Intrinsic Signal Amplification by Type-III CRISPR-Cas Systems Provides a Sequence-Specific Viral Diagnostic

Abstract

To combat viral pandemics, there is an urgent need for inexpensive new technologies that enable fast, reliable, and scalable detection of viruses. Here we repurposed the type III CRISPR-Cas system for sensitive and sequence specific detection of SARS-CoV-2 in an assay that can be performed in one hour or less. RNA recognition by type III systems triggers Cas10-mediated polymerase activity, which simultaneously generates pyrophosphates, protons and cyclic oligonucleotides. We show that amplified products of the Cas10-polymerase are detectable using colorimetric or fluorometric readouts.

An engineered CRISPR-Cas12a variant and DNA-RNA hybrid guides enable robust and rapid COVID-19 testing

“…Hence, the ideal situation is to utilize a thermophilic Cas enzyme that can function well at 60°C or above. However, we are not aware of such an enzyme besides the AapCas12b nuclease used in STOPCovid 22 and iSCAN 28 as well as the TtCsm complex used in CRISPR-Csm 39 . Fourth, for a rapid diagnostic test to be truly useful in a point-ofneed setting, it should be able to accept patient samples directly.…”

Accelerated RNA detection using tandem CRISPR nucleases

“…Detection takes advantage of the intrinsic enzymatic functions of CRISPR-Cas proteins from Type III and Type VI CRISPR-Cas systems, including the multisubunit effector Csm/Cmr, or the single-protein effector Cas13 1,2,9,10 . Target RNA recognition by the effector triggers multiple-turnover trans -cleavage of single-stranded RNA (ssRNA) by either the same protein, in the case of Cas13, or by a separate protein called Csm6, in the case of Csm/Cmr 1,2,11,12 .…”

“…Enzymatic RNA cleavage generates a fluorescent signal when directed towards a reporter oligonucleotide containing a quencher and dye pair 1,2,13 . Cas13 or Csm/Cmr is capable of reaching an RNA detection sensitivity in the attomolar range in under an hour when coupled to a target sequence amplification procedure, such as reverse transcription-recombinase polymerase amplification (RT-RPA) or reverse transcription-loop-mediated isothermal amplification (RT-LAMP) 1,2,9,10,14,15 . However, addition of RT-RPA or RT-LAMP requires multiple liquid handling steps and/or high-temperature incubation (55-65°C), procedures that are challenging for point-of-care testing 9,10,16–18 .…”

“…Cas13 or Csm/Cmr is capable of reaching an RNA detection sensitivity in the attomolar range in under an hour when coupled to a target sequence amplification procedure, such as reverse transcription-recombinase polymerase amplification (RT-RPA) or reverse transcription-loop-mediated isothermal amplification (RT-LAMP) 1,2,9,10,14,15 . However, addition of RT-RPA or RT-LAMP requires multiple liquid handling steps and/or high-temperature incubation (55-65°C), procedures that are challenging for point-of-care testing 9,10,16–18 . We reported that amplification-free RNA detection using L. buccalis Cas13 (LbuCas13) with three guide RNAs could rapidly identify SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) genomic RNA in patient samples with PCR-derived cycle threshold (Ct) values up to 22, corresponding to ∼1600 copies/µl in the assay 19 .…”

“…Csm6, a dimeric RNA endonuclease from Type III CRISPR-Cas systems, has the potential to boost RNA detection based on its endogenous function in signal amplification 9,10,20 . During CRISPR-Cas interference, activation of Csm or Cmr by viral RNA recognition triggers synthesis of cyclic tetra- or hexaadenylates (cA 4 or cA 6 ) that bind to the Csm6 CRISPR-associated Rossman fold (CARF) and activate its higher eukaryote/prokaryote nucleotide binding (HEPN) domain’s ribonuclease activity 7,11,12,21 .…”