Novel Isothermal Amplification Integrated with CRISPR/Cas13a and Its Applications for Ultrasensitive Detection of SARS-CoV-2

We herein developed an ultrasensitive and rapid strategy to identify genomic nucleic acids by integrating a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 13a (Cas13a) into our recently developed isothermal technique, nicking and extension chain reaction system-based amplification (NESBA) reaction. In this technique, named CESBA, the NESBA reaction isothermally produces a large amount of RNA amplicons from the initial target genomic RNA (gRNA). The RNA amplicons bind to the crispr RNA (crRNA) and activate the collateral cleavage activity of Cas13a, which would then cleave the reporter probe nearby, consequently producing the final signals. Based on this design principle, we successfully detected SARS-CoV-2 gRNA as a model target very sensitively down to even a single copy (0.05 copies/μL) in both fluorescence- and lateral flow assay (LFA)-based modes with excellent specificity against other human coronaviruses (H-CoVs). We further validated the clinical applicability of CESBA by testing the 20 clinical samples with 100% clinical sensitivity and specificity. This work represents a potent and innovative strategy for the identification of genomic nucleic acids in molecular diagnostics, delivering exceptional levels of sensitivity.