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.
Highlights
We present the latest advances in the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated proteins (Cas)-based nucleic acid detection platform for COVID-19, including strategies that were used to simplify the molecular workflow and to enhance the sensitivity and specificity of the CRISPR-Cas system
Given that reverse transcription polymerase chain reaction (rRT-PCR) is deemed as the standard diagnostic test for the confirmation of COVID-19, Huang et al [41] demonstrated the ease of coupling reverse transcription (RT)-PCR with a CRISPRCas12a assay, termed specific enhancer for detection of PCR-amplified nucleic acids (SENA), to improve the sensitivity and specificity of the technique for SARS-CoV-2 detection [41]
Many CRISPR-Cas13-based detections of SARS-CoV-2 described to date consist of a nucleic acid amplification step, during which a T7 RNA polymerase promoter is incorporated into the amplicons, followed by simultaneous T7 transcription and Cas13a (LwaCas13a) detection via a fluorescence reader or lateral flow device (LFD) [38,39,66,67]
Summary
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Cas9‐sgRNA complexes can be mature CRISPR RNAs (crRNAs), the spacer sequence will serve as guide for the Cas protein made to target ssRNA for site‐specific cleavage in a manner that is similar to PAM‐de‐. In the invading sequence is a prerequisite major characteristics of the Cas proteins used for CRISPR‐based SARS‐CoV‐2 detection is for the PAM-dependent system to target and cleave foreign nucleic while presented in Table 1,CRISPR-Cas including their targeting requirements Following the discovery of target-activated trans-cleavage activity in several Cas proteins, the applications of CRISPR-Cas systems for nucleic acid detection have continued to grow with each passing year. A comparison of major characteristics of the Cas proteins used for CRISPR-based SARS-CoV-2 detection is presented, including their targeting requirements (such as PAM and protospacer flanking sequence (PFS) and guide RNA requirements), cis- and trans-cleavage activities, and on- and off-target substrates A comparison of major characteristics of the Cas proteins used for CRISPR-based SARS-CoV-2 detection is presented in Table 1, including their targeting requirements (such as PAM and protospacer flanking sequence (PFS) and guide RNA requirements), cis- and trans-cleavage activities, and on- and off-target substrates
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