In Silico Evaluation of CRISPR-Based Assays for Effective Detection of SARS-CoV-2

Abstract

Coronavirus disease (COVID-19) caused by the SARS-CoV-2 has been an outbreak since late 2019 up to now. This pandemic causes rapid development in molecular detection technologies to diagnose viral infection for epidemic prevention. In addition to antigen test kit (ATK) and polymerase chain reaction (PCR), CRISPR-based assays for detection of SARS-CoV-2 have gained attention because it has a simple setup but still maintain high specificity and sensitivity. However, the SARS-CoV-2 has been continuing mutating over the past few years. Thus, molecular tools that rely on matching at the nucleotide level need to be reevaluated to preserve their specificity and sensitivity. Here, we analyzed how mutations in different variants of concern (VOC), including Alpha, Beta, Gamma, Delta, and Omicron strains, could introduce mismatches to the previously reported primers and crRNAs used in the CRISPR-Cas system. Over 40% of the primer sets and 15% of the crRNAs contain mismatches. Hence, primers and crRNAs in nucleic acid-based assays must be chosen carefully to pair up with SARS-CoV-2 variants. In conclusion, the data obtained from this study could be useful in selecting the conserved primers and crRNAs for effective detections against the VOC of SARS-CoV-2.

Keywords: COVID-19; CRISPR; SARS-CoV-2; variants of concern.

Figure 1

Structure of SARS-CoV-2 and summary of key mutations on spike proteins in SARS-CoV-2 variants of concern. ACE2: Angiotensin-converting enzyme 2 receptor. HR1: Heptad repeat 1. HR2: Heptad repeat 2. RBD: Receptor binding domain. TMPRSS2: Transmembrane protease serine 2. This figure was created with BioRender.com.

Figure 2

CRISPR-Cas detection system for SARS-CoV-2 RNA. Extracted RNAs from SARS-CoV-2 RNA were reverse-transcribed to cDNA and subsequently amplified by either PCR, LAMP, or RPA. The amplified DNA together with matched crRNA can activate collateral activity in the Cas12a enzyme. For Cas13, the amplified DNA is used as the IVT template to generate multiple copies of RNA targets, which can activate the collateral activity of Cas13 with matched crRNA. The collateral activity of Cas enzyme can cleave nucleotide reporter for fluorescent readout or lateral flow. This figure was created with BioRender.com.