Study Of A Gene Editing Technique—CRISPR/Cas9 In Lactobacillus Plantarum

Lactobacillus is a kind of microorganism closely related to human health.With the increasing number of lactic acid bacteria that complete genome-wide sequencing,the research of functional genes is becoming more and more important.CRISPR/Cas9——a new gene editing technology can be used to knock out or modify the genome accurately and efficiently due to its simple experiment,easy operation and time saving.It has been rapidly developed and widely used in different species.Glucosamine-6-phosphate synthase(GlmS)is a catalytic enzyme of the aminohexose metabolic pathway(HBP).The final product is N-acetyl-D-glucosamine(GlcNAc)and glucosamine(GlcN).The glmSl gene is an important gene encoding GlmS,and the glmS1 gene-deficient bacteria will quickly inactivate and die in the environment without GlcN and GlcNAc.In this study,glmSl gene was selected as the target gene of CRISPR/Cas9 gene editing technology,and the Lactobacillus plantarum WCFS1 gene editing system was constructed and validated,which laid a foundation for further editing of functional genes of lactic acid bacteria.In order to construct and validate the Lactobacillus plantarum WCFS1 CRISPR/Cas9 gene editing system,in this study,we used glmSl gene as the target gene of gene editing,laying a foundation for further editing of functional genes in Lactobacillus.We first successfully constructed two plasmids,pMT019 containing Cas9 and recT genes,ptracrRNA containing tracrRNA.Subsequently,the expression of Cas9 protein was optimized,we constructed three Cas9 expression-optimized plasmids.The expression of Cas9 gene and recT gene was highest in pMT023 plasmid by Real-Time PCR.When the pMT023 plasmid was induced for 1h,the relative expression of Cas9 gene and recT gene was up-reg?Lated by 29.58-fold and 26.11-fold respectively.At the same time,the preparation process of WCFS1 competent cells was optimized,and the optimal preparation condition was OD600=0.3,and the inducer SppIP was added at 30? for 1h.Finally,MT+GlcNAc(Cm1O+Ery10)and MT(Cm10+Ery10)were used to screen and verify.The selected strains are extracted from the whole genome and sequenced,and if correctly sequenced,the WCFS1-?glmS1 strain is obtained.However,the target recombinant has not been screened at present.