Method for improving homologous recombination efficiency based on CRISPR gene editing system

Abstract

The invention provides a method for improving homologous recombination efficiency based on a CRISPR gene editing system, which comprises the following steps: introducing a Cas9 plasmid fused with streptavidin and a corresponding homologous donor with biotin modification into a host cell, and further introducing an NHEJ fluorescent reporter plasmid into the host cell. According to the invention, a Cas9 plasmid fused with streptavidin, a corresponding biotin-modified homologous donor and an NHEJ fluorescent reporter plasmid are introduced into a CRISPR/Cas9 system for the first time to improve the homologous recombination efficiency, and the NHEJ-based fluorescent reporter plasmid can indicate all elements subjected to homologous recombination; preliminary flow analysis results show that the homologous recombination efficiency in enriched cells can be improved to 50-80%.

Description

Technical field [0001] The present invention belongs to the technical field of genetic engineering, particularly to a system based editing method of CRISPR genetic system to improve the efficiency of homologous recombination. Background technique [0002] CRISPR / Cas system as is currently the most widely applied gene editing technology, double-strand breaks in the genome specific sites via a single-stranded guide RNA (single-guide RNA, sgRNA) of the guide, followed by a non-host cell homologous end joining (Non-homology end joining, NHEJ) and homologous recombination (homology-directed repair, HDR) are two main ways to achieve repair genome editing. Although NHEJ occurs more efficient, but it tends to cause cleavage site generates a random nucleotide insertions and deletions, not accurate editing. In contrast, the HDR precise editing can be achieved almost all forms of gene insertion include, base substitutions, etc., in the case of providing a donor human template. Thus in pra...

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Problems solved by technology

However, the problem with this type of method is that it is necessary to apply drugs to adjust the growth state of cells, and we know that NHEJ and HDR are two important repair methods for cells to maintain their own genome and protect cells from damage. The method of inhibiting another repair method may reduce the ability of cells to resist damage and cause different degrees of safety hazards.

The other is to improve homologous recombination by changing the form of the donor. Although the double-stranded donor has the advantages of high efficiency and convenient acquisition, its naked 3′-OH will become the host cell nuclease recognition With the target of attack, the rate of homologous recombination is not high

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