Specific cleavage of 5' overhangs of non-target strand by Cas9 activated by target DNA binding

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has become a powerful genome-editing tool that uses RNA-DNA pairing to cleave target DNA with protospacer adjacent motif (PAM) sequences. While its primary function is well-studied, secondary activities remain poorly understood, causing unintended off-target effects. This study reports for the first time that Cas9 specifically cleaves the 5' overhang of the non-target strand (NTS) in target double-stranded DNA. This specific cleavage requires an additional PAM element at the NTS 5' region, is mediated by Cas9's RuvC domain, and is regulated by the HNH domain. It depends on the exact positioning of the NTS 5' end, but not on the overhang homopolymer sequence or overhang length. Adequate single-guide RNA-DNA complementarity is also essential. This discovery potentially advances our understanding of Cas9's enzymatic versatility to enhance genome-editing precision and efficacy and offers new nucleic acid detection strategies. Based on this cleavage, we developed a sensitive assay for Severe Acute Respiratory Syndrome Coronavirus 2 pseudovirus down to 2.4 copies μL-1, demonstrated extremely high sensitivity in diagnostic applications.