Functional characterization of CRISPR-associated Cas1 protein of E. coli

¹ Department for Molecular Biology of Bacteria, Heinrich-Heine-Universität Düsseldorf, Germany

² Institute of Biochemistry, Heinrich-Heine-Universität Düsseldorf, Germany

The recently discovered CRISPR/Cas defense system confers an acquired immunity against invasion by foreign genetic elements [1]. CRISPR arrays (clustered regularly interspaced short palindromic repeats) consist of repetitive short palindromic repeat sequences, separated by unique spacer sequences. Immunity is achieved by incorporation of short invader DNA pieces in the CRISPR array [2]. CRISPR arrays are associated with cas genes, encoding for diverse proteins, which mediate the processing of the pre-crRNA and the crRNA-guided targeting and inactivation of the invader DNA [3].

The Cas1 protein, found in nearly all CRISPR-containing organisms, likely mediates the acquisition of new spacers. We have analyzed E. coli Cas1 for its nuclease activity. Our preliminary results indicate that Cas1 cleaves single-stranded and double-stranded DNA in presence of divalent metal ions without sequence specificity. However, nuclease assays with DNA containing the CRISPR repeat sequences are preferentially cleaved within the repeats, suggesting a crucial activity for the integration of new spacers. Cas1 also exhibits a single-strand specific RNase activity. The possibility of small RNAs as spacer precursors will be discussed.