| CRISPR(clustered regularly interspaced short palindromic repeats)-Cas(CRISPR associated)genome editing technology has been rapidly developed in recent years,enabling rapid gene editing in a wide range of organisms.CRISPR/SpyCas9-based gene editing in Streptococcus pyogenes has been widely used in eukaryotes such as mammalian cells,plants,and yeast,but less in prokaryotes,especially in lactic acid bacteria.Lactobacillus is the largest genus in the lactic acid bacteria family,and its members are both similar and exhibit extraordinary diversity in genetic and evolutionary backgrounds,and the most widely used CRISPR/SpyCas9 gene editing technology is not well generalized when applied to the genus Lactobacillus.The development of a gene editing system for Lactobacillus-derivedCas9 will hopefully avoid the shortcomings of SpyCas9 for Lactobacillus applications.In this paper,Lactobacillus paracasei(L.paracasei)was selected as the target of this study,and the PAM sequence recognized by the most widespread LpCas9 protein in L.paracasei was experimentally verified based on the pre-laboratory stage,and the L.paracasei-derived CRISPR/LpCas9 gene editing system,which is expected to be a powerful gene editing tool for Lactobacillus paracasei.The main findings of this paper are as follows.1.PAM sequences recognized by LpCas9 protein were identified: the most widespreadCas9 direct homolog of L.paracasei,LpCas9,was used as the target of the study,and the candidate PAM library preestablished in the lab was experimentally validated by a dual fluorescence reporter system combined with flow cytometry.The flow cytometry results showed that 5’-TCAAAA-3’ and 5’-TGTAAA-3’ were the valid PAM sequences recognized by LpCas9.2.A CRISPR-LpCas9 gene editing system was established and the editing efficiency was evaluated.The CRISPR/LpCas9 gene editing system was established based on the experimentally validated PAM sequences recognized by LpCas9,and the efficiency of the CRISPR-LpCas9 gene editing system was evaluated using HEK293 T cells as the evaluation platform,and the experimental results showed that the wild-type LpCas9 editing efficiency was slightly lower than that of the now widely used wild-type SpyCas9 editing.The efficiency of wild-type LpCas9 gene editing was about 55.8%,but the predicted off-target sites of wild-type LpCas9 were much lower than those of wild-type SpyCas9.3.Broaden the CRISPR-LpCas9 PAM recognition spectrum.To address the shortcoming that the PAM sequence recognized by wild-type LpCas9 is too long,resulting in a smaller targeting genome range.The wild-type LpCas9 was optimized by replacing the PI(PAM Interaction,PI)structural domain of LpCas9 to achieve the replacement of the PAM sequence recognized by LpCas9,which increasing the target range of the CRISPR-LpCas9 exogenous gene editing system.The editing efficiency of mutanttype LpCas9 was about 54.3%.4.Assessment of L.paracasei CRISPR-Cas9 resistance to exogenous genetic elements: L.paracasei LC2 W strain was used as the study target,and three representative Spacer sequences on the CRISPR array on the genome of L.paracasei LC2 W strain were selected to evaluate the strain using plasmid interference experiments.The ability of the endogenous CRISPR/LpCas9 system to resist exogenous genetic elements was assessed using plasmid interference assays.The experimental results showed that the CRISPR-Cas9 system of L.paracasei LC2 W had strong resistance to exogenous genetic elements as a whole,with the most recently acquired Spacer sequences having the strongest resistance to exogenous genetic elements.This thesis focuses on the newly discovered LpCas9 protein.By analyzing the PAM sequence recognized by the protein to activate the nuclease function of the protein,a CRISPR/LpCas9 gene editing system is constructed,which can solve the current application of SpyCas9 in Lactobacillus paracasei.The above limitation is expected to become an efficient and rapid gene editing tool for Lactobacillus paracasei.The CRISPR/LpCas9 gene editing system established in this study will promote the rapid development of lactic acid bacteria genetic selection and breeding,and has broad application prospects. |
