Speaker: Phong Phan

Title: Arms Race Between Bacteria and Virus: Which Side Are You On?

Abstract: Type I-E CRISPR (clustered regularly interspaced short palindromic repeat) – Cas (CRISPR-associated protein) systems allow bacteria to fight off bacteriophage and other invader threats through an RNA-guided DNA “interference” mechanism. Target binding is initiated at the PAM (protospacer adjacent motif) site, which triggers DNA unwinding and formation of an RNA-DNA hybrid duplex between the guide RNA and the DNA target. Target unwinding occurs directionally away from the PAM and the first 8 base pairs that form with the guide RNA constitute a “seed sequence” is extremely important to ensure binding. Mutation within this seed sequence often reduces the level of CRISPR interference, leading to phage escape. These mutations can also promote an alternative “priming” mechanism, during which the CRISPR-Cas system can acquire short fragments of DNA from the invader to create new, fully functional guide RNAs. Our previous results have demonstrated that some spacers are more tolerant of target mutations, suggesting they may provide better immunity by decreasing the probability of phage escape. Here, we have investigated how the G/C content in guide RNA seed sequences affects CRISPR interference and priming activities. I will present high-throughput studies of the mutational tolerance for bacterial strains expressing varied guide RNA seed sequences, and my future plans to probe the mechanism of mutational tolerance.