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

Abstract

As the COVID-19 pandemic continues, people are becoming infected at an alarming rate, individuals are unknowingly spreading disease, and more lives are lost every day. There is an immediate need for a simple, rapid, early and sensitive point-of-care testing for COVID-19 disease. However, current testing approaches do not meet such need. Recently, clustered regularly interspaced short palindromic repeats (CRISPR)-based detection methods have received substantial attention for nucleic acid-based molecular testing due to their simplicity, high sensitivity and specificity. This review explores the various CRISPR-based COVID-19 detection methods and related diagnostic devices. As with any emerging technology, CRISPR/Cas-based nucleic acid testing methods have several challenges that must be overcome for practical applications in clinics and hospitals. More importantly, these detection methods are not limited to COVID-19 but can be applied to detect any type of pathogen, virus, and fungi that may threaten humans, agriculture, and food industries in resource-limited settings. CRISPR/Cas-based detection methods have the potential to become simpler, more reliable, more affordable, and faster in the near future, which is highly important for achieving point-of-care diagnostics.

Keywords: COVID-19; CRISPR-based nucleic acid detection; SARS-CoV-2; coronavirus; point-of-care testing.

Figure 1

Nucleic Acid Detection of SARS-CoV-2 Using CRISPR/Cas Assays. (A) Patient specimens can be collected from different types of clinical samples. (B) RNA is extracted from the specimen. (C) From the nucleic acid extraction, the DNA must be amplified. (D) The nucleic acid of SARS-CoV-2 can now be detected. If a person has COVID-19 (Scenario I), then the CRISPR/Cas complex will bind to the target region of the amplified nucleic acid and collateral cleavage activity can occur by cleaving the nearby fluorescence reporter nucleic acids. This can be detected by either by using the naked eye under specific light, a fluorescence plate reader, or a lateral flow assay that can indicate the presence of the virus’s nucleic acid. If a person does not have COVID-19 (Scenario II), then the CRISPR/Cas complex will not bind to the target region of the amplified nucleic acid and collateral cleavage activity will not be initiated; this means that there will not be any viral signal (glow) from the sample observed by the naked eye, a plate reader, or a lateral flow assay.

Figure 2

SARS-CoV-2 detection on microfluidic diagnostic devices using CRISPR/Cas technology. (A) Fluorescence images of digitization-enhanced CRISPR/Cas-assisted one-pot virus detection (deCOVID) on microfluidic digital chips for SARS-CoV-2 detection (Digital CRISPR/Cas-assisted assay for rapid and sensitive detection of SARS-CoV-2 by Park et al., 2020 is licensed under CC BY 4.0). (B) Working principle of isotachophoresis-CRISPR (ITP-CRISPR) assay. The microfluidic chip has two channels for ITP extraction of nucleic acids (mode 1) and ITP–CRISPR detection (mode 2). In the mode 1, when an electric field is applied, nucleic acids selectively focus within the electromigrating LE–TE interface, leaving behind impurities. After off-chip RT-LAMP of ITP-extracted nucleic acids, in mode 2, ITP is used to detect target DNA by using a CRISPR-Cas12. A positive sample shows a strong fluorescent signal, while negative control not (Electric field-driven microfluidics for rapid CRISPR-based diagnostics and its application to detection of SARS-CoV-2 by Ramachandran et al., 2020 is licensed under CC BY 4.0).