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Construction and application of a high-fidelity CRISPR/AsCpf1 mutant

A mutant and gene editing technology, applied in the field of high-fidelity CRISPR/AsCpf1 mutant construction, can solve the problem of few high-fidelity versions, and achieve good fidelity, good target cutting activity, and good specificity

Active Publication Date: 2021-09-07
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As for Cpf1, there are very few high-fidelity versions currently in development

Method used

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  • Construction and application of a high-fidelity CRISPR/AsCpf1 mutant
  • Construction and application of a high-fidelity CRISPR/AsCpf1 mutant
  • Construction and application of a high-fidelity CRISPR/AsCpf1 mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1 Recombinant expression plasmid construction

[0034] The sequence of pU6-CAG-AsCpf1-mCherry is shown in SEQ ID NO:5.

[0035]With wild-type pU6-CAG-AsCpf1-mCherry ( figure 1 ) as a carrier (its nucleotide sequence is shown in SEQ ID NO: 5), using Gibson Assembly principle technology, design corresponding mutation primers, perform PCR, and finally connect to obtain pU6-CAG-AsCpf1-KA-mCherry ( figure 2 ). details as follows:

[0036] First, the pU6-CAG-AsCpf1-mCherry vector was digested with PmI I and BamHI to obtain the backbone vector. Using pU6-CAG-AsCpf1-mCherry as a template, PCR amplified fragments containing mutated bases respectively. The sequences of PCR primers are as follows:

[0037] AsCpf1-Pml I-F: 5'-ACCAGCGACAAGTTCTTTTTCCACGTGCCTATCA-3'; (SEQ ID NO: 6)

[0038] AsCpf1-KA-R: 5'-CACAGACCAGGCCTGAGCGGCCGCCACCTTCTCTTCTCCCTGTG-3'; (SEQ ID NO: 7)

[0039] AsCpf1-KA-F: 5'-GAAGGAGAAGGTGGCGGCCGCTCAGGCCTGGTCTGTGGTGGGC-3'; (SEQ ID NO: 8)

[0040] A...

Embodiment 2

[0045] Example 2 specificity verification

[0046] In order to verify that the fidelity of the obtained mutant AsCpf1-KA (ie, the AsCpf1 mutant) is better than that of the wild-type AsCpf1, the following experiments were designed for specificity verification:

[0047] (1) According to literature reports (BP Kleinstiver, et al. Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells, Nartue.2016), wild-type AsCpf1 can cut some gRNAs even if they are not completely matched, especially 1 , 2, 8, 9, 19, 20, 21, 22, 23 and other positions. That is to say, the wild-type AsCpf1 can tolerate some base mismatches of gRNA, that is, the specificity is average. Therefore, we designed a gRNA targeting the site of DNMT1-site3 for verification, including a fully matched on-target site (ON), and mismatch 1 (mm1), mismatch 8 (mm8), and mismatch 9 (mm9), mismatch 19 (mm19), mismatch 20 (mm20) and other gRNAs, the specific sequence is as follows:

[0048] AsCpf1-gRNA-DNMT1-3-ON:...

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Abstract

The invention discloses the construction and application of a high-fidelity CRISPR / AsCpf1 mutant. The AsCpf1 mutant is to mutate the 951st arginine and / or the 955th arginine in the amino acid sequence of the AsCpf1 protein, and replace it with an amino acid that does not form a hydrogen bond with the DNA of the target site, and its amino acid sequence is as follows: Shown in SEQ ID NO: 1-3. The nucleotide sequence of the coding gene of the AsCpf1 mutant is shown in SEQ ID NO:4. Application of the coding gene in constructing a CRISPR / AsCpf1 gene editing system. A CRISPR / AsCpf1 gene editing system, comprising a gene encoding an AsCpf1 protein, and the AsCpf1 protein is the aforementioned AsCpf1 mutant. Application of CRISPR / AsCpf1 gene editing system in reducing off-target effects in gene editing. The novel AsCpf1 mutant constructed in the present invention not only retains the gene editing efficiency of wild-type AsCpf1, but also has higher specificity than wild-type AsCpf1.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the construction and application of a high-fidelity CRISPR / AsCpf1 mutant. Background technique [0002] CRISPR / Cpf1 is a DNA editing technology similar to the CRISPR / Cas9 system. Like CRISPR / Cas9, it belongs to the second class of CRISPR system endonucleases. However, compared with Cas9, Cpf1 is smaller and has a simpler structure, and Cpf1 also has some characteristics that Cas9 does not have, such as sticky ends formed by Cpf1 cutting, the PAM region is TTTN, and it has the characteristics of self-processing CrRNA. Therefore, Cpf1 can not only help to make up for some defects of the CRISPR / Cas9 system, but also may have advantages over CRISPR / Cas9 in some applications. At present, CRISPR systems are widely used, especially they are gradually applied to gene therapy of diseases and improvement of degenerative diseases. Although CRISPR systems can efficiently edit various cells, ti...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/22C12N15/55C12N15/63
CPCC12N9/22C12N15/63
Inventor 荣知立林瑛黄洪新单琳
Owner SOUTHERN MEDICAL UNIVERSITY