Equipment-free detection of SARS-CoV-2 and Variants of Concern using Cas13

Abstract

The COVID-19 pandemic, and the recent rise and widespread transmission of SARS-CoV-2 Variants of Concern (VOCs), have demonstrated the need for ubiquitous nucleic acid testing outside of centralized clinical laboratories. Here, we develop SHINEv2, a Cas13-based nucleic acid diagnostic that combines quick and ambient temperature sample processing and lyophilized reagents to greatly simplify the test procedure and assay distribution. We benchmarked a SHINEv2 assay for SARS-CoV-2 detection against state-of-the-art antigen-capture tests using 96 patient samples, demonstrating 50-fold greater sensitivity and 100% specificity. We designed SHINEv2 assays for discriminating the Alpha, Beta, Gamma and Delta VOCs, which can be read out visually using lateral flow technology. We further demonstrate that our assays can be performed without any equipment in less than 90 minutes. SHINEv2 represents an important advance towards rapid nucleic acid tests that can be performed in any location.

Multiplexed biosensor for point-of-care COVID-19 monitoring: CRISPR-powered unamplified RNA diagnostics and protein-based therapeutic drug management

“…The latter can achieve remarkably low LODs due to its capability of trans-cleaving approximately 10 4 (re)RNA molecules in its vicinity upon activation by recognition of the target sequence 60 . Nevertheless, most methods developed for the detection of SARS-CoV-2 genes employ either reverse transcription loop-mediated isothermal amplification (RT-LAMP; LOD: 100 copies/µl, 90 min sample-to-result time 61 ) or reverse transcription recombinase polymerase amplification (RT-RPA; LOD: 2.5-100 copies/µl, sample-to-result-time: 20 min - 1 hour 42,62,63 ) as a target-amplification strategy or even both (LOD: 82 copies/µl after ca. 50 min 64 ).…”

COVID-19 Variant Detection with a High-Fidelity CRISPR-Cas12 Enzyme

“…For example, the miSHERLOCK variant assay uses LbCas12a (NEB) with RPA pre-amplification to detect N501Y, E484K and Y144Del covering eight lineages (WA-1, Alpha, Beta, Gamma, Eta, Iota, Mu and Zeta) and was tested only on contrived samples (RNA spiked into human saliva) 21 . The SHINEv2 assay uses LwaCas13a with RPA pre-amplification to detect 69/70Del, K417N/T, L452R and 156/157Del + R158G covering eight lineages (WA-1, Alpha, Beta, Gamma, Delta, Epsilon, Kappa and Mu) and was tested with only the 69/70Del gRNAs on 20 Alpha-positive NP clinical samples 49 . Finally, the mCARMEN variant identification panel (VIP) uses 26 crRNA pairs with either the LwaCas13a or LbaCas13a and PCR pre-amplification to identify all current circulating lineages including Omicron; however, the VIP requires the Fluidigm Biomark HD system or similar, more complex instrumentation for streamlined execution 50 .…”

“…For example, the miSHERLOCK variant assay uses LbCas12a (NEB) with RPA preamplification to detect N501Y, E484K and Y144Del covering eight lineages (WA-1, Alpha, Beta, Gamma, Eta, Iota, Mu and Zeta) and was tested only on contrived samples (RNA spiked into human saliva) 21 . The SHINEv2 assay uses LwaCas13a with RPA pre-amplification to detect 69/70Del, K417N/T, L452R and 156/157Del + R158G covering eight lineages (WA-1, Alpha, Beta, Gamma, Delta, Epsilon, Kappa and Mu) and was tested with only the 69/70Del gRNAs on 20 Alpha-positive NP clinical samples 49 .…”

RT-PCR/MALDI-TOF diagnostic target performance reflects circulating SARS-CoV-2 variant diversity in New York City

“…A number of recent reports have focused on variant panels designed to target a finite number of emergent mutations to discern variant identity, particularly in the first months of 2021. Most panels utilize RT-PCR or novel isothermal technologies (e.g., RT-LAMP, CRISPR/Cas-based, massspectrometry) to target specific polymorphisms in the S gene that have been ascribed to each of these variants 25,27,45,[48][49][50] . However, a recent deep analysis of circulating variants across the globe revealed that most of these signature mutations are not under positive selection 1 .…”