The CRISPR system was recently identified as a bacterial defense mechanism against phages and plasmids. The CRISPR system is composed of DNA arrays containing short sequences identical to those present in phages and plasmids. These short DNAs are transcribed and processed by CRISPR associated proteins that also guide other CRISPR proteins to target the invading DNA. Only a few of the CRISPR components have been characterized to date and their mechanism of action is still largely unknown. Phage defense mechanisms probably have co-evolved against the CRISPR system, but none have yet been found. We propose to identify phage genes that counteract the CRISPR system.
We will utilize screens that make use of the bacterial version of the yeast two-hybrid genetic system, phage genomic libraries, and biochemical assays to identify phage genes that help phages evade the CRISPR system. The candidate genes will be characterized both genetically and biochemically to allow structural studies of their interactions with the CRISPR system. These genes will then be cloned into commercial phages that are used to kill bacterial pathogens so that the phages acquire resistance to the CRISPR defense mechanism. <P>
We also propose to identify the yet unknown E. coli proteins that participate in the activity of the CRISPR system by using genetic screens of transposon insertion mutant libraries. The identification of novel proteins that participate in the CRISPR system will constitute a significant step toward the ultimate goal of reconstituting the complete system from purified proteins. Finally, we propose to clone all the components of the E. coli CRISPR system and render them functional in heterologous prokaryotic organisms such as attenuated strains of Salmonella, Shigella and more distantly related species such as Streptococci. Cloning the genes of the E. coli CRISPR system into heterologous bacteria will allow us to genetically address the role of each gene.
Funded under 7th FWP (Seventh Framework Programme)