Primary Studies On Both The Type I-B-svi CRISPR-Cas System In Streptomyces Virginiae IBL14 And The Self-targeting Gene Editing To The Genes Involved In Penicillin Metabolism

Streptomyces virginiae IBL14(Streptomyces virginiae IBL14)is a Gram-positive strain discovered and isolated from sludge around pharmaceutical factory by our laboratory,which can efficiently transform and degrade various steroidal compounds,such as cortisol,cholesterol,ostrone and progesterone.In a previous antibiotic resistance screening study,IBL14 strain were found to be able to grow in medium containing higher concentrations of penicillin,which is typically produced by fungi.In order to reveal this phenomenon,the whole genome was sequenced by Illumina Hiseq2000 platform sequencing,and the data were filtered,assembled and evaluated.Then,the ORFs were annotated about its general functions by GO,KEGG and Swiss-Prot,as well as NR and COG databases.The results showed that seven genes related to penicillin metabolism were identified as sviipe,penicillin amidase(svipam1 and svipam2)and ?-lactamase(sviblal,svibla2,svibla3,svibla4).CRISPR-Cas system is an immune defense mechanism formed in the long-term evolution of prokaryotes.It is widely found in archaea and eubacteria.The CRISPR site is a multistage RS structure consisting of a series of highly conserved repeats and spacer sequences.A series of CRISPR association proteins(cas proteins)are present in the around of the site.According to the difference of Cas protein,they were divided into two classes and six types.Although there are nearly 2,000 species contain CRISPR-Cas system,but only CRISPR-Cas9 and Cpfl plays a gene editing role,which is currently the most mature application of a system.As a new gene editing tool,CRISPR-Cas system overcomes the shortcomings of traditional technology,such as cumbersome,time-consuming and costly,making gene editing more precise,efficient and fast.The whole genome analysis of IBL14 showed that CRISPR was present in chromosome.Cas protein,Cas7,Cas5,Cas3,Cas4,Cas1 and Cas2 were found on the chromosome,and these proteins were located on the same operon.Among them,Cas3 has DNA cutting effect.Therefore,the CRISPR-Cas system is named I-B-svi CRISPR-Cas system.In this paper,we first edited the gene of penicillin metabolism-related enzymes,including insertion of sviipe,single-knockout of svipam and svibla,double-knockout of svipam1-sviipe/sviblal by the CRISPR-Cas IB-SV14 system of IBL14 strain.At the same time,different PAM sites and homologous arm lengths were also selected.The rate of gene edited when it comes to the sequence of PAM,ttc is higher than tcc.The longer homologous arm also shows the higher rate of gene edited.But,the sequence of repeat shows the same result of gene edited.Then,the simple penicillin resistance test of the penicillin metabolism-related enzyme knockout strain was carried out to verify the correctness of the gene knockout and the function of the lactamase gene.In the process,only editing templatets and target constructs were designed to construct a gene editing plasmid containing the single-stranded RNA(RNA)and gene editing template,and then transformed into IBL14.The recombinants were amplified by PCR.The agarose gel electrophoresis showed that the bands of the PCR products had been reduced or increased to the expected number of bases.The PCR products were sequenced analysis.If the sequencing results showed that the sequence is the same with the sequence expected,indicating that the gene has been successfully knocked out.The method is simple,time-consuming and efficient,which not only provides a new direction for gene editing,but also provides a new idea and method for gene function identification.