DisTAL-Seq: A TALEN-specific adaptation of DISCOVER-Seq for off-target profiling

TL;DR

DisTAL-Seq is a new method to detect TALEN-induced DNA breaks in human cells, improving genome editing safety and precision.

Contribution

DisTAL-Seq adapts DISCOVER-Seq for TALENs by incorporating their unique binding and cleavage properties.

Findings

DisTAL-Seq identifies on- and off-target TALEN activity in human cells.

TALENs tolerate a specific number of mismatches at target sites.

DSB location relative to the target site is a key feature of TALEN activity.

Abstract

Programmable guided nucleases have revolutionized genome editing and biomedical research, with transformative potential for gene and cell therapy. Although the widespread adoption of the CRISPR-Cas system has provided deep insights into target recognition and specificity, the behavior of clinically relevant tools like transcription activator-like effector nucleases (TALENs) remains poorly characterized in human cells. To address this gap, we implemented DisTAL-Seq, a TALEN-specific adaptation of the DISCOVER-Seq pipeline, which detects MRE11 recruitment to double-strand breaks (DSBs). Based on the DISCOVER-Seq principle, DisTAL-Seq incorporates alignment logic tailored to TALEN-binding properties, including variable RVD specificity, cleavage offset, and dimerization behavior. Using DisTAL-Seq, we identified and validated on- and off-target sites across diverse TALENs and T cell donors. This unbiased approach revealed key features of TALEN activity in human cells, including number of tolerated mismatches to a target site and relative location of the induced DSB. DisTAL-Seq thus extends DISCOVER-Seq to the TALEN family and provides a robust platform for assessing modifications in enzyme architecture and application contexts on a genome-wide scale, supporting the development of safer and more effective genome editing tools.

DisTAL-Seq extends the DISCOVER-Seq platform to enable genome-wide detection of TALEN-induced double-strand breaks in human cells. By incorporating TALEN-specific dimer architecture and binding-score logic, it refines off-target identification and supports safer, more precise therapeutic genome engineering.

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