CSA3A Of CRISPR/Cas System Activates The Transcription Of ACas Genes In SULFOLOBUS

Clustered regularly interspaced short palindromic repeats (CRISPRs) along with Cas proteins is a widespread system across bacteria and archaea that causes interference against foreign nucleic acids. CRISPR/Cas systems have three basic functions:process and incorporate spacer sequence derived from foreign nucleic acids; target and cleave foreign nucleic acids with acquired spacer sequence. In this study, using Sulfolobus islandicus homologous double crossover knockout system csa3a、cmr2a、cmr2b and csa2genes in Sulfolobus islandicus E233S CRISPR loci were successfully deleted from the genome. Validating the CRISPR targeting function of Δcsa3a、Δcmr2a、Δcmr2b mutants, we find deleting csa3a、cmr2a、cmr2b does not affect targeting foreign invasions for the host. The result indicates that csa3a is not participate in transcription and interference phase of CRISPR system and deletion either of cmr2two copys do not affect CRISPR targeting function.Based on the deletion mutant Acsa3a and complementary OEcsa3a, transcription of csal、casl、cas2and cas4genes related to CRISPR immune adaptation phase (named aCas genes) was studied by reverse transcription quantitative PCR. The result indicates Csa3a is a transcription activation protein for aCas genes. Afterwards, EMSA analysis also confirm that csal promoter do interact with Csa3a protein. Through truncating csal promoter to narrow the range of binding site, we position a piece of sequence with24bases as Csa3a binding site. By sequence alignement, we find one site similar to Csa3a binding site on the cas2promoter and two sites on the casl promoter, one located in the upstream of casl transcription initiation site, another downstream. EMSA validated casl probe and Csa3a protein can interact with each other. They can form two complexes with different mobility. According to the results, we put forward a hypothesis. First of all, Csa3a bind to casl downstream site slightly revealing specific base binding site, then isometric Csa3a protein bind to upstream site toughly and active casl transcription.In summary, this study provide a strong support for revealing the activation way of the transcriptional regulator Csa3a protein and theoretical mechanism of spacer acquisition in Sulfolobus CRISPR immune adaptation phase.