CRISPR repeat sequences and relative spacing specify DNA integration by Pyrococcus furiosus Cas1 and Cas2.

Acquiring foreign spacer DNA into the CRISPR locus is an essential primary step of the CRISPR-Cas pathway in prokaryotes for developing host immunity to mobile genetic elements. Here, we investigate spacer integration in vitro using proteins from Pyrococcus furiosus and demonstrate that Cas1 and Cas2 are sufficient to accurately integrate spacers into a minimal CRISPR locus. Using high-throughput sequencing, we identified high frequency spacer integration occurring at the same CRISPR repeat border sites utilized in vivo, as well as at several non-CRISPR plasmid sequences which share features with repeats.