The Molecular Architecture for RNA-Guided RNA Cleavage by Cas13a

Abstract

Cas13a, a type VI-A CRISPR-Cas RNA-guided RNA ribonuclease, degrades invasive RNAs targeted by CRISPR RNA (crRNA) and has potential applications in RNA technology. To understand how Cas13a is activated to cleave RNA, we have determined the crystal structure of Leptotrichia buccalis (Lbu) Cas13a bound to crRNA and its target RNA, as well as the cryo-EM structure of the LbuCas13a-crRNA complex. The crRNA-target RNA duplex binds in a positively charged central channel of the nuclease (NUC) lobe, and Cas13a protein and crRNA undergo a significant conformational change upon target RNA binding. The guide-target RNA duplex formation triggers HEPN1 domain to move toward HEPN2 domain, activating the HEPN catalytic site of Cas13a protein, which subsequently cleaves both single-stranded target and collateral RNAs in a non-specific manner. These findings reveal how Cas13a of type VI CRISPR-Cas systems defend against RNA phages and set the stage for its development as a tool for RNA manipulation.

RNA Binding and HEPN-Nuclease Activation Are Decoupled in CRISPR-Cas13a

“…This was motived by the observation that that Cas13a’s HEPN-nuclease activity can be activated using a range crRNA guide sequence lengths (Abudayyeh et al, 2017; Abudayyeh et al, 2016; Cox et al, 2017; East-Seletsky et al, 2016; East-Seletsky et al, 2017; Gootenberg et al, 2017; Knott et al, 2017; Konermann et al, 2018; Liu et al, 2017a; Smargon et al, 2017; Yan et al, 2018), and led us to perform the same fluorescence-anisotropy binding assay with an analogous set of fluorophore-labeled mismatched activator-RNAs and a crRNA containing a 24-nt. guide sequence (Fig.…”

“…First, upon binding a precursor-crRNA (pre-crRNA), Cas13 cleaves within the crRNA direct repeat in a pre-crRNA array to form mature Cas13-crRNA complexes (East-Seletsky et al, 2016; East-Seletsky et al, 2017; Konermann et al, 2018; Smargon et al, 2017; Yan et al, 2018). Second, binding of an RNA target complementary to the crRNA (henceforth referred to as an activator-RNA) triggers Cas13 to cleave RNA non-specifically by activating the enzyme’s two HEPN-domains to form a single composite RNase active site (Abudayyeh et al, 2016; East-Seletsky et al, 2016; Konermann et al, 2018; Liu et al, 2017a; Liu et al, 2017b; Smargon et al, 2017; Yan et al, 2018). This HEPN-activated conformation of Cas13 is a general nuclease, which is capable of cleaving either the RNA molecule that it was activated by ( cis cleavage), or any other RNA molecule that it happens to encounter ( trans / collateral cleavage).…”

Implementation of the CRISPR-Cas13a system in fission yeast and its repurposing for precise RNA editing

“…This design proved effective and efficient in generating crRNA alone, or crRNA and pRNA molecules that were either separated or joined. Note that the reported intrinsic RNase activity for the processing of pre-crRNA by Cas13a could be used to simplify our system (8,42). However, in our study design, we were concerned that the inactive LshCas13a (dCas13a) (to be employed in constructing the RNA editing complex) might impair the pre-crRNA processing ability.…”

“…The reported crystal structure of LshCas13a in complex with crRNA suggests that nucleotide bases 24–28 of the crRNA are exposed on the outside of the LshCas13a structure (42). Furthermore, based on the crystal structure of another closely related Cas13a from Lachnospiraceae bacterium (LbaCas13a), the first 13 nucleotides (C1–G13) of the spacer in the LbaCas13a/crRNA complex are well ordered and sequestered within the LbaCas13a protein, whereas the rest 11 (G14–C24) were disordered and exposed in solvent (9).…”

“…For example, an excessive amount of crRNA–pRNA over dCas13a-hADAR2d molecules would prevent the dCas13a-hADAR2d/crRNA–pRNA complex from acting on target RNAs by pre-occupying their proto-spacer sites on target RNA. It was also found that the formation of a Cas13a and crRNA complex was a pre-requisite for their recognition and binding of target RNA (9,42). Further investigation is warranted to illustrate the mechanism underlying the specificity and efficiency of the Cas13-mediated RNA interference system.…”