Venue: Polo Ferrari 2, via Sommarive 9 (Povo) – Room B104
- Virginijus Siksnys - Vilnius University/Institute of Biotechnology, Vilnius, Lithuania
Bacteriophages infect bacteria in order to reproduce and often represent a lethal threat to bacteria. To counter fight phage attacks bacteria developed multiple defense barriers that interfere with nearly every step of virus life cycle. These multiple defense barriers constitute a primitive immune system that protects bacteria against invading viruses. In prokaryotes restriction-modification systems functions as innate immune system that uses methyl-tag to discriminate between self and non-self DNA. On the other hand, CRISPR-Cas system functions as an adaptive and heritable arm of the immune system. Deciphering of the molecular mechanisms of CRISPR-Cas immunity enabled development of novel tools for targeted genome engineering. Class2 effector nucleases that provide CRISPR-Cas immunity are abundant in prokaryotes. To explore this largely uncharacterized diversity of the Class2 nucleases for genome editing applications, we established a phylogeny-guided bioinformatic approach and developed biochemical screens for the rapid identification and characterization of the PAM and guide RNA requirements of new nucleases. This approach permitted the rapid characterization of more than 50 Cas9 orthologs with diverse PAM sequence requirements. Next, using the same biochemical screen we detected PAM and gRNA requirements for several miniature CRISPR-associated proteins coming from the Cas14 family and uncharacterized Type V CRISPR-Cas subtypes. Taken together, our results demonstrate that Class2 effector nucleases provide a rich source of biochemical and biophysical diversity that may be beneficial for genome editing.