Nature communicationsAug 11, 2026

UNCOVERseq allows precise detection of gene editing off-target effects across different CRISPR-Cas methods and systems

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗OA ↗

Updated Aug 13, 2026

Abstract

UNCOVERseq achieves 97.6% analytical sensitivity in identifying off-target gene editing events.

The workflow identifies rare off-target events using defined inputs and analytical process controls.
It outperforms existing nomination methods in terms of analytical sensitivity and precision.
Application of UNCOVERseq across 192 guide RNAs reveals six guides with varying specificity.
The method allows for relative risk assessment of different gene editing tools in specific cell types.
There is strong agreement between double-strand break and single-strand break nomination sites.

Simplified

The rapid expansion of CRISPR-Cas gene editing enables new therapeutic strategies but complicates assessment of unintended editing risks due to emerging modalities and unclear analytical standards. We present UNCOVERseq (Unbiased Nomination of CRISPR Off-target Variants using Enhanced RhPCR), an improved in cellulo off-target nomination workflow that sensitively identifies rare off-target events using defined inputs and analytical process controls. Using an inter-method off-target confirmation benchmarking dataset, UNCOVERseq demonstrates high analytical sensitivity (97.6%) and precision (78%), outperforming published nomination methods. We apply UNCOVERseq across 192 guide RNAs and identify six guides spanning a broad specificity range, enabling relative risk assessment across S. pyogenes Cas9, high-fidelity variants, and base editors in hematopoietic stem and progenitor cells. We further show that double-strand break nomination sites retain strong rank-order concordance with single-strand break-mediated base editing. Together, these results establish UNCOVERseq as a robust framework for informed off-target risk assessment in translational gene-editing systems.