Study On The Mechanisms Of CRISPR Immunity Adaptation And Interference Regulated By Sulfolobus Csa3 Family Proteins

CRISPR-Cas is an acquired immune system for prokaryotes to resist invading genetic elements,such as plasmids and viruses.Hyperthermophilic archaeon Sulfolobus islandicus REY15A encodes a subtype I-A and two subtype III-B?Cmr-?and Cmr-??CRISPR-Cas systems.The Csa3?Csa3a and Csa3b?family proteins are the only known regulatory factors associated with the CRISPR-Cas in archaea.Functional studies have shown that Csa3a activates CRISPR immune adaptation and Csa3b acts as an inhibitor of type I-A interference gene cassette.This study focused on the integrated pathway composed of Csa3a and Csa3b to regulate the immune adaptation of CRISPR and the regulatory mechanism of the subtype III-B CRISPR system RNA interference involving Csa3b.Studies have shown that the overexpression of csa3b seriously inhibited cell growth.The comparative transcriptome analysis of csa3b overexpression strain and wild-type strain shows that cmr genes of III-B system and other genes were up-regulated.DNase I Footprinting and Surface plasmon resonance?SPR?experiments confirmed that Csa3b protein specifically binds to the promoter of the adaptation cas operon and the promoter of Cmr-?and Cmr-?operon of subtype III-B.Compared with the wild-type strain,deletion of csa3b gene triggers new spacer acquisition from shuttle plasmid p Se SD,and the deletion strain hyperactively adapts spacers from virus after infected with the Sulfolobus virus STSV2.The results indicated that Csa3b inhibite the immune adaptation of CRISPR.Compared with the wild type,the RNA interference ability of the subtype III-B Cmr systems are significantly reduced in the csa3b deletion strain,demonstrating that Csa3b could activate Cmr-mediated RNA interference.In addition,DNA replication and repair genes?e.g.cdc6-2,tfb3?,DNA damage response genes?e.g.Ups and Ced system?were significantly up-regulated in csa3b overexpression strain,suggesting that Csa3b may play a role as a global transcription factor in cells.Cyclic oligoadenylates?c OA?have been found to play an important role in the regulation of CRISPR interference.c OA acts as signaling molecules that specifically binds to the CARF domain of the Csm6/Csx1 superfamily proteins then allosterically activates the HEPN for RNA degradation.However,the regulatory role of signaling molecules in CRISPR adaptation has not been reported.Csa3,the only CARF family links to type I system and subtype I-A in particular,lacks a C-terminal catalytic effector domain,but is fused with HTH DNA binding domain.Here,we show that Csa3b protein binds to an oligonucleotide 5'-CAAAA-3'?CA₄?in vitro and its CARF domain mutation reduces the binding ability to CA₄,and oligonucleic acid CA₄ can promote the binding ability of Csa3b protein to csa1 promoter.Moreover,overexpression of csa3b seriously retards cell growth,while overexpression of its CARF mutant could not cause growth delay.Meanwhile,Csa3a protein binds c OAs and oligonucleic acid CA₄ in vitro.Mmutations at CARF domain of Csa3a protein affect its binding with these signals in vitro and could not induce the expression of the adaptation cas genes,and therefore could not stimulate CRISPR immune adaptation in vivo.These results suggest that the CARF domain and binding of signaling molecules to the CARF domain are critical to the regulation of Csa3 proteins.In summary,this study revealed an allosteric model of CRISPR immune adaptation and interference mediated by Csa3a and Csa3b transcription factors in S.islandicus.