Off-target detection of CRISPR-Cas9 nuclease <i>in vitro</i> with CROFT-Seq

Abstract Programmable CRISPR-Cas9 nucleases have become invaluable tools for genome editing. However, off-target cleavage by these nucleases can lead to unintended changes in the edited genome. Detection of off-target sites is critical to make genome editing technology safe and predictable. Although current in vitro methods for off-target detection can identify these sites, they are time-consuming and complex. Here, we present CROFT-Seq (CRISPR nuclease off-target detection by sequencing), a sensitive, rapid, and affordable assay for the genome-wide detection of Cas9 off-target sites in vitro. CROFT-Seq performs comparably to the commonly used in vitro methods and serves as a valuable and efficient tool for the rapid assessment of genome-editing nuclease specificity. Notably, a high proportion of the top-ranked off-target sites identified by CROFT-Seq were validated in cells, highlighting its strong predictive performance.