Functional Analysis Of The Nucleic Acid Interference Core Domains In Cmr-? Complex Of Type ?-B CRISPR-Cas System In Sulfolobus Islandicus

Viruses and their hosts have established a co-evolutionary relationship over a long evolutionary period during which host cells,in order to defend themselves against viral invasions,have evolved various defense mechanisms whereas viruses have gained various molecular mechanisms to evade their host's defenses in order to survive.The CRISPR-Cas system encoded by bacteria and archaea and the anti-CRISPR protein(Acr)encoded by viruses are a typical result of their coevolution.Yet we have had very little understanding of the involved mechanisms as well as their coevolution.The aims of this study was(1)to reveal the functions of the two key conserved functional domains of Cmr-? in the plasmid interference and viral interference activities of the antiviral system,and(2)to systematically localize the key genes that escape or hinder the ?-B Cmr-? subtype antiviral response to archaeal viruses SMV1 and STSV2 to explore the mechanism of virus-host co-evolution.Main results obtained in this study are as following:First,this project was set to employ a mini CRISPR plasmid assay to study the influence of the HD structure domain of Cmr2(Cas10)protein in Sulfolobus islandicus as well as its DD motif of the Palm domain on DNA interference.Both HD and Palm domains were found to have an effect on the antiviral activity by the ?-B immune system,as reported by other researches.However,the influence of the Cas10 Palm domain on the ?-B immunity in S.islandicus was found to be 100-fold lower than that exerted from its HD domain.Based on this study,a CRISPR plasmid-based virus assay was established in which mini CRISPR plasmids targeting the genome of a virus were constructed and introduced into the archaeal host and infection of the virus was then employed to investigate whether three different nucleic acid interference activities of the type ?-B Cmr system could efficiently inhibit viral replication or viral growth and reproduction processes,respectively.Targeting various viral genes has revealed that the signal transduction pathway initiated by c OA is essential for virus clearance whereas the ss DNA cleavage is not.In addition,the ?-B immunity exhibits differential effects on early viral gene targeting and late gene targeting.Next,CRISPR plasmids were used to systematically screen viral genes in order to identify the anti ?-B genes(acr ?-B)present in the archaeal viruses SMV1 and STSV2.The selection criterion that any viral genes that facilitate CRIPSR plasmid transformation efficiency would be considered as candidate acr ?-B genes.A few candidates were identified,providing a basis for further characterization of their possible function in the anti-CRISPR activity as well as the involved mechanisms.