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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.<\/jats:p>","DOI":"10.1101\/2025.08.26.667396","type":"posted-content","created":{"date-parts":[[2025,8,27]],"date-time":"2025-08-27T04:30:13Z","timestamp":1756269013000},"source":"Crossref","is-referenced-by-count":0,"title":["An integrated enzymatic and computational pipeline for quantifying off-target base-editing"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1803-3623","authenticated-orcid":false,"given":"Alexander G.","family":"McFarland","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Soon-Keat","family":"Ooi","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Davin","family":"Tafuri","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Elahe","family":"Kamali","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Natalie","family":"Cooper","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Emma J.","family":"Cook","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shantan","family":"Reddy","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Danuta","family":"Jarocha","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nils","family":"Wellhausen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Saar I.","family":"Gill","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Aoife M.","family":"Roche","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Joseph A.","family":"Fraietta","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Frederic D.","family":"Bushman","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Friederike","family":"Herbst","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2025083002051039000_2025.08.26.667396v1.1","doi-asserted-by":"publisher","DOI":"10.1038\/nbt.4199"},{"key":"2025083002051039000_2025.08.26.667396v1.2","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-022-24184-8"},{"key":"2025083002051039000_2025.08.26.667396v1.3","doi-asserted-by":"publisher","DOI":"10.1126\/science.abb1390"},{"key":"2025083002051039000_2025.08.26.667396v1.4","doi-asserted-by":"publisher","DOI":"10.1038\/s41587-020-0453-z"},{"key":"2025083002051039000_2025.08.26.667396v1.5","doi-asserted-by":"publisher","DOI":"10.1038\/nature24644"},{"key":"2025083002051039000_2025.08.26.667396v1.6","doi-asserted-by":"publisher","DOI":"10.1038\/s41587-020-0491-6"},{"key":"2025083002051039000_2025.08.26.667396v1.7","doi-asserted-by":"publisher","DOI":"10.1038\/nature17946"},{"key":"2025083002051039000_2025.08.26.667396v1.8","doi-asserted-by":"publisher","DOI":"10.1038\/s41587-022-01533-6"},{"key":"2025083002051039000_2025.08.26.667396v1.9","doi-asserted-by":"publisher","DOI":"10.1038\/s41587-022-01611-9"},{"key":"2025083002051039000_2025.08.26.667396v1.10","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-019-1314-0"},{"key":"2025083002051039000_2025.08.26.667396v1.11","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-021-03609-w"},{"key":"2025083002051039000_2025.08.26.667396v1.12","doi-asserted-by":"publisher","DOI":"10.1161\/ATVBAHA.117.309881"},{"key":"2025083002051039000_2025.08.26.667396v1.13","doi-asserted-by":"crossref","unstructured":"Chadwick, A.C. & Musunuru, K . 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