TY - JOUR

T1 - Major and minor crRNA annealing sites facilitate low stringency DNA protospacer binding prior to Type I-A CRISPR-Cas interference in Sulfolobus

AU - Mousaei, Marzieh

AU - Deng, Ling

AU - She, Qunxin

AU - Garrett, Roger Antony

PY - 2016

Y1 - 2016

N2 - 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 centred 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 G+C 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-Bα and III-Bβ interference gene cassettes, which showed similar interference levels to those of the wild-type strain. Parallels are drawn to the involvement of two 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 amongst the Sulfolobales is considered.

AB - 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 centred 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 G+C 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-Bα and III-Bβ interference gene cassettes, which showed similar interference levels to those of the wild-type strain. Parallels are drawn to the involvement of two 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 amongst the Sulfolobales is considered.

U2 - 10.1080/15476286.2016.1229735

DO - 10.1080/15476286.2016.1229735

M3 - Journal article

C2 - 27618562

VL - 13

SP - 1166

EP - 1173

JO - R N A Biology

JF - R N A Biology

SN - 1547-6286

IS - 11

ER -