Trans-activating crRNA (tracrRNAs), a unique family of small non-coding RNAs with conserved function but no obvious structure or sequence conservation, are inherent and exclusive to the type II CRISPR-Cas systems and critical for maturation of precursor crRNA (pre-crRNA) and interference with invading sequences. Following coprocessing of tracrRNA and pre-crRNA by RNase III, dual-tracrRNA:crRNA guides the CRISPR-associated endonuclease Cas9 (Csn1) to cleave site-specifically cognate target DNA. TracrRNA is an essential component of the dual-RNA:Cas9 ternary silencer complex, which is proposed as an attractive programmable tool for site-specific genome modification. The large panel of various tracrRNA and Cas9 proteins should represent a valuable source of sequences to improve the design of dual-RNA:Cas9 and derived single-guide RNA:Cas9.
Trans-activating small RNA (tracrRNA) is a small trans-encoded RNA. It was first discovered in the human pathogen Streptococcus pyogenes. A genome-wide computational analysis revealed tracrRNA located upstream of the cas genes of a Type II-A CRISPR-Cas system on the opposite strand. As an atypical, small RNA family, tracrRNA family has no obvious conservation of structure, sequence or localization within type II CRISPR-Cas loci. Although diverse in these aspects, tracrRNA orthologs share the common feature to contain an anti-pre-crRNA repeat sequence (anti-repeat) and act in trans. Studies have shown that tracrRNAs are characterized by an anti-repeat sequence capable of base-pairing with each of the pre-crRNA repeats to form tracrRNA:pre-crRNA repeat duplexes that are cleaved by RNase III in the presence of Cas9 protein.
Fig 1. Location and structure of trans-activating crRNA (tracrRNA).
The sgRNA consists of crRNA- and tracrRNA-derived sequences connected by an artificial tetraloop. The crRNA sequence can be divided into guide (20-nt) and repeat (12-nt) regions, while the tracrRNA sequence can be divided into anti-repeat (14-nt) and three tracrRNA stem loops. As expected from the RNA-fold predictions based on the nucleotide sequence, the tracrRNA 3′ tail (nucleotides 68-81 and 82-96) forms stem loops 2 and 3 via four and six Watson-Crick base pairs (A69:U80-U72:A77 and G82:C96-G87:C91), respectively. In addition, nucleotides 52-62 form the newly detected stem loop (stem loop 1) via three Watson-Crick base pairs (G53:C61, G54:C60 and C55:G58), with U59 flipped out from the stem. Stem loop 1 is stabilized by the G62-G53:C61 stacking interaction and the G62-A51/A52 polar interactions. TracrRNAs play important roles in adaptive immune response.
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. Apart from Cas9, tracrRNA is the second signature of the Type II systems and both does not share any obvious similarity with the Type I and III systems. In Type II CRISPR-Cas systems, tracrRNA and pre-crRNA undergo coprocessing through base pairing of tracrRNA anti-repeat and pre-crRNA repeats to form a dual-RNA that is cleaved by the housekeeping RNase III in the presence of Cas9 protein. Both co-processed 75-nt tracrRNA and 66-nt intermediate crRNA species carry short overhangs at the 3' end, which is a part of the antirepeat:repeat region and is typical for cleavage by the endoribonuclease RNase III.
According to previous in silico predictions, in contrast to types I and III, type II CRISPR repeats are only weakly palindromic. Hence, they lack per se the distinct characteristic of types I and III repeats to form stem-loop structures required for Cas6-like cleavage of pre-crRNA within the repeats. Base pairing of tracrRNA with pre-crRNA repeats would compensate this deficiency by providing an intermolecular structure that activates a first processing of pre-crRNA by RNase III-mediated cleavage, leading to concomitant processing of tracrRNA itself. Following maturation, the dual-tracrRNA:crRNA structure associated with Cas9 endonuclease constitutes a ternary silencing complex that targets cognate invading DNA. These functional characteristics make the tracrRNA family distinct from other families of non-coding RNAs that ultimately affect the maintenance or function of either mRNAs or proteins.
TracrRNA-mediated crRNA maturation is conserved among different bacterial species. TracrRNA not only plays a key role in the processing of crRNA in Type II CRISPR-Cas systems but also forms an essential component of the Cas9 target recognition and cleavage complex. In particular, following a second maturation event, a mature duplex comprising both the crRNA and tracrRNA bound to Cas9 endonuclease and directs the protein to cleave almost arbitrary invading DNA sequence in a recognition process involving base-pairing complementarity between the guide crRNA sequence of the dual-RNA and the cognate target DNA sequence to introduce a double-strand break (DSB) in the target DNA. A chimeric sgRNA that combines the crRNA and tracrRNA into a single RNA transcript simplifies the system while retaining fully functional Cas9-mediated sequence-specific DNA cleavage.
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