CRISPR-mediated prokaryotic immunity

Bacteria and archaea have evolved resistance to viruses and plasmids using a nucleic acid-based system that has strong analogies to the RNA interference system in eukaryotes. This adaptive immune system is accomplished by integrating short fragments of foreign DNA into Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) into their genome. In E. coli, these CRISPR transcripts are processed into short RNAs that are incorporated into a large ribonucleoprotein surveillance complex called Cascade. This complex specifically targets and binds foreign nucleic acids in a sequence-specific manner, followed by degradation of the targeted substrate. In collaboration with the Jennifer Doudna lab, we used cryo-electron microscopy to determine the structure of Cascade before and after binding to a target substrate. These structures were solved to subnanometer resolution, and revealed that the CRISPR RNA (crRNA) is displayed along a helical arrangement of six CasC subunits. It was previously established that the Cascade complex engages invading nucleic acids through high-affinity base pairing interactions located near the 5’ end of the crRNA, but we determined in our studies that this binding triggers a concerted conformational change of the seahorse-shaped protein architecture. Together, these structures explain the molecular mechanism for target recognition and reveal a structural reorganization that likely serves as a signal for subsequent destruction of the invading DNA.

We are currently working towards a more complete structural understanding of the this CRISPR system in collaboration with the Doudna lab.