基因编辑技术对大肠杆菌yjjW基因点突变的两步法策略

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

摘要:

[目的] 为了实现对大肠杆菌靶基因的点突变，本研究将同源重组系统与CRISPR-Cas9技术相结合，探索一种高效、简捷的两步法策略。[方法] 将靶基因的上下游同源臂和标记基因（amp）与pKOV质粒连接，获得pKOV-HR重组质粒。将pKOV-HR转化至大肠杆菌，借助其自身RecA重组系统，介导DNA发生同源重组，获得靶基因敲除菌株。随后，靶基因的点突变采用pSGKP-km和pCasKP-apr双质粒系统。首先，设计与amp结合具有定向引导作用的spacer序列，并利用overlap PCR获得带有靶基因点突变的同源臂，将spacer和同源臂与pSGKP-km质粒连接，获得重组质粒pSGKP-km-spacer-HR；接着，将pSGKP-km-spacer-HR和pCasKP-apr质粒转化至上述敲除菌株；最后，利用质粒表达的Cas9切割蛋白和λ-Red重组蛋白，发生DNA同源重组，获得靶基因点突变菌株。[结果] 利用上述方法，既成功获得了大肠杆菌yjjW敲除菌株D7ΔyjjW：：amp^R，又实现了yjjW第24位碱基T到C的点突变，获得点突变菌株D7yjjW-24。[结论] 本研究建立了一种高效、简捷的大肠杆菌靶基因点突变方法，amp基因的插入提供了有效的筛选标记，此外该方法建立的重组质粒pSGKP-km-spacer，可应用于同种菌株其他靶基因的点突变，能够缩短后续实验操作，为基因编辑技术的发展提供了科学理论以及实验操作基础。

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.