An integrated enzymatic and computational pipeline for quantifying off-target base-editing

McFarland, A. G.; Ooi, S.-k.; Tafuri, D.; Kamali, E.; Cooper, N.; Cook, E. J.; Reddy, S.; Jarocha, D.; Wellhausen, N.; Gill, S. I.; Roche, A. M.; Fraietta, J. A.; Bushman, F. D.; Herbst, F.

doi:10.1101/2025.08.26.667396

bioRxiv · 10.1101/2025.08.26.667396

An integrated enzymatic and computational pipeline for quantifying off-target base-editing

McFarland, A. G.·Ooi, S.-k.·Tafuri, D.·Kamali, E.·Cooper, N.·Cook, E. J.·Reddy, S.·Jarocha, D.·Wellhausen, N.·Gill, S. I.·Roche, A. M.·Fraietta, J. A.·Bushman, F. D.·Herbst, F.

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Abstract

DNA base editing is increasingly used for human genetic modification, but methods for monitoring off-target editing are nascent. Here we present a simple model-independent workflow for identifying sites of off-target base-editing in relevant cell types on a genome-wide level. We report that sites of off-target editing by the ABE8e editor could be identified using an ABE8e derivative with restored DSB cleavage activity. This allows marking of enzyme-generated double-stranded (ds) DNA breaks by incorporation of dsDNA oligonucleotides that are transfected into primary target cells. DNA sequencing at sites of oligonucleotide incorporation reported both the genomic location of off-target cleavage and the extent of base-editing nearby. We present a platform combining this cellular (BEiGUIDE-Seq) and computational workflow (CRISPRito) to generate optimized amplicon panels for convenient monitoring of off-target base editing. This work introduces a generalizable strategy to evaluate off-target edits in patient-derived cells, addressing a critical safety gap for clinical base editing.

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McFarland, A. G., Ooi, S.-k., Tafuri, D., Kamali, E., Cooper, N., Cook, E. J., Reddy, S., Jarocha, D., Wellhausen, N., Gill, S. I., Roche, A. M., Fraietta, J. A., Bushman, F. D., Herbst, F.. 2025-08-26. An integrated enzymatic and computational pipeline for quantifying off-target base-editing. https://doi.org/10.1101/2025.08.26.667396

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