Comparative genomics and evolution of trans-activating RNAs in Class 2 CRISPR-Cas systems

Abstract

Trans-activating CRISPR (tracr) RNA is a distinct RNA species that interacts with the CRISPR (cr) RNA to form the dual guide (g) RNA in type II and subtype V-B CRISPR-Cas systems. The tracrRNA-crRNA interaction is essential for pre-crRNA processing as well as target recognition and cleavage. The tracrRNA consists of an antirepeat, which forms an imperfect hybrid with the repeat in the crRNA, and a distal region containing a Rho-independent terminator. Exhaustive comparative analysis of the sequences and predicted structures of the Class 2 CRISPR guide RNAs shows that all these guide RNAs share distinct structural features, in particular, the nexus stem-loop that separates the repeat-antirepeat hybrid from the distal portion of the tracrRNA and the conserved GU pair at that end of the hybrid. These structural constraints might ensure full exposure of the spacer for target recognition. Reconstruction of tracrRNA evolution for 4 tight bacterial groups demonstrates random drift of repeat-antirepeat complementarity within a window of hybrid stability that is, apparently, maintained by selection. An evolutionary scenario is proposed whereby tracrRNAs evolved on multiple occasions, via rearrangement of a CRISPR array to form the antirepeat in different locations with respect to the array. A functional tracrRNA would form if, in the new location, the antirepeat is flanked by sequences that meet the minimal requirements for a promoter and a Rho-independent terminator. Alternatively, or additionally, the antirepeat sequence could be occasionally 'reset' by recombination with a repeat, restoring the functionality of tracrRNAs that drift beyond the required minimal hybrid stability.

A natural single-guide RNA repurposes Cas9 to autoregulate CRISPR-Cas expression

“…We next investigated whether trL-mediated repression of Pcas9 is specific to S. pyogenes or conserved in other type II-A CRISPR-Cas systems. In a list of previously annotated short-form tracrRNA loci 56 , we examined 'branch 1' which contains 80 representatives from Streptococci, Listeria and Lactobacilli. We queried the presence of trL regulation by looking for 3 criteria: (i) an 11 bp or greater match between a putative trL and a region predicted to contain the Cas9 promoter, (ii) a 6 bp lower stem sequence (5'-GTTTTA-3') just downstream from the trL targeting site and (iii) a PAM sequence (5'-NGG-3') just downstream from the Pcas9 target site.…”

Identification and evolution of Cas9 tracrRNAs

“…This cutoff-value was based on the longest characterized Cas9 tracrRNA length plus a generous buffer. 25 Step3: Clustering tracrRNA candidates Putative tracrRNAs were next clustered at 95% sequence identity with a 90% sequence coverage cutoff using cd-hit-est v.4.7. 38 Sequence clusters that did not map back to at least five different assemblies were removed from further analysis unless they contained a putative tracrRNA from an assembly with only a single putative candidate (Figure 1, Step 3).…”

“…[17][18][19][20][21][22][23][24] In prokaryotes, thousands of Cas9s have been identified computationally. [25][26][27][28] In contrast, the gRNA solution for orthologous Cas9s may not be easily recognizable. This is mainly due to large variation in tracrRNA location, size and sequence identity.…”

CRISPR-Cas: more than ten years and still full of mysteries

“…The identification of tracrRNA has been rather challenging and Chyou and Brown now report a computational pipeline for the prediction of tracrRNAs [16]. In a second paper, Faure et al use a computational approach to perform a comprehensive analysis of the evolution of tracrRNA [17]. They present evidence that the anti-repeat regions of tracrRNAs might have multiple origins through repeat recruitment and/or recombination.…”