Research About In Vivo Nucleic Acid Targeting Of CRISPR-Cas Cmr-? Subtype In Sulfolobus Islandicus

CRISPR-Cas systems are an acquired immune system widely distributed in bacteria(40%)and archaea(90%),which are characterized by the division of repeated sequences from foreign nucleic acid by various spacers.Ribonucleoprotein complexes are guided by the base pairing of mature crRNAs and targets before cutting foreign nucleic acid.CRISPR-Cas system can be characterized into 2 classes,6 types and 19 subtypes according to the different structures of Cas complexes' core proteins during nucleic acid targeting.This immune system comprises a three-step process: acquisition of the spacer from foreign DNA;maturation of the transcripted CRISPR arrays into crRNAs;and comparison of the ribonucleoprotein complexes before nucleic acid targeting by the guide of base pairing between crRNA and target.Only Type III CRISPR-Cas system can target DNA and RNA.It has been reported in vitro that Cas10 cleaves ssDNA activated by the base pairing between cr RNA and target mRNA,while Cmr4 cleaves target mRNA every 6nt.Although the mechanism of Type III nucleic acid targeting is clear,there are still some problems,such as whether the length of crRNA and the deletion of the cmr gene would reduce the RNA targeting.Meanwhile,the effect of the nuclease out of the CRISPR-Cas systems during nucleic acid targeting is still unknown.Sulfolobus islandicus E233 S strain has 2 kinds of CRISPR-Cas systems,Type I-A and Type III-B(Cmr-? and Cmr-?).Type I-A has only DNA targeting activity,Type III-B Cmr-? has only RNA targeting activity,while Type III-B Cmr-? has both.With the use of S.islandicus ?cmr-? strain,this thesis has done some biochemical analysis and constructed few genotypes with the cmr-? gene-deleted strains.Some results are shown as follow:(1)It's reported that the repairing of nuclease Csx1 can recover the DNA targeting activity of Cmr-? system in the large deletion strain(?98kb E233S),which has no DNA targeting activity of Cmr-? system.But we found out that the strain still have nucleic acid targeting activity without csx1.Therefore we speculate that there are other nucleases which are involved in DNA targeting of Cmr-? system.(2)We discovered that the deletion of some Cas proteins has no impact on Cmr-? system's RNA interference from the result of RNA targeting activity of cmr6 gene-deleted strains in the background of ?cmr-? E233 S.(3)After transferring mini-CRISPR(the simplest CRISPR array)with designed different lengths spacers plasmids into the ?cmr-? E233 strain,we measured the RNA targeting activity of every treatment.We found that the shortest effective spacer is 32nt(wildtype is 40nt)and the overlong spacer cannot affect the RNA targeting activity of Cmr-? system.(4)After measuring the RNA targeting activity of the various mutations of spacer,we concluded that the seed sequence of Cmr-? system consists in 25nt~30nt of spacer.Seems that some nuclease is necessary for in vivo nucleic acid targeting by Cmr-? system of Sulfolobus islandicus,but nucleases Csx1 and Cmr subunit Cmr6 is unnecessary.And mismatch of seed sequence of crRNA would reduce the RNA targeting activity.