The Development Of A Gene Editing Method Based On CRISPR/Cas9 Applicable To Diverse Bacteria

In recent years,CRISPR/Cas9 gene editing systems have been widely used in eukaryotes and prokaryotes.In the process of gene editing in Escherichia coli(E.coli),the pCas/pTarget gene editing system is most frequently used.This system is able to achieve a positive gene editing rate of nearly 100%in E.coli K-12 series strains,but it is difficult to achieve successful gene editing in E.coli B series strains,mainly due to the existence of leaked expression of sgRNA used for plasmid elimination.pEcCas/pEcgRNA system replaces the more stringent pCas/pTarget system with a The pEcCas/pEcgRNA system,based on the pCas/pTarget system,replaces the more stringent rhamnose promoter to regulate the sgRNA for plasmid elimination and achieves the application in E.coli B series strains,but this method does not fundamentally solve the problem of leaked expression in transcriptional regulation.AgRNA is a small molecule-regulated sgRNA constructed by fusing a nucleic acid aptamer with sgRNA.the nucleic acid aptamer can affect the function of agRNA through conformational regulation.The nucleic acid aptamer can influence the function of agRNA through conformational regulation,and conformational regulation can avoid the problem of leaking expression by transcriptional regulation,thus enabling gene editing that is rigorously regulated by small molecules.However,this method also has some drawbacks,different N20 sequences can affect the function of agRNA.Based on this,we plan to construct a more general bacterial gene editing method based on the CRISPR/Cas9 system,optimize the pCas/pTarget system from the perspective of conformational regulation,by screening agRNAs targeting fixed sequences for the elimination process of the pTarget series plasmids,and at the same time avoid the problem of poor versatility of agRNAs and transcriptional level regulation of leaky expression problem,the new system has wider potential strain versatility.In this study,in order to screen agRNAs well regulated by small molecules,we first constructed a high-throughput screening method for agRNAs well regulated by small molecules,and obtained agRNAs well regulated by small molecules through a large number of screens.and constructed a self-eliminating plasmid by simultaneously constructing the preferred agRNAs with the corresponding target sequences on the pTarget plasmid pATarget,which carries both sgRNA for gene editing and agRNA for plasmid elimination.subsequently,after a series of optimizations of the pCas/pTarget system,a pEcmN/pATarget dual plasmid gene editing system was finally constructed,and the E.coli BL21(DE3)strains maeA,murQ,alkB,flgK,yggE and sodC in E.coli BL21(DE3)strain were selected for gene knockdown test,and all the target genes were effectively edited,among which the knockdown efficiency of murQ reached 55%and the knockdown efficiency of the remaining five genes were 100%.After the completion of gene editing,pATarget series plasmids were tested for elimination,and the efficiency of plasmid elimination was nearly 100%,achieving strict regulation of the plasmid elimination step during the whole gene editing process.To test the versatility of the pEcmN/pATarget system,we selected two genes,yqhD and dhaT,from Klebsiella pneumoniae for knockdown and inserted the ldhA gene at the same time,and the editing efficiencies of the two genes were 73%and 20%,respectively,successfully achieving the integration of heterologous genes while knockdown,and the elimination efficiency of the pATarget series plasmids could reach 100%.In summary,we designed a high-throughput screening method for agRNAs well regulated by small molecules,screened agRNAs well regulated by theophylline,and fixed their targeting sequences for pTarget series plasmid elimination.The use of conformational regulation instead of transcriptional regulation avoids the problem of leakage of expression by transcriptional level regulation.A pEcmN/pATarget dual plasmid gene editing system was finally constructed,which enables efficient gene editing in E.coli and Klebsiella pneumoniae with broader potential strain versatility.The system may provide some reference for future improvements of genome editing methods in other species.