Genetic Studies On CRISPR-Cas Functions In Antiviral Defense In Sulfolobus Islandicus

Many bacteria and most archaea are equipped with adaptive immune system called CRISPR-Cas(clustered regularly interspaced short palindromic repeat–CRISPRassociated). There are three major types of CRISPR-Cas systems, type I, II and III, including at least 11 subtypes. Sulfolobus islandicus Rey15 A encodes a type I-A(Cascade) and two type III-B(Cmr-α and Cmr-β) CRISPR-Cas systems where the cas gene functions have not been dissected yet. Before this Ph D project started, the by then already characterized Cmr complexes were implicated in in vitro RNA cleavage but in vivo RNA interference activity was not demonstrated for any of them. In this study, the functions of each type I Cas protein in CRISPR-Cas targeting, as well as the in vivo RNA interference activity of Cmr systems have been unambiguously demonstrated for S. islandicus.Knockouts of individual type I-A cas genes or each of other cas cassettes were constructed. Characterization of those mutants demonstrated that the Cas7, Cas5, Cas6 and Cas3 proteins are essential for the type I-A DNA interference activity, whereas Csa5, along with other Cas modules, is dispensable for the interference. Cas6 is found to be the only enzyme that is responsible for pre-cr RNA processing while Cas7, Cas5 and Cas3 also contribute to this process. Cas7 and Cas5 are required for stabilizing the processing intermediates and the mature cr RNAs, respectively, while depleting of Cas3 results in the accumulation of processing intermediates but the mechanism involved is currently unknown.An RNA interference assay has been developed in which a plasmid was constructed to express an artificial CRISPR(p AC) that carries a spacer derived from the lac S gene encoding a β-glycosidase. The cr RNA produced from the plasmid-borne AC reprogramed the targeting activity of the Cmr complexes on the m RNA of lac S. The targeting activity was investigated by measuring the residual β-glycosidase activity in the cellular extracts of p AC transformants. Testing RNA targeting activity in the knockouts of each Cmr locus and both loci showed that RNA targeting was lost only in the double Cmr loci mutant, indicating that each Cmr possesses RNA targeting activity in S. islandicus Rey15 A.Oligonucleotides carrying mutations at different positions of the spacer were designed and used to construct p AC plasmids to test their effects on RNA targeting activity. This identified a trinucleotide motif, located at the 3’ end of a cr RNA, is crucial for the RNA interference activity. Finally, cleavage of lac S m RNA was confirmed by evaluating the amount of uncut m RNA via real-time quantitative PCR(q PCT) and the cleavage sites were mapped within the S1 protospacer by rapid amplification of c DNA ends(RACE). Cmr-α and Cmr-β cleave target RNAs at defined positions and UA-like sites, respectively.