CRISPR technology-based method for site-directed mutation of E. coli genes

Abstract

The invention discloses a CRISPR technology-based method for site-directed mutation of E. coli genes. The method includes the steps: performing combination of an own homologous recombination system with pKOV plasmids, replacing target genes in an E. coli genome with resistance marker genes so as to obtain recombinant E. coli, and replacing the resistance marker genes in the recombinant E. coli genome with the target genes with mutation sites through a CRISPR / Cas9 system and a lambda-Red homologous recombinant system. The efficient and simple method for site-directed mutation of the target genes is established through combination of a traditional E. coli gene editing technology with the CRISPR system, and the recombinant plasmids pSGKP-km-spacer which are established in the process can be applied to site-directed mutation of other target genes. On the one hand, experimental operation is reduced greatly, on the other hand, a foundation is laid for study of subsequent realization of site-directed mutation of a large number of target genes.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for performing point mutations on Escherichia coli genes based on CRISPR technology. Background technique [0002] At present, there are two ways to modify the target gene at the chromosome level in Escherichia coli: one is to use the recombination protein encoded by its own RecA homologous recombination system to mediate DNA homologous recombination; the other is to introduce exogenous recombinase λ- Red or RecET increases homologous recombination efficiency. Both of the above two methods realize the editing of target genes through double crossover of DNA molecules. These traditional E. coli homologous recombination techniques usually require exogenous helper plasmids and screening marker genes, and experimental operations such as knockout and insertion of target genes, but when more precise operations such as point mutations are performed on target genes, these recombinat...

AI Technical Summary

Problems solved by technology

These traditional Escherichia coli homologous recombination techniques usually require exogenous helper plasmids and screening marker genes, and experimental operations such as knockout and insertion of target genes, but when more precise operations such as point mutations are performed on target genes, these recombination The technology has shortcomings such as low reorganization efficiency and cumbersome process, and it still faces huge challenges in large-scale application

[0003] The base editor modified by the CRISPR / Cas system is currently a widely used method for point mutation of nucleic acid bases. As a new gene editing tool, this technology still has problems such as high off-target rate and limited editing window

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