Clustered regularly interspaced short palindromic repeats (CRISPR) are a distinctive feature of the genomes of most Bacteria and Archaea and are thought to be involved in resistance to bacteriophages. We found that, after viral challenge, bacteria integrated new spacers derived from phage genomic sequences. Removal or addition of particular spacers modified the phage-resistance phenotype of the cell. Thus, CRISPR, together with associated cas genes, provided resistance against phages, and resistance specificity is determined by spacer-phage sequence similarity.

Clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide adaptive immunity against viruses and plasmids in bacteria and archaea. The silencing of invading nucleic acids is executed by ribonucleoprotein complexes preloaded with small, interfering CRISPR RNAs (crRNAs) that act as guides for targeting and degradation of foreign nucleic acid. Here, we demonstrate that the Cas9–crRNA complex of the Streptococcus thermophilus CRISPR3/Cas system introduces in vitro a double-strand break at a specific site in DNA containing a sequence complementary to crRNA. DNA cleavage is executed by Cas9, which uses two distinct active sites, RuvC and HNH, to generate site-specific nicks on opposite DNA strands. Results demonstrate that the Cas9–crRNA complex functions as an RNA-guided endonuclease with RNA-directed target sequence recognition and protein-mediated DNA cleavage. These findings pave the way for engineering of universal programmable RNA-guided DNA endonucleases.

CRISPR Defenses: Prokaryotes can be infected by parasites and pathogens and, like eukaryotes, have evolved systems to protect themselves. Horvath and Barrangou (p. 167 ) review a recently discovered prokaryotic “immune system” characterized by CRISPR—clustered regularly interspaced short palindromic repeats—found in most archaeal and many bacterial species. CRISPR loci harbor short sequences captured from viruses and invasive genetic elements. These sequences are transcribed, and the RNA is cleaved into short CRISPR RNAs (crRNAs) by one of a family of CRISPR-associated (cas) proteins. These crRNAs direct other cas family proteins to homologous nucleic acid targets to effect their destruction. Through its ability to impede the spread of specific nucleic acid sequences, the CRISPR/Cas systems might be exploited to block the dissemination of antibiotic-resistance markers.