Exploring molecular mechanisms of RNA-mediated gene regulation
Prokaryotes have evolved a nucleic acid-based immune system that shares some functional similarities with RNA interference in eukaryotes. Central to this system are DNA repeats called CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats). CRISPRs are genetic elements containing direct repeats separated by unique spacers, many of which are identical to sequences found in phage and other foreign genetic elements. Recent work has demonstrated the role of CRISPRs in adaptive immunity and shown that small RNAs derived from CRISPRs (crRNAs) are implemented as homing oligos for the targeted interference of foreign DNA.
Phylogenetic analysis of CRISPR-associated (Cas) proteins suggests there are at least seven distinct versions of this immune system. These systems can be extremely divergent mechanistically and provide a rich area to research RNA:protein interactions, including novel protein folds. To explore this diversity, we have determined the structures of diverse CRISPR-associated proteins, including the large E. coli CASCADE silencing complex (in collaboration with Eva Nogales, UC Berkeley, HHMI). This seahorse-shaped assembly shows how the CRISPR RNA is cradled by six repeating subunits and presented for DNA inspection. Further, we have solved the structure of the CasA subunit by X-ray crystallography, which revealed that it is poised to have a role in discriminating between “nonself” (foreign DNA) or “self” (host DNA) prior to targeting. This step is critical, as reckless silencing could prove lethal to the host.
Current work in the lab focuses on understanding the molecular basis of interaction between CRISPR targeting systems and their RNAs, the recognition mechanism of foreign nucleic acid and specificity, and dual-RNA CRISPR systems. Recent work has led to potential applications of CRISPR systems for programmable DNA cleavage, and RNA purification systems using the incredibly high affinity of CRISPR protein:RNA complexes.