Digenome-Detect: Accurate, statistics-based analysis software for identifying off-target cleavage sites in genome...

Off-target mutations pose major safety concerns in genome-editing therapy owing to their potential for leading to serious adverse events such as carcinogenesis. Thus, accurate prediction and evaluation of off-target mutations are critical in ensuring the safety of genome-editing therapeutics. Among the methods available for predicting off-target mutations, cell-free assays, which detect cleavage sites in extracted genomic DNA treated with genome-editing tools in vitro, are valuable because of their genome-wide, unbiased, and sensitive detection capabilities. However, cell-free assays are prone to identifying false-positive off-target cleavage sites, limiting their practical utility. To address this issue, we developed "Digenome-Detect," a highly accurate and sensitive data analysis software tool for Digenome-seq, which is the simplest cell-free assay. Digenome-Detect calculates a statistically derived score for genomic cleavage sites, followed by additional filters to reduce false positives. Compared with the current standard software, Digenome-toolkit, Digenome-Detect identified more off-target cleavage sites with fewer obvious false positives. Digenome-Detect enables accurate and sensitive prediction of off-target cleavage sites and can thereby contribute to ensuring the safety of genome-editing therapeutics.