Exploring molecular mechanisms of RNA-mediated gene regulation
CRISPR RNA-guided surveillance complexes target foreign DNA for degradation through RNA–DNA base-pairing and recognition of a unique sequence adjacent to the target DNA called the protospacer adjacent motif (PAM). Addressing how the DNA is unwound during this binding event, and how short 20–30 base-pair target sequences are efficiently located and recognized within entire genomes, has been a recent focus of our research. In collaboration with Eric Greene’s laboratory at Columbia University, we have applied a combination of single-molecule and bulk biochemical experiments to resolve the mechanism of DNA interrogation for two phylogenetically unrelated complexes: Cas9, the DNA-targeting protein found in Type II CRISPR–Cas systems (S. pyogenes), and Cascade, the DNA-targeting complex found in Type I-E CRISPR–Cas systems (E. coli). Our results have revealed that the target search is PAM-guided, and that these distinct RNA-guided complexes have converged on a common mechanism for target DNA recognition.
Type II CRISPR-Cas systems use an RNA-guided DNA endonuclease, Cas9, to generate double-strand breaks in invasive DNA during an adaptive bacterial immune response. Cas9-mediated cleavage is strictly dependent on the presence of a protospacer adjacent motif (PAM) in the target DNA. The ability to program Cas9 for DNA cleavage at specific sites defined by guide RNAs has led to its adoption as a versatile platform for genome engineering and gene regulation. To compare the architectures and domain organization of diverse Cas9 proteins, we have solved the atomic structures of Cas9 from Streptococcus pyogenes (SpyCas9) and Actinomyces naeslundii (AnaCas9), revealing the structural core shared by all Cas9 family members, and the structurally divergent regions, including the PAM recognition loops, are likely responsible for distinct guide RNA and PAM specificities. Our EM analysis further shows that by triggering a conformational rearrangement in Cas9, the guide RNA acts as a critical determinant of target DNA binding (in collaboration with Eva Nogales, UC Berkeley, HHMI).
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 (in collaboration with Eva Nogales, UC Berkeley, HHMI).
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. 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.