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bioRxiv · 10.1101/2025.09.25.678665

Sensitive, direct detection of non-coding off-target base editor unwinding and editing in primary cells

Wang, T.·Jessa, S.·Marinov, G. K.·Klemm, S.·Kundaje, A.·Greenleaf, W. J.
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Abstract

Base editors create precise nucleotide changes in DNA, but their off-target activity remains challenging to quantify. Here, we develop and deploy a direct, in cellulo sequencing assay that simultaneously measures both Cas9-mediated unwinding and deaminase editing of genomic DNA (beCasKAS). Our strategy nominates >460-fold more potential off-target sites than other methods by enriching for Cas9-dependent R-loops immediately preceding editing. Using beCasKAS in primary human T-cells, we observe that mRNA-encoded ABE8e and PAMless ABE8e-SpRY base editors have distinct off-target profiles that can be mitigated by optimizing mRNA dose. Finally, we combine beCasKAS with base-resolution deep learning models to risk-stratify off-target edits by their likelihood of epigenetic dysregulation. Collectively, beCasKAS offers a sensitive and facile tool to optimize the balance between base editor on- and off-target activity.

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Wang, T., Jessa, S., Marinov, G. K., Klemm, S., Kundaje, A., Greenleaf, W. J.. 2025-09-25. Sensitive, direct detection of non-coding off-target base editor unwinding and editing in primary cells. https://doi.org/10.1101/2025.09.25.678665

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