DNA Uptake into CRISPR Loci

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. Pyrococcus furiosus, a hyperthermophilic archaeaon, utilizes one set of adaptation proteins (Cas1, Cas2, Cas4-1 and Cas4-2) for spacer DNA uptake into each of its seven CRISPR loci. We have shown that the 200 different crRNAs naturally encoded by the seven CRISPR arrays are each incorporated into three functional crRNP effector complexes, Type I-A (Csa), Type I-G (Cst), and Type III-B (Cmr), to destroy matching plasmid DNA through either DNA- or RNA-targeting mechanisms. Recently, we found that Pyrococcus furiosus actively takes up spacers into each of its seven CRISPR loci under normal growth conditions and without the need for over-expressing adaptation proteins.

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. Analysis of non-CRISPR integration sites revealed that Cas1 and Cas2 are directed to catalyze full-site spacer integration at specific DNA stretches where guanines and/or cytosines are 30 base pairs apart and the intervening sequence harbors several positionally conserved bases. Moreover, assaying a series of CRISPR repeat mutations, followed by sequencing of the integration products, revealed that the specificity of integration is primarily directed by sequences at the leader-repeat junction as well as an adenine-rich sequence block in the mid-repeat. Together, our results indicate that P. furiosus Cas1 and Cas2 recognize multiple sequence features distributed over a 30 base pair DNA region for accurate spacer integration at the CRISPR repeat.