Ultra-sensitive profiling of CRISPR-Cas off-target effects with Tracking-seq2
- Tingting Cong
- Runda Xu
- Xuancheng Chen
- Junsong Yuan
- Zuomiao Lin
- Wenqin Yang
- Anji Ju
- Liren Wang
- Qingcan Wang
- Jiqin Zhang
- Yinqing Li
- Dali Li
- Ming Zhu
2026-08-11
Accurate detection of off-target activity in primary human cells is crucial for ensuring the safety of gene therapies, yet existing methods often lack sufficient sensitivity. To address this limitation, we develop Tracking-seq2, an advanced technology that integrates exogenous 5′ → 3′ exonuclease treatment and non-homologous end joining (NHEJ) pathway inhibitors with the original Tracking-seq. Tracking-seq2 exhibits enhanced sensitivity in profiling off-target sites of diverse genome editors—including Cas9, Cas12a, cytosine base editors (CBEs), adenine base editors (ABEs), and prime editors (PEs). Critically, Tracking-seq2 is directly applicable to clinically relevant primary human cell types, such as T cells and CD34 + hematopoietic stem and progenitor cells (HSPCs). Furthermore, our findings reveal that genomic variations drive distinct off-target heterogeneity across different individuals, highlighting the necessity for personalized safety assessment in clinical genome editing applications. Tracking-seq2 provides a robust platform for sensitive off-target detection in primary cells, with sensitivity comparable to or exceeding current state-of-the-art methods.