Mechanistic Studies of the Type I CRISPR-Cas system

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Project Summary Prokaryotes can acquire resistance to viruses and plasmids by integrating short fragments of foreign DNA, called prespacers, into clusters of regularly interspaced short palindromic repeats (CRISPR's). These repeats are then transcribed and processed into small guide RNA's that are used to direct the destruction of foreign nucleic acid. This mechanism has many parallels with eukaryotic RNA interference but the proteins that are associated with the CRISPR response are evolutionarily unrelated to their eukaryotic counterparts. Our long-term goal is to understand the biochemical and structural basis of CRISPR-mediated resistance in prokaryotes. The objectives here are to understand how changes in the target sequence modulate the immune response and provide insight into how the prespacers are incorporated into CRISPR arrays. Our objectives will combine structural, biochemical and cell based experiments. Successful completion of the proposed studies is significant because it will increase our understanding of bacterial resistance to viruses and plasmids, both of which play important roles in the genetics of pathogenic bacteria. It is also significant because these studies will help in the further development of CRISPR-based tools.