A CRISPR/Cas9 eraser strategy for contamination‐free PCR end‐point detection

Lin, Wei; Tian, Tian; Jiang, Yongzhong; Xiong, Erhu; Zhu, Debin; Zhou, Xiaoming

Polymerase chain reaction (PCR), a central technology for molecular diagnostics, is highly sensitive but susceptible to the risk of false positives caused by aerosol contamination, especially when an end‐point detection mode is applied. Here, we proposed a solution by designing a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 eraser strategy for eliminating potential contamination amplification. The CRISPR/Cas9 engineered eraser is firstly adopted into artpcr reverse‐transcription PCR (RT‐PCR) system to achieve contamination‐free RNA detection. Subsequently, we extended this CRISPR/Cas9 eraser to the PCR system. We engineered conventional PCR primers to enable the amplified products to contain an implanted NGG (protospacer adjacent motif, PAM) site, which is used as a code for specific CRISPR/Cas9 recognition. Pre‐incubation of Cas9/sgRNA with PCR mix leads to a selective cleavage of contamination amplicons, thus only the template DNA is amplified. The developed CRISPR/Cas9 eraser, adopted by both RT‐PCR and PCR systems, showed high‐fidelity detection of SARS‐CoV‐2 and African swine fever virus with a convenient strip test.