Yuvaraj Bhoobalan-Chitty:
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The popularity associated with CRISPR-Cas has naturally resulted in increased attention to the identification and characterization of anti-CRISPR proteins that inhibit CRISPR-Cas systems. Although studies on Acrs are in infantile stage, to date all identified Acrs belong to the bacterial domain. The substantially higher distribution of CRISPR-Cas systems across archaea in comparison to bacteria suggests an abundance of archaeal mobile genetic elements encoded Acrs. The purpose of this work was to study the immune systems in Sulfolobus and more importantly to study counteracting strategies evolved by viruses specifically archaeal lytic viruses. Existence of efficient gene manipulation systems in the hyperthermophilic acidophile Sulfolobus and the lytic nature of its virus made it the most suitable virus-host system to study in archaea.
This thesis describes the anti-CRISPR proteins identified from the virus SIRV2 and SIRV3 inhibiting subtype I-D and two subtype III-B CRISPR-Cas systems in Sulfolobus islandicus LAL14/1. We first isolated viruses susceptible to host immunity and then identified the gene necessary to restore viral infectivity by impeding the CRISPR-Cas systems. Using this approach we identified the first anti- CRISPR from archaea domain and also the first against the subtype I-D system. The Acr described, AcrID1, bound the large subunit (Cas10d) of the subtype I-D system.
Next, using this Acr as marker, a gene manipulation system was developed for Sulfolobus viruses along with a CRISPR based marker less modification system. This enabled us to remove all genetic entities not involved in virus propagation to obtain a minimal virus able to propagate only in the absence of host immune systems. Based on this gene manipulation system we were able to discover another Acr, AcrIIIB1, the first anti-CRISPR among both bacteria and archaea to inhibit type III-B system. Interestingly AcrIIIB1 was able to inhibit both subtype III-B systems in S. islandicus LAL14/1, with the type III-B systems differentiated based on their repeat sequence specificity. AcrIIIB1 inhibition was by direct binding to the type III-B complex and specific to spacers targeting late-expressed genes.
Toxin-Antitoxin systems are a group of functionally co-dependent loci ubiquitous in both bacterial and archaeal genomes. Among the abundant Toxin-Antitoxins in Sulfolobus we identified 4 non-toxic VapCs, among these we characterized an unusual TA pair with an RNase antitoxin with a PIN domain fold and a toxin with a DNA binding domain, requiring it to be re-classified as a type V TA system.