Engineered protein

Abstract

To provide engineered proteins usable as genome engineering tools.SOLUTION: Disclosed is a protein comprising at least one substitution at amino acid position 188 to histidine, and at position 195 to lysine in the amino acid sequence of AsCas12f. Preferably, the protein is a double mutant protein comprising at least a substitution at position 188 to histidine, or a double mutant protein comprising at least a substitution at position 195 to lysine. Preferably, the protein is a triple mutant protein comprising at least a substitution at position 188 to histidine, and a substitution at position 232 to alanine, or a triple mutant protein comprising at least a substitution at position 195 to lysine and a substitution at position 232 to alanine.SELECTED DRAWING: Figure 1-1

Description

[Technical field]

[0001] The present invention relates to engineered substances, such as engineered proteins. [Background technology]

[0002] It is known that the CRISPR-Cas systems of bacteria and archaea provide adaptive immunity against foreign nucleic acids. Patent Document 1 discloses that CRISPR-Cas systems are classified into two classes (classes 1 and 2) and six types (types I to VI). Class 2 systems include types II, V, and VI, and include single multi-domain effector Cas proteins such as Cas9 (type II) and Cas12 (type V). As disclosed in Patent Document 1, Cas9 and Cas12a exhibit strong nuclease activity in eukaryotic cells and are therefore widely used as versatile genome engineering tools. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication No. 2022 / 092317 Summary of the Invention [Problem to be solved by the invention]

[0004] Proteins used as genome engineering tools, such as Cas9 and Cas12, are required to have various properties. For example, SpCas9 derived from Streptococcus pyogenes is suitable as a genome engineering tool because of its strong activity. However, its molecular size is large at 1,367 amino acid residues (the gene is 4.1 kb). On the other hand, the space for a foreign gene in an AAV vector is less than 4.5 kb, so it is difficult to even load SpCas9 on an AAV vector and introduce it into animal cells, and its use as a genome engineering tool is limited by its large size. Therefore, there is a need to provide engineered proteins that can be used as genome engineering tools.

[0005] The present invention has been made under the above-mentioned background, and aims to provide an engineered substance that can be used as a genome engineering tool. [Means for solving the problem]

[0006] One embodiment of the present invention is a protein having, in the amino acid sequence represented by SEQ ID NO:1, 1) no mutation at amino acid position 195 (D195) and a mutation at amino acid position 188, or 2) no mutation at amino acid position S188 and a mutation at amino acid position 195. Effect of the Invention

[0007] According to the present invention, it is possible to provide an engineered substance that can be used as a genome engineering tool. [Brief description of the drawings]

[0008] [Figure 1-1] A shows the domain structure of AsCas12f, and B shows the complex structure analysis using cryo-electron microscopy. [Figure 1-2] C is a structural model of the AsCas12f-sgRNA-target DNA ternary complex. [Figure 1-3] D is a schematic diagram of sgRNA and target DNA. [Figure 2-1]A: Size exclusion chromatography profile, B: Illustrative diagram of a representative cryo-EM image recorded on TitanKrios. [Figure 2-2] C is an illustration of the single particle cryo-electron microscopy image processing workflow, and D is a graph showing the Fourier shell correlation (FSC) curves for 3D reconstruction. [Figure 2-3] E: Size exclusion chromatography profile, F: Illustration of a representative cryo-EM image recorded on TitanKrios. [Figure 2-4] G is an illustration of the single particle cryo-electron microscopy image processing workflow, and H is a graph showing Fourier shell correlation (FSC) curves for 3D reconstruction. [Figure 2-5] I: Size exclusion chromatography profile; J: Illustration of a representative cryo-EM image recorded on TitanKrios. [Figure 2-6] K is an illustration of the single particle cryo-electron microscopy image processing workflow, and L is a graph showing Fourier shell correlation (FSC) curves for 3D reconstruction. [Figure 3-1] A is an explanatory diagram comparing the structures of AsCas12f and UnCas12f. [Figure 3-2] B is an explanatory diagram of the dimer interface between AsCas12f.1 and AsCas12f.2 and the recognition site of the sgRNA scaffold, and C is an explanatory diagram of the superposition of AsCas12f.1 and AsCas12f.2 based on the REC lobe. [Figure 3-3] D is an explanatory diagram of the dimer interface between REC.1 and REC.2, E is the dimer interface between RuvC.1 and RuvC.2, F is the recognition of Stem2 by AsCas12f.2, and G is the recognition of Stem2 by AsCas12f.1. [Figure 4-1] A is a diagram of the recognition sites of the guide RNA and target DNA, and B is an illustration of the electrostatic surface potential of AsCas12f. [Figure 4-2] C–E are schematic diagrams of stem 1 and PK 1, stem 3, and the recognition of the guide RNA-target DNA heteroduplex, respectively. [Figure 4-3]F is an explanatory diagram comparing the structures of the TS, NTS, and RuvC active site of AsCas12f with those of UnCas12f derived from an uncultured archaea. [Figure 5-1] A) Schematic overview of the AsCas12f DMS library design and B) the DMS approach for genomic assessment of gene editing efficiency in the context of the GFP gene deletion. [Figure 5-2] C: Diagram of the effect of single mutations on genome editing activity; D: Diagram of the split-GFP-reconstitution assay to assess genome editing activity on the VEGF gene in HEK293T cells; E: Diagram of genome editing activity of AsCas12f mutations; F: Time course of GFP deletion induced by AsCas12f-HKRA and AsCas12f in HEK293T cells expressing d2EGFP. [Figure 6-1] A, Graph showing correlation of the effect of single mutants on GFP deletion. [Figure 6-2] B-1 is a list of single amino acid substitutions that increase editing efficiency by 20% or more compared to WT. [Figure 6-3] B-2 is a list of single amino acid substitutions that increase editing efficiency by 20% or more compared to WT. [Figure 6-4] C, D, and E are graphs showing the genome editing activity of AsCas12f mutants in double, triple, and quadruple configurations, respectively. [Figure 7-1] A is an illustration of the sequences of sgRNA mutants; B is an illustration depicting the genome editing activity of sgRNA mutants determined by flow cytometry; C is a graph depicting sgRNA expression quantified by qPCR for common sequence sites (n=3 means ±SD); D is a graph depicting the time course of GFP deletion. [Figure 7-2] E is a schematic diagram of the wild-type sgRNA, and F is a schematic diagram of sgRNA_ΔS3-5_v7. [Figure 7-3] G is an illustration of the structure of the wild-type sgRNA scaffold, and H is an illustration of the structure of the sgRNA_ΔS3-5_v7 scaffold. [Figure 7-4]I: Enlarged view of the PK1 region of the wild-type sgRNA, J: enlarged view of the PK1 region of sgRNA_ΔS3-5_v7, K: enlarged view of the PK2 region of the wild-type sgRNA, and L: enlarged view of the stem 3 region of sgRNA_ΔS3-5_v7. [Figure 8-1] A is an illustration of the target DNA and target DNA complex results obtained using cryo-electron microscopy. [Figure 8-2] B is the dimer interface of AsCas12f-YHAM, C is target DNA recognition by AsCas12f-YHAM, D is hydrophobic interaction of AsCas12f-YHAM, E is guide RNA-target DNA heteroduplex recognition by AsCas12f-HKRA, F is target DNA recognition, and G is an illustration of sgRNA scaffold recognition. [Figure 9] Illustration of the results of data collection, processing, model refinement and validation. [Figure 10] Illustration of the results of data collection, processing, model refinement and validation. [Figure 11-1] A is an explanatory diagram of the self-targeting library, and B is a graph showing the editing frequency. [Figure 11-2] C to E are graphs showing genome editing activity of various Cas. [Figure 12-1] A is a graph showing the comparison results of target editing efficiency, and B is an explanatory diagram of the PAM duplex recognition results. [Figure 12-2] C and D are graphs showing the indel efficiency at other target sites, etc. [Figure 12-3] E is a graph showing the indel efficiency at other target sites, etc. [Figure 13-1] A is a graph showing the effect of mismatches, and B is an explanatory diagram comparing the number of off-target edits. [Figure 13-2] C-1 is an illustration of off-target sites identified by guide sequences. [Figure 13-3] C-2 is an illustration of off-target sites identified by guide sequences. [Figure 13-4] C-3 is an illustration of off-target sites identified by guide sequences. [Figure 14-1]FIG. 1 is an explanatory diagram of the generation of muscular dystrophy model induced pluripotent stem cells (iPSCs) and their differentiation into cardiomyocytes (iPSC-CMs). (A) is an explanatory diagram of the confirmation of complete deletion of DMD exons by PCR, and (B) is an explanatory diagram of the genomic sequence of a DMD exon 44 deletion-iPS cell line. [Figure 14-2] FIG. 1 is an explanatory diagram of the generation of muscular dystrophy model induced pluripotent stem cells (iPSCs) and their differentiation into cardiomyocytes (iPSC-CMs). C is an explanatory diagram of α-actinin-mCherry flow cytometry analysis on day 14 of cardiac differentiation, and D is a diagram showing western blot analysis. [Figure 14-3] E is a graph showing a comparison of luciferase activity, F is a schematic diagram of the plasmid construct, and G is a graph showing an increase in luciferase activity in cells into which the plasmid vector was introduced. [Figure 15-1] A to F are diagrams illustrating treatment and knock-in using mutants. [Figure 15-2] G to J are diagrams illustrating treatment and knock-in using mutants. [Figure 16-1] A to F are diagrams illustrating knock-in of FIX (mouse) at the Alb locus using the AsCas12f mutant. [Figure 16-2] G to J are diagrams illustrating gene transcription activation using AsCas12f mutants. [Figure 17] A is an explanatory diagram showing luciferase expression due to gene transcription activation in mice, and B is a graph showing ROI values. [Figure 18] An illustration of the nucleic acid sequences used for structural analysis in connection with STAR Methods. [Figure 19] Illustration of nucleic acid sequences used for genome editing in the context of STAR Methods. [Figure 20-1] Diagram of primers and constructs used for genome editing in the context of STAR Methods. [Figure 20-2] Diagram of primers and constructs used for genome editing in the context of STAR Methods. [Figure 20-3]Diagram of primers and constructs used for genome editing in the context of STAR Methods. [Figure 20-4] Diagram of primers and constructs used for genome editing in the context of STAR Methods. [Figure 21-1] An explanation of key resources in STAR Methods. [Figure 21-2] An explanation of key resources in STAR Methods. [Figure 21-3] An explanation of key resources in STAR Methods. [Figure 21-4] An explanation of key resources in STAR Methods. [Figure 21-5] An explanation of key resources in STAR Methods. [Figure 21-6] An explanation of key resources in STAR Methods. [Figure 21-7] An explanation of key resources in STAR Methods. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] In the present specification, when a substitution mutation in an amino acid sequence is expressed, it may be expressed by the single letter code of the original amino acid, followed by a position number with one to three digits, and then the single letter code of the substituted amino acid. For example, when a mutation occurs at amino acid position 1022 in which aspartic acid (D) is replaced by asparagine (N), it is expressed as "D1022N", which is synonymous with "substitution of Asp at amino acid position 1022 by Asn". The -CO-NH- bond connecting amino acids is called a peptide bond. An amino acid bonded in this way is called an amino acid residue. In addition, "mutation" refers to a change that has occurred in the starting amino acid or nucleic acid sequence. This term is intended to include substitution, insertion, and deletion. In addition, in this embodiment, the engineered substance is a protein, for example, engineered AsCas12f. The engineered substance may be used as a composition together with other substances such as guide RNA and vectors.

[0010] Bacterial and archaeal CRISPR-Cas systems provide adaptive immunity against foreign nucleic acids and are divided into two classes (classes 1 and 2) and six types (types I–VI). Class 2 systems include types II, V, and VI, which contain single multidomain effector Cas proteins such as Cas9 (type II) and Cas12 (type V). The use of CRISPR-Cas is not limited to genome editing. Since Cas that has lost its cleavage activity functions as an RNA-dependent DNA binding protein, it is used in technologies such as transcriptional activity regulation and single base editing by fusing various effectors. Genome editing enzymes are introduced into target tissues using viral vectors such as AAV, but there is a limit to the size of the gene that can be carried, and Cas9 and Cas12a are difficult to carry due to their size.

[0011] Cas9 binds to dual RNA guides (CRISPR RNA [crRNA] and trans-activating crRNA [tracrRNA]) or single guide RNA (sgRNA) that is complementary to the 20 nt guide segment of the RNA guide and cleaves double-stranded DNA (dsDNA) targets at sequences adjacent to an NGG (where N is any nucleotide) proto-spacer adjacent motif (PAM).

[0012] Among the various type V Cas12s, type VA Cas12a (also known as Cpf1) binds to crRNA and cleaves dsDNA targets with TTTV (where V is A, G, or C) PAM. Cas9 contains two nuclease domains, HNH and RuvC, which cleave the target strand (TS) and non-target strand (NTS) of dsDNA targets, respectively.

[0013] In contrast, Cas12a cleaves both the TS and NTS with a single RuvC nuclease domain.Cas9 and Cas12a exhibit potent nuclease activity in eukaryotic cells and are therefore widely used as versatile genome engineering tools.

[0014] In addition, Cas9 (type II) (SpCas9) from Streptococcus pyogenes and, among the various type V Cas12s, Cas12a (AsCas12a) from Acidaminococcus sp. are widely used as genome editing tools in human cells.

[0015] However, these are relatively large in size, which is a limitation in delivery by adeno-associated virus vectors, which have cargo size limitations. On the other hand, type VF Cas12f (AsCas12f) derived from Acidibacillus sulfuroxidans is very compact (422 amino acids) and functions in human cells. Table 1 shows the size comparison results between AsCas12f and Cas reported so far. AsCas12f has the amino acid sequence shown in SEQ ID NO: 1, and is the smallest Cas currently reported at 422 residues, which is less than half the size of Cas9, Cas12a, Cas12b, Cas12e, etc. [Table 1] However, the nuclease activity of wild-type AsCas12a is relatively low. Therefore, in this embodiment, two types of AsCas12f activity-enhanced (enAsCas12f) variants were developed by structural analysis and mutant screening using a cryo-electron microscope.

[0016] In particular, the enAsCas12f mutant showed genome editing activity equal to or greater than that of SpCas9 and AsCas12a in human cells. Structural analysis revealed that the mutant stabilized dimer formation and enhanced interaction with nucleic acids, improving DNA cleavage activity. Furthermore, an all-in-one AAV vector co-packaged with partner genes showed efficient knock-in / knock-out activity and transcriptional activation in mice. Taken together, enAsCas12f may provide a minimal genome editing platform for in vivo gene therapy.

[0017] The clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (Cas) systems, which provide adaptive immunity against mobile genetic elements in bacteria and archaea, are divided into two classes (class 1 and class 2) and six types (types I-VI). SpCas9, accompanied by dual RNA guides (CRISPR RNA [crRNA] and trans-activating crRNA [tracrRNA], or artificially linked single guide RNA [sgRNA]), uses its HNH and RuvC nuclease domains to cleave double-stranded DNA (dsDNA) targets flanked by NGG (where N is any nucleotide) protospacer adjacent motifs (PAMs). In contrast, AsCas12a binds to crRNA and uses a single RuvC nuclease domain to cleave dsDNA targets with TTTV (where V is A, G, or C) PAMs.

[0018] SpCas9 and AsCas12a show robust nuclease activity in eukaryotic cells and are widely used as versatile genome engineering tools. However, the large gene size of both SpCas9 (1,368 amino acids) and AsCas12a (1,307 amino acids) makes it difficult to efficiently package them into a single adeno-associated virus (AAV) vector, hindering their clinical application in in vivo gene therapy.

[0019] VF-type Cas12 effectors have been shown to be very compact (400-700 amino acids) RNA-guided DNA endonucleases. Cas12f associates with dual crRNA:tracrRNA guides and cleaves target DNA with a T-rich PAM. Previous structural studies of Cas12f (UnCas12f) from an uncultured archaea with 529 amino acids have revealed that UnCas12f functions as a dimer to compensate for its small size. However, UnCas12f cleaves DNA targets only under low-salt conditions in vitro and lacks activity in human cells, limiting its application as a genome editing tool. On the other hand, as described below, the smallest Cas12Cf (AsCas12f) from Acidibacillus sulfuroxidans consists of only 422 amino acids and can cleave DNA targets with a TTR (R is A or G) PAM under physiological conditions in vitro. Furthermore, AsCas12f exhibits small but detectable genome editing activity in human cells, making it useful as a compact genome editing tool that can be packaged into a single AAV vector.

[0020] In this embodiment, we improved the AsCas12f system for genome editing. By combining structural analysis and DMS (Deep mutational scanning), we revealed the molecular basis and identified a detailed landscape of amino acid substitutions that significantly enhance the nuclease activity of AsCas12f. The synergistic effect of these mutations and guide RNA engineering significantly improved the genome editing efficiency of AsCas12f in human cells, to a level comparable to or even higher than that of SpCas9 and engineered Cas12a proteins. The size of AsCas12f is compact, which is very useful in AAV-deliverable gRNAs and partner genes such as base editors and epigenetic modifiers, making the AsCas12f system a promising genome editing platform.

[0021] <Cryo-electron microscopy structure of the AsCas12f-sgRNA-target DNA ternary complex> Figure 1-1 shows a diagram representing the domain structure of AsCas12f (A) and the cryo-electron microscopy map of the AsCas12f-sgRNA-target DNA ternary complex (B). Figure 1-2 shows a diagram representing the structural model of the AsCas12f-sgRNA-target DNA ternary complex (C). Figure 1-3 is a schematic diagram of sgRNA and target DNA (D), where the disordered region is enclosed by a dashed frame, TS is the target strand, NTS is the non-target strand, and PK represents a pseudoknot. To distinguish NTS from TS, NTS is denoted by an asterisk.

[0022] Figures 2-1 and 2-2 show cryo-electron microscopy analysis of AsCas12f-sgRNA-target DNA (A - D), Figures 2-3 and 2-4 show cryo-electron microscopy analysis of AsCas12f-YHAM-sgRNA_ΔS3-5_v7-target DNA (E - H), and Figures 2-5 and 2-6 show cryo-electron microscopy analysis of the AsCas12f-HKRA-sgRNA_ΔS3-5_v7-target DNA complex (I - L). Specifically, in Figures 2-1 to 2-6, A represents the size-exclusion chromatography profile of AsCas12f-sgRNA-target DNA, E represents the size-exclusion chromatography profile of EAsCas12f-YHAM-sgRNA_ΔS3-5_v7-target DNA, and I represents the size-exclusion chromatography profile of the AsCas12f-HKRA-sgRNA_ΔS3-5_v7-target DNA complex. The peak fractions were used for the following cryo-electron microscopy analysis. Also, in Figures 2-1 to 2-6, B, F, and J represent representative cryo-EM images recorded on a TitanKrios at 300 kV with a K3 camera. C, G, and K are illustrations of the single particle cryo-EM image processing workflow. D, H, and L are graphs showing the Fourier shell correlation (FSC) curves for 3D reconstruction with the gold-standard cut-off (FSC=0.143), shown as black dotted lines.

[0023] As shown in Figures 1-1-1-2 A-C, Figures 2-1-2 A-D, and Figures 9 and 10, which show the results of data collection, processing, model refinement, and validation, to understand the molecular mechanism of AsCas12f, we used cryo-electron microscopy (cryo-EM) to analyze the complex structure of AsCas12f with a 222-nucleotide (nt) sgRNA and a 38-nt dsDNA with the DNA backbone around the cleavage site phosphorothioate-modified with TTA PAM, and reconstructed it at a total resolution of 3.1 Å (angstroms). The structure revealed that an asymmetric homodimer was formed by the assembly of two AsCas12f molecules (AsCas12f.1 and AsCas12f.2) with one sgRNA molecule.

[0024] The Cas12f dimer has a bilobed structure with a recognition (REC) lobe and a nuclease (NUC) lobe, and the guide RNA-target DNA heteroduplex is bound to a central channel between the two lobes. The REC lobe is composed of the wedge (WED) and REC domains of AsCas12f.1 and AsCas12f.2 (WED.1 / WED.2 / REC.1 / REC.2), and the NUC lobe contains the RuvC and target nucleic acid binding (TNB) domains of AsCas12f.1 and AsCas12f.2 (RuvC.1 / RuvC.2 / TNB.1 / TNB.2). The AsCas12f-sgRNA-DNA complex structure was also found to function as a dimer, similar to UnCas12f.

[0025] As shown in Figure 1-3D, the sgRNA is composed of a 20-nt guide segment (G1 to C20) and a 202-nt sgRNA scaffold (A(-202) to C(-1)). The sgRNA scaffold has an unexpected architecture with two pseudoknots (PK1, PK2) and five stems (Stem1 to Stem5) that were not predicted from its primary sequence. Stem1 has a 6-base pair (bp) duplex (U(-192):A(-175) to G(-187):C(180)), and PK1 contains a 4-bp duplex (U(-185):A(-2) to A(-182):U(-5)).

[0026] Stem 2 contains a 16-bp duplex (from G(-171):U(-125)) with two non-canonical base pairs (G(-171):U(-125) to G(-161):U(-135)), and stem 3 consists of three base pairs (G(-118):C(-109) to C(-116):G(-111)). PK2 has two base pairs (G(-122):C(-88) and G(-121):C(-87)) connecting stems 2 and 3. Stem 4 contains a 16-bp duplex with two coaxially stacked duplexes (U(-102):A(-80) to C(-96):G(-86)). As shown in Figure 1-3D, in particular, C(-103) located between stems 3 and 4 is flipped out and inserted into stem 1 and PK1, forming a base pair with G(-181), thereby forming a continuous helix consisting of stem 1 and PK1. As shown in Figure 1-3, the 5' region (A(-202):A(-194)) and stem 5 (U(-70):U(-15)) are disordered in structure, suggesting their flexibility.

[0027] In Figures 3-1 to 3-3, A in Figure 3-1 shows a structural comparison between AsCas12f and UnCas12f derived from an uncultured archaea (PDBID: 7C7L). B in Figure 3-2 shows the dimer interface between AsCas12f.1 and AsCas12f.2 and the recognition site of the sgRNA scaffold. C in Figure 3-2 shows the superposition of AsCas12f.1 and AsCas12f.2 based on the REC lobe. D in Figure 3-3 shows the dimer interface between REC.1 and REC.2, and E in Figure 3-3 shows the dimer interface between RuvC.1 and RuvC.2. F in Figure 3-3 shows the recognition of Stem2 by AsCas12f.2, and G in Figure 3-3 shows the recognition of Stem2 by AsCas12f.1.

[0028] AsCas12f (422 residues) is more than 100 residues smaller than UnCas12f (529 residues), while its cognate sgRNA is approximately 40 nucleotides longer than UnCas12f. As shown in Figure 3-1A, structural comparison of AsCas12f and UnCas12f revealed that, although the domain organization is similar, AsCas12f lacks the zinc finger (ZF) domain inserted between the WED and REC domains found in UnCas12f. Instead, in the AsCas12f structure, the ZF domain is replaced by PK2 and Stem3, which are absent in UnCas12f. These findings explain the compactness of AsCas12f, which is compensated for by the longer sgRNA.

[0029] Small AsCas12f scaffolds associated with large sgRNAs As shown in Figures 3-2-3B-E, AsCas12f.1 and AsCas12f.2 interact with each other mainly through hydrophobic interactions at the REC.1 / REC.2 and RuvC.1 / RuvC.2 interfaces, thereby promoting the dimerization of AsCas12f, as observed with UnCas12f. As shown in Figures 3-3D-E, REC.1 and REC.2 form a symmetric interface in which they face each other in opposite directions, with W43, F48, and I116 playing central roles, while RuvC.1 and RuvC.2 form an asymmetric interface in which each protomer adopts a different conformation. In particular, the α1 helix and α1-α2 loop of RuvC.1 interact with the α1 helix and α2 helix of RuvC.2, as shown in Figure 3-3E. As shown in Figure 3F and G, in addition to protein-protein interactions, the lower and upper regions of stem 2 interacted extensively with AsCas12f.1 and AsCas12f.2, respectively, suggesting that protein-nucleic acid interactions also contribute to AsCas12f dimerization.

[0030] A in Figure 4-1 represents the recognition site of the guide RNA and the target DNA, and B in Figure 4-1 represents the electrostatic surface potential of AsCas12f. The heteroduplex of the sgRNA and the target DNA, and the stem 1, stem 2, and PK1 regions of the sgRNA scaffold are accommodated in the positively charged groove of the AsCas12f dimer. C to E in Figure 4-2 represent the recognition of stem 1 and PK1, stem 3, and the guide RNA-target DNA heteroduplex, respectively. F in Figure 4-3 represents a structural comparison of the TS, NTS, and RuvC active sites of AsCas12f with UnCas12f derived from an uncultured archaea (PDBID: 7C7L). The position of the RuvC.1 active site relative to the target DNA is similar in both structures, suggesting that AsCas12f also cleaves the target DNA using the RuvC.1 domain.

[0031] As shown in Figure 4-1A and B, the assembly of the sgRNA scaffold with AsCas12f is driven by both base-specific and non-specific interactions. As shown in Figure 4-2C, the continuous helix consisting of Stem1 and PK1 is recognized and accommodated in the groove formed mainly by the WED.1 and RuvC.1 domains. As shown in Figure 4-3F and G, stem 2 passes through the gap between AsCas12f.1 and AsCas12f.2 and interacts extensively with both protomers, which may enhance dimerization as described above. As shown in Figure 4-2D, PK2 is recognized by the WED.1 and REC.1 domains through sugar-phosphate backbone interactions and is further stabilized by the coordination of metal ions. In contrast, stems 3 and 4 are exposed to the solvent and have minimal interactions with AsCas12f.

[0032] As shown in Figure 4-1B and Figure 4-2E, the heteroduplex of the guide RNA and the target DNA is accommodated in the positively charged central channel and recognized by AsCas12f by non-specific base interactions. As shown in Figure 4-2E, the PAM-proximal region of the heteroduplex (G1:dC18 to G12:dC7) is primarily recognized by Cas12f.1, whereas the PAM-distal region of the heteroduplex (C13:dG6 to G18:dC1) is recognized by AsCas12f.2. As shown in Figure 4-2E, in particular, P240.2 of RuvC.2 stacks with the G18:dC1 base pair of the heteroduplex, indicating that the 18 nucleotides of the spacer sequence function as a guide segment. As shown in Figure 4-3F, structural comparison with UnCas12f revealed that the location of RuvC.1 in AsCas12f is similar to that of RuvC.1 in UnCas12f, which cleaves both the target and non-target strands. These structural observations suggest that AsCas12f.1 is responsible for cleaving the target DNA, while AsCas12f.2 plays an important role in recognizing the PAM-distal region of the heteroduplex.

[0033] <AsCas12f engineering to improve genome editing efficiency in mammalian cells> AsCas12f has been shown to exhibit limited genome editing activity in human cells. To expand the utility of this ultra-compact protein, we created AsCas12f mutants with enhanced activity. We performed deep mutational scanning (DMS) to examine how all amino acid substitutions affect genome editing efficiency in HEK293T cells. In detail, we used DMS to evaluate how mutations that changed all 422 residues of AsCas12f to 20 different amino acids affect the activity of GFP (Green Fluorescent Protein)-targeted genome editing (genome editing efficiency was determined by the disappearance of GFP fluorescence).

[0034] In Figure 5-1, A is an illustration of the DMS library design of AsCas12f, B is a diagram outlining the DMS approach for genomic evaluation of gene editing efficiency in the context of GFP gene deletion. In Figure 5-2, C is an illustration of how all single mutations affect genome editing activity. D is an illustration of a split-GFP-reconstitution assay for evaluating genome editing activity on the VEGF gene in HEK293T cells. Plasmid-encoded mCherry is co-infected to identify plasmid-infected cells. Also in Figure 5-2, E is a diagram depicting genome editing activity of AsCas12f mutants carrying editing efficiency-enhancing mutations as determined by flow cytometry (n=4), and F is a graph depicting the time course of GFP deletion induced by AsCas12f-HKRA and AsCas12f in HEK293T cells expressing d2EGFP (n=3, mean±SD). sgRNA_ΔS5 and sgRNA_ΔS3-5_v7 were transduced using single copy lentivirus infection. In the graphs in Figure 5-2F, (a) shows the results for HKRA+ΔS3-5_v7, (b) shows the results for HKRA+ΔS5, (c) shows the results for WT+ΔS3-5_v7, and (d) shows the results for WT+ΔS5.

[0035] As shown in Figure 5-1A, a plasmid expressing both EGFP and AsCas12f was designed to create an AsCas12f library covering all 20 amino acid substitutions in the entire sequence (M1-K422). As shown in Figure 5-1B, sgRNA targeting GFP was introduced into HEK293T cells, and then the AsCas12f library packaged in lentivirus was expressed at a multiplicity of infection (MOI) of less than 0.2, so that the number of mutant AsCas12f1 expressed per cell did not exceed 1. Since the full-length sgRNA did not have optimal genome editing efficiency, a stem 5 deletion mutant (sgRNA_ΔS5), which has enhanced genome editing in human cells, was used for screening.

[0036] On the second day after infection, GFP-positive cells were selected to select lentivirus-infected cells, and then cultured for another 5 days. As shown in Figure 5-1B, on the seventh day after infection, mRNA was extracted separately from GFP-positive and -negative cells, and deep sequencing analysis was performed to identify mutations. Figure 5-2C shows the genome editing efficiency as a bar graph and concentration distribution. As the genome editing efficiency increases compared to the wild type, the bar graph becomes longer and the color becomes darker in the concentration distribution. Note that in Figure 5-2C, the concentration distribution is schematic, and the editing efficiency is mainly represented by the bar graph. As shown in this figure, the editing efficiency of each mutation was defined as the ratio of GFP-negative read counts to the total read counts, and was normalized by the value of wild type AsCas12f (WT: wild type).

[0037] A in Figure 6-1 is a graph showing the correlation of the effect of single mutants on GFP deletion, determined from replicas of independent mutation libraries. B-1 in Figure 6-2 and B-2 in Figure 6-3 are a list of single amino acid substitutions that increase the editing efficiency by 20% or more compared to WT. C in Figure 6-4 is a graph showing the genome editing activity of AsCas12f mutants in double, triple, and quadruple cases.

[0038] As shown in A of FIG. 6-1, the DMS experiment was performed in duplicate, and similar results were obtained [R2 (coefficient of determination) = ~0.6]. As a result, as shown in B-1 and B-2 of FIG. 6-2 and B-3, and C of FIG. 6-4, more than 200 amino acid substitutions were identified that improved the editing efficiency by 20% or more compared to WT. In B-1 and B-2, the horizontal axis shows 208 mutations whose genome editing efficiency was increased by 1.2 times or more compared to the wild type, and the vertical axis shows the genome editing efficiency of each mutation when the genome editing efficiency of the wild type is set to 1. In B-1 to E of FIG. 6-2 to FIG. 6-4, the mutations (F48Y, I123H, S188H, D195K, D208R, V232A, E316M) contained in the final mutants are highlighted by diagonal lines.

[0039] Figures 6-4, C, D, and E and Figures 5-2, E and F show the results of cross-breeding of mutations and genome editing efficiency measurements. In these measurements, contrary to Figures 6-2 and 6-3, B-1 and B-2, GFP fluorescence was restored by genome editing in a gene in which GFP fluorescence had disappeared. Among these mutations, we focused on S188H and D195K. These are located close to the nucleic acid in this structure and may form additional interactions to enhance target DNA binding by AsCas12f. Therefore, by cross-breeding multiple mutations in S188H and D195K, respectively, we finally developed two highly active mutants (Figure 5-2, E).

[0040] Next, as shown in Figure 5-2D, we developed a split-GFP reporter system linked with a frame-shift linker containing the VEGFA sequence, and evaluated the genome editing activity of WT and several mutants by measuring the ratio of frame-shift GFP-positive cells. As shown in Figure 5-2E, consistent with the DMS analysis, the S188H and D195K mutants showed higher genome editing activity than the WT. To generate mutants with higher genome editing activity than the single S188H or D195K mutant, we combined the S188H or D195K substitution with other effective substitutions identified in the DMS analysis to generate more than 20 mutants.

[0041] As shown in Figure 5-2E and Figure 6-4C, some double mutants showed reduced genome editing activity, while S188H / V232A and D195K / V232A showed improved activity. To further improve activity, we introduced additional mutations that enhance activity when combined with S188H or D195K to create seven triple mutants. As shown in Figure 5-2E and Figure 6-4D, the triple mutants S188H / V232A / E316M and D195K / D208R / V232A further improved genome editing activity. Finally, we combined the triple mutants S188H / V232A / E316M or D195K / D208R / V232A with other effective substitutions identified by DMS analysis to construct 29 quadruple mutants and evaluate their genome editing activity. As shown in Figures 5E and 6E, most of these mutants showed activity comparable to or less than that of the triple mutant, but the quadruple mutants F48Y / S188H / V232A / E316M and I123H / D195K / D208R / V232A further improved genome editing activity. Herein, the F48Y / S188H / V232A / E316M and I123H / D195K / D208R / V232A mutants are named AsCas12f-YHAM and AsCas12f-HKRA, respectively.

[0042] Furthermore, as shown in the graph of FIG. 5-2E, the ratio of frame-shifted GFP-positive cells in S188H, an example in which there is no mutation at amino acid position 195 and there is a mutation at amino acid position 188 but no mutations at other amino acid positions, and in D195K, an example in which there is no mutation at amino acid position 188 and there is a mutation at amino acid position 195 but there are no mutations at other amino acid positions, is about 18%. Thus, the mutant in which amino acid position 188 is mutated has a significantly higher ratio of frame-shifted GFP-positive cells than the value in the WT, which is a comparative example. In particular, S188H in which amino acid position 188 is mutated to histidine (H) has a significantly higher ratio of frame-shifted GFP-positive cells than the WT. Furthermore, it was shown that the mutants with a mutation at amino acid position 195 had a higher ratio of frame-shifted GFP-positive cells than the WT. In particular, D195K, in which amino acid position 195 was mutated to lysine (K), showed a significantly higher ratio of frame-shifted GFP-positive cells than the WT.

[0043] In contrast, in the case of S188A / V232A, an example of a double mutant with a mutation at amino acid position 188 and an additional mutation at amino acid position 232, the number of frame-shifted GFP-positive cells was just under twice as high as that of S188H, a significant improvement, provided that there was no mutation at amino acid position 195. Furthermore, in the case of S188H / V232A / E316M, an example of a triple mutant with a mutation at amino acid position 316 in addition to mutations at amino acid positions 188 and 232, the number of frame-shifted GFP-positive cells was about 2.5 times as high as that of S188H, a further improvement. Furthermore, in the case of F48Y / S188H / V232A / E316M, an example of a quadruple mutant with a mutation at amino acid position 48 in addition to mutations at amino acid positions 188, 232, and 316, the number of frame-shifted GFP-positive cells was slightly less than three times that of S188H, and this value was further improved. Furthermore, from these results, it is strongly predicted that multiple mutants such as quintuple or sextuple mutants containing the F48Y / S188H / V232A / E316M mutation will also have a higher ratio of frame-shifted GFP-positive cells compared to WT. These results indicate that mutants with a mutation at amino acid position 188, particularly mutants containing only the S188H mutation, and any multiple mutants such as double, triple, and quadruple mutants containing at least the S188H mutation, are useful. Therefore, an important finding has been obtained that in the case of a mutant having no mutation at amino acid position 195 and a mutation at amino acid position 188, the ratio of frameshift GFP-positive cells tends to be significantly improved.

[0044] In addition, in the case of D195K / V232A, an example of a double mutant with a mutation at amino acid position 195 and an additional mutation at amino acid position 232, the number of frame-shifted GFP-positive cells was slightly less than twice as high as that of D195K, a significant improvement, provided that there was no mutation at amino acid position 188. Furthermore, in the case of D195K / D208R / V232A, an example of a triple mutant with a mutation at amino acid position 208 in addition to mutations at amino acid positions 195 and 232, the number of frame-shifted GFP-positive cells was about 2.5 times as high as that of D195K, a further improvement. Furthermore, in I123H / D195K / D208R / V232A, an example of a quadruple mutant with a mutation at amino acid position 123 in addition to mutations at amino acid positions 195, 232, and 208, the number of frame-shifted GFP-positive cells is slightly less than three times that of D195K, and this value is further improved. Furthermore, from these results, it is strongly speculated that multiple mutants such as quintuple or sextuple mutants containing the I123H / D195K / D208R / V232A mutations will also have a higher ratio of frame-shifted GFP-positive cells compared to WT. From these findings, it is shown that mutants with a mutation at amino acid position 195, particularly mutants containing only the D195K mutation, and any multiple mutants such as double, triple, and quadruple mutants containing at least the D195K mutation, are useful. Thus, it has been found that there is a tendency for the ratio of frame-shifted GFP-positive cells to be significantly improved in mutants with a mutation at amino acid position 195.

[0045] Thus, it is shown that the mutants containing at least the S188H mutation and the mutants containing at least the D195K mutation are particularly useful. In addition, as shown in the graph in FIG. 6-4C, the ratio of frame-shift GFP-positive cells in the double mutant (S188H / D195K) containing both the S188H and D195K mutations is about 25%, which is higher than the mutants containing only the S188H mutation and the mutants containing only the D195K mutation, as shown in FIG. 6-4C.

[0046] On the other hand, as shown in FIG. 6-4D, in triple mutants (S188H / V232A / E316M, etc.) that do not have a mutation at amino acid position 195 and contain at least the mutations S188H and V232A, the ratio of frame-shifted GFP-positive cells is about 40% to 50%. This value is significantly higher than the value in mutants with only the S188H mutation and the value in double mutants containing both the S188H and D195K mutations, indicating that these are particularly useful. Furthermore, as shown in Figure 6-4E, in the triple mutant containing S188H, V232A, and E316M mutations (S188H / V232A / E316M) and the quadruple mutant containing at least S188H, V232A, and E316M mutations (S188H / V232A / E316M / F48Y, etc.), the ratio of frame-shifted GFP-positive cells was approximately 20% to 50%, indicating that these are useful. In particular, S188H / V232A / E316M / I23K, S188H / V232A / E316M / F48Y, S188H / V232A / E316M / K80R, S188H / V232A / E316M / I123H, S188H / V232A / E316M / E246M, S188H / V232A / E316M / I262 The quadruple mutants S188H / V232A / E316M / E354T, S188H / V232A / E316M / D364Q, and S188H / V232A / E316M / I388N all had higher values ​​than the double mutants containing the S188H and D195K mutations, indicating that these are particularly useful.

[0047] Furthermore, as shown in FIG. 6-4D, in triple mutants (D195K / D208R / V232A, etc.) that do not have a mutation at amino acid position 188 and contain at least the mutations D195K and V232A, the ratio of frameshift GFP-positive cells is about 30% to 50% or more. This value is significantly higher than the value in mutants with only the D195K mutation and the value in double mutants containing the S188H and D195K mutations, indicating that these are particularly useful. Furthermore, as shown in Figure 6-4E, in the triple mutant containing D195K, V232A, and D208R mutations (D195K / D208R / V232A) and the quadruple mutant containing at least D195K, V232A, and D208R mutations (D195K / D208R / V232A / FI123H, etc.), the ratio of frame-shifted GFP-positive cells was approximately 25% to 50%, indicating that these are useful.

[0048] In addition, in FIG. 6-4E, the ratio of frame-shifted GFP-positive cells in the quadruple mutant D195K / D208R / V232A / S188R containing a mutation at amino acid position 188 is about 25%, which is the same value as that of a double mutant in which both S188H and D195K are mutated. However, in other quadruple mutants containing D195K / D208R / V232A mutations, as shown on the right side of FIG. 6-4E, the ratio of frame-shifted GFP-positive cells is as high as about 35% to about 50%. This indicates that quadruple mutants in which amino acid position 195 is mutated without amino acid position 188 mutation, particularly quadruple mutants containing at least D195K / D208R / V232A mutations without amino acid position 188 mutation, are useful. In addition to the quadruple mutant, quintuple mutants and sextuple mutants containing at least the D195K / D208R / V232A mutations without a mutation at amino acid position 188 are also considered to be useful. Moreover, the triple mutants shown in FIG. 6-4D are all triple mutants in which only one of amino acid positions 188 and 195 is mutated, and the ratio of frame-shifted GFP-positive cells in all of these exceeds 30%. Therefore, both 1) a triple mutant with no mutation at amino acid position 195 and at least S188H / V232A mutations, and 2) a triple mutant with no mutation at amino acid position 188 and at least D195K / V232A mutations are considered to be useful. In 1) and 2), not only triple mutants but also any multiple mutants such as quadruple mutants and quintuple mutants are considered to be useful.

[0049] These results show that the ratio of frame-shifted GFP-positive cells is significantly improved by a protein with a mutation at amino acid position 188 in the amino acid sequence of AsCas12f, and also that the ratio of frame-shifted GFP-positive cells is significantly improved by a protein with a mutation at amino acid position 195 in the amino acid sequence of AsCas12f.

[0050] More specifically, as shown on the left side of C in FIG. 6-4, in each of the double mutants S188H / I2Y to S188H / L337I, the ratio of frame-shifted GFP-positive cells is significantly higher than the value in the WT (approximately 3 to 4%), which is the comparative example. In particular, although the ratio of frame-shifted GFP-positive cells in S188 / D208R is the lowest at approximately 6%, it is clearly shown that the value is significantly higher than that in the WT. From this, it can be said that a double mutant that satisfies the condition that amino acid position 188 is mutated to histidine is useful. In addition, in the graph for S188H in FIG. 6-4C, the ratio of frame-shifted GFP-positive cells was higher in S188H / K80R, S188H / Y105T, S188H / I123H, S188H / D195K, S188H / V232A, S188H / E246M, S188H / E316M, and S188H / L337I than in the case where only S188H was mutated. Therefore, these are particularly useful double mutants.

[0051] Next, as shown on the right side of FIG. 6-4C, in each of the double mutants D195K / I22Y to D195K / L337I, the ratio of frame-shifted GFP-positive cells is significantly higher than the value (about 3 to 4%) in the WT, which is a comparative example. In particular, although the ratio of frame-shifted GFP-positive cells in D195K / Y105T is the lowest at about 16%, it is clearly shown that the value is significantly higher than that of the WT. From this, it can be said that a double mutant that satisfies the condition that amino acid position 195 is mutated to lysine is useful. In addition, in the graph for D195K in FIG. 6-4C, D195K / I2Y, D195K / N70Y, D195K / K80R, D195K / I123H, D195K / D208R, D195K / V232A, D195K / E246M, D195K / E316M, and D195K / L337I all have a higher ratio of frame-shift GFP-positive cells than when only S188H is mutated. Therefore, these are shown to be particularly useful double mutants.

[0052] Next, as shown in FIG. 6-4D, the ratio of frame-shifted GFP-positive cells in each triple mutant of S188H / V232A / I123H, S188H / V232A / E246M, S188H / V232A / E316M, and S188H / V232A / L337I was about 35% or more, which is significantly higher than the value in the WT as a comparison example, and is comparable to or higher than the S188H / V232A double mutant. In particular, the triple mutants of S188H / V232A / E246M and S188H / V232A / E316M obtained higher values ​​than the S188H / V232A double mutant, indicating that these triple mutants are useful. In addition, because the ratio of frame-shifted GFP-positive cells was stably high, triple mutants that satisfy the condition of containing S188H / V232A mutations are particularly useful. As shown in the left side of Figure 6-4E, quadruple mutants that satisfy the condition of containing S188H / V232A mutations also have high ratios of frame-shifted GFP-positive cells, indicating that not only triple mutants but also n-fold mutants (n is a natural number of 3 or more) that contain at least D195K / V232A mutations are useful.

[0053] In addition, the ratio of frame-shifted GFP-positive cells in each of the triple mutants D195K / V232A / N70Y, D195K / V232A / D206R, and D195K / V232A / E316M was about 30% or more, which was significantly higher than the value in the WT as a comparative example, and was comparable to or higher than the D195K / V232A double mutant. In particular, the triple mutants S188H / V232A / E246M and S188H / V232A / E316M obtained higher values ​​than the S188H / V232A double mutant, indicating that these triple mutants are useful. In addition, since such a high ratio of frame-shifted GFP-positive cells was obtained stably, it can be said that the triple mutant that satisfies the condition of including the D195K / V232A mutation is particularly useful. Furthermore, as shown on the right side of Figure 6-4E, the proportion of frameshift GFP-positive cells was high in all quadruple mutants that met the condition of containing the D195K / V232A mutations, indicating that not only triple mutants but also n-fold mutants (n is a natural number greater than or equal to 3) that contain at least the D195K / V232A mutations are useful.

[0054] In the quadruple mutant containing the S188H / V232A / E316M mutations shown on the left side of FIG. 6-4E, the ratio of frame-shifted GFP-positive cells is about 20% to about 50%, which is significantly higher than the value in the WT as a comparative example. Also, in FIG. 6-4E, the ratio of frame-shifted GFP-positive cells in the quadruple mutant of S188H / V232A / E316M / D206 is the lowest at about 20%, but it is clearly shown that the value is significantly higher than that in the WT. Since a high value is obtained for the ratio of frame-shifted GFP-positive cells stably in this way, it is shown that the quadruple mutant that satisfies the condition of containing the S188H / V232A / E316M mutations is particularly useful. Furthermore, the quadruple mutants S188H / V232A / E316M / F48Y, S188H / V232A / E316M / E246M, S188H / V232A / E316M / I262N, and S188H / V232A / E316M / I388N showed similar or higher ratios of frame-shifted GFP-positive cells than the triple mutant S188H / V232A / E316M, indicating that these are particularly useful quadruple mutants. These results strongly suggest that not only quadruple mutants containing at least the S188H / V232A / E316M mutations, but also m-mutants (m is a natural number of 4 or more) containing at least the S188H / V232A / E316M mutations are useful.

[0055] In the quadruple mutant containing the D195K / D208R / V232A mutation, which is shown on the right side of E in Fig. 6-4, the ratio of frame-shift GFP-positive cells is about 30% to about 50%, which is significantly higher than the value in WT, which is a comparative example. Also, in E of Fig. 6-4, although the ratio of frame-shift GFP-positive cells in the quadruple mutant of D195K / D208R / V232A / S188R is the lowest at about 30%, it is clearly shown that the value is significantly higher compared to WT. Thus, since a high value of the ratio of frame-shift GFP-positive cells is stably obtained, it is shown that the quadruple mutant satisfying the condition of containing the D195K / D208R / V232A mutation is particularly useful. Furthermore, in each of the quadruple mutants of D195K / D208R / V232A / I23K, D195K / D208R / V232A / K80R, D195K / D208R / V232A / Q93M, and D195K / D208R / V232A / I123H, the ratio of frame-shift GFP-positive cells is equal to or higher than that of the D195K / D208R / V232A triple mutant, indicating that these are particularly useful quadruple mutants. Also, from these, it is strongly presumed that not only the quadruple mutant containing D195K / D208R / V232A but also the m-fold mutant (m is a natural number of 4 or more) containing at least the D195K / D208R / V232A mutation is useful.

[0056] <Optimization of sgRNA for AsCas12f> A in Fig. 7-1 is an explanatory diagram of the sequence of the sgRNA mutant (sgRNA_ΔS3-5_v1-v7). To remove stems 3 and 4 and retain the original architecture of the remaining region, the length and sequence of the linker region have been modified. The linker region is surrounded by a red dotted line frame.

[0057] FIG. 7-1B is an illustration showing the genome editing activity of sgRNA mutants determined by flow cytometry. FIG. 7-1C is a graph showing the expression of sgRNA quantified by qPCR for common sequence sites (n=3 means ±SD). FIG. 7-1D is a graph showing the time course of GFP deletion caused by various Cas enzymes in HEK293T cells expressing d2EGFP (n=3 means ±SD). Each sgRNA was introduced by single copy lentiviral infection. GFP deletion by AsCas12f-HKRA in sgRNA_ΔS3-5_v7 is slightly lower than that by SpCas9, but is faster than the deletion rate observed in AsCas12a, enAsCas12a and Ultra. In Figure 7-1D, (a) represents the results for WT+ΔS5, (b) represents the results for WT+ΔS3-5_v7, (c) represents the results for HKRA+ΔS5, (d) represents the results for WHKRA+ΔS3-5_v7, (e) represents the results for AsCas12a, (f) represents the results for enAsCas12a, (g) represents the results for Ultra, and (h) represents the results for SpCas9.

[0058] Figure 7-2E is a schematic illustration of the wild-type sgRNA, and Figure 7-2F is a schematic illustration of sgRNA_ΔS3-5_v7. The disordered regions are represented in grey. PK1, PK2, stem 1 and stem 2 form extensive interactions with the AsCas12 protein, whereas stem 3 and stem 4 form little interactions with the protein, and stem 5 is disordered. Therefore, we designed sgRNA_ΔS3-5_v7 by removing stems 3-5, which are shown enclosed by dotted lines. The remaining regions retain the original architecture.

[0059] G in Figure 7-3 represents the structure of the wild-type sgRNA scaffold, and H in Figure 7-3 represents the structure of the sgRNA_ΔS3-5_v7 scaffold. Disordered regions are indicated by dotted lines. I in Figure 7-4 represents an enlarged view of the PK1 region of the wild-type sgRNA, and J in Figure 7-4 represents an enlarged view of the PK1 region of sgRNA_ΔS3-5_v7. K in Figure 7-4 represents the PK2 region of the wild-type sgRNA, and L in Figure 7-4 represents an enlarged view of the stem 3 region of sgRNA_ΔS3-5_v7.

[0060] In this embodiment, the structure shows that stem 5 is completely disordered. It also shows that PK1, PK2, stem 1, and stem 2 interact extensively with the AsCas12f protein, while stems 3 and 4 are exposed to the solvent and barely interact with the protein.

[0061] Therefore, as shown in Figure 7-1A, we further engineered the sgRNA by truncating these domains. Seven sgRNA mutants (sgRNA_ΔS3-5_v1 to sgRNA_ΔS3-5_v7) were designed to remove stem 3 and stem 4, while the remaining regions maintained the original structure. As a result, as shown in Figure 7-1B, all sgRNA mutants showed increased genome editing activity compared to sgRNA_ΔS5, and in particular, sgRNA_ΔS3-5_v7 was the most effective.

[0062] As shown in C of FIG. 7-1, the expression level of sgRNA_ΔS3-5_v7 in HEK293 cells was 4.5 times that of sgRNA_ΔS5. This indicates that shortening the sgRNA increases the expression level and enhances the genome editing activity. To further clarify the effect of sgRNA modification, sgRNA_ΔS5 and sgRNA_ΔS3-5_v7 targeting GFP were introduced by single-copy lentiviral infection, and the deletion rate of GFP was evaluated. As shown in F of FIG. 5-2 and D of FIG. 7-1, sgRNA_ΔS3-5_v7 significantly improved the GFP deletion efficiency of both AsCas12f-WT and AsCas12f-HKRA compared to sgRNA_ΔS5, suggesting that sgRNA_ΔS3-5_v7 is effective in situations where the expression level is limited, such as in vivo gene delivery.

[0063] <Structure of the AsCas12f mutant optimized for sgRNA measured by cryo-electron microscopy> A in FIG. 8-1 represents the structural models of the AsCas12f-sgRNA-target DNA (left), AsCas12f-YHAMsgRNA_ΔS3-5_v7-target DNA (center), and AsCas12f-HKRAsgRNA_ΔS3-5_v7-target DNA (right) complexes. The mutated residues are shown as CPK models.

[0064] B in FIG. 8-2 represents the dimer interface of AsCas12f-YHAM, C in FIG. 8-2 represents the target DNA recognition of AsCas12f-YHAM, and D in FIG. 8-2 represents the hydrophobic interaction of AsCas12f-YHAM. The mutated residues are highlighted in red and represented by the darker-colored parts in the figure. E in FIG. 8-2 represents the recognition of the guide RNA-target DNA heteroduplex of AsCas12f-HKRA, F in FIG. 8-2 represents the recognition of the target DNA, and G in FIG. 8 represents the recognition of the sgRNA scaffold. The mutated residues are highlighted in red and represented by the darker-colored parts in the figure.

[0065] As shown in A of Figure 8-1, EL of Figures 2-3 to 2-6, and Figures 9 and 10, which are illustrations of the results of data collection, processing, model improvement, and validation, to gain mechanistic insight into the enhanced DNA cleavage activity of the AsCas12f mutant, we measured the structures of the AsCas12f-YHAM-sgRNA_ΔS3-5_v7-target DNA and AsCas12f-HKRA-sgRNA_ΔS3-5_v7-target DNA complexes at a total resolution of 2.9 Å using cryo-electron microscopy. As a result, it was revealed that the overall structures of the AsCas12f-YHAM mutant and the AsCas12f-HKRA mutant are similar to the structure of wild-type AsCas12f, and the mutations introduced by the DMS experiment do not significantly affect the overall structure of the complex. In A of Figure 8, the introduced mutations are displayed in cpk (color-coded by atom type).

[0066] Figures 7-2 to 7-4 E, G, I, and K show diagrams of the original wild-type sgRNA, and correspondingly, Figures 7-2 to 7-4 F, H, J, and L show diagrams of the modified sgRNA mutant, sgRNA__ΔS3-5_v7. As shown, sgRNA_ΔS3-5_v7 is composed of a 20-nt guide segment (G1 to C20) and a 96-nt sgRNA scaffold (A(-96) to C(-1)). In sgRNA_ΔS3-5_v7, stems 3, 4, and 5 regions are deleted while maintaining stems 1, 2, and PK1 regions, and it interacts extensively with AsCas12f.

[0067] In particular, C(-6), which corresponds to the inverted C(-103) of the wild-type sgRNA, base pairs with G(-83) to link PK1 / stem 1 and form a continuous helix, as shown in I and J of Figure 7-4. As shown in K and L of Figure 7-4, the stem 3 region, which corresponds to the PK2 region of the wild type, maintains the G(-24):C(-8) and G(-23):C(-9) base pairs (G(-122):C(-88) and G(-121):C(-87) in the wild type), while G(-12) is inverted and stacks with Trp17, which resembles the interaction of G(-90) with Trp17 in the wild type. Unexpectedly, as shown in Figure 7-4L, A(-10) forms a non-canonical base pair with U(-22), spanning the base pairs G(-24):C(-8) to G(-23):C(-9) and C(-21):G(-14) to C(-20):G(-15) in stem 3, explaining that sgRNA_ΔS3-5_v7 is the optimal sgRNA variant.

[0068] In addition, stems 1 to 5 are all stem loops, and the optimal sgRNA mutants were created by adjusting the sequence and length of the linker region that connects the deleted parts. In this way, it was confirmed that genome editing activity is increased by deleting a part of the sgRNA in different Cas. One reason for the increase in genome editing activity is thought to be that shortening the length of the sgRNA improves the transcription amount of the sgRNA in the introduced mammalian cells, which makes it easier to form a complex with Cas. In particular, the structure of AsCas12f showed that stems 4 and 5 hardly interact with AsCas12f, so a short sgRNA mutant was created by deleting these parts.

[0069] B to G in Figure 8-2 are explanatory diagrams showing the introduced mutations. In B in Figure 8-2, the F48Y mutation shown by Y48.1 is present at the dimer interface, and by becoming Y, it forms a new interaction with another molecule, strengthening the assembly of the dimer. In 8C in Figure 8-2, S188H shown by H188.1 forms a new hydrophobic interaction with nucleic acid. In D in Figure 8-2, M316.1 in AsCas12f.1 and E316M shown by M316.2 in AsCas12f.2 are surrounded by hydrophobic residues, so that E becomes M, contributing to the stability of the protein. In E in Figure 8-2, I123H / D195K shown by H123.1 and K195.1 forms a new interaction with RNA. 208R, shown as DR208.1 in Figure 8-2 F and R208.2 in Figure 8-2 G, forms new interactions with DNA / RNA. The mutant has increased genome editing activity due to the formation and increased stability of these new interactions.

[0070] In detail, in the AsCas12f-YHAM structure, Tyr48.1 (F48Y.1, shown as Y48.1 in the figure), located at the dimer interface, is thought to form a hydrogen bond with the main chain of G57.2 in addition to the hydrophobic interaction observed in wild-type AsCas12f, promoting more stable dimerization. As shown in Figure 8-2C, the side chain of His188.1 (S188H.1, shown as H188.1 in the figure) is stabilized by Ile2 and interacts with the backbone phosphate group between dT19 and dC18, indicating that the S188H mutation promotes unwinding of the target DNA. As shown in Figure 8-2D, Met316.1 and Met316.2 (E316M.1 and E316M.2, shown as M316.1 and M316.2 in the figure) may form additional hydrophobic interactions with Thr239 and Ala241, thereby increasing overall stability.

[0071] As shown in FIG. 8-2E, in the AsCas12f-HKRA structure, His123.1 (H123.1 in the figure) and Lys195.1 (K195.1 in the figure) provide hydrophobic and electrostatic interactions with the ribose moiety of A5 and the backbone phosphate groups of A3 and A5, respectively, stabilizing the guide RNA-target DNA heteroduplex. As shown in FIG. 8-2F, Arg208.1 (R208.1 in the figure) forms hydrogen bonds and hydrophobic interactions with dT19 and Ile2, respectively. This is similar to His188.1 in AsCas12f-YHAM, thereby promoting initial heteroduplex formation. Furthermore, as shown in Figure 8-2 G, Arg208.2 of another subunit (shown as R208.2 in the figure) also forms electrostatic interactions with the phosphate backbone of G(-43) in the sgRNA scaffold, stabilizing the complex structure.

[0072] Although both AsCas12f-YHAM and AsCas12f-HKRA mutants contain the V232A mutation, our structural analysis did not provide clear insight into the contribution of the V232A mutation to the enhanced DNA cleavage activity of these mutants. These findings suggest that DMS-based engineering approaches have great potential for generating highly active mutants that cannot be predicted by structural information alone.

[0073] Characterization of AsCas12f mutants in human cells A in FIG. 11-1 is an explanatory diagram of the measurement of PAM specificity, and B is a graph showing the results. The wild type (WT) hardly causes genome editing with any PAM, but the two mutants according to this embodiment are shown to cause genome editing with TTR PAM. The vertical axis of B in FIG. 11-1 indicates the editing frequency (Editing frequency %), and the bar graphs show the values ​​in WT (wild type), AsCas12f-YHAM, and AsCas12f-HKRA from the left in the figure. C to E in FIG. 11-2 are graphs showing genome editing activity with various Cass, where SpCas9 is the most commonly used highly active Cas9, AsCas12a is the most commonly used highly active Cas12a (Cpf1), and Ultra and enAsCas12a represent variants of AsCas12a, respectively. These results indicate that, depending on the target gene, the AsCas12f mutants of this embodiment (AsCas12f-YHAM, AsCas12f-HKRA) exhibit higher activity than these commonly used Cas, or exhibit equal or greater activity.

[0074] Figure 12-1A is a graph showing the comparison of the target editing efficiency in AsCas12f-YHAM and AsCas12f-HKRA. As shown in Figure 11-1A and Figure 12-1A, in order to evaluate the PAM specificity of the enAsCas12f mutant for a wide range of targets, a self-targeting library containing both sgRNA and its target sequence was developed, and indel formations induced by wild-type AsCas12f (for simplicity of description, referred to as AsCas12f), AsCas12f-YHAM, and AsCas12f-HKRA were measured for 750 different spacer sequences with 16 NTTN PAMs. As shown in Figure 12-1B, the deep sequencing analysis showed that the three enzymes induced indels with NTTR PAM but not with NTTY (Y is T or C) PAM, indicating that they both recognize the NTTR sequence as a PAM. In other words, AsCas12f recognizes TTR (R is A or G) as PAM.

[0075] As shown in Figure 1B, cryo-EM structures revealed that the nucleobases of dT(-3*) and dT(-2*) in the PAM duplex form hydrophobic interactions with Tyr76.1, and N6 of dA(-1*) forms a hydrogen bond with His72.1. Modeling suggested that O6 of dG(-1*) forms a hydrogen bond with His72.1, providing a clear explanation for the NTTR PAM preference. As shown in Figure 1B, in NTTR PAMs, AsCas12f induced an average of 3.0% indels, while AsCas12f-YHAM and AsCas12f-HKRA induced an average of 40.8% and 44.7% indels, respectively.

[0076] These results revealed that the enAsCas12f mutant exhibited much higher genome editing activity than AsCas12f at various target sequences using NTTR PAM. Next, we compared the genome editing efficiency of AsCas12f and enAsCas12f mutants (variants) with that of Cas12a and Cas12a mutants (UltraCas12a and enAsCas12a) at five target sites using NTTG PAM using HEK 293T cells.

[0077] In Figure 11-2C, the bar graphs for each item such as VEGFA, HEXA, etc., from the left in the figure, represent the indel frequency (%) in AsCas12f, AsCas12f-YHAM, AsCas12f-HKRA, AsCas12a, UltraCas12a, and enAsCas12a. At these five sites, AsCas12f, AsCas12f-YHAM, AsCas12f-HKRA, AsCas12a, UltraCas12a, and enAsCas12a generated indels at an average frequency of 8.5%, 22.3%, 14.3%, 9.0%, 11.0%, and 11.1%, respectively. These results indicate that the enAsCas12f mutant can induce indels with the same or higher efficiency as Cas12a and Cas12a mutants, while AsCas12f does not induce indels with such efficiency. Also, in Figure 11-2 D and E, the bar graphs for each item show the measurement results for AsCas12f, AsCas12f-YHAM, AsCas12f-HKRA, and SpCas9, respectively, from the left in the figure.

[0078] As shown in Figure 11-2D, the genome editing efficiency was compared with that of SpCas9 at eight target sites in HEK293T cells. These eight target sites have the same spacer sequence as 5′-TTTG PAM for AsCas12f and NGG-3′ PAM for SpCas9. As shown, AsCas12f, AsCas12f-YHAM, AsCas12f-HKRA and SpCas9 generated indels at the eight target sites with an average frequency of 1.7%, 14.8%, 16.9% and 22.9%. In addition, the formation of indels in three therapeutic targets, namely PCSK9 and ANGPTL3 for atherosclerosis and TTR for transthyretin amyloidosis, was also measured in HEK 293T cells.

[0079] As shown in Figure 11-2E, higher indel frequencies were observed with AsCas12f-YHAM and AsCas12f-HKRA than with SpCas9 at two of the three target sites. As shown in Figures 12-2C and 12-3E, similar results were obtained at other target sites and in other human cell lines, including Huh-7 hepatoma cells and HT-1080 sarcoma cells, indicating that AsCas12f-YHAM and AsCas12f-HKRA efficiently induced indels regardless of the target site or cell line.

[0080] Next, to examine the specificity of the enAsCas12f mutant, we examined mismatch tolerance. Figure 13-1A is a graph showing the effect of mismatches (n=3) on EGFP activation mediated by AsCas12f-YHAM and AsCas12f-HKRA in a split-GFP-reframing assay using the TTR sequence. The "-" on the left side of the graph represents data obtained when a normal guide RNA that is completely complementary to the target DNA was used. "M01", "M02" ... refer to the locations where mismatches were inserted between the guide RNA and the target DNA, that is, the locations where the incorrect guide sequence was used to prevent base pairing. Therefore, "M01" refers to a mismatch at the first position of the guide RNA, and "M01 / 02" refers to a double-mismatched guide RNA with mismatches at the first and second positions of the guide RNA. In Figure 13-1A, the vertical axis represents indel efficiency (%), and in each bar graph of M01, M02, M03, etc., the bar graph on the left side of the figure shows the value for AsCas12f-YHAM, and the bar graph on the right side shows the value for AsCas12f-HKRA.

[0081] As shown in Figure 13-1A, both enAsCas12f variants showed broad tolerance to single mismatches, except for the PAM-distal region (positions 16-20), as observed with Cas12a and other Cas12fs, but the tolerance to double mismatches was negligible. Furthermore, genome-wide specificity of AsCas12f-HKRA and SpCas9 was examined using GUIDE-seq (genome-wide, unbiased identification of double-strand breaks by sequencing) at nine target sites. Figure 13-1B is an illustration of the number of off-target sites for AsCas12f-HKRA and SpCas9 detected by guide sequences at the same spectre site as in Figure 11-2D, and Figure 13-2C-1, Figure 13-3C-2, and Figure 13-4C-3 are all illustrations of off-target sites identified by guide sequences.

[0082] As shown in B of FIG. 13-1, C-1 of FIG. 13-2, C-2 of FIG. 13-3, and C-3 of FIG. 13-4, AsCas12f-HKRA and SpCas9 have a comparable number of off-target sites, and AsCas12f-HKRA tolerates mismatches in the PAM-distal 5-nt region, which is consistent with the mismatch experiment. These results indicate that the enAsCas12f variant with sgRNA_ΔS3-5_v7 exhibits equivalent or even higher genome editing activity and specificity compared to SpCas9 and Cas12a, despite its very compact size.

[0083] <Therapeutic potential of genome editing via AsCas12f> FIGS. 14-1 to 14-2 are explanatory diagrams of the generation of muscle dystrophy model induced pluripotent stem cells (iPSCs) and their differentiation into cardiomyocytes (iPSC-CMs). A in FIG. 14-1 shows the confirmation of the complete deletion of the DMD exon by PCR, and B in FIG. 14-1 is an explanatory diagram of the genomic sequence of the DMD exon 44 deletion-iPS cell line. C in FIG. 14-2 is a diagram representing the flow cytometry analysis of α-actinin-mCherry on day 14 of cardiomyocyte differentiation, indicating that all DMD exon 44 deletion strains differentiated efficiently into cardiomyocytes without purification. D in FIG. 14-2 is a diagram representing the Western blot analysis of AR and DMD iPSC cardiomyocytes (#2-16, 33, 66, and 84). The arrow line in the figure indicates the immunoreactive band of dystrophin, and the asterisk indicates the non-specific band. E in FIG. 14-3 is a graph showing the comparison of luciferase activities between dAsCas12f WT and dAsCas12f-HKRA (n = 3, mean ± SD). F in FIG. 14-3 is a schematic explanatory diagram of the plasmid constructs of dAsCas12f with VP64 or VPR bound to different sites. The gRNA targeting HEXA and the plasmid were transfected into Huh-7 cells that stably express luciferase under the control of the minimal CMV promoter with the HEXA gRNA binding site. G in FIG. 14-3 is a graph showing the increase in luciferase activity in cells transfected with the plasmid vector (n = 3, mean ± SD).

[0084] As shown in Figures 14-1 and 14-2 A to D, induced pluripotent stem cells (iPSCs) lacking Duchenne muscular dystrophy (DMD) exon 44 (hDMDΔEx44), the main mutation in DMD, were generated and differentiated into cardiomyocytes (iPSC-CMs) to evaluate the possibility of therapeutic application. Dystrophin deficiency causes myopathy and cardiomyopathy, and exon 45 skipping to restore DMD protein is being studied.

[0085] A to F in FIG. 15-1 and G to J in FIG. 15-2 are explanatory diagrams of treatment and knock-in using mutants. In detail, A to C in FIG. 15-1 are explanatory diagrams of treatment (iPSCs) using AsCas12f mutants against dystrophin. D to F in FIG. 15-1 are explanatory diagrams of treatment (administration to mice using AAV) using AsCas12f mutants against transthyretin. In FIG. 15-1 E, the vertical axis represents plasma transthyretin (μg / ml), and the horizontal axis represents the number of days (weeks) that have passed. In addition, (a) represents the plasma transthyretin value in 3.0×1011vg AsCas12f, (b) represents 1.0×1012vg AsCas12f, (c) represents 3.0×1011vg HKRA, and (d) represents 1.0×1012vg HKRA. In addition, in F of Figure 15-1, the vertical axis is the indel frequency (%), and on the horizontal axis, (a) is 3.0 × 10 11 vg AsCas12f, (b) is 1.0 × 10 12 vg AsCas12f, (c) is 3.0 × 10 11 vg HKRA, and (d) is 1.0 × 10 12 vg HKR. G to I of Figure 15-2 show GFP / FIX knock-in (mammalian cells) at the Alb locus using AsCas12f mutants. In I of Figure 15-2, the vertical axis shows the ratio (%) of EGFP-positive cells, and in J, the vertical axis shows FIX:C (%), which represents the increase in plasma factor IX activity (FIX:C).

[0086] As shown in Figure 15-1A, we expressed AsCas12f-HKRA and sgRNA targeting the DMD gene using an all-in-one AAV serotype 6 vector in WT iPSC-CMs, and found that AsCas12f-HKRA efficiently reduced the amount of dystrophin protein. Furthermore, as shown in Figure 15-1B and C, AsCas12f-HKRA paired with sgRNA designed to skip exon 45 partially restored dystrophin protein in hDMDΔEx44-iPSC-CMs, confirming the potential of AAV gene delivery for DMD treatment.

[0087] Next, to investigate the therapeutic potential of AsCas12f in vivo, AsCas12f-HKRA was applied to genome editing in mouse liver. As shown in Figure 15-1D, an AAV vector encoding AsCas12f (or AsCas12f-HKRA) under the HCRhAAT promoter and a promoter-driven sgRNA (sgRNA) targeting the TTR gene for transthyretin amyloidosis were constructed. 7-week-old mice were injected with 3 × 1011vg or 1 × 1012vg of liver-tropic AAV serotype 8, and plasma transthyretin expression levels were evaluated. As shown in Figure 15-1E, AsCas12f could not regulate plasma transthyretin levels, but AsCas12f-HKRA reduced them in a dose-dependent manner. Eight weeks after AAV-based gene delivery, targeted loci were analyzed by targeted amplicon deep sequencing, revealing that high editing rates (66.3%) were achieved with 1 x 1012 vg of AsCas12f-HKRA (Figure 15-1F).

[0088] Next, we evaluated the in vivo knock-in efficiency of the AsCas12f system, which inserts the EGFP gene into the mAlb 3′UTR. Knock-in of genes in the Alb locus is a platform that has been investigated for ectopically producing therapeutic proteins, including coagulation factors and lysosomal enzymes, from the liver. Two AAV vectors were prepared, one expressing AsCas12f or AsCas12f-HKRA and sgRNA targeting the mAlb 3′UTR, and the other providing a donor template to knock-in EGFP only at the DSBs by homology-directed repair (HDR) (Figure 15-2G). These two AAV vectors were injected intraperitoneally into C57BL / 6 wild-type neonatal mice, and EGFP expression in the liver 4 weeks after vector injection was evaluated by immunofluorescence microscopy.

[0089] As a result, as shown in H and I of Figure 15-2, a significant increase in EGFP-positive hepatocytes was confirmed by injection of the AAV vector carrying AsCas12f-HKRA. Next, the EGFP gene of the donor vector was replaced with coagulation factor IX (F9) cDNA carrying the Padua mutation, and the vector was injected into neonatal hemophilia B mice (F9-deficient mice). As shown in J of Figure 15-2, knock-in of the F9 gene at the Alb locus using AsCas12f-HKRA significantly increased plasma coagulation factor IX (FIX) activity beyond the therapeutic range compared to the donor-only case, but no such increase was observed with AsCas12f. These data indicate that AsCas12f mutants with optimal sgRNAQ can be used for in vivo gene therapy, for example, for hemophilia.

[0090] <Application of compact enAsCas12f> Due to its small size, the AsCas12f gene can be packaged into an all-in-one AAV vector together with multiple sgRNAs and large partner genes, enabling its application in genome editing therapy that was not possible with previous genome editing tools. Figure 16-1, A to F, is an explanatory diagram of FIX knock-in (mouse) at the Alb locus using an AsCas12f mutant. Figure 16-2, G to J, is an explanatory diagram of gene transcription activation (CRISPR activation) (mammalian cells) using an AsCas12f mutant. As shown in the figure, a significant increase in transcription activity was achieved by simultaneously introducing MS2.

[0091] As shown in Figure 16-1A, a single AAV vector encoding AsCas12f (or AsCas12f-HKRA) under the liver-tropic Ttr promoter, donor sequence, and sgRNA acting under the control of the U6 promoter was designed to insert F9 cDNA with Padua mutation into the Alb3′UTR locus. As shown in Figure 16-1B and C, when a single AAV serotype 8 vector was injected into neonatal hemophilia B mice, both plasma FIX activity (FIX:C) and antigen (FIX:Ag) were significantly increased by the AAV vector carrying AsCas12f-HKRA, but not by the AAV vector carrying AsCas12f. Consistent with these results, as shown in Figure 16-1D and E, the clotting time assessed by activated partial thrombin time (APTT) and the expression level of F9 mRNA assessed by quantitative RT-PCR were significantly improved. As shown in Figure 16-1F, amylose gel analysis of the mRNA PCR fragments revealed that cDNA insertion occurred via HDR, although insertion via non-homologous end joining (NHEJ) was also observed.

[0092] Finally, as shown in Figure 16-2G, to investigate the utility of enAsCas12f in epigenome editing, a transcription activation assay was performed using Huh-7 cells stably expressing luciferase driven by a minimal CMV promoter with two HEXA gRNA recognition sites. To enhance the transcription activity of enAsCas12f, an sgRNA was created with an MS2 aptamer inserted into its stem loop. As shown in Figure 16-2G, a plasmid expressing AsCas12f-HKRA (dead AsCas12f-HKRA, hereafter referred to as dAsCas12f-HKRA) that has lost DNA cleavage activity and is conjugated with VP64 and MS2 fusion activator (MS2-p65-HSF1) and an engineered sgRNA targeting HEXA were transfected into Huh-7 cells.

[0093] As shown in Figure 16-2 H and Figure 14-3 E, dAsCs12f-HKRA combined with MS2 aptamer-inserted sgRNA significantly enhanced luciferase expression. As shown in Figure 14-3 F and G, direct conjugation of VPR (VP64, p65, Rta) was unable to enhance transcription by MS2-p65-HSF1, whereas binding of VP64 at the end of dAsCas12f-HKRA was the most effective for transcription activation. Furthermore, as shown in Figure 16-2 I and J, transduction of Huh-7 cells with a single AAV serotype 6 vector encoding dAsCas12f-HKRA conjugated with JVP64, MS2-p65-HSF1, and sgRNA significantly increased luciferase expression in a dose-dependent manner. These results indicate that enAsCas12f can be used as a transcription activation tool.

[0094] FIG. 17A is an explanatory diagram of an example in which an AAV8 type vector expressing luciferase driven by a minimal CMV promoter having two HEXA gRNA recognition sites and an AAV8 type vector having the structure shown in FIG. 16-2 I were simultaneously intravenously administered to a wild-type mouse. This figure is a color heat map in which the count value increases from blue (50 counts) to red (200 counts), converted into grayscale. FIG. 17A shows that, compared to the four mice of AsCas12fWT (left side), the first mouse from the left of AsCas12fHKRA (right side) has a circular region with a count value of about 50 to 100 shown in dark gray, a circular region with a count value of about 100 to 170 shown in light gray inside, and a small region with a count value of about 170 to 200 shown in slightly dark gray inside. The second mouse from the left in the figure shows scattered dark gray regions with count values ​​of about 50 to 100 and light gray regions with count values ​​of about 100 to 170. The third and fourth mice from the left in the figure show a circular region with count values ​​of about 50 to 100 shown in dark gray, an inner circular region with count values ​​of about 100 to 170 shown in light gray, and a wider closed region with count values ​​of about 170 to 200 shown in slightly darker gray within that.

[0095] FIG. 17B is a graph showing the ROI values ​​(vertical axis) in WT and HKRA. As shown in the figure, luciferase activity increased about 10-fold in Cas12f-HKRA compared to wild-type AsCas12f. As shown in the figure, P=0.0474, which indicates statistical significance (p<0.05). Based on these results, it may be possible to increase the transcription of endogenous genes by about 10-fold. For example, it may be possible to treat Duchenne muscular dystrophy by increasing the transcription of utrophin, a form of fetal dystrophin.

[0096] FIG. 18 shows an explanatory diagram of the nucleic acid sequence used in the structural analysis in this embodiment. FIG. 19 shows an explanatory diagram of the nucleic acid sequence used in the structural analysis in relation to the STAR Methods. FIG. 20-1 to FIG. 20-4 show explanatory diagrams of primers and constructs used in genome editing in relation to the STAR Methods. FIG. 21-1 to FIG. 21-7 show explanatory diagrams of key resources in the STAR Methods. These are illustrative and do not limit the invention. In addition, in FIG. 19 to FIG. 20-4, 3A to 3B and 3C to 3F correspond to A to B in FIG. 5-1 and C to F in FIG. 5-2, respectively, 5A to 5B and 5C to 5E correspond to A to B in FIG. 11-1 and C to E in FIG. 11-2, respectively, and 6A to 6F and 6G to 6J correspond to A to F in FIG. 15-1 and G to J in FIG. 15-2, respectively. Moreover, S4A to S4D, S4E to S4F, S4G to S4H, and S4I to S4L correspond to A to D, E to F, G to H, and I to L in FIG. 7-1, respectively.

[0097] As described above, we have successfully created a compact AsCas12f mutant with improved activity by applying deep mutation scanning (DMS) technology to CRISPR-Cas effectors. The DMS approach combines comprehensive protein mutagenesis and functional screening with deep sequencing, making it possible to evaluate the effects of thousands of mutations in a single experiment. One typical application of DMS is the introduction of libraries into a yeast surface display system and the evaluation of the binding affinity of ligands, including antibodies and viral glycoproteins. In this embodiment, we applied the yeast screening system to a mammalian cell-based screen to evaluate genome editing activity. We constructed a library that covers all 20 single amino acid substitutions at each position of the entire sequence of AsCas12f (422 residues), and identified more than 200 effective mutations at various positions. To efficiently explore effective combinations of these mutants, it is advantageous to adopt a structural perspective. As shown in this embodiment, in the absence of an experimentally determined structure, a structural prediction model such as AlphaFold can be a useful approach to efficiently select mutations identified by DMS.

[0098] The amino acid sequence of AsCas12f is as shown in sequence number 1 of the attached sequence listing, and is written in one letter as follows: MIKVYRYEIVKPLDLDWKEFGTILRQLQQETRFALNKATQLAWEWMGFSSDYKDNHGEYPKSKDILGYTNVHGYAYHTIKTKAYRLNSGNLSQTIKRATDRFKAY QKEILRGDMSIPSYKRDIPLDLIKENISVNRMNHGDYIASLSLLSNPAKQEMNVKRKISVIIIVRGAGKTIMDRILSGEYQVSASQIIHDDRKNKWYLNISYDFEP QTRVLDLNKIMGIDLGVAVAVYMAFQHTPARYKLEGGEIENFRRQVESRRISMLRQGKYAGGARGGHGRDKRIKPIEQLRDKIANFRDTTNHRYSRYIVDMAIKEG CGTIQMEDLTNIRDIGSRFLQNWTYYDLQQKIIYKAEEAGIKVIKIDPQYTSQRCSECGNIDSGNRIGQAIFKCRACGYEANADYNAARNIAIPNIDKIIAESIK*

Claims

1. In the amino acid sequence represented by SEQ ID NO: 1, the amino acid sequence includes at least one of a substitution with histidine at amino acid position 188 and a substitution with lysine at amino acid position 195. protein.

2. The protein of claim 1, which is a double mutant that includes at least a substitution of amino acid 188 with histidine.

3. The protein of claim 1, which is a mutant that includes at least a substitution at amino acid position 188 with histidine and a substitution at amino acid position 232 with alanine.

4. The protein of claim 1, which is a triple mutant comprising at least a substitution at amino acid position 188 with histidine and a substitution at amino acid position 232 with alanine.

5. a substitution at amino acid position 188 with histidine and a substitution at amino acid position 232 with alanine; and The protein of claim 1, which is a triple mutant containing any one of the following substitutions: a substitution at amino acid position 123 with histidine, a substitution at amino acid position 246 with methionine, a substitution at amino acid position 316 with methionine, or a substitution at amino acid position 337 with leucine.

6. The protein of claim 1, which is a mutant that includes at least a substitution at amino acid position 188 with histidine, a substitution at amino acid position 232 with alanine, and a substitution at amino acid position 316 with methionine.

7. The protein of claim 1, which is a quadruple mutant comprising at least a substitution at amino acid position 188 with histidine, a substitution at amino acid position 232 with alanine, and a substitution at amino acid position 316 with methionine.

8. The protein of claim 1, which is a mutant that includes at least a substitution at amino acid position 188 with histidine, a substitution at amino acid position 232 with alanine, a substitution at amino acid position 316 with methionine, and a substitution at amino acid position 48 with tyrosine.

9. The protein of claim 1, which is a double mutant containing at least a substitution of lysine at amino acid position 195.

10. The protein of claim 1, which is a mutant that includes at least a substitution of lysine at amino acid position 195 and a substitution of alanine at amino acid position 232.

11. The protein of claim 1, which is a triple mutant comprising at least a substitution at amino acid position 195 with lysine and a substitution at amino acid position 232 with alanine.

12. a substitution at amino acid position 195 with lysine and a substitution at amino acid position 232 with alanine; and The protein of claim 1, which is a triple mutant containing any one of the following substitutions: a substitution at amino acid position 70 with tyrosine, a substitution at amino acid position 208 with arginine, or a substitution at amino acid position 316 with methionine.

13. The protein of claim 1, which is a mutant that includes at least a substitution at amino acid position 195 with lysine, a substitution at amino acid position 232 with alanine, and a substitution at amino acid position 208 with arginine.

14. The protein of claim 1, which is a quadruple mutant comprising at least a substitution at amino acid position 195 with lysine, a substitution at amino acid position 232 with alanine, and a substitution at amino acid position 208 with arginine.

15. The protein of claim 1, which is a mutant that includes at least a substitution at amino acid position 195 with lysine, a substitution at amino acid position 232 with alanine, a substitution at amino acid position 208 with arginine, and a substitution at amino acid position 123 with histidine.

16. 1) a triple mutant in which the amino acid at position 195 is not mutated and which contains at least a substitution of histidine at position 188 and a substitution of alanine at position 232, or 2) The protein according to claim 1, which is a triple mutant in which amino acid position 188 is not mutated and which at least includes a substitution of lysine at amino acid position 195 and a substitution of alanine at amino acid position 232.

17. The protein of claim 1, which is a quadruple mutant in which amino acid position 188 is not mutated, and which includes at least a substitution with lysine at amino acid position 195, a substitution with arginine at amino acid position 208, and a substitution with alanine at amino acid position 232.

18. The protein according to any one of claims 1 to 14, which forms an asymmetric homodimer and forms a complex together with a guide RNA.

19. The protein of claim 18, which forms a complex with the guide RNA in which stem loop 4 and stem loop 5 are deleted.

Images