Clustered regularly interspaced short palindromic repeats
(CRISPRs) are a class of repetitive elements found in many
bacterial and archaeal genomes. They consist of a few to hundreds
of direct repeats separated by diverse spacer sequences and are
generally adjacent to genes encoding CRISPR-associated (Cas)
proteins. Over the past five years, evidence has mounted to
support the hypothesis that the CRISPR and Cas proteins represent
a microbial immune system against virus (phage) infection
similar to the eukaryotic RNAi pathway. I propose to evaluate
this hypothesis and advance understanding how the CRISPR-Cas
system works by elucidating roles of the Cas proteins. Despite
their functional analogies to protein components of RNAi, none of
the Cas proteins have been characterized in this context. Using
recombinant proteins, I will test Cas proteins analogous to
enzymes in the RNAi pathway for their activities in vitro. I
will also employ several approaches to identify their molecular
partners. Three-dimensional structures of Cas proteins will be
determined by X-ray crystallography.
