Diagnostics of COVID-19 Based on CRISPR-Cas Coupled to Isothermal Amplification: A Comparative Analysis and Update

Abstract

The emergence of the COVID-19 pandemic prompted fast development of novel diagnostic methods of the etiologic virus SARS-CoV-2. Methods based on CRISPR-Cas systems have been particularly promising because they can achieve a similar sensitivity and specificity to the benchmark RT-qPCR, especially when coupled to an isothermal pre-amplification step. Furthermore, they have also solved inherent limitations of RT-qPCR that impede its decentralized use and deployment in the field, such as the need for expensive equipment, high cost per reaction, and delivery of results in hours, among others. In this review, we evaluate publicly available methods to detect SARS-CoV-2 that are based on CRISPR-Cas and isothermal amplification. We critically analyze the steps required to obtain a successful result from clinical samples and pinpoint key experimental conditions and parameters that could be optimized or modified to improve clinical and analytical outputs. The COVID outbreak has propelled intensive research in a short time, which is paving the way to develop effective and very promising CRISPR-Cas systems for the precise detection of SARS-CoV-2. This review could also serve as an introductory guide to new labs delving into this technology.

Keywords: CRISPR–Cas; SARS-CoV-2; comparative analysis; isothermal amplification; molecular diagnostics; nucleic acid detection.

Figure 1

(A) Structural and genome features of SARS-CoV-2 virion. Class 2 CRISPR–Cas proteins extensively used in genetic diagnostics: (B) LbCas12a (type V, PDB: 5XUS) [28] (left) and (C) LbuCas13a (type VI, PDB: 5XWP) [29] (right). Colors represent different domains of Cas proteins. LbCas12a: Wedge I, II, and III (yellow), REC1 (light gray), REC2 (dark gray), PI (wheat), RuvC-I, II, and II (cyan), BH (green lime), and Nuc (magenta). LbuCas13a: NTD (cyan), Helical-1 (wheat), HEPN1-I and II (green lime), Helical-2 (yellow), Linker (orange), and HEPN2 (magenta). Schematics depicting DNA in black and the primers used for isothermal amplification methods in colors: RT-LAMP (D) and RT-RPA (E) and the target sequence for CRISPR–Cas systems (green).

Figure 2

General workflow to detect SARS-CoV-2 with CRISPR-based test includes five general steps: (1) Clinical sample collection, (2) RNA preparation by extraction or release methods, (3) target sequence amplification, (4) target recognition and generation of molecular signal, and (5) signal read-out using fluorescence or lateral flow strips which could include cell phone detection.