KR20230007218A - Hypercompact base editing systems and use thereof - Google Patents

Abstract

The present invention relates to nucleic acid decomposing proteins with small TnpB-derived molecular weight used in base editing or functional analog thereof, a hypercompact base editing system comprising the same, and a method for editing a specific base within a target gene with another base using the hypercompact base editing system. In addition, the present invention relates to a base editing composition containing a hypercompact base editing construct or system used in base editing. The hypercompact base editing construct or system in the present invention is packaged in one AAV vector to be efficiently transmitted to a target area within a cell, identifies the editing of adenine or cytosine on a target sequence with other base, and confirms the base editing generated at a higher editing rate compared with existing adenine base editing genetic scissor (ABEs) or cytosine base editing genetic scissor (CBEs). Accordingly, the hypercompact base editing construct or system according to the present invention will be an excellent advanced genetic editing system in genetic disease treatment through base editing.

Description

Hypercompact base editing systems and use thereof {Hypercompact base editing systems and use thereof}

The present invention relates to hypercompact base editing systems (Hypercompact base editing systems) for base editing, and more specifically, TnpB-derived low molecular weight nucleic acid degrading protein used for base editing, ultracompact base editing structures and systems including the same, A method for correcting a specific base in a target gene with another base using the miniaturized base correction system and a base correction composition therefor.

Genome editing is a technology that freely corrects the genetic information of living organisms. This genome editing technology can effectively change desired genetic information not only in microorganisms but also in animals and plants including humans. Accordingly, genome editing technology is a key technology that will create new industries in the bio field, such as cell engineering, model animal production, transgenic plant production, and utilization for gene therapy for cancer, genetic diseases, and infectious diseases. Great use.

In genome editing technology, the gene scissors (CRISPR/Cas) system designed to precisely find the target gene sequence and cut the site plays an important role. The gene scissors (CRISPR/Cas) system is largely divided into Class I and Class2 according to the composition of the protein complex containing the nucleolytic protein that composes it, and typeI, typeIII, and typeIV (or higher) in detail according to the composition and number of Cas genes. Class1), typeII, typeV, and typeVI (Class2) (Makarova, A et al., 2011). Cas9 derived from Streptococcus pyogenes, which has been most actively studied to date, is a representative Class2/Type II nucleolytic protein.

The CRISPR/Cas system consists of a complex of Cas endonuclease and CRISPR RNA (crRNA) capable of recognizing a target gene sequence, and additionally binds to the Cas endonuclease while binding to crRNA. A transactivating CRISPR RNA (tracrRNA) to form an additional complex.

A single-stranded guide RNA (sgRNA) form in which the two RNAs are connected by a linker is mainly used, and this guide RNA is the target gene of the target gene to be cut by the Cas endonuclease of the CRISPR/Cas system. It serves as a guide to the double-stranded DNA sequence. Cas endonuclease located at the target gene site recognizes a protospacer adjacent motif (PAM) adjacent to the target gene sequence, and then cuts an internal or external nucleotide sequence of the target sequence.

The target DNA cut by the CRISPR/Cas system is repaired through a DNA repair mechanism of homology directed repair (HDR) or non-homologous end joining (NHEJ) process. Through the DNA repair mechanism of non-homologous end joining (NHEJ), insertion or deletion of random bases occurs between the cut DNA sites, or a combination thereof (insertion and deletion, indel), and as a result A frameshift mutation or a premature termination mutation occurs in the coding region of a gene to knock-out the target gene. On the other hand, the DNA repair mechanism of homologous recombination (HDR) requires donor DNA to repair the cut DNA, and the sequence of the target gene is elaborately modified using the sequence of the donor DNA as a template.

Currently, genome editing technology is a key technology for gene therapy development, and various technologies are being developed in terms of efficiency, safety, and delivery. The CRISPR/Cas system performs base editing, prime editing, or indel of target genes and is used for gene therapy for cancer, genetic diseases, and infectious diseases. In such gene correction, it is most important to efficiently deliver the CRISPR/Cas system to cells throughout the body, and an efficient mediator is required for such delivery.

Adeno-associated virus (AAV) is an FDA-approved vector for gene therapy due to its safety, persistence, and compatibility with mass production (Wang, D., Tai, P. W. L. & Gao, G. Adeno-associated virus vector as a platform for gene therapy 688 delivery. Nat Rev Drug Discov 18, 358-378 (2019)), a CRISPR/Cas system that can contain all components in one AAV vector for the treatment of genetic diseases It is recognized as an important tool (Yu, W. & Wu, Z. Use of AAV Vectors for CRISPR-Mediated In Vivo Genome Editing in the Retina. Methods Mol Biol 1950, 123-139 (2019)).

The gene size of the packaging capacity that can be delivered using the adeno-associated virus (AAV) vector is limited to less than 4.7 kb. When using AAV vectors as intracellular delivery media for gene therapy, most of the existing CRISPR/Cas systems generally exceed the size of about 4.7 kb, which limits clinical application. (Wu, Z., Yang, H. & Colosi, P. Effect of genome size on AAV vector packaging. Mol Ther 18, 80-692 86 (2010)). For this reason, SaCas9 (Ran, F. A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186-191 (2015)) and CjCas9 (Kim, E. et al. In vivo genome editing with a smaller molecular weight than Cas9) small Cas9 orthologue derived from Campylobacter jejuni. Nat Commun 8, 14500 (2017)) is being studied as a genome editing tool that can be delivered into cells via adeno-associated virus (AAV).

The CRISPR/Cas system for use in gene therapy goes beyond gene editing technology that simply causes indel mutations through double-stranded DNA cutting, and combines other effector proteins with the CRISPR/Cas system. It is also used for base editing, prime editing, or epigenetic regulation.

However, conventional base-editing gene scissors have also been used as systems capable of correcting base sequences by binding deaminase to Cas9 or Cpf1 protein based on CRISPR/Cas9 or CRISPR/Cas12a (Cpf1). Since the size of the Cas9 or Cpf1 protein itself is large, base-correction genetic scissors coupled with deaminase can be used for indel gene scissors (CRISPR) when delivered to the body through a delivery vehicle such as adeno-associated virus (AAV). /Cas) has greater difficulty than the system.

As such, gene editing using the gene scissors (CRISPR/Cas) system has the advantage of presenting a more fundamental and effective application means for gene therapy for cancer, genetic diseases, infectious diseases, and the like. However, existing nucleolytic proteins such as Cas9 or Cpf1 have difficulties in incorporating them into AAV vectors, which are clinically proven delivery means, due to their large molecular weight. In addition, SaCas9 and CjCas9, known as relatively smaller Cas9 proteins, also exceed the packaging capacity of adeno-associated virus (AAV) vectors by binding to deaminase or other effector proteins. It is still unsuitable for use as a gene therapy delivered in vivo.

In order to solve this problem, Cas9 or Cpf1 proteins, which are known to have excellent gene editing efficiency, or SaCas9 and CjCas9, which are known to be relatively small nucleic acid degradation proteins, are smaller in molecular weight but have high targeting efficiency and editing activity in cells. There is an urgent need for a protein and a novel CRISPR/Cas system containing the protein.

The present invention is to solve the above needs and overcome the problems of the prior art, deaminase (deaminase) conjugated TnpB-derived low molecular weight nucleolytic protein or functional analogues thereof; and guide RNA.

The present invention also relates to a deaminase-linked TnpB-derived low-molecular-weight nucleolytic protein or a functional analog thereof; It is an object of the present invention to provide a vector comprising a nucleic acid construct in which nucleic acid sequences encoding each component of a microminiature sequencing system including; and guide RNA are operably linked.

The present invention provides a small molecular weight nucleolytic protein derived from Cas12f1 or TnpB or a functional analog thereof for use in correcting a specific base with another base at a target site of a target nucleic acid or target gene; And a deaminase (deaminase); for the purpose of providing a fusion protein comprising a.

The present invention also provides an ultra-small base editing construct comprising a TnpB-derived low-molecular-weight nucleolytic protein or a functional analogue thereof coupled to deaminase; or a micro base correction system in which the guide RNA is included in the micro base correction structure;

In addition, an object of the present invention is to provide a base editing method comprising the step of contacting the miniaturized base editing system with a target site sequence of a target nucleic acid or target gene.

Other objects of the present application and technical problems to be achieved by the present invention are not limited to the above-mentioned objects, and will become more clear by the following description of the invention together with the claims and drawings described in this specification. In addition, the technical problem to be achieved by the present invention, which is not described in the present specification, will be clearly understood and inferred by those skilled in the art (hereinafter referred to as 'ordinary technician').

In order to achieve the above object, the following invention is provided.

The present invention is a low molecular weight nucleolytic protein derived from TnpB linked to deaminase or a functional analog thereof; And guide RNA; provides a mini base editing (base editing) system comprising a.

In one embodiment of the present invention, the TnpB-derived low-molecular-weight nucleolytic protein in the ultra-small base correction system may be Cas12f1 protein (SEQ ID NO: 1) belonging to the Cas12 family or TnpB consisting of the amino acid sequence of SEQ ID NO: 7. For example, the TnpB-derived nucleolytic protein having a small molecular weight or a functional analogue thereof may include dead TnpB (dTnpB) having lost DNA double-strand cleavage activity; dead Cas12f1 (dCas12f1); or a functional analogue of dTnpB or dCas12f1. More specifically, the TnpB-derived low-molecular-weight nucleolytic protein comprises any one amino acid sequence selected from SEQ ID NO: 3 to SEQ ID NO: 6, SEQ ID NO: 11 to SEQ ID NO: 18, and SEQ ID NO: 168 to SEQ ID NO: 175, or It may be a protein containing at least two amino acid mutations among D354A, E450A, R518A and D538A in SEQ ID NO: 7.

Also, as an example, in the miniaturized base correction system, the deaminase may be an adenosine deaminase and/or a cytidine deaminase linked to the N-terminus or C-terminus of the fusion protein. ) can be. More specifically, the adenosine deaminase is E. coli -derived tRNA adenosine deaminase (TadA), monomeric TadA (SEQ ID NO: 126), eTadA1 (evolved tRNA-specific adenosine deaminase 1, sequence number 127), dTadA (SEQ ID NO: 128), deTadA1 (SEQ ID NO: 129), eTadA2 (SEQ ID NO: 130), eTadA3 (SEQ ID NO: 131), eTadA4 (SEQ ID NO: 176), eTadA5 (SEQ ID NO: 177), eTadA6 (SEQ ID NO: 177) 178), eTadA7 (SEQ ID NO: 137), eTadA8 (SEQ ID NO: 138), eTadA9 (SEQ ID NO: 139), eTadA10 (SEQ ID NO: 140), or eTadA11 (SEQ ID NO: 141), or the heterodimer TadA-eTadA , eTadA-TadA, dTadA-eTadA or TadA-deTadA.

For example, the cytidine deaminase may be APOBEC1, APOBEC3A, APOBEC3B, CDA, AID or PmCDA1, but is not limited thereto. Here, APOBEC1 may include the amino acid sequence of SEQ ID NO: 21, APOBEC3A may include the amino acid sequence of SEQ ID NO: 22, and APOBEC3B may include the amino acid sequence of SEQ ID NO: 23.

For example, in the subminiature base editing system, the deaminase or the nucleolytic protein includes 0, 1, or 2 or more UGIs (Uracil Glycosylase Inhibitors) at the N-terminus or C-terminus, respectively, or , Zero or one or more GAMs (Gam proteins) may be included at the N-terminus or C-terminus of the nucleolytic protein, respectively. Here, the deaminase may be cytidine deaminase.

In another embodiment of the present invention, the hypercompact base editing system includes Cas12f1, TnpB or functional analogues thereof; and a fusion protein comprising a deaminase; And wild-type or engineered guide RNA (engineered guide RNA); may include. In addition, the fusion protein may be a nucleolytic protein to which a deaminase is bound, and the guide RNA may include two or more wild-type or engineered guide RNAs. Here, the engineered guide RNA includes engineered tracrRNA (transactivating CRISPR RNA) or engineered crRNA (CRISPR RNA).

For example, the engineered tracrRNA may be a modified tracrRNA so as not to include five or more contiguous uridine sequences. Alternatively, the engineered tracrRNA may be a tracrRNA modified not to include five or more contiguous uridine sequences and modified to have a shorter length than wild-type tracrRNA.

More specifically, the engineered tracrRNA may include a first region, a second region, a third region, and a fourth region in order from the 5'-end to the 3'-end.

In one embodiment, the first region (MS3 region, 1-21 region) may be a 5'-CUUCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 24) sequence or a partial sequence of SEQ ID NO: 24 sequence. The partial sequence of the sequence of SEQ ID NO: 24 may be a partial sequence sequentially removed from the 5'-end of the sequence of SEQ ID NO: 24 and the remaining 3'-end.

In addition, the second region (MS5 region, 22-71 region) may be a 5'-CCGCUUCACCAAAAGCUGU CCCUUAGGGGAUUAGAACUUGAGUGAAGGUG-3' (SEQ ID NO: 25) sequence or a partial sequence of SEQ ID NO: 25 sequence. Part of the sequence of SEQ ID NO: 25 may be a sequence in which at least one pair of nucleotides forming a complementary bond and/or at least one or more nucleotides not forming a complementary bond in the sequence of SEQ ID NO: 25 are deleted. At this time, the 5'-UUAG-3' sequence of the loop part included in the partial sequence of SEQ ID NO: 25 may be optionally substituted with the 5'-GAAA-3' sequence.

The third region (front part of MS1, 72-129 region) may be a sequence having at least 70% sequence identity or sequence similarity to the 5'-GGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAA-3' (SEQ ID NO: 26) sequence or SEQ ID NO: 26 sequence.

The fourth region (MS 1, 130-169 region) may be a 5'-CAAAUUCANNNVNCCUCUCCAAUUC UGCACAA-3' (SEQ ID NO: 27) sequence or a partial sequence of SEQ ID NO: 27 sequence. Each of N is independently A, C, G or U, and V may be A, C or G. Part of the sequence of SEQ ID NO: 27 may be a sequence that includes the 5'-CAAAUUCANNNVN-3' (SEQ ID NO: 28) sequence of the sequence of SEQ ID NO: 27 and does not include a partial sequence at the 3'-end. Preferably, the fourth sequence may be a sequence including a 5'-CAAAUUCANNNCN-3' (SEQ ID NO: 29) sequence but not including a part of the sequence at the 3'-end. wherein each N is independently A, C, G or U.

In addition, the fourth sequence may include a sequence in which 5'-NNNVN-3' in SEQ ID NO: 27 is substituted with 5'-NVNNN-3'. Here, each N is independently A, C, G or U, and V may be A, C or G.

In another embodiment, the crRNA may be a wild-type crRNA or an engineered crRNA. The wild-type crRNA may include a wild-type repeat sequence and a guide sequence in order from the 5'-end to the 3'-end. The wild-type repetitive sequence may be a 5'-GUUGCAGAACCCGAAUAGACGAAUGAAGGAAUGCAAC-3' (SEQ ID NO: 30) sequence.

The engineered crRNA may include a fifth region (including MS1 modification), a sixth region, and a guide sequence in order from the 5'-end to the 3'-end. Here, the fifth region may be a 5'-GUUGCAGAACCCGAAUAGNBNNNUGAAGGA-3' (SEQ ID NO: 31) sequence or a partial sequence of SEQ ID NO: 31 sequence. Each N may independently be A, C, G or U. The B may be U, C or G. Part of the sequence of SEQ ID NO: 31 may be a sequence that includes the 5'-NBNNNUGAAGGA-3' (SEQ ID NO: 32) sequence of the sequence of SEQ ID NO: 31 and does not include a partial sequence at the 3'-end. Each N may independently be A, C, G or U. The B may be U, C or G.

The sixth region may be a 5'-AUGCAAC-3' (SEQ ID NO: 33) sequence or a sequence having at least 70% or more sequence homology to the 5'-AUGCAAC-3' sequence.

The engineered crRNA according to the present invention may further include a U-rich tail sequence as a seventh region at the 3'-end of the crRNA. The U-rich tail sequence may be a 5'-(UaN)dUe-3' sequence, a 5'-UaVUaVUe-3' sequence, or a 5'-UaVUaVUaVUe-3' sequence. The N may be A, C, G or U. Each V may independently be A, C or G. The a may be an integer of 0 to 4. d may be an integer of 0 to 3. The e may be an integer from 0 to 10.

Also, as an example, the engineered guide RNA may be a dual guide RNA or a single guide RNA. When the engineered guide RNA is a single guide RNA, the engineered guide RNA may further include a linker sequence. In this case, the linker sequence may be located between the engineered tracrRNA and the crRNA.

In one embodiment, the engineered tracrRNA comprises/consists of any one nucleotide sequence selected from SEQ ID NO: 34 to SEQ ID NO: 37 and SEQ ID NO: 39 to SEQ ID NO: 42, and the engineered crRNA is SEQ ID NO: 38 or SEQ ID NO: 38 It may contain/consist of the base sequence of 43.

As another example, the engineered tracrRNA includes/consists of any one of SEQ ID NO: 44 to SEQ ID NO: 47 and SEQ ID NO: 49 to SEQ ID NO: 52, and the engineered crRNA is SEQ ID NO: 48 or SEQ ID NO: 48 or SEQ ID NO: 48. It may contain/consist of the nucleotide sequence of 53.

In another embodiment of the present invention, in the miniaturized base editing system, the nucleolytic protein is a protein comprising any one amino acid sequence selected from SEQ ID NO: 3 to SEQ ID NO: 6 and SEQ ID NO: 11 to SEQ ID NO: 18, and the guide RNA may include any one of nucleotide sequences selected from SEQ ID NO: 55 to SEQ ID NO: 59. Preferably, the nucleolytic protein consists of any one amino acid sequence selected from SEQ ID NO: 3 to SEQ ID NO: 6, SEQ ID NO: 11 to SEQ ID NO: 18 and SEQ ID NO: 168 to SEQ ID NO: 175, or SEQ ID NO: 7 to D354A, E450A, It is a protein having two or more amino acid mutations among R518A and D538A, and the guide RNA may consist of any one nucleotide sequence selected from SEQ ID NO: 55 to SEQ ID NO: 59.

In addition, the adenosine deaminase in the microbase correction system is a heterodimer TadA-eTadA tRNA adenosine deaminase derived from E. coli Or it is included in the structure of eTadA-TadA, and the cytosine deaminase is APOBEC1, APOBEC3B, APOBEC3C, CDA, AID or PmCDA1, and UGI (Uracil Glycosylase Inhibitor) is attached to the N-terminus or C-terminus of APOBEC1 or PmCDA1. Each may be one or two or more combined.

The present invention also relates to a low molecular weight nucleolytic protein derived from TnpB linked to deaminase or a functional analog thereof; And guide RNA; to provide a vector comprising a nucleic acid construct operably linked to the nucleic acid sequence encoding each component of the base editing (Base editing) system comprising a.

In one embodiment of the present invention, the vector may include a nucleic acid encoding an engineered guide RNA. The vector may further include nucleic acids encoding one or more of the guide RNAs. RNA engineered herein may include engineered tracrRNA and/or crRNA. In this case, the engineered guide RNA may have the same configuration as the previously described embodiment of the engineered guide RNA.

The vector may further include one or two or more promoters for nucleic acids encoding the engineered guide RNA. Specifically, the promoter may be a U6 promoter, H1 promoter or 7SK promoter.

In addition, the vector may be a plasmid, a linear PCR amplicon or a viral vector. Here, the viral vector is a retrovirus vector, a lentivirus vector, an adenovirus vector, an adeno-associated virus (AAV) vector, and a vaccinia virus vector. It may be at least one viral vector selected from the group consisting of a vaccinia virus vector, a poxvirus vector, and a herpes simplex virus vector.

The present invention also relates to a deaminase-linked TnpB-derived low-molecular-weight nucleolytic protein or a functional protein thereof for use in correcting a specific base with another base in a target site sequence of a target nucleic acid or target gene. Subminiature base editing constructs, including analogs, are provided.

Functional analogs of the nucleolytic protein include dead TnpB (dTnpB) that has lost DNA double-strand cleavage activity; dead Cas12f1 (dCas12f1); or a functional analogue of dTnpB or dCas12f1; Here, the nucleolytic protein may be a subminiature base editing construct comprising an amino acid sequence selected from SEQ ID NO: 3 to SEQ ID NO: 6 and SEQ ID NO: 11 to SEQ ID NO: 18. Preferably, the nucleolytic protein may be a subminiature base editing construct characterized in that it consists of any one amino acid sequence selected from SEQ ID NO: 3 to SEQ ID NO: 6 and SEQ ID NO: 11 to SEQ ID NO: 18.

In addition, the deaminase may be an adenosine deaminase and/or a cytidine deaminase, and may be a subminiature base editing construct.

For example, the adenosine deaminase is E. coli-derived tRNA adenosine deaminase (TadA) and/or the cytidine deaminase is APOBEC1, APOBEC3B, APOBEC3C, CDA, AID or PmCDA1. It may be a subminiature base correction structure.

In addition, the subminiature base correction construct includes the adenosine deaminase in the structure of a heterodimer TadA-TadA ^* or TadA ^* -TadA, and/or the cytosine deaminase APOBEC1, APOBEC3B, APOBEC3C, CDA, AID or It may be characterized in that one or two or more UGIs (Uracil Glycosylase Inhibitors) are respectively bound to the N-terminus or C-terminus of PmCDA1.

One or two or more UGIs (Uracil Glycosylase Inhibitors) are included at the N-terminus or C-terminus of the deaminase or the nucleolytic protein, or at the N-terminus or C-terminus of the nuclease. contains 0 or 1 or more GAM (Gam protein), respectively, wherein the deaminase is cytidine deaminase. .

As an example, the miniaturized scissors structure may be a miniaturized scissors structure characterized by including one or more nuclear localization sequences (NLS) sequences at the N-terminus or C-terminus. The NLS sequence may include/consist of any one amino acid sequence selected from SEQ ID NO: 65 to SEQ ID NO: 68, and may be a miniaturized scissors structure.

As another example, the deaminase and the TnpB-derived low-molecular-weight nucleolytic protein may be coupled to each other through a linker. The linker is 5'-GAAA-3', SEQ ID NO: 132 (GGGGS)n, (G)n, SEQ ID NO: 133 (EAAAK)n, (GGS)n, SEQ ID NO: 134 SGSETPGTSESATPES, SEQ ID NO: 135 SGGS , (XP)n or any combination thereof, where n is independently an integer from 1 to 30, and X can be any amino acid. Specifically, the linker may be a subminiature base correction construct comprising/consisting of any one amino acid sequence selected from SEQ ID NO: 62 to SEQ ID NO: 64.

The fusion protein may include dCas12f1 or dTnpB, a deamination enzyme, and optionally one or two or more UGIs in any order, and each protein may be linked by an arbitrary linker. Examples of fusion proteins according to one embodiment are as follows, but are not limited thereto:

[NH ₂ ]-[deaminase]-[optional linker]-[UGI (optionally)]-[optional linker]-[dCas12f1 or dTnpB]-[COOH];

[NH ₂ ]-[UGI (optionally)]-[optional linker]-[deaminase]-[optional linker]-[dCas12f1 or dTnpB]-[COOH];

[NH ₂ ]-[UGI (optionally)]-[optional linker]-[dCas12f1 or dTnpB]-[optional linker]-[deaminase]-[COOH];

[NH ₂ ]-[dCas12f1 or dTnpB]-[optional linker]-[UGI (optionally)]-[optional linker]-[deaminase]-[COOH];

[NH ₂ ]-[dCas12f1 or dTnpB]-[optional linker]-[deaminase]-[optional linker]-[UGI (optionally)]-[COOH];

[NH ₂ ]-[Cytidine deaminase]-[optional linker]-[adenosine deaminase]-[optional linker]-[dCas12f1 or dTnpB]-[optional linker]-[UGI (optional to)]-[COOH];

[NH ₂ ]-[UGI (optionally)]-[optional linker]-[UGI (optionally)]-[optional linker]-[dCas12f1 or dTnpB]-[optional linker]-[cytidine deamination senase]-[optional linker]-[adenosine deaminase]-[COOH].

The present invention also relates to a deaminase-linked TnpB-derived low-molecular-weight nucleolytic protein or a functional analog thereof; And guide RNA; provides a base correction composition comprising a micro base correction system comprising a.

In one embodiment, the base correction composition may include RNA engineered into the micro base correction system or a nucleic acid encoding the same; and TnpB-derived low-molecular-weight nucleolytic protein or a functional analogue thereof or a nucleic acid encoding the same.

For example, the engineered RNA may include engineered tracrRNA and/or engineered crRNA. Here, the engineered RNA may have the same configuration as the embodiment of the engineered RNA described above. The engineered RNA may include any one of nucleotide sequences selected from SEQ ID NO: 55 to SEQ ID NO: 59. Preferably, the engineered RNA may consist of any one nucleotide sequence selected from SEQ ID NO: 55 to SEQ ID NO: 59.

In another embodiment, the composition for base correction may include a vector. At this time, the composition is a nucleic acid encoding the engineered guide RNA; and TnpB-derived low-molecular-weight nucleolytic protein or a functional analogue thereof. In this case, the vector may be a plasmid, mRNA transcript, PCR amplicon or viral vector.

In another embodiment, the base correction composition may be in the form of a mixture of nucleic acid and protein. In this case, the composition may include an engineered guide RNA and TnpB-derived low molecular weight nucleic acid degrading protein or a functional analogue thereof. The composition may be in the form of a ribonucleoprotein (RNP) complex of an engineered guide RNA and a nucleolytic protein according to the present invention.

In one embodiment of the present invention, the base correction composition is a TnpB-derived deaminase-conjugated composition for use in correcting a specific base with another base in a target site sequence of a target nucleic acid or target gene. It may include hypercompact base editing constructs, including a low molecular weight nucleolytic protein or a functional analogue thereof.

For example, in the base correction composition, adenine (A) and/or cytosine (C) may be substituted with other bases. More specifically, adenine (A) can be corrected with guanine (G), and cytosine (C) can be corrected with thymine (T).

As another example, the editing window of the composition for base correction is 2nd to 8th, 15th to 19th, or 2nd or 20th from the 5'-end of the target sequence in the case of adenine base correction. It may be adenine (A) located, and in the case of cytosine base correction, it may be a cytosine (C) located 2nd to 8th from the 5'-end of the target sequence. Preferably, the editing window may be adenine (A) located 3rd to 4th from the 5'-end of the target sequence in the case of adenine base editing, and in the case of cytosine base editing, the target sequence It may be in the 3rd to 5th, 3rd to 4th, or 3rd to 4th cytosine (C) ranges from the 5'-end.

In addition, the present invention provides a base editing method comprising the step of contacting the base editing composition with a target sequence.

For example, it may be a base editing method comprising the step of contacting the miniaturized base editing system or the miniaturized base editing construct with a target site sequence of a target nucleic acid or target gene in a cell. More specifically, it may be a method of modifying a target nucleic acid or target gene present in a target cell using the miniaturized sequencing system.

In one embodiment, the base editing method may include treating a prokaryotic cell or eukaryotic cell in which a target nucleic acid or target gene is present with a composition including the miniaturized base editing system according to the present invention. Here, the eukaryotic cells may be yeast, plant cells, non-human-animal cells or human cells. In addition, the target nucleic acid or target gene may include double-stranded DNA comprising a target strand having a target sequence; single-stranded DNA or RNA; or hybrid double-stranded DNA and RNA.

In one embodiment, through the base editing method, the target nucleic acid or target gene has an editing window 2nd to 8th, 3rd to 4th, 15th to 19th from the 5'-end of the target sequence. It may be an adenine (A) located at the 2nd, 2nd, or 20th position of the target sequence, or a cytosine (C) located at the 2nd to 8th or 3rd to 5th position from the 5'-end of the target sequence.

In one embodiment, the base editing method may include injecting the subminiature base editing system into a cell to contact the target nucleic acid or target gene.

For example, the delivery may include injecting a vector including a nucleic acid construct in which a nucleic acid sequence encoding each component of the miniaturized sequencing system is operably linked.

In another embodiment, the miniaturized sequencing system is packaged in a viral vector selected from the group consisting of retrovirus, lentivirus, adenovirus, adeno-associated virus, vaccinia virus, poxvirus, and herpes simplex virus vector to target nucleic acid or target gene can be delivered to existing cells. Preferably, the viral vector is an adeno-associated viral vector.

The hypercompact base editing systems of the present invention are novel ultracompact base editing systems prepared by including a TnpB-derived ultracompact nucleolytic protein or a functional analogue thereof and a guide RNA engineered to suit the protein. The miniaturized base editing system according to the present invention is a system capable of loading all the gene scissors required for various genome editing including base editing into a single adeno-associated virus (AAV) vector. The miniaturized base editing system according to the present invention, which has the above advantages, is a genetic scissors system containing proteins such as 'Cas9 or Cpf1, which have been actively researched and used in the past, is clinically verified intracellular delivery due to its size. It is to present a new gene scissors system that overcomes the biggest problem of using the AAV vector as a packaging tool, which is a tool.

In addition, the hypercompact base editing constructs according to the present invention prepared through the combination of TnpB-derived micronucleolytic protein or a functional analog thereof with adenosine deaminase or cytidine deaminase are novel base editing constructs. Functioning as a base editor, the miniaturized base editing system of the present invention is efficiently delivered into cells using one AAV vector as a packaging tool to correct a specific base in a target nucleic acid or target gene target site sequence with another base. In addition, since multi-targeting and multi-base editing are possible, the result of the present invention will be useful for the treatment of genetic diseases and research of genetic diseases through base editing. It is expected that

Therefore, the minimal base editing system for base editing of the present invention and the composition containing the same broaden the range of applications for selecting the optimal base editing gene scissors according to various target genes, and the existing genome editing or intracellular delivery is difficult. It will be an excellent next-generation gene editing system that can efficiently achieve genetic mutations of any size through base correction.

1 is a schematic diagram of various examples of subminiature base editing structures for base editing according to the present invention.
2 shows the structure of a heterodimer of adenosine deaminase included in the ultra-small base correction construct for base correction provided in the present invention and its amino acid sequence.
(a) shows the structure of adenosine deaminase TadA-eTadA1. They are linked in the order of Linker-TadA-Linker-eTadA1-NLS, and the amino acid sequences of TadA, eTadA1, linker and NLS are shown. (b) shows the structure of adenosine deaminase eTadA1-Tad. They are linked in the order of Linker-eTadA1-Linker-TadA-NLS, and the amino acid sequences of TadA, eTadA1, linker and NLS are shown. Each sequence shown in the figure is an exemplary sequence, but is not limited thereto.
3 shows a cytidine deaminase module included in the ultra-small base correction construct for base correction provided in the present invention and its amino acid sequence.
(a) shows the module structure of cytidine deaminase APOBEC1. They are connected in the order of NLS-APOBEC1-Linker, and the amino acid sequences of APOBEC1, linker and NLS are shown. The first methionine (M) of APOBEC1 is omitted. (b) shows the module structure of cytidine deaminase APOBEC3A. They are connected in the order of APOBEC3A-Linker-NLS, and the amino acid sequences of APOBEC3A, linker and NLS are shown. (c) shows the cytidine deaminase APOBEC3B module structure. They are connected in the order of APOBEC3B-Linker-NLS, and the amino acid sequences of APOBEC3B, linker and NLS are shown. Each sequence shown in the figure is an exemplary sequence, but is not limited thereto.
4 shows a subminiature base correction structure for base correction provided by the present invention.
(a) shows the modular structures of ABE-N1, ABE-N2, ABE-C1 and ABE-C2 of the adenine base-corrected gene scissors of dCas12f1 and dTnpB, respectively. (b) shows the structure of the CBE-N1, CBE-N2, CBE-C1 and CBE-C2 modular scissors.
Figure 5 shows wild-type guide RNA for Cas12f1, and each region of Modification Site (MS) MS1 to MS5 for the engineered guide RNA provided by the present invention is indicated.
6 is a result of confirming the indel efficiency (%) of the CRISPR/Cas12f1 complex including the engineered guide RNA having modifications in each region of MS1 to MS5 in wild-type guide RNA.
Figure 7 shows the single guide RNA structure and RNA sequence of engineered TnpB or Cas12f1, a component of the ultraminiature base correction system of the present invention.
8 shows the structure of an exemplary adeno-associated virus (AAV) vector provided in the present invention.
FIG. 9 is a result of confirming the base proofreading rate (%) of the microbase proofreading system including the microbase proofreading structures ABE-N1, ABE-N2, ABE-C1 or ABE-C2 of FIG. 4 .
(a), (b) and (c) show the results according to each target sequence of the minibase correction system including dCas12f1, and (d) and (e) show the results of the minibase correction system including dTnpB, a functional analogue of Cas12f1. The base correction rate (%) according to the target sequence of the system is shown.
10 is a result of confirming the efficiency of adenine base correction according to the type of dead variant when using the guide RNA of ver4.1 in the base correction efficiency of the ultra-small base correction system according to the present invention.
(a) and (b) show the results of base correction rates in target sequence 3 of the dCas12f1 variant and the dTnpB variant, respectively.
11 is a result of confirming the effect of the type of promoter involved in the expression of dTnpB or dCas12f1 on the base editing efficiency of the miniaturized base editing system according to the present invention.
(a) and (b) show the results of base correction rates according to promoter types for target sequence 1 and target sequence 3, respectively.
12 is a result confirming the effect of the linker length on the base proofreading efficiency of the microbase proofreading system according to the present invention.
(a) shows the base correction rate according to the linker length for the target sequence 3 of the system including dCas12f1 and the TadA1eTadA1 module, and (b) and (c) show the base correction rate of the system including dTnpB and the TadA1eTadA1 module or the TadA1eTadA3 module, respectively. It shows the base correction rate according to the linker length for target sequence 3.
13 is a result of confirming the effect of nucleolytic protein variants on the base proofreading efficiency of the micro base proofreading system according to the present invention.
(a) and (b) are the results of base correction rates for the target sequence 3 of the dCas12f1 variant and the dTnpB variant combined with the TadA1eTadA1 module, respectively, (c) is the calibration window expansion result of the dTnpB variant, and (d) is the result of dTnpB It is the result of the indel activity of the mutant.
14 shows the results of 3 repetitions of base correction windows according to amino acid substitution variants of dCas12f1 or dTnpB.
(a) is the result of the amino acid substitution variant of dCas12f1, and (b) is the result of the amino acid substitution variant of dTnpB.
15 shows the result of verifying the base correction system using TadAeTadA3 in various target sequences.
16 is a result of confirming the effect of Tad types on the base proofreading efficiency of the micro base proofreading system according to the present invention.
(a) to (c) show the results of base correction rates according to the gRNA types GE-Ver3.0, GE-Ver4.0 and GE-Ver4.1 for target sequence 3 of dCas12f1, respectively, and (d) to (f) shows the results of base correction rates according to gRNA types GE-Ver3.0, GE-Ver4.0 and GE-Ver4.1 for target sequence 3 of dTnpB1, respectively.
FIG. 17 is a result of confirming the base correction rate (%) of the micro base correction system including the micro base correction constructs CBE-N1, CBE-N2, CBE-C1 or CBE-C2 of FIG. 4 .
(a) and (b) show the results according to the target sequence, respectively.
18 is a result of comparing and confirming the unwanted indel occurrence rate (%) of the ultra-small base editing system according to the present invention and the existing base editing gene scissors.
(a) shows the indel generation rate (%) result of adenine base editors (ABEs), and (b) shows the indel generation rate (%) result of cytosine base editors (CBEs) showed
19 is a result of confirming the intracellular base correction rate (%) of the micro base correction system according to the present invention.
(a) shows a schematic diagram of the transformed cell line for analysis. (b) shows a schematic diagram of the AAV vector rAAV-ABE-C2 containing the ultraminiature base editing system including the adenine base editing construct dCas12f1-ABE-C2 and a cell line transfected therewith. (c) shows the result of the adenine base correction rate (%) of the target sequence region present in the cell by the rAAV-ABE-C2.
20 is a result of confirming the intracellular base correction rate (%) of the micro base correction system including dTnpB-ABE-C2.
(a) shows a schematic diagram of the transformed cell line for analysis. (b) shows a schematic diagram of the AAV vector rAAV-TnpB-ABE-C2 containing the ultraminiature base-correction system containing the adenine base-correction construct dTnpB-ABE-C2 and a cell line transfected therewith. (c) shows the adenine base correction rate (%) of the intracellular target sequence by the rAAV-TnpB-ABE-C2 compared to rAAV-SpCas9 split ABE.
21 is a result of comparing and confirming the intracellular base correction rate (%) of the ultra-small base correction system according to the present invention and the existing base correction gene scissors.
(a) is ABE-C2+sgRNA or ABE-C2+sgRNA+Auxillary; and AAV loadable SpCas9n-based adenine base-editing gene scissors miniABEmax; presented by Nguyen Tran et al (Nature Commun, 2020); This is the result showing the calibration efficiency of the base ABE.
22 is a result of confirming multiple gene editing through AAV delivery of the micro base editing system according to the present invention.
(a) shows a schematic diagram of AAV vectors AAV-S1 and AAV-S2 containing one type of gRNA and AAV vectors AAV-S1/2 containing two different types of gRNAs. (b) is a result showing the base correction rate (%) by the AAV vectors at the target sequence site in the cell.

The present inventors have identified the Cas12f1 protein, which has a molecular weight of about 1/3 smaller than that of the Cas9 protein, which has been studied the most to date, and has significantly higher targeting efficiency to target nucleic acids or target genes than existing nucleolytic proteins. . In addition, the present inventors have proposed a system that can be easily loaded into an adeno-associated virus (AAV) vector for base-editing of intracellular genes and can be effectively delivered in vivo. A novel ultraminiature base editing system containing a nucleolytic protein derived from TnpB was constructed.

In addition, by confirming for the first time that the miniaturized base editing system has high efficiency in base editing for a specific base at the target site of a target nucleic acid or target gene in a cell, the new minibase base editing system can be used in a variety of The present invention was completed based on its availability for genome editing.

The present invention relates to a deaminase-linked TnpB-derived low-molecular-weight nucleolytic protein or functional analog thereof for use in correcting a specific base with another base in the nucleotide sequence of a target nucleic acid or target gene; And guide RNA; It relates to a mini base correction system comprising a.

In addition, the present invention provides an ultra-small base correction construct comprising a deaminase-linked TnpB-derived low-molecular-weight nucleolytic protein or a functional analogue thereof, using which a specific base in a target nucleic acid or target gene is converted to another base. It relates to a correction method and a composition for base correction.

Hereinafter, the present invention will be described in more detail through explanation of terms, specific examples and examples. It should be noted that the above specific examples and embodiments include some embodiments of the invention and do not include all embodiments. It is obvious that the content of the invention disclosed by this specification is not limited to the specific implementation examples and examples described herein, and includes that those skilled in the art can implement it in various ways in the technical field to which the present invention belongs. Therefore, it should be understood that the content of the invention disclosed in this specification is not limited to the specific embodiments described herein, and modifications and other embodiments thereof are also included within the scope of the claims.

[Explanation of terms]

Base Editors (BEs)

"Base Editors (BEs)" used in the present invention is a single base editing tool, and adenosine deaminase or cytidine deaminase (Cytidine deaminase) DNA double-strand break activity It is constructed by binding either to the dead Cas (dCas) protein or to the nick Cas (nCas) protein that cuts one of the DNA double strands. These are called Adenine Base Editors (ABEs) and Cytosine Base Editors (CBEs), respectively.

Base Editors (BEs) may be referred to as "BEs", and Adenine Base Editors (ABEs) and Cytosine Base Editors (CBEs) may be referred to as "ABEs", respectively. and "CBEs". Base editors (BEs) preferably complete base editing by replacing a base at a specific site with another base in a target base sequence without causing unwanted DNA double-strand breakage.

Adenine Base Editors (ABEs)

The "Adenine Base Editors (ABEs)" used in the present invention are to correct an adenine (A) base with a guanine (G) base, and the tRNA adenosine deaminase (TadA) derived from E. coli and its variant (TadA ^* or eTadA) are combined into a monomer or heterodimer TadA-TadA ^* or TadA ^* -TadA structure, respectively, and then the monomer or heterodimer TadA-TadA ^* or TadA ^* -TadA protein was prepared by binding to the N-terminus or C-terminus of dead Cas12f1 (dCas12f1) protein, dead TnpB (dTnpB) protein or a functional analog thereof.

In addition, adenine base editors (ABEs) are dead Cas12f1 (dCas12f1) protein, dead TnpB (dTnpB) protein or functional analogues thereof; And various types are possible depending on the type of adenine deaminase coupled thereto.

adenosine deaminase

As used herein, the term "adenosine deaminase" or "adenosine deaminase protein" refers to targeting adenine (A) in RNA/DNA and DNA duplexes to adenine or adenine-containing molecules (e.g., adenosine, Proteins of proteins or polypeptides involved in deamination reactions that hydrolyze adenine moieties of DNA, RNA) to hypoxanthine or hypoxanthine-containing molecules (e.g., inosine (I)). , polypeptides or functional domains thereof.

The adenosine deaminase is rarely found in higher animals, but is known to be present in a small amount in the muscles of cows, milk, and blood of mice, and is present in large quantities in the intestines of crayfish and insects. The adenosine deaminase of the present invention can be derived from mammalian, bird, frog, squid, fish, fly, worm and metazoan species. Adenosine deaminase includes, but is not limited to, naturally occurring adenosine deaminase, such as TadA from Escherichia coli, and a mutant variant of TadA (eTadA). Here, the mutant variant of TadA (eTadA) is an evolved tRNA-specific adenosine deaminase, and may be used interchangeably with "eTadA" or "eTadA1", and may be interpreted in the same meaning.

Cytosine Base Editors (CBEs)

"Cytosine Base Editors (CBEs)" used in the present invention are to correct cytosine (C) with thymine (T), and dead APOBEC1, APOBEC3A, APOBEC3B derived from mice or PmCDA1 derived from lampreys. Cas12f1 (dCas12f1) protein, nick Cas12f1 (nCas12f1) protein, or a functional analogue thereof was constructed by linking to the N-terminus or C-terminus. In this case, the cytosine base correction gene scissors may be one or two or more UGIs (Uracil Glycosylase Inhibitors) bound to the N-terminus or C-terminus, respectively.

Cytosine Base Editors (CBEs) are BE1 fused with APOBEC1 protein to the N-terminus of dCas9, and uracil DNA glycosylase inhibitor (UGI) fused to the C-terminus of BE1. BE2, which suppresses the base excision repair (BER) function and improves the proofreading efficiency, and BE3, in which the efficiency of proofreading with thymine is increased by replacing the dCas9 part in BE2 with nCas9, but is not limited thereto.

cytidine deaminase

As used herein, the term "cytidine deaminase" or "cytidine deaminase protein" targets cytosine (C), which deaminates and causes conversion to uracil (U). is an enzyme protein that When the amine group of cytosine is removed to form uracil, uracil is converted to thymine (T) by a series of intracellular repair mechanisms, and finally base proofreading of cytosine (C) base to thymine (T) base is completed.

Cytidine deaminase generally works on RNA, but some are known to work on single-stranded DNA (ssDNA) as well (Harris et al., 2002). For example, human activation-induced cytidine deaminase (AID), human APOBEC3G, rat APOBEC1, APOBEC3A, APOBEC3B, CDA, AID, and lamprey PmCDA1, but are not limited thereto.

Base Editing Activity or Base Editing Rate

As used herein, the term "base editing activity" or "base editing rate" refers to the ability of adenine base editing gene scissors (ABEs) to adenine (A) at a specific adenine site in a DNA double-stranded base sequence. This refers to the extent to which guanine (G) is edited. In addition, in the case of cytosine base editing gene scissors (CBEs), it means the degree to which cytosine (C) is edited into thymine (T) at a specific cytosine site.

Editing window

The term "editing window" according to the present invention refers to a specific range of single-stranded DNA formed as guide RNA binds when base editing scissors of a base editing composition correct a specific base in a target nucleic acid or target gene. It refers to the range of simultaneous deamination of several bases present in . This can result in simultaneous proofreading of up to 2, 3 or 4 bases around the adenine (A) or cytosine (C) base of the target site. Each base-editing gene scissors may have a correction window for correcting a specific range of bases at a specific position.

Target nucleic acid or target gene

As used herein, the term "target nucleic acid" or "target gene" refers to a cell that is a target of gene editing by the miniaturized base editing structure or the miniaturized base editing system including the same according to the present invention. Means my genes or nucleic acids.

The target nucleic acid or target gene may be used interchangeably and may refer to the same target. Unless otherwise specified, the target nucleic acid or target gene may refer to all genes or nucleic acids native to the target cell or externally derived genes or nucleic acids, and is not particularly limited as long as it can be subjected to gene editing. The target gene or target nucleic acid may be a DNA single-strand, a DNA double-strand, or a hybrid double-strand of DNA and RNA.

The target nucleic acid or target gene may be a nucleic acid or gene existing in a cell or synthesized artificially. When present in a cell, the target nucleic acid or target gene may refer to both an endogenous gene or nucleic acid or an exogenous gene or nucleic acid possessed by the cell, and CRISPR /Cas12f1 It is not particularly limited as long as it can be subject to editing by the system.

Target region or target sequence

"Target region" or "target sequence" according to the present invention is a sequence present in a target nucleic acid or target gene, and the miniaturized base editing construct or the miniaturized base editing system including the same according to the present invention It refers to a specific sequence that recognizes to edit a target gene or target nucleic acid. The target site or target sequence may be appropriately selected depending on the purpose.

Specifically, the target site or target sequence is a sequence included in a target gene or target nucleic acid sequence, and refers to a sequence complementary to a spacer sequence included in the guide RNA or engineered guide RNA of the present invention.

In general, the spacer sequence is determined considering the sequence of the target gene or target nucleic acid and the PAM sequence recognized by the effector protein of the CRISPR/Cas system. The target site or target sequence may refer to only a specific strand that complementarily binds to the guide RNA of the base correction system, or may refer to the entire target double strand including the specific strand, which may be appropriately interpreted depending on the context. do.

Genetic scissors (CRISPR/Cas) or nucleolytic proteins

As used herein, the term "cRISPR/Cas" or "nucleolytic protein" refers to a nuclease capable of recognizing and editing a specific position in a target nucleic acid (DNA or RNA) or gene.

In addition, in the present invention, the CRISPR/Cas or nucleic acid degradation protein refers to a base editing system or an effector protein constituting a base editing construct. Here, the effector protein may be a nucleolytic protein capable of binding to a CRISPR protein or guide RNA (gRNA), or a peptide fragment capable of binding to an oligonucleic acid capable of binding to a target nucleic acid or target gene.

Specifically, it may be Cas12f1, Cas9, Cpf1, C2c1, C2c2, C2c3 or a modified nucleolytic protein, such as dead nucleolytic protein (dead Cas) or nick nucleolytic protein, but is not limited thereto.

In addition, the term "nucleolytic protein", as used herein, "cRISPR/Cas", "CRISPR" protein, "CRISPR/nucleolytic protein", "CRISPR effector", " "CRISPR/Cas effector", "CRISPR enzyme", "CRISPR/Cas enzyme", etc. may be used interchangeably, and may be interpreted in the same meaning.

Base editing construct

The term "base editing construct" used in the present invention has a "nucleolysis protein" as an essential component, and another effector protein, for example, adenosine deaminase (adenosine deaminase) deaminase) or cytidine deaminase (cytidine deaminase) is a structure that can be additionally linked. The connection may be made directly or by a linker.

The base editing construct is a basic component of a gene editing system, and can be used interchangeably with the "CRISPR/Cas module", and is a fusion in which a nucleolytic protein and a deamination enzyme are combined. It can be interpreted as meaning including protein.

Gene scissors (CRISPR/Cas) system

As used herein, the term "CRISPR/Cas system" refers to a complex comprising a target nucleic acid capable of binding to a target nucleic acid or a target gene in a CRISPR/Cas construct. Here, the targeting nucleic acid may be represented by guide RNA (gRNA), but is not limited thereto.

The CRISPR/Cas system is used in the present invention in various types according to the CRISPR/nucleolysis protein and the guide molecule corresponding to the protein, and the CRISPR/Cas protein uses the corresponding guide molecule. It specifically binds to a target nucleic acid or target gene to perform DNA double-strand breakage or base correction.

In addition, CRISPR/Cas systems are largely divided into Class 1 and Class 2. Here, the "2 CRISPR/Cas system" is characterized in that the effector complex includes a large single protein with multiple domains, and a typical Class 2 CRISPR/Cas system used in the present invention is Type II It is a CRISPR/Cas9 system. The CRISPR/Cas system, which is being actively researched for gene editing, such as the CRISPR/Cpf1 system, generally belongs to Class 2.

Class 2 CRISPR/Cas systems are also divided into Type II, V, and VI. Among them, the miniaturized gene editing system provided by the present invention belongs to the Type V CRISPR/Cas system including TnpB-derived small molecular weight proteins such as Cas12f1 as nucleolytic proteins. The effector protein of the Type V CRISPR/Cas system is named Cas12, and is named Cas12a, Cas12b, etc. according to detailed classification.

The Cas12 protein has one nuclease domain (RuvC-like nuclease), which is distinguished from Type II effector proteins (eg, Cas9 protein) having two nuclease domains (HNH and RuvC domains) characteristic to be The type V CRISPR/Cas system discovered so far is divided into 11 subtypes, among which the CRISPR/Cas12f1 system provided by the present invention belongs to V-F1, a variant of one of the subtypes V-F (Makarova et al., Nature Reviews, Microbiology volume 18, 67 (2020)).

CRISPR/Cas12f1 system

As used herein, the term "CRISPR/Cas12f1 system" refers to a target nucleic acid or a target nucleic acid or a target nucleic acid degrading protein, which was first produced in the present invention, to a CRISPR/Cas construct containing TnpB-derived low molecular weight proteins such as Cas12f1. It refers to a complex containing a targeting nucleic acid or guide RNA capable of binding to a gene.

Various types of CRISPR/Cas systems exist in nature, and new CRISPR/Cas systems are still being discovered. Among them, the CRISPR/Cas12f1 system provided by the present invention specifically belongs to the V-F subtype among Class 2, type V CRISPR/Cas systems, and is further divided into variants of V-F1 to V-F3.

In addition, the CRISPR/Cas12f system is among the effector proteins named Cas14 in a previous study (Harrington et al., Programmed DNA destruction by miniature CRISPR-Cas14 enzymes, Science 362, 839-842 (2018)), Cas14a, Cas14b , and the CRISPR/Cas14 system comprising Cas14c variants. Among them, the CRISPR/Cas14a system containing the Cas14a effector protein is classified as the CRISPR/Cas12f1 system (Makarova et al., Nature Reviews, Microbiology volume 18, 67 (2020)).

However, the CRISPR/Cas12f1 is not only a system belonging to the Cas14a family (Harrington et al., Programmed DNA destruction by miniature CRISPR-Cas14 enzymes, Science 362, 839-842 (2018)), but also an effector protein to c2c10. Also includes the named CRISPR/Cas system (Karvelis et al., Nucleic Acids Research, Vol. 48, No. 9 5017 (2020), Makarova et al., Nature Reviews, Microbiology volume 18, 67 (2020)).

Therefore, in this specification, the CRISPR/Cas12f1 system is a concept encompassing both the CRISPR/Cas14a system and the CRISPR/c2c10 system, unless otherwise stated, and when referring to the CRISPR/Cas14a system, the "CRISPR/Cas14a system" Or, it is referred to as "the CRISPR/Cas12f1 system belonging to the Cas14 family". The term has a meaning that a person skilled in the art can appropriately interpret according to the context.

Cas12f1 (CRISPR associated protein 12f1) protein

As used herein, the term "Cas12f1 protein" is a component of the engineered CRISPR/Cas12f1 complex and is a nucleolytic protein. The Cas12f1 protein may be a wild-type Cas12f1 protein existing in nature. The sequence encoding the Cas12f1 protein may be a sequence encoding a wild-type Cas12f1 protein, or a human codon-optimized Cas12f1 sequence for the protein.

In addition, the Cas12f1 protein may have the same function as the wild-type Cas12f1 protein existing in nature. However, unless otherwise specified, when "Cas12f1 protein" is used herein, it can mean not only wild-type or codon-optimized Cas12f1 proteins, but also modified Cas12f1 proteins and Cas12f1 fusion proteins.

The Cas12f1 protein may also be referred to as one having the same function as the wild-type Cas12f1 protein existing in nature, as well as one in which all or part of the function is modified or lost, and/or an additional function is added. The meaning of the Cas12f1 protein may be appropriately interpreted according to the context, and is interpreted in the broadest sense unless there are special cases.

Guide RNA (gRNA)

The term "guide RNA (gRNA)" according to the present invention is capable of forming a complex with a CRISPR-nucleolytic protein, hybridizing with a target nucleic acid sequence and directing sequence-specific binding of the complex to a target nucleic acid sequence. Refers to an RNA-based molecule comprising a guide sequence with sufficient complementarity with a target nucleic acid sequence. Guide molecules or guide RNAs are used interchangeably herein.

In addition, in the present invention, the guide RNA is an RNA-based molecule having one or more chemical modifications, for example, modification by chemical linkage of two ribonucleotides or by replacement of one or more ribonucleotides with one or more deoxyribonucleotides. specifically includes

The specificity of a guide RNA (gRNA) corresponding to a nucleolytic protein is governed by the direct repeat sequence constituting the guide RNA (gRNA). Accordingly, when designing an optimal guide RNA for a nucleolytic protein, the direct repeating sequence is designed according to the origin of the nucleolytic protein.

Therefore, the engineered gRNA used in the present invention can be used to construct complexes or constructs specific for nucleolytic proteins. For example, Cas effector proteins, which are functional analogs, are delivered to a target nucleic acid or target gene together with an engineered gRNA according to each protein to effectively perform a desired function.

Therefore, the engineered guide RNA (gRNA) according to the present invention is modified in its length and sequence from the guide RNA (gRNA) found in nature in order to show the maximum activity specifically for each functional analogue of these nucleolytic proteins. allow

In addition, the engineered guide RNA (gRNA) may also include a modification to add a U-rich tail to the 3'-end in order to successfully bind to the target sequence and form a CRISPR/Cas complex.

Scaffold area

The term "scaffold region" according to the present invention refers to a part of guide RNA capable of interacting with a nucleolytic protein as a whole, and may refer to the remaining parts of guide RNA found in nature, excluding spacers. . The scaffold region includes tracrRNA and parts of crRNA, and does not necessarily refer to one molecule of RNA. The scaffold area may be further subdivided into a first area, a second area, a third area, a fourth area, a fifth area, and a sixth area. When the subdivided regions are described on tracrRNA and crRNA, the first to fourth regions are included in tracrRNA, and the fifth to sixth regions are included in crRNA, specifically crRNA repeat sequence portions.

spacer sequence

The term "spacer sequence" according to the present invention refers to a polynucleotide that hybridizes with a portion of a target sequence in the CRISPR/Cas system according to the present invention. The spacer sequence refers to 10 to 50 consecutive nucleotides near the 3'-end of the crRNA of the guide RNA in the CRISPR/Cas system according to the present invention.

The spacer is designed to correspond to a target sequence of a target nucleic acid or target gene to be edited using the CRISPR/Cas system. That is, the spacer may have various sequences depending on the target sequence of the target nucleic acid. In the present specification, a spacer sequence refers to a spacer sequence within a crRNA or an engineered crRNA included in the CRISPR/Cas system, unless otherwise specified.

tracrRNA and crRNA

The terms "tracrRNA and crRNA" according to the present invention include all meanings that can be recognized by a person skilled in the art in the field of CRISPR/Cas technology. Although this term is generally used to refer to each molecule of dual guide RNA found in nature, it can also be used to refer to each corresponding part of single guide RNA in which the tracrRNA and crRNA are connected by a linker. Unless otherwise stated, when only tracrRNA and crRNA are described, it means tracrRNA and crRNA constituting the CRISPR/Cas12f1 system.

transactivating CRISPR RNA (tracrRNA)

The term "tracrRNA" according to the present invention is an important component constituting the guide RNA of the CRISPR/Cas system together with crRNA. In this specification, tracrRNA refers to tracrRNA constituting the guide RNA of the CRISPR/Cas12f1 system, unless otherwise specified. At least a portion of the tracrRNA may complementarily bind with at least a portion of the crRNA to form a double strand. More specifically, some sequences of the tracrRNA have a sequence complementary to all or part of the CRISPR RNA repeat sequence included in the crRNA.

CRISPR RNA (crRNA)

The term "CRISPR RNA (crRNA)" according to the present invention is an important component constituting the guide RNA in the CRISPR/Cas system according to the present invention. The crRNA includes a CRISPR RNA repeat sequence that binds to tracrRNA and a spacer sequence that recognizes a target gene sequence. That is, the crRNA containing the spacer sequence is part of a guide RNA that binds to a target gene or target nucleic acid.

Dual guide RNA (dual guide RNA, dual gRNA)

The term "dual guide RNA" according to the present invention means that tracrRNA and crRNA form separate RNA molecules. A portion of the tracrRNA and a portion of the crRNA complementarily combine to form a double strand. In the dual guide RNA, a portion including the 3' end of the tracrRNA and a portion including the CRISPR RNA repeat sequence of the crRNA may form a double strand.

Single guide RNA (sgRNA)

The term "single guide RNA (sgRNA)" according to the present invention refers to a single-stranded guide RNA molecule in which the tracrRNA and the crRNA are connected by a linker so that the guide RNA is easily formed.

The guide RNA according to the present invention may be a single guide RNA, and in this case, the single guide RNA is one molecule of RNA including both the tracrRNA and the crRNA including the spacer sequence.

The engineered guide RNA (engineered sgRNA) provided herein may be a single guide RNA (sgRNA). The engineered guide RNA (engineered sgRNA) may be one molecule of RNA including all of the engineered tracrRNA, engineered crRNA and U-rich tail according to the present invention.

linker

The term "linker" according to the present invention means a linker connecting two molecules or components in the present invention. For example, the engineered Cas12f1 guide RNA may be a single guide RNA of one molecule, wherein the 3'-end of the fourth region of tracrRNA and the 5'-end of the fifth region of crRNA may be connected through a linker.

In addition, in the miniaturized cleaving structure according to the present invention, an adenine deaminase protein or a cytosine deaminase protein may be linked to a TnpB-derived low-molecular-weight nucleolytic protein or a functional analog thereof by a linker. The linker may be 3 or more RNA or DNA, and may be 4 or more amino acids. For example, the linker is 5'-GAAA-3' or 5'-SGGSSGGSSGSETPGTSESATPESSGGSSGGS-3' (SEQ ID NO: 62), 5'-SGGSKRTADGSEFE-3' (SEQ ID NO: 63), 5'-EASSPKKRKVEAS-3' (SEQ ID NO: 62) SEQ ID NO: 64), SEQ ID NO: 132 (GGGGS)n, (G)n, SEQ ID NO: 133 (EAAAK)n, (GGS)n, SEQ ID NO: 134 SGSETPGTSESATPES, SEQ ID NO: 135 SGGS, (XP)n or any combination thereof It may contain.

approximately

In accordance with the present invention, the term "about" means 30, 25, 20, 15, 10, 9, 8, 7, 6 relative to a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight or length. , the amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by as much as 5, 4, 3, 2 or 1%.

operably linked

The term "operably linked" according to the present invention means that in gene expression technology, a specific component is linked to another component so that the specific component can express or function in an intended manner. For example, when a promoter sequence is said to be operably linked to a coding sequence, it means that the promoter is linked to affect transcription and/or expression of the coding sequence in a cell.

Vector

Unless otherwise specified, the term "vector" according to the present invention refers collectively to all materials capable of delivering genetic material into cells. For example, the vector may be a target genetic material, for example, a DNA molecule including a nucleic acid encoding an effector protein and/or a nucleic acid encoding a guide RNA of the microscopy system according to the present invention, but is limited thereto. it is not going to be

Nuclear Localization Signal (NLS) sequence

The term "Nuclear Localization Signal (NLS) sequence" according to the present invention refers to a kind of "tag" attached to a protein to be transported when a material outside the cell nucleus is transported into the nucleus by nuclear transport. )" means a peptide or its sequence of a certain length that plays a role. For example, for application of the miniaturized base editing construct or system comprising the same according to the present invention in eukaryotic cells, CRISPR/nucleolytic proteins and/or adenosine deaminase or cytidine deaminase are preferably NLS is tagged.

Specifically, the NLS is the NLS of the SV40 virus large T-antigen having the amino acid sequence PKKKRKV (SEQ ID NO: 65); Nucleoplasmin bipartite NLS with KRPAATKKAGQAKKKK (SEQ ID NO: 66) as the NLS sequence from nucleoplasmin; It may be a c-myc NLS with the amino acid sequence PAAKRVKLD (SEQ ID NO: 67) or RQRRNELKRSP (SEQ ID NO: 68). Also hRNPA1 M9 NLS sequence; NLS sequence of IBB domain from importin-alpha, NLS sequence of myoma T protein and NLS sequence of human p53, NLS sequence of mouse c-abl IV; NLS derived from the NLS sequence of influenza virus NS1, the NLS sequence of hepatitis virus delta antigen, the NLS sequence of mouse Mx1 protein, the NLS sequence of human poly(ADP-ribose) polymerase or the NLS sequence of steroid hormone receptor (human) glucocorticoid It may be a sequence, but is not limited thereto.

Nuclear Export Sequence (NES) sequences

The term "NES (Nuclear Export Signal) sequence" according to the present invention acts as a kind of "tag" attached to a protein to be transported when a substance inside the cell nucleus is transported to the outside of the nucleus by nuclear transport. It refers to a peptide of a certain length or a nucleic acid sequence encoding the same.

Tag

The term "tag" according to the present invention collectively refers to functional domains added to easily track and/or separate and purify peptides or proteins. Specifically, the tag is a tag protein such as histidine (His) tag, V5 tag, FLAG tag, influenza hemagglutinin (HA) tag, Myc tag, VSV-G tag and thioredoxin (Trx) tag, green fluorescent protein (GFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), blue fluorescent protein (BFP), autofluorescent proteins such as HcRED and DsRed, and glutathione-S-transferase (GST), horseradish ( horseradish) peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta-galactosidase, beta-glucuronidase, luciferase, and the like reporter genes.

found in nature

The term "found in nature" according to the present invention means an unmodified object found in nature, and is used to distinguish it from an "engineered object" artificially modified. The gene, nucleic acid, DNA, RNA, etc. "found in nature" is used as a concept encompassing both wild-type and mature form (active form) genes, nucleic acids, DNA, and RNA.

engineered

The term "engineered" according to the present invention is a term used to distinguish materials, molecules, etc. having a configuration that already exists in nature, and means that artificial transformation has been applied to the materials, molecules, etc. For example, in the case of "engineered guide RNA", it means a guide RNA in which artificial changes have been made to the structure of guide RNA existing in nature.

A, T, C, G, and U

The terms "T, C, G, and U" according to the present invention may be appropriately interpreted as a base, nucleoside, or nucleotide in DNA or RNA, depending on context and technology.

For example, when meaning a base, adenine (A), thymine (T), cytosine (C), guanine (G), or uracil (U) per se, respectively. can be interpreted, and when meaning a nucleoside, adenosine (A), thymidine (T), cytidine (C), guanosine (G) or uridine, respectively (uridine; U). When referring to a nucleotide in a nucleic acid sequence, it should be interpreted as meaning a nucleotide including each of the above nucleosides.

All technical terms used in the present invention, unless otherwise defined, include all meanings that can be recognized by a person skilled in the art, are used with the same meaning as commonly understood, and are appropriately interpreted according to the context. It can be.

In addition, although preferred methods or samples are described in this specification, those similar or equivalent thereto are also included in the scope of the present invention.

[Subminiature Base Editing System for Base Editing]

The present invention provides a hypercompact base editing system that exhibits excellent activity in base editing and has a remarkably small size of effector protein compared to the CRISPR/Cas9 system.

This feature is due to the size of most of the previously studied Cas endonucleases and the existing adenine base editing gene scissors (ABEs) or cytosine base editing gene scissors (CBEs) including them, FDA approval as an intracellular delivery medium. This is an important factor in addressing the limitations of loading into received adeno-associated virus (AAV) vectors.

Furthermore, since the miniaturized base editing system specifically corrects a specific base of a target nucleic acid or target gene and has high editing specificity and editing efficiency, the miniaturized base editing system according to the present invention can be used as a therapeutic agent for gene-related diseases. The scope of application is wide.

Hereinafter, each component of the hypercompact base editing systems provided by the present invention will be described in detail.

1. Cas12f1 protein

Various types of CRISPR/Cas proteins exist in nature, and new CRISPR/Cas systems are still being discovered. Among them, the nucleolytic protein included in the subminiature base editing system according to the present invention may include Cas12f1 protein belonging to the V-F subtype of Class 2, type V CRISPR/nucleolytic proteins.

The Cas12f1 protein forms a dimer structure, which can be largely divided into a REC lobe and a nuclease lobe (NUC lobe). The REC lobe is composed of the WED domain, ZF domain, and REC domain of one Cas12f1 protein constituting a dimer, and the WED domain, ZF domain, and REC domain of another Cas12f1 protein.

The nuclease lobe is composed of the RuvC domain and TNB domain of one Cas12f1 protein and the RuvC domain and TNB domain of another Cas12f1 protein constituting a dimer. All or part of each domain of the Cas12f1 protein recognizes a specific part of the scaffold region of the Cas12f1 guide RNA, respectively. It is divided into an amino-terminal domain (NTD) and a carboxy-terminal domain (CTD), and has a structure in which the two domains are connected through a linker loop. The NTD is composed of wedge (WED), recognition (REC), and zinc finger (ZF) domains, and the CTD is composed of another ZF domain and a RuvC domain.

In particular, the present invention is characterized by using the Cas12f1 protein, which has a small size, rather than the Cas9 protein, which has been extensively studied. The Cas12f1 protein belongs to the group with the smallest molecular weight among existing nucleolytic proteins and has an excellent effect of targeting and editing a target nucleic acid or target gene by forming a complex with an engineered short guide RNA. Hypercompact base editing systems) have great advantages.

In addition, the small size of the Cas12f1 protein can greatly relieve limitations in the production of fusion protein constructs with adenosine deaminase or cytidine deaminase for base editing.

In addition, since the Cas12f1 protein has 5'-TTTTA-3' or 5'-TTTTG-3' as PAM, unlike Cas9 having 5'-NGG-3' as PAM, it targets sequences with a lot of thymine (T). By allowing selection of nucleic acids or target genes, the selection of nucleolytic proteins for genome editing is broadened.

In one embodiment, the Cas12f1 protein may be a wild-type Cas12f1 protein. In one embodiment, the Cas12f1 protein is an effector protein named Cas14 in a previous study (Harrington et al., Programmed DNA destruction by Hypercompact CRISPR-Cas14 enzymes, Science 362, 839-842 (2018)), It may be from the Cas14 family, which includes Cas14a, Cas14b, and Cas14c variants.

In one embodiment, the nucleolytic protein of the miniaturized gene editing system according to the present invention may be Cas14a1 (identical to Cas12f1) protein derived from uncultured archaeon. In addition, it may be a Cas12f1 protein variant in which one or more or 28 or less amino acids are additionally added to the N-terminus of the Cas14a1 protein.

For example, it may be a transposase accessory protein TnpB protein of the IS200/IS605 family, and more preferably, it may be a protein comprising any one of the amino acid sequences of SEQ ID NOs: 7 to 10.

In addition, the Cas12f1 protein included in the hypercompact base editing systems according to the present invention may have the same function as the wild-type Cas12f1 protein or may have a modified function compared to the wild-type Cas12f1 protein. More specifically, the alteration includes modification of all or part function, loss of all or part function, and/or addition of additional function. The Cas12f1 protein is not particularly limited as long as it is a modification that a person skilled in the art can apply to the nucleolytic protein of the CRISPR/Cas system.

In one embodiment, the Cas12f1 protein may be modified to cleave only one of the double strands of the target nucleic acid. It includes those modified to allow base editing or prime editing for strands that are not cut.

More preferably, since the hypercompact base editing systems according to the present invention perform base editing rather than the activity of cutting DNA double strands, the Cas12f1 protein included in the hypercompact base editing system according to the present invention is the target. A dead form or a nick form variant, in other words, dCas12f1 or nCas12f1, which is modified so that all double strands of the nucleic acid or target gene cannot be cut, is used.

In one embodiment, the dead form variant dCas12f1 may be one in which Asp (D) at position 326 in the Cas12f1 protein having the amino acid sequence of SEQ ID NO: 1 is substituted with Ala (A). In addition, in the Cas12f1 protein comprising the amino acid sequence of SEQ ID NO: 1, the 422nd Glu (E) is Ala (A), the 490th Arg (R) is Ala (A), and the 510th Asp (D) is Ala ( A) may be substituted with each. Specifically, the dead Cas12f1 protein includes any one amino acid sequence selected from SEQ ID NO: 3 to SEQ ID NO: 6. Preferably, the dead Cas12f1 protein is composed of any one amino acid selected from SEQ ID NO: 3 to SEQ ID NO: 6.

In addition, in one embodiment, the Cas12f1 protein is a functional analog of Cas12f1 and includes a dead form variant of a low molecular weight nucleolytic protein derived from TnpB. In the protein consisting of any one amino acid sequence selected from SEQ ID NO: 7 to SEQ ID NO: 10, the 326th Asp (D) in the Cas12f1 protein containing the amino acid sequence of SEQ ID NO: 1 is Ala (A), and the 422nd Glu(E) is replaced with Ala(A), Arg(R) at position 490 is replaced with Ala(A), and Asp(D) at position 510 is replaced with Ala(A), respectively.

In one embodiment, the dead form of TnpB-derived nucleolytic protein having a low molecular weight may be a protein comprising any one amino acid sequence selected from SEQ ID NO: 11 to SEQ ID NO: 18. Preferably, the dead form of TnpB-derived nucleolytic protein having a small molecular weight is a protein composed of any one amino acid sequence selected from SEQ ID NOs: 11 to 18.

In one embodiment, since the hypercompact base editing systems of the present invention modify adenine or cytosine to other bases at a target locus of a target nucleic acid or target gene, the target locus is located in the nucleus of a cell. to be characterized Accordingly, the nucleolytic protein and/or deaminase protein used in the present invention includes one or two or more nuclear localization signal sequences (NLS) that localize the protein into the nucleus.

In one example, one or more NLSs are sufficient to direct a nucleolytic protein to its target into the nucleus in an amount detectable in the nucleus of a eukaryotic cell. The strength of its activity can be derived from the number of NLSs in the nucleolytic protein, the specific NLS(s) used, or a combination of these factors.

In one embodiment, the protein has at or near the amino-terminus about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLS, at or near the carboxy-terminus about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLSs, or combinations thereof (e.g., zero or at least one NLS at the amino-terminus or zero or one or more NLSs at the carboxy-terminus) do. When more than one NLS is present, each can be selected independently of the others, such that a single NLS can be present in more than one copy and/or present in combination with more than one other NLS present in more than one copy. .

In one embodiment, the NLS used in connection with the present invention is heterologous to the protein, exemplified by, but not limited to, the following NLS.

In one embodiment, the NLS is the NLS of the SV40 virus large T-antigen having the amino acid sequence PKKKRKV (SEQ ID NO: 65); Nucleoplasmin bipartite NLS with KRPAATKKAGQAKKKK (SEQ ID NO: 66) as the NLS sequence from nucleoplasmin; It may be a c-myc NLS with the amino acid sequence PAAKRVKLD (SEQ ID NO: 67) or RQRRNELKRSP (SEQ ID NO: 68). Also hRNPA1 M9 NLS sequence; NLS sequence of IBB domain from importin-alpha; NLS sequence of myoma T protein; NLS sequence of human p53; NLS sequence of mouse c-abl IV; NLS sequence of influenza virus NS1; NLS sequence of hepatitis virus delta antigen; NLS sequence of mouse Mx1 protein; NLS sequence of human poly(ADP-ribose) polymerase; or the NLS sequence of a steroid hormone receptor (human) glucocorticoid;

In one embodiment, the subminiature base editing system provided by the present invention may include a subminiature base editing construct in which a deaminase is coupled to Cas12f1 protein. In this case, the subminiature base editing construct may be a fusion of adenosine deaminase and/or cytidine deaminase to wild-type or modified Cas12f1 protein. Here, one or two or more nuclear localization signal sequences (NLS) may be included at the 3'-end of the fusion protein. You can also include any tags you need.

For example, the adenosine deaminase is E. coli-derived tRNA adenosine deaminase (TadA), monomeric TadA or eTadA, heterodimer TadA-eTadA structure or eTadA-TadA may be a rescue. The adenosine deaminase may be connected by a linker. In addition, the cytidine deaminase (cytidine deaminase) is APOBEC1, APOBEC3A, APOBEC3B, CAD, AID or PmCDA1, and the APOBEC1, APOBEC3A, APOBEC3B, CAD, AID or PmCDA1 is attached to the N-terminus or C-terminus of UGI. (Uracil Glycosylase Inhibitor) may be combined with one or two or more, respectively.

As another example, in the miniaturized base editing system according to the present invention, one or more UGIs (Uracil Glycosylase Inhibitors) are included at the N-terminus or C-terminus of the deaminase or the nucleolytic protein, respectively. And, where the deaminase may include cytidine deaminase.

Exemplary modules of hypercompact base editing constructs in which deaminase is coupled to Cas12f1 protein according to the present invention are specifically illustrated in FIG. 1 .

Additionally, a fusion protein in which a deaminase is bound to the Cas12f1 protein or a functional analogue thereof includes a fusion of various enzymes that may be involved in gene expression in cells. At this time, the Cas12f1 protein to which the enzyme is fused can cause various quantitative and qualitative changes in gene expression in cells.

In one embodiment, the various enzymes to be additionally coupled may be DNMT, TET, KRAB, DHAC, LSD, p300, Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase or variants thereof. At this time, the Cas12f1 protein to which the reverse transcriptase is fused or a functional analog thereof can also function as a prime editor.

2. Cas12f1 protein - PAM sequence

In order for the base editing system according to the present invention to be located at the target locus of a target nucleic acid or target gene and perform base editing accurately, the following two conditions are required. First, there must be a nucleotide sequence of a certain length that can be recognized by the Cas12f1 protein in the target nucleic acid or target gene. In addition, there must be a sequence capable of complementary binding with the spacer sequence included in the guide RNA (gRNA) around the nucleotide sequence of the predetermined length.

In other words, when the Cas12f1 protein recognizes the nucleotide sequence of the predetermined length and the spacer sequence portion complementarily binds to the sequence portion around the nucleotide sequence of the predetermined length, the target base of the target nucleic acid or target gene is accurately corrected. can do. At this time, the nucleotide sequence of a certain length recognized by the Cas12f1 protein is referred to as a protospacer adjacent motif (PAM) sequence. The PAM sequence is a unique sequence determined according to the Cas12f1 protein, and when determining the target sequence of the CRISPR/Cas12f1 complex, there is a constraint that the target sequence must be determined within a sequence adjacent to the PAM sequence.

The PAM sequence of the Cas12f1 protein according to the present invention may be a T-rich sequence. More specifically, the PAM sequence of the Cas12f1 protein may be TTTN in order from the 5'-end to the 3'-end. In this case, N is one of deoxythymidine (T), deoxyadenosine (A), deoxycytidine (C), or deoxyguanosine (G).

In one embodiment, the PAM sequence of the Cas12f1 protein may be TTTA, TTTT, TTTC or TTTG in order from 5'-end to 3'-end. More preferably, the PAM sequence of the Cas12f1 protein may be TTTA or TTTG in order from 5'-end to 3'-end.

Also, in one embodiment, the PAM sequence of the Cas12f1 protein may be different from the PAM sequence of the wild-type Cas12f1 protein.

3. Engineered guide RNAs for ultra-small base editing systems

The present invention was derived to overcome the size-dependent limitations of Cas9 of the prior art.

Therefore, in addition to selecting Cas12f1 protein or TnpB-derived protein with a small molecular weight as the nucleolytic protein included in the base editing system of the present invention, the present inventors have investigated the Cas12f1 protein or TnpB-derived protein. Guide RNAs have been artificially engineered to be much shorter than those found in nature.

The engineered guide RNA of the Cas12f1 system is characterized by adding a new structure to the guide RNA found in nature and also modifying some of its structure, and including a new structure, a U-rich tail, at the 3'-end. Specifically, the engineered guide RNAs include an engineering tracrRNA sequence including modified scaffold first to fourth regions, an engineering crRNA sequence including modified scaffold fifth to sixth regions, and a modified seventh region U -Include rich tail sequences.

The fourth region and the fifth region are sites complementary to each other, and include modification site 1 (MS1) and modification site 4 (MS4), and the seventh region, U- The rich tail sequence corresponds to modification site 2 (MS2). The first region corresponds to modification site 3 (MS3), and the second region corresponds to modification site 5 (MS5). The present invention includes modifications in any one of MSs 1 to 5 above, and numerous combinations of modifications selected from among them (FIG. 5).

In one embodiment, the present inventors engineered to remove unnecessary scaffold sequences to optimize the length of tracrRNA and crRNA constituting the guide RNA. Manipulation in the scaffold sequence made it possible to produce a shortened guide RNA, and as a result, it was possible to reduce the guide RNA synthesis cost and secure additional loading space when inserted into a viral vector.

Above all, the base editing system using the optimized guide RNA increased the efficiency of editing or modifying the target nucleic acid or target gene, and could be more effective in the application of therapeutic agents using adeno-associated virus (AAV) vectors. there is.

또한, 자연에 존재하는 Cas12f1 단백질에 대한 야생형 가이드 RNA(gRNA)는 이를 구성하는 야생형 tracrRNA (서열번호 69, 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUUUCCUCUCCAAUUCUGCACAA-3')의 내부에 연속된 다섯 개의 유리딘(U) 서열을 포함 are doing This has a problem with the nucleotide sequence that acts as a transcription termination signal under specific conditions when wild-type tracrRNA is to be expressed in a cell using a vector or the like.

The action of the five contiguous uridine (U) sequences as termination signals inhibits the normal expression of tracrRNA and the formation of normal guide RNA, resulting in the formation of the base editing system of the present invention and This may reduce the editing or modification efficiency of the target nucleic acid or target gene.

Accordingly, the present inventors developed an engineered tracrRNA by artificially modifying the five contiguous uridine sequences of wild-type tracrRNA.

In addition, the engineered guide RNA according to the present invention is a guide RNA found in nature by adding a new structure and modifying some of its structure, and it is characterized by including a new structure, U-rich tail, at the 3'-end do. The engineered guide RNA containing the U-rich tail serves to increase the base editing rate for the target nucleic acid or target gene of the base editing system.

Among the engineered guide RNAs, the present inventors found that optimal guide RNAs to increase the editing or modification efficiency of target nucleic acids or target genes by forming complexes with Cas12f1 protein or TnpB-derived nucleolytic proteins was selected, and a hypercompact base editing system including it was completed.

The engineered guide RNA (engineered gRNA) according to the present invention is characterized in that at least a part of the scaffold region serving to interact with the Cas12f1 protein is modified. The scaffold region includes tracrRNA and parts of crRNA, and does not necessarily refer to one molecule of RNA.

The engineered guide RNA sequence may be an engineered tracrRNA sequence comprising modified scaffold first to fourth regions and/or an engineered crRNA sequence comprising modified scaffold fifth to sixth regions, and / or a modified seventh region, a U-rich tail sequence. In addition, the engineered guide RNA may further include a linker or tag, if necessary.

In other words, the engineered scaffold region included in the engineered guide RNA (engineered gRNA) provided by the present invention is modified in any one or more regions of the first to seventh regions described above in the scaffold region found in nature. This may be a combination.

In addition, the engineered crRNA may include a fifth region (including MS1 modification), a sixth region, and a guide sequence in order from the 5'-end to the 3'-end. Here, the fifth region may be a 5'-GUUGCAGAACCCGAAUAGNBNNNUGAAGGA-3' (SEQ ID NO: 31) sequence or a partial sequence of SEQ ID NO: 31 sequence. Each N may independently be A, C, G or U. The B may be U, C or G. Part of the sequence of SEQ ID NO: 31 may include a sequence of 5'-NBNNNUGAAGGA-3' (SEQ ID NO: 32) of the sequence of SEQ ID NO: 31, but may not include a partial sequence at the 3'-end. Each N may independently be A, C, G or U. The B may be U, C or G.

The engineered guide RNA may be a dual guide RNA or a single guide RNA. When the engineered guide RNA is a single guide RNA, the engineered guide RNA may further include a linker sequence. In this case, the linker sequence may be located between the engineered tracrRNA and the crRNA.

일 구체예로, 상기 엔지니어링된 tracrRNA(engineered trRNA)는 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCANNNCNCCUCUCCAAUUCUGCACAA-3' (서열번호 34)인 염기서열, 5'-ACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCANNNCNCCUCUCCAAUUCUGCACAA-3 ' (서열번호 35)인 염기서열, 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAUUAGUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCANNNCNCCUCUCCAAUUCUGCACAA -3' (SEQ ID NO: 36) or 5'-ACCGCUUCACCAAUUAGUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCANNNCNCCUCUCCAAUUCUGCACAA-3' (SEQ ID NO: 37). In addition, the engineered crRNA (engineered crRNA) may include a 5'-GUUGCAGAACCCGAAUAGNGNNNUGAAGGAAUGCAAC-3' (SEQ ID NO: 38) sequence and a guide sequence. In this case, each N may be independently A, C, G or U. Preferably, 5'-NNNCN-3' in any one of SEQ ID NOs: 34 to 37 is 5'-GUGCU-3', and 5'-NGNNN-3' in SEQ ID NO: 38 is 5'-AGCAA- 3'.

In one embodiment, the engineered tracrRNA may consist of the nucleotide sequence of any one of SEQ ID NOs: 39 to 42, and the engineered crRNA may consist of the nucleotide sequence of SEQ ID NO: 43.

다른 일 예로, 상기 엔지니어링된 tracrRNA(engineered trRNA)는 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCANNNCNCCUCUC-3' (서열번호 44)인 염기서열, 5'-ACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCANNNCNCCUCUC-3' (서열번호 45)인 서열, 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAUUAGUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCANNNCNCCUCUC-3 ' (SEQ ID NO: 46) or 5'-ACCGCUUCACCAAUUAGUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCANNNCNCCUCUC-3' (SEQ ID NO: 47). In this case, the engineered crRNA (engineered crRNA) is 5'-GAAUAGNGNNUAACGAAGGA3' (SEQ ID NO: 48) and a guide sequence. Here, each N may independently be A, C, G or U.

For example, in any one of SEQ ID NOs: 44 to 47, 5'-NNNCN-3' is 5'-GUGCU-3', and 5'-NGNNN-3' in SEQ ID NO: 48 is 5'-AGCAA- 3'.

In one embodiment, the engineered tracrRNA may consist of any one of the nucleotide sequences of SEQ ID NOs: 49 to 52, and the engineered crRNA may consist of the nucleotide sequence of SEQ ID NO: 53.

In one embodiment, sgRNACas12f_ge3.0 (SEQ ID NO: 57) with modifications at MS1/MS2/MS3, sgRNA Cas12f_ge4.0 (SEQ ID NO: 58) with modifications at MS2/MS3/MS4 and/or MS2/MS3/MS4/ It may be the sgRNA Cas12f_ge4.1 (SEQ ID NO: 59) with a modification at MS5.

An exemplary structure of the engineered guide RNA according to the present invention is shown in FIG. 7 .

4. Scaffold area

When functionally dividing the sequence of the engineered guide RNA provided by the present invention, a sequence portion that interacts with the Cas12f1 protein to form a CRISPR/Cas12f1 complex, and a sequence portion that allows the CRISPR/Cas12f1 complex to find a target nucleic acid and U-rich tail sequence parts. At this time, a sequence portion that interacts with the Cas12f1 protein to form a CRISPR/Cas12f1 complex may be referred to as a scaffold sequence. Specifically, the scaffold sequence may include a sequence of two or more molecules of RNA.

In one embodiment, when the engineered guide RNA is a dual guide RNA, the scaffold sequence may include a tracrRNA sequence among the engineered guide RNA sequences and a CRISPR RNA repeat sequence included in the crRNA. In one embodiment, the tracrRNA sequence may be all or part of a tracrRNA sequence found in nature modified. In one embodiment, the CRISPR RNA repeat sequence may be a modified version of all or part of the CRISPR RNA repeat sequence found in nature.

In addition, in the present invention, when the engineered guide RNA is a single guide RNA, the scaffold sequence may include the tracrRNA sequence, the linker sequence, and the CRISPR RNA repeat sequence included in the crRNA sequence. In one embodiment, the tracrRNA sequence may be all or part of a tracrRNA sequence found in nature modified.

Also, in one embodiment, the scaffold region according to the present invention includes tracrRNA and part of crRNA, and does not necessarily refer to one molecule of RNA. The scaffold area may be further subdivided into a first area, a second area, a third area, a fourth area, a fifth area, and a sixth area. When the subdivided regions are described on tracrRNA and crRNA, the first to fourth regions are included in tracrRNA, and the fifth to sixth regions are included in crRNA, specifically crRNA repeat sequence portions.

In one embodiment, the tracrRNA includes a first region, a second region, a third region, and a fourth region. In one embodiment, the tracrRNA has a first region, a second region, a third region, and a fourth region sequentially linked from the 5'-end to the 3'-end.

일 구체예로, 엔지니어링된 tracrRNA(engineered trRNA)는 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUU GCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUU-3' (서열번호 70)인 염기서열 또는 5'-CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCANNNNNCCUCUCCAAUUCUGCACAA-3' (서열번호 71)일 수 있다. Here, N may be A, C, G or U.

Also, in one embodiment, the engineered crRNA sequence includes a crRNA repeat sequence and a spacer sequence. In this case, the crRNA repeat sequence may be 5'-GAAUGAAGGAAUGCAAC-3' (SEQ ID NO: 72) or 5'-GGAAUGCAAC-3' (SEQ ID NO: 73). The crRNA repeat sequence includes a fifth region and a sixth region. The spacer sequence may vary depending on the target sequence, and generally includes 10 to 50 nucleotides.

For example, in the crRNA, a fifth region, a sixth region, and a spacer are sequentially connected from the 5'-end to the 3'-end.

In one embodiment, the engineered scaffold region is different from the scaffold region of a guide RNA found in nature, wherein a portion of the scaffold region is modified.

In one embodiment, the engineered scaffold region may be obtained by removing some of guide RNA scaffold regions found in nature. The engineered scaffold region may be obtained by removing one or more nucleotides included in the scaffold region of guide RNA found in nature. The scaffold region is a region containing parts of tracrRNA and crRNA, and functions to interact with the Cas12f1 protein.

In addition, the engineered guide RNA includes a U-rich tail sequence at the 3'-end of the crRNA sequence. However, at this time it is modified to not include more than five uridine (U) sequences of the contiguous MS 1 region.

The first region of the scaffold is a region including the 5'-end of tracrRNA, and the first region may include nucleotides forming a stem structure in the CRISPR/Cas12f1 complex and may include nucleotides adjacent thereto. The first region includes a region that does not interact with the Cas12f1 protein in the CRISPR/Cas12f1 complex.

In one embodiment, the first region may mean from the 1st nucleotide to the 21st nucleotide from the 5'-terminus of the wild-type tracrRNA comprising the nucleotide sequence of SEQ ID NO: 69.

For example, the nucleotide sequence of the first region may be 5'-CUUCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 24).

The scaffold second region refers to a region located in the 3'-end direction of the first region in tracrRNA. The second region may include nucleotides forming a Stem structure in the CRISPR/Cas12f1 complex and may include nucleotides adjacent thereto. At this time, the stem structure is different from the stem included in the first region. The second region includes Stem 2 part (Takeda et al., Structure of the miniature type V-F CRISPR-Cas effector enzyme, Molecular Cell 81, 1-13 (2021)).

The second region may include one or more nucleotides adjacent to the Stem 2 portion. The second region may include one or more nucleotides interacting with the RuvC domain of one Cas12f1 protein forming a dimer and/or the RuvC domain of another Cas12f1 protein forming a dimer in the CRISPR/Cas12f1 complex. The second region includes a region that does not interact with the Cas12f1 protein in the CRISPR/Cas12f1 complex.

In one embodiment, the second region may mean from the 22nd nucleotide to the 71st nucleotide from the 5'-end of the wild-type tracrRNA comprising the nucleotide sequence of SEQ ID NO: 69.

In one embodiment, the nucleotide sequence of the second region may be 5'-CCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUG-3' (SEQ ID NO: 25).

The scaffold third region refers to a region located in the 3'-end direction of the second region in tracrRNA. The third region may include nucleotides forming a complementary bond with nucleotides forming the Stem structure in the CRISPR/Cas12f1 complex and some nucleotides included in crRNA, and may include nucleotides adjacent thereto.

In one embodiment, the third region may mean from the 72nd nucleotide to the 129th nucleotide from the 5'-end of the wild-type tracrRNA comprising the nucleotide sequence of SEQ ID NO: 69.

For example, the nucleotide sequence of the third region may be 5'-GGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAA-3' (SEQ ID NO: 26).

The scaffold fourth region refers to a region located in the 3'-end direction of the third region of tracrRNA. The fourth region may include nucleotides capable of forming complementary bonds with some nucleotides included in crRNA in the CRISPR/Cas12f1 complex, and may include nucleotides adjacent thereto.

The fourth region includes nucleotides belonging to tracrRNA in Stem 5 (R: AR-2) (Takeda et al., Structure of the miniature type V-F CRISPR-Cas effector enzyme, Molecular Cell 81, 1-13 (2021) ). The fourth region may include one or more nucleotides adjacent to nucleotides belonging to tracrRNA in Stem 5 (R:AR-2).

The fourth region may include one or more nucleotides complementarily binding to one or more nucleotides included in the fifth region of crRNA. The fourth region includes a region that does not interact with the Cas12f1 protein in the CRISPR/Cas12f1 complex.

In one embodiment, the fourth region may mean from the 130th nucleotide to the 142nd nucleotide from the 5'-end of the wild-type tracrRNA comprising the nucleotide sequence of SEQ ID NO: 69.

In one embodiment, the fourth region may mean from the 130th nucleotide to the 162nd nucleotide from the 5'-end of the wild type tracrRNA comprising the nucleotide sequence of SEQ ID NO: 69.

For example, the sequence of the fourth region may be 5'-CAAAUUCANNNVN-3' (SEQ ID NO: 28) or 5'-CAAAUUCAUUUUUCCUCUCCAAUUCUGCACAA-3' (SEQ ID NO: 74). Here, N may be A, C, G or U.

The scaffold region 5 refers to the region containing the 5' end of the crRNA. The fifth region includes a nucleotide forming a complementary bond with one or more nucleotides of the fourth region in the CRISPR/Cas12f1 complex, and may include a nucleotide adjacent thereto.

The fifth region may include one or more nucleotides complementarily binding to one or more nucleotides included in the fourth region. The fifth region includes a region that does not interact with the Cas12f1 protein in the CRISPR/Cas12f1 complex.

In one embodiment, the fifth region may mean from the 1st nucleotide to the 30th nucleotide from the 5'-terminus of the wild-type crRNA comprising the nucleotide sequence of SEQ ID NO: 30.

For example, the sequence of the fifth region may be 5'-GUUGCAGAACCCGAAUAGNBNNNUGAAGGA-3' (SEQ ID NO: 31). Here, N may be A, C, G or U, and B may be U, C, or G. Preferably, it may be 5'-GUUGCAGAACCCGAAUAGACGAAUGAAGGA-3' (SEQ ID NO: 75).

In another embodiment, the fifth region may refer to the 21st nucleotide to the 30th nucleotide from the 5'-end of the wild-type crRNA comprising the nucleotide sequence of SEQ ID NO: 30.

For example, the sequence of the fifth region may be 5'-GAAUGAAGGA-3' (SEQ ID NO: 76).

The sixth region of the scaffold refers to a region located in the 3'-end direction of the fifth region in the crRNA. The sixth region includes a nucleotide forming a complementary bond with one or more nucleotides of the third region in the CRISPR/Cas12f1 complex, and may include a nucleotide adjacent thereto.

In one embodiment, the sixth region may refer to the 31st nucleotide to the 37th nucleotide from the 5'-end of the wild-type crRNA crRNA comprising the nucleotide sequence of SEQ ID NO: 30.

For example, the sequence of the sixth region may be 5'-AUGCAAC-3' (SEQ ID NO: 33).

The present invention also provides an engineered scaffold region that can be introduced to improve gene editing efficiency of the CRISPR/Cas12f1 system. The engineered scaffold region synergizes with the aforementioned U-rich tail to improve gene editing efficiency of the CRISPR/Cas12f1 system using the engineered guide RNA.

In one embodiment, the engineered guide RNA may include a U-rich tail rich in uridine (U) at the 3'-end portion. The U-rich tail sequence basically contains uridine in abundance, and includes a sequence in which one or more uridines are consecutive. The U-rich tail sequence may further include additional bases other than uridine, depending on the actual use environment and expression environment of the engineered CRISPR/Cas12f1 system, for example, the internal environment of eukaryotic cells or prokaryotic cells.

The U-rich tail sequence provided herein more preferably includes one ribonucleoside (A, C, G) other than uridine for every 1 to 5 repetitions of uridine (U). It may contain a modified uridine repeat sequence. The modified uridine contiguous sequence is particularly useful when designing a vector expressing an engineered crRNA.

In one embodiment, the U-rich tail sequence may include a repeating sequence of one or more UV, UUV, UUUV, UUUUV, and/or UUUUUV. At this time, the V is one of adenosine (A), cytidine (C), and guanosine (G). In a specific embodiment, the sequence of the U-rich tail may be expressed as (UaN)bUc. In this case, N is one of A, U, C, or G, a, b, and c are integers, a is 1 or more and 5 or less, b is 0 or more and 2 or less, and c is 1 or more and 10 or less. .

In one embodiment, the sequence of the U-rich tail is 5'-U-3', 5'-UU-3', 5'-UUU-3', 5'-UUUU-3', 5'-UUUUU- 3', 5'-UUUUUU-3', 5'-UUURUUU-3' (SEQ ID NO: 77), 5'-UUURUUURUUU-3' (SEQ ID NO: 78), 5'-UUUURU-3' (SEQ ID NO: 79), 5'-UUUUR UU-3' (SEQ ID NO: 80), 5'-UUUURUUU-3' (SEQ ID NO: 81), 5'-UUUURUUUU-3' (SEQ ID NO: 82), 5'-UUUURUUUUU-3' (SEQ ID NO: 81) 83), or 5'-UUUURUUUUUU-3' (SEQ ID NO: 84). Where R can be A or G.

In another embodiment, the U-rich tail sequence is any one of SEQ ID NOs: 85 to 92, wherein R is A in any one of SEQ ID NOs: 77 to 84. may be made up of In addition, the sequence of the U-rich tail may be one in which R is G in any one of SEQ ID NOs: 77 to 84, and may consist of any one of SEQ ID NOs: 93 to 100. Preferably, the sequence of the U-rich tail is 5'-UUUUAUUUU-3' (SEQ ID NO: 90), 5'-UUUUAUUUUUU-3' (SEQ ID NO: 92), 5'-UUUUGUUUUUU-3' (SEQ ID NO: 100), or 5'-UUUUUU-3' (SEQ ID NO: 101).

The U-rich tail sequence may be a U-rich tail sequence disclosed in the PCT/KR2020/014961 application. Hereinafter, when referring to a U-rich tail sequence in the present specification, it should be understood that the contents and experimental results of the U-rich tail sequence disclosed in the PCT/KR2020/014961 application are included.

5. Spacer Sequence - Relationship to Target Nucleic Acid and Target Sequence

The spacer sequence is a sequence complementary to the target sequence and linked to the 3'-end of the crRNA repeat sequence. The spacer sequence is a sequence homologous to a protospacer sequence adjacent to a PAM (Protospacer Adjacent Motif) sequence recognized by the Cas12f1 protein, and thymidine (T) of the protospacer sequence is converted to uridine (U). have a substituted sequence. At this time, the target sequence and the protospacer sequence are determined within a sequence adjacent to the PAM sequence included in the target nucleic acid, and the spacer sequence is determined accordingly.

In one embodiment, the spacer sequence portion of the crRNA may complementarily bind to the target nucleic acid. In one embodiment, the spacer sequence portion of the crRNA may complementarily bind to the target sequence portion of the target nucleic acid. In one embodiment, when the target nucleic acid is double-stranded DNA, the spacer sequence may be a sequence complementary to the target sequence included in the target strand of the double-stranded DNA. Here, when the target nucleic acid is double-stranded DNA, the spacer sequence may be a sequence homologous to a protospacer sequence included in a non-target strand of the double-stranded DNA.

Specifically, the spacer sequence may have the same nucleotide sequence as the protospacer sequence, but may have a sequence in which each of thymidine (T) included in the nucleotide sequence is substituted with uridine (U). In one embodiment, the spacer sequence may be an RNA sequence corresponding to the DNA sequence of the protospacer.

In one embodiment, the length of the spacer sequence may be 10 nucleotides to 40 nucleotides in length. Preferably, the length of the spacer sequence may be 17 nucleotides to 30 nucleotides in length. More preferably, the length of the spacer sequence may be 17 nucleotides to 23 nucleotides in length.

6. Single Guide RNA or Dual Guide RNA

The engineered guide RNA according to the present invention may be single guide RNA or dual guide RNA. The dual guide RNA means that the guide RNA is composed of two molecules of RNA, tracrRNA and crRNA. The single guide RNA means that the 3'-end of the engineered tracrRNA and the 5'-end of the engineered crRNA are connected through a linker.

In one embodiment, the engineered single guide RNA (sgRNA) further includes a linker sequence, and the tracrRNA sequence and the crRNA sequence may be linked through the linker sequence. Preferably, the 3'-end of the fourth region and the 5'-end of the fifth region included in the engineered scaffold may be connected through a linker. More preferably, the 3'-end of the fourth region and the 5'-end of the fifth region may be linked by a linker 5'-GAAA-3'.

In one embodiment, in the sequence of the single guide RNA, a tracrRNA sequence, a linker sequence, a crRNA sequence, and a U-rich tail sequence are sequentially connected from the 5'-end to the 3'-end. A portion of the tracrRNA sequence and all or a portion of the CRISPR RNA repeat sequence included in the crRNA sequence have sequences complementary to each other. More specifically, the single guide RNA may have a sequence selected from the group consisting of SEQ ID NOs: 55 to 59.

In addition, the engineered gRNA provided in the present invention may be a dual guide RNA in which the tracrRNA and the crRNA form separate RNA molecules. In this case, a part of the tracrRNA and a part of the crRNA may have complementary sequences to form a double-stranded RNA. More specifically, in the dual guide RNA, a portion including the 3'-end of the tracrRNA and a portion including the CRISPR RNA repeat sequence of the crRNA may form a double strand.

The engineered guide RNA can bind to the Cas12f1 protein to form a CRISPR/Cas12f1 complex, recognize a target sequence complementary to a spacer sequence included in the crRNA sequence, and edit a target nucleic acid containing the target sequence. do.

For example, the sequence of the tracrRNA may include a complementary sequence having 0 to 20 mismatches with the CRISPR RNA repeat sequence. Preferably, the tracrRNA sequence may include a complementary sequence having 0 to 8 or 8 to 12 mismatches with the CRISPR RNA repeat sequence.

7. Modifications to make single guide RNAs

The engineered guide RNA provided by the present invention may be a single guide RNA of one molecule. Thus, the engineered scaffold region may be one or more of each region modified, and additionally, the 3'-end of the fourth region of tracrRNA and the 5'-end of the fifth region of crRNA may be connected through a linker.

In one embodiment, the engineered scaffold region is modified at one or more places in the scaffold region found in nature, and the 3'-end of the fourth region and the 5'-end of the fifth region are linked via a linker. may be connected. At this time, the linker may be 5'-GAAA-3'

Further, in one embodiment, the engineered scaffold region includes a region corresponding to each part of a scaffold region found in nature. Specifically, the engineered scaffold region includes a first region, a second region, a third region, a fourth region, a fifth region, and a sixth region, which are included in scaffold regions found in nature. Areas 1 to 6 correspond to each other.

In one embodiment, the engineered scaffold region may not include a region corresponding to the first region and/or the second region among scaffold regions found in nature.

The modification in the engineered scaffold region is described in detail below.

7-1. Deformation in scaffold first region

In one embodiment, the engineered scaffold region included in the engineered Cas12f1 guide RNA may include one or more nucleotides included in the first region among scaffold regions found in nature are removed. More specifically, the removed nucleotide may be a nucleotide included in a portion forming a stem structure in the CRISPR/Cas12f1 complex among the first regions found in nature.

In one embodiment, the removed nucleotide is Stem 1 of the first region found in nature (Takeda et al., Structure of the miniature type V-F CRISPR-Cas effector enzyme, Molecular Cell 81, 1-13 (2021)) It may be a nucleotide belonging to In one embodiment, the removed nucleotide may be a nucleotide that does not interact with the Cas12f1 protein in the CRISPR/Cas12f1 complex in the first region found in nature.

In one embodiment, the modified first region (MS3 region, 1-21 region) may be a 5'-CUUCACUGAUAAAGUGGAGAA-3' (SEQ ID NO: 24) sequence or a partial sequence of SEQ ID NO: 24 sequence.

For example, from the 5'-end to the 3'-end, 5'-A-3', 5'-AA-3', 5'-GAA-3', 5'-AGAA-3', 5' Some sequences of the sequence of SEQ ID NO: 24, such as -GAGAA-3', 5'-GGAGAA-3', 5'-UGGAGAA-3', 5'-GUGG AGAA-3', 5'-AGUGGAGAA-3' The 5'-end of the sequence of SEQ ID NO: 24 is sequentially removed, and the remaining 3'-end may be a sequential partial sequence.

7-2. Deformation in Scaffold Second Region

In another embodiment, the engineered scaffold region may include a modified second region. In this case, the modified second region is one in which one or more nucleotides are removed from the second region of the scaffold region found in nature. In this case, the removed nucleotide is a nucleotide selected from a region forming a stem structure in the CRISPR/Cas12f1 complex.

In one embodiment, the removal of nucleotides may occur in a part forming a stem structure among the second regions found in nature, and nucleotides may be removed in base pair units. In one embodiment, the removed nucleotide may be a nucleotide included in a portion forming a stem structure in the CRISPR/Cas12f1 complex among the second regions found in nature.

In one embodiment, the modified second region (MS5 region, 22-71 region) of the engineered scaffold region is a 5'-CCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUG-3' (SEQ ID NO: 25) sequence or SEQ ID NO: 25 sequence. It may be some sequence. It may be one in which 1 to 50 nucleotides are removed from the second region of the scaffold region found in nature.

Part of the sequence of SEQ ID NO: 25 may be a sequence in which at least one pair of nucleotides forming a complementary bond and/or at least one or more nucleotides not forming a complementary bond in the sequence of SEQ ID NO: 25 are deleted. At this time, the 5'-UUAG-3' sequence of the loop part included in the partial sequence of SEQ ID NO: 25 may be optionally substituted with the 5'-GAAA-3' sequence.

In one embodiment, the modified second region is the second region of the scaffold region found in nature, based on the nucleotide sequence of SEQ ID NO: 25, nucleotides 1 to 22 from the 5'-end and / Alternatively, one or more of the 27th to 50th nucleotides may be removed. In one embodiment, the modified second region is located in the second region of the scaffold region found in nature, based on the sequence of SEQ ID NO: 11, 1st to 22nd nucleotides from the 5'-end, and/or 27th to 27th nucleotides. One or more of the 50th nucleotides may be removed, and the 23rd to 26th nucleotides may be substituted with other ones.

In addition, the engineered scaffold region provided by the present invention may be a scaffold region found in nature in which the second region is removed. In one embodiment, the engineered scaffold region may not have a region corresponding to a second region of a scaffold region found in nature.

In one embodiment, the sequence of the scaffold region from which the second region is removed may be 5'-CUUCACUGAUAAAGUGGAGAAGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUGAAAGAAUGAAGGAAUGCAAC-3' (SEQ ID NO: 102).

7-3. Deformation in scaffold third area

In one embodiment of the present invention, the engineered scaffold region may include a modified third region. In this case, the modified third region is one in which one or more nucleotides are removed from the third region of the scaffold region found in nature. In this case, the removed nucleotide is a nucleotide selected from a region forming a stem structure in the CRISPR/Cas12f1 complex.

In one embodiment, the modified third region of the engineered scaffold region (pre-MS1 region, regions 72-129) is at least 70% 5'-GGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAA-3' (SEQ ID NO: 26) sequence or SEQ ID NO: 26 sequence. It may be a sequence having more than one sequence homology. It may be one in which 1 to 20 nucleotides are removed from the third region of the scaffold region found in nature.

In one embodiment, the modified third region is nucleotides 28 to 37 from the 5'-end and/or 42 based on the nucleotide sequence of SEQ ID NO: 26 in the third region of the scaffold region found in nature. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive nucleotides may be removed from the nucleotide to the 51st.

In another embodiment, the modified third region is the third region of the scaffold region found in nature, nucleotides 27 to 36 from the 5'-end based on the nucleotide sequence of SEQ ID NO: 26, and 42 One or more pairs of nucleotides forming a base pair in the CRISPR/Cas12f1 complex may be removed from among nucleotides at nucleotides 1 to 51.

In another embodiment, the modified third region is the third region of the scaffold region found in nature, based on the nucleotide sequence of SEQ ID NO: 26, nucleotides 27 to 36 from the 5'-end and 42 One or more nucleotide pairs constituting a base pair in the CRISPR/Cas12f1 complex and/or one or more nucleotides not forming a base pair may be removed from the nucleotides to the 51st nucleotides.

For example, the modified third region is characterized by including 5'-GCUGCUUGCAUCAGCCUAAUGUCGAG-3' (SEQ ID NO: 103), 5'-UUCG-3', and 5'-CUCGA-3' sequences.

7-4. Modification in scaffold fourth and/or fifth region

The engineered scaffold region provided herein may be one in which the fourth region and the fifth region are modified from scaffold regions found in nature. Since the fourth region and the fifth region include portions constituting a stem by hybridizing with each other within the CRISPR/Cas12f1 complex, the corresponding portions may be modified together to constitute an engineered scaffold region.

The modified fourth region is characterized in that one or more nucleotides are removed from the fourth region of the scaffold region found in nature. The modified fifth region is characterized in that one or more nucleotides are removed from the fifth region of the scaffold region found in nature.

In one embodiment, the modified fourth region is characterized in that it has a 5'-AACAAA-3' sequence in the 5'-end direction. In addition, the modified fifth region is characterized by having a 5'-GGA-3' sequence in the 3'-end direction.

In another embodiment, the modified fourth region of the engineered scaffold region may be obtained by removing 1 to 7 nucleotides from the fourth region of the scaffold region found in nature. For example, the modified fourth region of the engineered scaffold region may be obtained by removing 1 to 28 nucleotides from the fourth region of the scaffold region found in nature.

In one embodiment, the modified fourth region (MS 1, 130-169 region) may be a 5'-CAAAUUCA NNNVNCCUCUCCAAUUCUGCACAA-3' (SEQ ID NO: 27) sequence or a partial sequence of SEQ ID NO: 27 sequence. Each of N is independently A, C, G or U, and V may be A, C or G. Part of the sequence of SEQ ID NO: 27 may be a sequence that includes the 5'-CAAAUUCANNNVN-3' (SEQ ID NO: 28) sequence of the sequence of SEQ ID NO: 27 and does not include a partial sequence at the 3' end. Preferably, the fourth sequence may be a sequence that includes a 5'-5'-CAAAUUCANNNCN-3' (SEQ ID NO: 29) sequence and does not include a partial sequence at the 3' end. wherein each N is independently A, C, G or U.

The modification in the fourth region may be one or more of the 9th to 15th nucleotides from the 5'-end, based on the nucleotide sequence of SEQ ID NO: 27, in the fourth region of the scaffold region found in nature. there is.

In one embodiment, the modified fourth region is one or more of the 9th to 36th nucleotides from the 5'-end, based on the nucleotide sequence of SEQ ID NO: 27, in the fourth region of the scaffold region found in nature. may have been removed.

In addition, in the present invention, the modified fifth region is one or more of the 1st to 7th nucleotides from the 5'-end, based on the nucleotide sequence of SEQ ID NO: 30, in the fifth region of the scaffold region found in nature. may have been removed.

In one embodiment, the modified fifth region is one or more of nucleotides 1 to 27 from the 5'-end, based on the nucleotide sequence of SEQ ID NO: 30, in the fifth region of the scaffold region found in nature. may have been removed.

In the engineered scaffold region provided herein, the modified fourth and fifth regions are based on the nucleotide sequence of SEQ ID NO: 27 in the fourth and fifth regions of the scaffold region found in nature. , Based on the 9th to 15th nucleotides from the 5'-end and the nucleotide sequence of SEQ ID NO: 30, one or more pairs of nucleotides constituting a base pair in the CRISPR / Cas12f1 complex among the 1st to 7th nucleotides from the 5'-end, and / or One or more nucleotides not forming a base pair may be removed.

In one embodiment, the modified fourth and fifth regions are 9 from the 5'-end, based on the nucleotide sequence of SEQ ID NO: 27, in the fourth and fifth regions of the scaffold region found in nature. One or more pairs of nucleotides forming a base pair in the CRISPR/Cas12f1 complex among nucleotides 1 to 7 from the 5'-end and/or one or more pairs of mismatched nucleotides based on the base sequence of nucleotides 1 to 15 and SEQ ID NO: 30 may have been removed.

In another embodiment, the modified fourth and fifth regions are 9 from the 5'-end based on the nucleotide sequence of SEQ ID NO: 27 in the fourth and fifth regions of the scaffold region found in nature. Based on nucleotides 36 to 36 and nucleotides 1 to 27 based on the nucleotide sequence of SEQ ID NO: 30, one or more pairs of nucleotides forming a base pair in the CRISPR/Cas12f1 complex and/or one or more nucleotides not forming a base pair are removed. it could be

In one embodiment, the sequence of the modified fourth region is 5'-AACAAA-3', 5'-AACAAAU-3', 5'-AACAAAUU-3', 5'-AACAAAUUC-3', 5'-AACAAAUUCA -3' (SEQ ID NO: 104), 5'-AACAA AUUCAU-3' (SEQ ID NO: 105) or 5'-AACAAAUUCAUU-3' (SEQ ID NO: 106). In addition, the sequence of the modified fifth region is 5'-GGA-3', 5'-AGGA-3', 5'-AAGGA-3', 5'-GAAGGA-3', 5'-UGAAGGA-3' , 5'-AUGAAGGA-3' or 5'-AAUGAAGGA-3'.

In another embodiment, the engineered scaffold region in which the fourth region and the fifth region are modified is 5'-AACAAA-3', 5'-AACAAAU-3', 5'-AACAAA-3', 5'-terminal to 3'-terminal direction. '-AACAAAUU-3' and 5'-AACAAAUUC-3', 5'-AACAAAUUCA-3' (SEQ ID NO: 104), 5'-AACAAAUUCAU-3' (SEQ ID NO: 105) and 5'-AACAAAUUCAUU-3' (SEQ ID NO: 104) No. 106) sequences selected from the group are linked; And from the 5'-end to the 3' end, 5'-GGA-3', 5'-AGGA-3', 5'-AAGGA-3', 5'-GAAGGA-3', 5'-UGAAGGA-3 A sequence selected from the group consisting of ', 5'-AUGAAGGA-3' and 5'-AAUGAAGGA-3'; And 5'-AUGCAAC-3' may be a nucleic acid comprising a linked sequence.

7-5. Deformation in scaffold region 6

In the engineered scaffold provided herein, the sixth region is a region including nucleotides belonging to crRNA among PK(R:AR-1) parts. As described above, the sixth region includes one or more nucleotides interacting with the WED domain, ZF domain and/or RuvC domain of one Cas12f1 protein constituting a dimer in the CRISPR/Cas12f1 complex. The sixth region of the engineered scaffold may be the same as the sixth region of the scaffold found in nature, or may be modified within the extent that the function of the sixth region is not damaged.

In one embodiment, the sixth region may be a 5'-AUGCAAC-3' (SEQ ID NO: 33) sequence or a sequence having at least 70% sequence identity or sequence similarity to the 5'-AUGCAAC-3' sequence.

7-6. Modifications in the 7th region of the guide RNA

Modification in the seventh region of the guide RNA according to the present invention includes providing a U-rich tail sequence at the 3'-end of the crRNA to improve gene editing efficiency of the CRISPR/Cas12f1 system. The U-rich tail sequence is basically characterized in that it contains uridine abundantly, and includes a sequence in which one or more uridines are contiguous.

The engineered crRNA according to the present invention may further include a U-rich tail sequence as a seventh region at the 3'-end of the crRNA. The Urich tail sequence may be a 5'-(UaN)dUe-3' sequence, a 5'-UaVUaVUe-3' sequence, or a 5'-UaVUaVUaVUe-3' sequence. The N may be A, C, G or U. Each V may independently be A, C or G. The a may be an integer of 0 to 4. d may be an integer of 0 to 3. The e may be an integer from 0 to 10.

In one embodiment, the U-rich tail sequence may include 1 to 10 uridine repeat sequences. The U-rich tail sequence may further include additional bases other than uridine, depending on the actual use environment and expression environment of the engineered CRISPR/Cas12f1 system, for example, the internal environment of eukaryotic cells or prokaryotic cells.

In one embodiment, the U-rich tail sequence may include a sequence in which one or more of UV, UUV, UUUV and/or UUUUV are repeated. At this time, the V is one of adenosine (A), cytidine (C), and guanosine (G). The U-rich tail sequence is characterized in that it is linked to the 3'-end of the crRNA sequence included in the CRISPR/Cas12f1 system.

The U-rich tail sequence serves to increase the efficiency of cleavage of the target nucleic acid of the engineered CRISPR/Cas12f1 complex provided in the present invention. In this case, the target nucleic acid may be single-stranded DNA, double-stranded DNA and/or RNA.

The term "tail sequence" used herein may mean not only the RNA sequence itself rich in uridine (U), but also the DNA sequence encoding it, which is appropriately interpreted depending on the context. The present inventors experimentally revealed the structure of the U-rich tail sequence and its effect in detail, and will be described in detail with specific embodiments below.

In one embodiment, the U-rich tail sequence may be expressed as Ux. The x may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. For example, the x may be an integer within a range of two values selected in the immediately preceding sentence. For example, x may be an integer between 1 and 6. In addition, x may be an integer between 1 and 20. For example, x may be an integer of 20 or greater.

In one embodiment, the U-rich tail sequence may be expressed as (UaN)nUb. In this case, N is one of adenosine (A), uracil (U), cytidine (C), and guanosine (G). In this case, a is an integer between 1 and 5, and n is an integer greater than or equal to 0. Also, n may be an integer between 0 and 2. The b may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Also, b may be an integer within two numerical ranges selected in the immediately preceding sentence. For example, b may be an integer between 1 and 6.

In one embodiment, the U-rich tail sequence may be expressed as (UaV)nUb. At this time, the V is one of adenosine (A), cytidine (C), and guanosine (G). In this case, a is an integer between 1 and 4, and n is an integer greater than or equal to 0. Also, n may be 1 or 2. b may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. Also, b may be an integer within two numerical ranges selected in the immediately preceding sentence. For example, the b may be an integer between 1 and 6, the b may be an integer between 1 and 20, and the b may be an integer greater than or equal to 20.

In one embodiment, the U-rich tail sequence may be a combination of a sequence represented by Ux and a sequence represented by (UaV)n. For example, the U-rich tail sequence may be expressed as U)n1-V1-(U)n2-V2-Ux. In this case, V1 and V2 are each one of adenine (A), cytidine (C), and guanine (G). In this case, n1 and n2 may each be an integer between 1 and 4. In this case, x may be an integer between 1 and 20.

In one embodiment, the length of the U-rich tail sequence is 1nt, 2nt, 3nt, 4nt, 5nt, 6nt, 7nt, 8nt, 9nt, 10nt, 11nt, 12nt, 13nt, 14nt, 15nt, 16nt, 17nt, 18nt, It may be 19nt or 20nt. In one embodiment, the length of the U-rich tail sequence may be 20 nt or more.

In one embodiment, the sequence of the U-rich tail is 5'-U-3', 5'-UU-3', 5'-UUU-3', 5'-UUUU-3', 5'-UUUUU- 3', 5'-UUUUUU-3', 5'-UUURUUU-3' (SEQ ID NO: 77), 5'-UUURUUURUUU-3' (SEQ ID NO: 78), 5'-UUUURU-3' (SEQ ID NO: 79), 5'-UUUURUU-3' (SEQ ID NO: 80), 5'-UUUURUUU-3' (SEQ ID NO: 81), 5'-UUUURUUUU-3' (SEQ ID NO: 82), 5'-UUUURUUUUU-3' (SEQ ID NO: 83 ), or 5'-UUUURUUUUUU-3' (SEQ ID NO: 84). Where R can be A or G.

Preferably, the sequence of the U-rich tail is one in which R is A in any one of SEQ ID NOs: 77 to 84, and may consist of any one of SEQ ID NOs: 85 to 92. there is.

In addition, the sequence of the U-rich tail is that the R is G in any one of the base sequences of SEQ ID NOs: 77 to 84, and may consist of any one of SEQ ID NOs: 93 to 100. .

Most preferably, the sequence of the U-rich tail is 5'-UUUUAUUUU-3' (SEQ ID NO: 90), 5'-UUUUAUUUUUU-3' (SEQ ID NO: 92), 5'-UUUUGUUUUUU-3' (SEQ ID NO: 100) or 5'-UUUUUU-3' (SEQ ID NO: 101).

8. Additional sequences

The engineered tracrRNA of the present invention may optionally further include an additional sequence. The additional sequence may be located at the 3'-end of the engineered tracrRNA. The additional sequence may be located at the 3'-end of the fourth region. The additional sequence may also be located at the 5'-end of the engineered tracrRNA. The additional sequence may be located at the 5'-end of the first region.

The additional sequence may be from 1 to 40 nucleotides.

In one embodiment, the additional sequence may be any nucleotide sequence or any arrangement of nucleotide sequences. For example, the additional sequence may be the sequence 5'-AUAAAGGUGA-3' (SEQ ID NO: 107).

The additional sequence may be a known nucleotide sequence.

In one embodiment, the additional sequence may be a hammerhead ribozyme nucleotide sequence. Here, the hammerhead ribozyme nucleotide sequence may be a 5'-CUGAUGAGUCCGUGAGGACGAAACGAGUAAGCUCGUC-3' (SEQ ID NO: 108) sequence or a 5'-CUGCUCGAAUGAGCAAGCAGGAGUGCCUGAGUAGUC-3' (SEQ ID NO: 109) sequence. The above example is a simple example, and is not limited thereto.

9. Chemical modification

The engineered tracrRNA or crRNA according to the present invention may optionally have at least one nucleotide chemically modified, if necessary. In this case, the chemical modification may be modification of various covalent bonds that may occur in bases and/or sugars of nucleotides.

For example, the chemical modification is methylation, halogenation, acetylation, phosphorylation, phosphorothioate linkage, locked nucleic acid (LNA), 2'-O-methyl 3'phosphorothioate (MS) or 2'-O-methyl 3'thioPACE (MSP) can be The above example is a simple example, and is not limited thereto.

When the engineered guide RNA according to the present invention is used in the CRISPR/Cas12f1 system, compared to the case of using a guide RNA found in nature, the base editing or modification of target nucleic acid or target gene in cells is dramatically improved. appear.

Above all, the above engineered RNA optimizes the length of guide RNA and thus reduces the cost of guide RNA synthesis, secures additional space (capacity) when inserted into a viral vector, normal expression of tracrRNA, increases expression of guide RNA capable of functioning (operating), guide RNA stability of the guide RNA-Cas12f1 protein complex, induction of effective guide RNA-Cas12f1 protein complex formation, increased cleavage efficiency of target nucleic acids by the CRISPR/Cas12f1 (Cas14a1) system, and target by the CRISPR/Cas12f1 (Cas14a1) system Increased effect of base correction or modification of a nucleic acid.

Accordingly, when the engineered Cas12f1 guide RNA is used, it is possible to edit genes with high efficiency in cells by overcoming the limitations of the prior art.

In addition, the engineered Cas12f1 guide RNA has a shorter length compared to guide RNAs found in nature, and thus has high potential for application in the field of gene editing technology. When the engineered Cas12f1 guide RNA is used, the advantage of the very small size of the CRISPR/Cas12f1 system can be sufficiently utilized for gene editing technology.

[Design of vectors for the expression of a mini base editing system]

In order to use the CRISPR/Cas system for gene editing including base correction, a vector containing a sequence encoding each component of the CRISPR/Cas system is introduced into a target cell, and the expression of the CRISPR/Cas system in the target cell is introduced. Methods that allow each construct to be expressed are widely used.

In addition, in the base editing system of the present invention for base editing, the adenosine deaminase protein or the cytidine deaminase protein is a targeting complex for the target sequence, the CRISPR/Cas complex, and each separate protein. It can be delivered to the cell as a cell or expressed in the cell. Preferably, in order to achieve excellent targeting efficiency, the adenosine deaminase protein or cytidine deaminase protein may be linked to the N-terminus or C-terminus of the CRISPR/Cas complex and included in one vector. Here, the adenosine deaminase protein or the cytidine deaminase protein may be linked to either a nucleolytic protein or a guide molecule to form a fused protein.

Cb5,

Cb8r,

Cb12r,

Cb23r, 7s 및 PRR1등이 포함될 수 있다. 또한, 상기 융합된 형태의 단백질은 하나 이상의 지질 나노입자를 통해 전달될 수 있다.For example, the fused form of the protein may include an orthogonal RNA-binding protein or adapter protein present in a bacteriophage coat protein. Here, the envelope protein is MS2, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205,

Cb5,

Cb8r,

Cb12r,

Cb23r, 7s and PRR1 may be included. In addition, the fused form of the protein can be delivered through one or more lipid nanoparticles.

In addition, in one embodiment, the nucleolytic protein, adenosine deaminase protein or cytidine deaminase protein and/or adapter protein corresponding to the components of the miniaturized base editing system of the present invention encode them. can be delivered to cells as one or more guide RNAs and one or more mRNA molecules. At this time, the RNA molecule may be delivered through one or more lipid nanoparticles.

In one embodiment, the components of the miniaturized base editing system of the present invention may be in the form of one or more DNA molecules. wherein the one or more DNA molecules may comprise a nucleolytic protein, a guide molecule and one or more regulatory elements operably configured to express an adenosine deaminase protein or a cytidine deaminase protein or a catalytic domain thereof. . Optionally, one or more regulatory elements include an inducible promoter. The DNA molecules constituting the miniaturized base editing system may be contained in one or more adeno-associated virus (AAV) vectors and delivered into cells. Preferably, all of the DNA molecules can be contained in a single adeno-associated virus (AAV) vector and delivered into cells.

More specifically, the components of the vector that allows the subminiature base editing system according to the present invention to be expressed in cells include the following.

1. Nucleic acids encoding components of the micro base editing system

Since the purpose of the vector is to express each component of the base editing system according to the present invention in cells, the sequence of the vector encodes each component of the base editing system. It must necessarily contain at least one of the nucleic acid sequences that

Specifically, the sequence of the vector includes a nucleic acid sequence encoding a guide RNA and/or a nucleolytic protein included in the subminiature base editing system to be expressed. At this time, the sequence of the vector includes not only a nucleic acid sequence encoding a wild-type guide RNA and a wild-type nucleolytic protein, but also a nucleic acid sequence encoding an engineered guide RNA and/or a codon-optimized nucleolytic protein, engineered It may include a nucleic acid sequence encoding a nucleolytic protein, or a nucleic acid sequence encoding a nucleolytic protein with lost or reduced DNA double-strand cleavage activity.

Here, the nucleic acid sequence encoding a nucleolytic protein having lost or reduced DNA double-strand cleavage activity includes a nucleic acid sequence encoding a dead Cas (dCas) or nick Cas (nCas) protein.

In the present invention, the vector may be configured to express the Cas12f1 protein or a functional variant thereof. Here, the Cas12f1 protein or a functional variant thereof may be a dead Cas12f1 (dCas12f1) or nick Cas12f1 (nCas12f1) protein, which is a nucleolytic protein with lost or reduced DNA double-strand cleavage activity.

In one embodiment, the vector may be configured to express the wild-type Cas12f1 protein. Here, the wild-type Cas12f1 protein may be Cas14a1.

In another embodiment, the vector may be configured to express a Cas12f1 protein modified to cleave only one of the double strands of the target nucleic acid.

In one embodiment, the sequence of the vector may include a sequence encoding a wild-type Cas12f1 protein or a functional variant thereof. Here, the wild-type Cas12f1 protein may be Cas14a1, and the functional variant may be a TnpB-derived small molecular weight nucleolytic protein.

Preferably, the sequence of the vector may include a human codon-optimized nucleic acid sequence encoding the Cas12f1 protein or a functional variant thereof. Here, the human codon-optimized nucleic acid sequence encoding the Cas12f1 protein may be a human codon-optimized nucleic acid sequence encoding the Cas14a1 protein, and the functional variant may be a TnpB-derived low molecular weight nucleolytic protein.

In addition, the sequence of the vector according to the present invention may include a sequence encoding a modified Cas12f1 protein or a Cas12f1 fusion protein.

In one embodiment, the modified Cas12f1 protein is modified so that only one strand of the double strands of the target nucleic acid can be cut, and base editing or prime editing can be performed on the strand that is not cut. it could be Specifically, the Cas12f1 protein may be modified so as not to cleave all of the double strands of the target nucleic acid.

In addition, the Cas12f1 protein cannot cleave all of the double strands of the target nucleic acid, and may be modified to perform base editing or prime editing or gene expression control functions for the target nucleic acid.

In one embodiment, the Cas12f1 protein cannot cleave all of the double strands of the target nucleic acid, and the Cas12f1 modified to perform base editing or prime editing or gene expression control function for the target nucleic acid. It may contain sequences encoding proteins.

In one embodiment, the sequence of the vector is capable of cutting only one strand of the double strands of the target nucleic acid, and de-adenine so that base editing or prime editing can be performed on the non-cutting strand. It may include a sequence encoding an aminoase protein or a Cas12f1 protein to which cytosine deaminase is bound.

In addition, the vector may be configured to express a guide RNA engineered to have optimal targeting efficiency for Cas12f1. The vectors may be configured to express one or more different engineered Cas12f1 guide RNAs.

In one embodiment, the engineered guide RNA sequence may include a scaffold sequence, a spacer sequence and a U-rich tail sequence. Specifically, the engineered guide RNA sequence may include a modified tracrRNA sequence and/or a modified crRNA sequence, and may include a U-rich tail sequence.

In one embodiment, the U-rich tail sequence may be expressed as (UaN)nUb. Here, N is one of adenosine (A), uracil (U), cytidine (C), and guanosine (G). Here, a is an integer of 1 or more and 4 or less, n is an integer of 0, 1, or 2, and b is an integer of 1 or more and 10 or less. In another embodiment, the Urich tail sequence may be expressed as (UaV)nUb. In this case, a, n, and b are integers, a may be 1 or more and 4 or less, n may be 0 or more, and b may be 1 or more and 10 or less.

In the present invention, the vector may also be configured to express two or more different engineered guide RNAs.

In one embodiment, the vector may be configured to express the first engineered guide RNA and the second engineered guide RNA. For example, the first engineered guide RNA sequence comprises a first scaffold sequence, a first spacer sequence, and a first U-rich tail sequence, and the second engineered guide RNA sequence comprises a second scaffold sequence. , a second spacer sequence, and a second U-rich tail sequence.

In addition, the vector may include a nucleic acid sequence encoding additional expression elements to be expressed by a person skilled in the art in addition to the components of the above-described base editing system.

For example, the additional expression element may be a tag. Specifically, the additional expression element is a herbicide resistance gene such as glyphosate, glufosinate ammonium or phosphinothricin, ampicillin, kanamycin, G418 , antibiotic resistance genes such as bleomycin, hygromycin, and chloramphenicol.

2. Regulating and/or controlling components

In order for the vector to be expressed in a cell, it must contain one or more regulatory and/or control elements. Specifically, the regulatory and/or control elements are promoters, enhancers, introns, polyadenylation signals, Kozak consensus sequences, Internal Ribosome Entry Sites (IRES), splice acceptors, 2A It may include a sequence and/or origin of replication, but is not limited thereto. Here, the origin of replication may be the f1 origin of replication, the SV40 origin of replication, the pMB1 origin of replication, the adeno origin of replication, the AAV origin of replication, and/or the BBV origin of replication, but is not limited thereto.

3. Promoter

In order to express the nucleic acid sequence encoding the microscopic gene scissors system according to the present invention contained in the vector in a cell, a promoter sequence is operably linked to the sequence encoding each component so that the RNA transcription factor can be expressed in the cell. It has to be activated. The promoter sequence can be designed differently depending on the corresponding RNA transcription factor or expression environment, and is not limited as long as it can properly express the components of the CRISPR/Cas system according to the present invention in cells.

For example, the promoter sequence may be a promoter that promotes transcription of RNA polymerase RNA Pol I, Pol II, or Pol III. Specifically, the promoter is a cytomegalovirus such as the SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter, adenovirus major late promoter (Ad MLP), herpes simplex virus (HSV) promoter, CMV immediate early promoter region (CMVIE) (CMV) promoter, chicken β-actin (CBA) promoter, rous sarcoma virus (RSV) promoter, human U6 small nuclear promoter (U6), enhanced U6 promoter, and human H1 promoter (H1).

4. Closing signal

When the vector sequence includes the promoter sequence, transcription of a sequence operably linked to the promoter is induced by an RNA transcription factor, and a termination signal that induces termination of transcription of the RNA transcription factor may be included. The termination signal may vary depending on the type of promoter sequence. Specifically, when the promoter is a U6 or H1 promoter, the promoter recognizes the TTTTTT(T6) sequence, which is a thymidine (T) sequence, as a termination signal.

The sequence of the engineered guide RNA provided in the present invention includes a U-rich tail sequence at its 3'-end. Accordingly, the sequence encoding the engineered guide RNA includes a T-rich sequence corresponding to the U-rich tail sequence at its 3'-end. As described above, some promoter sequences recognize a thymidine (T) contiguous sequence, for example, a sequence in which 5 or more thymidine (T) are consecutively linked, as a termination signal. In some cases, the T-rich sequence is recognized as a termination signal. can be recognized as

In other words, when the vector sequence provided herein includes a sequence encoding an engineered guide RNA, a sequence encoding a U-rich tail sequence included in the engineered guide RNA sequence may be used as a termination signal.

In one embodiment, when the vector sequence includes a U6 or H1 promoter sequence and a sequence encoding an engineered guide RNA operably linked thereto, a U-rich tail sequence included in the engineered guide RNA sequence A portion of the sequence encoding can be recognized as a termination signal. At this time, the U-rich tail sequence includes a sequence in which 5 or more uridin (U) are consecutively linked.

5. Additional Expression Elements

If necessary, the vector may be configured to express additional components such as NLS and/or tag protein.

In one embodiment, the additional component may be expressed independently of the Cas12f1, modified Cas12f1 and/or engineered Cas12f1 guide RNA.

In another embodiment, the additional component may be expressed directly or linked by a linker to the Cas12f1, modified Cas12f1, and/or engineered Cas12f1 guide RNA.

In one embodiment, the subminiature base editing construct according to the present invention comprises one or more nuclear localization sequences (NLS) sequences at the N-terminus or C-terminus, the subminiature base It may be a corrective structure. The NLS sequence may be a subminiature base editing structure comprising or consisting of any one amino acid sequence selected from SEQ ID NO: 65 to SEQ ID NO: 68. Here, the additional component may be a component that is generally expressed when the CRISPR/Cas system is to be expressed, and known technologies may be referred to.

In one embodiment, a nucleic acid contained in a vector or the like is provided to express the engineered gRNA according to the present invention or a nucleic acid encoding the same and/or components of a base editing system. Here, the nucleic acid may be DNA or RNA that exists in nature, or may be a modified nucleic acid in which some or all of the constituent nucleic acids are chemically modified.

In one embodiment, the constituent nucleic acids may be naturally occurring DNA and/or RNA. For example, the constituent nucleic acid may be one or more nucleotides chemically modified. In this case, the chemical modification may include all modifications of nucleic acids known to those skilled in the art.

6. Types and types of expression vectors

A vector according to the present invention may be a viral vector. More specifically, the viral vector may be one or more selected from the group consisting of retrovirus, lentivirus, adenovirus, adeno-associated virus, vaccinia virus, poxvirus, and herpes simplex virus. Preferably, the viral vector may be an adeno-associated viral vector.

In addition, the vector according to the present invention may be a non-viral vector. More specifically, the non-viral vector may be one or more selected from the group consisting of plasmid, phage, naked DNA, DNA complex, and mRNA. In one embodiment, the plasmids are pcDNA series, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, and pUC19. It may be selected from the group consisting of

In one embodiment, the phage may be M13, and the vector may be a PCR amplicon.

The vector according to the present invention may be designed in the form of a linear or circular vector. When the vector is a linear vector, RNA transcription is terminated at its 3'-end, even if the sequence of the linear vector does not separately include a termination signal.

However, when the vector is a circular vector, RNA transcription is not terminated unless the sequence of the circular vector separately includes a termination signal. Therefore, when a circular vector is used as the vector, a termination signal corresponding to a transcription factor related to each promoter sequence must be included in order to express the intended target.

[Gene editing method using engineered gRNA]

The present invention provides a method for editing a target nucleic acid or target gene in a target cell using an engineered gRNA that has optimal target editing activity for Cas12f1 protein, TnpB protein or a functional analogue thereof. The gene editing method may be a base editing method in which a specific base in a target site is substituted with another base. The target gene or target nucleic acid includes a target sequence. The target nucleic acid may be single-stranded DNA, double-stranded DNA and/or RNA.

In one embodiment, the gene editing method includes delivering an engineered guide RNA and a Cas12f1 protein or a nucleic acid encoding each of them into a target cell containing a target nucleic acid or target gene.

As a result, the engineered CRISPR/Cas12f1 complex is injected into the target cell, or the formation of the engineered CRISPR/Cas12f1 complex is induced, and the target gene is edited by the engineered CRISPR/Cas12f1 complex. Here, the Cas12f1 protein is wild-type Cas12f1 or wild-type TnpB protein or a functional analogue thereof, modified Cas12f1 or modified TnpB protein, or adenosine deaminase protein or cytidine deaminase-linked Cas12f1 protein, TnpB protein or a functional analogue thereof. may be analogues.

In another embodiment, the gene editing method comprises an engineered guide RNA or a nucleic acid encoding the same; and Cas12f1, TnpB protein or a functional analogue thereof, or a nucleic acid encoding the same; into a target cell. At this time, the engineered guide RNA sequence includes the altered scaffold region sequence, spacer sequence, and U-rich tail sequence. Here, the sequence of the altered scaffold region has the same characteristics and structure as those described in the above-described 'engineered guide RNA for a base editing system' and 'scaffold region' section.

In one embodiment, the engineered tracrRNA may include/consist of any one nucleotide sequence selected from SEQ ID NO: 39 to SEQ ID NO: 42, and the engineered crRNA may include/consist of the nucleotide sequence of SEQ ID NO: 43. .

In another embodiment, the engineered tracrRNA may include/consist of any one nucleotide sequence selected from SEQ ID NO: 49 to SEQ ID NO: 52, and the engineered crRNA may include/consist of the nucleotide sequence of SEQ ID NO: 53. there is.

Preferably, the engineered guide RNA may include/consist of any one nucleotide sequence selected from SEQ ID NO: 55 to SEQ ID NO: 59.

In addition, the spacer sequence may complementarily bind to a target gene or target nucleic acid contained in the target cell, and the U-rich tail sequence may be expressed as (UaV)nUb. Here, a, n, and b are integers, a is 1 or more and 4 or less, n is 0 or more, and b is 1 or more and 10 or less.

In another embodiment, the U-rich tail sequence may be expressed as (UaN)nUb. Here, N is one of adenosine (A), uracil (U), cytidine (C), and guanosine (G). Here, a is an integer of 1 or more and 4 or less, n is an integer of 0, 1, or 2, and b is an integer of 1 or more and 10 or less.

In one embodiment, the sequence of the U-rich tail is 5'-U-3', 5'-UU-3', 5'-UUU-3', 5'-UUUU-3', 5'-UUUUU- 3', 5'-UUUUUU-3', 5'-UUURUUU-3' (SEQ ID NO: 77), 5'-UUUR UUURUUU-3' (SEQ ID NO: 78), 5'-UUUURU-3' (SEQ ID NO: 79) , 5'-UUUURUU-3' (SEQ ID NO: 80), 5'-UUUURUUU-3' (SEQ ID NO: 81), 5'-UUUURUUUU-3' (SEQ ID NO: 82), 5'-UUUURUUUUU-3' (SEQ ID NO: 81) 83), or 5'-UUUURUUUUUU-3' (SEQ ID NO: 84). Where R can be A or G. Preferably, the sequence of the U-rich tail may be 5'-UUUUAUUUU-3' or 5'-UUUUGUUUU-3'.

Hereinafter, the steps of the gene editing method using the engineered gRNA will be described.

1. Determination of target cell, target sequence and spacer sequence

A target cell to be subjected to base editing or gene editing with the miniaturized base editing system according to the present invention may be a prokaryotic cell or a eukaryotic cell. More specifically, the eukaryotic cells may be plant cells, animal cells and/or human cells, but are not limited thereto.

The target nucleic acid, target gene, or target sequence may be determined in consideration of the purpose of gene editing, the target cell environment, the PAM sequence recognized by the Cas12f1 protein, and/or other variables. Here, if a target sequence having an appropriate length or a PAM sequence recognized by the Cas12f1 protein can be determined within the target nucleic acid or target gene, the method can be performed without particular limitation using known techniques.

After the target sequence is determined, a spacer sequence corresponding thereto is designed. The spacer sequence is designed as a sequence capable of complementary binding to the target sequence. In one embodiment, the spacer sequence is designed as a sequence capable of complementary binding to the target nucleic acid or target gene. In one embodiment, the spacer sequence may be designed as a sequence complementary to the target sequence included in the target strand sequence of the target nucleic acid.

In addition, the spacer sequence may be designed as an RNA sequence corresponding to the DNA sequence of the protospacer included in the non-target strand sequence of the target nucleic acid. Specifically, the spacer sequence may have the same nucleotide sequence as the protospacer sequence, and may be designed as a sequence in which all thymidines included in the nucleotide sequence are substituted with uridine.

In one embodiment, the spacer sequence may be a complementary sequence having 60% or more sequence identity with the target sequence. Preferably, the spacer sequence may be a complementary sequence having 60% to 90% sequence identity with the target sequence. More preferably, the spacer sequence may be a complementary sequence having 90% to 100% sequence identity with the target sequence.

In addition, the spacer sequence according to the present invention has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches with the target sequence. It may be a complementary sequence. In one embodiment, the spacer sequence may have 1 to 5 mismatches with the target sequence. In addition, the spacer sequence may have 6 to 10 mismatches with the target sequence.

2. Intracellular delivery of each component of the micro base editing system

The base editing and gene editing method provided in the present specification is a method in which the base editing structure according to the present invention and the base editing system including the same specifically recognize and edit a target sequence for a target nucleic acid or target gene. Use points that are active.

The gene editing method provided herein is a method in which a base editing system or a base editing structure containing an engineered Cas12f1 sgRNA in a target cell contacts a target sequence region of a target nucleic acid or target gene assuming that

Accordingly, the gene editing method of the present invention includes effective transfer of the base editing structure and/or the base editing system including the base editing structure into a target cell. Preferably, contacting or inducing contact of the microbase nucleotide editing construct and/or each component of the miniaturization system including the same with a target sequence site of a target nucleic acid or target gene in a target cell.

In one embodiment, the gene editing method comprises an engineered guide RNA or a nucleic acid encoding the same; and Cas12f1 protein or a functional analog thereof, or a nucleic acid encoding the same; into a target cell. In addition, the gene editing method includes an engineered guide RNA; and Cas12f1 or a functional analogue protein thereof; into a target cell.

In one embodiment, the gene editing method comprises a nucleic acid encoding an engineered guide RNA; and Cas12f1 protein or a protein functional analogue thereof; into a target cell.

In addition, in one embodiment, the gene editing method is an engineered guide RNA; and a nucleic acid encoding the Cas12f1 protein or a protein functional analogue thereof; into a target cell.

In one embodiment, the gene editing method comprises a nucleic acid encoding an engineered guide RNA; and a nucleic acid encoding the Cas12f1 protein or a protein functional analogue thereof; into a target cell.

engineered guide RNAs or nucleic acids encoding them; and the Cas12f1 protein or functional analogue protein thereof, or a nucleic acid encoding the same; can be delivered into a target cell in a variety of delivery formats and using a variety of delivery methods. Here, the induction is not particularly limited as long as the base editing system or the base editing structure containing the engineered Cas12f1 sgRNA is brought into contact with the target nucleic acid in the cell.

2-1. form of delivery

The subminiature base editing structure for the method of the present invention and the delivery form of the subminiature base editing system including the base editing system include a Cas12f1 guide RNA engineered into a cell or a nucleic acid encoding the same; and Cas12f1 protein or a functional analogue thereof, or a nucleic acid encoding the same; it is not particularly limited as long as it can be delivered into a cell in an appropriate delivery form.

As a delivery form of the miniaturized base editing structure and the miniaturized base editing system including the same for the method of the present invention, a ribonucleoprotein particle (RNP) to which an engineered Cas12f1 guide RNA and Cas12f1 protein are bound can be used. .

In one embodiment, the gene editing method may include injecting a CRISPR/Cas12f1 complex in which the engineered Cas12f1 guide RNA and the Cas12f1 protein are combined into a target cell.

In another delivery form, an engineered Cas12f1 guide RNA or a nucleic acid encoding the same; And a Cas12f1 protein or a functional analogue thereof, or a nucleic acid encoding the same; a non-viral vector comprising a can be used.

In one embodiment, the gene editing method may include injecting a non-viral vector including a nucleic acid sequence encoding an engineered Cas12f1 guide RNA and a nucleic acid sequence encoding a Cas12f1 protein into a target cell. Specifically, the non-viral vector may be a plasmid, naked DNA, DNA complex, or mRNA, but is not limited thereto.

In another embodiment, the gene editing method involves injecting a first non-viral vector comprising a nucleic acid sequence encoding an engineered Cas12f1 guide RNA and a second non-viral vector comprising a nucleic acid sequence encoding a Cas12f1 protein into a target cell. may include doing

Specifically, the first non-viral vector and the second non-viral vector may each be one selected from the group consisting of plasmid, naked DNA, DNA complex, and mRNA, but are not limited thereto.

In another form of delivery, a viral vector comprising a nucleic acid sequence encoding an engineered Cas12f1 guide RNA and a nucleic acid sequence encoding a Cas12f1 protein or functional analog thereof can be used.

In one embodiment, the gene editing method may include injecting a viral vector containing a nucleic acid sequence encoding an engineered Cas12f1 guide RNA and a nucleic acid sequence encoding a Cas12f1 protein or a functional analogue thereof into a target cell. .

Specifically, the viral vector may be one selected from the group consisting of retrovirus, lentivirus, adenovirus, adeno-associated virus, vaccinia virus, poxvirus, and herpes simplex virus, but is not limited thereto. Preferably, the viral vector may be an adeno-associated virus.

In another example, the gene editing method includes injecting a first viral vector comprising a nucleic acid sequence encoding an engineered Cas12f1 guide RNA and a second viral vector comprising a nucleic acid sequence encoding a Cas12f1 protein into a target cell. can do. Specifically, the first viral vector and the second viral vector may be one selected from the group consisting of retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, vaccinia viruses, poxviruses, and herpes simplex viruses, respectively. It is not limited.

In addition, the delivery form may include an engineered Cas12f1 guide RNA or a nucleic acid encoding the same; and Cas12f1 protein or a functional analog thereof, or a nucleic acid encoding the same; may be delivered using nanoparticles.

In one embodiment, the delivery method comprises a Cas12f1 protein or a nucleic acid encoding the same, a first engineered Cas12f1 guide RNA or a nucleic acid encoding the same, and/or a second engineered Cas12f1 guide RNA or a nucleic acid encoding the same using nanoparticles. it may be conveying

Here, the delivery method is a cationic liposome method, lithium acetate-DMSO, lipid-mediated transfection (transfection), calcium phosphate precipitation method (precipitation), lipofection, electroporation, gene gun, sonoporation, magnetofection ), and/or transient cell compression or squeezing PEI (Polyethyleneimine)-mediated transfection, DEAE-dextran-mediated transfection, or nanoparticle-mediated nucleic acid delivery, but is not limited thereto.

In addition, the Cas12f1 guide RNA engineered in the present invention or a nucleic acid encoding the same; and Cas12f1 protein or a functional analogue thereof, or a nucleic acid encoding the same; the delivery form of the method of editing a target gene by delivering into a cell may be performed by combining the above delivery forms.

For example, the gene editing method may include delivering an engineered Cas12f1 guide RNA or a nucleic acid encoding the same in a first delivery form, and delivering a Cas12f1 protein or a nucleic acid encoding the same in a second delivery form. In this case, the first delivery form and the second delivery form may each be any one of the above-described delivery forms.

In one embodiment, the gene editing method may include delivering the engineered Cas12f1 guide RNA or a nucleic acid encoding the same by a first delivery method, and delivering the Cas12f1 protein or a nucleic acid encoding the same by a second delivery method. In this case, the first delivery method and the second delivery method may each be any one of the above-described delivery methods.

In the present invention, the delivery form of the gene editing method is a target nucleic acid or target gene to be edited for the microbase nucleotide editing structure according to the present invention to be delivered to one vector and the components of the microbase nucleotide editing system including the same. It is not particularly limited as long as it is a delivery form capable of being delivered to the environment.

2-2. order of delivery

The gene editing method includes an engineered Cas12f1 guide RNA or a nucleic acid encoding the same; and Cas12f1 protein or a nucleic acid encoding the same; wherein the construct may be simultaneously delivered into the cell, but may be delivered sequentially with a time difference.

In one embodiment, the gene editing method comprises an engineered Cas12f1 guide RNA or a nucleic acid encoding the same; and the Cas12f1 protein or a nucleic acid encoding the same.

In one embodiment, the gene editing method may include delivering the engineered Cas12f1 guide RNA or the nucleic acid encoding the same into the cell, and then delivering the Cas12f1 protein or the nucleic acid encoding the same into the cell with a time difference.

In one embodiment, the gene editing method may include delivering the Cas12f1 protein or a nucleic acid encoding the Cas12f1 protein into the cell, and then delivering the engineered Cas12f1 guide RNA into the cell with a time difference.

In one embodiment, the gene editing method may include delivering a Cas12f1 protein-encoding nucleic acid into a cell and then delivering an engineered Cas12f1 guide RNA into the cell with a time difference.

In addition, the gene editing method provided by the present invention may include delivering a Cas12f1 protein or a nucleic acid encoding the Cas12f1 protein and two or more engineered Cas12f1 guide RNAs or a nucleic acid encoding the same into a target cell.

Through this method, two or more CRISPR/Cas12f1 complexes targeting different sequences can be injected into a target cell or formed in a target cell. As a result, two or more target genes or target nucleic acids contained in the cell can be edited.

In one embodiment, the gene editing method comprises combining a Cas12f1 protein or a nucleic acid encoding the same, a first engineered Cas12f1 guide RNA or a nucleic acid encoding the same, and a second engineered Cas12f1 guide RNA or a nucleic acid encoding the same into a target gene or a target nucleic acid. It includes delivery into target cells containing. In this case, each of the components may be delivered into cells using one or more of the above-described delivery forms and delivery methods. Here, two or more components may be delivered into cells simultaneously or sequentially with a time difference.

[Composition and method for base editing]

Disclosed in the present invention is a gene editing composition comprising each component of a mini base editing system.

In one embodiment, a Cas12f1, TnpB protein or a functional analogue thereof, or a nucleic acid encoding the same; And an engineered Cas12f1 guide RNA or a nucleic acid encoding the same; discloses a gene editing composition comprising a. Here, the Cas12f1 protein may be a Cas12f1 protein mutant (dCas12f1 or nCas12f1), a TnpB protein mutant (dTnpB or nTnpB), and a functional variant thereof having lost or reduced DNA double-strand break activity in wild-type Cas12f1, TnpB protein or a functional analog thereof. . the dCas12f1, nCas12f1, dTnpB or nTnpB; and functional variants thereof; may be a fusion protein in which adenosine deaminase or cytidine deaminase is linked to the N-terminus or C-terminus. The fusion protein may exhibit base proofreading activity.

In the present invention, based on the fusion protein, a subminiature base editing construct including various combinations including a nucleolytic protein derived from dCas12f1 or dTnpB exhibiting base editing activity was prepared. These are essential components of the miniaturized base editing system according to the present invention.

In addition, in one embodiment, the miniaturized base editing system includes an engineered guide RNA that maximizes the target activity or gene editing activity for Cas12f1, TnpB or a functional analogue thereof.

This may be any one or more described in the above-described 'engineered guide RNA for a small base editing system' and 'scaffold region' section. Preferably, sgRNACas12f_ge3.0 with modifications at MS1/MS2/MS3 (SEQ ID NO: 57), sgRNA Cas12f_ge4.0 with modifications at MS2/MS3/MS4 (SEQ ID NO: 58) and/or MS2/MS3/MS4/MS5 It may be sgRNA Cas12f_ge4.1 (SEQ ID NO: 59) with modifications.

In addition, it is apparent that the composition for gene editing of the present invention may further include appropriate materials required for gene editing use, in addition to each component of the miniaturized base editing system according to the present invention.

The present invention also provides a base editing method comprising the step of contacting a target sequence with the miniaturized base editing system or the composition including the base editing system according to the present invention. Here, the base correction may be a method of proofreading an adenine (A) base with guanine (G) or final proofreading a cytosine (C) base with thymine (T).

As a result, one or more specific bases in the target nucleic acid or target gene are changed to other bases as intended, unlike indels in which any base in the target nucleic acid or target gene is deleted or added. After all, 'intentional point mutation' is caused as a result of base substitution in a target sequence in a target nucleic acid or target gene.

In one embodiment, in the base correction method according to the present invention, the target sequence in the target nucleic acid or target gene may include a target base to be corrected, and the target base to be corrected is adenine associated with a disease or disease. (A) or cytosine (C) bases.

In addition, the target base is a base present in the stop codon, and through base correction, the stop codon is corrected with a codon encoding an amino acid, and the transcription stopped by the stop codon can be resumed. can be for Conversely, it may be a base at a site where a stop codon is to be made by correcting the target base. In this case, it may correspond to gene editing that can replace gene editing by indel.

In one embodiment, the gene editing method includes a base editing system in the form of ribonucleoprotein particles in which the engineered Cas12f1 guide RNA and the base editing structure protein according to the present invention are combined into eukaryotic cells. may include delivery. In this case, the delivery may be performed using electroporation or lipofection.

In another embodiment, the gene editing method preferably comprises a single adeno- comprising both a nucleic acid sequence encoding an engineered Cas12f1 guide RNA and a nucleic acid sequence encoding the base editing construct according to the present invention. It may include using an associated viral (AAV) vector to deliver the target nucleic acid or target gene into a eukaryotic cell.

Hereinafter, the invention provided by this specification will be described in more detail through examples. These examples are only for exemplifying the content disclosed by this specification, and those of ordinary skill in the art are not to be construed as limiting the scope of the content disclosed by this specification. It will be self-evident for

[Example]

Example 1. Manufacturing of each component of a mini base editing system

Example 1-1. Nucleic acid encoding human codon-optimized Cas12f1

To construct a subminiature base editing system expressed in human cells, the Cas12f1 protein (SEQ ID NO: 1) belonging to the Cas14 family was human codon-optimized using a codon optimization program. Add 5'-CCAAAGAAGAAGCGGAAGGTC-3' (SEQ ID NO: 60) and 5'-AAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAG-3' (SEQ ID NO: 61) as NLS sequences to the 5'-end and 3'-end of the codon-optimized gene, respectively, A human codon-optimized Cas12f1 protein coding sequence (SEQ ID NO: 2) was prepared by connecting a linker 5'-GGTATCCACGGAGTCCCAGCAGCC-3' (SEQ ID NO: 136) between the 5'-terminal NLS sequence and the start codon of Cas12f1.

PCR amplification is performed using the prepared gene as a template, and cloning is performed according to the desired cloning sequence into a vector having a promoter capable of expression in a eukaryotic system and a poly(A) signal sequence through the Gibson assembly method. proceeded. After cloning, the sequence of the obtained recombinant plasmid vector was finally confirmed through the Sanger sequencing method. The amino acid sequence of the Cas12f1 protein used for cloning and the human codon-optimized Cas12f1 base sequence encoding it are shown in [Table 1].

Label Sequence(5' to 3') SEQ ID
NO:
Cas12f1
protein
MAKNTITKTLKLRIVRPYNSAEVEKIVADEKNNREKIALEKNKDKVKEACSKHLKVAAYCTTQVERNACLFCKARKLDDKFYQKLRGQFPDAVFWQEISEIFRQLQKQAAEIYNQSLIELYYEIFIKGKGIANASSVEHYLSDVCYTRAAELFKNAAIASGLRSKIKSNFRLKELKNMKSGLPTTKSDNFPIPLVKQKGGQYTGFEISNHNSDFIIKIPFGRWQVKKEIDKYRPWEKFDFEQVQKSPKPISLLLSTQRRKRNKGWSKDEGTEAEIKKVMNGDYQTSYIEVKRGSKIGEKSAWMLNLSIDVPKIDKGVDPSIIGGIDVGVKSPLVCAINNAFSRYSISDNDLFHFNKKMFARRRILLKKNRHKRAGHGAKNKLKPITILTEKSERFRKKLIERWACEIADFFIKNKVGTVQMENLESMKRKEDSYFNIRLRGFWPYAEMQNKIEFKLKQYGIEIRKVAPNNTSKTCSKCGHLNNYFNFEYRKKNKFPHFKCEKCNFKENADYNAALNISNPKLKSTKEEP
One
Human codon-optimized Cas12f1
base sequence
ATGGCCAAGAACACAATTACAAAGACACTGAAGCTGAGGATCGTGAGACCATACAACAGCGCTGAGGTCGAGAAGATTGTGGCTGATGAAAAGAACAACAGGGAAAAGATCGCCCTCGAGAAGAACAAGGATAAGGTGAAGGAGGCCTGCTCTAAGCACCTGAAAGTGGCCGCCTACTGCACCACACAGGTGGAGAGGAACGCCTGTCTGTTTTGTAAAGCTCGGAAGCTGGATGATAAGTTTTACCAGAAGCTGCGGGGCCAGTTCCCCGATGCCGTCTTTTGGCAGGAGATTAGCGAGATCTTCAGACAGCTGCAGAAGCAGGCCGCCGAGATCTACAACCAGAGCCTGATCGAGCTCTACTACGAGATCTTCATCAAGGGCAAGGGCATTGCCAACGCCTCCTCCGTGGAGCACTACCTGAGCGACGTGTGCTACACAAGAGCCGCCGAGCTCTTTAAGAACGCCGCTATCGCTTCCGGGCTGAGGAGCAAGATTAAGAGTAACTTCCGGCTCAAGGAGCTGAAGAACATGAAGAGCGGCCTGCCCACTACAAAGAGCGACAACTTCCCAATTCCACTGGTGAAGCAGAAGGGGGGCCAGTACACAGGGTTCGAGATTTCCAACCACAACAGCGACTTTATTATTAAGATCCCCTTTGGCAGGTGGCAGGTCAAGAAGGAGATTGACAAGTACAGGCCCTGGGAGAAGTTTGATTTCGAGCAGGTGCAGAAGAGCCCCAAGCCTATTTCCCTGCTGCTGTCCACACAGCGGCGGAAGAGGAACAAGGGGTGGTCTAAGGATGAGGGGACCGAGGCCGAGATTAAGAAAGTGATGAACGGCGACTACCAGACAAGCTACATCGAGGTCAAGCGGGGCAGTAAGATTGGCGAGAAGAGCGCCTGGATGCTGAACCTGAGCATTGACGTGCCAAAGATTGATAAGGGCGTGGATCCCAGCATCATCGGAGGGATCGATGTGGGGGTCAAGAGCCCCCTCGTGTGCGCCATCAACAACGCCTTCAGCAGGTACAGCATCTCCGATAACGACCTGTTCCACTTTAACAAGAAGATGTTCGCCCGGCGGAGGATTTTGCTCAAGAAGAACCGGCACAAGCGGGCCGGACACGGGGCCAAGAACAAGCTCAAGCCCATCACTATCCTGACCGAGAAGAGCGAGAGGTTCAGGAAGAAGCTCATCGAGAGATGGGCCTGCGAGATCGCCGATTTCTTTATTAAGAACAAGGTCGGAACAGTGCAGATGGAGAACCTCGAGAGCATGAAGAGGAAGGAGGATTCCTACTTCAACATTCGGCTGAGGGGGTTCTGGCCCTACGCTGAGATGCAGAACAAGATTGAGTTTAAGCTGAAGCAGTACGGGATTGAGATCCGGAAGGTGGCCCCCAACAACACCAGCAAGACCTGCAGCAAGTGCGGGCACCTCAACAACTACTTCAACTTCGAGTACCGGAAGAAGAACAAGTTCCCACACTTCAAGTGCGAGAAGTGCAACTTTAAGGAGAACGCCGATTACAACGCCGCCCTGAACATCAGCAACCCTAAGCTGAAGAGCACTAAGGAGGAGCCC
2

Example 1-2. dead Cas12f1 (dCas12f1) protein variant

In order to manufacture a subminiature base editing system for base editing, a nucleolytic protein with reduced DNA double-strand cleavage activity should be used.

Accordingly, the present inventors prepared a dead Cas12f1 (dCas12f1) protein mutant in which the DNA double-strand break activity was lost in the Cas12f1 protein.

Specifically, dCas12f1 protein variants confirmed to have lost DNA double-strand break activity were dCas12f1 D326A mutant, dCas12f1 E422A mutant, dCas12f1 R490A mutant and dCas12f1 D510A mutant, which are shown in [Table 2]. Among the identified dCas12f1 protein variants, the dCas12f1 D326A variant was used as a representative nucleolytic protein of the ultra-miniature base correction construct and the ultra-miniature base correction system for base correction according to the present invention.

Label Sequence(5' to 3') SEQ ID
NO:
dCas12f1
D326A
MAKNTITKTLKLRIVRPYNSAEVEKIVADEKNNREKIALEKNKDKVKEACSKHLKVAAYCTTQVERNACLFCKARKLDDKFYQKLRGQFPDAVFWQEISEIFRQLQKQAAEIYNQSLIELYYEIFIKGKGIANASSVEHYLSDVCYTRAAELFKNAAIASGLRSKIKSNFRLKELKNMKSGLPTTKSDNFPIPLVKQKGGQYTGFEISNHNSDFIIKIPFGRWQVKKEIDKYRPWEKFDFEQVQKSPKPISLLLSTQRRKRNKGWSKDEGTEAEIKKVMNGDYQTSYIEVKRGSKIGEKSAWMLNLSIDVPKIDKGVDPSIIGGI A VGVKSPLVCAINNAFSRYSISDNDLFHFNKKMFARRRILLKKNRHKRAGHGAKNKLKPITILTEKSERFRKKLIERWACEIADFFIKNKVGTVQMENLESMKRKEDSYFNIRLRGFWPYAEMQNKIEFKLKQYGIEIRKVAPNNTSKTCSKCGHLNNYFNFEYRKKNKFPHFKCEKCNFKENADYNAALNISNPKLKSTKEEP
3
dCas12f1
E422A
MAKNTITKTLKLRIVRPYNSAEVEKIVADEKNNREKIALEKNKDKVKEACSKHLKVAAYCTTQVERNACLFCKARKLDDKFYQKLRGQFPDAVFWQEISEIFRQLQKQAAEIYNQSLIELYYEIFIKGKGIANASSVEHYLSDVCYTRAAELFKNAAIASGLRSKIKSNFRLKELKNMKSGLPTTKSDNFPIPLVKQKGGQYTGFEISNHNSDFIIKIPFGRWQVKKEIDKYRPWEKFDFEQVQKSPKPISLLLSTQRRKRNKGWSKDEGTEAEIKKVMNGDYQTSYIEVKRGSKIGEKSAWMLNLSIDVPKIDKGVDPSIIGGIAVGVKSPLVCAINNAFSRYSISDNDLFHFNKKMFARRRILLKKNRHKRAGHGAKNKLKPITILTEKSERFRKKLIERWACEIADFFIKNKVGTVQM A NLESMKRKEDSYFNIRLRGFWPYAEMQNKIEFKLKQYGIEIRKVAPNNTSKTCSKCGHLNNYFNFEYRKKNKFPHFKCEKCNFKENADYNAALNISNPKLKSTKEEP
4
dCas12f1
R490A
MAKNTITKTLKLRIVRPYNSAEVEKIVADEKNNREKIALEKNKDKVKEACSKHLKVAAYCTTQVERNACLFCKARKLDDKFYQKLRGQFPDAVFWQEISEIFRQLQKQAAEIYNQSLIELYYEIFIKGKGIANASSVEHYLSDVCYTRAAELFKNAAIASGLRSKIKSNFRLKELKNMKSGLPTTKSDNFPIPLVKQKGGQYTGFEISNHNSDFIIKIPFGRWQVKKEIDKYRPWEKFDFEQVQKSPKPISLLLSTQRRKRNKGWSKDEGTEAEIKKVMNGDYQTSYIEVKRGSKIGEKSAWMLNLSIDVPKIDKGVDPSIIGGIAVGVKSPLVCAINNAFSRYSISDNDLFHFNKKMFARRRILLKKNRHKRAGHGAKNKLKPITILTEKSERFRKKLIERWACEIADFFIKNKVGTVQMENLESMKRKEDSYFNIRLRGFWPYAEMQNKIEFKLKQYGIEIRKVAPNNTSKTCSKCGHLNNYFNFEY A KKNKFPHFKCEKCNFKENADYNAALNISNPKLKSTKEEP
5
dCas12f1
D510A
MAKNTITKTLKLRIVRPYNSAEVEKIVADEKNNREKIALEKNKDKVKEACSKHLKVAAYCTTQVERNACLFCKARKLDDKFYQKLRGQFPDAVFWQEISEIFRQLQKQAAEIYNQSLIELYYEIFIKGKGIANASSVEHYLSDVCYTRAAELFKNAAIASGLRSKIKSNFRLKELKNMKSGLPTTKSDNFPIPLVKQKGGQYTGFEISNHNSDFIIKIPFGRWQVKKEIDKYRPWEKFDFEQVQKSPKPISLLLSTQRRKRNKGWSKDEGTEAEIKKVMNGDYQTSYIEVKRGSKIGEKSAWMLNLSIDVPKIDKGVDPSIIGGIAVGVKSPLVCAINNAFSRYSISDNDLFHFNKKMFARRRILLKKNRHKRAGHGAKNKLKPITILTEKSERFRKKLIERWACEIADFFIKNKVGTVQMENLESMKRKEDSYFNIRLRGFWPYAEMQNKIEFKLKQYGIEIRKVAPNNTSKTCSKCGHLNNYFNFEYRKKNKFPHFKCEKCNFKENA A YNAALNISNPKLKSTKEEP
6

Example 1-3. Protein variants derived from dead TnpB (dTnpB)

In addition, in the present invention, a fusion protein for base editing, a mini base editing structure, or a nucleolytic protein of a mini base editing system including the same includes a TnpB protein and a TnpB-derived nucleic acid degrading protein having a small molecular weight. Preferably, the TnpB protein is a protein consisting of the amino acid sequence of SEQ ID NO: 7, and the TnpB-derived low molecular weight nucleolytic protein is CasX-Cas12f1 consisting of the amino acid sequence of SEQ ID NO: 8, 28aa- consisting of the amino acid sequence of SEQ ID NO: 9 28aa-extension Cas12f1 or 26aa-extension Cas12f1 consisting of the amino acid sequence of SEQ ID NO: 10 (26aa-extension Cas12f1).

Accordingly, for the TnpB protein and the TnpB-derived low-molecular-weight nucleolytic protein, dead nucleolytic protein mutants having lost DNA double-strand cleavage activity were prepared. Here, the dead nucleolytic protein variants for the TnpB protein and the TnpB-derived small molecular weight nucleolytic protein are D326A, E422A, and R490A of the Cas12f1 protein, as well as the dCas12f1 protein variants confirmed to have lost DNA double-strand cleavage activity. Alternatively, it was designed to have a mutation corresponding to the amino acid substitution of D510A, and a corresponding variant was produced.

More specifically, the dead nucleolytic protein variants are dTnpB D354A mutant (SEQ ID NO: 11), dTnpB E450A mutant (SEQ ID NO: 12), dTnpB R518A mutant (SEQ ID NO: 13), dTnpB D538A mutant (SEQ ID NO: 14), 28aa-extension dCas12f1 D354A variant (SEQ ID NO: 15), 28aa-extension dCas12f1 E450A variant (SEQ ID NO: 16), 28aa-extension dCas12f1 R518A variant (SEQ ID NO: 17) and 28aa-extension dCas12f1 D538A variant (SEQ ID NO: 18).

In addition, these are shown in [Table 3] and [Table 4]. In particular, the dTnpB D354A variant was used as an exemplary nucleic acid degradation protein of the ultra-small base editing system for base editing according to the present invention.

Label Sequence(5' to 3') SEQ ID
NO:
dTnpB
D354A
MGEKSSRRRRNGKSGAWTAAITSCVGGKMAKNTITKTLKLRIVRPYNSAEVEKIVADEKNNREKIALEKNKDKVKEACSKHLKVAAYCTTQVERNACLFCKARKLDDKFYQKLRGQFPDAVFWQEISEIFRQLQKQAAEIYNQSLIELYYEIFIKGKGIANASSVEHYLSDVCYTRAAELFKNAAIASGLRSKIKSNFRLKELKNMKSGLPTTKSDNFPIPLVKQKGGQYTGFEISNHNSDFIIKIPFGRWQVKKEIDKYRPWEKFDFEQVQKSPKPISLLLSTQRRKRNKGWSKDEGTEAEIKKVMNGDYQTSYIEVKRGSKIGEKSAWMLNLSIDVPKIDKGVDPSIIGGI A VGVKSPLVCAINNAFSRYSISDNDLFHFNKKMFARRRILLKKNRHKRAGHGAKNKLKPITILTEKSERFRKKLIERWACEIADFFIKNKVGTVQMENLESMKRKEDSYFNIRLRGFWPYAEMQNKIEFKLKQYGIEIRKVAPNNTSKTCSKCGHLNNYFNFEYRKKNKFPHFKCEKCNFKENADYNAALNISNPKLKSTKEEP
11
dTnpB
E450A
MGEKSSRRRRNGKSGAWTAAITSCVGGKMAKNTITKTLKLRIVRPYNSAEVEKIVADEKNNREKIALEKNKDKVKEACSKHLKVAAYCTTQVERNACLFCKARKLDDKFYQKLRGQFPDAVFWQEISEIFRQLQKQAAEIYNQSLIELYYEIFIKGKGIANASSVEHYLSDVCYTRAAELFKNAAIASGLRSKIKSNFRLKELKNMKSGLPTTKSDNFPIPLVKQKGGQYTGFEISNHNSDFIIKIPFGRWQVKKEIDKYRPWEKFDFEQVQKSPKPISLLLSTQRRKRNKGWSKDEGTEAEIKKVMNGDYQTSYIEVKRGSKIGEKSAWMLNLSIDVPKIDKGVDPSIIGGIAVGVKSPLVCAINNAFSRYSISDNDLFHFNKKMFARRRILLKKNRHKRAGHGAKNKLKPITILTEKSERFRKKLIERWACEIADFFIKNKVGTVQM A NLESMKRKEDSYFNIRLRGFWPYAEMQNKIEFKLKQYGIEIRKVAPNNTSKTCSKCGHLNNYFNFEYRKKNKFPHFKCEKCNFKENADYNAALNISNPKLKSTKEEP
12
dTnpB
R518AA
MGEKSSRRRRNGKSGAWTAAITSCVGGKMAKNTITKTLKLRIVRPYNSAEVEKIVADEKNNREKIALEKNKDKVKEACSKHLKVAAYCTTQVERNACLFCKARKLDDKFYQKLRGQFPDAVFWQEISEIFRQLQKQAAEIYNQSLIELYYEIFIKGKGIANASSVEHYLSDVCYTRAAELFKNAAIASGLRSKIKSNFRLKELKNMKSGLPTTKSDNFPIPLVKQKGGQYTGFEISNHNSDFIIKIPFGRWQVKKEIDKYRPWEKFDFEQVQKSPKPISLLLSTQRRKRNKGWSKDEGTEAEIKKVMNGDYQTSYIEVKRGSKIGEKSAWMLNLSIDVPKIDKGVDPSIIGGIAVGVKSPLVCAINNAFSRYSISDNDLFHFNKKMFARRRILLKKNRHKRAGHGAKNKLKPITILTEKSERFRKKLIERWACEIADFFIKNKVGTVQMENLESMKRKEDSYFNIRLRGFWPYAEMQNKIEFKLKQYGIEIRKVAPNNTSKTCSKCGHLNNYFNFEY A KKNKFPHFKCEKCNFKENADYNAALNISNPKLKSTKEEP
13
dTnpB
D538A
A YNAALNISNPKLKSTKEEP
14

Label Sequence(5' to 3') SEQ ID
NO
d28aa-
extend-
sion Cas12f1
D354A
MAGGPGAGSAAPVSSTSSLPLAALNMRVMAKNTITKTLKLRIVRPYNSAEVEKIVADEKNNREKIALEKNKDKVKEACSKHLKVAAYCTTQVERNACLFCKARKLDDKFYQKLRGQFPDAVFWQEISEIFRQLQKQAAEIYNQSLIELYYEIFIKGKGIANASSVEHYLSDVCYTRAAELFKNAAIASGLRSKIKSNFRLKELKNMKSGLPTTKSDNFPIPLVKQKGGQYTGFEISNHNSDFIIKIPFGRWQVKKEIDKYRPWEKFDFEQVQKSPKPISLLLSTQRRKRNKGWSKDEGTEAEIKKVMNGDYQTSYIEVKRGSKIGEKSAWMLNLSIDVPKIDKGVDPSIIGGI A VGVKSPLVCAINNAFSRYSISDNDLFHFNKKMFARRRILLKKNRHKRAGHGAKNKLKPITILTEKSERFRKKLIERWACEIADFFIKNKVGTVQMENLESMKRKEDSYFNIRLRGFWPYAEMQNKIEFKLKQYGIEIRKVAPNNTSKTCSKCGHLNNYFNFEYRKKNKFPHFKCEKCNFKENADYNAALNISNPKLKSTKEEP
15
d28aa-
extend-
sion Cas12f1
E450A
MAGGPGAGSAAPVSSTSSLPLAALNMRVMAKNTITKTLKLRIVRPYNSAEVEKIVADEKNNREKIALEKNKDKVKEACSKHLKVAAYCTTQVERNACLFCKARKLDDKFYQKLRGQFPDAVFWQEISEIFRQLQKQAAEIYNQSLIELYYEIFIKGKGIANASSVEHYLSDVCYTRAAELFKNAAIASGLRSKIKSNFRLKELKNMKSGLPTTKSDNFPIPLVKQKGGQYTGFEISNHNSDFIIKIPFGRWQVKKEIDKYRPWEKFDFEQVQKSPKPISLLLSTQRRKRNKGWSKDEGTEAEIKKVMNGDYQTSYIEVKRGSKIGEKSAWMLNLSIDVPKIDKGVDPSIIGGIAVGVKSPLVCAINNAFSRYSISDNDLFHFNKKMFARRRILLKKNRHKRAGHGAKNKLKPITILTEKSERFRKKLIERWACEIADFFIKNKVGTVQM A NLESMKRKEDSYFNIRLRGFWPYAEMQNKIEFKLKQYGIEIRKVAPNNTSKTCSKCGHLNNYFNFEYRKKNKFPHFKCEKCNFKENADYNAALNISNPKLKSTKEEP
16
28aa-
extend-
sion Cas12f1
R518A
MAGGPGAGSAAPVSSTSSLPLAALNMRVMAKNTITKTLKLRIVRPYNSAEVEKIVADEKNNREKIALEKNKDKVKEACSKHLKVAAYCTTQVERNACLFCKARKLDDKFYQKLRGQFPDAVFWQEISEIFRQLQKQAAEIYNQSLIELYYEIFIKGKGIANASSVEHYLSDVCYTRAAELFKNAAIASGLRSKIKSNFRLKELKNMKSGLPTTKSDNFPIPLVKQKGGQYTGFEISNHNSDFIIKIPFGRWQVKKEIDKYRPWEKFDFEQVQKSPKPISLLLSTQRRKRNKGWSKDEGTEAEIKKVMNGDYQTSYIEVKRGSKIGEKSAWMLNLSIDVPKIDKGVDPSIIGGIAVGVKSPLVCAINNAFSRYSISDNDLFHFNKKMFARRRILLKKNRHKRAGHGAKNKLKPITILTEKSERFRKKLIERWACEIADFFIKNKVGTVQMENLESMKRKEDSYFNIRLRGFWPYAEMQNKIEFKLKQYGIEIRKVAPNNTSKTCSKCGHLNNYFNFEY A KKNKFPHFKCEKCNFKENADYNAALNISNPKLKSTKEEP
17
d28aa-
extend-
sion Cas12f1
D538A
A YNAALNISNPKLKSTKEEP
18

Example 1-4. Adenosine deaminase

In the present invention, the adenosine deaminase coupled to the nucleolytic protein of the subminiature base editing system for base editing is a representative example of tRNA adenosine deaminase (TadA) derived from E. coli or its variant eTadA. include The adenosine deaminase targets adenine (A) at a target site in a target nucleic acid or target gene, deaminates the adenine (A) base, and performs base correction with a guanine (G) base.

In an embodiment of the present invention, naturally occurring adenosine deaminase (TadA, SEQ ID NO: 126) and the mutant variant eTadA1 (SEQ ID NO: 127) of TadA or SEQ ID NO: 126 to SEQ ID NO: 131 any monomer or direct or linker are connected through to form a heterodimer TadA-eTadA or eTadA-TadA structure, and this heterodimer adenine deaminase is included in the CRISPR/Cas system according to the present invention.

More preferably, the linker is SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 62), and NLS is bonded to the C-terminus of the adenine deaminase heterodimer thus linked. Here, the NLS consists of the amino acid sequence of SEQ ID NO: 66 (KRPAATKKAGQAKKKK). This is shown in detail in FIG. 2 .

Example 1-5. Cytidine deaminase

In the present invention, it is combined with a nucleolytic protein of a subminiature base editing system for base editing, targets cytosine (C) at a target site in a target nucleic acid or target gene, and deaminates cytosine to uracil (Uracil, U) include APOBEC1 (SEQ ID NO: 21), APOBEC3A (SEQ ID NO: 22) or APOBEC3B (SEQ ID NO: 23). In the base conversion process, when the amine group of cytosine is removed to form uracil, uracil is converted to thymine (T) by a series of intracellular repair mechanisms, and finally base correction is completed from cytosine (C) base to thymine (T) base. .

In an embodiment of the present invention, the APOBEC1, APOBEC3A or APOBEC3B; Alternatively, one or two or more UGIs (Uracil Glycosylase Inhibitors) may be bound to the N-terminus or C-terminus of the nucleolytic protein; and additionally, NLS may be coupled to the C-terminus. In this case, the connection is made directly or through a linker. Here, the linker is SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 62) or SGGSKRTADGSEFE (SEQ ID NO: 63), and the NLS consists of the amino acid sequence of PKKKRKV (SEQ ID NO: 65). This is shown exemplarily in FIG. 3 .

Example 1-6. Subminiature Base Editing Structures for Base Editing

In the present invention, a subminiature base editing structure that is one component of a base editing system for base editing and has an activity of correcting a specific base of a target site with another base in a base sequence of a target nucleic acid or target gene is A base editor module containing deaminase-linked TnpB-derived low-molecular-weight nucleic acid degrading protein or a functional analogue thereof was produced and completed.

The subminiature base editing structure is a structure in which adenosine deaminase or cytidine deaminase is linked directly or by a linker to a low molecular weight nucleolytic protein derived from TnpB or a functional analogue thereof, which is NLS and/or It is a structure that additionally includes one or two or more UGIs (Uracil Glycosylase Inhibitors).

The subminiature base editing structure is composed of adenosine deaminase or cytidine deaminase type; and types of TnpB-derived low-molecular-weight nucleolytic proteins or functional analogues thereof; and their arrangement sequence; Depending on the back, there may be numerous combinations.

In FIG. 1 , various exemplary microminiature proofreading constructs are shown in detail.

Preferably, the ultraminiature base editing construct of the present invention is one in which adenosine deaminase heterodimer TadA-eTadA or eTadA-TadA linked through a linker is bonded to the N-terminus or C-terminus of dCas12f1 or dTnpB, and additionally C- NLS is attached to the end. This is shown in detail in Figure 4a. Here, TadA may have the amino acid sequence of SEQ ID NO: 126, may be dTadA (SEQ ID NO: 128), eTadA may have the amino acid sequence of SEQ ID NO: 127, deTadA1 (SEQ ID NO: 129), eTadA2 (SEQ ID NO: 129) 130), eTadA3 (SEQ ID NO: 131), eTadA7 (SEQ ID NO: 137), eTadA8 (SEQ ID NO: 138), eTadA9 (SEQ ID NO: 139), eTadA10 (SEQ ID NO: 140) or eTadA11 (SEQ ID NO: 141), but are limited thereto. it is not going to be

In addition, the subminiature base editing construct of the present invention binds the cytidine deaminase APOBEC1 to the N-terminus or C-terminus of dCas12f1 through a linker, and then binds 2 to its N-terminus or C-terminus. It was prepared by connecting two UGIs with a linker and additionally attaching NLS to the C-terminus. This is shown in detail in Figure 4b.

4a and 4b show an exemplary ultraminiature base editing construct according to the present invention, and dTnpB or a functional analogue thereof may be included instead of the illustrated dCas12f1.

The subminiature base editing construct was prepared by the following method. Nucleic acid sequences encoding the subminiature base editing constructs used in the present invention include human codon-optimized dCas12f1 DNA or human codon-optimized dCas12f1 functional analog DNA sequences. The nucleic acid sequence was cloned into the pMAL-c2 plasmid vector and cloned. BL21(DE3) E. coli was transformed using the plasmid vector. The transformed E. coli colonies were grown in LB broth at 37°C until an optical density of 0.7 was reached. The transformed E. coli was cultured overnight at 18° C. in the presence of 0.1 mM isopropylthio-β-D-galactoside.

Then, the transformed E. coli was centrifuged at 3,500 g for 30 minutes and collected. The collected transformed E. coli was resuspended in 20 mM Tris-HCl (pH 7.6), 500 mM NaCl, 5 mM β-mercaptoethanol, and 5% glycerol. The resuspended E. coli was dissolved and disrupted through sonication. Samples containing disrupted E. coli were centrifuged at 15,000 g for 30 minutes, and the supernatant was filtered through a 0.45 μm syringe filter (Millipore). The dCas12f1 protein bound to adenosine deaminase or cytidine deaminase present in the filtered supernatant was loaded onto a Ni ²⁺ -affinity column using an FPLC purification system (KTA Purifier, GE Healthcare). The loaded dCas12f1 protein was eluted with a 80-400 mM imidazole, 20 mM Tris-HCl (pH 7.5) gradient.

The eluted protein was treated with TEV protease for 16 hours. The isolated protein was purified on a Heparin column with a linear gradient of 0.15-1.6 M NaCl. Recombinant Cas12f1 protein purified on a heparin column was dialyzed against 20 mM Tris pH 7.6, 150 mM NaCl, 5 mM β-mercaptoethanol, 5% glycerol. The dialyzed protein was purified by passing it through an MBP column and resuspended on a monoS column (GE Healthcare) or EnrichS with a linear gradient of 0.5-1.2 M NaCl. The resuspended proteins were collected and dialyzed against 20 mM Tris pH 7.6, 150 mM NaCl, 5 mM β-mercaptoethanol, and 5% glycerol to obtain a base editing construct. The concentration of the produced protein was quantified using Bradford quantification using bovine serum albumin (BSA) as a standard and measured electropheromerically on a coomassie blue-stained SDS-PAGE gel.

Example 1-7. Engineered guide RNA

In the present invention, as one component of a subminiature base editing system for base editing, maximum targeting efficiency and targeting efficiency and We wanted to create an engineered guide RNA that has gene editing efficiency.

The present inventors also estimated that Cas12f1-mediated base editing by the miniaturized base editing system according to the present invention would appear at a high level at DNA target sites with high Cas12f1 endonuclease activity. Accordingly, a Cas12f1 guide RNA engineered as follows was prepared and its activity was tested.

The engineered Cas12f1 guide RNA according to the present invention adds a new structure to the guide RNA found in nature, and also modifies some of its structure, and includes a new structure, a U-rich tail, at the 3'-end. to be Specifically, the engineered guide RNA is an engineering tracrRNA sequence comprising modified scaffold first to fourth regions, an engineering crRNA sequence comprising modified scaffold fifth to sixth regions, and/or a modified seventh region. contains a U-rich tail sequence.

The fourth region and the fifth region are sites complementary to each other, and include modification site 1 (MS1) and modification site 4 (MS4), and the seventh region, U- The rich tail sequence corresponds to modification site 2 (MS2). The first region corresponds to modification site 3 (MS3), and the second region corresponds to modification site 5 (MS5).

Exemplary structures of engineered guide RNAs are shown in detail in FIG. 5 .

In the present invention, an engineered Cas12f1 single guide RNA (engineered Cas12f1 sgRNA) including modifications in any one of MS1 to MS5 and composed of various combinations of modifications selected from among them is included. The gene editing efficiency of the engineered Cas12f1 sgRNA having various combinations thus constructed was confirmed by measuring the indel generation rate (%).

As a result, as shown in FIG. 6, in canonical sgRNA (SEQ ID NO: 54) existing in nature, sgRNAs having modifications in the MS2, MS3, MS4 and MS5 regions (MS2/MS3/MS4/MS5, SEQ ID NO: 59) ; sgRNA with modifications in the MS2, MS3 and MS4 regions (MS2/MS3/MS4, SEQ ID NO: 58); and sgRNAs having modifications in the MS1, MS2 and MS3 regions (MS1/MS2/MS3, SEQ ID NO: 57); all showed a gene editing rate of 95% or higher.

Based on the above results, as one component of the base editing system according to the present invention, an engineered Cas12f1 guide RNA (engineered Cas12f1 sgRNA) was selected as follows.

The engineered Cas12f1 sgRNA is a canonical sgRNA (SEQ ID NO: 54) with modifications in MS1/MS2/MS3, and was named Cas12f_ge3.0 (SEQ ID NO: 57) in the present invention. In addition, sgRNA Cas12f_ge4.0 (SEQ ID NO: 58) with modifications at MS2/MS3/MS4 and sgRNA Cas12f_ge4.1 (SEQ ID NO: 59) with modifications at MS2/MS3/MS4/MS5 were constructed. Preferably, sgRNA Cas12f_ge4.1 having the shortest length was selected as an exemplary single guide RNA (sgRNA) of the ultra-small base editing system according to the present invention and used in subsequent experiments.

The specific structures and nucleotide sequences of the engineered Cas12f1 sgRNAs are shown in detail in FIG. 7 and Table 5.

gRNAs
Engineering Sequence(5' to 3') SEQ ID
NO Canonical sgRNAs CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAUUUUUCCUCUCCAAUUCUGCACAAgaaaGUUGCAGAACCCGAAUAGacgaaUGAAGGAAUGCAACNNNNNNNNNNNNNNNN 54 MS1 CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAGUGCUCCUCUCCAAUUCUGCAAgaaaGUUGCAGAACCCGAAUAGAGCAAUGAAGGAAUGCAACNNNNNNNNNNNNNNNNNN 55 MS1/MS2 CUUCACUGAUAAAGUGGAGAACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAGUGCUCCUCUCCAAUUCUGCACAAgaaaGUUGCAGAACCCGAAUAGAGCAAUGAAGGAAUGCAACNNNNNNNNNNNNNNNNUUUAUUUUUU 56 MS1/MS2/
MS3
(Cas12f_ge3.0) ACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAUUCAGUGCUCCUCUCCAAUUCUGCACAAgaaaGUUGCAGAACCCGAAUAGAGCAAUGAAGGAAUGCAACNNNNNNNNNNNNNNNNNNUUUUAUUUUUU 57 MS2/MS3/
MS4
(Cas12f_ge4.0) ACCGCUUCACCAAAAGCUGUCCCUUAGGGGAUUAGAACUUGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAgaaaGGAAUGCAACNNNNNNNNNNNNNNNNNNNNUUUUAUUUUUU 58 MS2/MS3/
MS4/MS5
(Cas12f_ge4.1) ACCGCUUCACUUAGAGUGAAGGUGGGCUGCUUGCAUCAGCCUAAUGUCGAGAAGUGCUUUCUUCGGAAAGUAACCCUCGAAACAAAgaaaGGAAUGCAACNNNNNNNNNNNNNNNNNNNNUUUAUUUUUUU 59

The engineered Cas12f1 sgRNA designed above was prepared by the following method. To prepare the designed Cas12f1 sgRNA, a guide RNA was chemically synthesized to prepare a guide RNA, and then a PCR amplicon containing the designed guide RNA sequence and the T7 promoter sequence was prepared.

U-rich tail ligation to the 3'-end of the engineered Cas12f1 single guide RNA was performed using Pfu PCR Master Mix5 (Biofact) in the presence of a sequence-modified primer and Cas12f1 guide RNA plasmid vector. The PCR amplicons were purified using the HiGene ^™ Gel & PCR Purification System (Biofact).

In addition, modifications of the second, fourth and fifth regions of the engineered scaffold region of the engineered Cas12f1 single guide RNA are sequences modified in a vector encoding the linearized guide RNA using ApoI and BamHI restriction enzymes. It was performed by cloning synthetic oligonucleotides containing

Modification of the first region of the engineered scaffold region of the engineered Cas12f1 single guide RNA is performed using a forward primer targeting the 5'-end of tracrRNA and a reverse primer targeting the U6 promoter region. ) was performed by PCR amplification of canonical or engineered template plasmid vectors using

The PCR amplification was performed by Q5 Hot Start high-fidelity DNA polymerase (NEB), and the PCR products were ligated using KLD Enzyme Mix (NEB). The ligated PCR products were transformed into DH5α E.coli cells. Mutagenesis was confirmed by Sanger sequencing analysis.

The modified plasmid vector was purified using the NucleoBond®Xtra Midi EF kit (MN). One microgram of the purified plasmid was used as a template for mRNA synthesis using T7 RNA polymerase (NEB) and NTPs (Jena Bioscience). The engineered Cas12f1 guide RNA prepared above was purified using Monarch® RNA cleanup kit (NEB), aliquoted into cryogenic vials, and stored in liquid nitrogen.

To prepare guide RNA and engineered guide RNA amplicons, using KAPA HiFi HotStart DNA polymerase (Roche) or Pfu DNA polymerase (Biofact), the canonical guide RNA template DNA plasmid and the engineered guide RNA template DNA plasmid were U6 - PCR amplification was performed using a complementary forward primer and a protospacer sequence-complementary reverse primer. The PCR amplification products were purified using Higene ^™ Gel & PCR purification system (Biofact) to obtain guide RNA and engineered guide RNA amplicons.

In vitro transcription was performed using the PCR amplicon as a template and NEB T7 polymerase. The in vitro transcription result was treated with NEB DNase I, purified using Monarch RNA Cleanup Kit (NEB), and guide RNA was obtained. Thereafter, a plasmid vector containing the previously designed guide RNA sequence and the T7 promoter sequence was prepared according to the Tblunt plasmid cloning method.

The vector was purified by cutting both ends of the guide RNA sequence including the T7 promoter sequence (double cut), and the resultant was subjected to in vitro transcription using NEB T7 polymerase. The in vitro transcription result was treated with NEB DNase I, purified using Monarch RNA Cleanup Kit (NEB), and guide RNA was obtained.

Example 1-8. Manufacture of ribonucleoprotein particles (RNP)

The protein expressing the subminiature base correction construct purified in Examples 1-6 and the engineered Cas12f1 single guide RNA (engineered Cas12f1 sgRNA) prepared in Examples 1-7 were incubated at room temperature for 10 minutes at 300 nM and 900 nM, respectively. Incubation was performed to prepare ribonucleoprotein particles (RNP).

Example 2. Plasmid vector design and manufacturing

The dCas12f1 gene, the dTnpB gene, or a functional analog gene thereof was human codon-optimized for expression in human cells, and an oligonucleotide of the gene containing the codon-optimized nucleotide sequence was synthesized. In addition, while including the nucleotide sequence of the synthesized dCas12f1, dTnpB or its functional analogue gene, the codon-optimized base editing (Base editing) encoding the base editing construct prepared in Examples 1-6 of the present invention editing) polynucleotide of the structure was synthesized.

The polynucleotide of the codon-optimized subminiature base editing construct is a chicken β-actin (CBA) promoter, a nuclear localization signal (NLS) sequence at the 5'-end and 3'-end, and self-cleavage To a plasmid containing a sequence encoding eGFP linked to the T2A peptide (2A) or a plasmid containing a CMV enhancer, a CMV promoter, and a nuclear localization signal (NLS) sequence at the 5'-end or 3'-end It was operably linked and cloned.

In addition, a template DNA for the canonical guide RNA used in this experiment was synthesized (Twist Bioscience), and cloned into a pTwist Amp plasmid vector and cloned. Template DNA for the engineered guide RNA was prepared using an enzyme cloning technique, which was cloned and cloned into the pTwist Amp plasmid. In addition, using the plasmid as a template, guide RNA or engineered guide RNA amplicons were prepared using a U6-complementary forward primer and a protospacer sequence-complementary reverse primer. . If necessary, the prepared amplicon was cloned into T-blunt plasmid (Biofact) and cloned.

In addition, in order to prepare engineered dual guide RNA (Engineered dual guide RNA), engineered tracrRNA and oligonucleotides encoding engineered crRNA were digested with restriction enzymes BamHI and HindIII restriction enzymes (New England Biolabs), and pSilencer 2.0 (ThermoFisher Scientific ) and cloned into.

Template DNAs encoding Cas12f_ge3.0, Cas12f_ge4.0, and Cas12f_ge4.1, respectively engineered Cas12f1 sgRNAs according to the present invention, were synthesized and cloned into a pTwist Amp plasmid vector (Twist Bioscience). If necessary, the vector was used as a template for amplification of the guide RNA coding sequence, using a U6-complementary forward primer and a protospacer-complementary reverse primer.

By cloning the polynucleotide encoding the Cas12f1 sgRNA engineered into a vector containing the polynucleotide of the codon-optimized base editing construct using Gibson assembly, expressing the base editing system A vector was prepared.

Specifically, as a vector expressing the base editing system, 1) a chicken β-actin (CBA) promoter, a nuclear localization signal (NLS) sequence at the 5'- and 3'-ends, and Sequence encoding eGFP linked with self-cleaving T2A peptide (2A) or 1) -1 CMV enhancer, CMV promoter, 5'- or 3'-end nuclear localization signal (NLS) sequence, 2) the present invention and 3) an adeno-associated virus inverted terminal repeat plasmid vector (AAV inverted terminal repeat vector, in which the engineered Cas12f1 sgRNA according to the present invention is operably linked to the polynucleotide of the codon-optimized tiny genetic scissors (CRISPR/Cas) construct according to ) and vectors were made.

Here, the dCas12f1 or its functional analog and guide RNA are transcribed from the chicken β-actin (CBA) promoter and U6 promoter, respectively; or by the CMV promoter and the U6 promoter; In addition, the vectors and AAV vectors may be appropriately changed according to the purpose of gene editing, such as eGFP, the number of engineered Cas12f1 sgRNAs, and/or the addition of effector proteins.

The structure of an exemplary vector produced in the present invention is shown in detail in FIG. 8 .

To mass-produce the AAV, the AAV vector, helper plasmid, and RC plasmid were transduced into HEK 293T cells, and the transduced HEK 293T cells were cultured in DMEM medium containing 2% FBS. Recombinant pseudotyped AAV vector stocks were generated using PEIpro (Polyplus-transfection) and PEI coprecipitation using triple-transfection for plasmids at equal molar ratios. After 72 hours of culture, the cells were lysed, and the AAV was purified from the lysate by iodixanol (Sigma-Aldrich) stepgradient ultracentrifugation.

Example 3. Cell Transduction

HEK 293T (ATCC CRL-11268), HeLa (ATCC CLL-2), U-2OS (ATCC HTB-96) and K-562 (ATCC CCL-243) cells were cultured in 10% heat-inactivated FBS, 1% penicillin/strepto In DMEM medium supplemented with mycin and 0.1 mM non-essential amino acids, they were cultured at 37 °C and 5% CO ₂ conditions.

For cell transfection of the plasmid vector encoding the base editing construct prepared in Example 2 of the present invention and the engineered guide RNA, 1.0 x 10 ⁵ HEK 293T cells were seeded 1 day before transfection. . Cell transfection was performed by electroporation or lipofection.

In the case of electroporation, 2-5 μg each of DNA encoding the plasmid vector encoding the base editing construct and the engineered guide RNA was 4 X 10 ⁵ HEK 293 T using the Neon transfection system (Invitrogen). Cells were transfected. The electroporation was performed under 1300V, 10 mA, 3 pulse conditions.

For lipofection, 6-15 μl FuGene reagent (Promega) was mixed with 2-5 μg of a plasmid vector encoding the miniaturization construct and 1.5-5 μg of a PCR amplicon and 15 Mixed for minutes. This mixture (300 μl) was added to 1.5 ml DMEM medium in which 1×10 ⁶ cells were plated 1 day before transfection. The cells were cultured for 1 to 10 days in the presence of the mixture. After culturing, the cells were harvested and the genomic DNA of the cells was isolated using the PureHelix™ genomic DNA preparation kit (NanoHelix) or manually using the Maxwell RSC Cultured Cells DNA Kit (Promega).

1, 5, 10, 50, 100, 100, 1000 determined by quantitative PCR for cell transfection of the AAV vector containing the nucleic acid sequence encoding the subminiature base editing system prepared in Example 2 above. , Human HEK 293T cells were infected with the AAV vectors at different multiplicities of infection (MOI) of 10000, 50000 and 100000. The transfected HEK 293T cells were cultured in DMEM medium containing 2% FBS. Cells were harvested for isolation of genomic DNA at different time points, eg on days 1, 3, 5, 7, and 9.

In addition, the ribonucleoprotein particles (RNP) prepared according to Examples 1-8 are transfected using electroporation, or after transfection through the lipofection method, 1 day later, the prepared according to Examples 1-7 Engineered guide RNAs were transfected using electroporation.

Example 4. Analysis of results

Example 4-1. Gene editing efficiency analysis

Among genomic DNAs isolated from HEK 293T cells, PCR was performed on a region containing a protospacer in the presence of KAPA HiFi HotStart DNA polymerase (Roche) using target-specific primers. The amplification method followed the manufacturer's instructions.

PCR amplicons resulting from the above amplification containing Illumina TruSeq HT dual indexes were subjected to 150 bp pair-end sequencing using Illumina iSeq 100. Indel frequencies were calculated using MAUND. The MAUND is provided at https://github.com/ibscge/maund.

PCR products were obtained using BioFACT ^™ Lamp Pfu DNA polymerase. The PCR product (100-300 μg) was reacted with 10 units of T7E1 enzyme (NewEngland Biolabs) in a 25 μg reaction mixture at 37° C. for 30 minutes. 20 μl of the reaction mixture was directly loaded on a 10% acrylamide gel, and the digested PCR products were run in a TBE buffer system. After staining the gel image with an ethidium bromide solution, it was digitized using a Printgraph 2 M gel imaging system (Atto). The digitized result was analyzed to evaluate gene editing efficiency. In addition, in order to identify unwanted indels, analysis was performed through NGS.

Example 4-2. Intracellular base proofreading activity assay

Deamination analysis of an adenine (A) base or a cytosine (C) base located at a target site of a target nucleic acid or target gene in a cell was performed as follows.

The adeno-associated virus (AAV) vector constructed in Example 2 of the present invention was transduced into HEK 293T cells. After 3, 5 and 7 days, genomic DNA was obtained from the transfected HEK 293T cells and purified using a Genomic DNA prep kit (QIAGEN, Catalog #: 69504). After the target nucleic acid or the target region of the target gene was amplified by PCR in the purified product, the final PCR product was analyzed using targeted deep sequencing.

The target site was amplified for library generation using the KAPA HiFi HotStart PCR kit (KAPA Biosystems #: KK2501). This library was sequenced using a MiniSeq with TruSeq HT Dual Index system (Illumina).

Example 4-3. statistical analysis

Statistical significance verification by two-tailed Student's t-test was performed using Sigma Plot software (ver. 14.0). A p-value of less than 0.05 was considered statistically significant, and the p-value was shown in each figure. Error bars of all data are shown using Sigma plot, and mean the standard deviation value of each data. Sample size was not pre-determined based on statistical methods. Experiments for each experimental example were performed three times, and the average value of each value was used for analysis.

Example 5. Confirmation of base editing by a mini base editing system

Example 5-1. Adenine editing by a subminiature base editing construct containing the adenosine deaminase of the present invention

In order to investigate whether the base editing system according to the present invention has a base editing activity that replaces an adenine (A) base located in a target sequence of a target nucleic acid or gene with a guanine (G) base in a cell , 10 human endogenous DNA target sites containing the PAM sequence of Cas12f1 and containing a large number of adenine (A) within the calibration window were identified. The target sequences used in the experiment are shown in the following [Table 6].

Target name Target sequence (5' to 3') SEQ ID NO: Target-1 [TTTG]CACACACACAGTGGGCTACC 110 Target-2 [TTTG]CATCCCCAGGACACACACAC 111 Target-3 [TTTA]CAAAGACACTCACCCTGTTG 112 Target-4 [TTTA]AAGAAAGCTACAGGAAAGCA 113 Target-5 [TTTA]CAAAACCCAACTGATTCACC 114 Target-6 [TTTA]CAAAAGCTACCACACATAGC 115 Target-7 [TTTA]CAAAACTGTGGCCAATACAG 116 Target-8 [TTTG]GAAAACTGCAGGCAAGATTC 117 Target-9 [TTTG]CAAAACTGTACACGTGGGCC 118 Target-10 [TTTG]CAAAACGTGCACAATGTGCA 119

ABE-N1, ABE-N2, ABE-C1 or ABE-C2 according to the present invention, Cas12f_ge4.1 with the smallest size among the plasmids expressing the base editing constructs and the engineered sgRNA produced in the present invention A plasmid expressing the sgRNA was transfected into human HEK 293T cells, and genomic DNA was isolated after 3 days.

The target region of the base editing system according to the present invention was amplified by PCR method, and the amplified amplicon was processed by target deep sequencing.

Then, the ABE-N1, ABE-N2, ABE-C1 or ABE-C2 subminiature base editing constructs were tested for adenine base editing activity using a base editing analyzer.

To this end, for the 10 target sites listed in Table 6, N1 to N20 in the single guide RNA (sgRNA) binding region of the 5'-end target site, which is the expected range of editing windows (at the 1st nucleotide following the PAM sequence) 20th nucleotide) was analyzed for substitution of all bases with other bases at each nucleotide in the range.

As a result, as shown in FIG. 9, the mini base editing system including the ABE-N1, ABE-N2, ABE-C1 or ABE-C2 subminiature base editing structure according to the present invention is adenine within the correction window range. It was confirmed that (A) was effectively corrected with guanine (G). In particular, the subminiature base editing constructs ABE-N1, ABE-N2, ABE-C1 or ABE-C2 according to the present invention showed the highest efficiency in substituting adenine bases at positions A3 and A4 with guanine bases.

In the case of dCas12f1-ABE-N1, a subminiature base editing construct containing dCas12f1 (D326A), the frequency of correcting the A2 base of Target-1 with guanine is about 15% (FIG. 9a), and the Target-1 The frequency of proofreading the A3 and A4 bases of 3 with guanine was confirmed to be about 41% and about 35%, respectively (FIG. 9b). In addition, it was confirmed that A3 and A4 bases of Target-5, Target-7, and Target-8 were simultaneously corrected with guanine at a frequency of 30 to 40% (FIG. 9c).

In the case of dTnpB-ABE-C2, a subminiature base editing construct containing dTnpB (D354A), the frequency of editing the A4 base of Target-1 with guanine was about 14% (FIG. 9d). In addition, the frequency of correcting the A3 and A4 bases of Target-3 with guanine was confirmed to be about 47% and about 40%, respectively, and the frequency of correcting the A4 base of Target-4 with guanine was very high at about 37% ( Figure 9e).

In addition, dCas12f1 or dTnpB's dead variants dCas12f1 (D326A), dCas12f1 (E422A), dCas12f1 (R490A), dCas12f1 (D510A) and their corresponding dTnpB (D354A), dTnpB (E450A), dTnpB (R518A) , the base proofreading rate of dTnpB (D538A) was confirmed. Here, dCas12f1-TadAeTadA1 (FIG. 10A) and dTnpB-TadAeTadA3 (FIG. 10B) were used as subminiature base editing constructs.

As a result, as shown in FIGS. 10A and 10B, dCas12f1 (D326A), dCas12f1 (E422A), dCas12f1 (R490A), and dCas12f1 (D510A) were about 9%, about 13%, and about 3% respectively for 3A of Target-3. and about 5% of base proofreading rates, and 4A showed base proofreading rates of about 4%, about 5%, about 2%, and about 7%, respectively. In the case of dTnpB (D354A), dTnpB (E450A), dTnpB (R518A), and dTnpB (D538A), base correction rates of about 10%, about 14%, about 11%, and about 17% for 2A of Target-3 were obtained, respectively. 3A showed base correction rates of about 22%, about 25%, about 17%, and about 33%, respectively, and about 20%, about 22%, about 15%, and about 26% of bases for 4A, respectively. showed the correction rate.

As a result, it was confirmed that dTnpB had a better base editing efficiency than dCas12f1.

On the other hand, it was confirmed whether the base editing efficiency of the miniaturized base editing system according to the present invention is affected by the promoter, linker length, nuclease variant type or Tad type.

As a result, the mini base editing system including the mini base editing construct dTnpB-TadAeTadA2 showed excellent base editing efficiency for both the CBA promoter and the CMV promoter, especially when the CBV promoter was combined with gRNA Ver4.1. showed the best activity (FIG. 11).

The length of the linker between dCas12f1 or dTnpB and the deaminase all showed similar activity in the length of 10 to 40 amino acids, confirming that the linker length did not significantly affect the base editing efficiency (FIG. 12).

Variants of dCas12f1 were tested: dCas12f1 (D510A), dCas12f1 (I131W), dCas12f1 (S136Y) and dCas12f1 (D538A, I131W, S136Y) (Fig. 13a), and variants of dTnpB were dTnpB (D538A), dTnpB (D1538A) , dTnpB (D538A, S164Y) and dTnpB (D538A, I159W, S164Y) were selected (Fig. 13b). As a result, the tested mutants showed similar base correction rates for 3A and 4A of Target-3. In the case of the I159W and/or S164Y mutants, the base correction rate for 6A of Target-3 was significantly increased compared to the wild type (FIG. 13c). At this time, it was confirmed that the variants of dTnpB had no indel efficiency (FIG. 13d).

Figure 14 shows the result of the base correction window of the amino acid substitution variant of dCas12f1 or dTnpB extending from target sequence 1 to N2 to N18.

In addition, as an example of implementation, as a result of verifying the base correction system using TadAeTadA3 in various target sequences, it was confirmed that base correction windows are available from A2 to A8 and/or A15 to A20 in 25 different target sequences (FIG. 15).

As for the base editing efficiency according to the Tad variants, as shown in FIG. 16, TadA-eTadA2, eTadA2 and TadA-eTadA1 showed excellent base editing efficiency together with dCas12f1 or dTnpB. This means that Tad is an important factor affecting the base proofreading efficiency.

Next, the base-correction effect of adenine (A) to guanine (G) in the microbase-correction system containing TnpB protein and TnpB-derived nucleic acid degrading protein having a small molecular weight was confirmed.

To this end, as a dead variant of the TnpB protein or a functional analogue of Cas12f1, CasX (N-terminal 26 amino acids) -Cas12f1, including the respective dead variants for 26aa-extended Cas12f1 or 28aa-extended Cas12f1 and a miniaturized base-correction construct, ABE-C2, containing TadAeTadA1 as an adenosine deaminase. Then, to the prepared microbase correction construct, sgRNA Cas12f_ge4.0 (SEQ ID NO: 58) with modifications in MS2 / MS3 / MS4 or sgRNA Cas12f_ge4.1 (SEQ ID NO: 59) with modifications in MS2 / MS3 / MS4 / MS5 A micro-base correction system including a was fabricated.

The base proofreading effect of each of the miniaturized base proofreading systems is shown in [Table 7].

ABE Target-1 Target-3 2A 4A 6A 8A 2A 3A 4A 6A 8A dCas12f-ver4.0 2.22 4.74 0.60 0.73 2.40 19.92 13.49 2.95 0.57 dcas12f-ver4.1 2.08 12.12 0.69 0.59 3.35 30.29 18.29 2.95 0.60 dTnpB-ver4.0 1.20 22.36 0.60 0.83 5.33 56.88 34.52 3.73 0.53 dTnpB-ver4.1 2.47 23.86 0.68 0.73 5.69 59.29 26.71 4.08 0.55 dCasX-ver4.0 2.37 16.30 0.51 0.64 3.96 38.87 23.11 3.19 0.59 dCasX-ver4.1 3.03 30.12 0.66 0.64 3.15 46.81 22.08 3.63 0.53 d26ext-ver4.0 2.63 10.98 0.73 0.76 3.75 29.85 19.67 3.25 0.55 d26ext-ver4.1 2.52 30.44 0.69 0.66 3.32 40.15 21.24 3.40 0.48 d28ext-ver4.0 2.57 14.58 0.57 0.56 4.68 38.92 25.48 3.64 0.52 d28ext-ver4.1 2.87 32.98 0.64 0.56 5.59 53.20 27.88 3.49 0.56 wt 0.49 0.37 0.40 0.60 0.30 0.36 0.39 0.50 0.30

As a result, as shown in Table 7, the dTnpB protein, CasX (N-terminal 26 amino acids) -dCas12f1, 26aa-elongated dCas12f1 or 28aa-elongated dCas12f1; and sgRNA Cas12f_ge4.0 (SEQ ID NO: 58) having a modification in MS2/MS3/MS4, the frequency of correcting A4 adenine of Target-1 with guanine is about 22%, about 16%, and about 11%, respectively. and about 15%. This shows a much higher proofreading rate than the base proofreading rate of about 5% in the case of dCas12f1. In addition, when the sgRNA Cas12f_ge4.1 (SEQ ID NO: 59) having a modification in MS2/MS3/MS4/MS5 is included, the dTnpB protein, CasX-dCas12f1, 26aa-elongated dCas12f1 and 28aa-elongated dCas12f1 are about 24%, respectively. The base proofreading rate was 30%, about 30%, and about 33%, confirming that adenine was corrected with guanine at a proofreading rate more than twice as high as the base proofreading rate of about 12% for dCas12f1.

It was confirmed that the ultraminiature base editing system including the TnpB protein or a functional analog of Cas12f1 has a remarkable effect of simultaneously correcting A3 adenine, A4 adenine, and A6 adenine of Target-3 with guanine.

The dTnpB protein, CasX (N-terminal 26 amino acids) -dCas12f1, 26aa-elongated dCas12f1 or 28aa-elongated dCas12f1 combined with sgRNA Cas12f_ge4.0 (SEQ ID NO: 58) were about 57%, about 39%, about 30% and A3 adenine in Target-3 was corrected with guanine at a correction rate of about 39%, and A4 adenine in Target-3 was corrected with guanine at correction rates of about 35%, about 23%, about 20%, and about 25%. This is about twice as high as dCas12f1, which corrects A3 adenine and A4 adenine of Target-3 with guanine at a correction rate of about 20% and about 13%, respectively.

In addition, base correction rates of the dTnpB protein, CasX-dCas12f1, 26aa-elongated dCas12f1 or 28aa-elongated dCas12f1 combined with sgRNA Cas12f_ge4.1 (SEQ ID NO: 59) were confirmed.

The dTnpB protein, CasX (N-terminal 26 amino acids)-dCas12f1, 26aa-extended dCas12f1 or 28aa-extended dCas12f1, had correction rates of about 60%, about 47%, about 40%, and about 53%, respectively, for A3 of Target-3. Adenine was corrected with guanine, and A4 adenine in Target-3 was corrected with guanine at correction rates of about 27%, about 22%, about 21%, and about 28%. This indicates a proofreading efficiency about 1.5 to 2 times higher than that of dCas12f1, which corrects A3 adenine and A4 adenine of Target-3 with guanine at a proofreading rate of about 30% and about 18%, respectively.

Through the above results, the subminiature base editing structure containing adenine deaminase and the subminiature base editing system including the adenine deaminase according to the present invention are less than half the size of the existing adenine base editing and can be applied intracellularly. It was confirmed that it could be an easy new adenine base editor.

It was confirmed that the base editing system according to the present invention can perform base editing in a narrow range of only two bases and simultaneously replace two consecutive adenine bases with guanine bases.

In addition, the base editing system according to the present invention for adenine base editing has a correction window ranging from A2 to A8 and/or A15 to A20 depending on the type of nuclease and/or adenosine deaminase. It was confirmed that it can be extended to .

In summary, the base editing system according to the present invention can solve the problem of base editing that causes silent mutation, and in the case of the stop codon UAA, even if base editing occurs from the third adenine to guanine It is still a stop codon UAG and can overcome the problem of not showing the effect of base editing, and it is a new base editing gene scissors that has the advantage of solving the target site restriction by selectively using an ultra-small base editing system with a wide base editing window. (Base Editor).

Example 5-2. Cytosine editing by a subminiature base editing construct containing the cytidine deaminase of the present invention

In the present invention, it was tested whether the subminiature base editing system according to the present invention has cytosine base editing activity by substituting a cytosine base located in a target sequence of a target nucleic acid or gene in a cell. To confirm this, three human endogenous DNA target sites including the PAM sequence of Cas12f1 and containing a large number of cytosines within the correction window were identified. The target sequences used in the experiment are shown in the following [Table 8].

Target name Target sequence (5' to 3') SEQ ID No. Target-2 [TTTG]CATCCCCAGGACACACACAC 111 Target-11 [TTTA]CCCCCACAGGATTGTAATAA 120 Target-12 [TTTA]GGCCAAGTGCGAAGTCAGAG 121

In order to confirm the cytosine editing, among the plasmids expressing the CBE-N1, CBE-N2, CBE-C1 or CBE-C2 subminiature base editing constructs according to the present invention and the engineered sgRNA produced in the present invention A plasmid expressing the smallest Cas12f_ge4.1 sgRNA was transfected into human HEK 293T cells, and 3 days later, genomic DNA was isolated.

The target region of the base editing system according to the present invention was amplified by PCR method, and the amplified amplicon was processed by target deep sequencing. Thereafter, the cytosine base editing gene editing activity of the subminiature base editing construct containing the cytosine deaminase was tested using a base editing analyzer.

To this end, for the three target sites described in [Table 8], N2 to N20 in the single guide RNA (sgRNA) binding region of the 5'-end target site, which is within the editing window range (the 2nd nucleotide following the PAM sequence) At each nucleotide in the range (from the 20th nucleotide), substitution of all bases with other bases was analyzed.

As a result, as shown in FIG. 17, the base editing system including the base editing structures CBE-C1 and CBE-C2 according to the present invention corrects cytosine to thymine within the correction window. It was confirmed that

In the case of Target-2, both CBE-C1 and CBE-C2 subminiature base editing structures had the highest frequency of substituting cytosine bases at positions C4 and C5 with thymine bases (FIG. 17a). In addition, in the case of the CBE-C2 subminiature base editing construct, the frequencies of correcting the C3 and C4 bases of Target-12 with thymine were confirmed to be about 20% and about 16%, respectively (FIG. 17b).

From the above results, the subminiature base editing construct containing the cytidine deaminase of the present invention has two cytosines located consecutively from C3 to C5 following the PAM sequence, that is, C3 and C4; or C4 and C5; were confirmed to be able to be corrected with thymine at the same time.

As a result, it was confirmed that the ultra-small base correction system for cytosine base correction according to the present invention has a narrow correction window in the range of C3 to C5, and two consecutive cytosine bases within that range can be simultaneously corrected with thymine base.

Like the base editing system for adenine base editing according to the present invention, the base editing system for cytosine base editing according to the present invention is also a base editing system that causes silent mutations. The problem can be solved, and in the case of the stop codon UAA, even if the third adenine is base-corrected with guanine, it still becomes the stop codon UAG and has the advantage of overcoming the problem that the effect of base correction does not appear. editor).

Example 5-3. Testing for unwanted indels

On the other hand, existing adenine base editing scissors (ABEs) or cytosine base editing scissors (CBEs) in which adenosine deaminase or cytidine deaminase are linked to dCas9 or nCas9 proteins are used to modify target nucleic acids in addition to base correction. This is a problem because double-stranded DNA cleavage causes 'unwanted indels' in which bases are deleted or added in the target nucleic acid.

Accordingly, it was confirmed whether the base editing structure and the base editing system including the base editing structure according to the present invention cause unwanted indels in the gene editing process of base editing.

As a result, as shown in FIG. 18, both the base editing system ABE-C2 for adenine base editing and the base editing system CBE-C2 for cytosine base editing according to the present invention are desired. It was confirmed that almost no indels occurred.

However, in the existing dCas9 or nCas9 protein-based adenine base editing scissors (ABEs) ABE7.10 and ABE8e and cytosine base editing scissors (CBEs) BE4 and BE4-Gam, the ABE-C2 and CBE-C2 according to the present invention It was confirmed that each of them generated 10-fold or more unwanted indels compared to each other.

The above results are in stark contrast to the similar base proofreading activity of proofreading adenine (A) with guanine (G) or proofreading cytosine (C) with thymine (T). This indicates that the base editing structure completed in the present invention and the base editing system including the same are very useful in a situation where only specific base editing is required without generating indels. suggests

Furthermore, the results show that the base editing structure and the base editing system including the base editing system according to the present invention having a wide application range as the smallest base editing system have indel, base editing or prime editing ( It is a new confirmation that the target gene editing method, such as prime editing, can be selected without restrictions and can be performed effectively without side effects.

Example 5-4. Intracellular base editing of the ultra-small base editing system according to the present invention

The base editing system according to the present invention confirmed the efficiency of base editing by replacing an adenine (A) base or a cytosine (C) base located at a target site of a target nucleic acid or target gene in a cell with another base. .

In order to confirm the efficiency of the base editing, a transduced cell line was prepared to normally express eGFP but not to express the mRuby gene due to the stop codon present in the front part. First, 1) a chicken β-actin (CBA) promoter, a nuclear localization signal (NLS) sequence at the 5'- and 3'-ends, and a sequence encoding eGFP connected to the self-cleaved T2A peptide (2A); 2) a target sequence (Target) and / or an auxiliary target sequence (Auxillary) polynucleotide that can be recognized by the codon-optimized subminiature base editing construct according to the present invention and has a stop codon (TAG); and 3) a plasmid vector in which the mRuby gene sequence was operably linked. The vectors were transduced into HEK 293T cells. HEK 293T cells into which the plasmid vector was inserted normally express eGFP, but cannot express the mRuby gene due to the stop codon present in the front part of the HEK 293T cell. The plasmid vector structure and the target sequence are shown in detail in FIGS. 19A and 20A.

Next, the HEK 293T cells into which the plasmid vector was inserted were additionally transfected with AAV containing the nucleic acid encoding the base editing system of the present invention prepared in Example 2. Here, the AAV vector encodes a nucleic acid encoding ABE-C2, a subminiature base editing construct having adenine base editing activity prepared in Examples 1-6, and/or an auxiliary target sequence (Auxillary) or TnpB-ABE. A polynucleotide of a nucleic acid is included.

The AAV thus prepared is named rAAV-ABE-C2 vector and rAAV-TnpB-ABE vector in the present invention. The structure of the vector is shown in detail in FIGS. 19B and 20B, respectively.

In HEK293T cells transfected with the rAAV-ABE-C2 vector or the rAAV-TnpB-ABE vector, the base editing system according to the present invention replaces adenine (A) base with guanine (G) base. It was confirmed whether mRuby gene was expressed.

The reason for determining whether mRuby is expressed is that the vector is transferred into the transduced HEK 293T cells, recognizes the target sequence, is located at the site, and adenine of the stop codon T A G present in the target sequence (A) If the base is substituted with a guanine (G) base, the stop codon existing at the front of the mRuby gene is changed to T G G (Trp) and the stop codon disappears, so the mRuby gene will be normally expressed. .

As a result, as shown in FIG. 19c, it was confirmed that the base editing system according to the present invention included in the rAAV-ABE-C2 vector normally expresses the mRuby gene in the transduced HEK 293T cells. . The base correction rate was found to be 25.2%.

In addition, the base editing system according to the present invention included in the rAAV-TnpB-ABE vector shows a base editing rate of about 40% on the 6th day and 60% or more on the 9th day after transduction, HEK 293T It was confirmed that the mRuby gene was normally expressed in the cells (FIG. 20c).

Example 5-5. Comparison of intracellular base editing efficiency with existing base editing gene scissors (BEs)

In addition, the intracellular base editing activity of the ultra-small base editing system according to the present invention was compared with the adenine base editing gene scissors miniABEmax having a size capable of being transferred to an existing AAV vector.

To this end, an AAV vector (ABE-C2) containing a nucleic acid encoding the subminiature base editing construct ABE-C2 having an adenine base editing activity prepared in Examples 1-6, An AAV vector (ABE-C2 + sgRNA) containing nucleic acid encoding a base editing system and an AAV vector (ABE-C2 + sgRNA) containing an auxiliary target sequence (Auxillary) polynucleotide in the ABE-C2 + sgRNA + Auxillary).

In addition, a conventional spCas9n-based adenine base editing gene scissors AAV vector, miniABEmax, was prepared to compare base editing efficiency with the miniaturized base editing system according to the present invention.

The prepared vectors were transfected into HEK 293T cells, and 3 days later, genomic DNA was obtained from the infected HEK 293T cells. The PCR product thereof was analyzed for substitution of an adenine (A) base with a guanine (G) base using targeted deep sequencing.

As shown in FIG. 21a, the ABE-C2 + sgRNA and ABE-C2 + sgRNA + Auxillary, which are subminiature base editing systems according to the present invention, replace adenine (A) bases with guanine (G) bases. Base correction rates were found to be 30% and 40%, respectively.

On the other hand, the existing SpCas9n-based adenine nucleotide editing scissors miniABEmax showed low adenine nucleotide editing specificity, less than 5%.

The above results show that the subminiature base editing system containing dCas12f1 or its functional analogue according to the present invention has the advantage of a wide range of gene editing applications due to the small size described above, and the most existing research in base editing. was progressed, and it was confirmed that the base editing efficiency was significantly increased compared to those currently used as base editing gene scissors.

Next, the proofreading efficiency of the existing Cas9-based ABE and the Cas12f1 or TnpB-based ABE according to the present invention was compared.

As a result, as shown in FIG. 21b, TnpB-based ABE-C3.1 (same as dTnpB-TadAeTadA3) is A3 compared to Cas12f1-based ABEMINI (same as TadAeTadA1-dCas12f1) or Cas9-based ABE7.10, 8e, and 9. and A4 showed excellent base proofreading rates.

This means that the TnpB-based ABE exhibits the best ABE activity.

Example 5-6. Multiplex gene editing of microbases through AAV delivery

The greatest advantage of the ultra-small base editing system according to the present invention is that there is no restriction on the transfer size limit of AAV. Moreover, the base correction system developed through the present invention has an additional space within the AAV delivery limit when one guide RNA is inserted. By adding additional elements such as guide RNA or shRNA in this additional space, it has the advantage of being able to correct multiple genes simultaneously.

To prove this, as shown in FIG. 22a, AAV2 loaded with one different guide RNA and AAV2 loaded with the two guide RNAs were produced. Then, HEK293T cells were infected with AAV2 containing a nucleic acid encoding dTnpB-TadAeTadA3 (ABE-C3.1) at the same multiplicity of infection (MOI).

The prepared AAV2 vectors were transfected into HEK 293T cells, and 10 days later, genomic DNA was obtained from the infected HEK 293T cells. The PCR product thereof was analyzed for substitution of an adenine (A) base with a guanine (G) base using targeted deep sequencing.

As a result of the analysis, as shown in FIG. 22B, the efficiency of adenine base correction was confirmed for both targets in AAV2 loaded with two types of guide RNAs. Each efficiency was similar to the case of loading a single guide RNA. Through this, it was confirmed that the ultra-small base editing system is capable of multi-target base editing through the AAV delivery system.

Summarizing the results of the above examples according to the present invention, the Cas12f1 protein and functional analogs thereof of the present invention have excellent proofreading efficiency activity in base proofreading. In addition, due to the remarkably small size of the Cas12f1 protein and its functional analogues, functional domains such as base editors or prime editors can be included in one AAV vector, and furthermore, two or more gene editing constructs can be included together. there is.

This indicates that the scope of application of the miniaturized base editing system including dCas12f1 and its functional analogs according to the present invention is very wide in various fields, and ultimately, the next generation gene editing system is replaced by the ultraminiature base editing system of the present invention. will advance

The description of the present invention described above is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. There will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

<110> GenKOre Co., Ltd. Korea Research Institute of Bioscience and Biotechnology <120> Hypercompact base editing systems and use thereof <130> PN21407 <150> KR 10-2021-0087956 <151> 2021-07-05 <160> 178 <170> KoPatentIn 3.0 <210> 1 <211> 529 <212> PRT <213> unknown <220> <223> Cas12f1 <400> 1 Met Ala Lys Asn Thr Ile Thr Lys Thr Leu Lys Leu Arg Ile Val Arg 1 5 10 15 Pro Tyr Asn Ser Ala Glu Val Glu Lys Ile Val Ala Asp Glu Lys Asn 20 25 30 Asn Arg Glu Lys Ile Ala Leu Glu Lys Asn Lys Asp Lys Val Lys Glu 35 40 45 Ala Cys Ser Lys His Leu Lys Val Ala Ala Tyr Cys Thr Thr Gln Val 50 55 60 Glu Arg Asn Ala Cys Leu Phe Cys Lys Ala Arg Lys Leu Asp Asp Lys 65 70 75 80 Phe Tyr Gln Lys Leu Arg Gly Gln Phe Pro Asp Ala Val Phe Trp Gln 85 90 95 Glu Ile Ser Glu Ile Phe Arg Gln Leu Gln Lys Gln Ala Ala Glu Ile 100 105 110 Tyr Asn Gln Ser Leu Ile Glu Leu Tyr Tyr Glu Ile Phe Ile Lys Gly 115 120 125 Lys Gly Ile Ala Asn Ala Ser Ser Val Glu His Tyr Leu Ser Asp Val 130 135 140 Cys Tyr Thr Arg Ala Ala Glu Leu Phe Lys Asn Ala Ala Ile Ala Ser 145 150 155 160 Gly Leu Arg Ser Lys Ile Lys Ser Asn Phe Arg Leu Lys Glu Leu Lys 165 170 175 Asn Met Lys Ser Gly Leu Pro Thr Thr Lys Ser Asp Asn Phe Pro Ile 180 185 190 Pro Leu Val Lys Gln Lys Gly Gly Gln Tyr Thr Gly Phe Glu Ile Ser 195 200 205 Asn His Asn Ser Asp Phe Ile Ile Lys Ile Pro Phe Gly Arg Trp Gln 210 215 220 Val Lys Lys Glu Ile Asp Lys Tyr Arg Pro Trp Glu Lys Phe Asp Phe 225 230 235 240 Glu Gln Val Gln Lys Ser Pro Lys Pro Ile Ser Leu Leu Leu Ser Thr 245 250 255 Gln Arg Arg Lys Arg Asn Lys Gly Trp Ser Lys Asp Glu Gly Thr Glu 260 265 270 Ala Glu Ile Lys Lys Val Met Asn Gly Asp Tyr Gln Thr Ser Tyr Ile 275 280 285 Glu Val Lys Arg Gly Ser Lys Ile Gly Glu Lys Ser Ala Trp Met Leu 290 295 300 Asn Leu Ser Ile Asp Val Pro Lys Ile Asp Lys Gly Val Asp Pro Ser 305 310 315 320 Ile Ile Gly Gly Ile Asp Val Gly Val Lys Ser Pro Leu Val Cys Ala 325 330 335 Ile Asn Asn Ala Phe Ser Arg Tyr Ser Ile Ser Asp Asn Asp Leu Phe 340 345 350 His Phe Asn Lys Lys Met Phe Ala Arg Arg Arg Ile Leu Leu Lys Lys 355 360 365 Asn Arg His Lys Arg Ala Gly His Gly Ala Lys Asn Lys Leu Lys Pro 370 375 380 Ile Thr Ile Leu Thr Glu Lys Ser Glu Arg Phe Arg Lys Lys Leu Ile 385 390 395 400 Glu Arg Trp Ala Cys Glu Ile Ala Asp Phe Phe Ile Lys Asn Lys Val 405 410 415 Gly Thr Val Gln Met Glu Asn Leu Glu Ser Met Lys Arg Lys Glu Asp 420 425 430 Ser Tyr Phe Asn Ile Arg Leu Arg Gly Phe Trp Pro Tyr Ala Glu Met 435 440 445 Gln Asn Lys Ile Glu Phe Lys Leu Lys Gln Tyr Gly Ile Glu Ile Arg 450 455 460 Lys Val Ala Pro Asn Asn Thr Ser Lys Thr Cys Ser Lys Cys Gly His 465 470 475 480 Leu Asn Asn Tyr Phe Asn Phe Glu Tyr Arg Lys Lys Asn Lys Phe Pro 485 490 495 His Phe Lys Cys Glu Lys Cys Asn Phe Lys Glu Asn Ala Asp Tyr Asn 500 505 510 Ala Ala Leu Asn Ile Ser Asn Pro Lys Leu Lys Ser Thr Lys Glu Glu 515 520 525 Pro <210> 2 <211> 1587 <212> DNA <213> artificial sequence <220> <223> Human codon-optimized Cas12f1 <400> 2 atggccaaga acacaattac aaagacactg aagctgagga tcgtgagacc atacaacagc 60 gctgaggtcg agaagattgt ggctgatgaa aagaacaaca gggaaaagat cgccctcgag 120 aagaacaagg ataaggtgaa ggaggcctgc tctaagcacc tgaaagtggc cgcctactgc 180 accacacagg tggagaggaa cgcctgtctg ttttgtaaag ctcggaagct ggatgataag 240 ttttaccaga agctgcgggg ccagttcccc gatgccgtct tttggcagga gattagcgag 300 atcttcagac agctgcagaa gcaggccgcc gagatctaca accagagcct gatcgagctc 360 tactacgaga tcttcatcaa gggcaagggc attgccaacg cctcctccgt ggagcactac 420 ctgagcgacg tgtgctacac aagagccgcc gagctcttta agaacgccgc tatcgcttcc 480 gggctgagga gcaagattaa gagtaacttc cggctcaagg agctgaagaa catgaagagc 540 ggcctgccca ctacaaagag cgacaacttc ccaattccac tggtgaagca gaaggggggc 600 cagtacacag ggttcgagat ttccaaccac aacagcgact ttattattaa gatccccttt 660 ggcaggtggc aggtcaagaa ggagattgac aagtacaggc cctgggagaa gtttgatttc 720 gagcaggtgc agaagagccc caagcctatt tccctgctgc tgtccacaca gcggcggaag 780 aggaacaagg ggtggtctaa ggatgagggg accgaggccg agattaagaa agtgatgaac 840 ggcgactacc agacaagcta catcgaggtc aagcggggca gtaagattgg cgagaagagc 900 gcctggatgc tgaacctgag cattgacgtg ccaaagattg ataagggcgt ggatcccagc 960 atcatcggag ggatcgatgt gggggtcaag agccccctcg tgtgcgccat caacaacgcc 1020 ttcagcaggt acagcatctc cgataacgac ctgttccact ttaacaagaa gatgttcgcc 1080 cggcggagga ttttgctcaa gaagaaccgg cacaagcggg ccggacacgg ggccaagaac 1140 aagctcaagc ccatcactat cctgaccgag aagagcgaga ggttcaggaa gaagctcatc 1200 gagagatggg cctgcgagat cgccgatttc tttattaaga acaaggtcgg aacagtgcag 1260 atggagaacc tcgagagcat gaagaggaag gaggattcct acttcaacat tcggctgagg 1320 gggttctggc cctacgctga gatgcagaac aagattgagt ttaagctgaa gcagtacggg 1380 attgagatcc ggaaggtggc ccccaacaac accagcaaga cctgcagcaa gtgcgggcac 1440 ctcaacaact acttcaactt cgagtaccgg aagaagaaca agttcccaca cttcaagtgc 1500 gagaagtgca actttaagga gaacgccgat tacaacgccg ccctgaacat cagcaaccct 1560 aagctgaaga gcactaagga ggagccc 1587 <210> 3 <211> 529 <212> PRT <213> artificial sequence <220> <223> DeadCas12f1(D326A) <400> 3 Met Ala Lys Asn Thr Ile Thr Lys Thr Leu Lys Leu Arg Ile Val Arg 1 5 10 15 Pro Tyr Asn Ser Ala Glu Val Glu Lys Ile Val Ala Asp Glu Lys Asn 20 25 30 Asn Arg Glu Lys Ile Ala Leu Glu Lys Asn Lys Asp Lys Val Lys Glu 35 40 45 Ala Cys Ser Lys His Leu Lys Val Ala Ala Tyr Cys Thr Thr Gln Val 50 55 60 Glu Arg Asn Ala Cys Leu Phe Cys Lys Ala Arg Lys Leu Asp Asp Lys 65 70 75 80 Phe Tyr Gln Lys Leu Arg Gly Gln Phe Pro Asp Ala Val Phe Trp Gln 85 90 95 Glu Ile Ser Glu Ile Phe Arg Gln Leu Gln Lys Gln Ala Ala Glu Ile 100 105 110 Tyr Asn Gln Ser Leu Ile Glu Leu Tyr Tyr Glu Ile Phe Ile Lys Gly 115 120 125 Lys Gly Ile Ala Asn Ala Ser Ser Val Glu His Tyr Leu Ser Asp Val 130 135 140 Cys Tyr Thr Arg Ala Ala Glu Leu Phe Lys Asn Ala Ala Ile Ala Ser 145 150 155 160 Gly Leu Arg Ser Lys Ile Lys Ser Asn Phe Arg Leu Lys Glu Leu Lys 165 170 175 Asn Met Lys Ser Gly Leu Pro Thr Thr Lys Ser Asp Asn Phe Pro Ile 180 185 190 Pro Leu Val Lys Gln Lys Gly Gly Gln Tyr Thr Gly Phe Glu Ile Ser 195 200 205 Asn His Asn Ser Asp Phe Ile Ile Lys Ile Pro Phe Gly Arg Trp Gln 210 215 220 Val Lys Lys Glu Ile Asp Lys Tyr Arg Pro Trp Glu Lys Phe Asp Phe 225 230 235 240 Glu Gln Val Gln Lys Ser Pro Lys Pro Ile Ser Leu Leu Leu Ser Thr 245 250 255 Gln Arg Arg Lys Arg Asn Lys Gly Trp Ser Lys Asp Glu Gly Thr Glu 260 265 270 Ala Glu Ile Lys Lys Val Met Asn Gly Asp Tyr Gln Thr Ser Tyr Ile 275 280 285 Glu Val Lys Arg Gly Ser Lys Ile Gly Glu Lys Ser Ala Trp Met Leu 290 295 300 Asn Leu Ser Ile Asp Val Pro Lys Ile Asp Lys Gly Val Asp Pro Ser 305 310 315 320 Ile Ile Gly Gly Ile Ala Val Gly Val Lys Ser Pro Leu Val Cys Ala 325 330 335 Ile Asn Asn Ala Phe Ser Arg Tyr Ser Ile Ser Asp Asn Asp Leu Phe 340 345 350 His Phe Asn Lys Lys Met Phe Ala Arg Arg Arg Ile Leu Leu Lys Lys 355 360 365 Asn Arg His Lys Arg Ala Gly His Gly Ala Lys Asn Lys Leu Lys Pro 370 375 380 Ile Thr Ile Leu Thr Glu Lys Ser Glu Arg Phe Arg Lys Lys Leu Ile 385 390 395 400 Glu Arg Trp Ala Cys Glu Ile Ala Asp Phe Phe Ile Lys Asn Lys Val 405 410 415 Gly Thr Val Gln Met Glu Asn Leu Glu Ser Met Lys Arg Lys Glu Asp 420 425 430 Ser Tyr Phe Asn Ile Arg Leu Arg Gly Phe Trp Pro Tyr Ala Glu Met 435 440 445 Gln Asn Lys Ile Glu Phe Lys Leu Lys Gln Tyr Gly Ile Glu Ile Arg 450 455 460 Lys Val Ala Pro Asn Asn Thr Ser Lys Thr Cys Ser Lys Cys Gly His 465 470 475 480 Leu Asn Asn Tyr Phe Asn Phe Glu Tyr Arg Lys Lys Asn Lys Phe Pro 485 490 495 His Phe Lys Cys Glu Lys Cys Asn Phe Lys Glu Asn Ala Asp Tyr Asn 500 505 510 Ala Ala Leu Asn Ile Ser Asn Pro Lys Leu Lys Ser Thr Lys Glu Glu 515 520 525 Pro <210> 4 <211> 529 <212> PRT <213> artificial sequence <220> <223> DeadCas12f1(E422A) <400> 4 Met Ala Lys Asn Thr Ile Thr Lys Thr Leu Lys Leu Arg Ile Val Arg 1 5 10 15 Pro Tyr Asn Ser Ala Glu Val Glu Lys Ile Val Ala Asp Glu Lys Asn 20 25 30 Asn Arg Glu Lys Ile Ala Leu Glu Lys Asn Lys Asp Lys Val Lys Glu 35 40 45 Ala Cys Ser Lys His Leu Lys Val Ala Ala Tyr Cys Thr Thr Gln Val 50 55 60 Glu Arg Asn Ala Cys Leu Phe Cys Lys Ala Arg Lys Leu Asp Asp Lys 65 70 75 80 Phe Tyr Gln Lys Leu Arg Gly Gln Phe Pro Asp Ala Val Phe Trp Gln 85 90 95 Glu Ile Ser Glu Ile Phe Arg Gln Leu Gln Lys Gln Ala Ala Glu Ile 100 105 110 Tyr Asn Gln Ser Leu Ile Glu Leu Tyr Tyr Glu Ile Phe Ile Lys Gly 115 120 125 Lys Gly Ile Ala Asn Ala Ser Ser Val Glu His Tyr Leu Ser Asp Val 130 135 140 Cys Tyr Thr Arg Ala Ala Glu Leu Phe Lys Asn Ala Ala Ile Ala Ser 145 150 155 160 Gly Leu Arg Ser Lys Ile Lys Ser Asn Phe Arg Leu Lys Glu Leu Lys 165 170 175 Asn Met Lys Ser Gly Leu Pro Thr Thr Lys Ser Asp Asn Phe Pro Ile 180 185 190 Pro Leu Val Lys Gln Lys Gly Gly Gln Tyr Thr Gly Phe Glu Ile Ser 195 200 205 Asn His Asn Ser Asp Phe Ile Ile Lys Ile Pro Phe Gly Arg Trp Gln 210 215 220 Val Lys Lys Glu Ile Asp Lys Tyr Arg Pro Trp Glu Lys Phe Asp Phe 225 230 235 240 Glu Gln Val Gln Lys Ser Pro Lys Pro Ile Ser Leu Leu Leu Ser Thr 245 250 255 Gln Arg Arg Lys Arg Asn Lys Gly Trp Ser Lys Asp Glu Gly Thr Glu 260 265 270 Ala Glu Ile Lys Lys Val Met Asn Gly Asp Tyr Gln Thr Ser Tyr Ile 275 280 285 Glu Val Lys Arg Gly Ser Lys Ile Gly Glu Lys Ser Ala Trp Met Leu 290 295 300 Asn Leu Ser Ile Asp Val Pro Lys Ile Asp Lys Gly Val Asp Pro Ser 305 310 315 320 Ile Ile Gly Gly Ile Asp Val Gly Val Lys Ser Pro Leu Val Cys Ala 325 330 335 Ile Asn Asn Ala Phe Ser Arg Tyr Ser Ile Ser Asp Asn Asp Leu Phe 340 345 350 His Phe Asn Lys Lys Met Phe Ala Arg Arg Arg Ile Leu Leu Lys Lys 355 360 365 Asn Arg His Lys Arg Ala Gly His Gly Ala Lys Asn Lys Leu Lys Pro 370 375 380 Ile Thr Ile Leu Thr Glu Lys Ser Glu Arg Phe Arg Lys Lys Leu Ile 385 390 395 400 Glu Arg Trp Ala Cys Glu Ile Ala Asp Phe Phe Ile Lys Asn Lys Val 405 410 415 Gly Thr Val Gln Met Ala Asn Leu Glu Ser Met Lys Arg Lys Glu Asp 420 425 430 Ser Tyr Phe Asn Ile Arg Leu Arg Gly Phe Trp Pro Tyr Ala Glu Met 435 440 445 Gln Asn Lys Ile Glu Phe Lys Leu Lys Gln Tyr Gly Ile Glu Ile Arg 450 455 460 Lys Val Ala Pro Asn Asn Thr Ser Lys Thr Cys Ser Lys Cys Gly His 465 470 475 480 Leu Asn Asn Tyr Phe Asn Phe Glu Tyr Arg Lys Lys Asn Lys Phe Pro 485 490 495 His Phe Lys Cys Glu Lys Cys Asn Phe Lys Glu Asn Ala Asp Tyr Asn 500 505 510 Ala Ala Leu Asn Ile Ser Asn Pro Lys Leu Lys Ser Thr Lys Glu Glu 515 520 525 Pro <210> 5 <211> 529 <212> PRT <213> artificial sequence <220> <223> DeadCas12f1(R490A) <400> 5 Met Ala Lys Asn Thr Ile Thr Lys Thr Leu Lys Leu Arg Ile Val Arg 1 5 10 15 Pro Tyr Asn Ser Ala Glu Val Glu Lys Ile Val Ala Asp Glu Lys Asn 20 25 30 Asn Arg Glu Lys Ile Ala Leu Glu Lys Asn Lys Asp Lys Val Lys Glu 35 40 45 Ala Cys Ser Lys His Leu Lys Val Ala Ala Tyr Cys Thr Thr Gln Val 50 55 60 Glu Arg Asn Ala Cys Leu Phe Cys Lys Ala Arg Lys Leu Asp Asp Lys 65 70 75 80 Phe Tyr Gln Lys Leu Arg Gly Gln Phe Pro Asp Ala Val Phe Trp Gln 85 90 95 Glu Ile Ser Glu Ile Phe Arg Gln Leu Gln Lys Gln Ala Ala Glu Ile 100 105 110 Tyr Asn Gln Ser Leu Ile Glu Leu Tyr Tyr Glu Ile Phe Ile Lys Gly 115 120 125 Lys Gly Ile Ala Asn Ala Ser Ser Val Glu His Tyr Leu Ser Asp Val 130 135 140 Cys Tyr Thr Arg Ala Ala Glu Leu Phe Lys Asn Ala Ala Ile Ala Ser 145 150 155 160 Gly Leu Arg Ser Lys Ile Lys Ser Asn Phe Arg Leu Lys Glu Leu Lys 165 170 175 Asn Met Lys Ser Gly Leu Pro Thr Thr Lys Ser Asp Asn Phe Pro Ile 180 185 190 Pro Leu Val Lys Gln Lys Gly Gly Gln Tyr Thr Gly Phe Glu Ile Ser 195 200 205 Asn His Asn Ser Asp Phe Ile Ile Lys Ile Pro Phe Gly Arg Trp Gln 210 215 220 Val Lys Lys Glu Ile Asp Lys Tyr Arg Pro Trp Glu Lys Phe Asp Phe 225 230 235 240 Glu Gln Val Gln Lys Ser Pro Lys Pro Ile Ser Leu Leu Leu Ser Thr 245 250 255 Gln Arg Arg Lys Arg Asn Lys Gly Trp Ser Lys Asp Glu Gly Thr Glu 260 265 270 Ala Glu Ile Lys Lys Val Met Asn Gly Asp Tyr Gln Thr Ser Tyr Ile 275 280 285 Glu Val Lys Arg Gly Ser Lys Ile Gly Glu Lys Ser Ala Trp Met Leu 290 295 300 Asn Leu Ser Ile Asp Val Pro Lys Ile Asp Lys Gly Val Asp Pro Ser 305 310 315 320 Ile Ile Gly Gly Ile Asp Val Gly Val Lys Ser Pro Leu Val Cys Ala 325 330 335 Ile Asn Asn Ala Phe Ser Arg Tyr Ser Ile Ser Asp Asn Asp Leu Phe 340 345 350 His Phe Asn Lys Lys Met Phe Ala Arg Arg Arg Ile Leu Leu Lys Lys 355 360 365 Asn Arg His Lys Arg Ala Gly His Gly Ala Lys Asn Lys Leu Lys Pro 370 375 380 Ile Thr Ile Leu Thr Glu Lys Ser Glu Arg Phe Arg Lys Lys Leu Ile 385 390 395 400 Glu Arg Trp Ala Cys Glu Ile Ala Asp Phe Phe Ile Lys Asn Lys Val 405 410 415 Gly Thr Val Gln Met Glu Asn Leu Glu Ser Met Lys Arg Lys Glu Asp 420 425 430 Ser Tyr Phe Asn Ile Arg Leu Arg Gly Phe Trp Pro Tyr Ala Glu Met 435 440 445 Gln Asn Lys Ile Glu Phe Lys Leu Lys Gln Tyr Gly Ile Glu Ile Arg 450 455 460 Lys Val Ala Pro Asn Asn Thr Ser Lys Thr Cys Ser Lys Cys Gly His 465 470 475 480 Leu Asn Asn Tyr Phe Asn Phe Glu Tyr Ala Lys Lys Asn Lys Phe Pro 485 490 495 His Phe Lys Cys Glu Lys Cys Asn Phe Lys Glu Asn Ala Asp Tyr Asn 500 505 510 Ala Ala Leu Asn Ile Ser Asn Pro Lys Leu Lys Ser Thr Lys Glu Glu 515 520 525 Pro <210> 6 <211> 529 <212> PRT <213> artificial sequence <220> <223> DeadCas12f1(D510A) <400> 6 Met Ala Lys Asn Thr Ile Thr Lys Thr Leu Lys Leu Arg Ile Val Arg 1 5 10 15 Pro Tyr Asn Ser Ala Glu Val Glu Lys Ile Val Ala Asp Glu Lys Asn 20 25 30 Asn Arg Glu Lys Ile Ala Leu Glu Lys Asn Lys Asp Lys Val Lys Glu 35 40 45 Ala Cys Ser Lys His Leu Lys Val Ala Ala Tyr Cys Thr Thr Gln Val 50 55 60 Glu Arg Asn Ala Cys Leu Phe Cys Lys Ala Arg Lys Leu Asp Asp Lys 65 70 75 80 Phe Tyr Gln Lys Leu Arg Gly Gln Phe Pro Asp Ala Val Phe Trp Gln 85 90 95 Glu Ile Ser Glu Ile Phe Arg Gln Leu Gln Lys Gln Ala Ala Glu Ile 100 105 110 Tyr Asn Gln Ser Leu Ile Glu Leu Tyr Tyr Glu Ile Phe Ile Lys Gly 115 120 125 Lys Gly Ile Ala Asn Ala Ser Ser Val Glu His Tyr Leu Ser Asp Val 130 135 140 Cys Tyr Thr Arg Ala Ala Glu Leu Phe Lys Asn Ala Ala Ile Ala Ser 145 150 155 160 Gly Leu Arg Ser Lys Ile Lys Ser Asn Phe Arg Leu Lys Glu Leu Lys 165 170 175 Asn Met Lys Ser Gly Leu Pro Thr Thr Lys Ser Asp Asn Phe Pro Ile 180 185 190 Pro Leu Val Lys Gln Lys Gly Gly Gln Tyr Thr Gly Phe Glu Ile Ser 195 200 205 Asn His Asn Ser Asp Phe Ile Ile Lys Ile Pro Phe Gly Arg Trp Gln 210 215 220 Val Lys Lys Glu Ile Asp Lys Tyr Arg Pro Trp Glu Lys Phe Asp Phe 225 230 235 240 Glu Gln Val Gln Lys Ser Pro Lys Pro Ile Ser Leu Leu Leu Ser Thr 245 250 255 Gln Arg Arg Lys Arg Asn Lys Gly Trp Ser Lys Asp Glu Gly Thr Glu 260 265 270 Ala Glu Ile Lys Lys Val Met Asn Gly Asp Tyr Gln Thr Ser Tyr Ile 275 280 285 Glu Val Lys Arg Gly Ser Lys Ile Gly Glu Lys Ser Ala Trp Met Leu 290 295 300 Asn Leu Ser Ile Asp Val Pro Lys Ile Asp Lys Gly Val Asp Pro Ser 305 310 315 320 Ile Ile Gly Gly Ile Asp Val Gly Val Lys Ser Pro Leu Val Cys Ala 325 330 335 Ile Asn Asn Ala Phe Ser Arg Tyr Ser Ile Ser Asp Asn Asp Leu Phe 340 345 350 His Phe Asn Lys Lys Met Phe Ala Arg Arg Arg Ile Leu Leu Lys Lys 355 360 365 Asn Arg His Lys Arg Ala Gly His Gly Ala Lys Asn Lys Leu Lys Pro 370 375 380 Ile Thr Ile Leu Thr Glu Lys Ser Glu Arg Phe Arg Lys Lys Leu Ile 385 390 395 400 Glu Arg Trp Ala Cys Glu Ile Ala Asp Phe Phe Ile Lys Asn Lys Val 405 410 415 Gly Thr Val Gln Met Glu Asn Leu Glu Ser Met Lys Arg Lys Glu Asp 420 425 430 Ser Tyr Phe Asn Ile Arg Leu Arg Gly Phe Trp Pro Tyr Ala Glu Met 435 440 445 Gln Asn Lys Ile Glu Phe Lys Leu Lys Gln Tyr Gly Ile Glu Ile Arg 450 455 460 Lys Val Ala Pro Asn Asn Thr Ser Lys Thr Cys Ser Lys Cys Gly His 465 470 475 480 Leu Asn Asn Tyr Phe Asn Phe Glu Tyr Arg Lys Lys Asn Lys Phe Pro 485 490 495 His Phe Lys Cys Glu Lys Cys Asn Phe Lys Glu Asn Ala Ala Tyr Asn 500 505 510 Ala Ala Leu Asn Ile Ser Asn Pro Lys Leu Lys Ser Thr Lys Glu Glu 515 520 525 Pro <210> 7 <211> 557 <212> PRT <213> unknown <220> <223> TnpB <400> 7 Met Gly Glu Lys Ser Ser Arg Arg Arg Arg Asn Gly Lys Ser Gly Ala 1 5 10 15 Trp Thr Ala Ala Ile Thr Ser Cys Val Gly Gly Lys Met Ala Lys Asn 20 25 30 Thr Ile Thr Lys Thr Leu Lys Leu Arg Ile Val Arg Pro Tyr Asn Ser 35 40 45 Ala Glu Val Glu Lys Ile Val Ala Asp Glu Lys Asn Asn Arg Glu Lys 50 55 60 Ile Ala Leu Glu Lys Asn Lys Asp Lys Val Lys Glu Ala Cys Ser Lys 65 70 75 80 His Leu Lys Val Ala Ala Tyr Cys Thr Thr Gln Val Glu Arg Asn Ala 85 90 95 Cys Leu Phe Cys Lys Ala Arg Lys Leu Asp Asp Lys Phe Tyr Gln Lys 100 105 110 Leu Arg Gly Gln Phe Pro Asp Ala Val Phe Trp Gln Glu Ile Ser Glu 115 120 125 Ile Phe Arg Gln Leu Gln Lys Gln Ala Ala Glu Ile Tyr Asn Gln Ser 130 135 140 Leu Ile Glu Leu Tyr Tyr Glu Ile Phe Ile Lys Gly Lys Gly Ile Ala 145 150 155 160 Asn Ala Ser Ser Val Glu His Tyr Leu Ser Asp Val Cys Tyr Thr Arg 165 170 175 Ala Ala Glu Leu Phe Lys Asn Ala Ala Ile Ala Ser Gly Leu Arg Ser 180 185 190 Lys Ile Lys Ser Asn Phe Arg Leu Lys Glu Leu Lys Asn Met Lys Ser 195 200 205 Gly Leu Pro Thr Thr Lys Ser Asp Asn Phe Pro Ile Pro Leu Val Lys 210 215 220 Gln Lys Gly Gly Gln Tyr Thr Gly Phe Glu Ile Ser Asn His Asn Ser 225 230 235 240 Asp Phe Ile Ile Lys Ile Pro Phe Gly Arg Trp Gln Val Lys Lys Glu 245 250 255 Ile Asp Lys Tyr Arg Pro Trp Glu Lys Phe Asp Phe Glu Gln Val Gln 260 265 270 Lys Ser Pro Lys Pro Ile Ser Leu Leu Leu Ser Thr Gln Arg Arg Lys 275 280 285 Arg Asn Lys Gly Trp Ser Lys Asp Glu Gly Thr Glu Ala Glu Ile Lys 290 295 300 Lys Val Met Asn Gly Asp Tyr Gln Thr Ser Tyr Ile Glu Val Lys Arg 305 310 315 320 Gly Ser Lys Ile Gly Glu Lys Ser Ala Trp Met Leu Asn Leu Ser Ile 325 330 335 Asp Val Pro Lys Ile Asp Lys Gly Val Asp Pro Ser Ile Ile Gly Gly 340 345 350 Ile Asp Val Gly Val Lys Ser Pro Leu Val Cys Ala Ile Asn Asn Ala 355 360 365 Phe Ser Arg Tyr Ser Ile Ser Asp Asn Asp Leu Phe His Phe Asn Lys 370 375 380 Lys Met Phe Ala Arg Arg Arg Ile Leu Leu Lys Lys Asn Arg His Lys 385 390 395 400 Arg Ala Gly His Gly Ala Lys Asn Lys Leu Lys Pro Ile Thr Ile Leu 405 410 415 Thr Glu Lys Ser Glu Arg Phe Arg Lys Lys Leu Ile Glu Arg Trp Ala 420 425 430 Cys Glu Ile Ala Asp Phe Phe Ile Lys Asn Lys Val Gly Thr Val Gln 435 440 445 Met Glu Asn Leu Glu Ser Met Lys Arg Lys Glu Asp Ser Tyr Phe Asn 450 455 460 Ile Arg Leu Arg Gly Phe Trp Pro Tyr Ala Glu Met Gln Asn Lys Ile 465 470 475 480 Glu Phe Lys Leu Lys Gln Tyr Gly Ile Glu Ile Arg Lys Val Ala Pro 485 490 495 Asn Asn Thr Ser Lys Thr Cys Ser Lys Cys Gly His Leu Asn Asn Tyr 500 505 510 Phe Asn Phe Glu Tyr Arg Lys Lys Asn Lys Phe Pro His Phe Lys Cys 515 520 525 Glu Lys Cys Asn Phe Lys Glu Asn Ala Asp Tyr Asn Ala Ala Leu Asn 530 535 540 Ile Ser Asn Pro Lys Leu Lys Ser Thr Lys Glu Glu Pro 545 550 555 <210> 8 <211> 555 <212> PRT <213> artificial sequence <220> <223> CasX-Cas12f1 <400> 8 Met Glu Lys Arg Ile Asn Lys Ile Arg Lys Lys Leu Ser Ala Asp Asn 1 5 10 15 Ala Thr Lys Pro Val Ser Arg Ser Gly Pro Met Ala Lys Asn Thr Ile 20 25 30 Thr Lys Thr Leu Lys Leu Arg Ile Val Arg Pro Tyr Asn Ser Ala Glu 35 40 45 Val Glu Lys Ile Val Ala Asp Glu Lys Asn Asn Arg Glu Lys Ile Ala 50 55 60 Leu Glu Lys Asn Lys Asp Lys Val Lys Glu Ala Cys Ser Lys His Leu 65 70 75 80 Lys Val Ala Ala Tyr Cys Thr Thr Gln Val Glu Arg Asn Ala Cys Leu 85 90 95 Phe Cys Lys Ala Arg Lys Leu Asp Asp Lys Phe Tyr Gln Lys Leu Arg 100 105 110 Gly Gln Phe Pro Asp Ala Val Phe Trp Gln Glu Ile Ser Glu Ile Phe 115 120 125 Arg Gln Leu Gln Lys Gln Ala Ala Glu Ile Tyr Asn Gln Ser Leu Ile 130 135 140 Glu Leu Tyr Tyr Glu Ile Phe Ile Lys Gly Lys Gly Ile Ala Asn Ala 145 150 155 160 Ser Ser Val Glu His Tyr Leu Ser Asp Val Cys Tyr Thr Arg Ala Ala 165 170 175 Glu Leu Phe Lys Asn Ala Ala Ile Ala Ser Gly Leu Arg Ser Lys Ile 180 185 190 Lys Ser Asn Phe Arg Leu Lys Glu Leu Lys Asn Met Lys Ser Gly Leu 195 200 205 Pro Thr Thr Lys Ser Asp Asn Phe Pro Ile Pro Leu Val Lys Gln Lys 210 215 220 Gly Gly Gln Tyr Thr Gly Phe Glu Ile Ser Asn His Asn Ser Asp Phe 225 230 235 240 Ile Ile Lys Ile Pro Phe Gly Arg Trp Gln Val Lys Lys Glu Ile Asp 245 250 255 Lys Tyr Arg Pro Trp Glu Lys Phe Asp Phe Glu Gln Val Gln Lys Ser 260 265 270 Pro Lys Pro Ile Ser Leu Leu Leu Ser Thr Gln Arg Arg Lys Arg Asn 275 280 285 Lys Gly Trp Ser Lys Asp Glu Gly Thr Glu Ala Glu Ile Lys Lys Val 290 295 300 Met Asn Gly Asp Tyr Gln Thr Ser Tyr Ile Glu Val Lys Arg Gly Ser 305 310 315 320 Lys Ile Gly Glu Lys Ser Ala Trp Met Leu Asn Leu Ser Ile Asp Val 325 330 335 Pro Lys Ile Asp Lys Gly Val Asp Pro Ser Ile Ile Gly Gly Ile Asp 340 345 350 Val Gly Val Lys Ser Pro Leu Val Cys Ala Ile Asn Asn Ala Phe Ser 355 360 365 Arg Tyr Ser Ile Ser Asp Asn Asp Leu Phe His Phe Asn Lys Lys Met 370 375 380 Phe Ala Arg Arg Arg Ile Leu Leu Lys Lys Asn Arg His Lys Arg Ala 385 390 395 400 Gly His Gly Ala Lys Asn Lys Leu Lys Pro Ile Thr Ile Leu Thr Glu 405 410 415 Lys Ser Glu Arg Phe Arg Lys Lys Leu Ile Glu Arg Trp Ala Cys Glu 420 425 430 Ile Ala Asp Phe Phe Ile Lys Asn Lys Val Gly Thr Val Gln Met Glu 435 440 445 Asn Leu Glu Ser Met Lys Arg Lys Glu Asp Ser Tyr Phe Asn Ile Arg 450 455 460 Leu Arg Gly Phe Trp Pro Tyr Ala Glu Met Gln Asn Lys Ile Glu Phe 465 470 475 480 Lys Leu Lys Gln Tyr Gly Ile Glu Ile Arg Lys Val Ala Pro Asn Asn 485 490 495 Thr Ser Lys Thr Cys Ser Lys Cys Gly His Leu Asn Asn Tyr Phe Asn 500 505 510 Phe Glu Tyr Arg Lys Lys Asn Lys Phe Pro His Phe Lys Cys Glu Lys 515 520 525 Cys Asn Phe Lys Glu Asn Ala Asp Tyr Asn Ala Ala Leu Asn Ile Ser 530 535 540 Asn Pro Lys Leu Lys Ser Thr Lys Glu Glu Pro 545 550 555 <210> 9 <211> 557 <212> PRT <213> artificial sequence <220> <223> 28aa-extension Cas12f1 <400> 9 Met Ala Gly Gly Pro Gly Ala Gly Ser Ala Ala Pro Val Ser Ser Thr 1 5 10 15 Ser Ser Leu Pro Leu Ala Ala Leu Asn Met Arg Val Met Ala Lys Asn 20 25 30 Thr Ile Thr Lys Thr Leu Lys Leu Arg Ile Val Arg Pro Tyr Asn Ser 35 40 45 Ala Glu Val Glu Lys Ile Val Ala Asp Glu Lys Asn Asn Arg Glu Lys 50 55 60 Ile Ala Leu Glu Lys Asn Lys Asp Lys Val Lys Glu Ala Cys Ser Lys 65 70 75 80 His Leu Lys Val Ala Ala Tyr Cys Thr Thr Gln Val Glu Arg Asn Ala 85 90 95 Cys Leu Phe Cys Lys Ala Arg Lys Leu Asp Asp Lys Phe Tyr Gln Lys 100 105 110 Leu Arg Gly Gln Phe Pro Asp Ala Val Phe Trp Gln Glu Ile Ser Glu 115 120 125 Ile Phe Arg Gln Leu Gln Lys Gln Ala Ala Glu Ile Tyr Asn Gln Ser 130 135 140 Leu Ile Glu Leu Tyr Tyr Glu Ile Phe Ile Lys Gly Lys Gly Ile Ala 145 150 155 160 Asn Ala Ser Ser Val Glu His Tyr Leu Ser Asp Val Cys Tyr Thr Arg 165 170 175 Ala Ala Glu Leu Phe Lys Asn Ala Ala Ile Ala Ser Gly Leu Arg Ser 180 185 190 Lys Ile Lys Ser Asn Phe Arg Leu Lys Glu Leu Lys Asn Met Lys Ser 195 200 205 Gly Leu Pro Thr Thr Lys Ser Asp Asn Phe Pro Ile Pro Leu Val Lys 210 215 220 Gln Lys Gly Gly Gln Tyr Thr Gly Phe Glu Ile Ser Asn His Asn Ser 225 230 235 240 Asp Phe Ile Ile Lys Ile Pro Phe Gly Arg Trp Gln Val Lys Lys Glu 245 250 255 Ile Asp Lys Tyr Arg Pro Trp Glu Lys Phe Asp Phe Glu Gln Val Gln 260 265 270 Lys Ser Pro Lys Pro Ile Ser Leu Leu Leu Ser Thr Gln Arg Arg Lys 275 280 285 Arg Asn Lys Gly Trp Ser Lys Asp Glu Gly Thr Glu Ala Glu Ile Lys 290 295 300 Lys Val Met Asn Gly Asp Tyr Gln Thr Ser Tyr Ile Glu Val Lys Arg 305 310 315 320 Gly Ser Lys Ile Gly Glu Lys Ser Ala Trp Met Leu Asn Leu Ser Ile 325 330 335 Asp Val Pro Lys Ile Asp Lys Gly Val Asp Pro Ser Ile Ile Gly Gly 340 345 350 Ile Asp Val Gly Val Lys Ser Pro Leu Val Cys Ala Ile Asn Asn Ala 355 360 365 Phe Ser Arg Tyr Ser Ile Ser Asp Asn Asp Leu Phe His Phe Asn Lys 370 375 380 Lys Met Phe Ala Arg Arg Arg Ile Leu Leu Lys Lys Asn Arg His Lys 385 390 395 400 Arg Ala Gly His Gly Ala Lys Asn Lys Leu Lys Pro Ile Thr Ile Leu 405 410 415 Thr Glu Lys Ser Glu Arg Phe Arg Lys Lys Leu Ile Glu Arg Trp Ala 420 425 430 Cys Glu Ile Ala Asp Phe Phe Ile Lys Asn Lys Val Gly Thr Val Gln 435 440 445 Met Glu Asn Leu Glu Ser Met Lys Arg Lys Glu Asp Ser Tyr Phe Asn 450 455 460 Ile Arg Leu Arg Gly Phe Trp Pro Tyr Ala Glu Met Gln Asn Lys Ile 465 470 475 480 Glu Phe Lys Leu Lys Gln Tyr Gly Ile Glu Ile Arg Lys Val Ala Pro 485 490 495 Asn Asn Thr Ser Lys Thr Cys Ser Lys Cys Gly His Leu Asn Asn Tyr 500 505 510 Phe Asn Phe Glu Tyr Arg Lys Lys Asn Lys Phe Pro His Phe Lys Cys 515 520 525 Glu Lys Cys Asn Phe Lys Glu Asn Ala Asp Tyr Asn Ala Ala Leu Asn 530 535 540 Ile Ser Asn Pro Lys Leu Lys Ser Thr Lys Glu Glu Pro 545 550 555 <210> 10 <211> 555 <212> PRT <213> artificial sequence <220> <223> 26aa-extension Cas12f1 <400> 10 Met Ala Gly Gly Pro Gly Ala Gly Ser Ala Ala Pro Val Ser Ser Thr 1 5 10 15 Ser Ser Leu Pro Leu Ala Ala Leu Asn Met Met Ala Lys Asn Thr Ile 20 25 30 Thr Lys Thr Leu Lys Leu Arg Ile Val Arg Pro Tyr Asn Ser Ala Glu 35 40 45 Val Glu Lys Ile Val Ala Asp Glu Lys Asn Asn Arg Glu Lys Ile Ala 50 55 60 Leu Glu Lys Asn Lys Asp Lys Val Lys Glu Ala Cys Ser Lys His Leu 65 70 75 80 Lys Val Ala Ala Tyr Cys Thr Thr Gln Val Glu Arg Asn Ala Cys Leu 85 90 95 Phe Cys Lys Ala Arg Lys Leu Asp Asp Lys Phe Tyr Gln Lys Leu Arg 100 105 110 Gly Gln Phe Pro Asp Ala Val Phe Trp Gln Glu Ile Ser Glu Ile Phe 115 120 125 Arg Gln Leu Gln Lys Gln Ala Ala Glu Ile Tyr Asn Gln Ser Leu Ile 130 135 140 Glu Leu Tyr Tyr Glu Ile Phe Ile Lys Gly Lys Gly Ile Ala Asn Ala 145 150 155 160 Ser Ser Val Glu His Tyr Leu Ser Asp Val Cys Tyr Thr Arg Ala Ala 165 170 175 Glu Leu Phe Lys Asn Ala Ala Ile Ala Ser Gly Leu Arg Ser Lys Ile 180 185 190 Lys Ser Asn Phe Arg Leu Lys Glu Leu Lys Asn Met Lys Ser Gly Leu 195 200 205 Pro Thr Thr Lys Ser Asp Asn Phe Pro Ile Pro Leu Val Lys Gln Lys 210 215 220 Gly Gly Gln Tyr Thr Gly Phe Glu Ile Ser Asn His Asn Ser Asp Phe 225 230 235 240 Ile Ile Lys Ile Pro Phe Gly Arg Trp Gln Val Lys Lys Glu Ile Asp 245 250 255 Lys Tyr Arg Pro Trp Glu Lys Phe Asp Phe Glu Gln Val Gln Lys Ser 260 265 270 Pro Lys Pro Ile Ser Leu Leu Leu Ser Thr Gln Arg Arg Lys Arg Asn 275 280 285 Lys Gly Trp Ser Lys Asp Glu Gly Thr Glu Ala Glu Ile Lys Lys Val 290 295 300 Met Asn Gly Asp Tyr Gln Thr Ser Tyr Ile Glu Val Lys Arg Gly Ser 305 310 315 320 Lys Ile Gly Glu Lys Ser Ala Trp Met Leu Asn Leu Ser Ile Asp Val 325 330 335 Pro Lys Ile Asp Lys Gly Val Asp Pro Ser Ile Ile Gly Gly Ile Asp 340 345 350 Val Gly Val Lys Ser Pro Leu Val Cys Ala Ile Asn Asn Ala Phe Ser 355 360 365 Arg Tyr Ser Ile Ser Asp Asn Asp Leu Phe His Phe Asn Lys Lys Met 370 375 380 Phe Ala Arg Arg Arg Ile Leu Leu Lys Lys Asn Arg His Lys Arg Ala 385 390 395 400 Gly His Gly Ala Lys Asn Lys Leu Lys Pro Ile Thr Ile Leu Thr Glu 405 410 415 Lys Ser Glu Arg Phe Arg Lys Lys Leu Ile Glu Arg Trp Ala Cys Glu 420 425 430 Ile Ala Asp Phe Phe Ile Lys Asn Lys Val Gly Thr Val Gln Met Glu 435 440 445 Asn Leu Glu Ser Met Lys Arg Lys Glu Asp Ser Tyr Phe Asn Ile Arg 450 455 460 Leu Arg Gly Phe Trp Pro Tyr Ala Glu Met Gln Asn Lys Ile Glu Phe 465 470 475 480 Lys Leu Lys Gln Tyr Gly Ile Glu Ile Arg Lys Val Ala Pro Asn Asn 485 490 495 Thr Ser Lys Thr Cys Ser Lys Cys Gly His Leu Asn Asn Tyr Phe Asn 500 505 510 Phe Glu Tyr Arg Lys Lys Asn Lys Phe Pro His Phe Lys Cys Glu Lys 515 520 525 Cys Asn Phe Lys Glu Asn Ala Asp Tyr Asn Ala Ala Leu Asn Ile Ser 530 535 540 Asn Pro Lys Leu Lys Ser Thr Lys Glu Glu Pro 545 550 555 <210> 11 <211> 557 <212> PRT <213> artificial sequence <220> <223> dTnpB (D354A) <400> 11 Met Gly Glu Lys Ser Ser Arg Arg Arg Arg Asn Gly Lys Ser Gly Ala 1 5 10 15 Trp Thr Ala Ala Ile Thr Ser Cys Val Gly Gly Lys Met Ala Lys Asn 20 25 30 Thr Ile Thr Lys Thr Leu Lys Leu Arg Ile Val Arg Pro Tyr Asn Ser 35 40 45 Ala Glu Val Glu Lys Ile Val Ala Asp Glu Lys Asn Asn Arg Glu Lys 50 55 60 Ile Ala Leu Glu Lys Asn Lys Asp Lys Val Lys Glu Ala Cys Ser Lys 65 70 75 80 His Leu Lys Val Ala Ala Tyr Cys Thr Thr Gln Val Glu Arg Asn Ala 85 90 95 Cys Leu Phe Cys Lys Ala Arg Lys Leu Asp Asp Lys Phe Tyr Gln Lys 100 105 110 Leu Arg Gly Gln Phe Pro Asp Ala Val Phe Trp Gln Glu Ile Ser Glu 115 120 125 Ile Phe Arg Gln Leu Gln Lys Gln Ala Ala Glu Ile Tyr Asn Gln Ser 130 135 140 Leu Ile Glu Leu Tyr Tyr Glu Ile Phe Ile Lys Gly Lys Gly Ile Ala 145 150 155 160 Asn Ala Ser Ser Val Glu His Tyr Leu Ser Asp Val Cys Tyr Thr Arg 165 170 175 Ala Ala Glu Leu Phe Lys Asn Ala Ala Ile Ala Ser Gly Leu Arg Ser 180 185 190 Lys Ile Lys Ser Asn Phe Arg Leu Lys Glu Leu Lys Asn Met Lys Ser 195 200 205 Gly Leu Pro Thr Thr Lys Ser Asp Asn Phe Pro Ile Pro Leu Val Lys 210 215 220 Gln Lys Gly Gly Gln Tyr Thr Gly Phe Glu Ile Ser Asn His Asn Ser 225 230 235 240 Asp Phe Ile Ile Lys Ile Pro Phe Gly Arg Trp Gln Val Lys Lys Glu 245 250 255 Ile Asp Lys Tyr Arg Pro Trp Glu Lys Phe Asp Phe Glu Gln Val Gln 260 265 270 Lys Ser Pro Lys Pro Ile Ser Leu Leu Leu Ser Thr Gln Arg Arg Lys 275 280 285 Arg Asn Lys Gly Trp Ser Lys Asp Glu Gly Thr Glu Ala Glu Ile Lys 290 295 300 Lys Val Met Asn Gly Asp Tyr Gln Thr Ser Tyr Ile Glu Val Lys Arg 305 310 315 320 Gly Ser Lys Ile Gly Glu Lys Ser Ala Trp Met Leu Asn Leu Ser Ile 325 330 335 Asp Val Pro Lys Ile Asp Lys Gly Val Asp Pro Ser Ile Ile Gly Gly 340 345 350 Ile Ala Val Gly Val Lys Ser Pro Leu Val Cys Ala Ile Asn Asn Ala 355 360 365 Phe Ser Arg Tyr Ser Ile Ser Asp Asn Asp Leu Phe His Phe Asn Lys 370 375 380 Lys Met Phe Ala Arg Arg Arg Ile Leu Leu Lys Lys Asn Arg His Lys 385 390 395 400 Arg Ala Gly His Gly Ala Lys Asn Lys Leu Lys Pro Ile Thr Ile Leu 405 410 415 Thr Glu Lys Ser Glu Arg Phe Arg Lys Lys Leu Ile Glu Arg Trp Ala 420 425 430 Cys Glu Ile Ala Asp Phe Phe Ile Lys Asn Lys Val Gly Thr Val Gln 435 440 445 Met Glu Asn Leu Glu Ser Met Lys Arg Lys Glu Asp Ser Tyr Phe Asn 450 455 460 Ile Arg Leu Arg Gly Phe Trp Pro Tyr Ala Glu Met Gln Asn Lys Ile 465 470 475 480 Glu Phe Lys Leu Lys Gln Tyr Gly Ile Glu Ile Arg Lys Val Ala Pro 485 490 495 Asn Asn Thr Ser Lys Thr Cys Ser Lys Cys Gly His Leu Asn Asn Tyr 500 505 510 Phe Asn Phe Glu Tyr Arg Lys Lys Asn Lys Phe Pro His Phe Lys Cys 515 520 525 Glu Lys Cys Asn Phe Lys Glu Asn Ala Asp Tyr Asn Ala Ala Leu Asn 530 535 540 Ile Ser Asn Pro Lys Leu Lys Ser Thr Lys Glu Glu Pro 545 550 555 <210> 12 <211> 557 <212> PRT <213> artificial sequence <220> <223> dTnpB (E450A) <400> 12 Met Gly Glu Lys Ser Ser Arg Arg Arg Arg Asn Gly Lys Ser Gly Ala 1 5 10 15 Trp Thr Ala Ala Ile Thr Ser Cys Val Gly Gly Lys Met Ala Lys Asn 20 25 30 Thr Ile Thr Lys Thr Leu Lys Leu Arg Ile Val Arg Pro Tyr Asn Ser 35 40 45 Ala Glu Val Glu Lys Ile Val Ala Asp Glu Lys Asn Asn Arg Glu Lys 50 55 60 Ile Ala Leu Glu Lys Asn Lys Asp Lys Val Lys Glu Ala Cys Ser Lys 65 70 75 80 His Leu Lys Val Ala Ala Tyr Cys Thr Thr Gln Val Glu Arg Asn Ala 85 90 95 Cys Leu Phe Cys Lys Ala Arg Lys Leu Asp Asp Lys Phe Tyr Gln Lys 100 105 110 Leu Arg Gly Gln Phe Pro Asp Ala Val Phe Trp Gln Glu Ile Ser Glu 115 120 125 Ile Phe Arg Gln Leu Gln Lys Gln Ala Ala Glu Ile Tyr Asn Gln Ser 130 135 140 Leu Ile Glu Leu Tyr Tyr Glu Ile Phe Ile Lys Gly Lys Gly Ile Ala 145 150 155 160 Asn Ala Ser Ser Val Glu His Tyr Leu Ser Asp Val Cys Tyr Thr Arg 165 170 175 Ala Ala Glu Leu Phe Lys Asn Ala Ala Ile Ala Ser Gly Leu Arg Ser 180 185 190 Lys Ile Lys Ser Asn Phe Arg Leu Lys Glu Leu Lys Asn Met Lys Ser 195 200 205 Gly Leu Pro Thr Thr Lys Ser Asp Asn Phe Pro Ile Pro Leu Val Lys 210 215 220 Gln Lys Gly Gly Gln Tyr Thr Gly Phe Glu Ile Ser Asn His Asn Ser 225 230 235 240 Asp Phe Ile Ile Lys Ile Pro Phe Gly Arg Trp Gln Val Lys Lys Glu 245 250 255 Ile Asp Lys Tyr Arg Pro Trp Glu Lys Phe Asp Phe Glu Gln Val Gln 260 265 270 Lys Ser Pro Lys Pro Ile Ser Leu Leu Leu Ser Thr Gln Arg Arg Lys 275 280 285 Arg Asn Lys Gly Trp Ser Lys Asp Glu Gly Thr Glu Ala Glu Ile Lys 290 295 300 Lys Val Met Asn Gly Asp Tyr Gln Thr Ser Tyr Ile Glu Val Lys Arg 305 310 315 320 Gly Ser Lys Ile Gly Glu Lys Ser Ala Trp Met Leu Asn Leu Ser Ile 325 330 335 Asp Val Pro Lys Ile Asp Lys Gly Val Asp Pro Ser Ile Ile Gly Gly 340 345 350 Ile Asp Val Gly Val Lys Ser Pro Leu Val Cys Ala Ile Asn Asn Ala 355 360 365 Phe Ser Arg Tyr Ser Ile Ser Asp Asn Asp Leu Phe His Phe Asn Lys 370 375 380 Lys Met Phe Ala Arg Arg Arg Ile Leu Leu Lys Lys Asn Arg His Lys 385 390 395 400 Arg Ala Gly His Gly Ala Lys Asn Lys Leu Lys Pro Ile Thr Ile Leu 405 410 415 Thr Glu Lys Ser Glu Arg Phe Arg Lys Lys Leu Ile Glu Arg Trp Ala 420 425 430 Cys Glu Ile Ala Asp Phe Phe Ile Lys Asn Lys Val Gly Thr Val Gln 435 440 445 Met Ala Asn Leu Glu Ser Met Lys Arg Lys Glu Asp Ser Tyr Phe Asn 450 455 460 Ile Arg Leu Arg Gly Phe Trp Pro Tyr Ala Glu Met Gln Asn Lys Ile 465 470 475 480 Glu Phe Lys Leu Lys Gln Tyr Gly Ile Glu Ile Arg Lys Val Ala Pro 485 490 495 Asn Asn Thr Ser Lys Thr Cys Ser Lys Cys Gly His Leu Asn Asn Tyr 500 505 510 Phe Asn Phe Glu Tyr Arg Lys Lys Asn Lys Phe Pro His Phe Lys Cys 515 520 525 Glu Lys Cys Asn Phe Lys Glu Asn Ala Asp Tyr Asn Ala Ala Leu Asn 530 535 540 Ile Ser Asn Pro Lys Leu Lys Ser Thr Lys Glu Glu Pro 545 550 555 <210> 13 <211> 557 <212> PRT <213> artificial sequence <220> <223> dTnpB (R518A) <400> 13 Met Gly Glu Lys Ser Ser Arg Arg Arg Arg Asn Gly Lys Ser Gly Ala 1 5 10 15 Trp Thr Ala Ala Ile Thr Ser Cys Val Gly Gly Lys Met Ala Lys Asn 20 25 30 Thr Ile Thr Lys Thr Leu Lys Leu Arg Ile Val Arg Pro Tyr Asn Ser 35 40 45 Ala Glu Val Glu Lys Ile Val Ala Asp Glu Lys Asn Asn Arg Glu Lys 50 55 60 Ile Ala Leu Glu Lys Asn Lys Asp Lys Val Lys Glu Ala Cys Ser Lys 65 70 75 80 His Leu Lys Val Ala Ala Tyr Cys Thr Thr Gln Val Glu Arg Asn Ala 85 90 95 Cys Leu Phe Cys Lys Ala Arg Lys Leu Asp Asp Lys Phe Tyr Gln Lys 100 105 110 Leu Arg Gly Gln Phe Pro Asp Ala Val Phe Trp Gln Glu Ile Ser Glu 115 120 125 Ile Phe Arg Gln Leu Gln Lys Gln Ala Ala Glu Ile Tyr Asn Gln Ser 130 135 140 Leu Ile Glu Leu Tyr Tyr Glu Ile Phe Ile Lys Gly Lys Gly Ile Ala 145 150 155 160 Asn Ala Ser Ser Val Glu His Tyr Leu Ser Asp Val Cys Tyr Thr Arg 165 170 175 Ala Ala Glu Leu Phe Lys Asn Ala Ala Ile Ala Ser Gly Leu Arg Ser 180 185 190 Lys Ile Lys Ser Asn Phe Arg Leu Lys Glu Leu Lys Asn Met Lys Ser 195 200 205 Gly Leu Pro Thr Thr Lys Ser Asp Asn Phe Pro Ile Pro Leu Val Lys 210 215 220 Gln Lys Gly Gly Gln Tyr Thr Gly Phe Glu Ile Ser Asn His Asn Ser 225 230 235 240 Asp Phe Ile Ile Lys Ile Pro Phe Gly Arg Trp Gln Val Lys Lys Glu 245 250 255 Ile Asp Lys Tyr Arg Pro Trp Glu Lys Phe Asp Phe Glu Gln Val Gln 260 265 270 Lys Ser Pro Lys Pro Ile Ser Leu Leu Leu Ser Thr Gln Arg Arg Lys 275 280 285 Arg Asn Lys Gly Trp Ser Lys Asp Glu Gly Thr Glu Ala Glu Ile Lys 290 295 300 Lys Val Met Asn Gly Asp Tyr Gln Thr Ser Tyr Ile Glu Val Lys Arg 305 310 315 320 Gly Ser Lys Ile Gly Glu Lys Ser Ala Trp Met Leu Asn Leu Ser Ile 325 330 335 Asp Val Pro Lys Ile Asp Lys Gly Val Asp Pro Ser Ile Ile Gly Gly 340 345 350 Ile Asp Val Gly Val Lys Ser Pro Leu Val Cys Ala Ile Asn Asn Ala 355 360 365 Phe Ser Arg Tyr Ser Ile Ser Asp Asn Asp Leu Phe His Phe Asn Lys 370 375 380 Lys Met Phe Ala Arg Arg Arg Ile Leu Leu Lys Lys Asn Arg His Lys 385 390 395 400 Arg Ala Gly His Gly Ala Lys Asn Lys Leu Lys Pro Ile Thr Ile Leu 405 410 415 Thr Glu Lys Ser Glu Arg Phe Arg Lys Lys Leu Ile Glu Arg Trp Ala 420 425 430 Cys Glu Ile Ala Asp Phe Phe Ile Lys Asn Lys Val Gly Thr Val Gln 435 440 445 Met Glu Asn Leu Glu Ser Met Lys Arg Lys Glu Asp Ser Tyr Phe Asn 450 455 460 Ile Arg Leu Arg Gly Phe Trp Pro Tyr Ala Glu Met Gln Asn Lys Ile 465 470 475 480 Glu Phe Lys Leu Lys Gln Tyr Gly Ile Glu Ile Arg Lys Val Ala Pro 485 490 495 Asn Asn Thr Ser Lys Thr Cys Ser Lys Cys Gly His Leu Asn Asn Tyr 500 505 510 Phe Asn Phe Glu Tyr Ala Lys Lys Asn Lys Phe Pro His Phe Lys Cys 515 520 525 Glu Lys Cys Asn Phe Lys Glu Asn Ala Asp Tyr Asn Ala Ala Leu Asn 530 535 540 Ile Ser Asn Pro Lys Leu Lys Ser Thr Lys Glu Glu Pro 545 550 555 <210> 14 <211> 557 <212> PRT <213> artificial sequence <220> <223> dTnpB (D538A) <400> 14 Met Gly Glu Lys Ser Ser Arg Arg Arg Arg Asn Gly Lys Ser Gly Ala 1 5 10 15 Trp Thr Ala Ala Ile Thr Ser Cys Val Gly Gly Lys Met Ala Lys Asn 20 25 30 Thr Ile Thr Lys Thr Leu Lys Leu Arg Ile Val Arg Pro Tyr Asn Ser 35 40 45 Ala Glu Val Glu Lys Ile Val Ala Asp Glu Lys Asn Asn Arg Glu Lys 50 55 60 Ile Ala Leu Glu Lys Asn Lys Asp Lys Val Lys Glu Ala Cys Ser Lys 65 70 75 80 His Leu Lys Val Ala Ala Tyr Cys Thr Thr Gln Val Glu Arg Asn Ala 85 90 95 Cys Leu Phe Cys Lys Ala Arg Lys Leu Asp Asp Lys Phe Tyr Gln Lys 100 105 110 Leu Arg Gly Gln Phe Pro Asp Ala Val Phe Trp Gln Glu Ile Ser Glu 115 120 125 Ile Phe Arg Gln Leu Gln Lys Gln Ala Ala Glu Ile Tyr Asn Gln Ser 130 135 140 Leu Ile Glu Leu Tyr Tyr Glu Ile Phe Ile Lys Gly Lys Gly Ile Ala 145 150 155 160 Asn Ala Ser Ser Val Glu His Tyr Leu Ser Asp Val Cys Tyr Thr Arg 165 170 175 Ala Ala Glu Leu Phe Lys Asn Ala Ala Ile Ala Ser Gly Leu Arg Ser 180 185 190 Lys Ile Lys Ser Asn Phe Arg Leu Lys Glu Leu Lys Asn Met Lys Ser 195 200 205 Gly Leu Pro Thr Thr Lys Ser Asp Asn Phe Pro Ile Pro Leu Val Lys 210 215 220 Gln Lys Gly Gly Gln Tyr Thr Gly Phe Glu Ile Ser Asn His Asn Ser 225 230 235 240 Asp Phe Ile Ile Lys Ile Pro Phe Gly Arg Trp Gln Val Lys Lys Glu 245 250 255 Ile Asp Lys Tyr Arg Pro Trp Glu Lys Phe Asp Phe Glu Gln Val Gln 260 265 270 Lys Ser Pro Lys Pro Ile Ser Leu Leu Leu Ser Thr Gln Arg Arg Lys 275 280 285 Arg Asn Lys Gly Trp Ser Lys Asp Glu Gly Thr Glu Ala Glu Ile Lys 290 295 300 Lys Val Met Asn Gly Asp Tyr Gln Thr Ser Tyr Ile Glu Val Lys Arg 305 310 315 320 Gly Ser Lys Ile Gly Glu Lys Ser Ala Trp Met Leu Asn Leu Ser Ile 325 330 335 Asp Val Pro Lys Ile Asp Lys Gly Val Asp Pro Ser Ile Ile Gly Gly 340 345 350 Ile Asp Val Gly Val Lys Ser Pro Leu Val Cys Ala Ile Asn Asn Ala 355 360 365 Phe Ser Arg Tyr Ser Ile Ser Asp Asn Asp Leu Phe His Phe Asn Lys 370 375 380 Lys Met Phe Ala Arg Arg Arg Ile Leu Leu Lys Lys Asn Arg His Lys 385 390 395 400 Arg Ala Gly His Gly Ala Lys Asn Lys Leu Lys Pro Ile Thr Ile Leu 405 410 415 Thr Glu Lys Ser Glu Arg Phe Arg Lys Lys Leu Ile Glu Arg Trp Ala 420 425 430 Cys Glu Ile Ala Asp Phe Phe Ile Lys Asn Lys Val Gly Thr Val Gln 435 440 445 Met Glu Asn Leu Glu Ser Met Lys Arg Lys Glu Asp Ser Tyr Phe Asn 450 455 460 Ile Arg Leu Arg Gly Phe Trp Pro Tyr Ala Glu Met Gln Asn Lys Ile 465 470 475 480 Glu Phe Lys Leu Lys Gln Tyr Gly Ile Glu Ile Arg Lys Val Ala Pro 485 490 495 Asn Asn Thr Ser Lys Thr Cys Ser Lys Cys Gly His Leu Asn Asn Tyr 500 505 510 Phe Asn Phe Glu Tyr Arg Lys Lys Asn Lys Phe Pro His Phe Lys Cys 515 520 525 Glu Lys Cys Asn Phe Lys Glu Asn Ala Ala Tyr Asn Ala Ala Leu Asn 530 535 540 Ile Ser Asn Pro Lys Leu Lys Ser Thr Lys Glu Glu Pro 545 550 555 <210> 15 <211> 557 <212> PRT <213> artificial sequence <220> <223> 28aa-extension dCas12f1 (D354A) <400> 15 Met Ala Gly Gly Pro Gly Ala Gly Ser Ala Ala Pro Val Ser Ser Thr 1 5 10 15 Ser Ser Leu Pro Leu Ala Ala Leu Asn Met Arg Val Met Ala Lys Asn 20 25 30 Thr Ile Thr Lys Thr Leu Lys Leu Arg Ile Val Arg Pro Tyr Asn Ser 35 40 45 Ala Glu Val Glu Lys Ile Val Ala Asp Glu Lys Asn Asn Arg Glu Lys 50 55 60 Ile Ala Leu Glu Lys Asn Lys Asp Lys Val Lys Glu Ala Cys Ser Lys 65 70 75 80 His Leu Lys Val Ala Ala Tyr Cys Thr Thr Gln Val Glu Arg Asn Ala 85 90 95 Cys Leu Phe Cys Lys Ala Arg Lys Leu Asp Asp Lys Phe Tyr Gln Lys 100 105 110 Leu Arg Gly Gln Phe Pro Asp Ala Val Phe Trp Gln Glu Ile Ser Glu 115 120 125 Ile Phe Arg Gln Leu Gln Lys Gln Ala Ala Glu Ile Tyr Asn Gln Ser 130 135 140 Leu Ile Glu Leu Tyr Tyr Glu Ile Phe Ile Lys Gly Lys Gly Ile Ala 145 150 155 160 Asn Ala Ser Ser Val Glu His Tyr Leu Ser Asp Val Cys Tyr Thr Arg 165 170 175 Ala Ala Glu Leu Phe Lys Asn Ala Ala Ile Ala Ser Gly Leu Arg Ser 180 185 190 Lys Ile Lys Ser Asn Phe Arg Leu Lys Glu Leu Lys Asn Met Lys Ser 195 200 205 Gly Leu Pro Thr Thr Lys Ser Asp Asn Phe Pro Ile Pro Leu Val Lys 210 215 220 Gln Lys Gly Gly Gln Tyr Thr Gly Phe Glu Ile Ser Asn His Asn Ser 225 230 235 240 Asp Phe Ile Ile Lys Ile Pro Phe Gly Arg Trp Gln Val Lys Lys Glu 245 250 255 Ile Asp Lys Tyr Arg Pro Trp Glu Lys Phe Asp Phe Glu Gln Val Gln 260 265 270 Lys Ser Pro Lys Pro Ile Ser Leu Leu Leu Ser Thr Gln Arg Arg Lys 275 280 285 Arg Asn Lys Gly Trp Ser Lys Asp Glu Gly Thr Glu Ala Glu Ile Lys 290 295 300 Lys Val Met Asn Gly Asp Tyr Gln Thr Ser Tyr Ile Glu Val Lys Arg 305 310 315 320 Gly Ser Lys Ile Gly Glu Lys Ser Ala Trp Met Leu Asn Leu Ser Ile 325 330 335 Asp Val Pro Lys Ile Asp Lys Gly Val Asp Pro Ser Ile Ile Gly Gly 340 345 350 Ile Ala Val Gly Val Lys Ser Pro Leu Val Cys Ala Ile Asn Asn Ala 355 360 365 Phe Ser Arg Tyr Ser Ile Ser Asp Asn Asp Leu Phe His Phe Asn Lys 370 375 380 Lys Met Phe Ala Arg Arg Arg Ile Leu Leu Lys Lys Asn Arg His Lys 385 390 395 400 Arg Ala Gly His Gly Ala Lys Asn Lys Leu Lys Pro Ile Thr Ile Leu 405 410 415 Thr Glu Lys Ser Glu Arg Phe Arg Lys Lys Leu Ile Glu Arg Trp Ala 420 425 430 Cys Glu Ile Ala Asp Phe Phe Ile Lys Asn Lys Val Gly Thr Val Gln 435 440 445 Met Glu Asn Leu Glu Ser Met Lys Arg Lys Glu Asp Ser Tyr Phe Asn 450 455 460 Ile Arg Leu Arg Gly Phe Trp Pro Tyr Ala Glu Met Gln Asn Lys Ile 465 470 475 480 Glu Phe Lys Leu Lys Gln Tyr Gly Ile Glu Ile Arg Lys Val Ala Pro 485 490 495 Asn Asn Thr Ser Lys Thr Cys Ser Lys Cys Gly His Leu Asn Asn Tyr 500 505 510 Phe Asn Phe Glu Tyr Arg Lys Lys Asn Lys Phe Pro His Phe Lys Cys 515 520 525 Glu Lys Cys Asn Phe Lys Glu Asn Ala Asp Tyr Asn Ala Ala Leu Asn 530 535 540 Ile Ser Asn Pro Lys Leu Lys Ser Thr Lys Glu Glu Pro 545 550 555 <210> 16 <211> 557 <212> PRT <213> artificial sequence <220> <223> 28aa-extension dCas12f1 (E450A) <400> 16 Met Ala Gly Gly Pro Gly Ala Gly Ser Ala Ala Pro Val Ser Ser Thr 1 5 10 15 Ser Ser Leu Pro Leu Ala Ala Leu Asn Met Arg Val Met Ala Lys Asn 20 25 30 Thr Ile Thr Lys Thr Leu Lys Leu Arg Ile Val Arg Pro Tyr Asn Ser 35 40 45 Ala Glu Val Glu Lys Ile Val Ala Asp Glu Lys Asn Asn Arg Glu Lys 50 55 60 Ile Ala Leu Glu Lys Asn Lys Asp Lys Val Lys Glu Ala Cys Ser Lys 65 70 75 80 His Leu Lys Val Ala Ala Tyr Cys Thr Thr Gln Val Glu Arg Asn Ala 85 90 95 Cys Leu Phe Cys Lys Ala Arg Lys Leu Asp Asp Lys Phe Tyr Gln Lys 100 105 110 Leu Arg Gly Gln Phe Pro Asp Ala Val Phe Trp Gln Glu Ile Ser Glu 115 120 125 Ile Phe Arg Gln Leu Gln Lys Gln Ala Ala Glu Ile Tyr Asn Gln Ser 130 135 140 Leu Ile Glu Leu Tyr Tyr Glu Ile Phe Ile Lys Gly Lys Gly Ile Ala 145 150 155 160 Asn Ala Ser Ser Val Glu His Tyr Leu Ser Asp Val Cys Tyr Thr Arg 165 170 175 Ala Ala Glu Leu Phe Lys Asn Ala Ala Ile Ala Ser Gly Leu Arg Ser 180 185 190 Lys Ile Lys Ser Asn Phe Arg Leu Lys Glu Leu Lys Asn Met Lys Ser 195 200 205 Gly Leu Pro Thr Thr Lys Ser Asp Asn Phe Pro Ile Pro Leu Val Lys 210 215 220 Gln Lys Gly Gly Gln Tyr Thr Gly Phe Glu Ile Ser Asn His Asn Ser 225 230 235 240 Asp Phe Ile Ile Lys Ile Pro Phe Gly Arg Trp Gln Val Lys Lys Glu 245 250 255 Ile Asp Lys Tyr Arg Pro Trp Glu Lys Phe Asp Phe Glu Gln Val Gln 260 265 270 Lys Ser Pro Lys Pro Ile Ser Leu Leu Leu Ser Thr Gln Arg Arg Lys 275 280 285 Arg Asn Lys Gly Trp Ser Lys Asp Glu Gly Thr Glu Ala Glu Ile Lys 290 295 300 Lys Val Met Asn Gly Asp Tyr Gln Thr Ser Tyr Ile Glu Val Lys Arg 305 310 315 320 Gly Ser Lys Ile Gly Glu Lys Ser Ala Trp Met Leu Asn Leu Ser Ile 325 330 335 Asp Val Pro Lys Ile Asp Lys Gly Val Asp Pro Ser Ile Ile Gly Gly 340 345 350 Ile Asp Val Gly Val Lys Ser Pro Leu Val Cys Ala Ile Asn Asn Ala 355 360 365 Phe Ser Arg Tyr Ser Ile Ser Asp Asn Asp Leu Phe His Phe Asn Lys 370 375 380 Lys Met Phe Ala Arg Arg Arg Ile Leu Leu Lys Lys Asn Arg His Lys 385 390 395 400 Arg Ala Gly His Gly Ala Lys Asn Lys Leu Lys Pro Ile Thr Ile Leu 405 410 415 Thr Glu Lys Ser Glu Arg Phe Arg Lys Lys Leu Ile Glu Arg Trp Ala 420 425 430 Cys Glu Ile Ala Asp Phe Phe Ile Lys Asn Lys Val Gly Thr Val Gln 435 440 445 Met Ala Asn Leu Glu Ser Met Lys Arg Lys Glu Asp Ser Tyr Phe Asn 450 455 460 Ile Arg Leu Arg Gly Phe Trp Pro Tyr Ala Glu Met Gln Asn Lys Ile 465 470 475 480 Glu Phe Lys Leu Lys Gln Tyr Gly Ile Glu Ile Arg Lys Val Ala Pro 485 490 495 Asn Asn Thr Ser Lys Thr Cys Ser Lys Cys Gly His Leu Asn Asn Tyr 500 505 510 Phe Asn Phe Glu Tyr Arg Lys Lys Asn Lys Phe Pro His Phe Lys Cys 515 520 525 Glu Lys Cys Asn Phe Lys Glu Asn Ala Asp Tyr Asn Ala Ala Leu Asn 530 535 540 Ile Ser Asn Pro Lys Leu Lys Ser Thr Lys Glu Glu Pro 545 550 555 <210> 17 <211> 557 <212> PRT <213> artificial sequence <220> <223> 28aa-extension dCas12f1 (R518A) <400> 17 Met Ala Gly Gly Pro Gly Ala Gly Ser Ala Ala Pro Val Ser Ser Thr 1 5 10 15 Ser Ser Leu Pro Leu Ala Ala Leu Asn Met Arg Val Met Ala Lys Asn 20 25 30 Thr Ile Thr Lys Thr Leu Lys Leu Arg Ile Val Arg Pro Tyr Asn Ser 35 40 45 Ala Glu Val Glu Lys Ile Val Ala Asp Glu Lys Asn Asn Arg Glu Lys 50 55 60 Ile Ala Leu Glu Lys Asn Lys Asp Lys Val Lys Glu Ala Cys Ser Lys 65 70 75 80 His Leu Lys Val Ala Ala Tyr Cys Thr Thr Gln Val Glu Arg Asn Ala 85 90 95 Cys Leu Phe Cys Lys Ala Arg Lys Leu Asp Asp Lys Phe Tyr Gln Lys 100 105 110 Leu Arg Gly Gln Phe Pro Asp Ala Val Phe Trp Gln Glu Ile Ser Glu 115 120 125 Ile Phe Arg Gln Leu Gln Lys Gln Ala Ala Glu Ile Tyr Asn Gln Ser 130 135 140 Leu Ile Glu Leu Tyr Tyr Glu Ile Phe Ile Lys Gly Lys Gly Ile Ala 145 150 155 160 Asn Ala Ser Ser Val Glu His Tyr Leu Ser Asp Val Cys Tyr Thr Arg 165 170 175 Ala Ala Glu Leu Phe Lys Asn Ala Ala Ile Ala Ser Gly Leu Arg Ser 180 185 190 Lys Ile Lys Ser Asn Phe Arg Leu Lys Glu Leu Lys Asn Met Lys Ser 195 200 205 Gly Leu Pro Thr Thr Lys Ser Asp Asn Phe Pro Ile Pro Leu Val Lys 210 215 220 Gln Lys Gly Gly Gln Tyr Thr Gly Phe Glu Ile Ser Asn His Asn Ser 225 230 235 240 Asp Phe Ile Ile Lys Ile Pro Phe Gly Arg Trp Gln Val Lys Lys Glu 245 250 255 Ile Asp Lys Tyr Arg Pro Trp Glu Lys Phe Asp Phe Glu Gln Val Gln 260 265 270 Lys Ser Pro Lys Pro Ile Ser Leu Leu Leu Ser Thr Gln Arg Arg Lys 275 280 285 Arg Asn Lys Gly Trp Ser Lys Asp Glu Gly Thr Glu Ala Glu Ile Lys 290 295 300 Lys Val Met Asn Gly Asp Tyr Gln Thr Ser Tyr Ile Glu Val Lys Arg 305 310 315 320 Gly Ser Lys Ile Gly Glu Lys Ser Ala Trp Met Leu Asn Leu Ser Ile 325 330 335 Asp Val Pro Lys Ile Asp Lys Gly Val Asp Pro Ser Ile Ile Gly Gly 340 345 350 Ile Asp Val Gly Val Lys Ser Pro Leu Val Cys Ala Ile Asn Asn Ala 355 360 365 Phe Ser Arg Tyr Ser Ile Ser Asp Asn Asp Leu Phe His Phe Asn Lys 370 375 380 Lys Met Phe Ala Arg Arg Arg Ile Leu Leu Lys Lys Asn Arg His Lys 385 390 395 400 Arg Ala Gly His Gly Ala Lys Asn Lys Leu Lys Pro Ile Thr Ile Leu 405 410 415 Thr Glu Lys Ser Glu Arg Phe Arg Lys Lys Leu Ile Glu Arg Trp Ala 420 425 430 Cys Glu Ile Ala Asp Phe Phe Ile Lys Asn Lys Val Gly Thr Val Gln 435 440 445 Met Glu Asn Leu Glu Ser Met Lys Arg Lys Glu Asp Ser Tyr Phe Asn 450 455 460 Ile Arg Leu Arg Gly Phe Trp Pro Tyr Ala Glu Met Gln Asn Lys Ile 465 470 475 480 Glu Phe Lys Leu Lys Gln Tyr Gly Ile Glu Ile Arg Lys Val Ala Pro 485 490 495 Asn Asn Thr Ser Lys Thr Cys Ser Lys Cys Gly His Leu Asn Asn Tyr 500 505 510 Phe Asn Phe Glu Tyr Ala Lys Lys Asn Lys Phe Pro His Phe Lys Cys 515 520 525 Glu Lys Cys Asn Phe Lys Glu Asn Ala Asp Tyr Asn Ala Ala Leu Asn 530 535 540 Ile Ser Asn Pro Lys Leu Lys Ser Thr Lys Glu Glu Pro 545 550 555 <210> 18 <211> 557 <212> PRT <213> artificial sequence <220> <223> 28aa-extension dCas12f1 (D538A) <400> 18 Met Ala Gly Gly Pro Gly Ala Gly Ser Ala Ala Pro Val Ser Ser Thr 1 5 10 15 Ser Ser Leu Pro Leu Ala Ala Leu Asn Met Arg Val Met Ala Lys Asn 20 25 30 Thr Ile Thr Lys Thr Leu Lys Leu Arg Ile Val Arg Pro Tyr Asn Ser 35 40 45 Ala Glu Val Glu Lys Ile Val Ala Asp Glu Lys Asn Asn Arg Glu Lys 50 55 60 Ile Ala Leu Glu Lys Asn Lys Asp Lys Val Lys Glu Ala Cys Ser Lys 65 70 75 80 His Leu Lys Val Ala Ala Tyr Cys Thr Thr Gln Val Glu Arg Asn Ala 85 90 95 Cys Leu Phe Cys Lys Ala Arg Lys Leu Asp Asp Lys Phe Tyr Gln Lys 100 105 110 Leu Arg Gly Gln Phe Pro Asp Ala Val Phe Trp Gln Glu Ile Ser Glu 115 120 125 Ile Phe Arg Gln Leu Gln Lys Gln Ala Ala Glu Ile Tyr Asn Gln Ser 130 135 140 Leu Ile Glu Leu Tyr Tyr Glu Ile Phe Ile Lys Gly Lys Gly Ile Ala 145 150 155 160 Asn Ala Ser Ser Val Glu His Tyr Leu Ser Asp Val Cys Tyr Thr Arg 165 170 175 Ala Ala Glu Leu Phe Lys Asn Ala Ala Ile Ala Ser Gly Leu Arg Ser 180 185 190 Lys Ile Lys Ser Asn Phe Arg Leu Lys Glu Leu Lys Asn Met Lys Ser 195 200 205 Gly Leu Pro Thr Thr Lys Ser Asp Asn Phe Pro Ile Pro Leu Val Lys 210 215 220 Gln Lys Gly Gly Gln Tyr Thr Gly Phe Glu Ile Ser Asn His Asn Ser 225 230 235 240 Asp Phe Ile Ile Lys Ile Pro Phe Gly Arg Trp Gln Val Lys Lys Glu 245 250 255 Ile Asp Lys Tyr Arg Pro Trp Glu Lys Phe Asp Phe Glu Gln Val Gln 260 265 270 Lys Ser Pro Lys Pro Ile Ser Leu Leu Leu Ser Thr Gln Arg Arg Lys 275 280 285 Arg Asn Lys Gly Trp Ser Lys Asp Glu Gly Thr Glu Ala Glu Ile Lys 290 295 300 Lys Val Met Asn Gly Asp Tyr Gln Thr Ser Tyr Ile Glu Val Lys Arg 305 310 315 320 Gly Ser Lys Ile Gly Glu Lys Ser Ala Trp Met Leu Asn Leu Ser Ile 325 330 335 Asp Val Pro Lys Ile Asp Lys Gly Val Asp Pro Ser Ile Ile Gly Gly 340 345 350 Ile Asp Val Gly Val Lys Ser Pro Leu Val Cys Ala Ile Asn Asn Ala 355 360 365 Phe Ser Arg Tyr Ser Ile Ser Asp Asn Asp Leu Phe His Phe Asn Lys 370 375 380 Lys Met Phe Ala Arg Arg Arg Ile Leu Leu Lys Lys Asn Arg His Lys 385 390 395 400 Arg Ala Gly His Gly Ala Lys Asn Lys Leu Lys Pro Ile Thr Ile Leu 405 410 415 Thr Glu Lys Ser Glu Arg Phe Arg Lys Lys Leu Ile Glu Arg Trp Ala 420 425 430 Cys Glu Ile Ala Asp Phe Phe Ile Lys Asn Lys Val Gly Thr Val Gln 435 440 445 Met Glu Asn Leu Glu Ser Met Lys Arg Lys Glu Asp Ser Tyr Phe Asn 450 455 460 Ile Arg Leu Arg Gly Phe Trp Pro Tyr Ala Glu Met Gln Asn Lys Ile 465 470 475 480 Glu Phe Lys Leu Lys Gln Tyr Gly Ile Glu Ile Arg Lys Val Ala Pro 485 490 495 Asn Asn Thr Ser Lys Thr Cys Ser Lys Cys Gly His Leu Asn Asn Tyr 500 505 510 Phe Asn Phe Glu Tyr Arg Lys Lys Asn Lys Phe Pro His Phe Lys Cys 515 520 525 Glu Lys Cys Asn Phe Lys Glu Asn Ala Ala Tyr Asn Ala Ala Leu Asn 530 535 540 Ile Ser Asn Pro Lys Leu Lys Ser Thr Lys Glu Glu Pro 545 550 555 <210> 19 <211> 167 <212> PRT <213> artificial sequence <220> <223> Adenine deaminase Tad <400> 19 Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu 1 5 10 15 Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala 20 25 30 Val Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro 35 40 45 Ile Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg 50 55 60 Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu 65 70 75 80 Tyr Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His 85 90 95 Ser Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly 100 105 110 Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His 115 120 125 Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu 130 135 140 Leu Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys 145 150 155 160 Lys Ala Gln Ser Ser Thr Asp 165 <210> 20 <211> 167 <212> PRT <213> artificial sequence <220> <223> Adenine deaminase eTad <400> 20 Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu 1 5 10 15 Thr Leu Ala Lys Arg Ala Arg Asp Glu Arg Glu Val Pro Val Gly Ala 20 25 30 Val Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Ala 35 40 45 Ile Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg 50 55 60 Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu 65 70 75 80 Tyr Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His 85 90 95 Ser Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr Gly 100 105 110 Ala Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His 115 120 125 Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu 130 135 140 Leu Cys Tyr Phe Phe Arg Met Pro Arg Gln Val Phe Asn Ala Gln Lys 145 150 155 160 Lys Ala Gln Ser Ser Thr Asp 165 <210> 21 <211> 229 <212> PRT <213> artificial sequence <220> <223> cytidine deaminase APOBEC1 <400> 21 Met Ser Ser Glu Thr Gly Pro Val Ala Val Asp Pro Thr Leu Arg Arg 1 5 10 15 Arg Ile Glu Pro His Glu Phe Glu Val Phe Phe Asp Pro Arg Glu Leu 20 25 30 Arg Lys Glu Thr Cys Leu Leu Tyr Glu Ile Asn Trp Gly Gly Arg His 35 40 45 Ser Ile Trp Arg His Thr Ser Gln Asn Thr Asn Lys His Val Glu Val 50 55 60 Asn Phe Ile Glu Lys Phe Thr Thr Glu Arg Tyr Phe Cys Pro Asn Thr 65 70 75 80 Arg Cys Ser Ile Thr Trp Phe Leu Ser Trp Ser Pro Cys Gly Glu Cys 85 90 95 Ser Arg Ala Ile Thr Glu Phe Leu Ser Arg Tyr Pro His Val Thr Leu 100 105 110 Phe Ile Tyr Ile Ala Arg Leu Tyr His His Ala Asp Pro Arg Asn Arg 115 120 125 Gln Gly Leu Arg Asp Leu Ile Ser Ser Gly Val Thr Ile Gln Ile Met 130 135 140 Thr Glu Gln Glu Ser Gly Tyr Cys Trp Arg Asn Phe Val Asn Tyr Ser 145 150 155 160 Pro Ser Asn Glu Ala His Trp Pro Arg Tyr Pro His Leu Trp Val Arg 165 170 175 Leu Tyr Val Leu Glu Leu Tyr Cys Ile Ile Leu Gly Leu Pro Pro Cys 180 185 190 Leu Asn Ile Leu Arg Arg Lys Gln Pro Gln Leu Thr Phe Phe Thr Ile 195 200 205 Ala Leu Gln Ser Cys His Tyr Gln Arg Leu Pro Pro His Ile Leu Trp 210 215 220 Ala Thr Gly Leu Lys 225 <210> 22 <211> 199 <212> PRT <213> artificial sequence <220> <223> cytidine deaminase APOBEC3A <400> 22 Met Glu Ala Ser Pro Ala Ser Gly Pro Arg His Leu Met Asp Pro His 1 5 10 15 Ile Phe Thr Ser Asn Phe Asn Asn Gly Ile Gly Arg His Lys Thr Tyr 20 25 30 Leu Cys Tyr Glu Val Glu Arg Leu Asp Asn Gly Thr Ser Val Lys Met 35 40 45 Asp Gln His Arg Gly Phe Leu His Asn Gln Ala Lys Asn Leu Leu Cys 50 55 60 Gly Phe Tyr Gly Arg His Ala Glu Leu Arg Phe Leu Asp Leu Val Pro 65 70 75 80 Ser Leu Gln Leu Asp Pro Ala Gln Ile Tyr Arg Val Thr Trp Phe Ile 85 90 95 Ser Trp Ser Pro Cys Phe Ser Trp Gly Cys Ala Gly Glu Val Arg Ala 100 105 110 Phe Leu Gln Glu Asn Thr His Val Arg Leu Arg Ile Phe Ala Ala Arg 115 120 125 Ile Tyr Asp Tyr Asp Pro Leu Tyr Lys Glu Ala Leu Gln Met Leu Arg 130 135 140 Asp Ala Gly Ala Gln Val Ser Ile Met Thr Tyr Asp Glu Phe Lys His 145 150 155 160 Cys Trp Asp Thr Phe Val Asp His Gln Gly Cys Pro Phe Gln Pro Trp 165 170 175 Asp Gly Leu Asp Glu His Ser Gln Ala Leu Ser Gly Arg Leu Arg Ala 180 185 190 Ile Leu Gln Asn Gln Gly Asn 195 <210> 23 <211> 382 <212> PRT <213> artificial sequence <220> <223> cytidine deaminase APOBEC3B <400> 23 Met Asn Pro Gln Ile Arg Asn Pro Met Glu Arg Met Tyr Arg Asp Thr 1 5 10 15 Phe Tyr Asp Asn Phe Glu Asn Glu Pro Ile Leu Tyr Gly Arg Ser Tyr 20 25 30 Thr Trp Leu Cys Tyr Glu Val Lys Ile Lys Arg Gly Arg Ser Asn Leu 35 40 45 Leu Trp Asp Thr Gly Val Phe Arg Gly Gln Val Tyr Phe Lys Pro Gln 50 55 60 Tyr His Ala Glu Met Cys Phe Leu Ser Trp Phe Cys Gly Asn Gln Leu 65 70 75 80 Pro Ala Tyr Lys Cys Phe Gln Ile Thr Trp Phe Val Ser Trp Thr Pro 85 90 95 Cys Pro Asp Cys Val Ala Lys Leu Ala Glu Phe Leu Ser Glu His Pro 100 105 110 Asn Val Thr Leu Thr Ile Ser Ala Ala Arg Leu Tyr Tyr Tyr Trp Glu 115 120 125 Arg Asp Tyr Arg Arg Ala Leu Cys Arg Leu Ser Gln Ala Gly Ala Arg 130 135 140 Val Lys Ile Met Asp Tyr Glu Glu Phe Ala Tyr Cys Trp Glu Asn Phe 145 150 155 160 Val Tyr Asn Glu Gly Gln Gln Phe Met Pro Trp Tyr Lys Phe Asp Glu 165 170 175 Asn Tyr Ala Phe Leu His Arg Thr Leu Lys Glu Ile Leu Arg Tyr Leu 180 185 190 Met Asp Pro Asp Thr Phe Thr Phe Asn Phe Asn Asn Asp Pro Leu Val 195 200 205 Leu Arg Arg Arg Gln Thr Tyr Leu Cys Tyr Glu Val Glu Arg Leu Asp 210 215 220 Asn Gly Thr Trp Val Leu Met Asp Gln His Met Gly Phe Leu Cys Asn 225 230 235 240 Glu Ala Lys Asn Leu Leu Cys Gly Phe Tyr Gly Arg His Ala Glu Leu 245 250 255 Arg Phe Leu Asp Leu Val Pro Ser Leu Gln Leu Asp Pro Ala Gln Ile 260 265 270 Tyr Arg Val Thr Trp Phe Ile Ser Trp Ser Pro Cys Phe Ser Trp Gly 275 280 285 Cys Ala Gly Glu Val Arg Ala Phe Leu Gln Glu Asn Thr His Val Arg 290 295 300 Leu Arg Ile Phe Ala Ala Arg Ile Tyr Asp Tyr Asp Pro Leu Tyr Lys 305 310 315 320 Glu Ala Leu Gln Met Leu Arg Asp Ala Gly Ala Gln Val Ser Ile Met 325 330 335 Thr Tyr Asp Glu Phe Glu Tyr Cys Trp Asp Thr Phe Val Tyr Arg Gln 340 345 350 Gly Cys Pro Ser Gln Pro Trp Asp Gly Leu Glu Glu His Ser Gln Ala 355 360 365 Leu Ser Gly Arg Leu Arg Ala Ile Leu Gln Asn Gln Gly Asn 370 375 380 <210> 24 <211> 21 <212> RNA <213> artificial sequence <220> <223> The first region of tracrRNA for Cas12f1 gRNA <400> 24 cuucacugau aaaguggaga a 21 <210> 25 <211> 50 <212> RNA <213> artificial sequence <220> <223> The second region of tracrRNA for Cas12f1 gRNA <400> 25 ccgcuucacc aaaagcuguc ccuuagggga uuagaacuug agugaaggug 50 <210> 26 <211> 58 <212> RNA <213> artificial sequence <220> <223> The third region of tracrRNA for Cas12f1 gRNA <400> 26 58 <210> 27 <211> 32 <212> RNA <213> artificial sequence <220> <223> The fourth region of tracrRNA for Cas12f1 gRNA <400> 27 caaauucann nvnccucucc aauucugcac aa 32 <210> 28 <211> 13 <212> RNA <213> artificial sequence <220> <223> The fourth region of tracrRNA for Cas12f1 gRNA <400> 28 caaauucann nvn 13 <210> 29 <211> 13 <212> RNA <213> artificial sequence <220> <223> The fourth region of tracrRNA for Cas12f1 gRNA <400> 29 caaauucann ncn 13 <210> 30 <211> 37 <212> RNA <213> artificial sequence <220> <223> crRNA for Cas12f1 gRNA <400> 30 guugcagaac ccgaauagac gaaugaagga augcaac 37 <210> 31 <211> 30 <212> RNA <213> artificial sequence <220> <223> The fifth region for Cas12f1 gRNA <400> 31 guugcagaac ccgaauagnb nnnugaagga 30 <210> 32 <211> 12 <212> RNA <213> artificial sequence <220> <223> The fifth region for Cas12f1 gRNA <400> 32 nbnnnugaag ga 12 <210> 33 <211> 7 <212> RNA <213> artificial sequence <220> <223> The sixth region for Cas12f1 gRNA <400> 33 augcaac 7 <210> 34 <211> 161 <212> RNA <213> artificial sequence <220> <223> engineered tracrRNA for Cas12f1 gRNA <400> 34 cuucacugau aaaguggaga accgcuucac caaaagcugu cccuuagggg auuagaacuu 60 gagugaaggu gggcugcuug caucagccua augucgagaa gugcuuucuu cggaaaguaa 120 cccucgaaac aaauucannn cnccucucca auucugcaca a 161 <210> 35 <211> 141 <212> RNA <213> artificial sequence <220> <223> engineered tracrRNA for Cas12f1 gRNA <400> 35 accgcuucac caaaagcugu cccuuagggg auuagaacuu gagugaaggu gggcugcuug 60 caucagccua augucgagaa gugcuuucuu cggaaaguaa cccucgaaac aaauucannn 120 cnccucucca auucugcaca a 141 <210> 36 <211> 135 <212> RNA <213> artificial sequence <220> <223> engineered tracrRNA for Cas12f1 gRNA <400> 36 cuucacugau aaaguggaga accgcuucac cuuagaguga aggugggcug cuugcaucag 60 ccuaaugucg agaagugcuu ucuucggaaa guaacccucg aaacaaauuc annncnccuc 120 uccaauucug cacaa 135 <210> 37 <211> 115 <212> RNA <213> artificial sequence <220> <223> engineered tracrRNA for Cas12f1 gRNA <400> 37 accgcuucac cuuagaguga aggugggcug cuugcaucag ccuaaugucg agaagugcuu 60 ucuucggaaa guaacccucg aaacaaauuc annncnccuc uccaauucug cacaa 115 <210> 38 <211> 37 <212> RNA <213> artificial sequence <220> <223> engineered crRNA for Cas12f1 gRNA <400> 38 guugcagaac ccgaauagng nnnugaagga augcaac 37 <210> 39 <211> 161 <212> RNA <213> artificial sequence <220> <223> engineered tracrRNA for Cas12f1 gRNA <400> 39 cuucacugau aaaguggaga accgcuucac caaaagcugu cccuuagggg auuagaacuu 60 gagugaaggu gggcugcuug caucagccua augucgagaa gugcuuucuu cggaaaguaa 120 cccucgaaac aaauucagug cuccucucca auucugcaca a 161 <210> 40 <211> 141 <212> RNA <213> artificial sequence <220> <223> engineered tracrRNA for Cas12f1 gRNA <400> 40 accgcuucac caaaagcugu cccuuagggg auuagaacuu gagugaaggu gggcugcuug 60 caucagccua augucgagaa gugcuuucuu cggaaaguaa cccucgaaac aaauucagug 120 cuccucucca auucugcaca a 141 <210> 41 <211> 134 <212> RNA <213> artificial sequence <220> <223> engineered tracrRNA for Cas12f1 gRNA <400> 41 cuucacugau aaaguggaga accgcuucac uuagagugaa ggugggcugc uugcaucagc 60 120 ccaauucugc acaa 134 <210> 42 <211> 114 <212> RNA <213> artificial sequence <220> <223> engineered tracrRNA for Cas12f1 gRNA <400> 42 accgcuucac uuagagugaa ggugggcugc uugcaucagc cuaaugucga gaagugcuuu 60 cuucggaaag uaacccucga aacaaauuca gugcuccucu ccaauucugc acaa 114 <210> 43 <211> 37 <212> RNA <213> artificial sequence <220> <223> engineered crRNA for Cas12f1 gRNA <400> 43 guugcagaac ccgaauagag caaugaagga augcaac 37 <210> 44 <211> 148 <212> RNA <213> artificial sequence <220> <223> engineered tracrRNA for Cas12f1 gRNA <400> 44 cuucacugau aaaguggaga accgcuucac caaaagcugu cccuuagggg auuagaacuu 60 gagugaaggu gggcugcuug caucagccua augucgagaa gugcuuucuu cggaaaguaa 120 cccucgaaac aaauucannn cnccucuc 148 <210> 45 <211> 128 <212> RNA <213> artificial sequence <220> <223> engineered tracrRNA for Cas12f1 gRNA <400> 45 accgcuucac caaaagcugu cccuuagggg auuagaacuu gagugaaggu gggcugcuug 60 caucagccua augucgagaa gugcuuucuu cggaaaguaa cccucgaaac aaauucannn 120 cnccucuc 128 <210> 46 <211> 127 <212> RNA <213> artificial sequence <220> <223> engineered tracrRNA for Cas12f1 gRNA <400> 46 cuucacugau aaaguggaga accgcuucac caauuaguug agugaaggug ggcugcuugc 60 aucagccuaa ugucgagaag ugcuuucuuc ggaaaguaac ccucgaaaca aauucannnc 120 nccucuc 127 <210> 47 <211> 101 <212> RNA <213> artificial sequence <220> <223> engineered tracrRNA for Cas12f1 gRNA <400> 47 accgcuucac uuagagugaa ggugggcugc uugcaucagc cuaaugucga gaagugcuuu 60 cuucggaaag uaacccucga aacaaauuca nnncnccucu c 101 <210> 48 <211> 25 <212> RNA <213> artificial sequence <220> <223> engineered crRNA for Cas12f1 gRNA <400> 48 gaauagngnn nugaaggaau gcaac 25 <210> 49 <211> 147 <212> RNA <213> artificial sequence <220> <223> engineered tracrRNA for Cas12f1 gRNA <400> 49 cuucacugau aaaguggaga accgcuucac caaaagcugu cccuuagggg auuagaacuu 60 gagugaaggu gggcugcuug caucagccua augucgagaa gugcuuucuu cggaaaguaa 120 cccucgaaac aaauucgugc uccucuc 147 <210> 50 <211> 128 <212> RNA <213> artificial sequence <220> <223> engineered tracrRNA for Cas12f1 gRNA <400> 50 accgcuucac caaaagcugu cccuuagggg auuagaacuu gagugaaggu gggcugcuug 60 caucagccua augucgagaa gugcuuucuu cggaaaguaa cccucgaaac aaauucagug 120 128 <210> 51 <211> 127 <212> RNA <213> artificial sequence <220> <223> engineered tracrRNA for Cas12f1 gRNA <400> 51 cuucacugau aaaguggaga accgcuucac caauuaguug agugaaggug ggcugcuugc 60 aucagccuaa ugucgagaag ugcuuucuuc ggaaaguaac ccucgaaaca aauucagugc 120 127 <210> 52 <211> 107 <212> RNA <213> artificial sequence <220> <223> engineered tracrRNA for Cas12f1 gRNA <400> 52 accgcuucac caauuaguug agugaaggug ggcugcuugc aucagccuaa ugucgagaag 60 ugcuuucuuc ggaaaguaac ccucgaaaca aauucagugc uccucuc 107 <210> 53 <211> 25 <212> RNA <213> artificial sequence <220> <223> engineered crRNA for Cas12f1 gRNA <400> 53 gaauagagca augaaggaau gcaac 25 <210> 54 <211> 222 <212> RNA <213> artificial sequence <220> <223> Canonical sgRNA for Cas12f1 <400> 54 cuucacugau aaaguggaga accgcuucac caaaagcugu cccuuagggg auuagaacuu 60 gagugaaggu gggcugcuug caucagccua augucgagaa gugcuuucuu cggaaaguaa 120 cccucgaaac aaauucauuu uuccucucca auucugcaca agaaaguugc agaacccgaa 180 uagacgaaug aaggaaugca acnnnnnnnn nnnnnnnnnn nn 222 <210> 55 <211> 222 <212> RNA <213> artificial sequence <220> <223> Engineered sgRNA with modification of MS1 for Cas12f1 <400> 55 cuucacugau aaaguggaga accgcuucac caaaagcugu cccuuagggg auuagaacuu 60 gagugaaggu gggcugcuug caucagccua augucgagaa gugcuuucuu cggaaaguaa 120 cccucgaaac aaauucagug cuccucucca auucugcaca agaaaguugc agaacccgaa 180 uagagcaaug aaggaaugca acnnnnnnnn nnnnnnnnnn nn 222 <210> 56 <211> 233 <212> RNA <213> artificial sequence <220> <223> Engineered sgRNA with modification of MS1/MS2 for Cas12f1 <400> 56 cuucacugau aaaguggaga accgcuucac caaaagcugu cccuuagggg auuagaacuu 60 gagugaaggu gggcugcuug caucagccua augucgagaa gugcuuucuu cggaaaguaa 120 cccucgaaac aaauucagug cuccucucca auucugcaca agaaaguugc agaacccgaa 180 uagagcaaug aaggaaugca acnnnnnnnn nnnnnnnnnn nnuuuuauuu uuu 233 <210> 57 <211> 213 <212> RNA <213> artificial sequence <220> <223> Engineered sgRNA with modification of MS1/MS2/MS3 for Cas12f1 <400> 57 accgcuucac caaaagcugu cccuuagggg auuagaacuu gagugaaggu gggcugcuug 60 caucagccua augucgagaa gugcuuucuu cggaaaguaa cccucgaaac aaauucagug 120 cuccucucca auucugcaca agaaaguugc agaacccgaa uagagcaaug aaggaaugca 180 acnnnnnnnn nnnnnnnnnn nnuuuuauuu uuu 213 <210> 58 <211> 158 <212> RNA <213> artificial sequence <220> <223> Engineered sgRNA with modification of MS2/MS3/MS4 for Cas12f1 <400> 58 accgcuucac caaaagcugu cccuuagggg auuagaacuu gagugaaggu gggcugcuug 60 caucagccua augucgagaa gugcuuucuu cggaaaguaa cccucgaaac aaagaaagga 120 augcaacnnn nnnnnnnnnn nnnnnnnuuu uauuuuuu 158 <210> 59 <211> 131 <212> RNA <213> artificial sequence <220> <223> Engineered sgRNA with modification of MS2/MS3/MS4/MS5 for Cas12f1 <400> 59 accgcuucac uuagagugaa ggugggcugc uugcaucagc cuaaugucga gaagugcuuu 60 cuucggaaag uaacccucga aacaaagaaa ggaugcaac nnnnnnnnnn nnnnnnnnnn 120 uuuuauuuuu u 131 <210> 60 <211> 21 <212> DNA <213> artificial sequence <220> <223> NLS Sequence <400> 60 ccaaagaaga agcggaaggt c 21 <210> 61 <211> 48 <212> DNA <213> artificial sequence <220> <223> NLS Sequence <400> 61 aaaaggccgg cggccacgaa aaaggccggc caggcaaaaa agaaaaag 48 <210> 62 <211> 32 <212> PRT <213> artificial sequence <220> <223> linker <400> 62 Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr 1 5 10 15 Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser 20 25 30 <210> 63 <211> 14 <212> PRT <213> artificial sequence <220> <223> linker <400> 63 Ser Gly Gly Ser Lys Arg Thr Ala Asp Gly Ser Glu Phe Glu 1 5 10 <210> 64 <211> 13 <212> PRT <213> artificial sequence <220> <223> linker <400> 64 Glu Ala Ser Ser Pro Lys Lys Arg Lys Val Glu Ala Ser 1 5 10 <210> 65 <211> 7 <212> PRT <213> artificial sequence <220> <223> NLS <400> 65 Pro Lys Lys Lys Arg Lys Val 1 5 <210> 66 <211> 16 <212> PRT <213> artificial sequence <220> <223> NLS <400> 66 Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys 1 5 10 15 <210> 67 <211> 9 <212> PRT <213> artificial sequence <220> <223> NLS <400> 67 Pro Ala Ala Lys Arg Val Lys Leu Asp 1 5 <210> 68 <211> 11 <212> PRT <213> artificial sequence <220> <223> NLS <400> 68 Arg Gln Arg Arg Asn Glu Leu Lys Arg Ser Pro 1 5 10 <210> 69 <211> 161 <212> RNA <213> artificial sequence <220> <223> tracrRNA for Cas12f1 gRNA <400> 69 cuucacugau aaaguggaga accgcuucac caaaagcugu cccuuagggg auuagaacuu 60 gagugaaggu gggcugcuug caucagccua augucgagaa gugcuuucuu cggaaaguaa 120 cccucgaaac aaauucauuu uuccucucca auucugcaca a 161 <210> 70 <211> 140 <212> RNA <213> artificial sequence <220> <223> engineered tracrRNA for Cas12f1 gRNA <400> 70 cuucacugau aaaguggaga accgcuucac caaaagcugu cccuuagggg auuagaacuu 60 gagugaaggu gggcugcuug caucagccua augucgagaa gugcuuucuu cggaaaguaa 120 cccucgaaac aaauucauuu 140 <210> 71 <211> 161 <212> RNA <213> artificial sequence <220> <223> engineered tracrRNA for Cas12f1 gRNA <400> 71 cuucacugau aaaguggaga accgcuucac caaaagcugu cccuuagggg auuagaacuu 60 gagugaaggu gggcugcuug caucagccua augucgagaa gugcuuucuu cggaaaguaa 120 cccucgaaac aaauucannn nnccucucca auucugcaca a 161 <210> 72 <211> 17 <212> RNA <213> artificial sequence <220> <223> engineered crRNA for Cas12f1 gRNA <400> 72 gaaugaagga augcaac 17 <210> 73 <211> 10 <212> RNA <213> artificial sequence <220> <223> engineered crRNA for Cas12f1 gRNA <400> 73 ggaaugcaac 10 <210> 74 <211> 32 <212> RNA <213> artificial sequence <220> <223> The fourth region of tracrRNA for Cas12f1 gRNA <400> 74 caaauucauu uuuccucucc aauucugcac aa 32 <210> 75 <211> 30 <212> RNA <213> artificial sequence <220> <223> The fifth region for Cas12f1 gRNA <400> 75 guugcagaac ccgaauagac gaaugaagga 30 <210> 76 <211> 10 <212> RNA <213> artificial sequence <220> <223> The fifth region for Cas12f1 gRNA <400> 76 gaaugaagga 10 <210> 77 <211> 7 <212> RNA <213> artificial sequence <220> <223> U-rich tail <400> 77 uuuruuu 7 <210> 78 <211> 11 <212> RNA <213> artificial sequence <220> <223> U-rich tail <400> 78 uuuruuuruu u 11 <210> 79 <211> 6 <212> RNA <213> artificial sequence <220> <223> U-rich tail <400> 79 uuuuru 6 <210> 80 <211> 7 <212> RNA <213> artificial sequence <220> <223> U-rich tail <400> 80 uuuuruu 7 <210> 81 <211> 8 <212> RNA <213> artificial sequence <220> <223> U-rich tail <400> 81 uuuuruuu 8 <210> 82 <211> 9 <212> RNA <213> artificial sequence <220> <223> U-rich tail <400> 82 uuuuruuuu 9 <210> 83 <211> 10 <212> RNA <213> artificial sequence <220> <223> U-rich tail <400> 83 uuuuruuuuu 10 <210> 84 <211> 11 <212> RNA <213> artificial sequence <220> <223> U-rich tail <400> 84 uuuuruuuuu u 11 <210> 85 <211> 7 <212> RNA <213> artificial sequence <220> <223> U-rich tail <400> 85 uuuauuu 7 <210> 86 <211> 11 <212> RNA <213> artificial sequence <220> <223> U-rich tail <400> 86 uuuauuuuu u 11 <210> 87 <211> 6 <212> RNA <213> artificial sequence <220> <223> U-rich tail <400> 87 uuuuu 6 <210> 88 <211> 7 <212> RNA <213> artificial sequence <220> <223> U-rich tail <400> 88 uuuuauu 7 <210> 89 <211> 8 <212> RNA <213> artificial sequence <220> <223> U-rich tail <400> 89 uuuuauuu 8 <210> 90 <211> 9 <212> RNA <213> artificial sequence <220> <223> U-rich tail <400> 90 uuuuauuuu 9 <210> 91 <211> 10 <212> RNA <213> artificial sequence <220> <223> U-rich tail <400> 91 uuuuauuuuu 10 <210> 92 <211> 11 <212> RNA <213> artificial sequence <220> <223> U-rich tail <400> 92 uuuuauuuuu u 11 <210> 93 <211> 7 <212> RNA <213> artificial sequence <220> <223> U-rich tail <400> 93 uuuguuu 7 <210> 94 <211> 11 <212> RNA <213> artificial sequence <220> <223> U-rich tail <400> 94 uuuguuuguu u 11 <210> 95 <211> 6 <212> RNA <213> artificial sequence <220> <223> U-rich tail <400> 95 uuuugu 6 <210> 96 <211> 7 <212> RNA <213> artificial sequence <220> <223> U-rich tail <400> 96 uuuuguu 7 <210> 97 <211> 8 <212> RNA <213> artificial sequence <220> <223> U-rich tail <400> 97 uuuuguuu 8 <210> 98 <211> 9 <212> RNA <213> artificial sequence <220> <223> U-rich tail <400> 98 uuuuguuuu 9 <210> 99 <211> 10 <212> RNA <213> artificial sequence <220> <223> U-rich tail <400> 99 uuuuguuuuu 10 <210> 100 <211> 11 <212> RNA <213> artificial sequence <220> <223> U-rich tail <400> 100 uuuuguuuuu u 11 <210> 101 <211> 6 <212> RNA <213> artificial sequence <220> <223> U-rich tail <400> 101 uuuuuu 6 <210> 102 <211> 110 <212> RNA <213> artificial sequence <220> <223> Engineered Cas12f1 gRNA <400> 102 cuucacugau aaaguggaga agcugcuugc aucagccuaa ugucgagaag ugcuuucuuc 60 ggaaaguaac ccucgaaaca aauucauuug aaagaaugaa ggaaugcaac 110 <210> 103 <211> 26 <212> RNA <213> artificial sequence <220> <223> The third region of tracrRNA for Cas12f1 gRNA <400> 103 gcugcuugca ucagccuaau gucgag 26 <210> 104 <211> 10 <212> RNA <213> artificial sequence <220> <223> The fourth region of tracrRNA for Cas12f1 gRNA <400> 104 aacaaauuca 10 <210> 105 <211> 11 <212> RNA <213> artificial sequence <220> <223> The fourth region of tracrRNA for Cas12f1 gRNA <400> 105 aacaaauuca u 11 <210> 106 <211> 12 <212> RNA <213> artificial sequence <220> <223> The fourth region of tracrRNA for Cas12f1 gRNA <400> 106 aacaaauuca uu 12 <210> 107 <211> 10 <212> RNA <213> artificial sequence <220> <223> The additional sequence for Cas12f1 gRNA <400> 107 auaaagguga 10 <210> 108 <211> 37 <212> RNA <213> artificial sequence <220> <223> The additional sequence for Cas12f1 gRNA <400> 108 cugaugaguc cgugaggacg aaacgaguaa gcucguc 37 <210> 109 <211> 37 <212> RNA <213> artificial sequence <220> <223> The additional sequence for Cas12f1 gRNA <400> 109 cugcucgaau gagcaaagca ggagugccug aguaguc 37 <210> 110 <211> 24 <212> DNA <213> artificial sequence <220> <223> Target-1 sequence <400> 110 tttgcacaca cacagtgggc tacc 24 <210> 111 <211> 24 <212> DNA <213> artificial sequence <220> <223> Target-2 sequence <400> 111 tttgcatccc caggacacac acac 24 <210> 112 <211> 24 <212> DNA <213> artificial sequence <220> <223> Target-3 sequence <400> 112 tttacaaaga cactcaccct gttg 24 <210> 113 <211> 24 <212> DNA <213> artificial sequence <220> <223> Target-4 sequence <400> 113 tttaaagaaa gctacaggaa agca 24 <210> 114 <211> 24 <212> DNA <213> artificial sequence <220> <223> Target-5 sequence <400> 114 tttacaaaac ccaactgatt cacc 24 <210> 115 <211> 24 <212> DNA <213> artificial sequence <220> <223> Target-6 sequence <400> 115 tttacaaaag ctaccacaca tagc 24 <210> 116 <211> 24 <212> DNA <213> artificial sequence <220> <223> Target-7 sequence <400> 116 tttacaaaac tgtggccaat acag 24 <210> 117 <211> 24 <212> DNA <213> artificial sequence <220> <223> Target-8 sequence <400> 117 tttggaaaac tgcaggcaag attc 24 <210> 118 <211> 24 <212> DNA <213> artificial sequence <220> <223> Target-9 sequence <400> 118 tttgcaaaac tgtacacgtg ggcc 24 <210> 119 <211> 24 <212> DNA <213> artificial sequence <220> <223> Target-10 sequence <400> 119 tttgcaaaac gtgcacaatg tgca 24 <210> 120 <211> 24 <212> DNA <213> artificial sequence <220> <223> Target-11 sequence <400> 120 tttaccccca caggattgta ataa 24 <210> 121 <211> 24 <212> DNA <213> artificial sequence <220> <223> Target-12 sequence <400> 121 tttaggccaa gtgcgaagtc agag 24 <210> 122 <211> 24 <212> DNA <213> artificial sequence <220> <223> Target-13 sequence <400> 122 tttactagga cactcaccct gttg 24 <210> 123 <211> 24 <212> DNA <213> artificial sequence <220> <223> Target-14 sequence <400> 123 tttgctagca cacagtgggc agag 24 <210> 124 <211> 24 <212> DNA <213> artificial sequence <220> <223> Target-15 sequence <400> 124 tttactagga cactcaccct ggag 24 <210> 125 <211> 24 <212> DNA <213> artificial sequence <220> <223> Target-16 sequence <400> 125 tttactagtc ggccattcag agag 24 <210> 126 <211> 167 <212> PRT <213> artificial sequence <220> <223> TadA <400> 126 Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu 1 5 10 15 Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala 20 25 30 Val Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro 35 40 45 Ile Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg 50 55 60 Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu 65 70 75 80 Tyr Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His 85 90 95 Ser Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly 100 105 110 Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His 115 120 125 Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu 130 135 140 Leu Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys 145 150 155 160 Lys Ala Gln Ser Ser Thr Asp 165 <210> 127 <211> 167 <212> PRT <213> artificial sequence <220> <223> eTadA1 <400> 127 Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu 1 5 10 15 Thr Leu Ala Lys Arg Ala Arg Asp Glu Arg Glu Val Pro Val Gly Ala 20 25 30 Val Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Ala 35 40 45 Ile Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg 50 55 60 Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu 65 70 75 80 Tyr Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His 85 90 95 Ser Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr Gly 100 105 110 Ala Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His 115 120 125 Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu 130 135 140 Leu Cys Tyr Phe Phe Arg Met Pro Arg Gln Val Phe Asn Ala Gln Lys 145 150 155 160 Lys Ala Gln Ser Ser Thr Asp 165 <210> 128 <211> 167 <212> PRT <213> artificial sequence <220> <223> dTadA (E59A) <400> 128 Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu 1 5 10 15 Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala 20 25 30 Val Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro 35 40 45 Ile Gly Arg His Asp Pro Thr Ala His Ala Ala Ile Met Ala Leu Arg 50 55 60 Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu 65 70 75 80 Tyr Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His 85 90 95 Ser Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly 100 105 110 Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His 115 120 125 Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu 130 135 140 Leu Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys 145 150 155 160 Lys Ala Gln Ser Ser Thr Asp 165 <210> 129 <211> 167 <212> PRT <213> artificial sequence <220> <223> deTadA1 (E59A) <400> 129 Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu 1 5 10 15 Thr Leu Ala Lys Arg Ala Arg Asp Glu Arg Glu Val Pro Val Gly Ala 20 25 30 Val Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Ala 35 40 45 Ile Gly Leu His Asp Pro Thr Ala His Ala Ala Ile Met Ala Leu Arg 50 55 60 Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu 65 70 75 80 Tyr Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His 85 90 95 Ser Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr Gly 100 105 110 Ala Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His 115 120 125 Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu 130 135 140 Leu Cys Tyr Phe Phe Arg Met Pro Arg Gln Val Phe Asn Ala Gln Lys 145 150 155 160 Lys Ala Gln Ser Ser Thr Asp 165 <210> 130 <211> 167 <212> PRT <213> artificial sequence <220> <223> eTadA2 <400> 130 Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu 1 5 10 15 Thr Leu Ala Lys Arg Ala Arg Asp Glu Arg Glu Val Pro Val Gly Ala 20 25 30 Val Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Ala 35 40 45 Ile Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg 50 55 60 Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu 65 70 75 80 Tyr Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His 85 90 95 Ser Arg Ile Gly Arg Val Val Phe Gly Trp Arg Asn Ser Lys Arg Gly 100 105 110 Ala Ala Gly Ser Leu Met Asn Val Leu Asn Tyr Pro Gly Met Asn His 115 120 125 Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu 130 135 140 Leu Cys Asp Phe Tyr Arg Met Pro Arg Gln Val Phe Asn Ala Gln Lys 145 150 155 160 Lys Ala Gln Ser Ser Ile Asn 165 <210> 131 <211> 167 <212> PRT <213> artificial sequence <220> <223> eTadA3 <400> 131 Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu 1 5 10 15 Thr Leu Ala Lys Arg Ala Arg Asp Glu Arg Glu Val Pro Val Gly Ala 20 25 30 Val Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Ala 35 40 45 Ile Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg 50 55 60 Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu 65 70 75 80 Tyr Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His 85 90 95 Ser Arg Ile Gly Arg Val Val Phe Gly Trp Arg Gln Ser Lys Arg Gly 100 105 110 Ala Ala Gly Ser Leu Met Asn Val Leu Asn Tyr Pro Gly Met Asn His 115 120 125 Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu 130 135 140 Leu Cys Asp Phe Tyr Arg Met Pro Arg Gln Val Phe Asn Ala Gln Lys 145 150 155 160 Lys Ala Gln Ser Ser Ile Asn 165 <210> 132 <211> 5 <212> PRT <213> artificial sequence <220> <223> linker <400> 132 Gly Gly Gly Gly Ser 1 5 <210> 133 <211> 5 <212> PRT <213> artificial sequence <220> <223> linker <400> 133 Glu Ala Ala Ala Lys 1 5 <210> 134 <211> 16 <212> PRT <213> artificial sequence <220> <223> linker <400> 134 Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser 1 5 10 15 <210> 135 <211> 4 <212> PRT <213> artificial sequence <220> <223> linker <400> 135 Ser Gly Gly Ser One <210> 136 <211> 24 <212> DNA <213> artificial sequence <220> <223> linker <400> 136 ggtatccacg gagtcccagc agcc 24 <210> 137 <211> 167 <212> PRT <213> artificial sequence <220> <223> eTadA7 <400> 137 Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu 1 5 10 15 Thr Leu Ala Lys Arg Ala Arg Asp Glu Arg Glu Val Pro Val Gly Ala 20 25 30 Val Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Ala 35 40 45 Ile Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg 50 55 60 Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu 65 70 75 80 Tyr Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His 85 90 95 Ser Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ser Lys Arg Gly 100 105 110 Ala Ala Gly Ser Leu Met Asn Val Leu Asn Tyr Pro Gly Met Asn His 115 120 125 Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu 130 135 140 Leu Cys Asp Phe Tyr Arg Met Pro Arg Gln Val Phe Asn Ala Gln Lys 145 150 155 160 Lys Ala Gln Ser Ser Ile Asn 165 <210> 138 <211> 167 <212> PRT <213> artificial sequence <220> <223> eTadA8 <400> 138 Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu 1 5 10 15 Thr Leu Ala Lys Arg Ala Arg Asp Glu Arg Glu Val Pro Val Gly Ala 20 25 30 Val Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Ala 35 40 45 Ile Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg 50 55 60 Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu 65 70 75 80 Tyr Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His 85 90 95 Ser Arg Ile Gly Arg Val Val Phe Gly Trp Arg Asn Ala Lys Thr Gly 100 105 110 Ala Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His 115 120 125 Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu 130 135 140 Leu Cys Tyr Phe Phe Arg Met Pro Arg Gln Val Phe Asn Ala Gln Lys 145 150 155 160 Lys Ala Gln Ser Ser Thr Asp 165 <210> 139 <211> 167 <212> PRT <213> artificial sequence <220> <223> eTadA9 <400> 139 Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu 1 5 10 15 Thr Leu Ala Lys Arg Ala Arg Asp Glu Arg Glu Val Pro Val Gly Ala 20 25 30 Val Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Ala 35 40 45 Ile Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg 50 55 60 Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu 65 70 75 80 Tyr Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His 85 90 95 Ser Arg Ile Gly Arg Val Val Phe Gly Trp Arg Gln Ala Lys Thr Gly 100 105 110 Ala Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His 115 120 125 Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu 130 135 140 Leu Cys Tyr Phe Phe Arg Met Pro Arg Gln Val Phe Asn Ala Gln Lys 145 150 155 160 Lys Ala Gln Ser Ser Thr Asp 165 <210> 140 <211> 167 <212> PRT <213> artificial sequence <220> <223> eTadA10 <400> 140 Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu 1 5 10 15 Thr Leu Ala Lys Arg Ala Arg Asp Glu Arg Glu Val Pro Val Gly Ala 20 25 30 Val Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Ala 35 40 45 Ile Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg 50 55 60 Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Tyr Asp Ala Thr Leu 65 70 75 80 Tyr Ser Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His 85 90 95 Ser Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr Gly 100 105 110 Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His 115 120 125 Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu 130 135 140 Leu Cys Arg Phe Phe Arg Met Pro Arg Arg Val Phe Asn Ala Gln Lys 145 150 155 160 Lys Ala Gln Ser Ser Thr Asp 165 <210> 141 <211> 167 <212> PRT <213> artificial sequence <220> <223> eTadA11 <400> 141 Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu 1 5 10 15 Thr Leu Ala Lys Arg Ala Arg Asp Glu Arg Glu Val Pro Val Gly Ala 20 25 30 Val Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Ala 35 40 45 Ile Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg 50 55 60 Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu 65 70 75 80 Tyr Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His 85 90 95 Ser Arg Ile Gly Arg Val Val Phe Gly Trp Arg Asn Ala Lys Thr Gly 100 105 110 Ala Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His 115 120 125 Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu 130 135 140 Leu Cys Tyr Phe Phe Arg Met Pro Arg Gln Val Phe Asn Ala Gln Lys 145 150 155 160 Lys Ala Gln Ser Ser Thr Asp 165 <210> 142 <211> 24 <212> DNA <213> artificial sequence <220> <223> Target-17 sequence <400> 142 tttagcagtc ggaatggcgg atgg 24 <210> 143 <211> 24 <212> DNA <213> artificial sequence <220> <223> Target-18 sequence <400> 143 tttaagaaca catacccctg ggcc 24 <210> 144 <211> 24 <212> DNA <213> artificial sequence <220> <223> Target-19 sequence <400> 144 tttgcagtgt gtgcaggaac ggag 24 <210> 145 <211> 24 <212> DNA <213> artificial sequence <220> <223> Target-20 sequence <400> 145 tttaatacag aaatcctaaa tggt 24 <210> 146 <211> 24 <212> DNA <213> artificial sequence <220> <223> Target-21 sequence <400> 146 tttaaagaaa gctacaggaa agca 24 <210> 147 <211> 24 <212> DNA <213> artificial sequence <220> <223> Target-22 sequence <400> 147 tttaaataag tcttaccacg tgtc 24 <210> 148 <211> 24 <212> DNA <213> artificial sequence <220> <223> Target-23 sequence <400> 148 tttaacaaag aaaccagcag tggc 24 <210> 149 <211> 24 <212> DNA <213> artificial sequence <220> <223> Target-24 sequence <400> 149 tttaacaagt tcagaatcac ctta 24 <210> 150 <211> 24 <212> DNA <213> artificial sequence <220> <223> Target-25 sequence <400> 150 tttaaggact atgtgtggcc agtg 24 <210> 151 <211> 24 <212> DNA <213> artificial sequence <220> <223> Target-26 sequence <400> 151 tttacaaaga aatgtactgc ctta 24 <210> 152 <211> 24 <212> DNA <213> artificial sequence <220> <223> Target-27 sequence <400> 152 tttacaacag cctcaccagg aaca 24 <210> 153 <211> 24 <212> DNA <213> artificial sequence <220> <223> Target-28 sequence <400> 153 tttacacaag ggatctgaga cttg 24 <210> 154 <211> 24 <212> DNA <213> artificial sequence <220> <223> Target-29 sequence <400> 154 tttacacata ggccattcag aaac 24 <210> 155 <211> 24 <212> DNA <213> artificial sequence <220> <223> Target-30 sequence <400> 155 tttacagagt cccgggaaca agcc 24 <210> 156 <211> 24 <212> DNA <213> artificial sequence <220> <223> Target-31 sequence <400> 156 tttacataca gggctctgta ccca 24 <210> 157 <211> 24 <212> DNA <213> artificial sequence <220> <223> Target-32 sequence <400> 157 tttactgaga tttgcgaaga gtta 24 <210> 158 <211> 24 <212> DNA <213> artificial sequence <220> <223> Target-33 sequence <400> 158 tttacttagt agtctcagaa ccaa 24 <210> 159 <211> 24 <212> DNA <213> artificial sequence <220> <223> Target-34 sequence <400> 159 tttagaaata tgactggaag taaa 24 <210> 160 <211> 24 <212> DNA <213> artificial sequence <220> <223> Target-35 sequence <400> 160 tttagagaga ccgctcaggc tgga 24 <210> 161 <211> 24 <212> DNA <213> artificial sequence <220> <223> Target-36 sequence <400> 161 tttagcagta cacctgaggg aaca 24 <210> 162 <211> 24 <212> DNA <213> artificial sequence <220> <223> Target-37 sequence <400> 162 tttagcatta aggccagcgc tggg 24 <210> 163 <211> 24 <212> DNA <213> artificial sequence <220> <223> Target-38 sequence <400> 163 tttagccatg gtgaaggtga aatc 24 <210> 164 <211> 24 <212> DNA <213> artificial sequence <220> <223> Target-39 sequence <400> 164 tttaggcaag ggtcttgatg catc 24 <210> 165 <211> 24 <212> DNA <213> artificial sequence <220> <223> Target-40 sequence <400> 165 tttagtaggc tgctgttgga caga 24 <210> 166 <211> 24 <212> DNA <213> artificial sequence <220> <223> Target-41 sequence <400> 166 tttagtcaaa taaagaaaaa tacg 24 <210> 167 <211> 24 <212> DNA <213> artificial sequence <220> <223> Target-42 sequence <400> 167 tttaagagca gcgattgtaa ggag 24 <210> 168 <211> 555 <212> PRT <213> artificial sequence <220> <223> CasX-Cas12f1 (D352A) <400> 168 Met Glu Lys Arg Ile Asn Lys Ile Arg Lys Lys Leu Ser Ala Asp Asn 1 5 10 15 Ala Thr Lys Pro Val Ser Arg Ser Gly Pro Met Ala Lys Asn Thr Ile 20 25 30 Thr Lys Thr Leu Lys Leu Arg Ile Val Arg Pro Tyr Asn Ser Ala Glu 35 40 45 Val Glu Lys Ile Val Ala Asp Glu Lys Asn Asn Arg Glu Lys Ile Ala 50 55 60 Leu Glu Lys Asn Lys Asp Lys Val Lys Glu Ala Cys Ser Lys His Leu 65 70 75 80 Lys Val Ala Ala Tyr Cys Thr Thr Gln Val Glu Arg Asn Ala Cys Leu 85 90 95 Phe Cys Lys Ala Arg Lys Leu Asp Asp Lys Phe Tyr Gln Lys Leu Arg 100 105 110 Gly Gln Phe Pro Asp Ala Val Phe Trp Gln Glu Ile Ser Glu Ile Phe 115 120 125 Arg Gln Leu Gln Lys Gln Ala Ala Glu Ile Tyr Asn Gln Ser Leu Ile 130 135 140 Glu Leu Tyr Tyr Glu Ile Phe Ile Lys Gly Lys Gly Ile Ala Asn Ala 145 150 155 160 Ser Ser Val Glu His Tyr Leu Ser Asp Val Cys Tyr Thr Arg Ala Ala 165 170 175 Glu Leu Phe Lys Asn Ala Ala Ile Ala Ser Gly Leu Arg Ser Lys Ile 180 185 190 Lys Ser Asn Phe Arg Leu Lys Glu Leu Lys Asn Met Lys Ser Gly Leu 195 200 205 Pro Thr Thr Lys Ser Asp Asn Phe Pro Ile Pro Leu Val Lys Gln Lys 210 215 220 Gly Gly Gln Tyr Thr Gly Phe Glu Ile Ser Asn His Asn Ser Asp Phe 225 230 235 240 Ile Ile Lys Ile Pro Phe Gly Arg Trp Gln Val Lys Lys Glu Ile Asp 245 250 255 Lys Tyr Arg Pro Trp Glu Lys Phe Asp Phe Glu Gln Val Gln Lys Ser 260 265 270 Pro Lys Pro Ile Ser Leu Leu Leu Ser Thr Gln Arg Arg Lys Arg Asn 275 280 285 Lys Gly Trp Ser Lys Asp Glu Gly Thr Glu Ala Glu Ile Lys Lys Val 290 295 300 Met Asn Gly Asp Tyr Gln Thr Ser Tyr Ile Glu Val Lys Arg Gly Ser 305 310 315 320 Lys Ile Gly Glu Lys Ser Ala Trp Met Leu Asn Leu Ser Ile Asp Val 325 330 335 Pro Lys Ile Asp Lys Gly Val Asp Pro Ser Ile Ile Gly Gly Ile Ala 340 345 350 Val Gly Val Lys Ser Pro Leu Val Cys Ala Ile Asn Asn Ala Phe Ser 355 360 365 Arg Tyr Ser Ile Ser Asp Asn Asp Leu Phe His Phe Asn Lys Lys Met 370 375 380 Phe Ala Arg Arg Arg Ile Leu Leu Lys Lys Asn Arg His Lys Arg Ala 385 390 395 400 Gly His Gly Ala Lys Asn Lys Leu Lys Pro Ile Thr Ile Leu Thr Glu 405 410 415 Lys Ser Glu Arg Phe Arg Lys Lys Leu Ile Glu Arg Trp Ala Cys Glu 420 425 430 Ile Ala Asp Phe Phe Ile Lys Asn Lys Val Gly Thr Val Gln Met Glu 435 440 445 Asn Leu Glu Ser Met Lys Arg Lys Glu Asp Ser Tyr Phe Asn Ile Arg 450 455 460 Leu Arg Gly Phe Trp Pro Tyr Ala Glu Met Gln Asn Lys Ile Glu Phe 465 470 475 480 Lys Leu Lys Gln Tyr Gly Ile Glu Ile Arg Lys Val Ala Pro Asn Asn 485 490 495 Thr Ser Lys Thr Cys Ser Lys Cys Gly His Leu Asn Asn Tyr Phe Asn 500 505 510 Phe Glu Tyr Arg Lys Lys Asn Lys Phe Pro His Phe Lys Cys Glu Lys 515 520 525 Cys Asn Phe Lys Glu Asn Ala Asp Tyr Asn Ala Ala Leu Asn Ile Ser 530 535 540 Asn Pro Lys Leu Lys Ser Thr Lys Glu Glu Pro 545 550 555 <210> 169 <211> 483 <212> PRT <213> artificial sequence <220> <223> CasX-Cas12f1 (E448A) <400> 169 Lys Glu Ala Cys Ser Lys His Leu Lys Val Ala Ala Tyr Cys Thr Thr 1 5 10 15 Gln Val Glu Arg Asn Ala Cys Leu Phe Cys Lys Ala Arg Lys Leu Asp 20 25 30 Asp Lys Phe Tyr Gln Lys Leu Arg Gly Gln Phe Pro Asp Ala Val Phe 35 40 45 Trp Gln Glu Ile Ser Glu Ile Phe Arg Gln Leu Gln Lys Gln Ala Ala 50 55 60 Glu Ile Tyr Asn Gln Ser Leu Ile Glu Leu Tyr Tyr Glu Ile Phe Ile 65 70 75 80 Lys Gly Lys Gly Ile Ala Asn Ala Ser Ser Val Glu His Tyr Leu Ser 85 90 95 Asp Val Cys Tyr Thr Arg Ala Ala Glu Leu Phe Lys Asn Ala Ala Ile 100 105 110 Ala Ser Gly Leu Arg Ser Lys Ile Lys Ser Asn Phe Arg Leu Lys Glu 115 120 125 Leu Lys Asn Met Lys Ser Gly Leu Pro Thr Thr Lys Ser Asp Asn Phe 130 135 140 Pro Ile Pro Leu Val Lys Gln Lys Gly Gly Gln Tyr Thr Gly Phe Glu 145 150 155 160 Ile Ser Asn His Asn Ser Asp Phe Ile Ile Lys Ile Pro Phe Gly Arg 165 170 175 Trp Gln Val Lys Lys Glu Ile Asp Lys Tyr Arg Pro Trp Glu Lys Phe 180 185 190 Asp Phe Glu Gln Val Gln Lys Ser Pro Lys Pro Ile Ser Leu Leu Leu 195 200 205 Ser Thr Gln Arg Arg Lys Arg Asn Lys Gly Trp Ser Lys Asp Glu Gly 210 215 220 Thr Glu Ala Glu Ile Lys Lys Val Met Asn Gly Asp Tyr Gln Thr Ser 225 230 235 240 Tyr Ile Glu Val Lys Arg Gly Ser Lys Ile Gly Glu Lys Ser Ala Trp 245 250 255 Met Leu Asn Leu Ser Ile Asp Val Pro Lys Ile Asp Lys Gly Val Asp 260 265 270 Pro Ser Ile Ile Gly Gly Ile Asp Val Gly Val Lys Ser Pro Leu Val 275 280 285 Cys Ala Ile Asn Asn Ala Phe Ser Arg Tyr Ser Ile Ser Asp Asn Asp 290 295 300 Leu Phe His Phe Asn Lys Lys Met Phe Ala Arg Arg Arg Ile Leu Leu 305 310 315 320 Lys Lys Asn Arg His Lys Arg Ala Gly His Gly Ala Lys Asn Lys Leu 325 330 335 Lys Pro Ile Thr Ile Leu Thr Glu Lys Ser Glu Arg Phe Arg Lys Lys 340 345 350 Leu Ile Glu Arg Trp Ala Cys Glu Ile Ala Asp Phe Phe Ile Lys Asn 355 360 365 Lys Val Gly Thr Val Gln Met Ala Asn Leu Glu Ser Met Lys Arg Lys 370 375 380 Glu Asp Ser Tyr Phe Asn Ile Arg Leu Arg Gly Phe Trp Pro Tyr Ala 385 390 395 400 Glu Met Gln Asn Lys Ile Glu Phe Lys Leu Lys Gln Tyr Gly Ile Glu 405 410 415 Ile Arg Lys Val Ala Pro Asn Asn Thr Ser Lys Thr Cys Ser Lys Cys 420 425 430 Gly His Leu Asn Asn Tyr Phe Asn Phe Glu Tyr Arg Lys Lys Asn Lys 435 440 445 Phe Pro His Phe Lys Cys Glu Lys Cys Asn Phe Lys Glu Asn Ala Asp 450 455 460 Tyr Asn Ala Ala Leu Asn Ile Ser Asn Pro Lys Leu Lys Ser Thr Lys 465 470 475 480 Glu Glu Pro <210> 170 <211> 555 <212> PRT <213> artificial sequence <220> <223> CasX-Cas12f1 (R516A) <400> 170 Met Glu Lys Arg Ile Asn Lys Ile Arg Lys Lys Leu Ser Ala Asp Asn 1 5 10 15 Ala Thr Lys Pro Val Ser Arg Ser Gly Pro Met Ala Lys Asn Thr Ile 20 25 30 Thr Lys Thr Leu Lys Leu Arg Ile Val Arg Pro Tyr Asn Ser Ala Glu 35 40 45 Val Glu Lys Ile Val Ala Asp Glu Lys Asn Asn Arg Glu Lys Ile Ala 50 55 60 Leu Glu Lys Asn Lys Asp Lys Val Lys Glu Ala Cys Ser Lys His Leu 65 70 75 80 Lys Val Ala Ala Tyr Cys Thr Thr Gln Val Glu Arg Asn Ala Cys Leu 85 90 95 Phe Cys Lys Ala Arg Lys Leu Asp Asp Lys Phe Tyr Gln Lys Leu Arg 100 105 110 Gly Gln Phe Pro Asp Ala Val Phe Trp Gln Glu Ile Ser Glu Ile Phe 115 120 125 Arg Gln Leu Gln Lys Gln Ala Ala Glu Ile Tyr Asn Gln Ser Leu Ile 130 135 140 Glu Leu Tyr Tyr Glu Ile Phe Ile Lys Gly Lys Gly Ile Ala Asn Ala 145 150 155 160 Ser Ser Val Glu His Tyr Leu Ser Asp Val Cys Tyr Thr Arg Ala Ala 165 170 175 Glu Leu Phe Lys Asn Ala Ala Ile Ala Ser Gly Leu Arg Ser Lys Ile 180 185 190 Lys Ser Asn Phe Arg Leu Lys Glu Leu Lys Asn Met Lys Ser Gly Leu 195 200 205 Pro Thr Thr Lys Ser Asp Asn Phe Pro Ile Pro Leu Val Lys Gln Lys 210 215 220 Gly Gly Gln Tyr Thr Gly Phe Glu Ile Ser Asn His Asn Ser Asp Phe 225 230 235 240 Ile Ile Lys Ile Pro Phe Gly Arg Trp Gln Val Lys Lys Glu Ile Asp 245 250 255 Lys Tyr Arg Pro Trp Glu Lys Phe Asp Phe Glu Gln Val Gln Lys Ser 260 265 270 Pro Lys Pro Ile Ser Leu Leu Leu Ser Thr Gln Arg Arg Lys Arg Asn 275 280 285 Lys Gly Trp Ser Lys Asp Glu Gly Thr Glu Ala Glu Ile Lys Lys Val 290 295 300 Met Asn Gly Asp Tyr Gln Thr Ser Tyr Ile Glu Val Lys Arg Gly Ser 305 310 315 320 Lys Ile Gly Glu Lys Ser Ala Trp Met Leu Asn Leu Ser Ile Asp Val 325 330 335 Pro Lys Ile Asp Lys Gly Val Asp Pro Ser Ile Ile Gly Gly Ile Asp 340 345 350 Val Gly Val Lys Ser Pro Leu Val Cys Ala Ile Asn Asn Ala Phe Ser 355 360 365 Arg Tyr Ser Ile Ser Asp Asn Asp Leu Phe His Phe Asn Lys Lys Met 370 375 380 Phe Ala Arg Arg Arg Ile Leu Leu Lys Lys Asn Arg His Lys Arg Ala 385 390 395 400 Gly His Gly Ala Lys Asn Lys Leu Lys Pro Ile Thr Ile Leu Thr Glu 405 410 415 Lys Ser Glu Arg Phe Arg Lys Lys Leu Ile Glu Arg Trp Ala Cys Glu 420 425 430 Ile Ala Asp Phe Phe Ile Lys Asn Lys Val Gly Thr Val Gln Met Glu 435 440 445 Asn Leu Glu Ser Met Lys Arg Lys Glu Asp Ser Tyr Phe Asn Ile Arg 450 455 460 Leu Arg Gly Phe Trp Pro Tyr Ala Glu Met Gln Asn Lys Ile Glu Phe 465 470 475 480 Lys Leu Lys Gln Tyr Gly Ile Glu Ile Arg Lys Val Ala Pro Asn Asn 485 490 495 Thr Ser Lys Thr Cys Ser Lys Cys Gly His Leu Asn Asn Tyr Phe Asn 500 505 510 Phe Glu Tyr Ala Lys Lys Asn Lys Phe Pro His Phe Lys Cys Glu Lys 515 520 525 Cys Asn Phe Lys Glu Asn Ala Asp Tyr Asn Ala Ala Leu Asn Ile Ser 530 535 540 Asn Pro Lys Leu Lys Ser Thr Lys Glu Glu Pro 545 550 555 <210> 171 <211> 555 <212> PRT <213> artificial sequence <220> <223> CasX-Cas12f1 (D536A) <400> 171 Met Glu Lys Arg Ile Asn Lys Ile Arg Lys Lys Leu Ser Ala Asp Asn 1 5 10 15 Ala Thr Lys Pro Val Ser Arg Ser Gly Pro Met Ala Lys Asn Thr Ile 20 25 30 Thr Lys Thr Leu Lys Leu Arg Ile Val Arg Pro Tyr Asn Ser Ala Glu 35 40 45 Val Glu Lys Ile Val Ala Asp Glu Lys Asn Asn Arg Glu Lys Ile Ala 50 55 60 Leu Glu Lys Asn Lys Asp Lys Val Lys Glu Ala Cys Ser Lys His Leu 65 70 75 80 Lys Val Ala Ala Tyr Cys Thr Thr Gln Val Glu Arg Asn Ala Cys Leu 85 90 95 Phe Cys Lys Ala Arg Lys Leu Asp Asp Lys Phe Tyr Gln Lys Leu Arg 100 105 110 Gly Gln Phe Pro Asp Ala Val Phe Trp Gln Glu Ile Ser Glu Ile Phe 115 120 125 Arg Gln Leu Gln Lys Gln Ala Ala Glu Ile Tyr Asn Gln Ser Leu Ile 130 135 140 Glu Leu Tyr Tyr Glu Ile Phe Ile Lys Gly Lys Gly Ile Ala Asn Ala 145 150 155 160 Ser Ser Val Glu His Tyr Leu Ser Asp Val Cys Tyr Thr Arg Ala Ala 165 170 175 Glu Leu Phe Lys Asn Ala Ala Ile Ala Ser Gly Leu Arg Ser Lys Ile 180 185 190 Lys Ser Asn Phe Arg Leu Lys Glu Leu Lys Asn Met Lys Ser Gly Leu 195 200 205 Pro Thr Thr Lys Ser Asp Asn Phe Pro Ile Pro Leu Val Lys Gln Lys 210 215 220 Gly Gly Gln Tyr Thr Gly Phe Glu Ile Ser Asn His Asn Ser Asp Phe 225 230 235 240 Ile Ile Lys Ile Pro Phe Gly Arg Trp Gln Val Lys Lys Glu Ile Asp 245 250 255 Lys Tyr Arg Pro Trp Glu Lys Phe Asp Phe Glu Gln Val Gln Lys Ser 260 265 270 Pro Lys Pro Ile Ser Leu Leu Leu Ser Thr Gln Arg Arg Lys Arg Asn 275 280 285 Lys Gly Trp Ser Lys Asp Glu Gly Thr Glu Ala Glu Ile Lys Lys Val 290 295 300 Met Asn Gly Asp Tyr Gln Thr Ser Tyr Ile Glu Val Lys Arg Gly Ser 305 310 315 320 Lys Ile Gly Glu Lys Ser Ala Trp Met Leu Asn Leu Ser Ile Asp Val 325 330 335 Pro Lys Ile Asp Lys Gly Val Asp Pro Ser Ile Ile Gly Gly Ile Asp 340 345 350 Val Gly Val Lys Ser Pro Leu Val Cys Ala Ile Asn Asn Ala Phe Ser 355 360 365 Arg Tyr Ser Ile Ser Asp Asn Asp Leu Phe His Phe Asn Lys Lys Met 370 375 380 Phe Ala Arg Arg Arg Ile Leu Leu Lys Lys Asn Arg His Lys Arg Ala 385 390 395 400 Gly His Gly Ala Lys Asn Lys Leu Lys Pro Ile Thr Ile Leu Thr Glu 405 410 415 Lys Ser Glu Arg Phe Arg Lys Lys Leu Ile Glu Arg Trp Ala Cys Glu 420 425 430 Ile Ala Asp Phe Phe Ile Lys Asn Lys Val Gly Thr Val Gln Met Glu 435 440 445 Asn Leu Glu Ser Met Lys Arg Lys Glu Asp Ser Tyr Phe Asn Ile Arg 450 455 460 Leu Arg Gly Phe Trp Pro Tyr Ala Glu Met Gln Asn Lys Ile Glu Phe 465 470 475 480 Lys Leu Lys Gln Tyr Gly Ile Glu Ile Arg Lys Val Ala Pro Asn Asn 485 490 495 Thr Ser Lys Thr Cys Ser Lys Cys Gly His Leu Asn Asn Tyr Phe Asn 500 505 510 Phe Glu Tyr Arg Lys Lys Asn Lys Phe Pro His Phe Lys Cys Glu Lys 515 520 525 Cys Asn Phe Lys Glu Asn Ala Ala Tyr Asn Ala Ala Leu Asn Ile Ser 530 535 540 Asn Pro Lys Leu Lys Ser Thr Lys Glu Glu Pro 545 550 555 <210> 172 <211> 555 <212> PRT <213> artificial sequence <220> <223> 26aa-Extension dCas12f1 (D352A) <400> 172 Met Ala Gly Gly Pro Gly Ala Gly Ser Ala Ala Pro Val Ser Ser Thr 1 5 10 15 Ser Ser Leu Pro Leu Ala Ala Leu Asn Met Met Ala Lys Asn Thr Ile 20 25 30 Thr Lys Thr Leu Lys Leu Arg Ile Val Arg Pro Tyr Asn Ser Ala Glu 35 40 45 Val Glu Lys Ile Val Ala Asp Glu Lys Asn Asn Arg Glu Lys Ile Ala 50 55 60 Leu Glu Lys Asn Lys Asp Lys Val Lys Glu Ala Cys Ser Lys His Leu 65 70 75 80 Lys Val Ala Ala Tyr Cys Thr Thr Gln Val Glu Arg Asn Ala Cys Leu 85 90 95 Phe Cys Lys Ala Arg Lys Leu Asp Asp Lys Phe Tyr Gln Lys Leu Arg 100 105 110 Gly Gln Phe Pro Asp Ala Val Phe Trp Gln Glu Ile Ser Glu Ile Phe 115 120 125 Arg Gln Leu Gln Lys Gln Ala Ala Glu Ile Tyr Asn Gln Ser Leu Ile 130 135 140 Glu Leu Tyr Tyr Glu Ile Phe Ile Lys Gly Lys Gly Ile Ala Asn Ala 145 150 155 160 Ser Ser Val Glu His Tyr Leu Ser Asp Val Cys Tyr Thr Arg Ala Ala 165 170 175 Glu Leu Phe Lys Asn Ala Ala Ile Ala Ser Gly Leu Arg Ser Lys Ile 180 185 190 Lys Ser Asn Phe Arg Leu Lys Glu Leu Lys Asn Met Lys Ser Gly Leu 195 200 205 Pro Thr Thr Lys Ser Asp Asn Phe Pro Ile Pro Leu Val Lys Gln Lys 210 215 220 Gly Gly Gln Tyr Thr Gly Phe Glu Ile Ser Asn His Asn Ser Asp Phe 225 230 235 240 Ile Ile Lys Ile Pro Phe Gly Arg Trp Gln Val Lys Lys Glu Ile Asp 245 250 255 Lys Tyr Arg Pro Trp Glu Lys Phe Asp Phe Glu Gln Val Gln Lys Ser 260 265 270 Pro Lys Pro Ile Ser Leu Leu Leu Ser Thr Gln Arg Arg Lys Arg Asn 275 280 285 Lys Gly Trp Ser Lys Asp Glu Gly Thr Glu Ala Glu Ile Lys Lys Val 290 295 300 Met Asn Gly Asp Tyr Gln Thr Ser Tyr Ile Glu Val Lys Arg Gly Ser 305 310 315 320 Lys Ile Gly Glu Lys Ser Ala Trp Met Leu Asn Leu Ser Ile Asp Val 325 330 335 Pro Lys Ile Asp Lys Gly Val Asp Pro Ser Ile Ile Gly Gly Ile Ala 340 345 350 Val Gly Val Lys Ser Pro Leu Val Cys Ala Ile Asn Asn Ala Phe Ser 355 360 365 Arg Tyr Ser Ile Ser Asp Asn Asp Leu Phe His Phe Asn Lys Lys Met 370 375 380 Phe Ala Arg Arg Arg Ile Leu Leu Lys Lys Asn Arg His Lys Arg Ala 385 390 395 400 Gly His Gly Ala Lys Asn Lys Leu Lys Pro Ile Thr Ile Leu Thr Glu 405 410 415 Lys Ser Glu Arg Phe Arg Lys Lys Leu Ile Glu Arg Trp Ala Cys Glu 420 425 430 Ile Ala Asp Phe Phe Ile Lys Asn Lys Val Gly Thr Val Gln Met Glu 435 440 445 Asn Leu Glu Ser Met Lys Arg Lys Glu Asp Ser Tyr Phe Asn Ile Arg 450 455 460 Leu Arg Gly Phe Trp Pro Tyr Ala Glu Met Gln Asn Lys Ile Glu Phe 465 470 475 480 Lys Leu Lys Gln Tyr Gly Ile Glu Ile Arg Lys Val Ala Pro Asn Asn 485 490 495 Thr Ser Lys Thr Cys Ser Lys Cys Gly His Leu Asn Asn Tyr Phe Asn 500 505 510 Phe Glu Tyr Arg Lys Lys Asn Lys Phe Pro His Phe Lys Cys Glu Lys 515 520 525 Cys Asn Phe Lys Glu Asn Ala Asp Tyr Asn Ala Ala Leu Asn Ile Ser 530 535 540 Asn Pro Lys Leu Lys Ser Thr Lys Glu Glu Pro 545 550 555 <210> 173 <211> 555 <212> PRT <213> artificial sequence <220> <223> 26aa-Extension dCas12f1 (E448A) <400> 173 Met Ala Gly Gly Pro Gly Ala Gly Ser Ala Ala Pro Val Ser Ser Thr 1 5 10 15 Ser Ser Leu Pro Leu Ala Ala Leu Asn Met Met Ala Lys Asn Thr Ile 20 25 30 Thr Lys Thr Leu Lys Leu Arg Ile Val Arg Pro Tyr Asn Ser Ala Glu 35 40 45 Val Glu Lys Ile Val Ala Asp Glu Lys Asn Asn Arg Glu Lys Ile Ala 50 55 60 Leu Glu Lys Asn Lys Asp Lys Val Lys Glu Ala Cys Ser Lys His Leu 65 70 75 80 Lys Val Ala Ala Tyr Cys Thr Thr Gln Val Glu Arg Asn Ala Cys Leu 85 90 95 Phe Cys Lys Ala Arg Lys Leu Asp Asp Lys Phe Tyr Gln Lys Leu Arg 100 105 110 Gly Gln Phe Pro Asp Ala Val Phe Trp Gln Glu Ile Ser Glu Ile Phe 115 120 125 Arg Gln Leu Gln Lys Gln Ala Ala Glu Ile Tyr Asn Gln Ser Leu Ile 130 135 140 Glu Leu Tyr Tyr Glu Ile Phe Ile Lys Gly Lys Gly Ile Ala Asn Ala 145 150 155 160 Ser Ser Val Glu His Tyr Leu Ser Asp Val Cys Tyr Thr Arg Ala Ala 165 170 175 Glu Leu Phe Lys Asn Ala Ala Ile Ala Ser Gly Leu Arg Ser Lys Ile 180 185 190 Lys Ser Asn Phe Arg Leu Lys Glu Leu Lys Asn Met Lys Ser Gly Leu 195 200 205 Pro Thr Thr Lys Ser Asp Asn Phe Pro Ile Pro Leu Val Lys Gln Lys 210 215 220 Gly Gly Gln Tyr Thr Gly Phe Glu Ile Ser Asn His Asn Ser Asp Phe 225 230 235 240 Ile Ile Lys Ile Pro Phe Gly Arg Trp Gln Val Lys Lys Glu Ile Asp 245 250 255 Lys Tyr Arg Pro Trp Glu Lys Phe Asp Phe Glu Gln Val Gln Lys Ser 260 265 270 Pro Lys Pro Ile Ser Leu Leu Leu Ser Thr Gln Arg Arg Lys Arg Asn 275 280 285 Lys Gly Trp Ser Lys Asp Glu Gly Thr Glu Ala Glu Ile Lys Lys Val 290 295 300 Met Asn Gly Asp Tyr Gln Thr Ser Tyr Ile Glu Val Lys Arg Gly Ser 305 310 315 320 Lys Ile Gly Glu Lys Ser Ala Trp Met Leu Asn Leu Ser Ile Asp Val 325 330 335 Pro Lys Ile Asp Lys Gly Val Asp Pro Ser Ile Ile Gly Gly Ile Asp 340 345 350 Val Gly Val Lys Ser Pro Leu Val Cys Ala Ile Asn Asn Ala Phe Ser 355 360 365 Arg Tyr Ser Ile Ser Asp Asn Asp Leu Phe His Phe Asn Lys Lys Met 370 375 380 Phe Ala Arg Arg Arg Ile Leu Leu Lys Lys Asn Arg His Lys Arg Ala 385 390 395 400 Gly His Gly Ala Lys Asn Lys Leu Lys Pro Ile Thr Ile Leu Thr Glu 405 410 415 Lys Ser Glu Arg Phe Arg Lys Lys Leu Ile Glu Arg Trp Ala Cys Glu 420 425 430 Ile Ala Asp Phe Phe Ile Lys Asn Lys Val Gly Thr Val Gln Met Ala 435 440 445 Asn Leu Glu Ser Met Lys Arg Lys Glu Asp Ser Tyr Phe Asn Ile Arg 450 455 460 Leu Arg Gly Phe Trp Pro Tyr Ala Glu Met Gln Asn Lys Ile Glu Phe 465 470 475 480 Lys Leu Lys Gln Tyr Gly Ile Glu Ile Arg Lys Val Ala Pro Asn Asn 485 490 495 Thr Ser Lys Thr Cys Ser Lys Cys Gly His Leu Asn Asn Tyr Phe Asn 500 505 510 Phe Glu Tyr Arg Lys Lys Asn Lys Phe Pro His Phe Lys Cys Glu Lys 515 520 525 Cys Asn Phe Lys Glu Asn Ala Asp Tyr Asn Ala Ala Leu Asn Ile Ser 530 535 540 Asn Pro Lys Leu Lys Ser Thr Lys Glu Glu Pro 545 550 555 <210> 174 <211> 555 <212> PRT <213> artificial sequence <220> <223> 26aa-Extension dCas12f1 (R516A) <400> 174 Met Ala Gly Gly Pro Gly Ala Gly Ser Ala Ala Pro Val Ser Ser Thr 1 5 10 15 Ser Ser Leu Pro Leu Ala Ala Leu Asn Met Met Ala Lys Asn Thr Ile 20 25 30 Thr Lys Thr Leu Lys Leu Arg Ile Val Arg Pro Tyr Asn Ser Ala Glu 35 40 45 Val Glu Lys Ile Val Ala Asp Glu Lys Asn Asn Arg Glu Lys Ile Ala 50 55 60 Leu Glu Lys Asn Lys Asp Lys Val Lys Glu Ala Cys Ser Lys His Leu 65 70 75 80 Lys Val Ala Ala Tyr Cys Thr Thr Gln Val Glu Arg Asn Ala Cys Leu 85 90 95 Phe Cys Lys Ala Arg Lys Leu Asp Asp Lys Phe Tyr Gln Lys Leu Arg 100 105 110 Gly Gln Phe Pro Asp Ala Val Phe Trp Gln Glu Ile Ser Glu Ile Phe 115 120 125 Arg Gln Leu Gln Lys Gln Ala Ala Glu Ile Tyr Asn Gln Ser Leu Ile 130 135 140 Glu Leu Tyr Tyr Glu Ile Phe Ile Lys Gly Lys Gly Ile Ala Asn Ala 145 150 155 160 Ser Ser Val Glu His Tyr Leu Ser Asp Val Cys Tyr Thr Arg Ala Ala 165 170 175 Glu Leu Phe Lys Asn Ala Ala Ile Ala Ser Gly Leu Arg Ser Lys Ile 180 185 190 Lys Ser Asn Phe Arg Leu Lys Glu Leu Lys Asn Met Lys Ser Gly Leu 195 200 205 Pro Thr Thr Lys Ser Asp Asn Phe Pro Ile Pro Leu Val Lys Gln Lys 210 215 220 Gly Gly Gln Tyr Thr Gly Phe Glu Ile Ser Asn His Asn Ser Asp Phe 225 230 235 240 Ile Ile Lys Ile Pro Phe Gly Arg Trp Gln Val Lys Lys Glu Ile Asp 245 250 255 Lys Tyr Arg Pro Trp Glu Lys Phe Asp Phe Glu Gln Val Gln Lys Ser 260 265 270 Pro Lys Pro Ile Ser Leu Leu Leu Ser Thr Gln Arg Arg Lys Arg Asn 275 280 285 Lys Gly Trp Ser Lys Asp Glu Gly Thr Glu Ala Glu Ile Lys Lys Val 290 295 300 Met Asn Gly Asp Tyr Gln Thr Ser Tyr Ile Glu Val Lys Arg Gly Ser 305 310 315 320 Lys Ile Gly Glu Lys Ser Ala Trp Met Leu Asn Leu Ser Ile Asp Val 325 330 335 Pro Lys Ile Asp Lys Gly Val Asp Pro Ser Ile Ile Gly Gly Ile Asp 340 345 350 Val Gly Val Lys Ser Pro Leu Val Cys Ala Ile Asn Asn Ala Phe Ser 355 360 365 Arg Tyr Ser Ile Ser Asp Asn Asp Leu Phe His Phe Asn Lys Lys Met 370 375 380 Phe Ala Arg Arg Arg Ile Leu Leu Lys Lys Asn Arg His Lys Arg Ala 385 390 395 400 Gly His Gly Ala Lys Asn Lys Leu Lys Pro Ile Thr Ile Leu Thr Glu 405 410 415 Lys Ser Glu Arg Phe Arg Lys Lys Leu Ile Glu Arg Trp Ala Cys Glu 420 425 430 Ile Ala Asp Phe Phe Ile Lys Asn Lys Val Gly Thr Val Gln Met Glu 435 440 445 Asn Leu Glu Ser Met Lys Arg Lys Glu Asp Ser Tyr Phe Asn Ile Arg 450 455 460 Leu Arg Gly Phe Trp Pro Tyr Ala Glu Met Gln Asn Lys Ile Glu Phe 465 470 475 480 Lys Leu Lys Gln Tyr Gly Ile Glu Ile Arg Lys Val Ala Pro Asn Asn 485 490 495 Thr Ser Lys Thr Cys Ser Lys Cys Gly His Leu Asn Asn Tyr Phe Asn 500 505 510 Phe Glu Tyr Ala Lys Lys Asn Lys Phe Pro His Phe Lys Cys Glu Lys 515 520 525 Cys Asn Phe Lys Glu Asn Ala Asp Tyr Asn Ala Ala Leu Asn Ile Ser 530 535 540 Asn Pro Lys Leu Lys Ser Thr Lys Glu Glu Pro 545 550 555 <210> 175 <211> 555 <212> PRT <213> artificial sequence <220> <223> 26aa-Extension dCas12f1 (D536A) <400> 175 Met Ala Gly Gly Pro Gly Ala Gly Ser Ala Ala Pro Val Ser Ser Thr 1 5 10 15 Ser Ser Leu Pro Leu Ala Ala Leu Asn Met Met Ala Lys Asn Thr Ile 20 25 30 Thr Lys Thr Leu Lys Leu Arg Ile Val Arg Pro Tyr Asn Ser Ala Glu 35 40 45 Val Glu Lys Ile Val Ala Asp Glu Lys Asn Asn Arg Glu Lys Ile Ala 50 55 60 Leu Glu Lys Asn Lys Asp Lys Val Lys Glu Ala Cys Ser Lys His Leu 65 70 75 80 Lys Val Ala Ala Tyr Cys Thr Thr Gln Val Glu Arg Asn Ala Cys Leu 85 90 95 Phe Cys Lys Ala Arg Lys Leu Asp Asp Lys Phe Tyr Gln Lys Leu Arg 100 105 110 Gly Gln Phe Pro Asp Ala Val Phe Trp Gln Glu Ile Ser Glu Ile Phe 115 120 125 Arg Gln Leu Gln Lys Gln Ala Ala Glu Ile Tyr Asn Gln Ser Leu Ile 130 135 140 Glu Leu Tyr Tyr Glu Ile Phe Ile Lys Gly Lys Gly Ile Ala Asn Ala 145 150 155 160 Ser Ser Val Glu His Tyr Leu Ser Asp Val Cys Tyr Thr Arg Ala Ala 165 170 175 Glu Leu Phe Lys Asn Ala Ala Ile Ala Ser Gly Leu Arg Ser Lys Ile 180 185 190 Lys Ser Asn Phe Arg Leu Lys Glu Leu Lys Asn Met Lys Ser Gly Leu 195 200 205 Pro Thr Thr Lys Ser Asp Asn Phe Pro Ile Pro Leu Val Lys Gln Lys 210 215 220 Gly Gly Gln Tyr Thr Gly Phe Glu Ile Ser Asn His Asn Ser Asp Phe 225 230 235 240 Ile Ile Lys Ile Pro Phe Gly Arg Trp Gln Val Lys Lys Glu Ile Asp 245 250 255 Lys Tyr Arg Pro Trp Glu Lys Phe Asp Phe Glu Gln Val Gln Lys Ser 260 265 270 Pro Lys Pro Ile Ser Leu Leu Leu Ser Thr Gln Arg Arg Lys Arg Asn 275 280 285 Lys Gly Trp Ser Lys Asp Glu Gly Thr Glu Ala Glu Ile Lys Lys Val 290 295 300 Met Asn Gly Asp Tyr Gln Thr Ser Tyr Ile Glu Val Lys Arg Gly Ser 305 310 315 320 Lys Ile Gly Glu Lys Ser Ala Trp Met Leu Asn Leu Ser Ile Asp Val 325 330 335 Pro Lys Ile Asp Lys Gly Val Asp Pro Ser Ile Ile Gly Gly Ile Asp 340 345 350 Val Gly Val Lys Ser Pro Leu Val Cys Ala Ile Asn Asn Ala Phe Ser 355 360 365 Arg Tyr Ser Ile Ser Asp Asn Asp Leu Phe His Phe Asn Lys Lys Met 370 375 380 Phe Ala Arg Arg Arg Ile Leu Leu Lys Lys Asn Arg His Lys Arg Ala 385 390 395 400 Gly His Gly Ala Lys Asn Lys Leu Lys Pro Ile Thr Ile Leu Thr Glu 405 410 415 Lys Ser Glu Arg Phe Arg Lys Lys Leu Ile Glu Arg Trp Ala Cys Glu 420 425 430 Ile Ala Asp Phe Phe Ile Lys Asn Lys Val Gly Thr Val Gln Met Glu 435 440 445 Asn Leu Glu Ser Met Lys Arg Lys Glu Asp Ser Tyr Phe Asn Ile Arg 450 455 460 Leu Arg Gly Phe Trp Pro Tyr Ala Glu Met Gln Asn Lys Ile Glu Phe 465 470 475 480 Lys Leu Lys Gln Tyr Gly Ile Glu Ile Arg Lys Val Ala Pro Asn Asn 485 490 495 Thr Ser Lys Thr Cys Ser Lys Cys Gly His Leu Asn Asn Tyr Phe Asn 500 505 510 Phe Glu Tyr Arg Lys Lys Asn Lys Phe Pro His Phe Lys Cys Glu Lys 515 520 525 Cys Asn Phe Lys Glu Asn Ala Ala Tyr Asn Ala Ala Leu Asn Ile Ser 530 535 540 Asn Pro Lys Leu Lys Ser Thr Lys Glu Glu Pro 545 550 555 <210> 176 <211> 159 <212> PRT <213> artificial sequence <220> <223> eTadA4 <400> 176 Met Val Glu Phe Ser Asp Glu Tyr Trp Met Arg His Ala Leu Thr Leu 1 5 10 15 Ala Lys Arg Ala Arg Asp Glu Gly Glu Val Pro Val Gly Ala Val Leu 20 25 30 Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Ala Ile Gly 35 40 45 Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln Gly 50 55 60 Gly Gln Val Leu Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr Val 65 70 75 80 Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser Arg 85 90 95 Ile Lys Arg Val Val Phe Gly Trp Arg Asn Ser Lys Arg Gly Ala Ala 100 105 110 Gly Ser Leu Met Asn Val Leu Asn His Pro Gly Met Asn His Arg Val 115 120 125 Glu Ile Thr Glu Gly Val Leu Ala Asp Glu Cys Ala Ala Leu Leu Ser 130 135 140 Asp Phe Phe Arg Met Arg Arg Gln Gln Lys Lys Ala Gln Lys Lys 145 150 155 <210> 177 <211> 159 <212> PRT <213> artificial sequence <220> <223> eTadA5 <400> 177 Met Val Glu Phe Ser Asp Glu Tyr Trp Met Arg His Ala Leu Thr Leu 1 5 10 15 Ala Lys Arg Ala Arg Asp Glu Gly Glu Val Pro Val Gly Ala Val Leu 20 25 30 Val Leu Asn Asn Gln Val Ile Gly Glu Gly Trp Asn Arg Ser Ile Ser 35 40 45 Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln Gly 50 55 60 Gly Gln Val Leu Gln Asn Tyr Arg Leu Ile Asp Cys Thr Leu Tyr Val 65 70 75 80 Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser Arg 85 90 95 Ile Lys Arg Val Val Phe Gly Trp Arg Asn Pro Lys Arg Gly Ala Ala 100 105 110 Gly Ser Leu Met Asn Val Leu Asn His Pro Gly Met Asn His Arg Ile 115 120 125 Glu Ile Thr Glu Gly Val Leu Ala Asp Glu Cys Ala Ala Met Leu Ser 130 135 140 Asp Phe Phe Arg Met Arg Arg Gln Gln Lys Lys Ala Gln Lys Lys 145 150 155 <210> 178 <211> 159 <212> PRT <213> artificial sequence <220> <223> eTadA6 <400> 178 Met Val Glu Phe Ser Asp Glu Tyr Trp Met Arg His Ala Leu Thr Leu 1 5 10 15 Ala Lys Arg Ala Arg Asp Glu Gly Glu Val Pro Val Gly Ala Val Leu 20 25 30 Val Leu Asn Asn Gln Val Ile Gly Glu Gly Trp Asn Arg Ser Ile Thr 35 40 45 His His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln Gly 50 55 60 Gly Gln Val Leu Gln Asn Tyr Arg Leu Ile Asp Cys Thr Leu Tyr Val 65 70 75 80 Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ala Arg 85 90 95 Ile Lys Arg Val Val Phe Gly Trp Arg Asn Pro Lys Arg Gly Ala Ala 100 105 110 Gly Ser Val Met Asn Val Leu Asn His Pro Gly Met Asn His Arg Ile 115 120 125 Glu Ile Thr Glu Gly Val Leu Ala Asp Glu Cys Ala Ala Met Leu Ser 130 135 140 Asp Phe Phe Arg Met Arg Arg Gln Gln Lys Lys Ala Gln Lys Lys 145 150 155

Claims (31)

Cas12f1, TnpB or functional analogues thereof; and a deaminase.
The method of claim 1, wherein the Cas12f1, TnpB or functional analogues thereof are dead TnpB (dTnpB) having lost DNA double-strand cleavage activity; dead Cas12f1 (dCas12f1); or a functional analog of dTnpB or dCas12f1; characterized in that, the fusion protein.
The method of claim 1, wherein the Cas12f1, TnpB or a functional analogue thereof comprises any one amino acid sequence selected from SEQ ID NO: 3 to SEQ ID NO: 6, SEQ ID NO: 11 to SEQ ID NO: 18, and SEQ ID NO: 168 to SEQ ID NO: 175 or at least two amino acid mutations among D354A, E450A, R518A and D538A in SEQ ID NO: 7.
The method of claim 1, wherein the deaminase is an adenosine deaminase and/or cytidine deaminase linked to the N-terminus or C-terminus of the fusion protein. Characterized in that, the fusion protein.
The method of claim 4, wherein the adenosine deaminase is TadA (tRNA-specific adenosine deaminase) and/or eTadA (evolved tRNA-specific adenosine deaminase), or cytidine deaminase ) are APOBEC1 (apolipoprotein B mRNA-editing complex 1), APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3F, APOBEC3G, APOBEC3H, activation-induced deaminase (AID), FERNY deaminase, CDA1 Arabidopsis A fusion protein, characterized in that it is cytidine deaminase 1 (AtCDA1) from Arabidopsis thaliana and/or cytidine deaminase 1 (PmCDA1) from Petromyzon marinus.
The method of claim 5, wherein the adenosine deaminase (adenosine deaminase) is monomeric TadA (SEQ ID NO: 126), eTadA1 (evolved tRNA-specific adenosine deaminase1, SEQ ID NO: 127), dTadA (SEQ ID NO: 128), deTadA1 (SEQ ID NO: 128) 129), eTadA2 (SEQ ID NO: 130), eTadA3 (SEQ ID NO: 131), eTadA4 (SEQ ID NO: 176), eTadA5 (SEQ ID NO: 177), eTadA6 (SEQ ID NO: 178), eTadA7 (SEQ ID NO: 137), eTadA8 (SEQ ID NO: 138 ), eTadA9 (SEQ ID NO: 139), eTadA10 (SEQ ID NO: 140), or eTadA11 (SEQ ID NO: 141), or the heterodimer TadA-eTadA , eTadA-TadA, dTadA-eTadA or TadA-deTadA, characterized in that the fusion protein.
The fusion protein according to claim 1, further comprising UGI (Uracil Glycosylase Inhibitor) or GAM (Gam protein).
The method of claim 1, wherein the Cas12f1, TnpB, or functional analogues thereof and the deaminase are 5'-GAAA-3', SEQ ID NO: 132 (GGGGS)n, (G)n, SEQ ID NO: 133 (EAAAK) n, (GGS)n, SGSETPGTSESATPES of SEQ ID NO: 134, SGGS of SEQ ID NO: 135, (XP)n, or a combination thereof, characterized in that connected by a linker.
A nucleic acid encoding the fusion protein of any one of claims 1 to 8.
The nucleic acid according to claim 9, further comprising at least one nuclear localization signal (NLS) sequence and/or nuclear export signal (NES) sequence.
The fusion protein of claim 1 or a nucleic acid encoding the same; And guide RNA; subminiature base editing (Base editing) system containing.
The method of claim 11, wherein the fusion protein is a deaminase-linked nucleolytic protein, and the guide RNA includes one or two or more wild-type or engineered guide RNAs. , a microbase correction system.
[Claim 12] The subminiature base correction system according to claim 11, wherein the guide RNA is wild-type or engineered guide RNA.
[Claim 12] The subminiature base correction system according to claim 11, wherein the guide RNA includes an engineered tracrRNA (transactivating CRISPR RNA) or an engineered crRNA (CRISPR RNA) and is a single-stranded or double-stranded guide RNA.
15. The method of claim 14, wherein the engineered tracrRNA is a tracrRNA modified not to include five or more contiguous uridine sequences and modified to be shorter than wild-type tracrRNA, wherein the engineered crRNA is SEQ ID NO: 38 or SEQ ID NO: 38 Characterized in that it comprises a part of the sequence of, the ultra-small base correction system.
12. The microbase correction system according to claim 11, wherein the guide RNA comprises a nucleic acid sequence selected from SEQ ID NO: 55 to SEQ ID NO: 59.
A vector comprising a nucleic acid construct in which a nucleic acid sequence encoding a component of the miniaturization system according to claim 11 is operably linked.
The vector according to claim 17, wherein the vector further comprises nucleic acids encoding one or two or more guide RNAs and one or two or more promoters therefor.
The vector according to claim 18, wherein the promoter is a U6 promoter, H1 promoter or 7SK promoter.
The vector according to claim 17, characterized in that the vector is RNA, plasmid, linear PCR amplicon, viral vector or ribonucleoprotein (RNP).
21. The vector according to claim 20, characterized in that the viral vector is a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a vaccinia virus vector, a poxvirus vector or a herpes simplex virus vector.
A base editing composition comprising the subminiature base editing system according to any one of claims 11 to 16 or the vector according to any one of claims 17 to 21.
[Claim 23] The method of claim 22, wherein the base correction composition is engineered guide RNA; and TnpB-derived low-molecular-weight nucleic acid degrading protein or a functional analogue thereof; characterized in that it is in the form of a ribonucleoprotein (RNP), a composition for base correction.
23. The composition for base correction according to claim 22, characterized in that adenine (A) and/or cytosine (C) are substituted with other bases.
The method of claim 22, wherein the composition for base editing comprises adenine (A) whose editing window is located 2nd to 8th, 15th to 19th, or 2nd to 20th from the 5'-end of the target sequence. and/or a cytosine (C) located at the 2nd to 7th or 3rd to 5th positions of the target sequence.
A method for correcting bases, comprising contacting the miniaturized base correction system according to any one of claims 11 to 16 and the vector according to any one of claims 17 to 21 with a target nucleic acid or a target gene.
The method of claim 26, wherein the target nucleic acid or the target gene is a double-stranded DNA comprising a target strand having a target sequence; single-stranded DNA or RNA; or hybrid double-stranded DNA and RNA.
27. The method of claim 26, wherein the method is performed in a prokaryotic or eukaryotic cell in which the target nucleic acid or target gene is present.
29. The method of claim 28, wherein the eukaryotic cell is a yeast, plant cell, non-human-animal cell or human cell.
The method of claim 26, wherein the base editing is carried out with adenine (A) and / Or a cytosine (C) located at the 2nd to 8th or 3rd to 5th positions, characterized in that, a method for correcting bases.
27. The method of claim 26, wherein the miniaturization system, the vector or the minibase editing construct is from the group consisting of retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, vacciniaviruses, poxviruses and herpes simplex virus vectors. A method for correcting a base, characterized in that it is packaged in a selected viral vector and delivered into a prokaryotic or eukaryotic cell.

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