Cas4 nucleases can effect specific integration of CRISPR spacers

CRISPR-Cas systems incorporate short DNA fragments from invasive genetic elements into host CRISPR arrays in order to generate host immunity.Recently, we demonstrated that the Csa3a regulator protein triggers CCN PAM-dependent CRISPR spacer acquisition in the subtype I-A CRISPR-Cas system of Sulfolobus islandicus. However, the mechanisms underlying specific protospacer selection and spacer insertion remained unclear. Here, we demonstrate that two Cas4 family proteins (Cas4 and Csa1) have essential roles (a) in recognizing the 59 PAM and 39 nucleotide motif of protospacers and (b) in determining both the spacer length and its orientation. Furthermore, we identify uncovered amino acid residues of the Cas4 proteins that facilitate these functions. Overexpression of the Cas4 and Csa1 proteins, and also of an archaeal virus-encoded Cas4 protein, resulted in strongly reduced adaptation efficiency and the formers yielded a high incidence of PAM-dependent atypical spacer integration, or of PAM-independent spacer integration. We further demonstrated that, in the plasmid challenging experiments, overexpressed Cas4-mediated defective spacer acquisition, in turn, potentially enabled targeted DNA to escape subtype I-A CRISPR-Cas interference. In summary, these results define the specific involvement of diverse Cas4 proteins in in vivoCRISPR spacer acquisition. Furthermore, we provide support for an anti-CRISPR role for virus-encoded Cas4 proteins that involves compromising CRISPR-Cas interference activity by hindering spacer acquisition.