ABSTRACT
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 5’ PAM and 3’ 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 vivo CRISPR 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.
Importance The Cas4 family endonuclease is an essential component of the adaptation module in many variants of CRISPR-Cas adaptive immunity systems. The Crenarchaeota Sulfolobus islandicus REY15A encodes two cas4 genes (cas4 and csa1) linked to the CRISPR arrays. Here, we demonstrate that Cas4 and Csa1 are essential to CRISPR spacer acquisition in this organism. Both proteins specify the upstream and downstream conserved nucleotide motifs of the protospacers and define the spacer length and orientation in the acquisition process. Conserved amino acid residues, in addition to the recently reported, were identified to be important for above functions. More importantly, overexpression of the Sulfolobus viral Cas4 abolished spacer acquisition, providing support for an anti-CRISPR role for virus-encoded Cas4 proteins that inhibit spacer acquisition.