UC Berkeley

CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems are a form of prokaryotic adaptive immunity against invading viruses or plasmids. In these systems, Cas nuclease effectors recognize and cleave foreign nucleic acids containing sequences that are complementary to their associated CRISPR RNA (crRNA) to trigger degradation of targeted sequences. For successful CRISPR interference to occur, the sequences encoding crRNAs must be first acquired from foreign genetic material. These acquired DNA sequences are then transcribed into pre-crRNAs and processed into mature crRNAs that can guide interference against the specific invaders. The type V CRISPR-Cas12c (V-C) system is a compact system that has unique 3’ pre-crRNA processing activity and lacks a cas2 gene that was previously thought to be essential during the acquisition stage for all CRISPR systems. Although the initial activities of this system have been analyzed, the mechanisms by which this system acquires functional spacer, produces its guide RNA, and restricts viral infection are not fully defined. For my graduate work, I addressed the fundamental mechanisms of the type V-C system by determining the mechanisms of Cas12c-mediated pre-crRNA processing and interference. My work has revealed that Cas12c is an RNA-guided DNA binder that does not cleave target DNA. The repurposed RuvC domain of Cas12c exclusively processes pre-crRNA, and maturation of crRNA is essential for efficient Cas12c DNA targeting. Importantly, targeted Cas12c DNA binding can repress transcription and restrict phage infection, accomplishing antiviral interference without chemical attack on the invader. Additionally, I attempted to reconstitute type V-C adaptation in vivo in a heterologous host Escherichia coli with the goal of eventually identifying determinants for adaptational protospacer adjacent motif (PAM) selectivity and looking for evidence of primed type V-C adaptation. Together the investigation described here in this dissertation sheds light on the mechanisms of the compact V-C CRISPR system, allowing for comparison with more complex systems and provides insight into the evolutionary history of CRISPR systems and functions.