Cas13d Is a Compact RNA-Targeting Type VI CRISPR Effector Positively Modulated by a WYL-Domain-Containing Accessory Protein

Abstract

Bacterial class 2 CRISPR-Cas systems utilize a single RNA-guided protein effector to mitigate viral infection. We aggregated genomic data from multiple sources and constructed an expanded database of predicted class 2 CRISPR-Cas systems. A search for novel RNA-targeting systems identified subtype VI-D, encoding dual HEPN domain-containing Cas13d effectors and putative WYL-domain-containing accessory proteins (WYL1 and WYL-b1 through WYL-b5). The median size of Cas13d proteins is 190 to 300 aa smaller than that of Cas13a-Cas13c. Despite their small size, Cas13d orthologs from Eubacterium siraeum (Es) and Ruminococcus sp. (Rsp) are active in both CRISPR RNA processing and targeting, as well as collateral RNA cleavage, with no target-flanking sequence requirements. The RspWYL1 protein stimulates RNA cleavage by both EsCas13d and RspCas13d, demonstrating a common regulatory mechanism for divergent Cas13d orthologs. The small size, minimal targeting constraints, and modular regulation of Cas13d effectors further expands the CRISPR toolkit for RNA manipulation and detection.

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

“…Type VI CRISPR-Cas systems include a single protein, Cas13 (formerly C2c2), that when assembled with a CRISPR-RNA (crRNA) forms a crRNA-guided RNA-targeting effector complex (Abudayyeh et al, 2016; East-Seletsky et al, 2016; Konermann et al, 2018; Shmakov et al, 2015; Smargon et al, 2017; Yan et al, 2018). 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).…”

“…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).…”

“…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).…”

“…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). …”

“…Versions of Cas13 where the HEPN-domains have been catalytically inactivated (Abudayyeh et al, 2016; East-Seletsky et al, 2016; Konermann et al, 2018; Smargon et al, 2017; Yan et al, 2018) (deactivated Cas13; dCas13) have shown to be useful as programmable RNA binding proteins for RNA imaging (Abudayyeh et al, 2017), RNA editing (Cox et al, 2017) and regulating specific transcripts RNA in other ways (e.g. regulating pre-mRNA splicing (Konermann et al, 2018)).…”