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bioRxiv · 10.1101/2025.02.17.638614

Off-target detection of CRISPR-Cas9 nuclease in vitro with CROFT-Seq

Toliusis, P.·Grybauskas, A.·Sinkunas, T.·Karvelis, T.·Sasnauskas, G.·Zaremba, M.
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Abstract

Programmable CRISPR-Cas9 nucleases have become invaluable tools for genome editing. However, off-target cleavage by these nucleases could 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, complex, and relatively costly. Here, we present CROFT-Seq (CRISPR nuclease off-target detection by sequencing), a sensitive, rapid, and cost-effective assay for the genome-wide detection of Cas9 off-target sites in vitro. CROFT-Seq performs comparably to the common currently 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-targets identified by CROFT-Seq were validated in cells, highlighting its effectiveness as a predictor of off-target sites.

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Toliusis, P., Grybauskas, A., Sinkunas, T., Karvelis, T., Sasnauskas, G., Zaremba, M.. 2025-02-17. Off-target detection of CRISPR-Cas9 nuclease in vitro with CROFT-Seq. https://doi.org/10.1101/2025.02.17.638614

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