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Gene editing tool formed by mutant with high activity, preparation method and method for repairing congenital retinopathy disease-causing gene

A technology of gene editing and mutants, which is applied in genetic engineering, botany equipment and methods, biochemical equipment and methods, etc., can solve the problem that the selection range of Cpf1 editing efficiency cannot be divided to meet scientific research work, and achieve flexibility, Effect of high DNA cleavage activity

Pending Publication Date: 2021-06-22
WENZHOU MEDICAL UNIV
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  • Abstract
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Problems solved by technology

However, at some sites, the editing efficiency of Cpf1 and the selection range of PAM cannot meet the needs of scientific research.

Method used

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  • Gene editing tool formed by mutant with high activity, preparation method and method for repairing congenital retinopathy disease-causing gene
  • Gene editing tool formed by mutant with high activity, preparation method and method for repairing congenital retinopathy disease-causing gene
  • Gene editing tool formed by mutant with high activity, preparation method and method for repairing congenital retinopathy disease-causing gene

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preparation example Construction

[0061] The invention provides a method for preparing an eaFnCpf1 gene editing tool composed of an FnCpf1 mutant with high activity in human cells, comprising the following steps:

[0062] A. The highly active eaFnCpf1 is derived from the FnCpf1 mutant plasmid library, and the construction of the FnCpf1 mutant plasmid library:

[0063] (1) Targeted mutagenesis of FnCpf1 by an error-prone PCR kit, changing the WED and REC domains;

[0064] (2) The FnCpf1 after the base mutation was seamlessly cloned to construct an expression vector, and three mutation libraries of Library I, LibraryII and Library III were obtained, which were prepared for editing cell genes;

[0065] B. Systematic screening of highly active mutants:

[0066] (1) Construct the expression vector pJET-U6-crRNA-site A targeting GFP and a 21nt long crRNA (later renamed pJET-U6-crRNA-site 5 according to the position sequence of its targeting GFP);

[0067] (2) 293-SC1 cells were seeded in a 24-well plate after coun...

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Abstract

The invention discloses an eaFnCpf1 gene editing tool formed by an FnCpf1 mutant with high activity in a human cell, a preparation method thereof and a method for repairing an X-linked congenital retina splitting disease-causing gene, and the eaFnCpf1 gene editing tool is characterized in that the human cell is used for carrying out in-vitro directed evolution screening to obtain an FnCpf1 mutant Q125R with high activity, and an expression vector of the FnCpf1 mutant Q125R is Addgene plasmid # 69976; the guide of the target DNA is cut into crRNA, and an expression vector of the crRNA is pJET-U6-crRNAs; the reporter cell lines for gene editing are 293-SC1 and 293-RS1; and a homologous recombination repair template of the mRS1 gene is synthesized single-stranded deoxyribonucleotide. The preparation method of the FnCpf1 mutant with the high activity comprises the following steps: establishing an FnCpf1 mutant library, screening a mutant eaFnCpf1 with the characteristics of high editing activity and low off-target, and editing an endogenous gene by using the eaFnCpf1. The mutant eaFnCpf1 with the high activity not only has relatively high editing activity, but also can identify PAM in a wider range, so that the skill of a gene editing tool is enriched to a certain extent, and the application of gene editing in the field of biomedicine is expanded.

Description

technical field [0001] The invention relates to a novel genome editing technology, which has broad application prospects in life sciences (including medicine), and specifically relates to an eaFnCpf1 gene editing tool composed of an FnCpf1 mutant with high activity in human cells. The present invention also relates to a preparation method of the above-mentioned eaFnCpf1 gene editing tool composed of an FnCpf1 mutant with high activity in human cells. The present invention also relates to a method for repairing the pathogenic gene of X-linked congenital retinoschisis by using the above-mentioned eaFnCpf1 gene editor. Background technique [0002] Gene editing technology refers to the purposeful modification of specific genes using gene editing tools. At present, gene editing tools mainly include zinc finger nuclease (zinc-finger nuclease, ZFN), transcription activator-like effector nuclease (transcription activator-like effector nuclease, TALEN) and regular clustered intersp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/65C12N15/55C12N9/22C12N15/10C12N15/12
CPCC12N15/85C12N15/65C12N9/22C12N15/1031C12N15/1058C12N15/102C07K14/47C12N2800/107C12Q2531/113C12Q2521/327C12Q2525/161
Inventor 谷峰刘写写
Owner WENZHOU MEDICAL UNIV
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