Biogenesis pathways of RNA guides in archaeal and bacterial CRISPR-Cas adaptive immunity

Abstract

CRISPR-Cas is an RNA-mediated adaptive immune system that defends bacteria and archaea against mobile genetic elements. Short mature CRISPR RNAs (crRNAs) are key elements in the interference step of the immune pathway. A CRISPR array composed of a series of repeats interspaced by spacer sequences acquired from invading mobile genomes is transcribed as a precursor crRNA (pre-crRNA) molecule. This pre-crRNA undergoes one or two maturation steps to generate the mature crRNAs that guide CRISPR-associated (Cas) protein(s) to cognate invading genomes for their destruction. Different types of CRISPR-Cas systems have evolved distinct crRNA biogenesis pathways that implicate highly sophisticated processing mechanisms. In Types I and III CRISPR-Cas systems, a specific endoribonuclease of the Cas6 family, either standalone or in a complex with other Cas proteins, cleaves the pre-crRNA within the repeat regions. In Type II systems, the trans-acting small RNA (tracrRNA) base pairs with each repeat of the pre-crRNA to form a dual-RNA that is cleaved by the housekeeping RNase III in the presence of the protein Cas9. In this review, we present a detailed comparative analysis of pre-crRNA recognition and cleavage mechanisms involved in the biogenesis of guide crRNAs in the three CRISPR-Cas types.

Keywords: Cas5d; Cas6; Cas9; RNase III; crRNA biogenesis; tracrRNA.

Figure 1.

cas gene composition of the CRISPR-Cas systems. Loci from Types I-A to I-F, Types II-A to II-C and Types III-A and II-B CRISPR-Cas systems are represented. The CRISPR arrays are composed of a series of repeats (black diamonds) interspaced by invading genome-targeting spacers (colored diamonds). An operon of cas genes is located in the close vicinity of the CRISPR array. The Cas proteins involved in the crRNA biogenesis in Types I-A, I-B, I-D, I-E and I-F and Types III-A and III-B belong to the Cas6 family. An exception is the gene product Cas5d responsible for the processing of pre-crRNA in Type I-C. In Type II systems, tracrRNA, and the proteins Cas9 and RNase III are the three components responsible for pre-crRNA maturation.

Figure 2.

crRNA processing pathways in Type I CRISPR-Cas systems. In Type I systems, the palindromic repeats in the pre-crRNA are either unstructured (Cascade/I-A, Cascade/I-B) or form hairpin structures (Cascade/I-C, Cascade/I-D, Cascade/I-E, Cascade/I-F) that are recognized by the nuclease Cas6 (Cas6a, Cascade/I-A; Cas6b, Cascade/I-B; Cas6d, Cascade/I-D; Cas6e, Cascade/I-E; Cas6f, Cascade/I-F) or Cas5 (Cas5d, Cascade/I-C). After cleavage, the crRNA hairpin remains associated with Cas6 or Cas5 whilst other subunits bind the 5^′ handle and spacer, which is used for the recognition of cognate genetic element sequences by the respective Cascade complexes.

Figure 3.

crRNA processing pathways in Type II CRISPR-Cas systems. In Type II systems, the precursor transcript of the CRISPR repeat-spacer array forms duplexes with the trans-activating tracrRNA through pre-crRNA repeat:tracrRNA anti-repeat interactions. The duplex RNAs stabilized by the protein Cas9 are recognized and cleaved by the bacterial endoribonuclease III (RNase III). A second processing by unknown nucleases (trimming by an exonuclease and/or cleavage by an endoribonuclease) generates the mature crRNAs. An alternative pathway for the production of mature crRNAs was described in a Type II-C of N. meningitidis. Here, the transcription of short crRNAs occurs directly from promoters contained within the repeats of the array, and thus independently of cleavage by RNase III. The mature dual tracrRNA:crRNAs complexed with the protein Cas9 form the interference complex that target and cleave site specifically double-stranded DNA.

Figure 4.

crRNA processing pathways in Type III CRISPR-Cas systems. In Type III-A and III-B systems, the standalone Cas6 endonuclease binds unstructured pre-crRNA and cleaves within each repeat to generate intermediate crRNAs with 5^′ and 3^′ repeat-derived termini. The crRNAs are loaded into the Csm (Type III-A) or Cmr (Type III-B) complex and undergo further maturation through trimming of the 3^′ repeat-derived sequence by nucleases that are yet to be identified.