CRISPR–Cas mediated cleavage of invading nucleic acids (360G-Wellcome-208604_Z_17_Z)

My lab focuses on structural and functional studies of the CRISPR-Cas system and prokaryotic Ago, anti-virus defense systems mediated by small RNA or DNA. We are interested in spacer acquisition in the CRISPR-Cas system and have shown how Cas1-Cas2 captures foreign DNA. We plan to solve the structure of the complete integrase complex to understand how it binds target DNA and inserts protospacers into the CRISPR locus. Cas4 is also involved in spacer acquisition in some CRISPR types. We will identify the structural basis of acquisition in Cas4-containing systems and elucidate the roles of Cas4. In addition, we plan to identify the unknown RNase that trims crRNA from its 3’-end, and address the molecular mechanism of RNA-induced silencing in type III systems. Our previous study suggested that bacteria have a DNA interference system. We demonstrated the molecular mechanism of DNA-guided DNA interference using structural and functional studies. We will now focus on the guide DNA biogenesis pathway. Ago also has potential to be a genome editing tool due to its sequence-specific DNA cleavage activity. We will optimize the TtAgo sequence to screen for mutants which have high cleavage activity at 37°C.