# US 2023/0056843 A1 · Construction of high-fidelity crispr/ascpf1 mutant and uses thereof

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- Number: US 2023/0056843 A1
- Document ID: US20230056843A1
- Status: Published application
- Kind: A1
- Application number: 17/794,263
- Filed: 2019-08-19
- Published: 2023-02-23
- Earliest claimed: 2019-08-19
- Assignees: Southern Medical University
- Applicants: Southern Medical University
- Inventors: Zhili RONG; Ying LIN; Hongxin HUANG; Lin SHAN
- Web page: https://patentgrep.com/patent/US20230056843A1

## CPC

- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12N 15/102: {Mutagenizing nucleic acids}
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 15/11: DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity}
- C12N 15/1137: {against enzymes (viral enzymes C12N15/1131; receptors C12N15/1138)}
- C12N 15/85: for animal cells
- C12N 15/90: Stable introduction of foreign DNA into chromosome
- C12N 15/907: {in mammalian cells}
- C12Y 201/01037: DNA (cytosine-5-)-methyltransferase (2.1.1.37)
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2800/107: for mammalian
- C12N 2800/80: Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
- C12R 2001/01: Bacteria or Actinomycetales {; using bacteria or Actinomycetales}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES
- C12R: INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS

## Abstract

A high-fidelity AsCpf1 mutant is obtained by performing a mutation on arginine at position 951 and/or 955 of a AsCpf1 protein amino acid sequence and replacing the same with an amino acid free of forming a hydrogen bond with DNA of a target site; and the amino acid sequence thereof is shown in SEQ ID NOS: 1-3. The encoding gene of the AsCpf1 mutant has a nucleotide sequence as shown in SEQ ID NO: 4 and can be used in the construction of a CRISPR/AsCpf1 gene editing system. A CRISPR/AsCpf1 gene editing system includes a gene encoding a AsCpf1 protein, and the AsCpf1 protein is the AsCpf1 mutant mentioned above. The CRISPR/AsCpf1 gene editing system can be used in lowering an off-target effect of gene editing. The novel AsCpf1 mutant not only retains the gene editing efficiency of wild-type AsCpf1, but also has a higher specificity than the wild-type AsCpf1.

## Claims

### Claim 1

An AsCpf1 mutant, wherein
the AsCpf1 mutant is obtained by mutating arginine at position 951 of an amino acid sequence of AsCpf1 protein and replacing the arginine at the position 951 with a specific amino acid, and the specific amino acid does not form a hydrogen bond with DNA of a target site;
or the AsCpf1 mutant is obtained by mutating arginine at position 955 of the amino acid sequence of the AsCpf1 protein and replacing the arginine at the position 955 with the specific amino acid;
or an AsCpf1 mutant is obtained by mutating the arginine at the position 951 and the arginine at the position 955 of the amino acid sequence of the AsCpf1 protein and replacing the arginine at the position 951 and the arginine at the position 955 with the specific amino acid.

### Claim 2 (depends on 1)

The AsCpf1 mutant according to claim 1, wherein the AsCpf1 mutant is obtained by mutating the arginine at the position 951 and the arginine at the position 955 of the amino acid sequence of the AsCpf1 protein and replacing the arginine at the position 951 and the arginine at the position 955 with the specific amino acid.

### Claim 3 (depends on 1)

The AsCpf1 mutant according to claim 1, wherein the AsCpf1 mutant is obtained by mutating the arginine at the position 951 of the amino acid sequence of the AsCpf1 protein into lysine; and the AsCpf1 mutant has an amino acid sequence as shown in SEQ ID NO: 1;
or the AsCpf1 mutant is obtained by mutating the arginine at the position 955 of the amino acid sequence of the AsCpf1 protein into alanine; and the AsCpf1 mutant has an amino acid sequence as shown in SEQ ID NO: 2;
or the AsCpf1 mutant is obtained by mutating the arginine at the position 951 of the amino acid sequence of the AsCpf1 protein into lysine and the arginine at the position 955 of the amino acid sequence of the AsCpf1 protein into alanine; and the AsCpf1 mutant has an amino acid sequence as shown in SEQ ID NO: 3.

### Claim 4 (depends on 3)

The AsCpf1 mutant according to claim 3, wherein the AsCpf1 mutant is obtained by mutating the arginine at the position 951 of the amino acid sequence of the AsCpf1 protein into lysine and the arginine at the position 955 of the amino acid sequence of the AsCpf1 protein into alanine; and the AsCpf1 mutant has the amino acid sequence as shown in SEQ ID NO: 3.

### Claim 5 (depends on 1)

An encoding gene of the AsCpf1 mutant according to claim 1.

### Claim 6 (depends on 5)

The encoding gene according to claim 5, wherein a nucleotide sequence of the encoding gene is shown in SEQ ID NO: 4.

### Claim 7 (depends on 5)

A method of constructing a CRISPR/AsCpf1 gene editing system, comprising a step of using the encoding gene of claim 5.

### Claim 8 (depends on 1)

A CRISPR/AsCpf1 gene editing system, comprising a gene encoding an AsCpf1 protein, wherein the AsCpf1 protein is the AsCpf1 mutant of claim 1.

### Claim 9 (depends on 8)

The CRISPR/AsCpf1 gene editing system according to claim 8, wherein the CRISPR/AsCpf1 gene editing system further comprises a U6 promoter used for initiating an sgRNA expression of an encoding gene of the AsCpf1 mutant, a necessary element CrRNA for AsCpf1 gene editing, a eukaryotic promoter used for initiating an expression of the encoding gene of the AsCpf1 mutant and a splicing peptide sequence P2A.

### Claim 10 (depends on 8)

A method of reducing off-target effect of gene editing, comprising a step of using the CRISPR/AsCpf1 gene editing system according to claim 8 for the gene editing.

### Claim 11 (depends on 5)

The encoding gene according to claim 5, wherein the AsCpf1 mutant is obtained by mutating the arginine at the position 951 and the arginine at the position 955 of the amino acid sequence of the AsCpf1 protein and replacing the arginine at the position 951 and the arginine at the position 955 with the specific amino acid.

### Claim 12 (depends on 5)

The encoding gene according to claim 5, wherein the AsCpf1 mutant is obtained by mutating the arginine at the position 951 of the amino acid sequence of the AsCpf1 protein into lysine; and the AsCpf1 mutant has an amino acid sequence as shown in SEQ ID NO: 1;
or the AsCpf1 mutant is obtained by mutating the arginine at the position 955 of the amino acid sequence of the AsCpf1 protein into alanine; and the AsCpf1 mutant has an amino acid sequence as shown in SEQ ID NO: 2;
or the AsCpf1 mutant is obtained by mutating the arginine at the position 951 of the amino acid sequence of the AsCpf1 protein into lysine and the arginine at the position 955 of the amino acid sequence of the AsCpf1 protein into alanine; and the AsCpf1 mutant has an amino acid sequence as shown in SEQ ID NO: 3.

### Claim 13 (depends on 12)

The encoding gene according to claim 12, wherein the AsCpf1 mutant is obtained by mutating the arginine at the position 951 of the amino acid sequence of the AsCpf1 protein into lysine and the arginine at the position 955 of the amino acid sequence of the AsCpf1 protein into alanine; and the AsCpf1 mutant has the amino acid sequence as shown in SEQ ID NO: 3.

### Claim 14 (depends on 7)

The method according to claim 7, wherein a nucleotide sequence of the encoding gene is shown in SEQ ID NO: 4.

### Claim 15 (depends on 8)

The CRISPR/AsCpf1 gene editing system according to claim 8, wherein the AsCpf1 mutant is obtained by mutating the arginine at the position 951 and the arginine at the position 955 of the amino acid sequence of the AsCpf1 protein and replacing the arginine at the position 951 and the arginine at the position 955 with the specific amino acid.

### Claim 16 (depends on 8)

The CRISPR/AsCpf1 gene editing system according to claim 8, wherein the AsCpf1 mutant is obtained by mutating the arginine at the position 951 of the amino acid sequence of the AsCpf1 protein into lysine; and the AsCpf1 mutant has an amino acid sequence as shown in SEQ ID NO: 1;
or the AsCpf1 mutant is obtained by mutating the arginine at the position 955 of the amino acid sequence of the AsCpf1 protein into alanine; and the AsCpf1 mutant has an amino acid sequence as shown in SEQ ID NO: 2;
or the AsCpf1 mutant is obtained by mutating the arginine at the position 951 of the amino acid sequence of the AsCpf1 protein into lysine and the arginine at the position 955 of the amino acid sequence of the AsCpf1 protein into alanine; and the AsCpf1 mutant has an amino acid sequence as shown in SEQ ID NO: 3.

### Claim 17 (depends on 16)

The CRISPR/AsCpf1 gene editing system according to claim 16, wherein the AsCpf1 mutant is obtained by mutating the arginine at the position 951 of the amino acid sequence of the AsCpf1 protein into lysine and the arginine at the position 955 of the amino acid sequence of the AsCpf1 protein into alanine; and the AsCpf1 mutant has the amino acid sequence as shown in SEQ ID NO: 3.

### Claim 18 (depends on 10)

The method according to claim 10, wherein the CRISPR/AsCpf1 gene editing system further comprises a U6 promoter used for initiating an sgRNA expression of an encoding gene of the AsCpf1 mutant, a necessary element CrRNA for AsCpf1 gene editing, a eukaryotic promoter used for initiating an expression of the encoding gene of the AsCpf1 mutant and a splicing peptide sequence P2A.