CAREER: Exploring novel interactions between CRISPR-Cas systems and temperate bacteriophages

Bacterial protein Cas9, part of the CRISPR-Cas immunity system, has emerged as a transformative gene editing tool with applications in basic research, agriculture, and healthcare. However, despite its widespread adoption in biotechnology, many questions remain regarding how Cas9 functions in nature. Cas9 main biological function is to protect bacteria from viral invaders known as bacteriophages, or phages for short.

These CRISPR-Cas9 systems acquire memories, in the form of DNA fragments, from prior phage infections, allowing bacterial cells to rapidly kill the same phage when it is encountered again in a later infection. This project will explore the hypothesis that a class of phages called “temperate phages” that are often neglected in CRISPR-Cas studies hold the key to understanding CRISPR-Cas memory acquisition.

These results will inform new generations of Cas9 technologies, including molecular recording devices, and highlight how CRISPR-Cas systems and temperate phages together influence the evolution of bacterial communities. The project also details an educational plan that will provide undergraduate students an opportunity to learn more about the arms race between bacteria and their phages and careers in microbiological research.

A central question in CRISPR-Cas memory creation is how cells survive a lytic phage infection long enough to create a new memory and utilize it for defense. Intriguingly, many CRISPR systems target temperate phages, which are capable of entering a program called ‘lysogeny’, in which their DNA is inserted into the bacterial chromosome, where it may remain in a dormant form.

This proposal tests the hypothesis that CRISPR-Cas systems can exploit the temperate lifecycle by acquiring memories about those phages under non-lytic conditions. Specifically, the proposal examines whether new memories can be made during the initiation of lysogeny (aim 1) and/or after lysogeny has occurred and the phage has integrated into the chromosome as a prophage (aim 2).

Finally, the proposal tests whether prophages can influence the ability of CRISPR-Cas systems to acquire memories from other infecting phages (aim 3). Temperate phages are understudied in CRISPR-Cas laboratory experiments, because the rates of lysogeny are significantly higher than spacer acquisition. This proposal will employ new genetic tools to thoroughly dissect the interactions between CRISPR-Cas systems and their natural temperate phage targets, revealing a new paradigm for how CRISPR-Cas immunity is established.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.