Immunology and Microbiology Seminar: CRISPR-Cas mining: from novel Cas9s to signaling molecules
Speaker: Virginijus Siksnys
Speaker address: Institute of Biotechnology, Vilnius University, Lithuania
Host: Ute Römling
Virginijus Siksnys studied chemistry at Vilnius University and obtained his Ph.D. in 1982 from Moscow State University before returning to Vilnius, where he moved through different research ranks at the Institute of Applied Enzymology/Institute of Biotechnology. He holds the position of Professor of Vilnius University since 2002 and is Chief scientist/Department head at the Institute of Biotechnology of Vilnius University. Dr. Siksnys has made a major and sustained contribution to the understanding of the structure and function of restriction enzymes. Together with colleagues he contributed nearly one third of restriction enzymes structures available at PDB. Since 2007 he switched to mechanistic studies of CRISPR-Cas antiviral defense systems. His research on the CRISPR-Cas has had a major impact on the field. His studies of the Cas9 protein paved the way for development of novel tools for genome editing applications. He is a member of Lithuania Academy of Sciences and was elected to the EMBO associate membership and Federation of European Microbiological societies (FEMS). His work has been recognized with several awards including the Lithuania National Science Prize, Warren Alpert Prize (2016) and Novozymes Prize (2017).
"CRISPR-Cas mining: from novel Cas9s to signaling molecules"
Institute of Biotechnology, Vilnius University, Vilnius
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
Ref: Kazlauskiene, M., Kostiuk, G., Venclovas, Č., Tamulaitis, G., Siksnys, V. Science 357, 605 (2017)Contact person: Ute Römling