RNA Targeting by Functionally Orthogonal Type VI-A CRISPR-Cas Enzymes

Abstract

CRISPR adaptive immunity pathways protect prokaryotic cells against foreign nucleic acids using CRISPR RNA (crRNA)-guided nucleases. In type VI-A CRISPR-Cas systems, the signature protein Cas13a (formerly C2c2) contains two separate ribonuclease activities that catalyze crRNA maturation and ssRNA degradation. The Cas13a protein family occurs across different bacterial phyla and varies widely in both protein sequence and corresponding crRNA sequence conservation. Although grouped phylogenetically together, we show that the Cas13a enzyme family comprises two distinct functional groups that recognize orthogonal sets of crRNAs and possess different ssRNA cleavage specificities. These functional distinctions could not be bioinformatically predicted, suggesting more subtle co-evolution of Cas13a enzymes. Additionally, we find that Cas13a pre-crRNA processing is not essential for ssRNA cleavage, although it enhances ssRNA targeting for crRNAs encoded internally within the CRISPR array. We define two Cas13a protein subfamilies that can operate in parallel for RNA detection both in bacteria and for diagnostic applications.

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.…”

“…Taken together, these similarities suggest broad conservation of activator-RNA binding and HEPN activation mechanisms (and their sensitivity to mismatches) across the U-cleaving Cas13a family. Subtle variation in the magnitude or position of these effects may exist between homologs, given the broad range of activator sensitivities and rates of trans -ssRNA cleavage observed across this family (Abudayyeh et al, 2016; East-Seletsky et al, 2016; East-Seletsky et al, 2017; Gootenberg et al, 2018; Gootenberg et al, 2017). …”

“…Cas13 proteins are classified into distinct subfamilies (Cas13a-d), and all Cas13 proteins studied to date possess two enzymatically distinct RNase activities that are required for optimal interference (Abudayyeh et al, 2016; East-Seletsky et al, 2016; Konermann et al, 2018; Smargon et al, 2017; Yan et al, 2018). 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). All Cas13 proteins characterized to date exhibit these behaviors (Abudayyeh et al, 2016; East-Seletsky et al, 2016; East-Seletsky et al, 2017; Gootenberg et al, 2018; Gootenberg et al, 2017; Konermann et al, 2018; Smargon et al, 2017; Yan et al, 2018). …”

Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6

“…Many applications require detection of more than one target molecule in a single reaction, and we therefore sought to create a multiplex platform that relies on unique cleavage preferences of Cas enzymes(2–5). To identify possible candidate enzymes compatible with multiplexing, we biochemically characterized three members of the CRISPR-Cas13a family and fourteen members of the CRISPR-Cas13b family(6, 7) (fig.…”