Viral detection using Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein and Argonaute nucleases.

Abstract

Viral pandemics pose severe threats to human health and societal stability, exemplified by the COVID-19 outbreak in 2019. Conventional viral detection methods such as Polymerase chain reaction (PCR) typically require trained personnel, expensive equipment, and 2-4 h for processing. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) and Argonaute (Ago) system-based detection methods achieve attomolar sensitivity or single-copy detection limits with single-base specificity within 1 h, without requiring complex or costly instruments. This review firstly introduces the mechanisms and functions of CRISPR/Cas systems (Cas9, Cas12, Cas13) and Ago systems. It also introduces viruses with significant social impact, and continued with reviewing applications of these systems in single and multiplex virus detection. Single viral detection includes recently developed DNA/RNA-activated Cas9 detection (DACD/RACD) using Cas9 trans-cleavage activity, Cas12-based DNA Endonuclease-targeted CRISPR Trans Reporter (DETECTR) with attomolar sensitivity, CRISPR/Cas13a-based Fluorescent Nanoparticle SARS-CoV-2 (CFNS) achieving 1 copy/mL sensitivity with quantum dot reporters, and amplification-free mobile phone detection detecting 31 copies/μL without amplification. Multiplex viral detection includes Microfluidic Device Integrated with CRISPR/Cas12a and Multiplex Recombinase Polymerase Amplification (MiCaR) enabling 30-plex detection through microfluidic chips with spatial discrimination, PfAgo-mediated Nucleic acid Detection (PAND) utilizing Ago-produced guide sequences for 5-plex detection, Specific High-Sensitivity Enzymatic Reporter UnLOCKing v2 (SHERLOCKv2) achieving 4-plex detection with multi-enzyme single-reaction systems, and Multiplexed Evaluation of Nucleic acids (CARMEN) supporting over 100 target assays. Finally, this review discusses challenges in CRISPR/Cas and Ago-based detection methods, including Protospacer Adjacent Motif (PAM) sequence requirements for Cas9/12, prolonged reaction times due to nucleic acid extraction/amplification, and instability of core components like nucleases and crRNAs. Detection specificity and multiplex capabilities could be further improved. Future directions are outlined for improving detection specificity, developing multiplex capabilities and advancing POCT. Developing diagnostic tools using CRISPR/Cas and Ago systems could transform molecular diagnostics, such tools promise to be easily accessible worldwide. They are essential for precise identification and strategic containment of infectious disease transmission.