CRISPR‐Mediated Profiling of Viral RNA at Single‐Nucleotide Resolution

Abstract

AbstractMass pathogen screening is critical to preventing the outbreaks and spread of infectious diseases. The large‐scale epidemic of COVID‐19 and the rapid mutation of the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) virus have put forward new requirements for virus detection and identification techniques. Here, we report a CRISPR‐based Amplification‐free Viral RNA Electrical Detection platform (CAVRED) for the rapid detection and identification of SARS‐CoV‐2 variants. A series of CRISPR RNA assays were designed to amplify the CRISPR‐Cas system‘s ability to discriminate between mutant and wild RNA genomes with a single‐nucleotide difference. The identified viral RNA information was converted into readable electrical signals through field‐effect transistor biosensors for the achievement of highly sensitive detection of single‐base mutations. CAVRED can detect the SARS‐CoV‐2 virus genome as low as 1 cp μL−1 within 20 mins without amplification, and this value is comparable to the detection limit of real‐time quantitative polymerase chain reaction. Based on the excellent RNA mutation detection ability, an 8‐in‐1 CAVRED array was constructed and realized the rapid identification of 40 simulated throat swab samples of SARS‐CoV‐2 variants with a 95.0 % accuracy. The advantages of accuracy, sensitivity, and fast speed of CAVRED promise its application in rapid and large‐scale epidemic screening.