Method for site-specific integration of large DNA fragment in mammalian cell through CRISPR/Cas9

A technology of mammals and cells, applied in the field of genetic engineering, can solve problems such as unfavorable physical distance homologous recombination exchange, small exogenous DNA fragments, and low efficiency of double exchange homologous recombination

Inactive Publication Date: 2017-09-08
SUN YAT SEN UNIV
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Problems solved by technology

However, this technology has the following disadvantages: the efficiency of double-exchange homologous recombination is often low, generally 10 -6 ; The exogenous DNA fragments that use this technology to achieve site-specific integration are relati

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  • Method for site-specific integration of large DNA fragment in mammalian cell through CRISPR/Cas9
  • Method for site-specific integration of large DNA fragment in mammalian cell through CRISPR/Cas9
  • Method for site-specific integration of large DNA fragment in mammalian cell through CRISPR/Cas9

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Embodiment 1

[0028] 1. CCR5 gene target site selection: According to the CCR5 gene sequence and the basic design principles of gRNA, and with the help of software analysis (http: / / crispr.mit.edu / ), design gRNA targeting the human CCR5 gene. The specific sequence is shown in Table 1 .

[0029] Table 1. Sequence information of gRNA targeting CCR5 gene

[0030] name Sequence (5'to 3') CRISPR / Cas9 Targeting Sites CATCCTGATAAACTGCAAA

[0031] 2. HEK293T cell culture and transfection: DMEM high-glucose medium with 10% fetal bovine serum was used for HEK293T cell culture, and the cells were cultured in about 1×10 5 Cells / mL were inoculated in 24-well cell culture plates for use. When the confluence of the cells reaches about 90%, it can be used for transfection. For transfection, 150ng pX330-CCR5 plasmid, 100ngCCR5-EGFP-Donor plasmid (pEGFP-CCR5-HR), 1μL Lipofectamine 3000, 1μLP3000TM and 50μL Opti-MEM per well The medium formula was added for transfection, and a single-...

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Abstract

The invention discloses a method for site-specific integration of a large DNA fragment in a mammalian cell through CRISPR/Cas9. The method comprises the following steps: (1) constructing a CRISPR/Cas9 targeting system of a target CCR5 gene, wherein the targeting system comprises a Cas9/sgRNA expression vector and a targeting donor, the targeting donor comprises a promotor, a fluorescence labelling gene and a design, and the design of the targeting donor comprises a gRNA target sequence at the middle part and shares a homologous sequence with a target gene; (2) enabling the CRISPR/Cas9 targeting system to cotransfer the mammalian cell, separating the cell stably expressing fluorescence after cotransfecting, and conducting single cell clone culture; (3) detecting the site-specific integration efficiency through single cell clone PCR, and acquiring the efficient site-specific site-detected integration mammalian cell. The method can realize the site-specific site-detected integration of the large DNA fragment more than 6 kb in the mammalian cell.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a method for realizing site-specific integration of large fragments of DNA in mammalian cells by using CRISPR / Cas9. Background technique [0002] The site-directed integration of large fragments of foreign genes in mammalian cell genomes usually adopts the method of double crossover homologous recombination. The implementation steps of this technology are to construct a targeting donor containing two homology arms on both sides of the target gene , using two homologous arms to perform double-crossover homologous recombination with the target gene, thereby replacing the target gene with an exogenous DNA fragment between the two homologous arms. However, this technology has the following disadvantages: the efficiency of double-exchange homologous recombination is often low, generally 10 -6 ; Using this technology to achieve site-specific integration of foreign DNA frag...

Claims

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Application Information

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IPC IPC(8): C12N15/90C12N9/22
CPCC12N9/22C12N15/102C12N15/907
Inventor 黄翔何祖勇刘小凤刘小红陈瑶生
Owner SUN YAT SEN UNIV
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