Digital CRISPR/Cas‐Assisted Assay for Rapid and Sensitive Detection of SARS‐CoV‐2

Abstract

The unprecedented demand for rapid diagnostics in response to the COVID‐19 pandemic has brought the spotlight onto clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated systems (Cas)‐assisted nucleic acid detection assays. Already benefitting from an elegant detection mechanism, fast assay time, and low reaction temperature, these assays can be further advanced via integration with powerful, digital‐based detection. Thus motivated, the first digital CRISPR/Cas‐assisted assay—coined digitization‐enhanced CRISPR/Cas‐assisted one‐pot virus detection (deCOViD)—is developed and applied toward SARS‐CoV‐2 detection. deCOViD is realized through tuning and discretizing a one‐step, fluorescence‐based, CRISPR/Cas12a‐assisted reverse transcription recombinase polymerase amplification assay into sub‐nanoliter reaction wells within commercially available microfluidic digital chips. The uniformly elevated digital concentrations enable deCOViD to achieve qualitative detection in <15 min and quantitative detection in 30 min with high signal‐to‐background ratio, broad dynamic range, and high sensitivity—down to 1 genome equivalent (GE) µL−1 of SARS‐CoV‐2 RNA and 20 GE µL−1 of heat‐inactivated SARS‐CoV‐2, which outstrips its benchtop‐based counterpart and represents one of the fastest and most sensitive CRISPR/Cas‐assisted SARS‐CoV‐2 detection to date. Moreover, deCOViD can detect RNA extracts from clinical samples. Taken together, deCOViD opens a new avenue for advancing CRISPR/Cas‐assisted assays and combating the COVID‐19 pandemic and beyond.

A Warm-start Digital CRISPR-based Method for the Quantitative Detection of Nucleic Acids

“…The copyright holder for this preprint this version posted June 15, 2021. ; https://doi.org/10.1101/2021.06.10.21258725 doi: medRxiv preprint was a strong linear relationship (R 2 = 0.99) between the target concentrations and the percentage of positive partitions in both DNA and RNA samples (Figures 4C and 4E). The measured concentration of WS-RADICA based on Poisson distribution was lower than the input target concentration, which can possibly be attributed to "molecular dropout" or low filling rate of the microwells, as has been previously reported for dPCR, dRPA, and dLAMP reactions 3,24,25,31 . Nevertheless, WS-RADICA has a single reaction temperature and faster reaction time, which are important advantages.…”

“…To achieve rapid quantification, our group and three other groups recently have developed digital CRISPR-based methods for nucleic acid quantification [24][25][26][27][28] . Most of these methods were performed at 37ºC or 42ºC, with the sample preparation at low temperature to prevent target amplification or target cleavage before loading on the chip 24,[26][27][28] . Another method elevated the reaction temperature by coupling low-temperature reverse transcription dualpriming isothermal amplification (RT-DAMP) with Cas12a, which allows quantification at 52ºC but has low sensitivity.…”

Point-of-care CRISPR-Cas-assisted SARS-CoV-2 detection in an automated and portable droplet magnetofluidic device

“…Consistent with our previous DM devices ( Shin et al, 2018 , 2017 ), we employed ChargeSwitch magnetic beads and binding buffer as our magnetic bead buffer. For SARS-CoV-2 amplification and detection, a fluorescence-based CRISPR-Cas12a-assisted RT-RPA is adopted and modified from AIOD-CRISPR ( Ding et al, 2020 ) and our recently developed deCOViD ( Park et al, 2021 ) that used a more sensitive, Alexa647 fluorophore-based ssDNA fluorogenic reporter ( Hsieh et al, 2020 ) (Table S1). For developing this new assay, we mixed SARS-CoV-2 RNA with the magnetic bead buffer and used a magnetic rack to pellet and wash the magnetic beads and bound RNA.…”

“…Despite their seminal status, both DETECTR-based ( Broughton et al, 2020 ) and SHERLOCK-based SARS-CoV-2 detection ( Patchsung et al, 2020 ) are ill-suited for POC use because they involve multiple manual assay steps and can only be performed with input SARS-CoV-2 RNA that is already extracted and purified by benchtop instruments. More recent assays ( Arizti-Sanz et al, 2020 ; Ding et al, 2020 ; Fozouni et al, 2021 ; Joung et al, 2020 ; Ning et al, 2021 ; Park et al, 2021 ) are more tenable for POC use but have yet to fully meet these requirements. For example, AIOD-CRISPR ( Ding et al, 2020 ), a one-step CRISPR-Cas12a-assisted reverse transcription recombinase polymerase amplification ( Piepenburg et al, 2006 ) (RT-RPA) assay, and the amplification-free CRISPR-Cas13a assay ( Fozouni et al, 2021 ) have incorporated portable detection modalities (e.g., mobile phone), but both still require benchtop-extracted SARS-CoV-2 RNA as the input and separate heating modules for incubation.…”

CRISPR-Based COVID-19 Testing: Toward Next-Generation Point-of-Care Diagnostics

“…With a similar goal of achieving faster, more accurate quantitative analysis, digitization-enhanced CRISPR/Casassisted one-pot virus detection (deCOVID) was developed using a similar approach as the devices mentioned above (Figure 2A). This device is faster but requires more components like WS-CRISPR (Park et al, 2020). The researchers used 0.1% Tween-20 to reduce the viscosity of the reaction mix to be compatible with digital PCR.…”