CRISPR Diversity Analysis Of Streptococcus Thermoophilus And Initial Construction Of Its CRISPR Editing Vector

S.thermophilus is an important lactic acid bacterium with both the starter exploitation value and the biological research value,and widely used in the production of fermented milk.CRISPR is a piece of clustered short-interspaced palindromic non-coding sequences,in which the invasive foreign DNA can be memorized by integrating new spacer sequences,while Cas protein is responsible for supporting CRISPR transcription products-cr RNA(CRISPR RNA)to splice foreign DNA.CRISPR-Cas system,based on the cooperation of the two segments,plays a role in resisting exogenous DNA infection.In recent years,the construction of the CRISPR editing system has become a hotspot research topic.This study takes S.thermophilus as the research object,and makes a systematic and in-depth study on the classification and diversity of its CRISPR-Cas system,and constructs two kinds of gene editing vectors based on conventional homologous recombination and CRISPR gene editing technology,in order to reveal the diversity of S.thermophilus CRISPR-Cas system and lay foundations for the CRISPR gene editing system for S.thermophilus.Firstly,in this study,CRISPRs of 22 S.thermophilus strains separated from traditional fermented milk in Chinese traditional pasturing area were identified,and the secondary structures of repeats as well as the distributions and sequence characteristics of spacers were also analyzed.The remaining strains contain four CRISPR types,except for 3-Q,CS21 and CS22 lacking CRISPR3.Prediction of CRISPR direct repeat(DR)secondary structures showed that all of four DRs could form stem-loop structures,but the stem or ring size together with the stabilities were different.Homology comparisons of spacers implied that most spacers are derived from partial sequences of exogenous DNA including various phages and plasmids.Of note,a large number of novel spacers were found in this study,indicating the unique phage environment they have undergone,especially 3-Q strain.In addition,the phylogenetic analysis of 22 S.thermophilus cas genes showed that the homology of the same cas gene in different strains was pretty high.However,the homology of the same cas gene in different CRISPRs is relatively low,which can provide references for studying the genetic relationships among strains.Next,in this study,three pairs of TCSs in S.thermophilus(hpk1/rr1,hpk2/rr2,and hpk5/rr5)were used as the target genes,and p RV300 recombinant vectors that could be used for S.thermophilus knockout were designed and constructed.They are able to lay foundations for the construction of S.thermophilus mutants.In this experiment,the upstream and downstream fragments of the target gene were respectively ligated to the empty p RV300 vectors.Ultimately,the recombinant plasmids p R1D(12),p H2D(25),p R2D(10),and p R5D(16)with the upstream and downstream fragments ligated on them were obtained.In addition,two vectors,p H1U(10)and p H5U(10),which are only ligated with upstream fragments of the target gene,were constructed.Finally,based on the CRISPR-Cas9 gene editing technology,this study designed and constructed an empty p Cas Sth vector for S.thermophilus gene editing,which contains an origin of replication,promoters,Cas9,resistance genes and the rep A gene used for plasmid elimination,so that it can be used for the knockout of specific genes after connecting the spacers and the homologous upstream and downstream arms of the target gene.After designing and synthesizing the spacer sequence according to the target gene and linking it to p Cas Sth,we obtained recombinant plasmids p H1(1)-spacer,p R1(9)-spacer,p H2(12)-spacer,p R2(4)-spacer,p H5(17)-spacer,p R5(1)-spacer.Next,in this experiment,the upstream and downstream fragments of the target gene were ligated to the p Cas Sth recombination vector containing the spacers,and a complete knockout vector p Cas Sth-H2 was obtained.These results lay the foundations for construction of CRISPR gene editing system of S.thermophilus.