Major and minor crRNA annealing sites facilitate low stringency DNA protospacer binding prior to Type I-A CRISPR-Cas interference in Sulfolobus

Abstract

The stringency of crRNA-protospacer DNA base pair matching required for effective CRISPR-Cas interference is relatively low in crenarchaeal Sulfolobus species in contrast to that required in some bacteria. To understand its biological significance we studied crRNA-protospacer interactions in Sulfolobus islandicus REY15A which carries multiple, and functionally diverse, interference complexes. A range of mismatches were introduced into a vector-borne protospacer that was identical to spacer 1 of CRISPR locus 2, with a cognate CCN PAM sequence. Two important crRNA annealing regions were identified on the 39 bp protospacer, a strong primary site centered on nucleotides 3 -7 and a weaker secondary site at nucleotides 21 -25. Multiple mismatches introduced into remaining protospacer regions did not seriously impair interference. Extending the study to different protospacers demonstrated that the efficacy of the secondary site was greatest for protospacers with higher GCC contents. In addition, the interference effects were assigned specifically to the type I-A dsDNA-targeting module by repeating the experiments with mutated protospacer constructs that were transformed into an S. islandicus mutant lacking type III-Ba and III-Bb interference gene cassettes, which showed similar interference levels to those of the wild-type strain. Parallels are drawn to the involvement of 2 annealing sites for microRNAs on some eukaryal mRNAs which provide enhanced binding capacity and specificity. A biological rationale for the relatively low crRNA-protospacer base pairing stringency among the Sulfolobales is considered.

Diversified local CRISPR-Cas immunity to viruses ofSulfolobus islandicus

“…Finally, we used the location of mismatches within a protospacer to investigate whether there is directed virus evolution to evade CRISPR immunity. Mutations in the PAM and in the seed region, which are the seven nucleotides nearest to PAM (for S. islandicus ), have the greatest effect on the efficiency of CRISPR interference [71,77,78]. Bacteriophage CRISPR escape mutants accumulate mutations in the seed region of protospacers [21].…”

“…We found that mutations in the five bases nearest to the PAM occur more frequently than in even the most extreme value obtained in 100 simulations of 2037 random mutations in a 40 nucleotide protospacer (figure 6 c ). Additionally, we found a relatively high number of mismatches between positions 21 and 25 of the protospacer, which may be important for spacer targeting specificity in S. islandicus [78]. We also performed this analysis on the 169 mismatches between spacers and Mutnovsky SSVs, but the distribution of mismatches was no different than random ( χ 2 (7, N = 169) = 4.804, p = 0.6839).…”

Stable maintenance of the rudivirus SIRV3 in a carrier state in Sulfolobus islandicus despite activation of the CRISPR-Cas immune response by a second virus SMV1

“…The presence of multiple SIRV3-matching spacers in CRISPR loci of strain REY15A (Table 2), together with the new spacers added in the coinfected cultures after 3 dpi (Table 5), were expected to provide a robust viral defence [32,33]. A likely explanation for the survival of the active viruses is that CRISPR-Cas interference was inhibited by Acr proteins encoded by SIRV3.…”

Heavily Armed Ancestors: CRISPR Immunity and Applications in Archaea with a Comparative Analysis of CRISPR Types in Sulfolobales

“…In the selection-independent virus approach employing shuttle vectors based on the lysogenic virus SSV1 [206], interference efficiency was directly quantified by counting transfected cells in plaque assays [205]. Both of these strategies were applied in various follow-up in vivo studies to further characterize PAM requirements for type I targeting [207], protospacer-crRNA matches needed for proper interference [129,208], crRNA processing and transcription regulation [207,209] and type III-mediated RNA recognition and interference [119,120]. A significant in vivo finding, already foreshadowing a link between CARF-domain nucleases and the type III-B immune response, was the demonstration of transcription-dependent DNA interference against a plasmid to be dependent on csx1/csm6 locus and an intact type III-B system in S. islandicus [120].…”