Dynamic properties of the <i>Sulfolobus</i> CRISPR/Cas and CRISPR/Cmr systems when challenged with vector‐borne viral and plasmid genes and protospacers

TLDR

The authors challenged Sulfolobus CRISPR/Cas and CRISPR/Cmr systems by cloning viral and plasmid genes and protospacers—matched to CRISPR spacers with varying mismatches—into pyrE/pyrF vectors and transforming them into uracil‑auxotrophic Sulfolobus solfataricus P2 and Sulfolobus islandicus REY15A strains. Most constructs yielded few viable transformants, with deletions of CRISPR loci or modules occurring independently of transcription; the protospacer CC motif promoted deletions in family I loci, and the high incidence of single‑spacer deletions in S.

Abstract

The adaptive immune CRISPR/Cas and CRISPR/Cmr systems of the crenarchaeal thermoacidophile Sulfolobus were challenged by a variety of viral and plasmid genes, and protospacers preceded by different dinucleotide motifs. The genes and protospacers were constructed to carry sequences matching individual spacers of CRISPR loci, and a range of mismatches were introduced. Constructs were cloned into vectors carrying pyrE/pyrF genes and transformed into uracil auxotrophic hosts derived from Sulfolobus solfataricus P2 or Sulfolobus islandicus REY15A. Most constructs, including those carrying different protospacer mismatches, yielded few viable transformants. These were shown to carry either partial deletions of CRISPR loci, covering a broad spectrum of sizes and including the matching spacer, or deletions of whole CRISPR/Cas modules. The deletions occurred independently of whether genes or protospacers were transcribed. For family I CRISPR loci, the presence of the protospacer CC motif was shown to be important for the occurrence of deletions. The results are consistent with a low level of random dynamic recombination occurring spontaneously, either inter-genomically or intra-genomically, at the repeat regions of Sulfolobus CRISPR loci. Moreover, the relatively high incidence of single-spacer deletions observed for S. islandicus suggests that an additional more directed mechanism operates in this organism.