Exploring molecular mechanisms of RNA-mediated gene regulation
CRISPR-Cas is a prokaryotic defense system against invading genetic elements. In a collaboration with John van der Oost’s laboratory, we are studying the structure and function of the effector complex of the Type III-A CRISPR-Cas system of Thermus thermophilus: the Csm complex (TtCsm). Recently, we showed that multiple Cas proteins and a crRNA guide assemble to recognize and cleave invader RNAs at multiple sites . Our negative stain EM structure of the TtCsm complex exhibits the characteristic architecture of Type I and Type III CRISPR-associated ribonucleoprotein complexes, suggesting a model for cleavage of the target RNA at periodic intervals (in collaboration with Eva Nogales, UC Berkeley, HHMI).
Cascade is composed of Cse1, Cse2, Cas7, Cas5e, and Cas6e subunits and one crRNA, forming a structure that binds and unwinds dsDNA to form an R-loop in which the target strand of the DNA base pairs with the 32-nt crRNA guide sequence. Recently, we used single-particle electron microscopy reconstructions of dsDNA-bound Cascade with and without Cas3 to reveal that Cascade positions the PAM-proximal end of the DNA duplex at the Cse1 subunit and near the site of Cas3 association. The finding that the DNA target and Cas3 colocalize with Cse1 implicates this subunit in a key target-validation step during DNA interference. We show biochemically that base pairing of the PAM region is unnecessary for target binding but critical for Cas3-mediated degradation. In addition, the L1 loop of Cse1, previously implicated in PAM recognition, is essential for Cas3 activation following target binding by Cascade. Together, these data show that the Cse1 subunit of Cascade functions as an essential partner of Cas3 by recognizing DNA target sites and positioning Cas3 adjacent to the PAM to ensure cleavage (in collaboration with Eva Nogales, UC Berkeley, HHMI).
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.