Research On The Mechanism Of Transcriptional Regulation Of Type I-A CRISPR-Cas System

CRISPR-Cas(Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated genes)system discovered in recent years provides the adaptive immunity against invading plasmids and viruses in prokaryotes.Sulfolobus islandicus REY15 A encodes Type I-A and Type III-B CRISPR-Cas system.The adaptation module of Type I-A takes up DNAs from invasive viruses and plasmids and incorporates them into the CRISPR arrays to serve as the memory providing adaptive defense against specific foreign invasive genetic elements.Spacer acquisition(adaptation)is the first and an important step,which requires Cas1,Cas2 and other proteins to process and integrate new spacers from non-self DNA into CRISPR arrays.However,proteins involved in this process and the mechanism of regulation remains poorly understood.Firstly,we demonstrated the transcriptional regulation mechanism of de novo spacer acquisition of Type I-A system in S.islandicus REY15 A.Csa3a regulator was found to specifically bind to the csa1 and cas1 promoters.Importantly,it was demonstrated that the transcription level of csa1,cas1,cas2 and cas4 was significantly enhanced in a csa3a-overexpression strain and,moreover,the Csa1 and Cas1 protein levels were up-regulated in this strain.Furthermore,we demonstrated the hyperactive uptake of unique spacers within both CRISPR loci in the presence of the csa3 a overexpression vector.The spacer acquisition process is dependent on the CCN PAM sequence and protospacer selection is random and non-directional.These results infer a regulation mechanism of de novo spacer acquisition that Csa3 a transcriptional regulator senses the presence of an invading genetic element and then activates the expression of adaptation genes leading to de novo spacer uptake from the invader.This study further demonstrated the transcriptional regulation mechanism of Csa3 a for intensive CRISPR adaptation and interference by activating the transcription of CRISPR RNA and the expression of DNA repair genes.Here,we found that de novo spacer acquisition required Csa1,Cas1,Cas2 and Cas4 proteins of the Sulfolobus islandicus Type I-A system,and disruption of genes implicated in cr RNA maturation or DNA interference led to a significant accumulation of acquired spacers,mainly derived from host genomic DNA.Transcriptome and proteome analyses showed that Csa3 a activated the expression of adaptation cas genes,CRISPR RNAs,and DNA repair genes,including her A helicase,nur A nuclease and DNA polymerase II genes.EMSA experiments indicated that Csa3 a specifically bound to the promoters of the above DNA repair genes in vitro,suggesting that they were directly activated by Csa3 a for adaptation.The Csa3 a regulator also specifically bound to the leader sequence to activate CRISPR RNA transcription in vivo.Overall,our research indicated a regulation mechanism of Type I-A CRISPR-Cas system in S.islandicus.The Csa3 a regulator couples transcriptional activation of CRISPR–Cas system adaptation and DNA repair genes for spacer adaptation and CRISPR RNA for efficient interference of invading genetic elements.