Short mature CRISPR RNAs (crRNAs) are critical elements in the adaptive immune response against invading foreign genetic elements. A CRISPR array composed of a set of cas genes, and repeats interspaced by spacer sequences acquired from invading DNA or RNA is transcribed as a precursor crRNA (pre-crRNA) molecule from a promoter located in the leader sequence, an AT-rich region located upstream of the CRISPR array. And then the pre-crRNA undergoes one or two maturation steps to generate the mature crRNAs which can guide CRISPR-associated (Cas) protein(s) to cognate invading genomes for their destruction. The Cas6 superfamily, Cas5d subclass, and the housekeeping RNase III endoribonucleases are responsible for the maturity of crRNA.
As we all known, CRISPR-Cas systems are categorized into three major types, namely Type I, Type II and Type III, each of which is further divided into subtypes. Sophisticated processing mechanisms of crRNAs have been discovered in different CRISPR-Cas systems. In types I (except for I-C) 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 CRISPR-Cas 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 endogenous RNase III in the presence of the protein Cas9.
The spacers originate from invader mobile genetic elements (MGEs) memorized upon a first infection, and enable recognition of the invading elements upon a second infection. The maturation of the crRNAs is critical for the activity of the CRISPR system and the biogenesis of mature crRNAs can be generally divided into three steps. Firstly, a long primary transcript or precursor crRNA (pre-crRNA) is generated from a promoter located within the leader sequence that precedes the CRISPR repeat-spacer array. Nextly, primary cleavage of the pre-crRNA occurs at a specific site within the repeats to yield crRNAs that consist of the entire spacer sequence flanked by partial repeat sequences. In some cases, an additional secondary cleavage step is required to generate the active mature crRNAs.
Fig 1. crRNA processing pathways in Type I CRISPR-Cas systems. A. Type I-C CRISPR system. B. Type I-E CRISPR system.
A common theme among the CRISPR-Cas types is the transcription of the pre-crRNA and the first processing event within the repeats. In Types I and III, a protein of the Cas6 family or alternatively Cas5d catalyzes this step. Each member of the Cas6 family of endoribonucleases recognizes a unique RNA sequence. Cas5d is the second distinct class of endoribonucleases responsible for processing crRNA in CRISPR-Cas systems that lack Cas6. In Type II, a trans-acting small RNA with 24 nucleotide complementarity to the repeat regions of crRNA precursor transcripts directs pre-crRNA dicing by housekeeping endoribonuclease III-mediated cleavage within the repeats in the presence of Cas9 protein. RNase III cleaves both strands of the dsRNA with a two base pair separation, resulting in a cleavage intermediate further processed by the Cas9 class protein.
Type II CRISPR-Cas systems use a unique crRNA biogenesis pathway distinct from Type I and III CRISPR-Cas systems that involve the coordinated action of three novel factors: the trans-acting small RNA (tracrRNA), the host-encoded endoribonuclease RNase III and the Cas9 protein. Genetic and dRNA-seq analysis have shown that tracrRNA and pre-crRNA undergo coprocessing through the double-stranded substrate formed by the base pairing of tracrRNA anti-repeat and pre-crRNA repeats. Further genetic and biochemical analysis confirms that the endogenous RNase III-a general RNA processing factor in bacteria-is recruited to cleave the duplex RNA which was stabilized by the Cas9 protein which will generate predictable dsDNA breaks into the target sequence. RNase III serves as a host factor in tracrRNA-mediated crRNA maturation.
Fig 1. crRNA processing pathways in Type II CRISPR-Cas systems. A. Type II-A+II-B CRISPR system. B. Type II-C CRISPR system.
Both co-processed 75nt tracrRNA and 66nt intermediate crRNA species carry short overhangs at the 3' end, typical for cleavage by the housekeeping RNase III. A second processing by unknown nucleases (trimming by an exonuclease and / or cleavage by an endoribonuclease) generates the mature crRNAs. And then Cas9 utilizes the 20-nt segment at the 5' end of the mature crRNA to bind cognate DNA sequences and deploys its HNH and RuvC nuclease domains to cleave the double-stranded DNA target. 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 CRISPR 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.
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