The CRISPR-Cas System Found In Streptomyces Viginiae IBL-14 And Its Gene Editing Procedures

CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRJSPR associated) system widely existed in bacteria and archaea is an immunity system formed in the long term of evolutionary process. Similar to restriction modification of some other defense systems, it is based on the self-non-self discrimination principle, but the act against invading genetic elements are mediated by guide-RNA. Now, CRISPR-Cas system has become into a powerful gene editing tool by designing guide RNA(s) and editing template(s), which recruit the Cas proteins to identify targeting sites and activate homologous recombination of repair mechanisms inside cells.Streptomyces Virginiae IBL14 was isolated from waste sludge from a steroid manfacture by our laboratory and was found to degrade a range of steroidal compounds. In this study, we carrieed out the BLAST of the complete genome sequence of the strain IBL14 and found 18 candidate CRISPR loci. Among them, three are identified as CRISPR sites. Also, seven Cas proteins named as Cas6, DevR (Cas7), Cas5, Cas3, Cas4, Cas 1, and Cas 2 in sequence between the CRISPR loci of SV01 and SV02 were found. This CRISPR-Cas systems belonging to type I-B was named CRISPR I-SV14B system.In this study, the gene svm016 in IBL14 was selected as a target gene, also the nucleotide sequence TAC selected as PAM (protospacer adjacent motif) site according to the features of CRISPR-Cas system (type I-B). The guide DNA and homologous arms we designed were respectively integrated into plasmid pKC1139. Finally we transformed the recombinatnt plasmid(s) to the protoplasts of the strain IBL14. Analysis results show that the svu016 gene was successfully knocked out. In the meantime, the knockout of svu016 gene in a traditional way was carried out by transforming pKC1139 harboring the homologous arms and chloramphenicol resistance gene, into the strain IBL14. After a few days of culture, we got one clone of recombinant IBL14 strain in which svu016 gene was knocked out. Experimental results show that, the use of CRISPR-Cas genes editing system efficiency was above 70%, while the efficiency of the conventional method of editing was only about 3%.The CRISPR-Cas Ⅰ-SV14B system can be used to genetic editing, and probably become a powerful genetic editing tool like CRISPR-Cas Ⅱ.