Cell strain capable of stably expressing Cas9 protein as well as preparation method and application thereof

A stable expression and cell line technology, which is applied in the field of genetic engineering and genetic modification, can solve problems affecting gene editing efficiency, low transfection efficiency, and reduced success rate of gene editing cell lines/gene editing model animals, etc. Editing efficiency, high-efficiency cutting, and high-efficiency cutting effects

Inactive Publication Date: 2020-04-03
湖南普拉特网络科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Since the Cas9 protein coding gene is relatively large, close to 4000bp, the transfection efficiency is often low due to the large plasmid during gene editing, which seriously affects the efficiency of gene editing, making the construction of gene editing cell li

Method used

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  • Cell strain capable of stably expressing Cas9 protein as well as preparation method and application thereof
  • Cell strain capable of stably expressing Cas9 protein as well as preparation method and application thereof
  • Cell strain capable of stably expressing Cas9 protein as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0043] Example: Construction of a cell line capable of stably expressing Cas9 protein

[0044] 1. Design and construct a site-directed cutting vector capable of expressing target sgRNA 1

[0045] 1.1 Extract the original plasmid of the cutting vector

[0046] Using the plasmid mini-extraction kit (purchased from Kangwei Century, Cat. No. CW0511C), a small amount of the original plasmid of site-directed cleavage vector 1 (purchased from addgene, cat.

[0047] 1.2 Restriction cutting the vector original plasmid

[0048] Using an endonuclease (purchased from Neb, catalog number R0539L), the plasmid extracted in 1.1 was digested according to the experimental procedure of the product manual, and the digested product was recovered.

[0049] 1.3 Screening suitable sgRNA target sequence fragments

[0050] 1.3.1 Selection of sgRNA target sites

[0051] According to the genome sequence of the human gene AAVS1 site given on the NCBI website, multiple target sites were selected for test...

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Abstract

The invention discloses a cell strain capable of stably expressing Cas9 protein as well as a preparation method and an application thereof. According to the method, a Cas9 protein expression gene, anantibiotic selection marker gene and a fluorescence marker gene are introduced into a 293T cell of a tool cell through a CRISPR/Cas9 system; after positive clone cell strains are obtained through resistance and fluorescence screening, the screening marker is cut off through the CRISPR/Cas9 system, and finally the cell strains which can stably express Cas9 protein and do not have any screening marker are obtained. The cell strain constructed by the invention is constructed on the basis of a CRISPR/Cas9 gene editing system and a homologous recombination principle, and is integrated at a safety site-AAVS1 site in the tool cell genome in a fixed point mode; the Cas9 protein can be stably expressed and does not screening marker is obtained, so that the tool cell strain of a subsequent screeningexperiment is not influenced.

Description

technical field [0001] The invention belongs to the field of genetic engineering and genetic modification, and in particular relates to a cell line capable of stably expressing Cas9 protein, a preparation method and application thereof. Background technique [0002] CRISPR (clustered regularly interspaced short palindromic repeats) / Cas (CRISPR-associated) system is an immune system against exogenous genetic material possessed by prokaryotes. Through RNA-mediated targeting of specific targets with specific sequences, the CRISPR system can cut exogenous DNA, including phages and exogenous plasmids, resulting in the loss or partial loss of the target gene function. CRISPR / Cas system can be used as a fixed-point gene editing system, which has the characteristics of simple operation, low cost and high efficiency. It is a new gene editing technology that has been widely used in basic research. [0003] The CRISPR / Cas system is a powerful tool for gene editing and can precisely ed...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/66C12N15/90C12N9/22C12N5/10
CPCC12N15/85C12N15/66C12N15/907C12N9/22C12N5/0636C12N2510/02
Inventor 蒋明贵刘军鹏方娟曾炳蔚刘彩云许澎屈飞
Owner 湖南普拉特网络科技有限公司
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