A Sensitive, Rapid, and Portable CasRx-based Diagnostic Assay for SARS-CoV-2

Abstract

Since its first emergence from China in late 2019, the SARS-CoV-2 virus has spread globally despite unprecedented containment efforts, resulting in a catastrophic worldwide pandemic. Successful identification and isolation of infected individuals can drastically curtail virus spread and limit outbreaks. However, during the early stages of global transmission, point-of-care diagnostics were largely unavailable and continue to remain difficult to procure, greatly inhibiting public health efforts to mitigate spread. Furthermore, the most prevalent testing kits rely on reagent- and time-intensive protocols to detect viral RNA, preventing rapid and cost-effective diagnosis. Therefore the development of an extensive toolkit for point-of-care diagnostics that is expeditiously adaptable to new emerging pathogens is of critical public health importance. Recently, a number of novel CRISPR-based diagnostics have been developed to detect COVID-19. Herein, we outline the development of a CRISPR-based nucleic acid molecular diagnostic utilizing a Cas13d ribonuclease derived from Ruminococcus flavefaciens (CasRx) to detect SARS-CoV-2, an approach we term SENSR (Sensitive Enzymatic Nucleic-acid Sequence Reporter). We demonstrate SENSR robustly detects SARS-CoV-2 sequences in both synthetic and patient-derived samples by lateral flow and fluorescence, thus expanding the available point-of-care diagnostics to combat current and future pandemics.

Harnessing CRISPR-Cas to Combat COVID-19: From Diagnostics to Therapeutics

“…A PPA of 57% and NPA of 100% were obtained when the performance of the SENSR targeting the N gene was evaluated with 21 positive and 21 negative SARS-CoV-2 clinical samples. This proof-of-concept work by Brogan et al [71] demonstrated the potential of utilizing Cas13d in CRISPR-Dx and highlights the possibility of combining Cas13d with other Cas proteins that lack poly-U preference for multiplex detection [71]. However, the low diagnostic sensitivity of SENSR indicated that further optimization is required.…”

“…The sensitive enzymatic nucleic-acid sequence reporter (SENSR) differed from the abovementioned CRISPR-Cas13-based assays for SARS-CoV-2 detection as the platform uses RfxCas13d (CasRx) from Ruminococcus flavefaciens. Similar to LwaCas13a, Cas13d is an RNA-guided RNA targeting Cas protein that does not require PFS and exhibits collateral cleavage activity upon target RNA binding, but Cas13d is ~20% smaller than Cas13a-Cas13c effectors [71]. SENSR is a two-step assay that consists of RT-RPA to amplify the target N or E genes of SARS-CoV-2 followed by T7 transcription and CasRx assay.…”

A Novel Miniature CRISPR-Cas13 System for SARS-CoV-2 Diagnostics

“… 20 , 22 − 24 , 27 Different Cas13 variants are used for nucleic acid detection. For example, Cas13a, 21 , 39 Cas13b, 40 and Cas13d 41 exhibit collateral cleavage activities and work for nucleic acid detection. Here, we sought to expand the Cas13-based toolbox for diagnostic applications by identifying and characterizing novel CRISPR/Cas13 effectors.…”

A field-deployable method for single and multiplex detection of DNA or RNA from pathogens using Cas12 and Cas13

“…Cas13d has a remarkably compact protein size, and one ortholog derived from Ruminococcus flavefaciens XPD3002 (RfxCas13d) has highly efficient, robust, specific RNA interference activity in both mammals and plants (Konermann et al, 2018;Kushawah et al, 2020;Mahas et al, 2019;Yan et al, 2018;Zhou et al, 2020). The collateral activity of RfxCas13d was recently developed as a diag-nostic tool for SARS-CoV-2 (Brogan et al, 2020).…”