CRISPR-Based Approaches for Detection of SARS-CoV-2

Abstract and Introduction

Abstract

An outbreak of COVID-19, caused by infection with SARS-CoV-2 in Wuhan, China in December 2019, spread throughout the country and around the world, quickly. The primary detection technique for SARS-CoV-2, the reverse-transcription polymerase chain reaction (RT-PCR)–based approach, requires expensive reagents and equipment and skilled personnel. In addition, for SARS-CoV-2 detection, specimens are usually shipped to a designated laboratory for testing, which may extend the diagnosis and treatment time of patients with COVID-19. The latest research shows that clustered regularly interspaced short palindromic repeats (CRISPR)–based approaches can quickly provide visual, rapid, ultrasensitive, and specific detection of SARS-CoV-2 at isothermal conditions. Therefore, CRISPR-based approaches are expected to be developed as attractive alternatives to conventional RT-PCR methods for the efficient and accurate detection of SARS-CoV-2. Recent advances in the field of CRISPR-based biosensing technologies for SARS-CoV-2 detection and insights into their potential use in many applications are reviewed in this article.

Introduction

Coronaviruses (CoVs), with 4 major structural proteins including spike, membrane, envelope, and nucleoprotein, are positive-sense, single-strand RNA viruses.^[1,2] Before SARS-CoV-2, there were 6 CoVs that were known to be pathogenic to humans: HCoV-OC43, HCoV-NL63, HCoV-HKU1, HCoV-229E, SARS-CoV, and MERS-CoV,^[3–5] with the latter 2 being highly transmissible and pathogenic. SARS-CoV-2 (previously named 2019-nCoV) is a new coronavirus causing COVID-19, which was first observed in December 2019 in Wuhan, China.^[6,7] As of August 17, 2020, based on the data provided by the World Health Organization, 7,716,255 people were confirmed to be infected globally, with 774,413 deaths. According to a response plan recently shared by the US government with the New York Times, the SARS-CoV-2 pandemic may continue for more than 18 months. According to this document, a "multi-wave disease" may occur in the next year and a half. Therefore, there is an urgent need for a point-of-care diagnosis method that can be used for SARS-CoV-2 screening.

Currently, nucleic-acid-based tests have been widely used as the standard method for the detection of SARS-CoV-2. Metagenomic next-generation sequencing (mNGS) and reverse-transcription polymerase chain reaction (RT-PCR) are 2 molecular methods that are frequently used for the diagnosis of SARS-CoV-2.^[8–11] Originally used for the identification of this new viral species, mNGS is considered one of the most important methods of detection. However, its wider application is limited by its cost and longer detection time of nearly a day. Therefore, mNGS is not suitable for large-scale screening for SARS-CoV-2.^[11,12] In addition, RT-PCR assay for the detection of SARS-CoV-2 is faster and more affordable in comparison than mNGS-based approaches. Nevertheless, the need for a thermocycler for RT-PCR-based diagnostics hinders its use in low-resource settings and curbs the assay throughput. In addition, currently available RT-PCR kits are variable, offering sensitivities ranging between 45% and 60%. Thus, in the early course of an infection, repeat testing may be required to reach a diagnosis.^[13] Consequently, RT-PCR and mNGS-based approaches are not suitable for the point-of-care diagnosis of SARS-CoV-2.

Aside from a lower demand for sophisticated temperature controlling instruments, isothermal molecular methods are advantageous beause of faster nucleic acid amplification.^[14,15] Clustered regularly interspaced short palindromic repeats (CRISPR) is a biotechnologic technique well-known for its use in gene editing. In addition, CRISPR has been recently used for the in vitro detection of nucleic acids. The latest research shows that CRISPR-based approaches can rapidly and efficiently detect SARS-CoV-2 with high sensitivity and specificity at isothermal conditions.^[16,17] Therefore, CRISPR-based approaches, emerging as a powerful and precise tool for SARS-CoV-2 diagnosis, are expected to be used for SARS-CoV-2 screening in homes and primary hospitals.