Sequence-specific capture and concentration of viral RNA by type III CRISPR system enhances diagnostic

Abstract

Type-III CRISPR-Cas systems have recently been adopted for sequence-specific detection of SARS-CoV-2. Here, we make two major advances that simultaneously limit sample handling and significantly enhance the sensitivity of SARS-CoV-2 RNA detection directly from patient samples. First, we repurpose the type III-A CRISPR complex from Thermus thermophilus (TtCsm) for programmable capture and concentration of specific RNAs from complex mixtures. The target bound TtCsm complex primarily generates two cyclic oligoadenylates (i.e., cA3 and cA4) that allosterically activate ancillary nucleases. To improve sensitivity of the diagnostic, we identify and test several ancillary nucleases (i.e., Can1, Can2, and NucC). We show that Can1 and Can2 are activated by both cA3 and cA4, and that different activators trigger changes in the substrate specificity of these nucleases. Finally, we integrate the type III-A CRISPR RNA-guided capture technique with the Can2 nuclease for 90 fM (5x104 copies/ul) detection of SARS-CoV-2 RNA directly from nasopharyngeal swab samples.

Cas12a2 elicits abortive infection through RNA-triggered destruction of dsDNA

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

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 .…”

CRISPR-based nucleic acid diagnostics for pathogens

“…Several nucleic acid sensing strategies were reported by utilizing different auxiliary CRISPR-associated enzymes, all of which can be activated by cOA with ring sizes ranging from cA3 to cA6, such as Csm6, Can1, Can2 and NucC [25,[33][34][35][36][37]. A particular type III-A system present in Lactobacillus delbrueckii subsp.…”

“…Nucleic acid diagnostics using subtype III-B systems were alike to subtype III-A, mainly performing the cOA signaling that generally using TTHB144 [38] and NucC [34]. Furthermore, subtype III-A system from Thermus thermophilus (TtCsm) can simultaneously generate cA3 and cA4 that can be respectively recognized by NucC and Can2, thus combining with two ancillary nucleases could improve sensitivity of the nucleic acid diagnostic [33].…”

“…Besides, harnessing the changes in pH and pyrophosphate concentration that occur during cyclic nucleic polymerization could realize the colorimetric or visible fluorometric detection of SARS-CoV-2 [35]. Inspired by the FIND-IT assay using activator variant for TtCsm6 [62], Wiedenheft group adopted the Can2 nuclease to assist the signaling of type III-A CRISPR capture technique [33]. Use of Can2 lacking the ring nuclease activity can sustain the high level of cA4 activator, contributing to sensitivity improvement.…”