Herbst, F. | bioRxiv Science

bioRxiv2025

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

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.

molecular biology↗

bioRxiv2024

TET2 regulates early and late transitions in exhausted CD8+ T-cell differentiation and limits CAR T-cell function

CD8+ T-cell exhaustion hampers disease control in cancer and chronic infections and limits efficacy of T-cell-based therapies, such as CAR T-cells. Epigenetic reprogramming of CAR T-cells by targeting TET2, a methylcytosine dioxygenase that mediates active DNA demethylation, has shown therapeutic potential; however, the role of TET2 in exhausted T-cell (TEX) development is unclear. In CAR T-cell exhaustion models and chronic LCMV infection, TET2 drove the conversion from stem cell-like, self-renewing TEX progenitors towards terminally differentiated and effector (TEFF)-like TEX. In mouse T-cells, TET2-deficient terminally differentiated TEX retained aspects of TEX progenitor biology, alongside decreased expression of the transcription factor TOX, suggesting that TET2 potentiates terminal exhaustion. TET2 also enforced a TEFF-like terminally differentiated CD8+ T-cell state in the early bifurcation between TEFF and TEX, indicating a broad role for TET2 in mediating the acquisition of an effector biology program that could be exploited therapeutically. Finally, we developed a clinically actionable strategy for TET2- targeted CAR T-cells, using CRISPR/Cas9 editing and site-specific adeno-associated virus transduction to simultaneously knock-in a CAR at the TRAC locus and a functional safety switch within TET2. Disruption of TET2 with this safety switch in CAR T-cells restrained terminal TEX differentiation in vitro and enhanced anti-tumor responses in vivo. Thus, TET2 regulates pivotal fate transitions in TEX differentiation and can be targeted with a safety mechanism in CAR T-cells for improved tumor control and risk mitigation. One Sentence SummaryModulation of exhausted CD8+ T-cell differentiation by targeting TET2 improves therapeutic potential of CAR T-cells in cancer.

immunology↗