Clustered regularly interspaced short palindromic repeats (CRISPR) locus and the CRISPR associated (cas) genes constitute an inheritable immune system that defends against foreign invaders in most archaea and many bacteria. Currently, there are six different types of CRISPRCas systems (type I~Ⅵ) belonging to two classes, which are different from each other with their cas genes. Thermophilic crenarchaeon Sulfolobus islandicus Rey15A is a well-characterized archaeon, which contains one type I-A (Cascade) and two type III-B interference systems (denoted as SisCmr-α and SisCmr-β), with additional two CRISPR arrays of identical repeats interspaced by 114 spacers and 92 spacers, respectively. Several viruses could infect this archaeon, i.e., Sulfolobus monocaudavirus 1(SMV1), however, SMV1 virus is much resistant to active S. islandicus CRISPR-Cas systems, in spite of the existence of multiple CRISPR-Cas systems and a matching spacer from the host CRISPR array in this organism, nevertheless, the tolerance of SMV1 to CRISPR immunity has not been tested in this archaeon. Furthermore, from the host perspective, autoimmunity avoidance requires a sufficient self vs. non-self discrimination strategy, and it is known that this autoimmunity for type III CRISPR-Cas system occurs at the RNA level: mismatches between the 5’-repeat tag of crRNAs and the corresponding 3’-flanking sequences of target transcripts (anti-tag), however, it remained elusive as how to achieve this stringent autoimmunity avoidance.
For the first part of this thesis, S. islandicus Rey15A strains carrying plasmid-borne mini-CRISPR arrays targeting SMV1 gene gp11 that is coding a coat protein, and the antiviral immunity was programmed from these strains constitutively expressing from either type I-A, or type III-B, or both type I-A and type III-B CRISPR-Cas systems against SMV1 infection. The tolerance of SMV1 to the host CRISPR-Cas immunity was investigated by collecting the infected cultures at different time course points. The results showed that type III-B CRISPR-Cas systems license robust resistance against SMV1, while the type I-A CRISPR immunity could not suppress viral proliferation, but the virus tolerance does not result from escape mutations in the PAM or protospacer sequences to type I-A CRISPR immunity.
In the second part of this thesis, I am interested in investigating how the target RNA (tgRNA) activates the type III-B antiviral immunity and how self-immunity is avoided using Cmr-α, one S.islandicus type III-B CRISPR-Cas system. Specifically, an array of tgRNAs was generated and tested their capability of activating the Cmr-α DNase and cyclic oligoadenylate (cOA) synthesis. Additionally, amino acid residues interacting with RNA were subjected to site-directed mutagenesis, and Cmr-α crRNPs carrying different Cmr3α mutants were purified and characterized. These results indicated that each of the four available nucleobases (4th ~ 7th) of crRNA plays a critical role in avoiding the autoimmunity, while the 7th nucleobase is more significant than the others, and Cmr3α regulates type III-B Cmr-α activities upon varying degrees of tag complementarity.
Collectively, virus challenge experiments presented here directly illustrated the arms race between the antiviral immunity from an archaeal host and the targeting viral genome, and for type III-B CRISPR-Cas systems, Cmr3α regulates the self vs. non-self discrimination, possibly because Cmr3α could interact with either crRNA or tgRNA to facilitate the immune response or avoid autoimmunity.