SSA (single-strand annealing) repair-based gene seamless editing method utilizing CRISPR/Cas9 technology

A genetic, seamless technology, applied in the field of seamless gene editing based on SSA repair using CRISPR/Cas9 technology

Inactive Publication Date: 2016-10-12
NORTHWEST A & F UNIV
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Problems solved by technology

But the PiggyBac transposon easily integrates into other sites in the genome

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  • SSA (single-strand annealing) repair-based gene seamless editing method utilizing CRISPR/Cas9 technology
  • SSA (single-strand annealing) repair-based gene seamless editing method utilizing CRISPR/Cas9 technology
  • SSA (single-strand annealing) repair-based gene seamless editing method utilizing CRISPR/Cas9 technology

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Embodiment 1

[0027] A method for seamless gene editing based on SSA repair using CRISPR / Cas9 technology. The seamless gene editing method first constructs the CCR5.CRISPR / Cas9 targeting the CCR5 gene and the eGFP.CRISPR / Cas9 expression vector targeting eGFP, and Construction of the donor CCR5-Donor vector with pXL-BACII-L-arm-CAG-TK-PGK-Puro R -T2A-eGFP-bGH pA-R-arm structure, where L-arm and R-arm are left and right homology arms, CAG-TK-PGK-Puro in the middle of L-arm and R-arm R -T2A-eGFP-bGH pA sequence is the structure of screening marker components; through two transfections, two CRISPR / Cas9 technologies are used to target the CCR5 gene and eGFP target sites and the intracellular HR and SSA repair mechanism, through the positive Negative screening for seamless editing.

[0028] Construction of CCR5.CRISPR / Cas9 targeting CCR5 gene and eGFP.CRISPR / Cas9 targeting eGFP expression vector:

[0029] Taking the construction of CRISPR / Cas9 expression vector as the prior art, the structure o...

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Abstract

The invention mainly belongs to the technical field of gene editing and particularly relates to an SSA (single-strand annealing) repair-based gene seamless editing method utilizing a CRISPR/Cas9 technology. According to the gene seamless editing method, CRISPR/Cas9 expression vectors CCR5.CRISPR/Cas9 and eGFP.CRISPR/Cas9 targeting CCR5 and eGFP are constructed firstly, a donor CCR5-Donor vector is constructed, a CCR5-Donor plasmid vector adopts the pXL-BACII-L-arm-CAG-TK-PGK-Puro<R>-T2A-eGFP-bGH pA-R-arm structure, L-arm and R-arm are left and right homologous arms, and CAG-TK-PGK-Puro<R>-T2A-eGFP-bGH pA between L-arm and R-arm is a screening marking component; through two times of transfection, seamless editing is completed by using HR and SSA repair mechanisms in the CRISPR-Cas9 system and cells through positive and negative screening of puromycin and ganciclovir of the cells.

Description

technical field [0001] The invention mainly belongs to the technical field of gene editing, and in particular relates to a method for seamless gene editing based on SSA repair using CRISPR / Cas9 technology. Background technique [0002] Gene editing technology is the key to people's understanding of gene function. Early research on gene function was mainly through gene editing through homologous recombination. This method relies too much on natural recombination. For mammals, the efficiency is extremely low, only one in ten million First, it cannot meet the increasing scientific research needs of human beings. Later, it was found in research that if double-strand breaks (Double Strand Breaks, DSBs) are introduced at the genomic DNA target site, the corresponding homologous recombination efficiency in mammalian cells is greatly improved compared with spontaneous homologous recombination, reaching 3-5 ×10 -2 . Three artificial nucleases that have emerged in recent years: zin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85
CPCC12N15/85C12N2800/107C12N2800/80
Inventor 张智英白义春徐坤魏泽辉和林洁任充华邵斯旻吴芸刘中天
Owner NORTHWEST A & F UNIV
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