Review
Cutting it close: CRISPR-associated endoribonuclease structure and function

https://doi.org/10.1016/j.tibs.2014.10.007Get rights and content

Highlights

  • We define the key structural features shared by CRISPR-associated endoRNases.

  • We explain how Cas6 enzymes recognize RNA with high affinity and specificity.

  • We review the cleavage mechanisms of different CRISPR-associated endoribonucleases.

  • We describe the roles of Cas5 and Cas6 proteins in CRISPR interference complexes.

  • We discuss future work and development of Cas6-based biotechnological applications.

Many bacteria and archaea possess an adaptive immune system consisting of repetitive genetic elements known as clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins. Similar to RNAi pathways in eukaryotes, CRISPR–Cas systems require small RNAs for sequence-specific detection and degradation of complementary nucleic acids. Cas5 and Cas6 enzymes have evolved to specifically recognize and process CRISPR-derived transcripts into functional small RNAs used as guides by interference complexes. Our detailed understanding of these proteins has led to the development of several useful Cas6-based biotechnological methods. Here, we review the structures, functions, mechanisms, and applications of the enzymes responsible for CRISPR RNA (crRNA) processing, highlighting a fascinating family of endonucleases with exquisite RNA recognition and cleavage activities.

Section snippets

CRISPR–Cas systems and crRNA biogenesis

Most prokaryotes employ an RNA-based adaptive immune system known as CRISPR–Cas to identify and eliminate genetic parasites (reviewed in 1, 2, 3, 4, 5). Upon detecting viral or plasmid DNA in the cell, bacteria and archaea with active CRISPR systems respond by integrating short fragments of foreign DNA into the host chromosome at one end of the CRISPR locus (Figure 1) 6, 7, 8. Such loci serve as molecular vaccination cards by maintaining a genetic record of prior encounters with foreign

Core structural elements of CRISPR endoribonucleases

Cas6 family members share surprisingly limited sequence homology. Nevertheless, the many Cas6 crystal structures (Table 1) reveal a common overall fold as well as specific structural features important for crRNA binding. Cas6 enzymes consist solely of two repeat-associated mysterious protein (RAMP) domains that form ferredoxin-like or RNA recognition motif (RRM) folds, a common feature of Cas proteins 4, 9, 27, 46. Each domain has a βαββαβ secondary structure arrangement, forming a

Structural basis of RNA binding and specificity

A hallmark feature of Cas6 enzymes is the high affinity and specificity with which they bind RNA. Binding affinity appears substantially lower for Cas5c enzymes, and there are currently no available structures of Cas5c proteins bound to substrate or product RNA [22]. The structural basis of sequence- and structure-specific pre-crRNA binding by Cas6 enzymes, however, has been well characterized. The RNA-binding regions of Cas6s are positively charged, allowing the proteins to make extensive

Mechanism of cleavage and active site plasticity

In addition to highly specific RNA binding, Cas5c and Cas6 enzymes cleave RNA exclusively because their catalytic mechanism requires nucleophilic attack of the scissile phosphate by the 2′ hydroxyl group of the 5′ upstream nucleotide 14, 29, 56, 57. Structured pre-crRNAs are cleaved 3′ of the stem loop, at or near its base. Replacing the upstream nucleotide with a deoxyribonucleotide prevents cleavage but permits tight binding, making a singly deoxy-substituted pre-crRNA substrate ideal for

Interference complex assembly and crRNA loading

In the extensively studied Type I-E and I-F systems, Cas6 is an integral subunit of the targeting complex, Cascade. Recently, work on the Type I-B system showed that Cas6 is also part of the Cascade/I-B complex [34]. Cas6e and Cas6f remain tightly bound to the crRNA stem loop after cleavage, serving as a nucleation point for subsequent complex assembly 16, 29, 30, 56. In the P. aeruginosa Type I-F system, pre-crRNA processing is the requisite first step in Cascade/I-F formation 33, 57. In

Concluding remarks

Cas5c and Cas6 proteins make up a class of endoribonucleases required for the generation of mature crRNAs, which are used to target complementary nucleic acids for destruction as part of the CRISPR–Cas adaptive immune system in prokaryotes. Cas6 is the dedicated endoRNase for Type III and most Type I systems. Cas5 proteins are non-catalytic components of the Cascade interference complex in all Type I systems except the I-C subtype, in which the Cas5c variant has evolved to perform the enzymatic

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