Two-step strategy for point mutation of yjjW in Escherichia coli by gene editing

Abstract:

[Objective] In order to investigate the target genes in Escherichia coli, we used the CRISPR-Cas9 tool combining with homologous recombination to develop a two-step strategy for point mutation. [Methods] First, we assembled the up- and down-stream homologous arms of a gene and the amp gene with the pKOV plasmid to construct the pKOV-HR plasmid. Then the pKOV-HR plasmid was transformed into E. coli and the gene knock-out mutants were obtained by the RecA recombination system of E. coli itself. Subsequently, we applied two plasmids system (pSGKP-km and pCasKP-apr) to handle the point mutations of the target gene. Firstly, we designed a spacer sequence of the amp gene which was able to guide Cas9 proteins. The overlap PCR was used to produce the homologous arm containing point mutations of the target gene. Then the spacer and homologous arm were connected by pSGKP-km plasmid and the plasmid pSGKP-km-spacer-HR was obtained. Next, we transformed this plasmid and pCasKP-apr plasmid into the above-mentioned knock-out mutants. Finally, we got the point mutants by the DNA-cleaving of Cas9 proteins and λ-Red recombinant proteins which were produced by the two plasmids. [Results] By this two-step strategy, we obtained yjjW knockout strains named D7ΔyjjW::amp^R, and point mutants named D7yjjW-24 (from T to C the 24th base) successfully. [Conclusion] The study established an efficient method for point mutations of target genes in E. coli. The insertion of amp gene provided an effective screening marker. The recombinant plasmid pSGKP-km-spacer established in this process can be applied to point mutations of other target genes, which will greatly shorten experimental processes. It contributes the scientific theory and practice for the development of gene editing technology.