Diversity Analysis And PAM Verification Of Lactobacillus Paracasei CRISPR-Cas System

CRISPR-Cas system is an adaptive immune system of bacteria that can target characteristic fragments through nucleases to resist foreign genes invasion.It has been developed as a widely used gene editing tool with the gradual analysis of the resistance mechanism to foreign genes.Lactobacillus paracasei is a widely used lactic acid bacteria with important scientific value.At present,the application of the CRISPR system in lactic acid bacteria has certain limitations,but the prospects are broad.In our research,bioinformatics analysis and biological experiments were used to carry out an in-depth analysis of CRISPR system and focus on the elements of the CRISPR interference stage in L.paracasei.Finally,the PAM sequence recognized by the most widespread Cas9 protein were predicted and validated.The main conclusions of this article are as follows:First,the CRISPR systems of 58 L.paracasei strains in the NCBI database were searched and located,and repeats and spacers were analyzed.The results showed that the incidence of CRISPR systems was about 43%,and most of them were type-? CRISPR systems.Within the same subtype,the length,nucleotide sequence and secondary structure of the repeats were all relatively conserved.CRISPR systems could be divided into 5 unique type-I CRISPR patterns and 16 unique type-? CRISPR patterns by aligning and typing the spacer sequences.The search results of the protospacer sequences showed that the phage genes targeted by the spacer were mostly the genes related to phage replication and survival.Second,the in-depth analysis of the interference phase elements in the type-? CRISPR system is particularly important for the development of gene editing tools.The results showed that the position of tracrRNA was relatively conserved,all of which were located between cas9 and cas1 gene,and the transcription direction was opposite to the cas gene.A total of six unique tracrRNA sequences were found,they could form a stable secondary structure,and the differences mainly occurred near the terminator.By analyzing the flanks of the protospacer sequence,the PAM sequence recognized by the Cas9 protein was predicted.Most proteins recognized the A-rich sequence.Finally,the Cas9 protein,which is the most widespread in L.paracasei,was selected as the research object,and the PAM sequence was verified by the double fluorescence reporting system experiment.The Cas9 protein could be expressed normally in HEK293 T cells after codon optimization.CGAA was used as a linker to connect crRNA and tracrRNA,and the last hairpin of tracrRNA was removed.The designed sgRNA and optimized Cas9 protein were cloned into the same vector for expression.PAM library with a total of 25 sequences was established according to PAM prediction results,the results showed that 5'-TCAAAA-3' and 5'-TGTAAA-3' were effective PAM sequences recognized by lpCas9.Our research provided a large amount of data for CRISPR research in L.paracasei,which is of great significance for the screening of commercial strains,transformation of excellent traits in strains and development of gene editing tools.