Specificity and sensitivity of an RNA targeting type III CRISPR complex coupled with a NucC endonuclease effector

Abstract

Type III CRISPR systems detect invading RNA, resulting in the activation of the enzymatic Cas10 subunit. The Cas10 cyclase domain generates cyclic oligoadenylate (cOA) second messenger molecules, activating a variety of effector nucleases that degrade nucleic acids to provide immunity. The prophage-encoded Vibrio metoecus type III-B (VmeCmr) locus is uncharacterised, lacks the HD nuclease domain in Cas10 and encodes a NucC DNA nuclease effector that is also found associated with Cyclic-oligonucleotide-based anti-phage signalling systems (CBASS). Here we demonstrate that VmeCmr is activated by target RNA binding, generating cyclic-triadenylate (cA3) to stimulate a robust NucC-mediated DNase activity. The specificity of VmeCmr is probed, revealing the importance of specific nucleotide positions in segment 1 of the RNA duplex and the protospacer flanking sequence (PFS). We harness this programmable system to demonstrate the potential for a highly specific and sensitive assay for detection of the SARS-CoV-2 virus RNA with a limit of detection (LoD) of 2 fM using a commercial plate reader without any extrinsic amplification step. The sensitivity is highly dependent on the guide RNA used, suggesting that target RNA secondary structure plays an important role that may also be relevant in vivo.

Cas12a2 elicits abortive infection via RNA-triggered destruction of double-stranded DNA

“…Specifically, the mechanism exhibited by Cas12a2 is reminiscent of the recently described CBASS defense system, which relies on the indiscriminate double-stranded DNAse NucC that degrades the host cell DNA and kills the cell 12 . Interestingly, some type III systems encode NucC enzymes, suggesting that other CRISPR-Cas systems have convergently evolved to use a similar DNA degrading Abi mechanism 13, 48 .…”

“…Interestingly, some type III systems encode NucC enzymes, suggesting that other CRISPR-Cas systems have convergently evolved to use a similar DNA degrading Abi mechanism 13,48 .…”

“…In each case, the indiscriminate RNase activities cleave both viral and host RNAs, reducing virus replication and causing cellular dormancy as an abortive phenotype [3][4][5] . It has been proposed that CRISPR-mediated DNase activity may also cause Abi through indiscriminate dsDNases (e.g., NucC) activated by type III system secondary messengers 12,13 , or indiscriminate ssDNase activity from type V Cas12a effectors 14 . However, type III CRISPR-mediated dsDNase activity has yet to be examined in vivo, and the ssDNase activity of Cas12a has not been shown to cause Abi 15 .…”

Type III CRISPR–Cas provides resistance against nucleus-forming jumbo phages via abortive infection

“…The copyright holder for this preprint this version posted June 20, 2022. ; https://doi.org/10.1101/2022.06.20.496707 doi: bioRxiv preprint accessory nucleases (Grüschow et al, 2021;Kazlauskiene et al, 2017;McMahon et al, 2020;Niewoehner et al, 2017;Røstol et al, 2021;Rouillon et al, 2018), and (3) the specific RNA bound to the interference complex is cleaved by each Cas7 (Csm3/Cmr4) subunit (Samai et al, 2015b;Staals et al, 2014). This multi-step pathway provides a powerful response to clear phage infection and remains active until the phage DNA is cleared and no longer transcribes any 'activating' target RNA (Jiang et al, 2016;Røstol and Marraffini, 2019;Rouillon et al, 2018).…”

Structural principles of CRISPR-Cas enzymes used in nucleic acid detection

“…The ability to re-engineer Type III systems towards the detection of nucleic acids has been demonstrated in recent studies towards diagnosing SARS-CoV-2, either by employing a Csm-Csm6 system ( Sridhara et al, 2021 ; Santiago-Frangos et al, 2021 ), a Cmr-TTHB144 system ( Steens et al, 2021 ) or a Cmr-NucC hybrid system ( Gruschow et al, 2021 ). This application depends on the viral RNA-stimulated dual collateral activities in these Type III systems ( Figs.…”