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

Abstract

The coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a global threat with an ever-increasing death toll even after a year on. Hence, the rapid identification of infected individuals with diagnostic tests continues to be crucial in the on-going effort to combat the spread of COVID-19. Viral nucleic acid detection via real-time reverse transcription polymerase chain reaction (rRT-PCR) or sequencing is regarded as the gold standard for COVID-19 diagnosis, but these technically intricate molecular tests are limited to centralized laboratories due to the highly specialized instrument and skilled personnel requirements. Based on the current development in the field of diagnostics, the programmable clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system appears to be a promising technology that can be further explored to create rapid, cost-effective, sensitive, and specific diagnostic tools for both laboratory and point-of-care (POC) testing. Other than diagnostics, the potential application of the CRISPR-Cas system as an antiviral agent has also been gaining attention. In this review, we highlight the recent advances in CRISPR-Cas-based nucleic acid detection strategies and the application of CRISPR-Cas as a potential antiviral agent in the context of COVID-19.

Keywords: COVID-19; CRISPR-Dx; antiviral; coronavirus; isothermal amplification.

Figure 1

Molecular mechanism of the CRISPR-Cas system. When a virus attacks a bacterium, a fragment of the genetic material from the invader will be acquired and integrated as a spacer into the host’s CRISPR locus (1). The CRISPR array is transcribed and further processed into crRNA (2) and upon subsequent attack by the same invader, the spacer will guide the Cas protein to cleave the invading nucleic acid sequence (3), thereby protecting the host.

Figure 2

(A) Typical workflow of various CRISPR-Dx for COVID-19 starting from RNA extraction, reverse transcription, amplification, Cas assay, and detection of collateral cleavage activity. (B) Various strategies for the detection of collateral cleavage activity which can be divided into fluorescent-based and colorimetric-based detection. (C) Detection of fluorescein-biotin reporter following Cas assay with a LFD in which the reporter is either cleaved in a positive reaction or remains intact in a negative reaction. Ab: antibody; AuNP: gold nanoparticles; CL: control line; LAMP: loop-mediated isothermal amplification; RAA: recombinase-aided amplification; RPA; recombinase polymerase amplification; TL: test line.

Figure 3

Labeling strategies employed in dCas9-based CRISPR-Dx using LFD for detection. (A) The sgRNA is labeled with fluorescein. (B) The dCas9 is labeled with biotin. In both (A,B), the recognition of labeled target amplicons by labeled dCas9-sgRNA results in the formation of a complex containing both biotin and fluorescein labels, allowing the complex to be captured and visualized on an LFD. (C) The biotinylated and digoxigeninylated amplicons are specifically captured at different test lines on an LFD. DNA conjugated AuNPs are used as universal label and bind to sgRNA of dCas9-sgRNA. Ab: antibody; AuNP: gold nanoparticles; CL: control line; TL: test line.