Professor Virginijus Siksnys, Vilnius University, Vilniu.
CRISPR-Cas systems in prokaryotes provide an adaptive immunity against invading nucleic acids. CRISPR-Cas systems are very diverse and are categorized into five main types depending on the number and arrangement of Cas genes. Each Type is specified by the so-called signature protein, which is conserved in the particular Type, for example, Cas9 in Type II and Cas10 in Type III. In the Type II systems Cas9-guide RNA complex alone provides immunity against invading DNA. Cas9 protein guided by crRNA binds to the target sequence and Cas9 protein cuts both DNA strands. The initiation of the target site binding by the Cas9 critically depends on a short sequence motif called PAM located in the vicinity of the target sequence complimentary to crRNA. In the gene editing experiments PAM sequence requirement may limit target site selection if genome specific target sites are desired. Cas9 orthologues with distinct PAM specificities may help expand the sequence space targeted by Cas9. To explore the space of Cas9 orthologs, we established a phylogeny-guided bioinformatics approach coupled with a rapid biochemical screen that allowed to identify new Cas9 variants. In contrast to Cas9 that acts as a stand-alone protein that cuts invading DNA, the Type III CRISPR-Cas system provide immunity against invading nucleic acids through the coordinated degradation of transcriptionally-active DNA and its transcripts. Three catalytic domains are required to mount an immune response. Ribonuclease domain guided by crRNA recognizes viral RNA transcript and initiates its degradation. The deoxyribonuclease domain of Cas10 becomes activated upon target RNA binding and launches simultaneous degradation of DNA template. We recently showed that target RNA binding also triggers Cas10 Palm domain dependent synthesis of cyclic oligoadenylates (cOA) from ATP. We further showed that cOAs act as signaling molecules that couple Type III immunity and Csm6 ribonuclease thereby demonstrating a novel cyclic oligonucleotide-based signaling pathway in prokaryotic antiviral defense systems.