In bacteria and archaea, CRISPR-Cas constitutes an adaptive RNA-mediated defence system which targets invading phages or plasmids in three steps: adaptation via integration of viral or plasmid DNA-derived spacers into the CRISPR locus; expression of short guide CRISPR RNAs (crRNAs) consisting of unique single repeat-spacer units and interference with the invading cognate foreign genomes. Short mature crRNAs are key elements in the interference step of the immune pathway. CRISPR-Cas systems are categorized into three major types (I, II and III) and several subtypes. Types I (except for I-C) and III employ the Cas6-mediated crRNAs processing while the type II CRISPR-Cas systems use the tracrRNA-guided processing mechanism with endogenous RNase III. Type I-C is associated with the Cas5d-mediated processing.

CRISPR RNA (crRNA) Maturation

crRNA maturation is an important process of CRISPR immunity. CRISPR arrays are comprised of a set of cas genes and identical repeats interspaced by spacer sequences acquired from invading mobile genomes. The repeat-spacer array is transcribed as a precursor crRNA (pre-crRNA) molecule. This pre-crRNA undergoes one or two maturation steps to generate the mature crRNAs that function as guides in destruction of invading DNA or RNA. Generally, 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. The maturation of the crRNAs is critical for the activity of the system.

Pathways and enzymes of CRISPR RNA processing
Pathways and enzymes of CRISPR RNA processing

Fig 1. Pathways and enzymes of CRISPR RNA processing. A. Type I and III CRISPR-Cas systems; B. Type II CRISPR-Cas system

CRISPR RNA (crRNA) Maturation in Type I and III CRISPR Systems

A common theme among the CRISPR-Cas types is the transcription of the pre-crRNA and the first muturation processing event within the repeats. In Types I and III, a protein of the Cas6 family or alternatively Cas5d catalyzes this step. The processed crRNAs from Types I-C, I-E and I-F do not undergo further maturation, whereas in at least Types I-A, I-B and I-D, as well as in Types II and III, a second maturation step produces the active crRNAs. In Types I and III, cleavage within each repeat by Cas6 releases the spacers bearing portions of the repeat on its 5' and 3' ends. The 5' flanking repeat of the crRNAs is the last 8 nts of the preceding repeat and the 3' flanking repeat is the remaining sequence of the downstream repeat. In some systems, the 3' flanking repeat sequences are further processed by uncharacterized exonucleases.

Each member of the Cas6 family of endoribonucleases recognizes a unique RNA sequence and collectively, Cas6 nucleases process a wide range of substrates of different sequences and secondary structures. Cas5d is the second distinct class of endoribonucleases responsible for processing crRNA in CRISPR system that lacks Cas6, such as Type I-C. Similar to Cas6, Cas5d also recognizes specific features of and cleaves within the repeat, resulting in crRNAs containing spacer sequences flanked by repeat sequences. While Cas5d has been shown functionally similar to Cas6, other subtypes of Cas5 have no roles in crRNA maturation. Rather, they play a key role in assembly of surveillance or effector complexes.

CRISPR RNA (crRNA) Maturation by tracrRNA and RNase III

Two Cas proteins, Cas6 and Cas5d, have been identified as endoribonucleases that cleave within the repeat sequences of pre-crRNA to generate the mature crRNAs. However, their homologues are missing in many CRISPR-Cas subtypes, suggesting the existence of alternate crRNA maturation pathways involving other Cas proteins and/or fundamentally different RNA processing events. Another pathway of CRISPR activation has been uncovered. In Type II CRISPR-Cas systems, a unique crRNA maturation pathway which distinct from the Type I and III, involves the coordinated action of three novel factors: the trans-acting small RNA (tracrRNA), the host-encoded endoribonuclease RNase III and the Cas9 protein.

TracrRNA and pre-crRNA undergo coprocessing through the double-stranded substrate formed by the base pairing of tracrRNA anti-repeat and pre-crRNA repeats. And then, 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, to generate predictable dsDNA breaks into the target sequence. 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. RNase III which serves as a host factor in tracrRNA-mediated crRNA maturation, is an evolutionarily conserved endoribonuclease involved in many biological processes.

Functions of CRISPR RNA (crRNA)

In the interference step, crRNAs combine with Cas proteins to form an effector complex which recognizes the target sequence in the invasive nucleic acid by base pairing to the complementary strand and induces sequence-specific cleavage, thereby preventing proliferation and propagation of foreign genetic elements. The structural organization and function of effector ribonucleoprotein (RNP) complexes involved in crRNA-mediated silencing of foreign nucleic acids differ between distinct CRISPR-Cas types. In the type I-E systems of E. coli and Streptococcus thermophilus CRISPR4-Cas, crRNAs are incorporated into a multisubunit effector complex called Cascade (CRISPR-associated complex for antiviral defense), which binds to the target DNA and triggers degradation by the signature Cas3 protein.

In type III CRISPR-Cas systems of Sulfolobus solfataricus and Pyrococcus furiosus, RNP complexes of Cas RAMP (Cmr) proteins and crRNA recognize and cleave synthetic RNA in vitro, while the CRISPR-Cas system of Staphylococcus epidermidis targets DNA in vivo. In type II CRISPR-Cas systems, as exemplified by S. pyogenes and S. thermophilus CRISPR3-Cas (St-CRISPR3-Cas), a single Cas9 protein, instead of a multisubunit Cascade or Csm/Cmr protein complex, provides DNA silencing. In fact, type II effector complex functions as an RNA-guided endonuclease which achieves target sequence recognition by crRNA and employs Cas9 protein for DNA cleavage within the target protospacer.

CRISPR RNA (crRNA) Related References

1. Deltcheva et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature. 2011 March 31; 471(7340): 602–607.
2. Karvelis et al. crRNA and tracrRNA guide Cas9-mediated DNA interference in Streptococcus thermophilus. RNA Biology. May 2013; 10(5): 841–851.
3. Haurwitz et al. Sequence- and structure-specific RNA processing by a CRISPR endonuclease. Science. 2010 September 10; 329(5997): 1355–1358.
4. Hong Li. Structural principles of CRISPR RNA processing. Structure. 2015 January 6; 23(1): 13–20.
5. Charpentier et al. Biogenesis pathways of RNA guides in archaeal and bacterial CRISPR-Cas adaptive immunity. FEMS Microbiology Reviews. fuv023, 39, 2015, 428–441.