Bacteria guard against viral infection by acquiring immunity to bacteriophage and plasmids. This research focuses on elucidating three critical aspects of this bacterial immune system: 1) how viral DNA sequences are incorporated into the host genome; 2) how these genomic inserts are used to produce small anti-sense RNAs; and 3) how foreign genetic elements are selectively silenced or destroyed. The longer-term goal of the project is to use these insights to engineer acquired immunity for selective microbial gene knockdown. This project has the potential to transform the way we understand gene regulation in bacteria, and to reveal the molecular basis for a previously unknown and unanticipated mechanism of anti-viral defense. The Doudna laboratory has a successful history of training students at all levels in the creative dissection of molecular mechanisms involving RNA and RNA-protein complexes using a variety of experimental methods. The project is being conducted in a highly collaborative environment at UC Berkeley, which has become a de facto center for CRISPR research encompassing ongoing work in the laboratories of Jill Banfield, Phil Hugenholtz and Adam Arkin. Quarterly joint group meetings with the Banfield lab are attended and given by all students working on CRISPR-related projects as a means of receiving outside input. An annual CRISPR conference at UC Berkeley attracts scientists from around the world to share their latest results. Students attend and present their work at this meeting, providing an outstanding opportunity for learning and discussing new results.
– Doudna Clustered regularly interspaced short palindromic repeats (CRISPRs) are components of nucleic-acid-based adaptive immune systems that are widespread in bacteria and archaea. These systems rely on small RNA molecules to detect and destroy foreign nucleic acids, including the DNA found in viruses and plasmids. Understanding how small RNAs are used to find and destroy foreign nucleic acids will provide new insights into the diverse mechanisms of RNA-controlled genetic silencing systems. During the previous project period, we made substantial progress in determining how RNA molecules function in bacterial adaptive immunity. We discovered the enzyme, Csy4, responsible for CRISPR transcript (pre-crRNA) processing in Pseudomonas aeruginosa. A 1.8 angstrom resolution crystal structure of Csy4 bound to its cognate RNA reveals that Csy4 makes sequence-specific interactions with the crRNA repeat structure. Together with electrostatic contacts to the phosphate backbone, these enable Csy4 to bind selectively and cleave pre-crRNAs using evolutionarily conserved serine and histidine residues in the active site. The RNA recognition mechanism we identified explains sequence- and structure-specific processing by a large family of CRISPR-specific enzymes. The CRISPR genetic locus in Pseudomonas aeruginosa includes four genes that are unique to and conserved in microorganisms harboring the Csy-type (CRISPR system yersinia) immune system. We found that the Csy proteins (Csy1-4) assemble into a 350 kDa ribonucleoprotein complex that facilitates target recognition by enhancing sequence-specific hybridization between the CRISPR RNA and complementary target sequences. Structural analysis of the complex by small-angle X-ray scattering and single particle electron microscopy revealed a crescent-shaped particle that bears striking resemblance to the architecture of a large CRISPR-associated complex from Escherichia coli, termed Cascade. Although similarity between these two complexes is not evident at the sequence level, their unequal subunit stoichiometry and quaternary architecture reveal conserved structural features that may be common among diverse CRISPR-mediated defense systems. We also found that another CRISPR enzyme, Cse3, binds to a hairpin structure and residues downstream of the cleavage site within the repetitive segment of cognate CRISPR RNA. Co-crystal structures of Cse3-RNA complexes revealed an RNA-induced conformational change in the enzyme active site that aligns the RNA strand for site-specific cleavage. These studies provide insight into a catalytically essential RNA recognition mechanism by a large class of CRISPR-related enzymes. Overall, the project outcomes provided extensive new insights into RNA-mediated bacterial immunity, leading to multiple patent filings and many opportunities to train students in various experimental techniques.
