Cas12i2 polypeptide contains one or more amino acid substitutions listed in Table 2. In some embodiments, the mutant Cas12i2 polypeptide contains at least one of the D581, G624, F626, D835, L836, P868, S879, D911, I926, V1020, V1030, E1035, and S1046 substitutions. In some embodiments, the mutant Cas12i2 polypeptide contains at least one of the D581R, G624R, F626R, D835R, L836R, P868R, S879R, D911R, I926R, V1020R, V1030R, E1035R, and S1046R substitutions. In some embodiments, the mutant Cas12i2 polypeptide includes at least one of the D581G, F626G, D835G, L836G, P868G, S879G, D911G, I926G, V1020G, V1030G, E1035G, and S1046G substitutions. In some embodiments, the mutant Cas12i2 polypeptide includes at least one of the D581R, G624R, F626R, D835R, L836R, P868T, S879R, D911R, I926R, V1020G, V1030G, E1035R, and S1046G substitutions, as well as at least one additional substitution listed in Table 2. In some embodiments, the mutant Cas12i2 polypeptide includes one of the SEQ ID NOs. 3-146 and 495-512. In some embodiments, the mutant Cas12i2 polypeptide includes one of SEQ ID NOs. 3-146 and 495-512, as well as at least one additional substitution listed in Table 2. In some embodiments, the mutant Cas12i2 polypeptide includes the amino acid sequence of SEQ ID NO. 3. In some embodiments, the mutant Cas12i2 polypeptide includes the amino acid sequence of SEQ ID NO. 4. In some embodiments, the mutant Cas12i2 polypeptide includes the amino acid sequence of SEQ ID NO. 5. In some embodiments, the mutant Cas12i2 polypeptide includes the amino acid sequence of SEQ ID NO. 495. In some embodiments, the mutant Cas12i2 polypeptide includes the amino acid sequence of SEQ ID NO. 496.

[0471] [Table 4]

[0472] Table 5

[0473] Table 6

[0474] Table 7

[0475] Table 8

[0476] Table 9

[0477] Table 10

[0478] Table 11

[0479] Table 12

[0480] Table 13

[0481] Table 14

[0482] Table 15

[0483] Table 16

[0484] Table 17

[0485] Table 18

[0486] Table 19

[0487] Table 20

[0488] Table 21

[0489] Table 22

[0490] Table 23

[0491] Table 24

[0492] [Table 25]

[0493] [Table 26]

[0494] In some embodiments, the mutant Cas12i2 polypeptide contains one or more of the amino acid substitutions listed in Table 2. In some embodiments, the mutant Cas12i2 polypeptide contains the sequence shown in one of the sequence codes 3-5, 495, or 496 illustrated in Figure 1. In some embodiments, the mutant Cas12i2 polypeptide contains the sequence shown in one of the sequence codes 6-146.

[0495] In some embodiments, the compositions described herein comprise one or more individual (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or more) variant Cas12i2 polypeptides. In such embodiments, the individual variant polypeptides may independently comprise one or more of the amino acid substitutions listed in Table 2. In some embodiments, the individual variant Cas12i2 polypeptide comprises the sequence shown in any one of SEQ ID NOs: 3-5, 495, or 496 illustrated in Figure 1. In some embodiments, the individual variant Cas12i2 polypeptide comprises the sequence shown in any one of SEQ ID NOs: 6-146.

[0496] In some embodiments, the mutant Cas12i2 polypeptide includes the mutations or sets of mutations shown in Table 3, where the mutations are based on the sequence of SEQ ID NO: 2.

[0497] [Table 27]

[0498] [Table 28]

[0499] [Table 29]

[0500] [Table 30]

[0501] [Table 31]

[0502] In some embodiments, the mutant Cas12i2 polypeptide is the polypeptide shown in Table 3. The substitutions in Table 3 are based on the sequence of SEQ ID NO: 2. In some embodiments, the mutant Cas12i2 polypeptide contains one or more of the amino acid substitutions listed in Table 3.

[0503] In some embodiments, the variant Cas12i2 polypeptide of the present invention comprises a polypeptide sequence having 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any one of SEQ ID NOs: 3-146 and 495-512. In some embodiments, the variant Cas12i2 polypeptide of the present invention comprises a polypeptide sequence having 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any one of SEQ ID NOs: 3-146 and 495-512.

[0504] In some embodiments, one or more amino acids among amino acids 597-607 of SEQ ID NO: 2 are altered or mutated. In some embodiments, one or more amino acids among amino acids 597-604 of SEQ ID NO: 2 are altered or mutated. In some embodiments, one or more amino acids among amino acids 830-833 of SEQ ID NO: 2 are altered or mutated. In some embodiments, one or more amino acids among amino acids 829-835 of SEQ ID NO: 2 are altered or mutated. In some embodiments, one or more amino acids among amino acids 882-888 of SEQ ID NO: 2 are altered or mutated. In some embodiments, one or more amino acids among amino acids 883-889 of SEQ ID NO: 2 are altered or mutated. See Table 2.

[0505] In some embodiments, one or more amino acids among amino acids 600-607 of SEQ ID NO: 2 are altered or mutated. In some embodiments, one or more amino acids among amino acids 833-871 of SEQ ID NO: 2 are altered or mutated. In some embodiments, one or more amino acids among amino acids 877-885 of SEQ ID NO: 2 are altered or mutated. See Table 2.

[0506] In some embodiments, the mutant Cas12i2 polypeptide contains at least one RuvC domain. In some embodiments, the mutant Cas12i2 polypeptide contains at least one RuvC motif (e.g., one, two, or three RuvC motifs). The domains of the Cas12i2 polypeptide disclosed herein are illustrated in Figure 19. The Wedge domain contains residues 1-14 and 442-586 of the Cas12i2 polypeptide. The Rec1 domain contains residues 15-176 and 270-441 of the Cas12i2 polypeptide. Within the Rec1 domain, the Helix I domain contains residues 15-176 and 270-327, and the Helix II domain contains residues 328-441. The PI domain contains residues 177-269 of the Cas12i2 polypeptide. The Rec2 domain contains residues 638-828 of the Cas12i2 polypeptide. The Nuc domain contains residues 880-1017 of the Cas12i2 polypeptide. The RuvC motif contains residues 587-637 (RuvC1), 829-879 (RuvC2), and 1018-1054 (RuvC3) of the Cas12i2 polypeptide.

[0507] In some embodiments, the variant Cas12i2 polypeptide having the characteristics described herein includes an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any one of SEQ ID NOs: 3-146 and 495-512.

[0508] The modifications described herein may be changes to one or more amino acids, but modifications to the mutant Cas12i2 polypeptide may also be of structural or substantive nature, such as the fusion of polypeptides as amino-terminus and / or carboxyl-terminus extensions. For example, the mutant Cas12i2 polypeptide may contain additional peptides, e.g., one or more peptides. Examples of additional peptides include epitope peptides for labeling, such as polyhistidine tags (His tags), Myc, and FLAG. In some embodiments, the mutant Cas12i2 polypeptide described herein may be fused with a detectable moiety, such as a fluorescent protein (e.g., green fluorescent protein (GFP) or yellow fluorescent protein (YFP)).

[0509] In some embodiments, the mutant Cas12i2 polypeptide comprises at least one (e.g., two, three, four, five, six or more) nuclear localization signals (NLS). In some embodiments, the mutant Cas12i2 polypeptide comprises at least one (e.g., two, three, four, five, six or more) nuclear export signals (NES). In some embodiments, the mutant Cas12i2 polypeptide comprises at least one (e.g., two, three, four, five, six or more) NLS and at least one (e.g., two, three, four, five, six or more) NES.

[0510] In some embodiments, the variant Cas12i2 polypeptides described herein may be self-inactivating. See Epstein et al., "Engineering a Self-Inactivating CRISPR System for AAV Vectors", Mol.Ther., 24(2016):S50 (as incorporated by reference in whole).

[0511] In some embodiments, the nucleotide sequences encoding the variant Cas12i2 polypeptide described herein may be codon-optimized for use in specific host cells or organisms. For example, nucleic acids may be codon-optimized for any non-human eukaryote, including mice, rats, rabbits, dogs, livestock, or non-human primates. Codon usage tables are readily available, for example, in the "Codon Usage Database" available at www.kazusa.orjp / codon / , and these tables can be adapted in several ways. See Nakamura et al. Nucl. Acids Res. 28:292 (2000) (referred to herein by reference in whole). Computer algorithms for codon-optimizing specific sequences for expression in specific host cells are also available, such as Gene Forge (Aptagen; Jacobus, PA).

[0512] Function of mutant polypeptides As used herein, “biologically active portion” is a portion (e.g., “minimal” or “core” domain) that retains at least one function of the parent polypeptide (e.g., completely, partially, or minimally). In some embodiments, the mutant Cas12i2 polypeptide retains enzymatic activity that is at least as high as that of the parent polypeptide. Thus, in some embodiments, the mutant Cas12i2 polypeptide has higher enzymatic activity than the parent polypeptide.

[0513] Furthermore, the present invention also provides a variant Cas12i2 polypeptide that has enzymatic activity, for example, nuclease or endonuclease activity, and when aligned using any of the alignment methods described above, has an amino acid sequence that differs from the amino acid sequence of any one of the parent polypeptide and SEQ ID NO: 2 by only 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue. In some embodiments, the enzyme-active mutant Cas12i2 polypeptide, when aligned using any of the alignment methods described above, contains an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of either the parent polypeptide or SEQ ID NO: 2.

[0514] In some embodiments, the mutant Cas12i2 polypeptide comprises at least one change or mutation that enhances the enzyme activity, RNA guide complex (binary complex) formation, RNA guide binding activity, RNA guide affinity, RNA guide binding specificity, protein-RNA interaction, protein-DNA interaction, protein stability, ternary complex formation, on-target binding activity, on-target binding specificity, and / or stability in the ternary complex. In some embodiments, the mutant Cas12i2 polypeptide of the present invention has at least one of the enzyme activity, RNA guide complex (binary complex) formation, RNA guide binding activity, RNA guide affinity, RNA guide binding specificity, protein-RNA interaction, protein-DNA interaction, protein stability, ternary complex formation, on-target binding activity, on-target binding specificity, and / or stability in the ternary complex that is equal to or better than that of the parent polypeptide. In some embodiments, the mutant Cas12i2 polypeptide comprises at least one change or mutation that enhances enzyme activity, RNA guide complex (binary complex) formation, RNA guide binding activity, RNA guide affinity, RNA guide binding specificity, protein-RNA interaction, protein-DNA interaction, protein stability, ternary complex formation, on-target binding activity, on-target binding specificity, and / or stability in the ternary complex, and the mutant Cas12i2 polypeptide comprises an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 4.

[0515] In some embodiments, the mutant Cas12i2 polypeptide comprises enhanced enzyme activity, RNA guide complex (binary complex) formation, RNA guide binding activity, RNA guide affinity, RNA guide binding specificity, protein-RNA interaction, protein-DNA interaction, protein stability, ternary complex formation, on-target binding activity, on-target binding specificity, and / or stability in the ternary complex, and the mutant Cas12i2 polypeptide comprises one of the amino acid sequences of SEQ ID NOs: 3-5, 495, or 496.

[0516] In some embodiments, the mutant Cas12i2 polypeptide comprises enhanced enzyme activity, RNA guide complex (binary complex) formation, RNA guide binding activity, RNA guide affinity, RNA guide binding specificity, protein-RNA interaction, protein-DNA interaction, protein stability, ternary complex formation, on-target binding activity, on-target binding specificity, and / or stability in the ternary complex, and the mutant Cas12i2 polypeptide comprises one of the amino acid sequences of SEQ ID NOs: 3-146 and 495-512.

[0517] In some embodiments of the present invention, the variant Cas12i2 polypeptide includes substitutions that alter the polypeptide's ability to interact with (e.g., bind to or form complexes with) nucleic acids (e.g., RNA guides or DNA). In some embodiments of the present invention, the variant Cas12i2 polypeptide includes substitutions that alter the polypeptide's affinity for nucleic acids (e.g., RNA guides or DNA).

[0518] In some embodiments, the mutant Cas12i2 polypeptide containing the D581R substitution exhibits enhanced enzyme activity. In some embodiments, the mutant Cas12i2 polypeptide containing the D581R substitution interacts with NTS. In some embodiments, the mutant Cas12i2 polypeptide containing the D581R substitution interacts with the PAM sequence backbone. In some embodiments, the mutant Cas12i2 polypeptide containing the D581R substitution exhibits enhanced electrostatic interaction with DNA in the PAM sequence. In some embodiments, the mutant Cas12i2 polypeptide containing the D581R substitution reduces repulsive interactions with nucleic acids. In some embodiments, the mutant Cas12i2 polypeptide containing the D581R substitution enhances R-loop stability. See Figure 20A.

[0519] In some embodiments, the mutant Cas12i2 polypeptide containing the V1030G substitution exhibits enhanced enzyme activity. In some embodiments, the mutant Cas12i2 polypeptide containing the V1030G substitution interacts with NTS. In some embodiments, the mutant Cas12i2 polypeptide containing the V1030G substitution is located near the Cas12i2 active site. See Figure 20B.

[0520] In some embodiments, the Cas12i2 polypeptide mutant containing the I926R substitution exhibits enhanced enzyme activity. In some embodiments, the Cas12i2 polypeptide mutant containing the I926R substitution interacts with single-stranded DNA near the Cas12i2 active site. In some embodiments, the Cas12i2 polypeptide mutant containing the I926R substitution stabilizes single-stranded DNA. See Figure 20B.

[0521] In some embodiments, the Cas12i2 polypeptide variant containing the G624R substitution exhibits enhanced enzyme activity. In some embodiments, the Cas12i2 polypeptide variant containing the G624R substitution interacts with NTS. In some embodiments, the Cas12i2 polypeptide variant containing the G624R substitution enhances R-loop stability.

[0522] In some embodiments, the mutant Cas12i2 polypeptide containing the F626R substitution exhibits enhanced enzyme activity. In some embodiments, the mutant Cas12i2 polypeptide containing the F626R substitution interacts with NTS. In some embodiments, the mutant Cas12i2 polypeptide containing the F626R substitution enhances R-loop stability.

[0523] In some embodiments, the Cas12i2 mutant polypeptide of the present invention is suitable for temperatures ranging from about 20°C to about 90°C, for example, about 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 5 At any one of 8°C, 59°C, 60°C, 65°C, or above, the mutant Cas12i2 polypeptide has at least one of the following characteristics: enzyme activity, RNA guide complex (binary complex) formation, RNA guide binding activity, RNA guide affinity, RNA guide binding specificity, protein-RNA interaction, protein-DNA interaction, protein stability, ternary complex formation, on-target binding activity, on-target binding specificity, and / or stability in the ternary complex. In some embodiments, the mutant Cas12i2 polypeptide of the present invention has at least one of the following characteristics at temperatures of about 20°C to about 25°C or about 37°C: enzyme activity, RNA guide complex (binary complex) formation, RNA guide binding activity, RNA guide affinity, RNA guide binding specificity, protein-RNA interaction, protein-DNA interaction, protein stability, ternary complex formation, on-target binding activity, on-target binding specificity, and / or stability in the ternary complex.

[0524] In some embodiments, the mutant Cas12i2 polypeptide exhibits enhanced enzyme activity, RNA guide complex (binary complex) formation, RNA guide binding activity, RNA guide affinity, RNA guide binding specificity, protein-RNA interaction, protein-DNA interaction, protein stability, ternary complex formation, on-target binding activity, on-target binding specificity, and / or stability in the ternary complex compared to the parent polypeptide in a buffer having a pH in the range of about 7.3 to about 8.6 (e.g., any value within the range of 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, or any combination of these values)

[0525] In some embodiments, the mutant Cas12i2 polypeptide is the T of the mutant Cas12i2 polypeptide. m The value is T of the parent polypeptide. m When the temperature is at least 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, or 20°C higher than the value, it exhibits at least one of the following compared to the parent polypeptide: enzyme activity, RNA guide complex (binary complex) formation, RNA guide binding activity, RNA guide affinity, RNA guide binding specificity, protein-RNA interaction, protein-DNA interaction, protein stability, ternary complex formation, on-target binding activity, on-target binding specificity, and / or stability in the ternary complex. In one embodiment, the mutant Cas12i2 polypeptide exhibits the T of the mutant Cas12i2 polypeptide. m The value is T of the parent polypeptide. m When the temperature is at least 8°C higher than the specified value, the enzyme activity, RNA guide complex (binary complex) formation, RNA guide binding activity, RNA guide affinity, RNA guide binding specificity, protein-RNA interaction, protein-DNA interaction, protein stability, ternary complex formation, on-target binding activity, on-target binding specificity, and / or stability in the ternary complex are enhanced.

[0526] In some embodiments, the variant Cas12i2 polypeptide of the present invention exhibits an increase in at least one of the following compared to the parent polypeptide over one incubation period of at least about 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, or more, at about 37°C: enzyme activity, RNA guide complex (binary complex) formation, RNA guide binding activity, RNA guide affinity, RNA guide binding specificity, protein-RNA interaction, protein-DNA interaction, protein stability, ternary complex formation, on-target binding activity, on-target binding specificity, and / or stability in the ternary complex. In some embodiments, the variant Cas12i2 polypeptide of the present invention exhibits, over a certain range of incubation times, an increase in at least one of the following compared to the parent polypeptide: enzyme activity, RNA guide complex (binary complex) formation, RNA guide binding activity, RNA guide affinity, RNA guide binding specificity, protein-RNA interaction, protein-DNA interaction, protein stability, ternary complex formation, on-target binding activity, on-target binding specificity, and / or stability in the ternary complex.

[0527] Furthermore, the present invention also provides a variant Cas12i2 polypeptide that exhibits dissociation from the RNA guide (dissociation of the binary complex), dissociation from the target nucleic acid (dissociation of the ternary complex), off-target binding to a non-target nucleic acid and / or a decrease in activity at a non-target locus of the target nucleic acid, and when aligned using any of the aforementioned alignment methods, contains an amino acid sequence that differs from the amino acid sequence of either the parent polypeptide or SEQ ID NO: 2 by only 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue. In some embodiments, mutant Cas12i2 polypeptides exhibiting dissociation from the RNA guide (dissociation of the binary complex), dissociation from the target nucleic acid (dissociation of the ternary complex), off-target binding to non-target nucleic acids and / or reduced activity at non-target loci of the target nucleic acid, when aligned using any of the aforementioned alignment methods, contain an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the amino acid sequence of either the parent polypeptide or SEQ ID NO: 2.

[0528] In some embodiments, the mutant Cas12i2 polypeptide comprises at least one alteration or mutation that reduces the mutant Cas12i2 polypeptide's dissociation from the RNA guide (binary complex dissociation), dissociation from the target nucleic acid (ternary complex dissociation), off-target binding to a non-target nucleic acid and / or activity at a non-target locus of the target nucleic acid. In some embodiments, the mutant Cas12i2 polypeptide comprises at least one alteration or mutation that reduces the dissociation from the RNA guide (binary complex dissociation), dissociation from the target nucleic acid (ternary complex dissociation), off-target binding to a non-target nucleic acid and / or activity at a non-target locus of the target nucleic acid, and the mutant Cas12i2 polypeptide comprises an amino acid sequence having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with SEQ ID NO: 4.

[0529] In some embodiments, the mutant Cas12i2 polypeptide comprises at least one alteration or mutation compared to the parent polypeptide that reduces dissociation from the RNA guide (binary complex dissociation), dissociation from the target nucleic acid (ternary complex dissociation), off-target binding to non-target nucleic acids and / or activity at non-target loci of the target nucleic acid. In some embodiments, the mutant Cas12i2 polypeptide comprises, compared to the parent polypeptide, dissociation from the RNA guide (binary complex dissociation), dissociation from the target nucleic acid (ternary complex dissociation), off-target binding to non-target nucleic acids and / or reduced activity at non-target loci of the target nucleic acid, and the mutant Cas12i2 polypeptide comprises one of the amino acid sequences of SEQ ID NOs: 3-5, 495, or 496.

[0530] In some embodiments, the mutant Cas12i2 polypeptide exhibits dissociation from the RNA guide (binary complex dissociation), dissociation from the target nucleic acid (ternary complex dissociation), off-target binding to non-target nucleic acids and / or reduced activity at non-target loci of the target nucleic acid compared to the parent polypeptide, and the mutant Cas12i2 polypeptide contains one of the amino acid sequences of SEQ ID NOs: 3-146 and 495-512.

[0531] In some embodiments, the variant Cas12i2 polypeptide of the present invention exhibits at least one of the following at a level equal to or lower than that of the parent polypeptide: dissociation from the RNA guide (dissociation of a binary complex), dissociation from the target nucleic acid (dissociation of a ternary complex), off-target binding to a non-target nucleic acid, and / or activity at a non-target locus of the target nucleic acid. In some embodiments, the variant Cas12i2 polypeptide of the present invention exhibits equivalent or reduced dissociation from the RNA guide (binary complex dissociation), dissociation from the target nucleic acid (ternary complex dissociation), off-target binding to non-target nucleic acids and / or activity at non-target loci of the target nucleic acid in a temperature range of about 20°C to about 90°C, for example, about 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 65°C or above. In some embodiments, the variant Cas12i2 polypeptide of the present invention performs at least one of the following at temperatures of about 20°C to about 25°C or about 37°C: dissociation from the RNA guide (dissociation of a binary complex), dissociation from the target nucleic acid (dissociation of a ternary complex), off-target binding to a non-target nucleic acid and / or activity at a non-target locus of the target nucleic acid.

[0532] In some embodiments, the mutant Cas12i2 polypeptide exhibits reduced activity compared to the parent polypeptide in buffers having a pH in the range of approximately 7.3 to approximately 8.6 (e.g., any value within the range of 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6 or any combination thereof), including dissociation from the RNA guide (binary complex dissociation), dissociation from the target nucleic acid (ternary complex dissociation), off-target binding to non-target nucleic acids, and / or reduced activity at non-target loci of the target nucleic acid.

[0533] In some embodiments, the mutant Cas12i2 polypeptide is the T of the mutant Cas12i2 polypeptide. m The value is T of the parent polypeptide. m When the temperature is at least 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, or 20°C higher than the value, it exhibits at least one of the following compared to the parent polypeptide: dissociation from the RNA guide (dissociation of the binary complex), dissociation from the target nucleic acid (dissociation of the ternary complex), off-target binding to non-target nucleic acids and / or activity at non-target loci of the target nucleic acid. In one embodiment, the mutant Cas12i2 polypeptide exhibits the T m The value is T of the parent polypeptide. m When the temperature is at least 8°C higher than the specified value, it exhibits dissociation from the RNA guide (dissociation of the binary complex), dissociation from the target nucleic acid (dissociation of the ternary complex), off-target binding to non-target nucleic acids, and / or reduced activity at non-target loci of the target nucleic acid.

[0534] Increased RNA guide interactions In some embodiments, the mutant Cas12i2 polypeptide includes changes that increase the interaction and / or affinity between the mutant Cas12i2 polypeptide and the RNA guide compared to the parent polypeptide. See Figure 20C. In some embodiments, the changes that increase the interaction and / or affinity between the mutant Cas12i2 polypeptide and the RNA guide involve substituting one or more amino acids with arginine, lysine, glutamine, asparagine, histidine, serine, or tyrosine residues. In some embodiments, the mutant Cas12i2 polypeptide includes substitution of one or more amino acids at the RNA binding interface with arginine, lysine, glutamine, asparagine, histidine, serine, or tyrosine residues. In some embodiments, the mutant Cas12i2 polypeptide includes substitution of one or more amino acids 441-586 or 637-828 with any one of the arginine, lysine, glutamine, asparagine, histidine, serine, or tyrosine residues. In some embodiments, the variant Cas12i2 polypeptide includes changes from one or more amino acids in at least one domain (e.g., the Wedge domain or the Rec2 domain) to arginine, lysine, glutamine, asparagine, histidine, serine, or tyrosine residues.In some embodiments, one or more RNA-binding interface substitutions improve RNA guide binding or RNA guide binding affinity by approximately 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, and 55% compared to the parent polypeptide. 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89 Increase by %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or more, or any percentage in between.

[0535] In some embodiments, substitutions increase RNA guide complex (binary complex) formation compared to the parent polypeptide. Table 4 shows non-limiting examples of substitutions that can alter the ability of the mutant Cas12i2 polypeptide to interact with the direct repeat sequence of the RNA guide. In some embodiments, mutant Cas12i2 polypeptides containing one or more substitutions listed in Table 4 exhibit enhanced RNA guide complex (binary complex) formation compared to the parent polypeptide. In some embodiments, mutant Cas12i2 polypeptides containing one or more substitutions listed in Table 4 form a more stable binary complex with the RNA guide compared to the binary complex containing the parent polypeptide.

[0536] [Table 32]

[0537] In some embodiments, one of the variant Cas12i2 polypeptides from SEQ ID NOs: 3-146 and 495-512 further comprises one or more substitutions listed in Table 4. In some embodiments, the variant Cas12i2 polypeptide comprises one or more substitutions listed in Tables 2 and 4.

[0538] In some embodiments, mutant Cas12i2 polypeptides exhibiting enhanced RNA guide complex (binary complex) formation contain two or more substitutions, for example, L654K Q658A, L520K Q496N, or L520K Q496S. In some embodiments, one mutant Cas12i2 polypeptide from sequence numbers 3-146 and 495-512 further contains the L654K Q658A, L520K Q496N, or L520K Q496S substitution. In some embodiments, one mutant Cas12i2 polypeptide from sequence numbers 3-146 and 495-512 further contains the L654K Q658A, L520K Q496N, or L520K Q496S substitution and one or more substitutions listed in Table 4.

[0539] In some embodiments, one of the mutant Cas12i2 polypeptides from SEQ ID NOs. 3-146 and 495-512, containing one or more substitutions listed in Table 4, exhibits increased enzyme activity. In some embodiments, one of the mutant Cas12i2 polypeptides from SEQ ID NOs. 3-146 and 495-512, further containing the L654K Q658A, L520K Q496N, or L520K Q496S substitution and one or more substitutions listed in Table 4, exhibits increased enzyme activity. In some embodiments, the mutant Cas12i2 polypeptide exhibits lower enzyme activity compared to the parent polypeptide (e.g., about 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%). It exhibits an increase of 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or more, or any percentage in between.

[0540] Increased double-stranded DNA interactions In some embodiments, the mutant Cas12i2 polypeptide includes modifications that increase interaction with double-stranded DNA compared to the parent polypeptide. In some embodiments, the increased interaction with double-stranded DNA is an increase in electrostatic interaction. In some embodiments, the mutant Cas12i2 polypeptide includes modifications that increase the affinity between the mutant Cas12i2 polypeptide and double-stranded DNA compared to the parent polypeptide. In some embodiments, the modifications that increase the interaction and / or affinity between the mutant Cas12i2 polypeptide and double-stranded DNA increase the binding of the mutant Cas12i2 polypeptide to the PAM sequence.

[0541] In some embodiments, the changes that increase the interaction and / or affinity between the mutant Cas12i2 polypeptide and double-stranded DNA are the substitution of one or more amino acids with arginine, lysine, glutamine, asparagine, histidine, or serine residues. In some embodiments, the mutant Cas12i2 polypeptide includes substitution of one or more amino acids at the double-stranded DNA binding interface with arginine, lysine, glutamine, asparagine, histidine, or serine residues. In some embodiments, the mutant Cas12i2 polypeptide includes substitution of one or more amino acids 140-190, 220-300, 440-480, or 560-570 with one of the arginine, lysine, glutamine, asparagine, histidine, or serine residues. In some embodiments, the variant Cas12i2 polypeptide includes a change from one or more amino acids in at least one domain (e.g., the Rec1 domain, PI domain, or Wedge domain) to an arginine, lysine, glutamine, asparagine, histidine, or serine residue.In some embodiments, one or more double-stranded DNA binding interface substitutions improve double-stranded DNA interaction and / or affinity by approximately 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 5%. 6%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% Increase by 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or more, or any percentage in between.In some embodiments, one or more double-stranded DNA binding interface substitutions increase the binding of the mutant Cas12i2 polypeptide to the PAM sequence by approximately 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 8 Increase by 9%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or more, or any percentage in between.

[0542] In some embodiments, substitutions that increase double-stranded DNA interaction increase ternary complex formation compared to the parent polypeptide. Table 5 shows non-limiting examples of substitutions that can alter the ability of the mutant Cas12i2 polypeptide to interact with double-stranded DNA. In some embodiments, mutant Cas12i2 polypeptides containing one or more substitutions listed in Table 5 exhibit increased double-stranded DNA interaction (ternary complex formation) compared to the parent polypeptide. In some embodiments, mutant Cas12i2 polypeptides containing one or more substitutions listed in Table 5 form a more stable ternary complex compared to the parent polypeptide.

[0543] [Table 33]

[0544] In some embodiments, one of the variant Cas12i2 polypeptides from SEQ ID NOs: 3-146 and 495-512 further comprises one or more substitutions listed in Table 5. In some embodiments, the variant Cas12i2 polypeptide comprises one or more substitutions listed in Tables 2 and 5.

[0545] In some embodiments, mutant Cas12i2 polypeptides exhibiting increased double-stranded DNA interaction include two or more substitutions, such as T562R E563K, T562R E563K N448K, I451R L452K, I451R L452K T562R E563R, I451R L452K Y472R, N229R Q224N, or N229K Q224N. In some embodiments, mutant Cas12i2 polypeptides exhibiting increased ternary complex formation / stability include two or more substitutions, such as T562R E563K, T562R E563K N448K, I451R L452K, I451R L452K T562R E563R, I451R L452K Y472R, N229R Q224N, or N229K Q224N. In some embodiments, any one of the variant Cas12i2 polypeptides of SEQ ID NOs. 3-146 and 495-512 further includes T562R E563K, T562R E563K N448K, I451R L452K, I451R L452K T562R E563R, I451R L452K Y472R, N229R Q224N, or N229K Q224N substitutions. In some embodiments, any one of the variant Cas12i2 polypeptides of SEQ ID NOs. 3-146 and 495-512 further comprises the T562R E563K, T562R E563K N448K, I451R L452K, I451R L452K T562R E563R, I451R L452K Y472R, N229R Q224N, or N229K Q224N substitutions, as well as one or more substitutions listed in Table 4 and / or Table 5. In some embodiments, the variant Cas12i2 polypeptide comprises any one or more substitutions listed in Table 4 and / or Table 5.In some embodiments, mutant Cas12i2 polypeptides having one or more of the substitutions listed in Table 4 and / or Table 5 have higher double-stranded DNA interaction and / or affinity (e.g., about 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 5%) compared to the parent polypeptide. 3%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88 It exhibits an increase of %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or more, or any percentage in between.In some embodiments, mutant Cas12i2 polypeptides having one or more of the substitutions listed in Table 4 and / or Table 5 exhibit improved ternary complex formation and / or ternary complex stability compared to the parent polypeptide (e.g., about 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%). 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 8 It exhibits an increase of 8%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or more, or any percentage in between.

[0546] In some embodiments, any one of the mutant Cas12i2 polypeptides from SEQ ID NOs. 3-146 and 495-512, containing one or more substitutions listed in Table 4 and / or Table 5, exhibits increased enzyme activity. In some embodiments, any one of the mutant Cas12i2 polypeptides from SEQ ID NOs. 3-146 and 495-512, further containing T562R E563K, T562R E563K N448K, I451R L452K, I451R L452K T562R E563R, I451R L452K Y472R, N229R Q224N or N229K Q224N substitutions, and any one or more substitutions listed in Table 4 and / or Table 5, exhibits increased enzyme activity. In some embodiments, the mutant Cas12i2 polypeptide exhibits lower enzyme activity compared to the parent polypeptide (e.g., about 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%). It exhibits an increase of 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or more, or any percentage in between.

[0547] Increased single-strand DNA interactions In some embodiments, the mutant Cas12i2 polypeptide includes modifications that increase interaction with single-stranded DNA compared to the parent polypeptide. In some embodiments, the mutant Cas12i2 polypeptide includes modifications that increase affinity between the mutant Cas12i2 polypeptide and double-stranded DNA compared to the parent polypeptide. In some embodiments, the single-stranded DNA includes a non-target strand (NTS). In some embodiments, the increased interaction with single-stranded DNA (e.g., NTS) is the interaction between the PAM sequence and the active site of the mutant Cas12i2 polypeptide. In some embodiments, the single-stranded DNA includes single-stranded DNA that interacts with the mutant Cas12i2 polypeptide at or near the active site of the mutant Cas12i2 polypeptide. In some embodiments, the modifications that increase interaction and / or affinity between the mutant Cas12i2 polypeptide and single-stranded DNA stabilize the R-loop. As used herein, “R-loop” refers to a nucleic acid comprising an RNA guide paired with a target strand (TS) and a single-stranded non-target strand (NTS).

[0548] In some embodiments, the changes that increase the interaction and / or affinity between the mutant Cas12i2 polypeptide and single-stranded DNA are the substitution of one or more amino acids with arginine, lysine, glutamine, asparagine, histidine, or alanine residues. In some embodiments, the mutant Cas12i2 polypeptide includes substitution of one or more amino acids at the single-stranded DNA binding interface with arginine, lysine, glutamine, asparagine, histidine, or alanine residues. In some embodiments, the mutant Cas12i2 polypeptide includes substitution of one or more amino acids 230-260, 350-400, 580-630, 700-760, 830-900, or 920-1035 with one of the arginine, lysine, glutamine, asparagine, histidine, or alanine residues. In some embodiments, the variant Cas12i2 polypeptide includes a change from one or more amino acids in at least one domain / motif (e.g., PI domain, Rec1 domain, Wedge domain, RuvC1 motif, Rec2 domain, RuvC2 motif, Nuc domain, or RuvC3 motif) to an arginine, lysine, glutamine, asparagine, histidine, or alanine residue.In some embodiments, one or more single-stranded DNA binding interface substitutions improve single-stranded DNA interaction and / or affinity by approximately 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 5%. 6%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% Increase by 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or more, or any percentage in between.

[0549] In some embodiments, substitutions that increase single-stranded DNA interaction increase ternary complex formation compared to the parent polypeptide. Table 6 shows non-limiting examples of substitutions that can alter the ability of the mutant Cas12i2 polypeptide to interact with single-stranded DNA. In some embodiments, mutant Cas12i2 polypeptides containing one or more substitutions listed in Table 6 exhibit increased single-stranded DNA interaction (ternary complex formation) compared to the parent polypeptide. In some embodiments, mutant Cas12i2 polypeptides containing one or more substitutions listed in Table 6 form a more stable ternary complex compared to the parent polypeptide.

[0550] [Table 34]

[0551] In some embodiments, one of the variant Cas12i2 polypeptides from SEQ ID NOs: 3-146 and 495-512 further comprises one or more substitutions listed in Table 6. In some embodiments, the variant Cas12i2 polypeptide comprises one or more substitutions listed in Tables 2 and 6.

[0552] In some embodiments, mutant Cas12i2 polypeptides exhibiting increased single-strand DNA interaction include two or more substitutions, e.g., G587R T588R, G587R T588K, G587R T588K Q590R, or G587R T588R Q590K. In some embodiments, mutant Cas12i2 polypeptides exhibiting increased ternary complex formation / stability include two or more substitutions, e.g., G587R T588R, G587R T588K, G587R T588K Q590R, or G587R T588R Q590K. In some embodiments, any one of the variant Cas12i2 polypeptides of SEQ ID NOs: 3-146 and 495-512 further comprises the G587R T588R, G587R T588K, G587R T588K Q590R, or G587R T588R Q590K substitution. In some embodiments, any one of the variant Cas12i2 polypeptides of SEQ ID NOs: 3-146 and 495-512 further comprises the G587R T588R, G587R T588K, G587R T588K Q590R, or G587R T588R Q590K substitution and one or more substitutions listed in Table 4 and / or Table 5 and / or Table 6. In some embodiments, the variant Cas12i2 polypeptide comprises one or more substitutions listed in Table 4 and / or Table 5 and / or Table 6.In some embodiments, mutant Cas12i2 polypeptides having one or more substitutions listed in Table 4 and / or Table 5 and / or Table 6 have a higher single-strand DNA interaction and / or affinity (e.g., about 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 5%) compared to the parent polypeptide. 2%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, It exhibits an increase of 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or more, or any percentage in between.In some embodiments, mutant Cas12i2 polypeptides having one or more of the substitutions listed in Table 4 and / or Table 5 and / or Table 6 exhibit improved ternary complex formation and / or ternary complex stability compared to the parent polypeptide (e.g., about 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%). 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% It exhibits an increase of 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or more, or any percentage in between.

[0553] In some embodiments, the mutant Cas12i2 polypeptide contains substitutions that increase single-stranded DNA stability (for example, substitutions that increase electrostatic interactions between single-stranded DNA and the active site of the mutant Cas12i2 polypeptide). In some embodiments, the mutant Cas12i2 polypeptide exhibits approximately 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, and 57% single-strand DNA stability compared to the parent polypeptide. Increase by 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or more, or any percentage in between. Table 6 shows non-limiting examples of substitutions that can alter the ability of the mutant Cas12i2 polypeptide to stabilize single-stranded DNA. In some embodiments, mutant Cas12i2 polypeptides containing one or more substitutions listed in Table 6 exhibit increased single-stranded DNA stability compared to the parent polypeptide.

[0554] In some embodiments, any one of the mutant Cas12i2 polypeptides of SEQ ID NOs. 3-146 and 495-512, containing one or more substitutions listed in Table 4 and / or Table 5 and / or Table 6, exhibits increased enzyme activity. In some embodiments, any one of the mutant Cas12i2 polypeptides of SEQ ID NOs. 3-146 and 495-512, further containing the G587R T588R, G587R T588K, G587R T588K Q590R, or G587R T588R Q590K substitution and one or more substitutions listed in Table 4 and / or Table 5 and / or Table 6, exhibits increased enzyme activity. In some embodiments, the mutant Cas12i2 polypeptide exhibits lower enzyme activity compared to the parent polypeptide (e.g., about 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%). It exhibits an increase of 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or more, or any percentage in between.

[0555] Increased heterodouble-chain interaction In some embodiments, the mutant Cas12i2 polypeptide includes substitutions that increase interaction with the DNA / RNA hybrid molecule compared to the parent polypeptide. In some embodiments, the mutant Cas12i2 polypeptide includes changes that increase affinity between the mutant Cas12i2 polypeptide and the DNA / RNA hybrid compared to the parent polypeptide. In some embodiments, the DNA / RNA hybrid molecule is a heteroduplex. As used herein, “heteroduplex” refers to a double helix formed by an RNA guide spacer and a target strand (TS). As used herein, the term “seed region” refers to the TS portion immediately downstream of the PAM sequence in the heteroduplex. The seed region contains a first base that pairs with the RNA guide in the heteroduplex and is necessary for RNA-DNA binding and TS substitution. In some embodiments, changes that increase interaction and / or affinity between the mutant Cas12i2 polypeptide and the heteroduplex increase nonspecific nucleic acid contact. In some embodiments, changes that increase interaction and / or affinity between the mutant Cas12i2 polypeptide and the heteroduplex increase ternary complex formation / stability compared to the parent polypeptide.

[0556] In some embodiments, the changes that increase the interaction and / or affinity between the mutant Cas12i2 polypeptide and the heteroduplex are the substitution of one or more amino acids with arginine, lysine, glutamine, asparagine, histidine, or serine residues. In some embodiments, the mutant Cas12i2 polypeptide includes substitution of one or more amino acids in contact with the heteroduplex to arginine, lysine, glutamine, asparagine, histidine, or serine residues. In some embodiments, the mutant Cas12i2 polypeptide includes substitution of one or more amino acids 110-130, 150-170, 250-320, 340-400, 420-450, 670-720, 770-810, or 830-870 to one of the arginine, lysine, glutamine, asparagine, histidine, or serine residues. In some embodiments, the variant Cas12i2 polypeptide includes changes from one or more amino acids in at least one domain / motif (e.g., Rec1 domain, PI domain, Rec2 domain, or RuvC2 motif) to arginine, lysine, glutamine, asparagine, histidine, or serine residues.In some embodiments, one or more nucleic acid interface substitutions improve heteroduplex interactions and / or affinity by approximately 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, Increase by 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or more, or any percentage in between.

[0557] In some embodiments, substitutions that increase heteroduplex interaction increase ternary complex formation / stability compared to the parent polypeptide. Table 7 shows non-limiting examples of substitutions that can alter the ability of the mutant Cas12i2 polypeptide to interact with heteroduplexes. In some embodiments, mutant Cas12i2 polypeptides containing one or more substitutions listed in Table 7 exhibit increased heteroduplex interaction (ternary complex formation) compared to the parent polypeptide. In some embodiments, mutant Cas12i2 polypeptides containing one or more substitutions listed in Table 7 form a more stable ternary complex compared to the parent polypeptide.

[0558] [Table 35]

[0559] [Table 36]

[0560] In some embodiments, one of the variant Cas12i2 polypeptides from SEQ ID NOs: 3-146 and 495-512 further comprises one or more substitutions listed in Table 7. In some embodiments, the variant Cas12i2 polypeptide comprises one or more substitutions listed in Tables 2 and 7.

[0561] In some embodiments, mutant Cas12i2 polypeptides exhibiting increased heterodouble-chain interactions include two or more substitutions, such as E691R A695R, S78K V438G, S78K V438A, S346R E343S, D782R D793N, S78R V438G, S78R V438A, S346K E343S, or D782K D793N. In some embodiments, mutant Cas12i2 polypeptides exhibiting increased ternary complex formation / stability include two or more substitutions, such as E691R A695R, S78K V438G, S78K V438A, S346R E343S, D782R D793N, S78R V438G, S78R V438A, S346K E343S, or D782K D793N. In some embodiments, any one of the variant Cas12i2 polypeptides of SEQ ID NOs. 3-146 and 495-512 further includes substitutions of E691R A695R, S78K V438G, S78K V438A, S346R E343S, D782R D793N, S78R V438G, S78R V438A, S346K E343S, or D782K D793N. In some embodiments, any one of the variant Cas12i2 polypeptides of SEQ ID NOs: 3-146 and 495-512 further comprises the E691R A695R, S78K V438G, S78K V438A, S346R E343S, D782R D793N, S78R V438G, S78R V438A, S346K E343S, or D782K D793N substitutions, as well as one or more substitutions listed in Table 4 and / or Table 5 and / or Table 6 and / or Table 7. In some embodiments, the variant Cas12i2 polypeptide comprises any one or more substitutions listed in Table 4 and / or Table 5 and / or Table 6 and / or Table 7.In some embodiments, mutant Cas12i2 polypeptides having one or more substitutions listed in Table 4 and / or Table 5 and / or Table 6 and / or Table 7 have higher heterodouble-chain interaction and / or affinity (e.g., about 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%) compared to the parent polypeptide. %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87 It exhibits an increase of %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or more, or any percentage in between.In some embodiments, mutant Cas12i2 polypeptides having one or more substitutions listed in Table 4 and / or Table 5 and / or Table 6 and / or Table 7 exhibit improved ternary complex formation and / or ternary complex stability compared to the parent polypeptide (e.g., about 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 5%). 1%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87 It exhibits an increase of %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or more, or any percentage in between.

[0562] In some embodiments, one of the variant Cas12i2 polypeptides from SEQ ID NOs: 3-146 and 495-512, containing one or more substitutions listed in Table 4 and / or Table 5 and / or Table 6 and / or Table 7, exhibits increased enzyme activity. In some embodiments, one of the variant Cas12i2 polypeptides of SEQ ID NOs. 3-146 and 495-512, further comprising the E691R A695R, S78K V438G, S78K V438A, S346R E343S, D782R D793N, S78R V438G, S78R V438A, S346K E343S, or D782K D793N substitutions, as well as one or more substitutions listed in Table 4 and / or Table 5 and / or Table 6 and / or Table 7, exhibits increased enzyme activity. In some embodiments, the mutant Cas12i2 polypeptide exhibits lower enzyme activity compared to the parent polypeptide (e.g., about 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%). It exhibits an increase of 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or more, or any percentage in between.

[0563] Increased stability of double-stranded and heterodouble-stranded DNA. During ternary complex formation, the double-stranded DNA downstream of the PAM sequence melts (e.g., unwinds) into a target strand (TS) and a non-target strand (NTS). An RNA guide spacer binds to the TS, forming a double helix called a heteroduplex. The PAM sequence remains intact as intact double-stranded DNA. As a result, these terminal PAM dsDNA base pairs are exposed to the environment and proteins, which can be energetically disadvantageous. Similarly, the terminal base pairs of the heteroduplex are also exposed, which can also be energetically disadvantageous. See Figure 21. In some embodiments, changes in the mutant Cas12i2 polypeptide that increase aromatic, hydrophobic, van der Waals, and / or cation-π interactions between the exposed double-stranded DNA terminal PAM bases or the heteroduplex terminal bases increase the stability of the DNA that melts during ternary complex formation. In some embodiments, changes that increase aromatic, hydrophobic, van der Waals, and / or cation-π interactions between the mutant Cas12i2 polypeptide and the exposed bases of the double-stranded DNA or heteroduplex increase R-loop stability during ternary complex formation. In some embodiments, changes that increase aromatic, hydrophobic, van der Waals, and / or cation-π interactions between the mutant Cas12i2 polypeptide and the exposed bases of the double-stranded DNA or heteroduplex increase ternary complex formation. In some embodiments, changes that increase aromatic, hydrophobic, van der Waals, and / or cation-π interactions between the mutant Cas12i2 polypeptide and the exposed bases of the double-stranded DNA or heteroduplex increase ternary complex stability. See Figure 20D.

[0564] In some embodiments, changes that increase aromaticity, hydrophobicity, van der Waals, and / or cation-π interactions involve substituting one or more residues with arginine, lysine, tryptophan, phenylalanine, tyrosine, methionine, histidine, glutamine, threonine, or valine residues. In some embodiments, changes that increase aromaticity, hydrophobicity, van der Waals, and / or cation-π interactions involve substituting one or more residues in contact with the double helix and / or heterodouble helix of double-stranded DNA with arginine, lysine, tryptophan, phenylalanine, tyrosine, methionine, histidine, glutamine, threonine, or valine residues. In some embodiments, changes that increase aromaticity, hydrophobicity, van der Waals, and / or cation-π interactions are the substitutions listed in Table 8. In some embodiments, mutant Cas12i2 polypeptides containing the substitutions listed in Table 8 exhibit increased aromatic, hydrophobic, van der Waals, and / or cation-π interactions between the mutant Cas12i2 polypeptide and the exposed bases of the double-stranded DNA or heteroduplex compared to the parent polypeptide. In some embodiments, the changes involve creating more energetically favorable conditions for the double-stranded DNA and heteroduplex by substituting an amino acid adjacent to the terminal double-stranded base pair with a positively charged, aromatic, hydrophobic, or branched-chain amino acid (e.g., arginine, lysine, tryptophan, phenylalanine, tyrosine, methionine, histidine, glutamine, threonine, isoleucine, or valine).

[0565] [Table 37]

[0566] In some embodiments, one of the variant Cas12i2 polypeptides from SEQ ID NOs: 3-146 and 495-512 further comprises one or more substitutions listed in Table 8. In some embodiments, the variant Cas12i2 polypeptide comprises one or more substitutions listed in Tables 2 and 8.

[0567] In some embodiments, mutant Cas12i2 polypeptides exhibiting increased ternary complex formation and / or ternary complex stability (e.g., by stabilizing the melting of DNA and / or the R-loop) include two or more substitutions, e.g., Q163N N164W, Q163M N164W, Q163M N164Q, Q163N N164Q, I5G P577Y, I5G P577F, I5G P577H, or I5M P577L. In some embodiments, any one of the mutant Cas12i2 polypeptides of SEQ ID NOs. 3-146 and 495-512 further includes the substitution Q163N N164W, Q163M N164W, Q163M N164Q, Q163N N164Q, I5G P577Y, I5G P577F, I5G P577H, or I5M P577L. In some embodiments, the mutant Cas12i2 polypeptide containing any one of SEQ ID NOs. 3-146 and 495-512 further comprises the Q163N N164W, Q163M N164W, Q163M N164Q, Q163N N164Q, I5G P577Y, I5G P577F, I5G P577H, or I5M P577L substitutions, as well as one or more substitutions from Tables 4, 5, 6, 7, and / or 8. In some embodiments, the mutant Cas12i2 polypeptide contains any one or more substitutions from Tables 4, 5, 6, 7, and / or 8.In some embodiments, mutant Cas12i2 polypeptides having one or more substitutions listed in Tables 4, 5, 6, 7, and / or 8 show improved ternary complex formation and / or ternary complex stability compared to the parent polypeptide (e.g., about 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%) 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% It exhibits an increase of 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or more, or any percentage in between.

[0568] In some embodiments, any one mutant Cas12i2 polypeptide from SEQ ID NOs. 3-146 and 495-512, containing one or more substitutions listed in Tables 4, 5, 6, 7, and / or 8, exhibits increased enzyme activity. In some embodiments, any one mutant Cas12i2 polypeptide from SEQ ID NOs. 3-146 and 495-512, further containing the Q163N N164W, Q163M N164W, Q163M N164Q, Q163N N164Q, I5G P577Y, I5G P577F, I5G P577H, or I5M P577L substitutions, and any one or more substitutions listed in Tables 4, 5, 6, 7, and / or 8, exhibits increased enzyme activity. In some embodiments, the mutant Cas12i2 polypeptide exhibits lower enzyme activity compared to the parent polypeptide (e.g., about 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%). It exhibits an increase of 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or more, or any percentage in between.

[0569] Increased conformational changes Conformational changes, such as those resulting from the binding of an RNA guide or target DNA, affect the function of the mutant Cas12i2 polypeptide. For example, conformational changes can alter the reaction rate of the mutant Cas12i2 polypeptide. During ternary complex formation, the Rec1 (helix II) domain of Cas12i2 moves and rotates to adapt to DNA binding. See Figures 22A and 22B. In some embodiments, changes that increase the movement (e.g., mobility or conformational changes) of the helix II domain increase DNA binding / DNA binding affinity. In some embodiments, substitutions that increase the mobility of the helix II domain, such as substitutions from bulk amino acids to amino acids with smaller or even smaller side chains (alanine, valine, glycine, or serine residues), increase ternary complex formation. In some embodiments, changes that increase the movement (e.g., mobility or conformational changes) of the helix II domain increase ternary complex stability. In some embodiments, a change that increases conformational variation of the helix II domain involves substituting one or more residues with alanine, valine, glycine, or serine residues. In some embodiments, a change that increases the mobility of the helix II domain involves substituting one or more residues with alanine, valine, glycine, or serine residues. In some embodiments, the mutant Cas12i2 polypeptide includes a change in one or more amino acids near the helix II domain. In some embodiments, the mutant Cas12i2 polypeptide includes a change from one or more amino acids near the helix II domain to alanine, valine, glycine, or serine. In some embodiments, the mutant Cas12i2 polypeptide includes a substitution from one or more amino acids 327-330 to one of alanine, valine, glycine, or serine. In some embodiments, the mutant Cas12i2 polypeptide includes the substitutions shown in Table 9.

[0570] [Table 38]

[0571] In some embodiments, one of the variant Cas12i2 polypeptides from SEQ ID NOs: 3-146 and 495-512 further comprises one or more substitutions listed in Table 9. In some embodiments, the variant Cas12i2 polypeptide comprises one or more substitutions listed in Tables 2 and 9.

[0572] In some embodiments, the changes that increase helix II domain mobility are the substitutions listed in Table 9. In some embodiments, mutant Cas12i2 polypeptides having one or more of the substitutions listed in Table 9 exhibit approximately 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, and 54% helix II domain mobility compared to the parent polypeptide. , 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, The percentages increase to 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or more, or any percentage increase in between. In some embodiments, the changes that increase DNA binding / DNA affinity are the substitutions listed in Table 9.In some embodiments, mutant Cas12i2 polypeptides having one or more of the substitutions listed in Table 9 exhibit DNA binding / DNA affinity of approximately 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, compared to the parent polypeptide. 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 8 The percentages are 9%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or more, or any percentage increase in between.In some embodiments, mutant Cas12i2 polypeptides containing the substitutions listed in Table 9 exhibit improved ternary complex formation and / or ternary complex stability compared to the parent polypeptide (e.g., about 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 5%). 4%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, It exhibits an increase of 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or more, or any percentage in between.

[0573] In some embodiments, the variant Cas12i2 polypeptide exhibiting increased helix II domain mobility includes two or more substitutions, e.g., L327V N328S N329G L330A, L327A N328S N329G L330A, L327V N328G N329S L330A, or L327V N328G N329G L330A. In some embodiments, the variant Cas12i2 polypeptide exhibiting increased DNA binding / affinity includes two or more substitutions, e.g., L327V N328S N329G L330A, L327A N328S N329G L330A, L327V N328G N329S L330A, or L327V N328G N329G L330A. In some embodiments, mutant Cas12i2 polypeptides exhibiting increased ternary complex formation / stability include two or more substitutions, e.g., L327V N328S N329G L330A, L327A N328S N329G L330A, L327V N328G N329S L330A, or L327V N328G N329G L330A. In some embodiments, any one of the mutant Cas12i2 polypeptides of SEQ ID NOs. 3-146 and 495-512 further includes the L327V N328S N329G L330A, L327A N328S N329G L330A, L327V N328G N329S L330A, or L327V N328G N329G L330A substitution. In some embodiments, any one of the variant Cas12i2 polypeptides of SEQ ID NOs: 3-146 and 495-512 further comprises the L327V N328S N329G L330A, L327A N328S N329G L330A, L327V N328G N329S L330A, or L327V N328G N329G L330A substitutions, as well as one or more substitutions listed in Table 4 and / or Table 5 and / or Table 6 and / or Table 7 and / or Table 8 and / or Table 9. In some embodiments, the variant Cas12i2 polypeptide comprises any one or more substitutions listed in Table 4 and / or Table 5 and / or Table 6 and / or Table 7 and / or Table 8 and / or Table 9.In some embodiments, mutant Cas12i2 polypeptides having one or more substitutions listed in Table 4 and / or Table 5 and / or Table 6 and / or Table 7 and / or Table 8 and / or Table 9 have DNA binding / affinity (e.g., about 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%) compared to the parent polypeptide. , 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 8 It exhibits an increase of 7%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or more, or any percentage in between.In some embodiments, mutant Cas12i2 polypeptides having one or more substitutions listed in Table 4 and / or Table 5 and / or Table 6 and / or Table 7 and / or Table 8 and / or Table 9 exhibit superior ternary complex formation and / or ternary complex stability compared to the parent polypeptide (e.g., approximately 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%). %, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86 It exhibits an increase of %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or more, or any percentage in between.

[0574] In some embodiments, one of the variant Cas12i2 polypeptides from SEQ ID NOs: 3-146 and 495-512, containing one or more substitutions listed in Table 4 and / or Table 5 and / or Table 6 and / or Table 7 and / or Table 8 and / or Table 9, exhibits increased enzyme activity. In some embodiments, any one variant Cas12i2 polypeptide of SEQ ID NOs. 3-146 and 495-512, further comprising the L327V N328S N329G L330A, L327A N328S N329G L330A, L327V N328G N329S L330A, or L327V N328G N329G L330A substitution, as well as one or more substitutions listed in Table 4 and / or Table 5 and / or Table 6 and / or Table 7 and / or Table 8 and / or Table 9, exhibits increased enzyme activity. In some embodiments, the mutant Cas12i2 polypeptide exhibits lower enzyme activity compared to the parent polypeptide (e.g., about 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%). It exhibits an increase of 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or more, or any percentage in between.

[0575] In some embodiments, modifications that increase the coupling of the interface between Nuc and helix II, which is formed when the target single-stranded DNA is at the active site of the Cas12i2 polypeptide, increase the transition from the binary to the ternary complex. In some embodiments, modifications that increase the coupling of the interface between Nuc and helix II increase ternary complex formation. See Figure 22B. In some embodiments, modifications that increase the coupling of the interface between Nuc and helix II increase ternary complex stability. In some embodiments, modifications that increase the coupling of the interface between Nuc and helix II involve substituting one or more residues with aspartic acid, glutamic acid, arginine, or lysine residues. In some embodiments, the mutant Cas12i2 polypeptide includes substitutions of one or more amino acids 380-390 or 910-930 with one of aspartic acid, glutamic acid, arginine, or lysine. In some embodiments, the mutant Cas12i2 polypeptide includes the substitutions shown in Table 10.

[0576] [Table 39]

[0577] In some embodiments, one of the variant Cas12i2 polypeptides from SEQ ID NOs. 3-146 and 495-512 further comprises one or more substitutions listed in Table 10. In some embodiments, the variant Cas12i2 polypeptide comprises one or more substitutions listed in Tables 2 and 10.

[0578] In some embodiments, the substitutions shown in Table 10 increase the interface coupling between Nuc and helix II. In some embodiments, mutant Cas12i2 polypeptides having one or more of the substitutions listed in Table 10 show approximately 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 5% 4%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% Increase by 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or more, or any percentage in between.In some embodiments, mutant Cas12i2 polypeptides containing the substitutions listed in Table 10 exhibit improved ternary complex formation and / or ternary complex stability compared to the parent polypeptide (e.g., about 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%). 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% It exhibits an increase of 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or more, or any percentage in between.

[0579] In some embodiments, mutant Cas12i2 polypeptides exhibiting increased interface linkage between Nuc and helix II are substituted with two or more others, e.g., V383D D387E, V383D D386E, V383E D387E, V383E D386E, Q919K D387E, Q919K D383D, Q919K V383E, D386E D387E, N925D D362R K365R, N925E D362R K365R, N925D D362K K365R, N925E D362K K365R, N925D D362R, N925E D362R, N925D D362K, N925E D362K, N925D K365R, N925E Includes K365R or D362E N925K. In some embodiments, mutant Cas12i2 polypeptides exhibiting increased ternary complex formation / stability are those with two or more substitutions, for example, V383D D387E, V383D D386E, V383E D387E, V383E D386E, Q919K D387E, Q919K D383D, Q919K V383E, D386E D387E, N925D D362R K365R, N925E D362R K365R, N925D D362K K365R, N925E D362K K365R, N925D D362R, N925E D362R, N925D D362K, N925E D362K, N925D K365R, N925E Includes K365R or D362E N925K. In some embodiments, any one of the variant Cas12i2 polypeptides from SEQ ID NOs: 3-146 and 495-512 is V383D D387E, V383D D386E, V383E D387E, V383E D386E, Q919K D387E, Q919K D383D, Q919K V383E, D386E D387E, N925D D362R K365R, N925E D362R K365R, N925D D362K K365R, N925E D362K K365R, N925D D362R, N925E D362R, N925D D362K, N925E D362K, N925D K365R, N925E Further includes substitution of K365R or D362E N925K.In some embodiments, any one of the variant Cas12i2 polypeptides from SEQ ID NOs: 3-146 and 495-512 is V383D D387E, V383D D386E, V383E D387E, V383E D386E, Q919K D387E, Q919K D383D, Q919K V383E, D386E D387E, N925D D362R K365R, N925E D362R K365R, N925D D362K K365R, N925E D362K K365R, N925D D362R, N925E D362R, N925D D362K, N925E D362K, N925D K365R, N925E The variant Cas12i2 polypeptide further includes the K365R or D362E N925K substitution and one or more substitutions listed in Table 4 and / or Table 5 and / or Table 6 and / or Table 7 and / or Table 8 and / or Table 9 and / or Table 10. In some embodiments, the variant Cas12i2 polypeptide includes one or more substitutions from Table 4 and / or Table 5 and / or Table 6 and / or Table 7 and / or Table 8 and / or Table 9 and / or Table 10.In some embodiments, mutant Cas12i2 polypeptides having one or more substitutions from Table 4 and / or Table 5 and / or Table 6 and / or Table 7 and / or Table 8 and / or Table 9 and / or Table 10 have a higher degree of interface linkage between Nuc and Helix II compared to the parent polypeptide (e.g., about 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%) %, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, It exhibits an increase of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or more, or any percentage in between.In some embodiments, mutant Cas12i2 polypeptides having one or more substitutions from Table 4 and / or Table 5 and / or Table 6 and / or Table 7 and / or Table 8 and / or Table 9 and / or Table 10 exhibit superior ternary complex formation and / or ternary complex stability compared to the parent polypeptide (e.g., about 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%). %, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, It exhibits an increase of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or more, or any percentage in between.

[0580] In some embodiments, one of the variant Cas12i2 polypeptides from SEQ ID NOs: 3-146 and 495-512, containing one or more substitutions listed in Table 4 and / or Table 5 and / or Table 6 and / or Table 7 and / or Table 8 and / or Table 9 and / or Table 10, exhibits increased enzyme activity. In some embodiments, V383D D387E, V383D D386E, V383E D387E, V383E D386E, Q919K D387E, Q919K D383D, Q919K V383E, D386E D387E, N925D D362R K365R, N925E D362R K365R, N925D D362K K365R, N925E D362K K365R, N925D D362R, N925E D362R, N925D D362K, N925E D362K, N925D K365R, N925E K365R or D362E One of the variant Cas12i2 polypeptides of SEQ ID NOs: 3-146 and 495-512, further comprising the N925K substitution and one or more substitutions listed in Table 4 and / or Table 5 and / or Table 6 and / or Table 7 and / or Table 8 and / or Table 9 and / or Table 10, exhibits increased enzyme activity.In some embodiments, the mutant Cas12i2 polypeptide exhibits lower enzyme activity compared to the parent polypeptide (e.g., about 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%). It exhibits an increase of 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or more, or any percentage in between.

[0581] In some embodiments, the modification reduces the interfacial linkage between Nuc and helix II. In some embodiments, the modification that reduces the interfacial linkage between Nuc and helix II increases ternary complex formation. In some embodiments, the modification that reduces the interfacial linkage between Nuc and helix II is the substitution of one or more residues with asparagine or serine residues. In some embodiments, the mutant Cas12i2 polypeptide includes substitutions of one or more amino acids 386, 387, 915, and 956 with asparagine or serine. In some embodiments, the mutant Cas12i2 polypeptide includes D386N R915S, D387N R956S, D386N D387N R915S R956S, or D386N D387N R915S R922S R956S substitutions. In some embodiments, the mutant Cas12i2 polypeptide containing any one of SEQ ID NOs.3-146 and 495-512 further comprises the D386N R915S, D387N R956S, D386N D387N R915S R956S, or D386N D387N R915S R922S R956S substitutions.

[0582] Increased fidelity In some embodiments, the mutant Cas12i2 polypeptide includes modifications that increase on-target specificity compared to the parent polypeptide. In some embodiments, the mutant Cas12i2 polypeptide includes modifications that increase on-target binding compared to the parent polypeptide. In some embodiments, the mutant Cas12i2 polypeptide includes modifications that increase the interaction (e.g., affinity) between the mutant Cas12i2 polypeptide and the on-target DNA compared to the parent polypeptide.

[0583] In some embodiments, the change that increases on-target specificity is the substitution of one or more amino acids with alanine, serine, valine, glutamine, or asparagine residues. In some embodiments, the change that increases on-target specificity is the truncation of residues that contact the spacer sequence of the RNA guide (e.g., the substitution of residues that contact the spacer sequence with residues having smaller side chains). In some embodiments, the change that increases on-target specificity is the truncation of residues that contact the spacer sequence of the RNA guide, e.g., substitution with alanine, serine, or valine. In some embodiments, the variant Cas12i2 polypeptide includes the substitution of one or more amino acids that contact the spacer sequence of the RNA guide with alanine, serine, valine, glutamine, or asparagine residues. In some embodiments, the mutant Cas12i2 polypeptide includes substitution of one or more amino acids from 290-320, 340-360, 390-450, 550-580, 710-720, 760-810, or 830-870 to one of alanine, serine, valine, glutamine, or asparagine residues. In some embodiments, the mutant Cas12i2 polypeptide includes substitution of one or more amino acids from at least one domain / motif (e.g., Wedge domain, Rec1 domain, Rec2 domain, or RuvC2 motif) to one of alanine, serine, valine, glutamine, or asparagine residues. In some embodiments, truncated substitution in the helix II domain results in increased on-target binding specificity of the mutant Cas12i2 polypeptide. In some embodiments, one or more of the following helix II residues are truncated: E348, E349, E395, I397, R398, N399, Y351, H356, H357, K394, and R428.In some embodiments, one or more of these substitutions improve the on-target specificity of the mutant Cas12i2 polypeptide by approximately 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, and 55% compared to the parent polypeptide. 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89 Increase by %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or more, or any percentage in between.In some embodiments, one or more of these substitutions increase the on-target binding of the mutant Cas12i2 polypeptide by approximately 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 5%. 6%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% Increase by 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or more, or any percentage in between.In some embodiments, one or more of these substitutions increase the on-target binding affinity of the mutant Cas12i2 polypeptide by approximately 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, and 55% compared to the parent polypeptide. 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89 Increase by %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or more, or any percentage in between.

[0584] Non-limiting examples of substitutions that can alter the ability of a mutant Cas12i2 polypeptide to selectively bind to on-target DNA are those listed in Table 11. In some embodiments, mutant Cas12i2 polypeptides containing one or more substitutions listed in Table 11 exhibit increased on-target specificity compared to the parent polypeptide. In some embodiments, mutant Cas12i2 polypeptides containing one or more substitutions listed in Table 11 exhibit increased on-target binding compared to the parent polypeptide. In some embodiments, mutant Cas12i2 polypeptides containing one or more substitutions listed in Table 11 exhibit increased on-target binding affinity compared to the parent polypeptide. As used herein, the term “TS seed” refers to a seed sequence that forms a double helix with an RNA guide, and the term “guide seed” refers to an RNA guide that pairs with the TS seed sequence.

[0585] [Table 40]

[0586] [Table 41]

[0587] In some embodiments, changes that increase on-target specificity (e.g., substitutions listed in Table 11) further increase on-target ternary complex formation and / or on-target ternary complex stability (e.g., on-target ternary complex formation / stability). In some embodiments, the changes that increase on-target specificity improve on-ternary formation and / or on-target ternary complex stability by approximately 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 5% compared to the parent polypeptide. 5%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 8 Increase by 9%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or more, or any percentage in between.

[0588] In some embodiments, the mutant Cas12i2 polypeptide includes modifications that reduce off-target specificity compared to the parent polypeptide. In some embodiments, the mutant Cas12i2 polypeptide includes modifications that reduce off-target binding compared to the parent polypeptide. In some embodiments, the mutant Cas12i2 polypeptide includes modifications that reduce the interaction (e.g., affinity) between the mutant Cas12i2 polypeptide and off-target DNA compared to the parent polypeptide.

[0589] In some embodiments, the change that reduces off-target specificity is the substitution of one or more amino acids with alanine, serine, valine, glutamine, or asparagine residues. In some embodiments, the change that reduces off-target specificity is the truncation of residues that contact the spacer sequence of the RNA guide (e.g., the substitution of residues that contact the spacer sequence with residues having smaller side chains). In some embodiments, the change that reduces off-target specificity is the truncation of residues that contact the spacer sequence of the RNA guide, e.g., substitution with alanine, serine, or valine. In some embodiments, the variant Cas12i2 polypeptide includes the substitution of one or more amino acids that contact the spacer sequence of the RNA guide with alanine, serine, valine, glutamine, or asparagine residues. In some embodiments, the mutant Cas12i2 polypeptide includes substitution of one or more amino acids from 290-320, 340-360, 390-450, 550-580, 710-720, 760-810, or 830-870 to one of alanine, serine, valine, glutamine, or asparagine residues. In some embodiments, the mutant Cas12i2 polypeptide includes substitution of one or more amino acids from at least one domain / motif (e.g., Wedge domain, Rec1 domain, Rec2 domain, or RuvC2 motif) to one of alanine, serine, valine, glutamine, or asparagine residues. In some embodiments, truncated substitution in the helix II domain results in the mutant Cas12i2 polypeptide exhibiting reduced off-target binding specificity. In some embodiments, one or more of the following helix II residues are truncated: E348, E349, E395, I397, R398, N399, Y351, H356, H357, K394, and R428.In some embodiments, one or more of these substitutions reduce the off-target specificity of the mutant Cas12i2 polypeptide by approximately 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, and 45% compared to the parent polypeptide. Reduce by %, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, one or more of these substitutions reduce off-target binding of the mutant Cas12i2 polypeptide by approximately 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, and 45% compared to the parent polypeptide. Reduce by 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.In some embodiments, one or more of these substitutions increase the off-target binding affinity of the mutant Cas12i2 polypeptide by approximately 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, and 45% compared to the parent polypeptide. Reduce by %, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0590] Non-limiting examples of substitutions that can alter the ability of the mutant Cas12i2 polypeptide to bind to off-target DNA are those listed in Table 11. In some embodiments, mutant Cas12i2 polypeptides containing one or more substitutions listed in Table 11 exhibit reduced off-target specificity compared to the parent polypeptide. In some embodiments, mutant Cas12i2 polypeptides containing one or more substitutions listed in Table 11 exhibit reduced off-target binding compared to the parent polypeptide. In some embodiments, mutant Cas12i2 polypeptides containing one or more substitutions listed in Table 11 exhibit reduced off-target binding affinity compared to the parent polypeptide.

[0591] In some embodiments, the on-target specificity of the mutant Cas12i2 polypeptide i...

Claims

1. A mutant Cas12i2 polypeptide comprising a sequence having at least 95% identity with the sequence shown in SEQ ID NO: 4, wherein the mutant Cas12i2 polypeptide comprises the D581R, I926R, and V1030G substitutions in the amino acid sequence of SEQ ID NO: 2, and (i) The mutant Cas12i2 polypeptide forms a more stable complex than the complex formed by the parent polypeptide having the amino acid sequence of SEQ ID NO: 2, the RNA guide, and the target nucleic acid in the presence of the RNA guide and the target nucleic acid, or (ii) The mutant Cas12i2 polypeptide exhibits higher enzyme activity than the parent polypeptide having the amino acid sequence of Sequence ID No.

2. The Cas12i2 mutant polypeptide.

2. A variant Cas12i2 polypeptide comprising a sequence having at least 99% identity with the sequence shown in SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 495, or SEQ ID NO: 496, wherein the variant Cas12i2 polypeptide comprises the D581R, I926R, and V1030G substitutions in the amino acid sequence of SEQ ID NO:

2.

3. A composition comprising a variant Cas12i2 polypeptide according to claim 1 or 2, or a nucleic acid encoding the variant Cas12i2 polypeptide, further comprising an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide comprises a direct repeat sequence and a spacer sequence.

4. The composition according to claim 3, (i) The direct repeat sequence includes a nucleotide sequence having at least 95% sequence identity with any one of sequence numbers 492 to 494, and optionally the direct repeat sequence includes a nucleotide sequence shown in any one of sequence numbers 492 to 494. (ii) The spacer sequence comprises about 11 to about 50 nucleotides, optionally comprising about 15 to about 35 nucleotides, and / or (iii) The spacer sequence is bound to the target nucleic acid sequence, and the target nucleic acid sequence is adjacent to the 5'-NTTN-3' sequence. composition.

5. A variant Cas12i2 polypeptide according to claim 1 or 2, or a composition according to claim 3 or 4, wherein the variant Cas12i2 polypeptide is (i) at least one nuclear localization signal (NLS), at least one nuclear export signal (NES), or at least one NLS and at least one NES, and / or (ii) Peptide tags, fluorescent proteins, base editing domains, DNA methylation domains, histone residue modification domains, localization factors, transcription modifiers, light-dependent regulators, chemically inducible factors, or chromatin visualization factors A variant Cas12i2 polypeptide or composition further comprising the above.

6. The composition according to any one of claims 3 to 5, which is present in a delivery system comprising nanoparticles, liposomes, exosomes, microvesicles, or gene guns.

7. A nucleic acid molecule encoding the mutant Cas12i2 polypeptide according to any one of claims 1, 2, or 5.

8. A cell comprising a variant Cas12i2 polypeptide according to any one of claims 1, 2, or 5, a composition according to any one of claims 3 to 6, or a nucleic acid molecule according to claim 7.

9. The cell according to claim 8, which is a eukaryotic cell, optionally a mammalian cell (e.g., a human cell), or a plant cell.

10. A composition or formulation comprising the mutant Cas12i2 polypeptide according to any one of claims 1, 2, or 5, and optionally, (i) an RNA guide or (ii) an RNA guide and cells.

11. A composition comprising a mutant Cas12i2 polypeptide according to any one of claims 1, 2, or 5, or a nucleic acid encoding the mutant Cas12i2 polypeptide, and an RNA guide, wherein the mutant Cas12i2 polypeptide and the RNA guide form a complex. (i) The mutant Cas12i2 polypeptide exhibits increased complex formation with the RNA guide compared to the parent polypeptide, and the parent polypeptide contains the amino acid sequence of SEQ ID NO:

2. (ii) The variant Cas12i2 polypeptide exhibits increased binding affinity to the RNA guide compared to the parent polypeptide, and / or (iii) The mutant Cas12i2 polypeptide and the RNA guide exhibit increased protein-RNA interaction compared to the parent polypeptide and the RNA guide. composition.

12. The mutant Cas12i2 polypeptide is the mutant Cas12i2 polypeptide according to claim 1 or 5, comprising at least one of the G624R, F626G, F626R, D835G, D835R, L836G, L836R, P868G, P868R, P868T, S879G, S879R, D911G, D911R, V1020G, V1020R, E1035G, E1035R, S1046G, and S1046R substitutions in the amino acid sequence of SEQ ID NO: 2, or the composition according to any one of claims 3 to 6, 10, or 11.

13. An ex vivo method for achieving a deletion in the DNA of a cell, comprising contacting the DNA of the cell with a composition according to any one of claims 3 to 6 or 10 to 12.

14. A composition comprising a mutant Cas12i2 polypeptide according to any one of claims 1, 2, 5, or 12, or a nucleic acid encoding the mutant Cas12i2 polypeptide, for use in achieving a deletion in the DNA of a cell.

15. A method for preparing the composition according to any one of claims 3 to 6, 10 to 12, or 14.

16. A method for complexing a mutant Cas12i2 polypeptide according to any one of claims 1, 2, 5, or 12 with an RNA guide.

17. A method for complexing a mutant binary complex prepared according to the method of claim 16 with a target nucleic acid.

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