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

Mass 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 mu L-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.

Automated summary

Mass pathogen screening is crucial in preventing outbreaks and transmission of infectious diseases. A CRISPR-based Amplification-free Viral RNA Electrical Detection platform (CAVRED) is developed for rapid identification of SARS-CoV-2 variants. CAVRED utilizes CRISPR RNA assays to amplify the CRISPR-Cas system's ability to detect single-nucleotide differences between mutant and wild RNA genomes. With highly sensitive field-effect transistor biosensors, CAVRED can detect SARS-CoV-2 virus genome as low as 1 cp mu L-1 within 20 mins without amplification, offering a comparable detection limit to real-time quantitative polymerase chain reaction. An 8-in-1 CAVRED array achieves 95.0% accuracy in rapid identification of 40 simulated throat swab samples of SARS-CoV-2 variants, promising its application in rapid and large-scale epidemic screening.