Compositions containing Cas12i2 mutant polypeptides and their use

Abstract

Providing a composition containing a Cas12i2 mutant polypeptide and its use. [Solution] The present invention relates to a mutant Cas12i2 polypeptide, a method for producing a mutant Cas12i2 polypeptide, a process for characterizing a mutant Cas12i2 polypeptide, cells containing a mutant Cas12i2 polypeptide, and a method for using a mutant Cas12i2 polypeptide. The present invention further relates to a complex containing a mutant Cas12i2 polypeptide, a method for producing a complex, a process for characterizing a complex, cells containing a complex, and a method for using a complex.

Description

[Technical Field]

[0001] Sequence List This application includes a sequence listing submitted electronically in ASCII format, and this sequence listing is in its entirety. The aforementioned ASCII was incorporated herein by reference on March 31, 2021. The copy has the name 51451-008WO5_Sequence_Listing_03 It is named _30_2021_ST25 and has a size of 1,589,401 bytes. [Background technology]

[0002] Clustered and regularly arranged short palindromic repeats (CRISPR) and CRIS PR-related (Cas) genes, collectively known as CRISPR-Cas or CRISPR / Cas genes. Stems, also known as stems, are genetic elements that differentiate a particular species from exogenous genetic elements in archaea and bacteria. It is a defensive, adaptive immune system. [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 disclosed herein is not limited to specific advantages or functions, but the present invention is As shown in any one of sequence numbers 3-146 or any one of sequence numbers 495-512 A variant Cas12i2 polypeptide containing a sequence that has at least 95% identity with the original sequence. We will provide the service.

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

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

[0007] In another embodiment of the mutant Cas12i2 polypeptide, the mutant Cas12i2 polypeptide Petido contains the sequence shown in sequence number 495.

[0008] In another embodiment of the mutant Cas12i2 polypeptide, the mutant Cas12i2 polypeptide Petitdo contains the sequence shown in sequence number 496.

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

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

[0011] The present invention relates to a set comprising the variant Cas12i2 polypeptide as described herein. Further products are provided, and this composition is an RNA guide or a nucleus encoding an RNA guide. The RNA guide further contains acid, and includes direct repeat sequences and spacer sequences.

[0012] In one embodiment of the composition, the direct repeat sequence is one of the sequence numbers 492 to 494. It contains a nucleotide sequence that has at least 95% sequence identity with any one of them.

[0013] In another embodiment of the composition, the direct repeat sequence is sequence numbers 492-494. It contains the nucleotide sequence shown in one of the following.

[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 binds to a target nucleic acid sequence, and this target nucleic acid The sequence is adjacent to the 5'-NTTN-3' sequence.

[0017] In another embodiment of the mutant Cas12i2 polypeptide or composition, mutant Cas12 i2 polypeptide has at least one nuclear localization signal (NLS) and at least one Nuclear export signal (NES) or at least one NLS and at least one NES It also includes.

[0018] In another embodiment of the mutant Cas12i2 polypeptide or composition, mutant Cas12 i2 polypeptide contains peptide tags, fluorescent proteins, base editing domains, and DNA methylation. Modification domains, histone residue modification domains, localization factors, transcription modifiers, light-dependent regulatory factors The following further include chemical-inducible factors or chromatin-visualizing factors.

[0019] In another embodiment of the composition, the composition comprises nanoparticles, liposomes, exosomes, and microvesicles. Alternatively, they exist in delivery systems, including gene guns.

[0020] The present invention encodes the variant Cas12i2 polypeptide as described herein. Further nucleic acid molecules are provided.

[0021] The present invention relates to the composition or variant Cas12i2 polypeptide as described herein. We will continue to provide cells containing [the substance].

[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 relates to the variant Cas12i2 polypeptide as described herein, and optional Further, compositions or formulations comprising, by selection, RNA guides and / or cells are provided.

[0026] The present invention further provides a composition comprising a mutant Cas12i2 polypeptide and an RNA guide. Furthermore, the Cas12i2 mutant polypeptide and the RNA guide form a complex. This variant Cas12i2 polypeptide exhibits differences in RNA guidance and performance compared to the parent polypeptide. It exhibits increased complex formation.

[0027] The present invention further provides a composition comprising a mutant Cas12i2 polypeptide and an RNA guide. Furthermore, the Cas12i2 mutant polypeptide and the RNA guide form a complex. This Cas12i2 mutant polypeptide, compared to the parent polypeptide, interacts with the RNA guide. It exhibits increased binding affinity.

[0028] The present invention further provides a composition comprising a mutant Cas12i2 polypeptide and an RNA guide. Furthermore, the Cas12i2 mutant polypeptide and the RNA guide form a complex. And Cas12i2 mutant polypeptides and RNA guides are the parent polypeptide and RNA Compared to the guide, it exhibits increased protein-RNA interactions.

[0029] In one embodiment of the composition, the variant Cas12i2 polypeptide is used at a certain temperature range, for example. For example, increased complex formation and increased binding affinity to RNA guides occurred between 20°C and 65°C. It exhibits increased stability and / or improved performance.

[0030] In another embodiment of the composition, the variant Cas12i2 polypeptide is used in a range of inks. Over the duration of the experiment, increased complex formation and increased binding affinity to the RNA guide were observed. And / or exhibit increased stability.

[0031] In another embodiment of the composition, the variant Cas12i2 polypeptide is approximately 7.3 to approximately 8. In a buffer with a pH in the range of 6, increased complex formation and increased RNA guide It exhibits improved binding affinity and / or increased stability.

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

[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 is combined with the parent polypeptide. It exhibits equivalent or superior enzyme activity.

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

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

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

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

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

[0040] In another embodiment of the composition, the variant Cas12i2 polypeptide is a modified ONTER It exhibits a GET join.

[0041] In another embodiment of the composition, the variant Cas12i2 polypeptide is modified off the It exhibits a GET join.

[0042] The present invention further provides a composition comprising a mutant Cas12i2 polypeptide and an RNA guide. Furthermore, the Cas12i2 mutant polypeptide and RNA guide are provided as a mutant binary complex. This mutant binary complex is formed by the parent polypeptide and the RNA guide. It exhibits reduced complex dissociation compared to the complex.

[0043] The present invention further provides a composition comprising a mutant Cas12i2 polypeptide and an RNA guide. Furthermore, the Cas12i2 mutant polypeptide and RNA guide are provided as a mutant binary complex. In mutant C, this RNA guide is reduced compared to the RNA guide of the parent complex. It exhibits dissociation from the as12i2 polypeptide.

[0044] The present invention further provides a composition comprising a mutant Cas12i2 polypeptide and an RNA guide. Furthermore, the Cas12i2 mutant polypeptide and RNA guide are provided as a mutant binary complex. This mutant binary complex is formed by the parent polypeptide and the RNA guide. It exhibits increased stability compared to the complex.

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

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

[0047] In another embodiment of the composition, the mutant binary complex has a pH in the range of about 7.3 to about 8.6 It exhibits increased stability in the buffer solution it contains.

[0048] In another embodiment of the composition, the mutant binary complex is the T of the mutant binary complex. m Value is parent composite Body T m When the temperature is at least 8°C higher than the value, ternary complex formation increases, and the target nucleic acid increases. It exhibits improved binding affinity 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 is combined with the parent polypeptide. It exhibits equivalent or superior enzyme activity.

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

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

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

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

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

[0056] In another embodiment of the composition, the variant Cas12i2 polypeptide is a modified ONTER It exhibits a GET join.

[0057] In another embodiment of the composition, the variant Cas12i2 polypeptide is modified off the It exhibits a GET join.

[0058] The present invention relates to the mutant Cas12i2 polypeptide as described herein, The RNA guide, for example, the method of complexing with the RNA guide, is described in the manual. We will also offer more.

[0059] The present invention comprises a variant Cas12i2 polypeptide, an RNA guide, and a target nucleic acid. The composition further provides a variant Cas12i2 polypeptide and an RNA guide, It forms a variant binary complex, and this variant binary complex, compared to the parent binary complex, targets the nucleic acid It exhibits increased ternary complex formation.

[0060] The present invention comprises a variant Cas12i2 polypeptide, an RNA guide, and a target nucleic acid. The composition further provides that the Cas12i2 mutant polypeptide and RNA guide are modified It forms a variant binary complex, and this variant binary complex, compared to the parent binary complex, targets the nucleic acid It exhibits increased binding affinity to [the substance].

[0061] The present invention comprises a variant Cas12i2 polypeptide, an RNA guide, and a target nucleic acid. The composition further provides that the Cas12i2 mutant polypeptide and RNA guide are modified It forms a heterozygous binary complex, and this mutant binary complex is increased compared to the parent binary complex. It exhibits protein-RNA interactions.

[0062] The present invention comprises a variant Cas12i2 polypeptide, an RNA guide, and a target nucleic acid. The composition further provides that the Cas12i2 mutant polypeptide and RNA guide are modified It forms a heterozygous binary complex, and this mutant binary complex is increased compared to the parent binary complex. It exhibits protein-DNA interactions.

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

[0064] In another embodiment of the composition, the mutant binary complex undergoes a certain range of incubation time. Over the course of the process, increased ternary complex formation, increased binding affinity to target nucleic acids and / or increased It exhibits stability.

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

[0066] In another embodiment of the composition, the mutant binary complex is the T of the binary complex. m The value is the parent binary complex T m When the temperature is at least 8°C higher than the value, ternary complex formation increases, and the target nucleic acid increases. It exhibits increased binding affinity 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 aspect of the composition, the mutant binary complex exhibits enzymatic activity equal to or greater than that of the parent binary complex. It exhibits this characteristic.

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

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

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

[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 mutant Cas12i2 polypeptide has increased RNA It exhibits id complex formation, RNA guide binding activity, and / or RNA guide binding specificity.

[0075] In another embodiment of the composition, the mutant binary complex has increased target nucleic acid complex formation, target It exhibits 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. ru.

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

[0078] The present invention comprises a variant Cas12i2 polypeptide, an RNA guide, and a target nucleic acid. The composition further provides that the Cas12i2 mutant polypeptide and RNA guide are modified It forms a heterobinary complex, and the mutant binary complex and the target nucleic acid form a mutant ternary complex. This mutant ternary complex exhibits reduced complex dissociation compared to the parent ternary complex.

[0079] The present invention comprises a variant Cas12i2 polypeptide, an RNA guide, and a target nucleic acid. The composition further provides that the Cas12i2 mutant polypeptide and RNA guide are modified It forms a heterobinary complex, and the mutant binary complex and the target nucleic acid form a mutant ternary complex. This target nucleic acid exhibits dissociation from the mutant ternary complex, which is reduced compared to the parent ternary complex. do.

[0080] The present invention comprises a variant Cas12i2 polypeptide, an RNA guide, and a target nucleic acid. The composition further provides that the Cas12i2 mutant polypeptide and RNA guide are modified It forms a heterobinary complex, and the mutant binary complex and the target nucleic acid form a mutant ternary complex. This mutant ternary complex exhibits increased stability compared to the parent ternary complex.

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

[0082] In another embodiment of the composition, the mutant ternary complex undergoes a certain incubation period. It exhibits increased stability over time.

[0083] In another embodiment of the composition, the mutant ternary complex has a pH in the range of about 7.3 to about 8.6 It exhibits increased stability in the buffer solution it contains.

[0084] In another embodiment of the composition, the mutant ternary complex is the T of the mutant ternary complex. m The value is parent sangen T of the 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 is combined with the parent polypeptide. It exhibits equivalent or superior enzyme activity.

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

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

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

[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 mutant Cas12i2 polypeptide has increased RNA It exhibits id complex formation, RNA guide binding activity, and / or RNA guide binding specificity.

[0094] In another embodiment of the composition, the mutant binary complex has increased target nucleic acid complex formation, target It exhibits 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. ru.

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

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

[0098] The present invention further provides a composition comprising a mutant Cas12i2 polypeptide and an RNA guide. Furthermore, the mutant Cas12i2 polypeptide and RNA guide are provided as a mutant binary complex. This mutant binary complex forms an increased bond to the target nucleic acid compared to the parent binary complex. It exhibits affinity.

[0099] The present invention further provides a composition comprising a mutant Cas12i2 polypeptide and an RNA guide. Furthermore, the Cas12i2 mutant polypeptide and RNA guide are provided as a mutant binary complex. This mutant binary complex forms a target gene locus of the target nucleic acid, compared to the parent binary complex. It exhibits increased target binding affinity.

[0100] In one embodiment of the composition, the mutant binary complex is heated to a certain temperature range, for example, 20°C to 65°C. It exhibits increased ternary complex formation and / or increased stability over a range of °C.

[0101] In another embodiment of the composition, the mutant binary complex undergoes a certain range of incubation time. Over this period, it exhibits increased ternary complex formation and / or increased stability.

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

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

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

[0105] In another aspect of the composition, the variant binary complex has enzyme activity equal to or higher than that of the parent binary complex .

[0106] In another aspect of the composition, the equal or higher enzyme activity is within a temperature range of about 20°C to about 90°C observed.

[0107] In another aspect of the composition, the variant binary complex has increased stability and / or protei n-RNA interaction.

[0108] In another aspect of the composition, the variant binary complex has increased stability and / or protei n-DNA interaction.

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

[0110] In another aspect of the composition, the variant binary complex further exhibits increased enzymatic activity.

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

[0112] In another aspect of the composition, the variant binary complex has increased target nucleic acid complex formation, target It exhibits 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. ru.

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

[0115] This invention relates to multiple variant Cas12i2 polypeptides and two or more distinct RNA guides. The present invention further provides a composition comprising, and each RNA guide is individually, separately, distinct variant Ca It forms a mutant binary complex with the s12i2 polypeptide, and these mutant binary complexes are It specifically binds to two or more target gene loci of the target nucleic acid.

[0116] This invention relates to multiple variant Cas12i2 polypeptides and two or more distinct RNA guides. The present invention further provides a composition comprising, and each RNA guide is individually, separately, distinct variant Ca The s12i2 polypeptide forms a mutant binary complex, and these mutant binary complexes are parent Compared to the binary complex, the on-target complex shows increased on-target activity at two or more target gene loci of the target nucleic acid. It presents a consensus.

[0117] This invention relates to multiple variant Cas12i2 polypeptides and two or more distinct RNA guides. The present invention further provides a composition comprising, and each RNA guide is individually, separately, distinct variant Ca The s12i2 polypeptide forms a mutant binary complex, and these mutant binary complexes are parent Compared to the binary complex, increased on-target activity with two or more target gene loci of the target nucleic acid. It exhibits a bond.

[0118] This invention relates to multiple variant Cas12i2 polypeptides and two or more distinct RNA guides. The present invention further provides a composition comprising, and each RNA guide is individually, separately, distinct variant Ca It forms a mutant binary complex with the s12i2 polypeptide, and these mutant binary complexes are It exhibits on-target ternary complex formation with two or more target gene loci of the target nucleic acid.

[0119] This invention relates to multiple variant Cas12i2 polypeptides and two or more distinct RNA guides. The present invention further provides a composition comprising, and each RNA guide is individually, separately, distinct variant Ca The s12i2 polypeptide forms a mutant binary complex, and these mutant binary complexes are parent Compared to binary complexes, ternary complex formation is increased with two or more target gene loci of the target nucleic acid. It exhibits this characteristic.

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

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

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

[0123] In another embodiment of the composition, the mutant binary complex is the T of the binary complex. m The value is the parent binary complex T mWhen the temperature is at least 8°C higher than the value, increased ternary complex formation with the target nucleic acid and / or It exhibits 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 is combined with the parent polypeptide. It exhibits equivalent or superior enzyme activity.

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

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

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

[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 mutant Cas12i2 polypeptide has increased RNA It exhibits id complex formation, RNA guide binding activity, and / or RNA guide binding specificity.

[0132] In another embodiment of the composition, the mutant binary complex increases target nucleic acid ternary complex formation. It exhibits 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. ru.

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

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

[0136] The present invention further provides a composition comprising a mutant Cas12i2 polypeptide and an RNA guide. Furthermore, the Cas12i2 mutant polypeptide and RNA guide are provided as a mutant binary complex. This mutant binary complex forms a target gene locus of the target nucleic acid, compared to the parent binary complex. It exhibits increased on-target binding affinity to the target.

[0137] The present invention further provides a composition comprising a mutant Cas12i2 polypeptide and an RNA guide. Furthermore, the mutant Cas12i2 polypeptide and RNA guide are provided as a mutant binary complex. This mutant binary complex forms a non-target gene of the target nucleic acid compared to the parent binary complex. It exhibits reduced binding affinity to the site.

[0138] The present invention further provides a composition comprising a mutant Cas12i2 polypeptide and an RNA guide. Furthermore, the mutant Cas12i2 polypeptide and RNA guide are provided as a mutant binary complex. This mutant binary complex forms a more on-target complex of target nucleic acids compared to the parent binary complex. It exhibits increased activity at the t gene locus.

[0139] The present invention further provides a composition comprising a mutant Cas12i2 polypeptide and an RNA guide. Furthermore, the mutant Cas12i2 polypeptide and RNA guide are provided as a mutant binary complex. This mutant binary complex forms a non-target gene of the target nucleic acid compared to the parent binary complex. It exhibits reduced activity at the site.

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

[0141] In another embodiment of the composition, the mutant binary complex undergoes a certain range of incubation time. This results in increased ternary complex formation and / or increased stability at the target gene locus. do.

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

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

[0144] In another embodiment of the composition, the parent binary complex comprises a parent poly(SEQ ID NO: 2) containing the amino acid sequence of SEQ ID NO: 2. Contains peptides.

[0145] In another embodiment of the composition, the variant Cas12i2 polypeptide is combined with the parent polypeptide. It exhibits equivalent or superior enzyme activity.

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

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

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

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

[0150] This invention relates to multiple variant Cas12i2 polypeptides and two or more distinct RNA guides. The present invention further provides a composition comprising, and each RNA guide is individually, separately, distinct variant Ca The s12i2 polypeptide forms a binary complex with multiple mutants, and these multiple mutant binary complexes The complex increases the number of target nucleic acids at two or more target gene loci compared to multiple parental binary complexes. It exhibits on-target binding.

[0151] This invention relates to multiple variant Cas12i2 polypeptides and two or more distinct RNA guides. The present invention further provides a composition comprising, and each RNA guide is individually, separately, distinct variant Ca The s12i2 polypeptide forms a binary complex with multiple mutants, and these multiple mutant binary complexes The complex reduces the target nucleic acid to two or more non-target gene loci compared to multiple parental binary complexes. It exhibits some off-target binding.

[0152] This invention relates to multiple variant Cas12i2 polypeptides and two or more distinct RNA guides. The present invention further provides a composition comprising, and each RNA guide is individually, separately, distinct variant Ca The s12i2 polypeptide forms a binary complex with multiple mutants, and these multiple mutant binary complexes The complex, compared to multiple parental binary complexes, is located at two or more target gene loci of the target nucleic acid. It exhibits increased on-target activity.

[0153] This invention relates to multiple variant Cas12i2 polypeptides and two or more distinct RNA guides. The present invention further provides a composition comprising, and each RNA guide is individually, separately, distinct variant Ca The s12i2 polypeptide forms a binary complex with multiple mutants, and these multiple mutant binary complexes The complex, compared to multiple parental binary complexes, is located at two or more non-target gene loci of the target nucleic acid. It exhibits reduced off-target activity.

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

[0155] In another embodiment of the composition, multiple mutant binary complexes undergo a range of incubation Over time, increased ternary complex formation and / or increase at the target gene locus of the target nucleic acid. It exhibits enhanced stability.

[0156] In another embodiment of the composition, the multiple mutant binary complexes are in the range of about 7.3 to about 8.6 In a buffer solution with pH, ​​the increased ternary complex shape of the target nucleic acid at the target gene locus It exhibits and / or increased stability.

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

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

[0159] In another embodiment of the composition, the variant Cas12i2 polypeptide is combined with the parent polypeptide. It exhibits equivalent or superior enzyme activity.

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

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

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

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

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

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

[0166] A mutation occurs in one embodiment of the composition described herein or the Cas12i2 polypeptide. The Cas12i2 polypeptide has amino acid sequences D581, G624, F in SEQ ID NO: 2. 626, D835, L836, P868, S879, D911, I926, V1020, Includes at least one of the V1030, E1035, and S1046 substitutions.

[0167] In another embodiment of the composition or Cas12i2 polypeptide, a variant Cas12i2 polypeptide The lipeptide consists of amino acids D581G, D581R, G624R, and F6 in the amino acid sequence of SEQ ID NO: 2. 26G, F626R, D835G, D835R, L836G, L836R, P868G, P868R, P868T, S879G, S879R, D911G, D911R, I926 G, I926R, V1020G, V1020R, V1030G, V1030R, E103 Includes at least one of 5G, E1035R, S1046G, and S1046R substitutions.

[0168] In another embodiment of the composition or Cas12i2 polypeptide, a variant Cas12i2 polypeptide The lipeptide consists of D581R, G624R, F626R, and P8 amino acids in the amino acid sequence of SEQ ID NO. 2. Replacement for 68T, D911R, I926R, V1030G, E1035R and S1046G Includes at least one.

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

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

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

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

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

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

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

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

[0177] In another aspect of the composition, the target nucleic acid is present in a cell.

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

[0179] The present invention still further provides a cell comprising the composition as described herein.

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

[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 composition as described herein. .

[0183] The present invention relates to a kit comprising a composition or one or more components thereof as described herein. Alternatively, we will continue to provide the system.

[0184] In one embodiment of the composition described herein, the RNA guide is 43 nucleotides or It contains or consists of approximately 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 Cas12i2 polypeptide, a variant Cas12i2 polypeptide Lipeptides exhibit enzymatic activity approximately 40 times higher than that of the parent polypeptide.

[0187] In another embodiment of the composition or Cas12i2 polypeptide, a variant Cas12i2 polypeptide Lipeptides exhibit increased on-target specificity compared to their parent polypeptides.

[0188] In another embodiment of the composition or Cas12i2 polypeptide, a variant Cas12i2 polypeptide Lipeptides exhibit reduced off-target specificity compared to their parent polypeptides.

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

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

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

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

[0193] In another aspect of the composition, the deletion is in a gene of a cell.

[0194] In another aspect of the composition, the deletion is up to about 40 nucleotides in length.

[0195] In another aspect of the composition, the deletion is from about 4 nucleotides to 40 nucleotides in length.

[0196] In another aspect of the composition, the deletion is from about 4 nucleotides to 25 nucleotides in length.

[0197] In another aspect of the composition, the deletion is from about 10 nucleotides to 25 nucleotides in length .

[0198] In another aspect of the composition, the deletion is from about 10 nucleotides to 15 nucleotides in length .

[0199] In another aspect of the composition, the deletion starts within about 5 nucleotides to about 15 nucleotides of the 5'-NTTN-3' sequence.

[0200] In another aspect of the composition, the deletion is approximately 5 nucleotides in the 5'-NTTN-3' sequence ~ approximately It begins within 10 nucleotides.

[0201] In another aspect of the composition, the deletion is approximately 10 nucleotides in the 5'-NTTN-3' sequence. It begins within approximately 15 nucleotides.

[0202] In another aspect of the composition, the deletion is approximately 5 nucleotides downstream of the 5'-NTTN-3' sequence. It starts within approximately 15 nucleotides.

[0203] In another aspect of the composition, the deletion is approximately 5 nucleotides downstream of the 5'-NTTN-3' sequence. It starts within approximately 10 nucleotides.

[0204] In another aspect of the composition, the deletion is approximately 10 nucleos downstream of the 5'-NTTN-3' sequence. The cytoplasm begins within approximately 15 nucleotides.

[0205] In another aspect of the composition, the deletion is approximately 20 nucleotides in the 5'-NTTN-3' sequence. It ends within approximately 30 nucleotides.

[0206] In another aspect of the composition, the deletion is approximately 20 nucleotides in the 5'-NTTN-3' sequence. It finishes within approximately 25 nucleotides.

[0207] In another aspect of the composition, the deletion is approximately 25 nucleotides in the 5'-NTTN-3' sequence. It ends within approximately 30 nucleotides.

[0208] In another aspect of the composition, the deletion is located about 20 nucleos downstream of the 5'-NTTN-3' sequence. The nucleotide sequence ends within approximately 30 nucleotides.

[0209] In another aspect of the composition, the deletion is located about 20 nucleos downstream of the 5'-NTTN-3' sequence. The nucleotide sequence ends within approximately 25 nucleotides.

[0210] In another aspect of the composition, the deletion is approximately 25 nucleos downstream of the 5'-NTTN-3' sequence. The nucleotide sequence ends within approximately 30 nucleotides.

[0211] In another aspect of the composition, the deletion is approximately 5 nucleotides downstream of the 5'-NTTN-3' sequence. It starts within approximately 15 nucleotides and is located approximately 20 nucleotides downstream of the 5'-NTTN-3' sequence. The rheotide ends within approximately 30 nucleotides.

[0212] In another aspect of the composition, the deletion is approximately 5 nucleotides downstream of the 5'-NTTN-3' sequence. It starts within approximately 15 nucleotides and is located approximately 20 nucleotides downstream of the 5'-NTTN-3' sequence. The rheotide ends within approximately 25 nucleotides.

[0213] In another aspect of the composition, the deletion is approximately 5 nucleotides downstream of the 5'-NTTN-3' sequence. It starts within approximately 15 nucleotides and is located approximately 25 nucleotides downstream of the 5'-NTTN-3' sequence. The rheotide ends within approximately 30 nucleotides.

[0214] In another aspect of the composition, the deletion is approximately 5 nucleotides downstream of the 5'-NTTN-3' sequence. It starts within approximately 10 nucleotides and is located approximately 20 nucleotides downstream of the 5'-NTTN-3' sequence. The rheotide ends within approximately 30 nucleotides.

[0215] In another aspect of the composition, the deletion is approximately 5 nucleotides downstream of the 5'-NTTN-3' sequence. It starts within approximately 10 nucleotides and is located approximately 20 nucleotides downstream of the 5'-NTTN-3' sequence. The rheotide ends within approximately 25 nucleotides.

[0216] In another aspect of the composition, the deletion is approximately 5 nucleotides downstream of the 5'-NTTN-3' sequence. It starts within approximately 10 nucleotides and is located approximately 25 nucleotides downstream of the 5'-NTTN-3' sequence. The rheotide ends within approximately 30 nucleotides.

[0217] In another aspect of the composition, the deletion is approximately 10 nucleos downstream of the 5'-NTTN-3' sequence. It begins within approximately 15 nucleotides and is located approximately 20 nucleotides downstream of the 5'-NTTN-3' sequence. Cleotide ends within approximately 30 nucleotides.

[0218] In another aspect of the composition, the deletion is approximately 10 nucleos downstream of the 5'-NTTN-3' sequence. It begins within approximately 15 nucleotides and is located approximately 20 nucleotides downstream of the 5'-NTTN-3' sequence. Cleotide ends within approximately 25 nucleotides.

[0219] In another aspect of the composition, the deletion is approximately 10 nucleos downstream of the 5'-NTTN-3' sequence. It starts within approximately 15 nucleotides of the 5'-NTTN-3' sequence and is located approximately 25 nucleotides downstream of the 5'-NTTN-3' sequence. Cleotide ends within approximately 30 nucleotides.

[0220] In another aspect 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' (where Y is C or T) Yes, B is any nucleotide except A, and D is any nucleotide except C. Yes, and R is either A or G.

[0221] In another aspect 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'- It is either GTTA-3' or 5'-GTTG-3'.

[0222] In another embodiment of the composition, the deletion is of a gene, e.g., B2M, TRAC, PDCD1 It's located in Exxon.

[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. 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. do.

[0230] In another embodiment of the composition, the deletion is induced in or originates 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 is a stem cell (e.g., totipotent stem cell). ) / omnipotent stem cells, pluripotent stem cells, multipotent stem cells, oligopotent stem cells They are induced into cytoplasm or unipotent stem cells, differentiated cells, or terminally differentiated cells.

[0235] In another embodiment of the composition, two or more (e.g., multiplexed targeted defects) Loss / deletion is induced.

[0236] The present invention further provides a method for achieving deletion in cells, and this method is described in the present specification. The mutant Cas12i2 polypeptide or complex as described in the book is used with the cell's DNA. This includes making contact.

[0237] The present invention relates to the mutant Cas12i2 polypeptide as described herein, and RN Further, the present invention provides a composition or formulation comprising Guide A and cells.

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

[0239] The present invention relates to the mutant Cas12i2 polypeptide as described herein, Further details regarding the RNA guide described in the manual, such as the method of complexing with the RNA guide, are still being proposed. To provide.

[0240] The present invention complexes a mutant binary complex with a target nucleic acid as described herein. Further methods will be provided.

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

[0242] The present invention further provides compositions comprising the mutant Cas12i2 polypeptide, The mutant Cas12i2 polypeptide, compared to the parent polypeptide, shows differences in the mutant Cas12i This includes substitutions that increase the interaction between polypeptides and nucleic acids.

[0243] In one embodiment of the composition, the variant Cas12i2 polypeptide is shown in SEQ ID NO: 4 Includes an array.

[0244] In another embodiment of the composition, the variant Cas12i2 polypeptide is sequence number 3, sequence number 3. It includes the sequence shown in number 5, sequence number 495, and sequence number 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 aspect of the composition, the interactions are aromatic, hydrophobic, van der Waals and / Alternatively, it is a cation-π interaction.

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

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

[0250] In another embodiment of the composition, the direct repeat sequence is sequence numbers 492-494. It contains a nucleotide sequence that has at least 95% sequence identity with one of the others.

[0251] In another embodiment of the composition, the direct repeat sequence is sequence numbers 492-494. It contains the nucleotide sequence shown in one of the following.

[0252] In another embodiment of the composition, the substitution is direct with the variant Cas12i2 polypeptide. Increases interaction with repeat sequences.

[0253] In another embodiment of the composition, the substitution increases binary complex formation compared to the parent polypeptide. To add.

[0254] In another embodiment of the composition, a binary complex comprising the variant Cas12i2 polypeptide is Compared to the parental binary complex, it exhibits increased stability.

[0255] In another embodiment of the composition, the substitutions are arginine, lysine, glutamine, and asparagine. The substitutions are histidine, tyrosine, or serine.

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

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

[0258] In another embodiment of the composition, the substitutions are those listed in Table 4.

[0259] In another embodiment of the composition, the variant Cas12i2 polypeptide is used in SEQ ID NOs: 3-14 The sequence shown in any one of 6 or any one of sequence numbers 495-512, and at least It also contains sequences with 95% identity.

[0260] In another embodiment of the composition, the variant Cas12i2 polypeptide is used in SEQ ID NOs: 3-14 The sequence shown in any one of 6 or any one of sequence numbers 495-512, and at least It also has 95% identity and includes sequences that further have the substitutions listed in Table 4.

[0261] In another embodiment of the composition, the variant Cas12i2 polypeptide is used in SEQ ID NOs: 3-14 It includes any one of the six sequences or any one of the sequences shown in sequence numbers 495 to 512.

[0262] In another embodiment of the composition, the variant Cas12i2 polypeptide is used in SEQ ID NOs: 3-14 It includes any one of 6 or any one of sequence numbers 495 to 512, and It further includes the substitutions listed in Table 4.

[0263] In another embodiment of the composition, the direct repeat sequence is shown in SEQ ID NO: 492, and The variant Cas12i2 polypeptide contains the sequence shown in Sequence ID No. 4.

[0264] In another embodiment of the composition, the direct repeat sequence is shown in SEQ ID NO: 492, and The variant Cas12i2 polypeptide is represented by SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 495 and Contains the sequence shown in one of the sequences of sequence number 496.

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

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

[0267] In another embodiment of the composition, the substitution involves the mutant Cas12i2 polypeptide and double-stranded DN Increase the interaction with A.

[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. To add.

[0270] In another embodiment of the composition, a ternary complex comprising the variant Cas12i2 polypeptide is Compared to the parent ternary complex, it exhibits increased stability.

[0271] In another embodiment of the composition, the substitutions are arginine, lysine, glutamine, and asparagine. , is substituted with histidine or serine.

[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 the Rec1 domain, the PI domain, or the Wedge This is a domain substitution.

[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 is used in SEQ ID NOs: 3-14 The sequence shown in any one of 6 or any one of sequence numbers 495-512, and at least It also contains sequences with 95% identity.

[0276] In another embodiment of the composition, the variant Cas12i2 polypeptide is used in SEQ ID NOs: 3-14 The sequence shown in any one of 6 or any one of sequence numbers 495-512, and at least It also has 95% identity and includes sequences that further have the substitutions listed in Table 5.

[0277] In another embodiment of the composition, the variant Cas12i2 polypeptide is used in SEQ ID NOs: 3-14 It includes any one of the six sequences or any one of the sequences shown in sequence numbers 495 to 512.

[0278] In another embodiment of the composition, the variant Cas12i2 polypeptide is used in SEQ ID NOs: 3-14 It includes any one of 6 or any one of sequence numbers 495 to 512, and It further includes the substitutions listed in Table 5.

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

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

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

[0282] In another embodiment of the composition, the substitution increases ternary complex formation compared to the parent polypeptide. To add.

[0283] In another embodiment of the composition, a ternary complex comprising the variant Cas12i2 polypeptide is Compared to the parent ternary complex, it exhibits increased stability.

[0284] In another embodiment of the composition, the substitutions are arginine, lysine, glutamine, and asparagine. , it is a histidine or alanine substitution.

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

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

[0287] In another embodiment of the composition, the substitutions are those listed in Table 6.

[0288] In another embodiment of the composition, the variant Cas12i2 polypeptide is used in SEQ ID NOs: 3-14 The sequence shown in any one of 6 or any one of sequence numbers 495-512, and at least It also contains sequences with 95% identity.

[0289] In another embodiment of the composition, the variant Cas12i2 polypeptide is used in SEQ ID NOs: 3-14 The sequence shown in any one of 6 or any one of sequence numbers 495-512, and at least It also has 95% identity and includes sequences that further have the substitutions listed in Table 6.

[0290] In another embodiment of the composition, the variant Cas12i2 polypeptide is used in SEQ ID NOs: 3-14 It includes any one of the six sequences or any one of the sequences shown in sequence numbers 495 to 512.

[0291] In another embodiment of the composition, the variant Cas12i2 polypeptide is used in SEQ ID NOs: 3-14 It includes any one of 6 or any one of sequence numbers 495 to 512, and It further includes the substitutions listed in Table 6.

[0292] In another embodiment of the composition, the substitution involves the mutant Cas12i2 polypeptide and DNA / R Increases interaction with NA hybrid molecules.

[0293] In another embodiment of the composition, the DNA / RNA hybrid molecule is an RNA guide. It is a heteroduplex containing a surgeon 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. To add.

[0296] In another embodiment of the composition, a ternary complex comprising the variant Cas12i2 polypeptide is Compared to the parent ternary complex, it exhibits increased stability.

[0297] In another embodiment of the composition, the substitutions are arginine, lysine, glutamine, and asparagine. , is substituted with histidine or serine.

[0298] In another embodiment of the composition, the substitution involves the Rec1 domain, the PI domain, and the Rec2 domain. This is a substitution in the in 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 is used in SEQ ID NOs: 3-14 The sequence shown in any one of 6 or any one of sequence numbers 495-512, and at least It also contains sequences with 95% identity.

[0301] In another embodiment of the composition, the variant Cas12i2 polypeptide is used in SEQ ID NOs: 3-14 The sequence shown in any one of 6 or any one of sequence numbers 495-512, and at least It also has 95% identity and includes sequences that further have the substitutions listed in Table 7.

[0302] In another embodiment of the composition, the variant Cas12i2 polypeptide is used in SEQ ID NOs: 3-14 It includes any one of the six sequences or any one of the sequences shown in sequence numbers 495 to 512.

[0303] In another embodiment of the composition, the variant Cas12i2 polypeptide is used in SEQ ID NOs: 3-14 It includes any one of 6 or any one of sequence numbers 495 to 512, and It further includes the substitutions listed in Table 7.

[0304] In another embodiment of the composition, the substitution is with the mutant Cas12i2 polypeptide, and (a) two The main strand of DNA double helix and / or (b) RNA guide spacer sequence and target strand It increases the interaction between the double hemisphere and the bases.

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

[0306] In another aspect of the composition, the interactions are aromatic, hydrophobic, van der Waals and / Alternatively, it is a cation-π interaction.

[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. To add.

[0309] In another embodiment of the composition, a ternary complex comprising the variant Cas12i2 polypeptide is Compared to the parent ternary complex, it exhibits increased stability.

[0310] In another embodiment of the composition, the substitutions are arginine, lysine, tryptophan, and phenyl Alanine, tyrosine, methionine, histidine, glutamine, threonine, or valine substitution That is the case.

[0311] In another embodiment of the composition, the substitution is the Wedge domain, the Rec1 domain, or Ru This is a substitution in the vC 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 is used in SEQ ID NOs: 3-14 The sequence shown in any one of 6 or any one of sequence numbers 495-512, and at least It also contains sequences with 95% identity.

[0314] In another embodiment of the composition, the variant Cas12i2 polypeptide is used in SEQ ID NOs: 3-14 The sequence shown in any one of 6 or any one of sequence numbers 495-512, and at least It also has 95% identity and includes sequences that further have the substitutions listed in Table 8.

[0315] In another embodiment of the composition, the variant Cas12i2 polypeptide is used in SEQ ID NOs: 3-14 It includes any one of the six sequences or any one of the sequences shown in sequence numbers 495 to 512.

[0316] In another embodiment of the composition, the variant Cas12i2 polypeptide is used in SEQ ID NOs: 3-14 It includes any one of 6 or any one of sequence numbers 495 to 512, and It further includes 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 variant Cas12i2 polypeptide is combined with the parent polypeptide. In comparison, it exhibits increased enzyme activity.

[0319] In another embodiment of the composition, the composition comprises a direct repeat sequence and a spacer sequence. It also contains RNA guides.

[0320] In another embodiment of the composition, the direct repeat sequence is sequence numbers 492-494. It contains a nucleotide sequence that has at least 95% sequence identity with one of the others.

[0321] In another embodiment of the composition, the direct repeat sequence is sequence numbers 492-494. It contains the nucleotide sequence shown in one of the following.

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

[0323] 239. In another embodiment of the composition, the direct repeat sequence is shown in SEQ ID NO: 492 Furthermore, the variant Cas12i2 polypeptide is represented by SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 4 Includes the sequence shown in either 95 or sequence number 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 compositions comprising the mutant Cas12i2 polypeptide, The mutant Cas12i2 polypeptide shows mutations in DNA binding compared to the parent polypeptide. This includes substitutions that increase the mobility of the Cas12i2 polypeptide.

[0327] In one embodiment of the composition, the variant Cas12i2 polypeptide is shown in SEQ ID NO: 4 Includes an array.

[0328] In another embodiment of the composition, the Cas12i2 polypeptide is represented by SEQ ID NO: 3, SEQ ID NO: 5 , including the sequence shown in either sequence number 495 or sequence number 496.

[0329] In another embodiment of the composition, the substitution is the mutant Cas12i2 polypeptide to the DNA. Increase the number of bonds.

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

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

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

[0333] In another embodiment of the composition, a ternary complex comprising the variant Cas12i2 polypeptide is Compared to the parent ternary complex, it exhibits increased stability.

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

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

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

[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 is used in SEQ ID NOs: 3-14 The sequence shown in any one of 6 or any one of sequence numbers 495-512, and at least It also contains sequences with 95% identity.

[0339] In another embodiment of the composition, the variant Cas12i2 polypeptide is used in SEQ ID NOs: 3-14 The sequence shown in any one of 6 or any one of sequence numbers 495-512, and at least It also has 95% identity and includes sequences that further have the substitutions listed in Table 9.

[0340] In another embodiment of the composition, the variant Cas12i2 polypeptide is used in SEQ ID NOs: 3-14 It includes any one of the six sequences or any one of the sequences shown in sequence numbers 495 to 512.

[0341] In another embodiment of the composition, the variant Cas12i2 polypeptide is used in SEQ ID NOs: 3-14 It includes any one of 6 or any one of sequence numbers 495 to 512, and It further includes the substitutions listed in Table 9.

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

[0343] In one embodiment of the composition, the variant Cas12i2 polypeptide is shown in SEQ ID NO: 4 Includes an array.

[0344] In another embodiment of the composition, the variant Cas12i2 polypeptide is sequence number 3, sequence number 3. It includes the sequence shown in number 5, sequence number 495, and sequence number 496.

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

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

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

[0348] In another embodiment of the composition, a ternary complex comprising the variant Cas12i2 polypeptide is Compared to the parent ternary complex, it exhibits increased stability.

[0349] In another embodiment of the composition, the substitution is aspartic acid, glutamic acid, arginine or This is a 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 is used in SEQ ID NOs: 3-14 The sequence shown in any one of 6 or any one of sequence numbers 495-512, and at least It also contains sequences with 95% identity.

[0352] In another embodiment of the composition, the variant Cas12i2 polypeptide is used in SEQ ID NOs: 3-14 The sequence shown in any one of 6 or any one of sequence numbers 495-512, and at least It also has 95% identity and includes sequences that further have the substitutions listed in Table 10.

[0353] In another embodiment of the composition, the variant Cas12i2 polypeptide is used in SEQ ID NOs: 3-14 It includes any one of the six sequences or any one of the sequences shown in sequence numbers 495 to 512.

[0354] In another embodiment of the composition, the variant Cas12i2 polypeptide is used in SEQ ID NOs: 3-14 It includes any one of 6 or any one of sequence numbers 495 to 512, and It further includes 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 variant Cas12i2 polypeptide is combined with the parent polypeptide. In comparison, it exhibits increased enzyme activity.

[0357] In another embodiment of the composition, the composition comprises a direct repeat sequence and a spacer sequence. It also contains RNA guides.

[0358] In another embodiment of the composition, the direct repeat sequence is sequence numbers 492-494. It contains a nucleotide sequence that has at least 95% sequence identity with one of the others.

[0359] In another embodiment of the composition, the direct repeat sequence is sequence numbers 492-494. It contains the nucleotide sequence shown in one of the following.

[0360] In another embodiment of the composition, the direct repeat sequence is shown in SEQ ID NO: 492, and The variant Cas12i2 polypeptide contains the sequence shown in Sequence 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 is represented by SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 495 and Contains the sequence shown in one of the sequences of sequence number 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 compositions comprising the mutant Cas12i2 polypeptide, The mutant Cas12i2 polypeptide, compared to the parent polypeptide, shows differences in the mutant Cas12i Includes substitutions that increase the on-target specificity of 2 polypeptides.

[0365] In one embodiment of the composition, the variant Cas12i2 polypeptide is shown in SEQ ID NO: 4 Includes an array.

[0366] In another embodiment of the composition, the variant Cas12i2 polypeptide is sequence number 3, sequence number 3. It includes the sequence shown in number 5, sequence number 495, and sequence number 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 is on-target ternary compared to the parent polypeptide. Increases complex formation.

[0370] In another embodiment of the composition, an on-target comprising a variant Cas12i2 polypeptide is provided. The ternary complex exhibits increased stability compared to the parental ternary complex.

[0371] In another embodiment of the composition, the substitution involves an amino acid in contact with the spacer sequence of the RNA guide. This is acid substitution.

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

[0373] In another embodiment of the composition, the substitution is alanine, serine, valine, glutamine or as This is a paragine substitution.

[0374] In another embodiment of the composition, the substitution is Wedge domain, Rec1 domain, Rec This is a substitution in two domains or the 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 is used in SEQ ID NOs: 3-14 The sequence shown in any one of 6 or any one of sequence numbers 495-512, and at least It also contains sequences with 95% identity.

[0378] In another embodiment of the composition, the variant Cas12i2 polypeptide is used in SEQ ID NOs: 3-14 The sequence shown in any one of 6 or any one of sequence numbers 495-512, and at least It also has 95% identity and includes sequences that further have the substitutions listed in Table 11.

[0379] In another embodiment of the composition, the variant Cas12i2 polypeptide is used in SEQ ID NOs: 3-14 It includes any one of the six sequences or any one of the sequences shown in sequence numbers 495 to 512.

[0380] In another embodiment of the composition, the variant Cas12i2 polypeptide is used in SEQ ID NOs: 3-14 It includes any one of 6 or any one of sequence numbers 495 to 512, and It further includes the substitutions listed in Table 11.

[0381] In another embodiment of the composition, substitution is used to catalyze the reaction rate of the mutant Cas12i2 polypeptide. Decrease the degree (Kcat).

[0382] In another embodiment of the composition, the substitutions are alanine, serine, threonine, valine, leucine. The substitutions are methionine, asparagine, or isoleucine.

[0383] In another embodiment of the composition, the substitution is Wedge domain, Rec1 domain, Rec This is a substitution in two domains or the 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 is used in SEQ ID NOs: 3-14 The sequence shown in any one of 6 or any one of sequence numbers 495-512, and at least It also contains sequences with 95% identity.

[0387] In another embodiment of the composition, the variant Cas12i2 polypeptide is used in SEQ ID NOs: 3-14 The sequence shown in any one of 6 or any one of sequence numbers 495-512, and at least It also has 95% identity and includes sequences that further have the substitutions listed in Table 12.

[0388] In another embodiment of the composition, the variant Cas12i2 polypeptide is used in SEQ ID NOs: 3-14 It includes any one of the six sequences or any one of the sequences shown in sequence numbers 495 to 512.

[0389] In another embodiment of the composition, the variant Cas12i2 polypeptide is used in SEQ ID NOs: 3-14 It includes any one of 6 or any one of sequence numbers 495 to 512, and It further includes the substitutions listed in Table 12.

[0390] In another embodiment of the composition, the variant Cas12i2 polypeptide is combined with the parent polypeptide. In comparison, it exhibits increased on-target enzyme activity.

[0391] In another embodiment of the composition, the variant Cas12i2 polypeptide is combined with the parent polypeptide. In comparison, it exhibits reduced off-target enzyme activity.

[0392] In another embodiment of the composition, the variant Cas12i2 polypeptide is used in on-target editing. It exhibits off-target editing, with less than 10% of the content being edited.

[0393] In another embodiment of the composition, the variant Cas12i2 polypeptide is used in on-target editing. It exhibits off-target editing, with less than 5% of the content being edited.

[0394] In another embodiment of the composition, the composition comprises a direct repeat sequence and a spacer sequence. It also contains RNA guides.

[0395] In another embodiment of the composition, the direct repeat sequence is sequence numbers 492-494. It contains a nucleotide sequence that has at least 95% sequence identity with one of the others.

[0396] In another embodiment of the composition, the direct repeat sequence is sequence numbers 492-494. It contains the nucleotide sequence shown in one of the following.

[0397] In another embodiment of the composition, the direct repeat sequence is shown in SEQ ID NO: 492, and The variant Cas12i2 polypeptide contains the sequence shown in Sequence 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 is represented by SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 495 and Contains the sequence shown in one of the sequences of sequence number 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 is Cas9 polypeptide Compared to cytoplasm, it exhibits a higher ratio of on-target binding to off-target binding. .

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

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

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

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

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

[0407] In another embodiment of the composition, off-target effects are provided by the variant Cas12i2 polypeptide. Binding, off-target activity, and / or off-target editing are performed by the Cas9 polypeptide. Less than off-target binding, off-target activity, and / or off-target editing. Both are 10% less.

[0408] In another embodiment of the composition, off-target effects are provided by the variant Cas12i2 polypeptide. Binding, off-target activity, and / or off-target editing are performed by the Cas9 polypeptide. Less than off-target binding, off-target activity, and / or off-target editing. Both are 20% less.

[0409] In another embodiment of the composition, off-target effects are provided by the variant Cas12i2 polypeptide. Binding, off-target activity, and / or off-target editing are performed by the Cas9 polypeptide. Less than off-target binding, off-target activity, and / or off-target editing. Both are 30% less.

[0410] In another embodiment of the composition, off-target effects are provided by the variant Cas12i2 polypeptide. Binding, off-target activity, and / or off-target editing are performed by the Cas9 polypeptide. Less than off-target binding, off-target activity, and / or off-target editing. Both are 40% less.

[0411] In another embodiment of the composition, off-target effects are provided by the variant Cas12i2 polypeptide. Binding, off-target activity, and / or off-target editing are performed by the Cas9 polypeptide. Less than off-target binding, off-target activity, and / or off-target editing. Both are 50% less.

[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 figures, The present invention is not limited thereto, but is limited only by the claims, as shown below. Unless otherwise specified, terms used are generally understood to have their common meaning. ru.

[0415] As used herein, the term “activity” refers to biological activity. In some embodiments In this context, activity refers to effector activity. In some embodiments, activity includes enzyme activity. Activity includes, for example, the catalytic ability of an effector. For example, activity includes nuclease activity. This may include. In some embodiments, the activity may include binding activity, for example, by an effector. This includes binding activity to RNA guides and / or target nucleic acids.

[0416] As used herein, the term “adjacent to” means a nucleotide or an amino acid. This refers to a sequence being in close proximity to another nucleotide or amino acid sequence. In some embodiments, In this context, one nucleotide sequence separates the two nucleotide sequences. They are adjacent when there is no nucleotide. In some embodiments, the nucleotide sequence is For a different nucleotide sequence, those two sequences are divided into a small number of nucleotides (for example, about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 Adjacent when separated (18, 19, or 20 nucleotides). Some embodiments In this case, the first sequence is relative to the second sequence, and the two sequences are approximately 5, 6, 7, 8, 9 Adjacent when separated by 10, 11, 12, 13, 14, or 15 nucleotides. In some embodiments, the term "adjacent to" means that the protein residues are separate. It is used to refer to interacting with protein residues. In some embodiments, The phrase "adjacent to" means that a protein residue interacts with a nucleotide or nucleic acid. It is used to refer to... In some embodiments, the term "adjacent to..." refers to... This refers to the interaction of one protein domain or motif with another protein domain or motif. It is used as follows. In some embodiments, the term "adjacent to" means protein This term is used to refer to the interaction of the main or motif with a nucleotide or nucleic acid sequence. When used herein, the term “adjacent to ~” means the modified cells of this disclosure. It is used to refer to the position of an indel (insertion / deletion).

[0417] As used herein, the term “complex” refers to an assembly of two or more molecules. In this embodiment, the complexes are interacting with each other (e.g., bound, in contact). It contains polypeptides and nucleic acid molecules that are in a state of being attached or bonded.

[0418] As used herein, the term “binary complex” means a combination of two molecules (e.g., polypeptide). This refers to an aggregate of (a and nucleic acid molecules). In some embodiments, the binary complex is a polypeptide complex. This refers to an assembly of a cydoid and a targeting portion (e.g., an RNA guide). (Some embodiments) In this context, the binary complex refers to ribonucleoprotein (RNP). At that time, the term "mutant binary complex" refers to the mutant Cas12i2 polypeptide and RNA guide This refers to a combination of the parent and the polypeptide. When used herein, the term "parent binary complex" means a combination of the parent and the polypeptide. This refers to an assembly of a reference polypeptide and an RNA guide, or a reference polypeptide and an RNA guide.

[0419] As used herein, the term “ternary complex” refers to a complex of three molecules (e.g., polypeptide). It refers to an aggregate of (and two nucleic acid molecules). In some embodiments, the "ternary complex" is This refers to an assembly of polypeptides, RNA molecules, and DNA molecules. In some embodiments, A ternary complex consists of a polypeptide, a targeting portion (e.g., an RNA guide), This refers to an aggregate with a target nucleic acid (e.g., a target DNA molecule). In some embodiments, "three The "original complex" consists of a binary complex (e.g., ribonucleoprotein) and a third molecule (e.g., target nucleus). Refers to an aggregate with an acid. When used herein, the terms "mutant ternary complex" and "mutant" are used. The "Cas12i2 ternary complex" and the "Cas12i2 mutant ternary complex" are mutant Ca The s12i2 polypeptide, the RNA guide, and the target nucleic acid (e.g., the Cas12 variant) i2 refers to the assembly of ribonucleoprotein and target nucleic acid. When used herein, the term "Parental ternary complex", "Parental Cas12i2 ternary complex", and "Cas12i2 parental ternary complex" " is a relationship between the parent polypeptide, the RNA guide, and the target nucleic acid (for example, the parent ribonucleoprotein (and the target nucleic acid) or a reference polypeptide, an RNA guide, and the target nucleic acid (for example, reference This refers to the aggregate of teribonucleoproteins and target nucleic acids.

[0420] As used herein, the term “deletion” means the loss or removal of a nucleotide from a nucleic acid sequence. It refers to deletion. A deletion can be a deletion in the genome of an organism. A deletion can be a deletion in a cell. It is possible. The deletion could be a DNA sequence. The deletion could be an RNA sequence. The deletion could be a f This may be a frame-shift mutation or a non-frame-shift mutation as described herein. Cas12i2-induced deletion refers to the loss of approximately 4 to 100 nucleotides from a nucleic acid molecule. Approximately 4 nucleotides to 50 nucleotides, approximately 4 nucleotides to 40 nucleotides, approximately 4 nucleotides Cleotide ~25 nucleotides, approximately 10 nucleotides ~25 nucleotides, approximately 10 nucleotides Ocides can refer to deletions of up to approximately 100 nucleotides, such as 15 nucleotides. In some embodiments, the Cas12i2-inducible deletion described herein is 5'- It appears downstream of the NTTN-3' sequence. In some embodiments, the term "Cas12i2 inducer" is used. "Induction" is a result of DNA cleavage being induced by the Cas12i2 polypeptide. This refers to a deletion. In some embodiments, the term "Cas12i2-induced" means Cas12 DNA breakage is induced by i2 polypeptide and repaired by the cell's DNA repair mechanism. This refers to the deletion that results from this process.

[0421] As used herein, the term “domain” refers to the distinct functional and / Alternatively, it refers to a structural unit. In some embodiments, a domain is a conserved amino acid sequence. It may include.

[0422] As used herein, the terms “editing efficiency” and “indel activity” refer to enzymes (e.g., The ability of the mutant Cas12i2 polypeptide to introduce indels (insertions / deletions) into the sequence. This refers to, for example, in some embodiments, an indel is introduced to each of the 10 target gene loci. The enzyme used exhibits 100% editing efficiency. It is used in 5 out of 10 target gene loci. The enzyme that introduces Dell exhibits a 50% editing efficiency. In another example, 50% of multiple cells In % of cases, the enzyme that introduces indels into the target gene locus exhibits a 50% editing efficiency. When used in detail, the terms "editing efficiency," "indel activity," and "on-target editing" are used. This means that the enzyme (for example, the mutant Cas12i2 polypeptide) creates indels at the target gene locus. This refers to the ability to selectively introduce genes. In some embodiments, the editing efficiency at the target gene locus is The editing efficiency is compared with that at a non-target locus. In some embodiments, at the target locus... Editing at this site is compared to editing at a non-target gene locus.

[0423] As used herein, the term “effector activity” refers to biological activity. In this embodiment, effector activity includes enzyme activity, such as the catalytic ability of the effector. This includes, for example, effector activity may include nuclease activity.

[0424] As used herein, the term “interface” refers to a nucleic acid molecule or a variant Cas12i2 poly Contact with a separate domain / motif of the peptide or a portion of a separate domain / motif. (For example, a variant Cas12i that interacts with or is adjacent to it) 2. One of the polypeptides (e.g., a domain / motif or a part of a domain / motif) This refers to the residues mentioned above. In some embodiments, the interface is embedded between adjacent domains or motifs. This is the surface area that is leaking. In some embodiments, the interface is between a polypeptide and a ligand (for example) The surface area where the polypeptide and ligand are in contact with each other (DNA or RNA) Yes. When used herein, the term “nucleic acid interface” means a nucleic acid sequence (e.g., DNA sequence). (or RNA sequence) is in close proximity to (e.g., adjacent to) or interacts with it This refers to residues of the variant Cas12i2 polypeptide. When used herein, the term The "RNA binding interface" is connected to the RNA guide (for example, the direct repeat of the RNA guide). A Cas1 mutant that is in close proximity (e.g., adjacent to it) or interacts with it. This refers to residues of the 2i2 polypeptide. When used herein, the term "double-stranded DNA bond" is used. The "interface" is adjacent to (for example, adjacent to) double-stranded DNA and / or it This refers to residues of the Cas12i2 polypeptide mutant that interact with it. At that time, the term "single-stranded DNA binding interface" refers to an interface that is in close proximity to single-stranded DNA (for example, to Residues of the mutant Cas12i2 polypeptide that are adjacent to and / or interact with it This refers to the interface between domains. When used herein, the term "interdomain interface" means that one domain is an interface between two domains. This refers to something that is close to the main (for example, adjacent to it). In some embodiments... Furthermore, interdomain interfaces (for example, the helix II domain-Nuc domain interface) are multiple It is formed during the formation of a composite (for example, during the formation of a ternary complex).

[0425] As used herein, the terms “parent,” “parent polypeptide,” and “parent sequence” are used in this specification. To create the new variant Cas12i2 polypeptide, the original polypeptide undergoes a change. Refers to a starter polypeptide (e.g., a starting polypeptide). In some embodiments, the parent is one or more A mutant with one or more mutations at a specified position has the same amino acid sequence as a polypeptide. In exemplary embodiments, a mutation is a change in an amino acid within a polypeptide sequence. Further details are provided. The parent molecule may be a naturally occurring (wild-type) polypeptide. In this case, the parent has at least 60%, at least 61%, and less than the polypeptide of Sequence ID No. 2. At least 62%, at least 63%, at least 64%, at least 65%, at least 7 0%, at least 72%, at least 73%, at least 74%, at least 75%, small 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%, and at least Polypeptide having 97%, at least 98%, at least 99%, or 100% identity It is a cydo. In some embodiments, mutations include joining, fusion, or other modifications of the parent. This may include structural modifications that do not alter the amino acid sequence. In some embodiments, The parent polypeptide sequence contains amino acid mutations and structural modifications.

[0426] As used herein, “multiple mutant binary complex” refers to multiple mutant Cas12 This refers to a binary complex containing multiple i2 polypeptides. It includes multiple parent polypeptides (e.g., sequence Multiple binary complexes, including the wild-type Cas12i2 polypeptide parent (number 2), are described herein. This is referred to as a "multiple parent binary complex." Multiple Cas12i2 polypeptides, for example, multiple A number of variant Cas12i2 polypeptides and two or more distinct targeting parts, for example If two or more separate RNA guides form two or more binary complexes (for example, multiple It forms a binary complex of the same. In such embodiments, complex formation is achieved with a single composition. This can sometimes be achieved by separate compositions, or independently.

[0427] When used herein, the terms “protospacer adjacent motif” or “PAM” are defined as follows: , targeting polypeptides (e.g., enzymes such as Cas12i2 or their variants) A binary complex containing a portion (e.g., an RNA guide) binds to DNA adjacent to the target sequence. Refers to the sequence. In some embodiments, PAM is required for enzyme activity. Double-stranded target site. In combination, the targeting portion (e.g., RNA guide) binds to the first strand of the target, as specified herein. The PAM sequence described in the second complementary strand is present. For example, in some embodiments In this configuration, the RNA guide binds to the target strand (e.g., the spacer complementary strand), as described herein. The PAM sequence is present in the non-target strand (i.e., the non-spacer complementary strand) as described. In the application configuration, 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,” and “reference” are used in this specification. The "reference complex" and "reference element" are negative controls or parental elements (e.g., parental sequence, parental protein, wild-type sequence). This refers to a control, such as a complex containing an protein or parent sequence. For example, a reference molecule is a mutant C This refers to the Cas12i2 polypeptide that is used as a comparison for the as12i2 polypeptide. The reference RNA guide refers to the targeting portion of the modified RNA guide that is used for comparison. The mutant or modified molecule has a sequence that is X% identical to the reference molecule (for example, the mutant or modified molecule has X% sequence identity). (may have sex or homology), thermal stability or activity (for example, a mutant or modified molecule may be a reference molecule) It can be compared to a reference molecule based on a reference molecule that may have X% of the activity of C. For example, if the reference molecule is C If it is an as12i2 polypeptide, then the variant or modified molecule is a reference Cas12i2 polypeptide. It can be characterized as having less than 10% activity of the peptide, or reference Cas12i2 It can be characterized as having at least 10% higher activity than the lipeptide. (See Cas12i) Examples of polypeptides include the naturally occurring unmodified Cas12i2 polypeptide, for example. Examples include naturally occurring Cas12i2 polypeptides derived from archaea or other bacterial species. In certain embodiments, the reference Cas12i2 polypeptide is used for comparison. A naturally occurring molecule with the closest sequence identity or homology to the mutant Cas12i2 polypeptide. This is a Cas12i2 polypeptide. In certain embodiments, refer to Cas12i2 The polypeptide undergoes mutations to obtain the mutant Cas12i2 polypeptide. It is a parent molecule that has a naturally occurring sequence or a known sequence.

[0429] When used herein, the terms “RNA guide” or “RNA guide sequence” are used in accordance with the foregoing. Any RNA that facilitates the targeting of polypeptides described in the document to target nucleic acids. It refers to a molecule. For example, an RNA guide recognizes a target nucleic acid (for example, it binds to it). It can be a molecule. RNA guides are designed to be complementary to specific nucleic acid sequences. This is possible. RNA guides use DNA targeting sequences and direct repeats (DR). ) Includes sequences. Terms CRISPR RNA (crRNA), precrRNA and mature In this specification, crRNA also refers to RNA guides.

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

[0431] As used herein, the term “targeting portion” refers to a targeting of another molecule or component. Molecules or components that facilitate targeting of nucleic acids (e.g., nucleic acids and / or RNA) This refers to (d). In some embodiments, the targeting portion specifically interacts with the target nucleic acid. To act upon or associate with something.

[0432] When used herein, the terms “target nucleic acid,” “target sequence,” “target substrate,” and “o A "target gene locus" is a locus where the targeting region (e.g., RNA guide) is specifically linked. Refers to matching nucleic acid sequences. In some embodiments, RNA guide DNA targeting The sequence binds to the target nucleic acid. The binding of the binary complex to the target gene locus is referred to herein as "O This is called "target bond".

[0433] As used herein, the terms “non-targeted” and “off-target” refer to targeting Nucleic acid sequences other than those to which the lig portion specifically binds or sequences to which it is intended to specifically bind Refers to a row. Non-target loci are not intended for targeting regions (e.g., RNA guides). It is an off-target target. The binding of the binary complex to a non-target gene locus is referred to herein as "off-target". This is referred to as "binding". In some embodiments, the non-target locus is a locus on the target nucleic acid. In some embodiments, non-target gene loci are nucleic acids other than the target nucleic acid (e.g., non It is a gene locus on the target nucleic acid.

[0434] As used herein, the terms “upstream” and “downstream” refer to a single nucleus in a nucleic acid molecule. Refers to the relative position within an acid (e.g., DNA) sequence. When used herein, the term "Upstream" and "downstream" refer to the relative positions of two sequences. The "flow" refers to the orientation from 5' to 3' where RNA transcription occurs. The first sequence is the first When the 3' end of the first sequence appears before the 5' end of the second sequence, it is upstream of the second sequence. The first sequence is defined as follows when the 5' end of the first sequence appears after the 3' end of the second sequence. It is located downstream of the array. When used herein, the terms “upstream” and “downstream” mean 5'- This term is used to refer to the relative position of the deletion relative to the NTTN'-3' sequence. When used, the 5'-NTTN-3' sequence is upstream of the Cas12i2-induced deletion, and The Cas12i2-induced deletion is located downstream of the 5'-NTTN-3' sequence.

[0435] When used herein, the terms “mutant Cas12i2 polypeptide” and “mutant” are used. "Effector polypeptides" have changes at one or more residue positions compared to the parent polypeptide. For example, this refers to polypeptides including substitution, insertion, deletion and / or fusion. When used, the terms "mutant Cas12i2 polypeptide" and "mutant effector polypeptide" are used. "Petido" refers to a polypeptide that contains changes compared to the polypeptide in Sequence 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 spacer sequence binds to the target nucleic acid sequence, and the target nucleic acid sequence is 5'-NTTN A composition according to any one of items 8 to 12, adjacent to the -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 C The as12i2 polypeptide is a composition that 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 aforementioned Cas12i2 polypeptide variant exhibits increased RNA guide complex formation, RNA guide binding activity, and / or RNA guide binding specificity, as per items 23-35. The composition described in item one. (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 mutant binary complex exhibits increased ternary complex formation, increased binding affinity to the target nucleic acid, and / or increased affinity in a buffer solution having a pH in the range of approximately 7.3 to 8.6. A composition that exhibits qualitative properties, as described in any one of items 56 to 61. (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, as described in 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, one of items 95-105. The composition described in the section. (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 mutant binary complex exhibits increased ternary complex formation and / or increased stability with the target nucleic acid over a certain incubation period, as described in items 112-117. The composition described in any one of the items. (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, The described mutant binary complex is a composition that 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 aforementioned mutant Cas12i2 polypeptide exhibits increased binary complex formation and RNA-guided binding. A composition according to any one of items 133 to 145, exhibiting synactivation 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 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 aforementioned mutant Cas12i2 polypeptide exhibits increased on-target specificity compared to the parent polypeptide, items 23-54, 56-93, 95-131, 133- A composition according to any one of the following items: 160, 163-175, or 181-183. (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 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 deletion is located approximately 5 to 10 nucleotides downstream of the 5'-NTTN-3' sequence. A composition according to any one of items 186 to 199, beginning within 10. (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 aforementioned 5'-NTTN-3' sequence is 5'-NTTY-3', 5'-NTTC-3', 5'-N TTT-3', 5'-NTTA-3', 5'-NTTB-3', 5'-NTTG-3', 5'-CTTY-3', 5'-DTTR'3', 5'-CTTR-3', 5'-DTTT-3', 5'-A TTN-3' or 5'-GTTN-3' (wherein Y is C or T, and B is any other than A) A composition according to any one of items 186 to 216, wherein D is any nucleotide other than C, and R is A or G. (Item 218) The aforementioned 5'-NTTN-3' sequence is 5'-CTTT-3', 5'-CTTC-3', 5'-G A composition according to any one of items 186 to 217, wherein the composition is TTT-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 item 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 aforementioned variant Cas12i2 polypeptide comprises the sequence shown in any one of SEQ ID NOs. 3, SEQ ID NOs. 5, SEQ ID NOs. 495, and SEQ ID NOs. 496, as described in item 239. (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 substitution is located at or adjacent to the nucleic acid interface, as described in item 239. (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 containing 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 mutant Cas12i2 polypeptide comprises the sequence shown in any one of SEQ ID NOs. 3 to 146 or any one of SEQ ID NOs. 495 to 512, and further comprises the substitutions listed in Table 5, as described in any one of items 239 to 245 or 262 to 270. (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 aforementioned mutant Cas12i2 polypeptide comprises the sequence shown in any one of SEQ ID NOs. 3 to 146 or any one of SEQ ID NOs. 495 to 512, and further comprises the substitutions listed in Table 6, as described in any one of items 239 to 245 or 276 to 283. (Item 289) The aforementioned substitution is performed on the mutant Cas12i2 polypeptide and the DNA / RNA hybrid. A composition according to any one of items 239 to 245 that increases interaction with the child. (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 aforementioned mutant Cas12i2 polypeptide comprises the sequence shown in any one of SEQ ID NOs. 3 to 146 or any one of SEQ ID NOs. 495 to 512, and further comprises the substitutions listed in Table 7, as described in any one of items 239 to 245 or 289 to 295. (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 interaction is aromatic, hydrophobic, van der Waals and / or cation-π interaction, as described in any one of items 239-245 or 301 or 302. . (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 mutant Cas12i2 polypeptide comprises the sequence shown in any one of SEQ ID NOs. 3 to 146 or any one of SEQ ID NOs. 495 to 512, and further comprises the substitutions listed in Table 8, as described in any one of items 239 to 245 or 301 to 308. (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 aforementioned variant Cas12i2 polypeptide comprises the sequence shown in any one of SEQ ID NOs. 3, SEQ ID NOs. 5, SEQ ID NOs. 495, and SEQ ID NOs. 496, as described in item 339. (Item 342) The interdomain interface is formed when single-stranded DNA comes into contact with the active site of the mutant Cas12i2 polypeptide, as described in any one of items 339 to 341. (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 aforementioned variant Cas12i2 polypeptide has 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. A composition according to any one of items 339 to 346, comprising the sequence. (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 aforementioned variant Cas12i2 polypeptide is represented by SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 495 The composition according to item 361, comprising the sequence shown in either of the sequences shown in or 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 aforementioned mutant Cas12i2 polypeptide exhibits fewer off-target bindings compared to the Cas9 polypeptide, as described in any one of items 365 to 395. (Item 400) The aforementioned mutant Cas12i2 polypeptide exhibits less off-target activity compared to the Cas9 polypeptide, as described in any one of items 365 to 396. (Item 401) The aforementioned variant Cas12i2 polypeptide exhibits less off-target editing compared to the Cas9 polypeptide, as described in any one of items 365-397. (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) Off-target binding and off-target activity by the aforementioned mutant Cas12i2 polypeptide. The composition according to any one of items 365 to 404, wherein the off-target activity and / or off-target editing by the Cas9 polypeptide is at least 40% less than the off-target binding, off-target activity and / or off-target editing 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 with 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 Sequence ID No. 2, as well as methods for their preparation and use. This disclosure relates to a complex comprising a variant of the effector of SEQ ID NO: 2, as well as its preparation and use. Further relating to the method. In some embodiments, this specification describes composites having one or more properties. A composition containing the following is described. In some embodiments, a method for delivering a composition containing the complex is described. It will be listed.

[0438] composition In some embodiments, the present invention described herein relates to a complex (e.g., a binary complex) The present invention includes a composition comprising Cas12 The composition includes a complex comprising an i2 polypeptide and a targeting moiety. Morphologically, the composition of the present invention comprises a mutant Cas12i2 polypeptide and an RNA guide. The variant Cas12i2 polypeptide, compared to the parent polypeptide, exhibits RNA-guided This shows an increase in complex formation with [the other party].

[0439] In some embodiments, the composition of the present invention comprises a mutant Cas12i2 polypeptide and RNA The complex includes a guide, where Cas12i2 mutant polypeptide and RNA The id has a higher binding affinity compared to the parent polypeptide and RNA guide.

[0440] In some embodiments, the composition of the present invention comprises a mutant Cas12i2 polypeptide and RNA The complex includes a guide, where Cas12i2 mutant polypeptide and RNA The id provides a stronger protein-RNA phase compared to the parent polypeptide and RNA guide. They have interactions. In some embodiments, protein-RNA interactions are ion-to-ion interactions. It is an action.

[0441] In some embodiments, the composition of the present invention comprises a mutant Cas12i2 polypeptide and RNA It includes a complex containing a guide, and this complex is formed by the parent polypeptide and the RNA guide. It is more stable than the complex that is formed.

[0442] In some embodiments, the present invention described herein relates to a complex (e.g., a ternary complex) The present invention includes a composition containing the variant Cas12i It contains two polypeptides, an RNA guide, and a target nucleic acid, and the mutant Cas12i2 polypeptide Compared to parent polypeptides, tides form complexes with RNA guides and target nucleic acids (for example). It shows an increase in (ternary complex formation).

[0443] In some embodiments, the composition of the present invention comprises a mutant Cas12i2 polypeptide and RNA The composition comprises a binary complex including a guide, and further comprises a target nucleic acid. Some embodiments In this context, the binary complex shows an increased tendency to form ternary complexes with target nucleic acids compared to the parental binary complex. This indicates.

[0444] In some embodiments, the compositions of the present invention include a mutant Cas12i2 polypeptide and RN A includes a guide and a target nucleic acid, where Cas12i2 variant polypeptide and RNA The guide exhibits higher binding to target nucleic acids compared to parent polypeptide and RNA guides. It has affinity.

[0445] In some embodiments, the composition of the present invention comprises a mutant Cas12i2 polypeptide and RNA The composition comprises a binary complex including a guide, and further comprises a target nucleic acid. Some embodiments In this case, the binary complex has a higher binding affinity to the target nucleic acid compared to the parent binary complex. It has a sexual nature.

[0446] In some embodiments, the compositions of the present invention include a mutant Cas12i2 polypeptide and RN The ternary complex comprises a guide A and a target nucleic acid, where the ternary complex is composed of parental polypeptides It is more stable than the complex formed by the RNA guide and the target nucleic acid.

[0447] In some embodiments, the composition of the present invention comprises a mutant Cas12i2 polypeptide and RNA The composition comprises a binary complex including a guide, and further comprises a target nucleic acid. Some embodiments In this context, the binary complex is formed by the parent binary complex and the target nucleic acid, rather than the ternary complex. It also forms a ternary complex with a highly stable target nucleic acid.

[0448] In some embodiments, the present invention described herein relates to a complex (e.g., a ternary complex) The present invention includes a composition containing the variant Cas12i This variant Cas12i2 polynucleotide comprises two polypeptides, an RNA guide, and a target nucleic acid. Peptide and RNA guides exhibit higher binding to target nucleic acids compared to the parent binary complex. They form a binary complex of mutants with affinity.

[0449] In some embodiments, the composition of the present invention comprises a mutant Cas12i2 polypeptide and RNA The composition comprises a binary complex including a guide, and further comprises a target nucleic acid. Some embodiments In this case, the binary complex, compared to the parental binary complex, is more effective with respect to the target gene locus of the target nucleic acid. It has a very high target binding affinity.

[0450] In some embodiments, the composition of the present invention comprises a plurality of variant Cas12i2 polypeptides and , including two or more separate RNA guides. In some embodiments, separate RNA guides Each of these molecules individually forms a mutant binary complex with a separate mutant Cas12i2 polypeptide. This mutant binary complex has two or more target gene loci of the target nucleic acid compared to the parent binary complex. It exhibits higher on-target binding.

[0451] In some embodiments, the composition of the present invention comprises a plurality of variant Cas12i2 polypeptides and , including two or more separate RNA guides. In some embodiments, separate RNA guides Each of these molecules individually forms a mutant binary complex with a separate mutant Cas12i2 polypeptide. This mutant binary complex has two or more target gene loci of the target nucleic acid compared to the parent binary complex. It shows higher on-target ternary complex formation.

[0452] In some embodiments, the composition of the present invention comprises a plurality of variant Cas12i2 polypeptides and , including two or more separate RNA guides. In some embodiments, separate RNA guides Each of these molecules individually forms a mutant binary complex with a separate mutant Cas12i2 polypeptide. This mutant binary complex has two or more target gene loci of the target nucleic acid compared to the parent binary complex. This forms a more stable ternary complex.

[0453] In some embodiments, the present invention described herein includes compositions comprising a complex. In one embodiment of the present invention, the mutant Cas12i2 polypeptide and an RNA guide It includes a binary complex that includes [something].

[0454] In some embodiments, the present invention involves a mutant Cas12i2 polypeptide and RNA gas It comprises a ternary complex including the id and the target gene locus of the target nucleic acid. In some embodiments, The composition of the present invention comprises multiple variant Cas12i2 polypeptides and two or more distinct RNAs. This separate RNA guide includes a distinct variant of Cas12i2 poly(A12i2) It forms a peptide-mutant binary complex.

[0455] In some embodiments, the composition of the present invention comprises a mutant Cas12i2 polypeptide and RNA This variant Cas12i2 polypeptide and RNA guide include a guide, and the parent binary compound Compared to fusion, it provides higher on-target binding affinity to the target gene locus of the target nucleic acid. It forms a mutant binary complex containing multiple Compared to the parental binary complex, it has a higher Onta for two or more target gene loci of the target nucleic acid. It contains multiple mutant binary complexes that have GET bonds.

[0456] In some embodiments, the composition of the present invention comprises a mutant Cas12i2 polypeptide and RNA It includes a binary complex that includes a guide, and this binary complex, compared to the parent binary complex, the target nucleus It has lower binding affinity to non-target gene loci of the acid. In some embodiments, The composition of the invention, compared to multiple parental binary complexes, has two or more non-target gene loci of the target nucleic acid. This includes several variant binary complexes that exhibit lower off-target binding to [the target molecule].

[0457] In some embodiments, the composition of the present invention comprises a mutant Cas12i2 polypeptide and RNA It includes a binary complex that includes a guide, and this binary complex, compared to the parent binary complex, the target nucleus It exhibits higher activity at the on-target gene locus of the acid. In some embodiments, The composition of the present invention, compared to multiple parental binary complexes, provides two or more target gene loci of the target nucleic acid. It includes multiple mutant binary complexes that exhibit higher on-target activity.

[0458] In some embodiments, the composition of the present invention comprises a mutant Cas12i2 polypeptide and RNA It includes a binary complex that includes a guide, and this binary complex, compared to the parent binary complex, the target nucleus It exhibits lower activity at non-target gene loci of the acid. In some embodiments, the present invention The composition, compared to multiple parental binary complexes, has two or more non-target gene loci of the target nucleic acid. It includes multiple mutant binary complexes with lower off-target activity.

[0459] Cas12i2 polypeptide variant In some embodiments, the composition of the present invention is the variant Cas12 described herein. Includes i2 polypeptides.

[0460] In some embodiments, the polypeptide of the present invention is a variant of the parent polypeptide, Here, the parent is coded by a polynucleotide containing a nucleotide sequence such as SEQ ID NO: 1. It contains amino acid sequences such as SEQ ID NO: 2 or similar.

[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 are reference nucleic acid sequences, for example It may be substantially identical to Sequence ID No. 1. In some embodiments, the variant Cas12i2 A polypeptide is a nucleic acid sequence that encodes a reference nucleic acid sequence, for example, a nucleic acid sequence that encodes a parent polypeptide, for example Sequence ID 1 and at least approximately 60%, at least approximately 65%, at least approximately 70%, at least approximately 7 5%, at least about 80%, at least about 85%, at least about 90%, at least about 9 1%, at least about 92%, at least about 93%, at least about 94%, at least about 9 5%, at least about 96%, at least about 97%, at least about 98%, at least about 9 Encoded by nucleic acids containing sequences with 9% or at least approximately 99.5% sequence identity The percentage identity between two such nucleic acids is the same as that between two optimally aligned nucleic acids. This can be done by manually examining the sequence or by using a software program that employs standard parameters. Alternatively, use an algorithm (e.g., BLAST, ALIGN, CLUSTAL). This can be determined by the following. One indicator that two nucleic acid sequences are substantially identical is the sequence of st Under lingent conditions (e.g., within the range of moderate to high stringency) This involves the hybridization of the nucleic acid molecule into the complementary sequence of the other molecule.

[0466] In some embodiments, the mutant Cas12i2 polypeptide is a reference nucleic acid sequence, for example If so, the nucleic acid sequence encoding the parent polypeptide, for example, SEQ ID NO: 1, is at least about 60% and less At least about 65%, at least about 70%, at least about 75%, at least about 80%, little At least 85%, at least 90%, at least 91%, at least 92%, At least about 93%, at least about 94%, at least about 95%, at least about 96%, little At least 97%, at least 98%, at least 99%, or higher sequence identity. It is encoded by nucleic acid sequences that have sex, but not 100% sequence identity.

[0467] In some embodiments, the variant Cas12i2 polypeptide of the present invention is represented by Sequence ID No. 2 and 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, or 99%, but not 100% It contains polypeptide sequences that have no identity. In some embodiments, variants of the present invention Cas12i2 polypeptide is available in 50%, 60%, 65%, 70%, and 75% of the original value (SEQ ID NO: 2). 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% polypeptide sequences having identity higher than 98% or 99%, but not 100%. Includes.

[0468] In some embodiments, the present invention relates to one or more reference polypeptides, for example, parent polypeptides For Tide, a specified degree of amino acid sequence identity, for example, the amino acid sequence of SEQ ID NO: 2 At least 60%, at least 65%, at least 70%, at least 75% for each column , at least 80%, at least 85%, at least 90%, at least 91%, less 92%, at least 93%, at least 94%, at least 95%, at least 96% %, at least 97%, at least 98%, or even more than 99%, but 100% This section describes a variant Cas12i2 polypeptide that has sequence identity but is not homologous. Sex or identity is as described herein, for example, BLAST, ALIGN, or C Determined by amino acid sequence alignment using programs such as LUSTAL. It is possible.

[0469] In some embodiments, the mutant Cas12i2 polypeptide is one of the parent polypeptides. It involves changes in one or more (for example, several) amino acids, where at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 1 9, 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, 5 9, 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, 9 9, 100, 101, 102, 103, 104, 105, 106, 107, 108, 10 9, 110, 111, 112, 113, 114, 115, 116, 117, 118, 11 9, 120, 121, 122, 123, 124, 125, 126, 127, 128, 12 9, 130, 131, 132, 133, 134, 135, 136, 137, 138, 13 9, 140, 141, 142, 143, 144, 145, 146, 147, 148, 14 9, 150, 151, 152, 153, 154, 155, 156, 157, 158, 15 9, 160, 161, 162, 162, 164, 164, 165, 166, 167, 16 8, 169, 170, 171, 172, 173, 174, 175, 176, 177, 17 8, 179, 180, 181, 182, 183, 184, 185, 186, 187, 18 8, 189, 190, 191, 193, 194, 195, 196, 197, 198, 19 9. 200 or more items have changed.

[0470] In some embodiments, the mutant Cas12i2 polypeptide is as shown in Table 2. It contains one or more amino acid substitutions. In some embodiments, the mutant Cas12i2 polypept Chido is D581, G624, F626, D835, L836, P868, S879, D At least 911, I926, V1020, V1030, E1035 and S1046 are replaced. It also includes one. In some embodiments, the variant Cas12i2 polypeptide is D58 1R, G624R, F626R, D835R, L836R, P868R, S879R, D 911R, I926R, V1020R, V1030R, E1035R and S1046R Includes at least one variant of Cas12i2. In some embodiments, the variant Cas12i2 polypeptide The models are D581G, F626G, D835G, L836G, P868G, S879G, D 911G, I926G, V1020G, V1030G, E1035G and S1046G Includes at least one variant of Cas12i2. In some embodiments, the variant Cas12i2 polypeptide The models are D581R, G624R, F626R, D835R, L836R, P868T, S 879R, D911R, I926R, V1020G, V1030G, E1035R and S At least one of the 1046G substitutions and at least one additional substitution listed in Table 2 Includes. In some embodiments, the mutant Cas12i2 polypeptide is used in SEQ ID NOs: 3-1 Includes one of 46 and 495-512. In some embodiments, mutant Cas The 12i2 polypeptide is one of the sequence numbers 3-146 and 495-512, and Includes at least one additional substitution as listed in Table 2. In some embodiments, variants The Cas12i2 polypeptide contains the amino acid sequence of SEQ ID NO: 3. In some embodiments, The mutant Cas12i2 polypeptide contains the amino acid sequence of SEQ ID NO: 4. In one embodiment, the mutant Cas12i2 polypeptide has the amino acid sequence of SEQ ID NO: 5 Includes. In some embodiments, the variant Cas12i2 polypeptide is SEQ ID NO: 495 The amino acid sequence includes the following. In some embodiments, the mutant Cas12i2 polypeptide is It contains the amino acid sequence of sequence number 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 is as shown in Table 2. It contains one or more amino acid substitutions. In some embodiments, the mutant Cas12i2 polypept Tide is shown in one of the following sequence numbers: 3-5, 495, or 496, as illustrated in Figure 1. Includes sequence. In some embodiments, the mutant Cas12i2 polypeptide is sequence number Contains the sequence shown in one of the numbers from 6 to 146.

[0495] In some embodiments, the compositions described herein include one or more individual (e.g.) , 2, 3, 4, 5, 6, 7, 8, 9 or more mutant Cas12i2 polypeptides Contains tide. In such embodiments, individual mutant polypeptides are independently listed in Table 2. It may contain one or more of the listed amino acid substitutions. In some embodiments, individual mutant C The as12i2 polypeptide is represented by sequence numbers 3-5, 495, or 496 as shown in Figure 1. The sequence includes one of the following. In some embodiments, a separate variant Cas12i The 2 polypeptide contains the sequence shown in one of sequence numbers 6 through 146.

[0496] In some embodiments, the variant Cas12i2 polypeptide is as shown in Table 3. This includes one mutation or a set of mutations, where the mutation is based on the sequence of SEQ ID NO: 2. This is the result.

[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 polypropylene shown in Table 3. It is a peptide. The substitutions in Table 3 are based on the sequence of SEQ ID NO: 2. Partial implementation Morphologically, the mutant Cas12i2 polypeptide exhibits the amino acid substitutions listed in Table 3. Includes one or more.

[0503] In some embodiments, the variant Cas12i2 polypeptide of the present invention is represented by Sequence ID No. 3. ~146 and one of 495~512, plus 50%, 60%, 65%, 70%, 75% 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , containing polypeptide sequences having 98%, 99%, or 100% identity. Some implementations In this state, the variant Cas12i2 polypeptide of the present invention is represented by SEQ ID NOs: 3-146 and 4 One of 95-512 and 50%, 60%, 65%, 70%, 75%, 80%, 85% %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 Contains polypeptide sequences with a higher percentage or 100% identity.

[0504] In some embodiments, one or more amino acids from amino acids 597 to 607 of SEQ ID NO: 2 The amino acids of SEQ ID NO: 2 are altered or mutated. In some embodiments, amino acids 597- One or more amino acids of 604 are altered or mutated. In some embodiments, One or more amino acids between amino acids 830 and 833 in sequence number 2 have been altered or mutated. In some embodiments, one or more amino acids from amino acids 829-835 of SEQ ID NO: It is altered or mutated. In some embodiments, amino acids 882-8 of SEQ ID NO: 2 One or more of the 88 amino acids are altered or mutated. In some embodiments, the sequence One or more amino acids between amino acids 883 and 889 of amino acid number 2 are altered or mutated. (Table) See section 2.

[0505] In some embodiments, one or more amino acids from amino acids 600 to 607 of SEQ ID NO: 2 The amino acids 833- of SEQ ID NO: 2 are altered or mutated. In some embodiments, amino acids 833- of SEQ ID NO: 2 are altered or mutated. One or more of the 871 amino acids are altered or mutated. In some embodiments, One or more amino acids between amino acids 877 and 885 in sequence number 2 have been altered or mutated. Please refer to Table 2.

[0506] In some embodiments, the mutant Cas12i2 polypeptide has at least one R It contains a uvC domain. In some embodiments, the mutant Cas12i2 polypeptide is , at least one RuvC motif (e.g., 1, 2, or 3 RuvC motifs) Includes. The domains of the Cas12i2 polypeptide disclosed herein are illustrated in Figure 19. The wedge domain consists of residues 1-14 and 442-58 of the Cas12i2 polypeptide. Includes 6. The Rec1 domain consists of residues 15-176 and 2 of the Cas12i2 polypeptide. It includes residues 70-441. Within the Rec1 domain, the helix I domain consists of residues 15-176. The PI domain includes residues 270-327, and the helix II domain includes residues 328-441. The domain contains residues 177-269 of the Cas12i2 polypeptide. Rec2 domain The n contains residues 638-828 of the Cas12i2 polypeptide. The Nuc domain is C Contains residues 880-1017 of the as12i2 polypeptide. The RuvC motif is Cas 12i2 polypeptide residues 587-637 (RuvC1), residues 829-879 (Ru It includes vC2) and residues 1018-1054 (RuvC3).

[0507] In some embodiments, a variant Cas1 having the characteristics described herein 2i2 polypeptides are limited to one of the following sequence numbers: 3-146 and 495-512. Approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 9 Having 3%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity Contains amino acid sequence.

[0508] The modifications described herein may involve changes to one or more amino acids, but the variant Cas The modifications to the 12i2 polypeptide include the amino-terminated and / or carboxyl-terminated extensions. It can also be of structural or substantive nature, such as the fusion of polypeptides. For example, The variant Cas12i2 polypeptide contains additional peptides, such as one or more peptides. Possible. Examples of additional peptides include polyhistidine tags (His tags), Myc, and Examples of labeling epitope peptides include FLAG. Furthermore, the variant Cas12i2 polypeptide described herein is a fluorescent protein (e.g. For example, it can detect green fluorescent protein (GFP) or yellow fluorescent protein (YFP). It can be integrated with the Noh aspect.

[0509] In some embodiments, the mutant Cas12i2 polypeptide has at least one ( For example, including 2, 3, 4, 5, 6 or more nuclear localization signals (NLS). In some embodiments, the mutant Cas12i2 polypeptide has at least one (e.g.) For example, including 2, 3, 4, 5, 6 or more nuclear export signals (NES). In some embodiments, the mutant Cas12i2 polypeptide contains at least one (e.g.) (e.g., 2, 3, 4, 5, 6 or more) NLS and at least one (e.g., 2) Includes 3, 4, 5, 6 or more NES units.

[0510] In some embodiments, the variant Cas12i2 polypeptide described herein is It may be a self-inactivating type. Epstein et al., "Engineerin ga Self-Inactivating CRISPR System for "AAV Vectors", Mol. Ther., 24 (2016): S50 (overall) See (which is referenced by the reference).

[0511] In some embodiments, the variant Cas12i2 polypeptide described herein is used The encoded nucleotide sequence is codon-optimized for use in specific host cells or organisms. It can be transformed. For example, nucleic acids can be used in mice, rats, rabbits, dogs, livestock, or non-human primates. Codon optimization can be performed for any non-human eukaryote, including, for example, w "Codon Usage" available at www.kazusa.orjp / codon / These tables are readily available in the "Database" and can be fitted in several ways. This is possible. Nakamura et al. Nucleo Acids Res. 28: See 292(2000) (as a whole, incorporated herein by reference). Computer algorithms for codon optimization of a specific sequence for expression in a particular host cell. The rhythms are also used by Gene Forge (Aptagen; Jacobus, PA), etc. It is possible.

[0512] Function of mutant polypeptides As used herein, “biologically active portion” means less than or equal to the parent polypeptide. Both retain a single function (for example, completely, partially, or minimally) in part (for example, This is the "minimum" or "core" domain. In some embodiments, the variant Cas12i The two polypeptides retain enzymatic activity that is at least as high as that of the parent polypeptide. Therefore, in some embodiments, the mutant Cas12i2 polypeptide is the parent polypeptide. It has higher enzymatic activity than peptides.

[0513] Furthermore, it has enzymatic activity, for example, nuclease or endonuclease activity, and as mentioned above. When aligned using any of the alignment methods, the parent polypeptide and One of the amino acid sequences in SEQ ID NO: 2 and 50, 40, 35, 30, 25, 20, 19 , 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, The present invention includes a variant Cas12i2 containing an amino acid sequence that differs by only 3, 2, or 1 amino acid residue. Polypeptides are also provided. In some embodiments, a mutant Cas1 having enzymatic activity is provided. The 2i2 polypeptide is aligned using one of the alignment methods described above. When this happens, the amino acid sequence of either the parent polypeptide or SEQ ID NO: 2 is at least about 6 0%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 9 Amino acids having 4%, 95%, 96%, 97%, 98%, 99%, or 100% identity Includes arrays.

[0514] In some embodiments, the mutant Cas12i2 polypeptide is the mutant Cas12i2 polypeptide. Lipeptide enzymatic activity, RNA guide complex (binary complex) formation, RNA guide binding activity RNA guide affinity, RNA guide binding specificity, protein-RNA interaction, protein Protein-DNA interaction, protein stability, ternary complex formation, on-target binding activity, At least enhances on-target binding specificity and / or stability in the ternary complex. Includes one change or mutation. In some embodiments, the present invention variant Cas12i Polypeptide 2 exhibits enzymatic activity equal to or greater than that of the parent polypeptide, and the RNA guide complex (binary polypeptide) (Combination) Formation, RNA guide binding activity, RNA guide affinity, RNA guide binding specificity, Protein-RNA interaction, protein-DNA interaction, protein stability, ternary complex Regarding complex formation, on-target binding activity, on-target binding specificity, and / or ternary complex formation It has at least one of the following stability characteristics. In some embodiments, the variant Cas12i2 Polypeptides exhibit enzyme activity, RNA guide complex (binary complex) formation, and 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 Fewer Both contain one change or mutation, and the mutant Cas12i2 polypeptide has a sequence number Number 4 and at least approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% Contains an amino acid sequence that has uniformity.

[0515] In some embodiments, the mutant Cas12i2 polypeptide exhibits enzyme activity and RNA metabolism. RNA guide binding activity, RNA guide affinity, RNA guide Id binding specificity, protein-RNA interaction, protein-DNA interaction, protein Quality stability, ternary complex formation, on-target binding activity, on-target binding specificity, and / This includes enhanced stability in the ternary complex, and the mutant Cas12i2 polypeptide is , containing one of the amino acid sequences of sequence numbers 3-5, 495, or 496.

[0516] In some embodiments, the mutant Cas12i2 polypeptide exhibits enzyme activity and RNA metabolism. RNA guide binding activity, RNA guide affinity, RNA guide Id binding specificity, protein-RNA interaction, protein-DNA interaction, protein Quality stability, ternary complex formation, on-target binding activity, on-target binding specificity, and / This includes enhanced stability in the ternary complex, and the mutant Cas12i2 polypeptide is , containing one of the amino acid sequences from sequence numbers 3-146 and 495-512.

[0517] In some embodiments of the present invention, the mutant Cas12i2 polypeptide is a polypeptide Interacts with nucleic acids (e.g., RNA guide or DNA) (e.g., binds or complexes) This includes substitutions that alter the ability to form bodies. In some embodiments of the present invention, mutant Ca The s12i2 polypeptide is a polypeptide for nucleic acids (e.g., RNA guide or DNA). This includes substitutions that alter the affinity of the cytoplasm.

[0518] In some embodiments, the mutant Cas12i2 polypeptide containing the D581R substitution is , exhibiting enhanced enzyme activity. In some embodiments, mutant Ca containing the D581R substitution is used. The s12i2 polypeptide interacts with NTS. In some embodiments, D581 The Cas12i2 polypeptide variant containing the R substitution interacts with the PAM sequence backbone. In one embodiment, the mutant Cas12i2 polypeptide containing the D581R substitution is P It exhibits enhanced electrostatic interaction with DNA in the AM sequence. In some embodiments, D The Cas12i2 polypeptide variant containing the 581R substitution reduces repulsive interactions with nucleic acids. To cause. In some embodiments, the mutant Cas12i2 polypeptide containing the D581R substitution Chido enhances R-loop stability. See Figure 20A.

[0519] In some embodiments, the variant Cas12i2 polypeptide containing the V1030G substitution This exhibits enhanced enzyme activity. In some embodiments, mutants containing the V1030G substitution are present. The Cas12i2 polypeptide interacts with NTS. In some embodiments, V1 The Cas12i2 polypeptide mutant containing the 030G substitution is located near the Cas12i2 active site. It is located in [location]. Please refer to Figure 20B.

[0520] In some embodiments, the mutant Cas12i2 polypeptide containing the I926R substitution is , exhibiting enhanced enzyme activity. In some embodiments, mutant Ca containing the I926R substitution is used. The s12i2 polypeptide interacts with single-stranded DNA near the Cas12i2 active site. In some embodiments, the variant Cas12i2 polypeptide containing the I926R substitution is used. This stabilizes single-stranded DNA. See Figure 20B.

[0521] In some embodiments, the variant Cas12i2 polypeptide containing the G624R substitution is , exhibiting enhanced enzyme activity. In some embodiments, a mutant Ca containing the G624R substitution is used. The s12i2 polypeptide interacts with NTS. In some embodiments, G624 The Cas12i2 polypeptide mutant, which includes an R substitution, enhances R-loop stability.

[0522] In some embodiments, the variant Cas12i2 polypeptide containing the F626R substitution is , exhibiting enhanced enzyme activity. In some embodiments, mutant Ca containing the F626R substitution is used. The s12i2 polypeptide interacts with NTS. In some embodiments, F626 The Cas12i2 polypeptide mutant, which includes an R substitution, enhances R-loop stability.

[0523] In some embodiments, the variant Cas12i2 polypeptide of the present invention is used at approximately 20°C~ A temperature range of approximately 90°C, for example, approximately 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 2 6℃, 27℃, 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 3 6℃, 37℃, 38℃, 39℃, 40℃, 41℃, 42℃, 43℃, 44℃, 45℃, 5 0℃, 51℃, 52℃, 53℃, 54℃, 55℃, 56℃, 57℃, 58℃, 59℃, 6 Enzyme activity, RNA guide complex at 0°C, 65°C, or above. Body (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 ternary The complex has at least one stability. In some embodiments, the present invention is modified. The variant Cas12i2 polypeptide is found at temperatures of approximately 20°C to 25°C or approximately 37°C. At different temperatures, enzyme activity, RNA guide complex (binary complex) formation, and RNA guide binding activity are affected. Sex, RNA guide affinity, RNA guide binding specificity, protein-RNA interaction, tan Protein-DNA interaction, protein stability, ternary complex formation, on-target binding activity , possessing at least one of on-target binding specificity and / or stability in the ternary complex do.

[0524] In some embodiments, the mutant Cas12i2 polypeptide is approximately 7.3 to approximately 8.6 pH range (e.g., 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 In a buffer having any value within the range of 5, 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-D NA interaction, protein stability, ternary complex formation, on-target binding activity, ON-T It exhibits enhanced GET binding specificity and / or stability in the ternary complex.

[0525] In some embodiments, the mutant Cas12i2 polypeptide is the mutant Cas12i 2 polypeptide T m The value is T of the parent polypeptide. m At least 1°C, 2°C, or 3°C above the value. 4℃, 5℃, 6℃, 7℃, 8℃, 9℃, 10℃, 11℃, 12℃, 13℃, 14℃, 15 When the temperature is 16°C, 17°C, 18°C, 19°C, or 20°C higher, compared to the parent polypeptide, Enzyme activity, RNA guide complex (binary complex) formation, RNA guide binding activity, RNA guide DNA affinity, RNA guide binding specificity, protein-RNA interaction, protein-DNA Interaction, protein stability, ternary complex formation, on-target binding activity, on-target It exhibits at least one of the following: 3-binding specificity and / or stability in the ternary complex. In this state, the mutant Cas12i2 polypeptide is the mutant Cas12i2 polypeptide. T m The value is T of the parent polypeptide. m When the temperature is at least 8°C higher than the value, enzyme activity and RNA gas RNA guide binding activity, RNA guide affinity, RNA guide Id binding specificity, protein-RNA interaction, protein-DNA interaction, protein Quality stability, ternary complex formation, on-target binding activity, on-target binding specificity, and / Alternatively, it exhibits enhanced stability in the ternary complex.

[0526] In some embodiments, the variant Cas12i2 polypeptide of the present invention is used at approximately 37°C. At least approximately 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes One of the following time periods: minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, or more. Over the incubation period, compared to the parent polypeptide, enzyme activity and RNA guidance were observed. Complex (binary complex) formation, RNA guide binding activity, RNA guide affinity, RNA guide Binding specificity, protein-RNA interaction, protein-DNA interaction, protein safety Qualitative, ternary complex formation, on-target binding activity, on-target binding specificity and / or It exhibits at least one increase in stability in the ternary complex. In some embodiments, The variant Cas12i2 polypeptide of the present invention can be incubated for a certain range of incubation times. Furthermore, 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-R NA interaction, protein-DNA interaction, protein stability, ternary complex formation, ON Target binding activity, on-target binding specificity and / or stability in the ternary complex It exhibits at least one increase.

[0527] Furthermore, dissociation from the RNA guide (dissociation of the binary complex), and dissociation from the target nucleic acid (ternary complex). Body dissociation, off-target binding to non-target nucleic acids and / or non-target gene loci of target nucleic acids. It shows a decrease in activity and is aligned using one of the alignment methods described above. When combined, the amino acid sequence of either the parent polypeptide or SEQ ID NO: 2 is 50, 4 0, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 , amino acid sequences that differ by only 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residue The present invention also includes a variant Cas12i2 polypeptide. In some embodiments, Dissociation from RNA guide (dissociation of binary complex), dissociation from target nucleic acid (dissociation of ternary complex) (Disconnection), off-target binding to non-target nucleic acids and / or at non-target gene loci of target nucleic acids The Cas12i2 polypeptide mutant showing reduced activity was used in the alignment method described above. When alignment is performed using the offset, either the parent polypeptide or SEQ ID NO: 2 The amino acid sequence and at least approximately 60%, 65%, 70%, 75%, 80%, 85%, 90% %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 1 Contains amino acid sequences with 00% identity.

[0528] In some embodiments, the mutant Cas12i2 polypeptide is the mutant Cas12i2 polypeptide. Dissociation of the lipeptide from the RNA guide (dissociation of the binary complex), dissociation from the target nucleic acid (ternary complex) Dissociation of the complex, off-target binding to non-target nucleic acids and / or non-target genes of target nucleic acids. Includes at least one change or mutation that reduces activity at the locus. Some embodiments In this process, the mutant Cas12i2 polypeptide dissociates from the RNA guide (binary complex). Dissociation of the target nucleic acid (dissociation of the ternary complex), off-target bonding to non-target nucleic acids. At least one change that reduced the activity of the combined and / or target nucleic acid at non-target gene loci. or include mutations, and the mutant Cas12i2 polypeptide is at least as well as Sequence ID No. 4. Approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93% A having identity of %, 94%, 95%, 96%, 97%, 98%, 99%, or 100% Contains a mino acid sequence.

[0529] In some embodiments, the mutant Cas12i2 polypeptide compared to the parent polypeptide Then, dissociation from the RNA guide (dissociation of the binary complex), dissociation from the target nucleic acid (dissociation of the ternary complex) Dissociation), off-target binding to non-target nucleic acids and / or at non-target gene loci of target nucleic acids This includes at least one change or mutation that reduces the activity. In some embodiments, the change The variant Cas12i2 polypeptide, compared to the parent polypeptide, shows a different response from the RNA guide. Dissociation (dissociation of the binary complex), dissociation from the target nucleic acid (dissociation of the ternary complex), and dissociation from non-target nucleic acids. This includes, and The mutant Cas12i2 polypeptide is one of the following: SEQ ID NOs: 3-5, 495, or 496. It contains a sequence of amino acids.

[0530] In some embodiments, the mutant Cas12i2 polypeptide compared to the parent polypeptide Then, dissociation from the RNA guide (dissociation of the binary complex), dissociation from the target nucleic acid (dissociation of the ternary complex) Dissociation), off-target binding to non-target nucleic acids and / or at non-target gene loci of target nucleic acids This includes a decrease in activity, and the mutant Cas12i2 polypeptide is associated with SEQ ID NOs: 3-146 and It contains one of the amino acid sequences from 495 to 512.

[0531] In some embodiments, the variant Cas12i2 polypeptide of the present invention is used in RNA guides. Dissociation from the target nucleic acid (dissociation of the binary complex), dissociation from the target nucleic acid (dissociation of the ternary complex), non-target Off-target binding to nucleic acids and / or low activity at non-target gene loci of target nucleic acids Both exhibit at a level equivalent to or lower than that of the parent polypeptide. In some embodiments, this The new Cas12i2 polypeptide mutant can thrive in a temperature range of approximately 20°C to approximately 90°C, for example, approximately 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℃, 50℃, 51℃, 52℃, 53℃, 54℃, 55℃, 56℃, 57℃, 58℃, 59℃, 60℃, 65℃ or above In any one of the following, equivalent or reduced dissociation from the RNA guide (binary complex Dissociation, dissociation from target nucleic acids (dissociation of the ternary complex), off-target binding to non-target nucleic acids. and / or activity at non-target gene loci of the target nucleic acid. In some embodiments, The mutant Cas12i2 polypeptide of the present invention is used at a temperature of about 20°C to about 25°C or At a temperature of approximately 37°C, dissociation from the RNA guide (dissociation of the binary complex) occurs, and from the target nucleic acid... Dissociation of (dissociation of the ternary complex), off-target binding to non-target nucleic acids and / or target nucleic acids It performs at least one of the activities at a non-target gene locus.

[0532] In some embodiments, the mutant Cas12i2 polypeptide is approximately 7.3 to approximately 8.6 pH range (e.g., 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 In a buffer having any value within the range of 5, compared to the parent polypeptide Then, dissociation from the RNA guide (dissociation of the binary complex), dissociation from the target nucleic acid (dissociation of the ternary complex) Dissociation), off-target binding to non-target nucleic acids and / or at non-target gene loci of target nucleic acids It exhibits reduced activity.

[0533] In some embodiments, the mutant Cas12i2 polypeptide is the mutant Cas12i 2 polypeptide T m The value is T of the parent polypeptide. m At least 1°C, 2°C, or 3°C above the value. 4℃, 5℃, 6℃, 7℃, 8℃, 9℃, 10℃, 11℃, 12℃, 13℃, 14℃, 15 When the temperature is 16°C, 17°C, 18°C, 19°C, or 20°C higher, 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 activation of target nucleic acids at non-target gene loci. It exhibits at least one of the sexes. In one embodiment, the mutant Cas12i2 polypeptide This is the T of the mutant Cas12i2 polypeptide. m The value is T of the parent polypeptide. m Less than the value When both temperatures are 8°C higher, dissociation from the RNA guide (dissociation of the binary complex) and dissociation from the target nucleic acid occur. (dissociation of the ternary complex), off-target binding to non-target nucleic acids and / or off-target binding of target nucleic acids. It exhibits reduced activity at the gene locus.

[0534] Increased RNA guide interactions In some embodiments, the mutant Cas12i2 polypeptide is compared to the parent polypeptide. In comparison, the interaction between the mutant Cas12i2 polypeptide and the RNA guide and / or This includes changes that increase affinity. See Figure 20C. In some embodiments, Increase the interaction and / or affinity between the heterogeneous Cas12i2 polypeptide and the RNA guide. The change involves adding one or more amino acids: arginine, lysine, glutamine, and asparagine. This involves substitution with histidine, serine, or tyrosine residues. In some embodiments, this involves substitution with histidine, serine, or tyrosine residues. The mutant Cas12i2 polypeptide has one or more amino acids at the RNA binding interface. Arginine, lysine, glutamine, asparagine, histidine, serine or tyrosine residue This includes substitution of the group. In some embodiments, the mutant Cas12i2 polypeptide is 1 Arginine, lysine, glutamate, and more than 441-586 or 637-828 amino acids This includes substitution of one of the following residues: n, asparagine, histidine, serine, or tyrosine. In some embodiments, the variant Cas12i2 polypeptide has at least one drug One or more amino acids in the main domain (e.g., Wedge domain or Rec2 domain) From acid, arginine, lysine, glutamine, asparagine, histidine, serine or tyrosine This includes changes to RNA residues. In some embodiments, one or more such RNA-binding bounds Surface substitution improves RNA guide binding or RNA guide binding affinity compared to the parent polypeptide. Approximately 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 1 5%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 2 5%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 3 5%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 4 5%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 5 5%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 6 5%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 7 5%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 8 5%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 9 5%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 1 40%, 150%, 160%, 170%, 180%, 190%, 200%, or the above. Increase by any percentage that exceeds or lies between them.

[0535] In some embodiments, the substitution is compared to the parent polypeptide in the RNA guide complex (binary) The mutant Cas12i2 polypeptide increases complex formation. This shows non-restrictive examples of substitutions that can change the ability to interact with a triplet sequence. As shown in 4. In some embodiments, mutant Ca containing one or more substitutions listed in Table 4. The s12i2 polypeptide has a different RNA guide complex (binary complex) compared to the parent polypeptide. It exhibits enhanced formation. In some embodiments, it includes one or more substitutions listed in Table 4. The mutant Cas12i2 polypeptide, compared to the binary complex containing the parent polypeptide, It forms a more stable binary complex with the NA guide.

[0536] [Table 32]

[0537] In some embodiments, one of sequence numbers 3-146 and 495-512 is modified. The variant Cas12i2 polypeptide further includes one or more substitutions listed in Table 4. In the embodiments described above, the variant Cas12i2 polypeptide is shown in Tables 2 and 4. Includes one or more substitutions.

[0538] In some embodiments, mutations exhibiting enhanced RNA guide complex (binary complex) formation are observed. The Cas12i2 polypeptide can have two or more substitutions, for example, L654K Q658A. Includes L520K Q496N or L520K Q496S. In some embodiments, One of the variant Cas12i2 polypeptides from sequence numbers 3-146 and 495-512 Chido is L654K Q658A, L520K Q496N, or L520K Q496S Further includes substitution. In some embodiments, the sequences 3-146 and 495-512 Either one of the Cas12i2 polypeptide mutants is L654K Q658A, L520 Substitutions of K Q496N or L520K Q496S and one or more substitutions listed in Table 4 It also includes.

[0539] In some embodiments, sequence numbers 3-146 include one or more substitutions listed in Table 4. And any one of the 495-512 variant Cas12i2 polypeptides has enzyme activity It shows an increase. In some embodiments, mutants include one or more substitutions listed in Table 4. The Cas12i2 polypeptide exhibits increased enzyme activity. In some embodiments, L 654K Q658A, L520K Q496N, or L520K Q496S replacement and table Sequence IDs 3-146 and 495-512, which include one or more substitutions listed in 4. Any one of the Cas12i2 polypeptide mutants exhibits increased enzyme activity. Morphologically, the mutant Cas12i2 polypeptide, compared to the parent polypeptide, is enzyme-based. Activity (for example, about 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 1%) 3%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 2 3%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 3 3%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 4 3%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 5 3%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 6 3%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 7 3%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 8 3%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 9 3%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120 %, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200 (An increase of %) or more, or any percentage in between. ru.

[0540] Increased double-stranded DNA interactions In some embodiments, the mutant Cas12i2 polypeptide is different from the parent polypeptide. This includes changes that increase interaction with double-stranded DNA. In some embodiments, double-stranded D Increased interaction with NA is an increase in electrostatic interaction. In some embodiments, mutation The parent Cas12i2 polypeptide is different from the mutant Cas12i2 polypeptide. This includes changes that increase the affinity between plutide and double-stranded DNA. In some embodiments, , interaction and / or affinity between the mutant Cas12i2 polypeptide and double-stranded DNA Changes that increase the binding of the mutant Cas12i2 polypeptide to the PAM sequence increase. To add.

[0541] In some embodiments, between the mutant Cas12i2 polypeptide and double-stranded DNA Changes that increase interaction and / or affinity involve adding one or more amino acids, such as arginine and lydine. This involves substitution with a residue of , glutamine, asparagine, histidine, or serine. In this embodiment, the mutant Cas12i2 polypeptide is used at the double-stranded DNA binding interface. From one or more amino acids, arginine, lysine, glutamine, asparagine, histidine This includes substitutions to n or serine residues. In some embodiments, the mutant Cas12i2 poly Peptides consist of one or more amino acids, 140-190, 220-300, 440-480 or From 560-570, arginine, lysine, glutamine, asparagine, histidine or ce Includes substitution of any one of the phosphorus residues. In some embodiments, the mutant Cas12i 2. Polypeptides have at least one domain (e.g., Rec1 domain, PI domain) Arginine, lysine, and gluten are derived from one or more amino acids in the (or Wedge domain). This includes changes to thamine, asparagine, histidine, or serine residues. In some embodiments... Furthermore, one or more double-stranded DNA binding interface substitutions, compared to the parent polypeptide, result in double-stranded DNA. DNA interaction and / or affinity at approximately 4%, 5%, 6%, 7%, 8%, 9%, 10%, 1% 1%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 2 1%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 3 1%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 4 1%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 5 1%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 6 1%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 7 1%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 8 1%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 9 1%, 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. Only the number of double-stranded DNA binding interfaces is increased. In some embodiments, one or more double-stranded DNA binding interface substitutions are performed. Compared to the parent polypeptide, the mutant Cas12i2 polypeptide is involved in the PAM sequence. The bonding occurs at approximately 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, and 1%. 4%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 2 4%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 3 4%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 4 4%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 5 4%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 6 4%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 7 4%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 8 4%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 9 4%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 13 0%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, etc. It increases by any percentage exceeding or between them.

[0542] In some embodiments, substitutions that increase double-stranded DNA interactions are performed on parent polypeptides. Compared to the other variant, it increases ternary complex formation. The mutant Cas12i2 polypeptide has double-stranded DN Table 5 shows non-restrictive examples of substitutions that can change the ability to interact with A. In the embodiment, the mutant Cas12i2 poly(V) includes one or more substitutions listed in Table 5. The peptide exhibits increased double-stranded DNA interaction (ternary complex formation) compared to the parent polypeptide. In some embodiments, the mutant Cas includes one or more substitutions listed in Table 5. The 12i2 polypeptide forms a more stable ternary complex compared to the parent polypeptide. To accomplish.

[0543] [Table 33]

[0544] In some embodiments, one of sequence numbers 3-146 and 495-512 is modified. The variant Cas12i2 polypeptide further includes one or more substitutions listed in Table 5. In the embodiments, the variant Cas12i2 polypeptide is shown in Tables 2 and 5. Includes one or more substitutions.

[0545] In some embodiments, the Cas12i2 mutant exhibits increased double-strand DNA interaction. Polypeptides can have two or more substitutions, for example, T562R E563K, T562R E5 63K N448K, I451R L452K, I451R L452K T562R E563R, I451R, L452K, Y472R, N229R, Q224N, or N22 Contains 9K Q224N. In some embodiments, it exhibits increased ternary complex formation / stability. The resulting Cas12i2 polypeptide has two or more substitutions, for example, T562R E5 63K, T562R E563K N448K, I451R L452K, I451R L452K T562R E563R, I451R L452K Y472R, N229 Includes R Q224N or N229K Q224N. In some embodiments, Sequence ID One of the variant Cas12i2 polypeptides, either 3-146 or 495-512, T562R E563K, T562R E563K N448K, I451R L452 K, I451R L452K T562R E563R, I451R L452K Y4 Further includes substitutions of 72R, N229R Q224N, or N229K Q224N. In the application morphology, one of the Cas mutants from sequence numbers 3-146 and 495-512 is used. 12i2 polypeptide is T562R E563K, T562R E563K N448 K, I451R L452K, I451R L452K T562R E563R, I4 51R L452K Y472R, N229R Q224N or N229K Q224N Substitutions and further include one or more substitutions listed in Table 4 and / or Table 5. Some embodiments In this case, the mutant Cas12i2 polypeptide is one of the following listed in Table 4 and / or Table 5. Includes one or more substitutions. In some embodiments, one or more substitutions are included in Table 4 and / or Table 5. The mutant Cas12i2 polypeptide having the above characteristics, compared to the parent polypeptide, has double-stranded D NA interaction and / or affinity (e.g., about 4%, 5%, 6%, 7%, 8%, 9%, 1%) 0%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 2 0%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 3 0%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 4 0%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 5 0%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 6 0%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 7 0%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 8 0%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 9 0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 1 00%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 1 80%, 190%, 200%, or more, or any percentage in between. Exhibits an increase in cents. In some embodiments, substitutions are found in Tables 4 and / or 5. The Cas12i2 polypeptide variant having one or more of the following characteristics is compared to the parent polypeptide: Ternary complex formation and / or ternary complex stability (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% 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 in between. It exhibits an increase of any percentage.

[0546] In some embodiments, the sequence includes one or more substitutions listed in Table 4 and / or Table 5. One of the Cas12i2 polypeptide variants numbered 3-146 and 495-512 This exhibits increased enzyme activity. In some embodiments, the following are shown in Tables 4 and / or 5. The Cas12i2 polypeptide variant containing one or more substitutions exhibits increased enzyme activity. In some embodiments, T562R E563K, T562R E563K N44 8K, I451R L452K, I451R L452K T562R E563R, I 451R L452K Y472R, N229R Q224N or N229K Q224 Sequence IDs 3-1 further include N substitutions and one or more substitutions listed in Table 4 and / or Table 5. One of the variant Cas12i2 polypeptides, 46 and 495-512, is enzyme-active. It exhibits increased sex. In some embodiments, the mutant Cas12i2 polypeptide is parent Compared to polypeptides, enzyme activity is (for example, 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%, 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. It shows an increase (in percentage).

[0547] Increased single-strand DNA interactions In some embodiments, the mutant Cas12i2 polypeptide is compared to the parent polypeptide. All involve changes that increase interaction with single-stranded DNA. In some embodiments, mutations are present. The parent Cas12i2 polypeptide is different from the mutant Cas12i2 polypeptide. This includes changes that increase the affinity between plutide and double-stranded DNA. In some embodiments, Single-stranded DNA includes a non-target strand (NTS). In some embodiments, single-stranded DNA Increased interaction with (for example, NTS) between PAM sequences and the mutant Cas12i2 polyp This is an interaction with the active site of the peptide. In some embodiments, single-stranded DNA is In or near the active site of the mutant Cas12i2 polypeptide, mutant Cas12 It contains single-stranded DNA that interacts with the i2 polypeptide. In some embodiments, variants The interaction and / or affinity between the Cas12i2 polypeptide and single-stranded DNA is increased. The resulting change stabilizes the R-loop. As used herein, "R-loop" refers to a standard This refers to nucleic acids containing RNA guides paired with the target strand (TS) and single-stranded non-target strand (NTS).

[0548] In some embodiments, between the mutant Cas12i2 polypeptide and single-stranded DNA Changes that increase interaction and / or affinity involve adding one or more amino acids, such as arginine and lydine. This involves substituting with a residue of , glutamine, asparagine, histidine, or alanine. In one embodiment, the mutant Cas12i2 polypeptide is used at the single-stranded DNA binding interface. From one or more amino acids, arginine, lysine, glutamine, asparagine, histamine This includes substitutions to din or alanine residues. In some embodiments, the mutant Cas12i2 Polypeptides consist of one or more amino acids, ranging from 230 to 260, 350 to 400, and 580 to 630. Arginine, lysine, and glutamate from 700-760, 830-900, or 920-1035. This includes substitution of one of the following residues: tamine, asparagine, histidine, or alanine. In one embodiment, the mutant Cas12i2 polypeptide is at least one domain N / motif (e.g., PI domain, Rec1 domain, Wedge domain, Ruv C1 motif, Rec2 domain, RuvC2 motif, Nuc domain, or RuvC3 From one or more amino acids in the motif, arginine, lysine, glutamine, asparagus This includes changes to silver, histidine, or alanine residues. In some embodiments, one or Multiple single-strand DNA binding interface substitutions, compared to the parent polypeptide, improve single-strand DNA interaction. and / or 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%, 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%, 110%, 12 0%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 20 Increase by 0%, or more than 0%, or any percentage in between. ru.

[0549] In some embodiments, substitutions that increase single-stranded DNA interactions are parent polypeptides. Compared to the other variant, it increases ternary complex formation. The mutant Cas12i2 polypeptide has single-stranded DN Table 6 shows non-restrictive examples of substitutions that can change the ability to interact with A. In one embodiment, the mutant Cas12i2 poly(V) containing one or more substitutions listed in Table 6 The peptide exhibits increased single-strand DNA interaction (ternary complex formation) compared to the parent polypeptide. In some embodiments, the mutant Cas includes one or more substitutions listed in Table 6. The 12i2 polypeptide forms a more stable ternary complex compared to the parent polypeptide. To accomplish.

[0550] [Table 34]

[0551] In some embodiments, one of sequence numbers 3-146 and 495-512 is modified. The variant Cas12i2 polypeptide further includes one or more substitutions listed in Table 6. In the embodiment, the mutant Cas12i2 polypeptide is shown in Tables 2 and 6. Includes one or more substitutions.

[0552] In some embodiments, the Cas12i2 mutant exhibits increased single-strand DNA interactions. polypeptide can have two or more substitutions, for example, G587R T588R, G587R T5 88K, G587R T588K Q590R or G587R T588R Q590K Includes. In some embodiments, mutant Cas exhibits increased ternary complex formation / stability. 12i2 polypeptide can have two or more substitutions, for example, G587R T588R, G587 R T588K, G587R T588K Q590R or G587R T588R Q Includes 590K. In some embodiments, sequence numbers 3-146 and 495-512 Either one of the Cas12i2 polypeptide mutants is G587R, T588R, or G587 R T588K, G587R T588K Q590R or G587R T588R Q Further includes 590K substitution. In some embodiments, SEQ ID NOs. 3-146 and 495-5 Any one of the 12 Cas12i2 polypeptide variants is G587R, T588R, G587R T588K, G587R T588K Q590R or G587R T58 8R Q590K substitution and one or more of the following listed in Table 4 and / or Table 5 and / or Table 6 Further substitutions are included. In some embodiments, the mutant Cas12i2 polypeptide is shown in Table 4. and / or include one or more substitutions from Table 5 and / or Table 6. Some embodiments include In this context, a mutant Cas having one or more substitutions listed in Table 4 and / or Table 5 and / or Table 6 The 12i2 polypeptide exhibits better single-stranded DNA interaction and / or interaction compared to the parent polypeptide. Affinity (for example, 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%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 12 0%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 20 An increase of 0% or more, or any percentage in between. In some embodiments, one or more of the substitutions listed in Table 4 and / or Table 5 and / or Table 6 are used. The mutant Cas12i2 polypeptide having the above characteristics is a ternary complex compared to the parent polypeptide. Body formation and / or ternary complex stability (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%, 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. It shows an increase (in percentage).

[0553] In some embodiments, the mutant Cas12i2 polypeptide exhibits single-stranded DNA stability. This includes substitutions that increase the single-stranded DNA and the mutant Cas12i2 polyp (Increases electrostatic interaction with the active site of the plutide). In some embodiments, mutation The Cas12i2 polypeptide exhibits approximately greater single-strand DNA stability compared to the parent polypeptide. 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%, 110%, 120%, 130%, 14 0%, 150%, 160%, 170%, 180%, 190%, 200%, or more than that. Increase by any percentage between or equal to the value obtained. (Cas12 variant) Substitutions that can alter the ability of i2 polypeptides to stabilize single-stranded DNA A limited example is shown in Table 6. In some embodiments, one or more substitutions listed in Table 6 are used. The included Cas12i2 polypeptide exhibits greater single-strand DNA stability compared to the parent polypeptide. It shows an increase.

[0554] In some embodiments, one or more items listed in Table 4 and / or Table 5 and / or Table 6 One of the Cas12i mutants containing substitutions, specifically those with sequence numbers 3-146 and 495-512. Polypeptides exhibit increased enzyme activity. In some embodiments, see Table 4 and / or The variant Cas12i2 polypeptides containing one or more substitutions listed in Table 5 and / or Table 6 The enzyme exhibits increased activity. In some embodiments, G587R T588R, G 587R T588K, G587R T588K Q590R or G587R T588 R Q590K replacement and one or more placements listed in Table 4 and / or Table 5 and / or Table 6 Cas12 mutants containing one of sequence numbers 3-146 and 495-512 further include the substitution. The i2 polypeptide exhibits increased enzyme activity. In some embodiments, the mutant Cas The 12i2 polypeptide exhibits lower enzyme activity (e.g., about 4%, 5%) compared to the parent polypeptide. %, 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%, 110%, 120%, 130%, 140%, 1 50%, 160%, 170%, 180%, 190%, 200%, or more. Or it exhibits an increase of any percentage between these.

[0555] Increased heterodouble-chain interaction In some embodiments, the mutant Cas12i2 polypeptide is compared to the parent polypeptide. All include substitutions that increase interaction with DNA / RNA hybrid molecules. Morphologically, the mutant Cas12i2 polypeptide is different from the parent polypeptide in mutant C Modifications that increase affinity between as12i2 polypeptide and DNA / RNA hybrids This includes the compounding. In some embodiments, the DNA / RNA hybrid molecule is heteroduplex. When used herein, "heteroduplex" refers to the spacer of the RNA guide and This refers to the double helix formed by the target chain (TS). When used herein, The term "seed region" refers to the TS portion immediately downstream of the PAM sequence in a heteroduplex. The seed region contains the first base that pairs with the RNA guide in the heteroduplex, and RNA-D It is necessary for NA binding and TS substitution. In some embodiments, the mutant Cas12i2 Changes that increase the interaction and / or affinity between polypeptides and heteroduplexes are non Increases specific nucleic acid contact. In some embodiments, mutant Cas12i2 polypeptide Changes that increase the interaction and / or affinity between the cytoplasm and the heteroduplex are related to the parent polypeptide. Compared to tide, it increases the formation and stability of the ternary complex.

[0556] In some embodiments, between the mutant Cas12i2 polypeptide and the heteroduplex Changes that increase interaction and / or affinity involve adding one or more amino acids, such as arginine and lydine. This involves substitution with a residue of , glutamine, asparagine, histidine, or serine. In this embodiment, the mutant Cas12i2 polypeptide contacts the heteroduplex 1 Arginine, lysine, glutamine, asparagine, histidine or This includes substitutions of serine residues. In some embodiments, the mutant Cas12i2 polypeptide D is one or more amino acids 110-130, 150-170, 250-320, 340- From 400, 420-450, 670-720, 770-810, or 830-870 One of the following residues: ginine, lysine, glutamine, asparagine, histidine, or serine This includes substitution to [a certain value]. In some embodiments, the variant Cas12i2 polypeptide is less [description of the substitution]. At least one domain / motif (for example, Rec1 domain, PI domain, Rec2 Arginine and lysine are derived from one or more amino acids in the domain or RuvC2 motif. This includes changes to glutamine, asparagine, histidine, or serine residues. Some implementations In this state, one or more nucleic acid interface substitutions, compared to the parent polypeptide, result in a heteroduplex. Interaction and / or affinity at 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%, 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. Increase.

[0557] In some embodiments, substitutions that increase heterodouble-chain interactions are performed on the parent polypeptide. Compared to the original, it increases ternary complex formation / stability. The mutant Cas12i2 polypeptide is Table 7 shows non-restrictive examples of substitutions that can alter the ability to interact with the tero double helix. In some embodiments, the Cas12 mutant includes one or more substitutions listed in Table 7. i2 polypeptides exhibit heterodouble-chain interactions (ternary complex formation) compared to their parent polypeptides. This exhibits an increase in [a certain value]. In some embodiments, mutations include one or more substitutions listed in Table 7. The Cas12i2 polypeptide is a more stable ternary polypeptide compared to the parent polypeptide. They form a combined form.

[0558] [Table 35]

[0559] [Table 36]

[0560] In some embodiments, one of sequence numbers 3-146 and 495-512 is modified. The variant Cas12i2 polypeptide further includes one or more substitutions listed in Table 7. In the embodiment, the variant Cas12i2 polypeptide is shown in Tables 2 and 7. Includes one or more substitutions.

[0561] In some embodiments, the Cas12i2 mutant exhibits increased heterodouble-chain interaction. Polypeptides can have two or more substitutions, for example, E691R A695R, S78K V43 8G, S78K V438A, S346R E343S, D782R D793N, S7 8R V438G, S78R V438A, S346K E343S or D782K D Contains 793N. In some embodiments, mutations exhibiting increased ternary complex formation / stability. The Cas12i2 polypeptide can have two or more substitutions, for example, E691R A695R. S78K V438G, S78K V438A, S346R E343S, D782R D793N, S78R V438G, S78R V438A, S346K E343S or This includes D782K and D793N. In some embodiments, SEQ ID NOs: 3-146 and 4 Any one of the 95-512 variant Cas12i2 polypeptides is E691R A6 95R, S78K V438G, S78K V438A, S346R E343S, D7 82R D793N, S78R V438G, S78R V438A, S346K E3 Further includes substitutions of 43S or D782K D793N. In some embodiments, SEQ ID NO: One of the variant Cas12i2 polypeptides, either 3-146 or 495-512, E691R A695R, S78K V438G, S78K V438A, S346R E343S, D782R D793N, S78R V438G, S78R V438A, Substitution of S346K E343S or D782K D793N and Tables 4 and / or Table 5 and / or further includes one or more substitutions listed in Table 6 and / or Table 7. In some embodiments Furthermore, the mutant Cas12i2 polypeptide is shown in Table 4 and / or Table 5 and / or Table 6 and / or include one or more substitutions listed in Table 7. In some embodiments, Table 4 and / or This is a Cas12i mutant having one or more substitutions listed in Table 5 and / or Table 6 and / or Table 7. Polypeptide 2 exhibits heterodouble-chain interactions and / or affinity compared to the parent polypeptide. (For example, 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%, 89%, 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200% younger It exhibits an increase of (or more than that, or any percentage between these). In some embodiments, substitutions are made as shown in Table 4 and / or Table 5 and / or Table 6 and / or Table 7. The Cas12i2 polypeptide variant having one or more of the following characteristics is compared to the parent polypeptide: Ternary complex formation and / or ternary complex stability (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% 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 in between. It exhibits an increase of any percentage.

[0562] In some embodiments, Tables 4 and / or 5 and / or 6 and / or 7 are listed. One of the mutations in sequence numbers 3-146 and 495-512 that includes one or more substitutions. The Cas12i2 polypeptide exhibits increased enzyme activity. In some embodiments, Changes including one or more substitutions listed in Table 4 and / or Table 5 and / or Table 6 and / or Table 7 The variant Cas12i2 polypeptide exhibits increased enzyme activity. In some embodiments, , E691R A695R, S78K V438G, S78K V438A, S346R E343S, D782R D793N, S78R V438G, S78R V438A , substitution of S346K E343S or D782K D793N and Table 4 and / or Table 5 and Sequence IDs 3-146 further include one or more substitutions listed in Tables 6 and / or 7. And any one of the 495-512 variant Cas12i2 polypeptides has enzyme activity It shows an increase. In some embodiments, the mutant Cas12i2 polypeptide is parent poly Compared to peptides, enzyme activity is (for example, about 4%, 5%, 6%, 7%, 8%, 9%, 1%) 0%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 2 0%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 3 0%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 4 0%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 5 0%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 6 0%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 7 0%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 8 0%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 9 0%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 1 00%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 1 80%, 190%, 200%, or more, or any percentage in between. It shows an increase in cents.

[0563] Increased stability of double-stranded and heterodouble-stranded DNA. During ternary complex formation, the double-stranded DNA downstream of the PAM sequence melts (for example, unwinds). The RNA guide spacer becomes the target strand (TS) and the non-target strand (NTS). It binds to form a double helix called a heteroduplex. The PAM sequence does not melt, It remains as contact double-stranded DNA. As a result, these terminal PAM dsDNA base pairs This would expose them to the environment and proteins, which could be energetically disadvantageous. Similarly, the terminal base pairs of the heteroduplex are also exposed, which can be energetically disadvantageous. See Figure 21. I want to be illuminated. In some embodiments, the mutant Cas12i2 polypeptide and exposed Aromatic and hydrophobic interactions between the terminal PAM bases of a double-stranded DNA double helix or the terminal bases of a heteroduplex. Changes that increase the sex, van der Waals, and / or cation-π interactions are ternary complex It increases the stability of DNA that melts during body formation. In some embodiments, mutant Ca Between the s12i2 polypeptide and the exposed bases of the double helix or heterodouble helix of double-stranded DNA Changes that increase aromatic, hydrophobic, van der Waals, and / or cation-π interactions This increases R-loop stability during ternary complex formation. In some embodiments, mutant Cas12i2 polypeptide and exposed bases of double-stranded DNA double helix or heterodouble helix Increases aromatic, hydrophobic, van der Waals, and / or cation-π interactions between them. The change increases ternary complex formation. In some embodiments, the mutant Cas12i2 Aromatic and sparse interactions between polypeptides and exposed bases of double-stranded DNA or heterodouble-stranded DNA. Changes that increase aqueous, van der Waals, and / or cation-π interactions are ternary compound This increases the stability of the fusion. See Figure 20D.

[0564] In some embodiments, aromatic, hydrophobic, van der Waals and / or cationic- Changes that increase π interactions involve adding one or more residues of arginine, lysine, and tryptophan. , phenylalanine, tyrosine, methionine, histidine, glutamine, threonine or The substitution involves valine residues. In some embodiments, aromatic, hydrophobic, and foundational materials are used. Changes that increase Ruwas and / or cation-π interactions affect the double helix and double strand of double-stranded DNA. One or more residues in contact with the heteroduplex are arginine, lysine, tryptoph Phenylalanine, tyrosine, methionine, histidine, glutamine, threonine Alternatively, substitution with a valine residue. In some embodiments, aromatic, hydrophobic, and fa Changes that increase der Waals and / or cation-π interactions are listed in Table 8. This is a substitution. In some embodiments, the Cas12i variant includes the substitutions listed in Table 8. Polypeptide 2 differs from the parent polypeptide in terms of the mutant Cas12i2 polypeptide and 2 Aromatic, hydrophobic, van der between exposed bases of the double helix or heterodouble helix of this DNA strand. It exhibits increased Waals and / or cation-π interactions. In some embodiments, For chemical reactions, amino acids adjacent to the terminal double-strand base pair are positively charged, aromatic, hydrophobic, or branched amino acids. No acids (for example, arginine, lysine, tryptophan, phenylalanine, tyrosine, melanin) Replace with thionine, histidine, glutamine, threonine, isoleucine, or valine. This creates more energetically favorable conditions for double-stranded DNA and heterodouble helix. This includes taking something out.

[0565] [Table 37]

[0566] In some embodiments, one of sequence numbers 3-146 and 495-512 is modified. The variant Cas12i2 polypeptide further includes one or more substitutions listed in Table 8. In the embodiment, the variant Cas12i2 polypeptide is shown in Tables 2 and 8. Includes one or more substitutions.

[0567] In some embodiments, ternary complex formation and / or ternary complex stability are increased (for example) A variant Cas1 exhibiting (by stabilizing the melting of DNA and / or the R-loop) 2i2 polypeptides can have two or more substitutions, for example, Q163N, N164W, Q163M N164W, Q163M N164Q, Q163N N164Q, I5G P577Y , including I5G P577F, I5G P577H, or I5M P577L. Some implementations Morphologically, one of the Cas1 mutants from sequence numbers 3-146 and 495-512 2i2 polypeptides include Q163N N164W, Q163M N164W, and Q163M N164Q, Q163N N164Q, I5G P577Y, I5G P577F, I Further includes 5G P577H or I5M P577L substitution. In some embodiments, The variant Cas12i2 polyp containing one of the following row numbers: 3-146 and 495-512 Petit Do is Q163N N164W, Q163M N164W, Q163M N164Q , Q163N N164Q, I5G P577Y, I5G P577F, I5G P57 7H or I5M P577L substitution and 1 from Table 4, Table 5, Table 6, Table 7 and / or Table 8 The further includes one or more substitutions. In some embodiments, the variant Cas12i2 polypeptide This includes one or more substitutions found in Tables 4, 5, 6, 7 and / or 8. In the embodiment, one or more substitutions are found in Tables 4, 5, 6, 7 and / or 8. The mutant Cas12i2 polypeptide forms a ternary complex compared to the parent polypeptide. and / or ternary complex stability (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%, 89%, 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 10 0%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 18 0%, 190%, 200%, or more than these percentages, or any percentage in between. It shows an increase in the vintage.

[0568] In some embodiments, one or more of the following are listed in Tables 4, 5, 6, 7 and / or 8. One of the Cas1 mutants from sequence numbers 3-146 and 495-512 containing the above substitution The 2i2 polypeptide exhibits increased enzyme activity. In some embodiments, see Tables 4 and 5. , a variant Cas12i2 containing one or more substitutions listed in Tables 6, 7 and / or 8 Lipeptides exhibit increased enzyme activity. In some embodiments, Q163N N16 4W, Q163M N164W, Q163M N164Q, Q163N N164Q, I 5G P577Y, I5G P577F, I5G P577H, or I5M P577L This further includes substitutions and one or more substitutions listed in Tables 4, 5, 6, 7 and / or 8. One of the variant Cas12i2 polypeptides from sequence numbers 3-146 and 495-512 The cydore exhibits increased enzyme activity. In some embodiments, the mutant Cas12i2 poly Peptides exhibit enzyme activity at a lower level 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%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 16 0%, 170%, 180%, 190%, 200%, or more than these It exhibits an increase of any percentage in between.

[0569] Increased conformational changes Conformational changes, such as those resulting from the binding of RNA guides or target DNA, This affects the function of the mutant Cas12i2 polypeptide, for example, conformational changes. This can alter the reaction rate of the mutant Cas12i2 polypeptide during ternary complex formation. The Rec1 (helix II) domain of Cas12i2 moves and rotates, and then binds to DNA. Applicable to. See Figures 22A and 22B. In some embodiments, Herrick Changes that increase the movement of the S2 domain (e.g., mobility or conformational changes) are , increases DNA binding / DNA binding affinity. In some embodiments, helix I Substitutions that increase mobility in the I domain, for example, from bulk amino acids, with smaller side chains Substitutions to a smaller amino acid (alanine, valine, glycine, or serine residue) are: Increases ternary complex formation. In some embodiments, the movement of the helix II domain. Changes that increase (for example, mobility or conformational changes) affect the stability of the ternary complex. To increase. In some embodiments, conformational changes of the helix II domain. Changes that increase the amount involve replacing one or more residues with alanine, valine, glycine, or serine residues. The goal is to replace the helix II domain. In some embodiments, the mobility of the helix II domain is increased. This change involves substituting one or more residues with alanine, valine, glycine, or serine residues. In some embodiments, the variant Cas12i2 polypeptide is a helix This includes changes in one or more amino acids near the II domain. In some embodiments, The variant Cas12i2 polypeptide has one or more near the helix II domain. This includes the conversion of amino acids to alanine, valine, glycine, or serine. In some embodiments, In this case, the mutant Cas12i2 polypeptide has one or more amino acids from 327 to 330 Includes substitution with one of alanine, valine, glycine, or serine. Some embodiments In this study, the mutant Cas12i2 polypeptide contains the substitutions shown in Table 9.

[0570] [Table 38]

[0571] In some embodiments, one of sequence numbers 3-146 and 495-512 is modified. The variant Cas12i2 polypeptide further includes one or more substitutions listed in Table 9. In the embodiment, the mutant Cas12i2 polypeptide is shown in Tables 2 and 9. Includes one or more substitutions.

[0572] In some embodiments, changes that increase helix II domain mobility are shown in Table 9. These are the substitutions listed. In some embodiments, one or more of the substitutions listed in Table 9 are included. The mutant Cas12i2 polypeptide exhibits a different helix II compared to the parent polypeptide. Domain mobility of approximately 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 1% 3%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 2 3%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 3 3%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 4 3%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 5 3%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 6 3%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 7 3%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 8 3%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 9 3%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120 %, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200 An increase of % or more, or any percentage in between. In some embodiments, the changes that increase DNA binding / DNA affinity are shown in Table 9. The substitution is one or more of the substitutions listed in Table 9. The mutant Cas12i2 polypeptide exhibits a higher DNA binding rate compared to the parent polypeptide. Affinity of approximately 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 1 4%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 2 4%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 3 4%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 4 4%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 5 4%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 6 4%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 7 4%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 8 4%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 9 4%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 13 0%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, etc. It exhibits an increase of any percentage greater than or between these values. In the embodiment, the variant Cas12i2 polypeptide containing the substitutions listed in Table 9 is Compared to the parent polypeptide, the ternary complex formation and / or ternary complex stability (for example, 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%, 89%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 14 0%, 150%, 160%, 170%, 180%, 190%, 200%, or more than that. It exhibits an increase of (or any percentage between) these values.

[0573] In some embodiments, a variant Cas exhibiting increased helix II domain mobility 12i2 polypeptides can have two or more substitutions, for example, L327V N328S N329 G L330A, L327A N328S N329G L330A, L327V N3 28G N329S L330A or L327V N328G N329G L330A Includes. In some embodiments, a variant Cas12 exhibiting increased DNA binding / affinity. i2 polypeptides can have two or more substitutions, for example, L327V N328S N329G L330A, L327A N328S N329G L330A, L327V N328 Includes G N329S L330A or L327V N328G N329G L330A Hmm. In some embodiments, the Cas12 mutant exhibits increased ternary complex formation / stability. i2 polypeptides can have two or more substitutions, for example, L327V N328S N329G L330A, L327A N328S N329G L330A, L327V N328 Includes G N329S L330A or L327V N328G N329G L330A In some embodiments, one of sequence numbers 3-146 and 495-512 The variant Cas12i2 polypeptides are L327V, N328S, N329G, and L330. A, L327A N328S N329G L330A, L327V N328G N3 Further includes replacement of 29S L330A or L327V N328G N329G L330A. In some embodiments, one of sequence numbers 3-146 and 495-512 The variant Cas12i2 polypeptides are L327V, N328S, N329G, and L330. A, L327A N328S N329G L330A, L327V N328G N3 29S L330A or L327V N328G N329G L330A replacement and table Tables 4 and / or 5 and / or 6 and / or 7 and / or 8 and / or 9 are listed. The further includes one or more substitutions. In some embodiments, the variant Cas12i2 polyp Petit de is in Table 4 and / or Table 5 and / or Table 6 and / or Table 7 and / or Table 8 and / or Includes one or more substitutions from Table 9. In some embodiments, Tables 4 and / or Table 9. 5 and / or one or more of the substitutions in Table 6 and / or Table 7 and / or Table 8 and / or Table 9 The mutant Cas12i2 polypeptide exhibits better DNA binding / Affinity (for example, 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%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 12 0%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 20 An increase of 0% or more, or any percentage in between. In some embodiments, Tables 4 and / or 5 and / or 6 and / or 7 and / or a variant Cas12i2 polypeptide having one or more substitutions listed in Table 8 and / or Table 9 Compared to the parent polypeptide, tide exhibits superior ternary complex formation and / or ternary complex stability (e.g., For example, 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%, 89%, 90%, 91%, 92%, 93%, 94% %, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130 %, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or It exhibits an increase (of a percentage greater than or between these two values).

[0574] In some embodiments, Table 4 and / or Table 5 and / or Table 6 and / or Table 7 and / or This includes sequence numbers 3-146 and 495, which contain one or more substitutions listed in Table 8 and / or Table 9. Any one of the ~512 variant Cas12i2 polypeptides exhibits increased enzyme activity. In some embodiments, Table 4 and / or Table 5 and / or Table 6 and / or Table 7 and / Or a variant Cas12i2 polyp containing one or more substitutions listed in Table 8 and / or Table 9 Butide exhibits increased enzyme activity. In some embodiments, L327V N328S N329G L330A, L327A N328S N329G L330A, L32 7V N328G N329S L330A or L327V N328G N329G Replacement of L330A and Tables 4 and / or 5 and / or 6 and / or 7 and / or 8 and / or Sequence IDs 3-146 and 495-, which further include one or more substitutions listed in Table 9. Any one of the 512 Cas12i2 polypeptide mutants exhibits increased enzyme activity. In some embodiments, the mutant Cas12i2 polypeptide is compared to the parent polypeptide. In comparison, enzyme activity (for example, 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%, 89%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 11 0%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 19 0%, 200%, or more than that, or any percentage in between. ) shows an increase.

[0575] In some embodiments, the target single-stranded DNA is the active site of the Cas12i2 polypeptide. A change that increases the connectivity of the interface between Nuc and Helix II when it is in this state is, Increases the transition from a binary complex to a ternary complex. In some embodiments, Nuc and Changes that increase the interface connectivity with Rix II increase ternary complex formation. (Figure) See 22B. In some embodiments, the interface between Nuc and Helix II. Changes that increase the linkage increase the stability of the ternary complex. In some embodiments, Changes that increase the interface linkage between Nuc and Helix II involve one or more residues as This involves substitution with a paratic acid, glutamic acid, arginine, or lysine residue. In the application morphology, the mutant Cas12i2 polypeptide has one or more amino acids 380-3 From 90 or 910-930, aspartic acid, glutamic acid, arginine or lysine This includes substitution with one of the variant Cas12i2 peptides. In some embodiments, the variant Cas12i2 peptide The substitutions include those shown in Table 10.

[0576] [Table 39]

[0577] In some embodiments, one of sequence numbers 3-146 and 495-512 is modified. The variant Cas12i2 polypeptide further includes one or more substitutions listed in Table 10. In some embodiments, the variant Cas12i2 polypeptide is shown in Tables 2 and 10. Includes one or more substitutions.

[0578] In some embodiments, the substitutions shown in Table 10 are at the interface between Nuc and Helix II. Increases the number of connections. In some embodiments, the mutation has one or more of the substitutions listed in Table 10. The variant Cas12i2 polypeptide has Nuc and helix I compared to the parent polypeptide. The interface connection with I is 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%, 89%, 90%, 91%, 92% %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, ...

Claims

[Claim 1] The invention described in the specification.

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