Tracking-seq reveals the heterogeneity of off-target effects in CRISPR-Cas9-mediated genome editing PMID: 38956324

ADC Targets in Oncology

2026-04-10

Tracking-seq reveals the heterogeneity of off-target effects in CRISPR-Cas9-mediated genome editing

Nat Biotechnol. 2024 Jul 2. doi: 10.1038/s41587-024-02307-y.

Ming Zhu ^# ¹ ² ³ , Runda Xu ^# ⁴ ⁵ ⁶ , Junsong Yuan ^# ⁴ ⁷ , Jiacheng Wang ^# ⁴ ⁶ ⁸ , Xiaoyu Ren ⁴ ⁷ , Tingting Cong ⁴ ⁷ , Yaxian You ⁴ ⁵ ⁶ , Anji Ju ⁴ ⁵ ⁶ , Longchen Xu ⁴ ⁸ , Huimin Wang ⁴ ⁵ , Peiyuan Zheng ⁴ ⁷ , Huiying Tao ⁵ ⁹ , Chunhua Lin ⁹ , Honghao Yu ⁵ ¹⁰ , Juanjuan Du ⁴ ⁷ , Xin Lin ⁴ ⁵ , Wei Xie ⁴ ⁸ , Yinqing Li ¹¹ ¹² , Xun Lan ¹³ ¹⁴ ¹⁵

Affiliations

1. Tsinghua-Peking Joint Center for Life Sciences, Tsinghua University, Beijing, China. [email protected].
2. Department of Basic Medical Sciences, School of Medicine, Tsinghua University, Beijing, China. [email protected].
3. MOE Key Laboratory of Bioinformatics, State Key Laboratory of Molecular Oncology, Tsinghua University, Beijing, China. [email protected].
4. Tsinghua-Peking Joint Center for Life Sciences, Tsinghua University, Beijing, China.
5. Department of Basic Medical Sciences, School of Medicine, Tsinghua University, Beijing, China.
6. MOE Key Laboratory of Bioinformatics, State Key Laboratory of Molecular Oncology, Tsinghua University, Beijing, China.
7. IDG-McGovern Institute for Brain Research, Center for Synthetic and Systems Biology, School of Pharmaceutical Sciences, Tsinghua University, Beijing, China.
8. School of Life Sciences, Tsinghua University, Beijing, China.
9. Department of Urology, Affiliated Yantai Yuhuangding Hospital of Qingdao University, Yantai, China.
10. Key Laboratory of Medical Biotechnology and Translational Medicine, Guilin Medical University, Guilin, China.
11. MOE Key Laboratory of Bioinformatics, State Key Laboratory of Molecular Oncology, Tsinghua University, Beijing, China. [email protected].
12. IDG-McGovern Institute for Brain Research, Center for Synthetic and Systems Biology, School of Pharmaceutical Sciences, Tsinghua University, Beijing, China. [email protected].
13. Tsinghua-Peking Joint Center for Life Sciences, Tsinghua University, Beijing, China. [email protected].
14. Department of Basic Medical Sciences, School of Medicine, Tsinghua University, Beijing, China. [email protected].
15. MOE Key Laboratory of Bioinformatics, State Key Laboratory of Molecular Oncology, Tsinghua University, Beijing, China. [email protected].
# Contributed equally.

Abstract

The continued development of novel genome editors calls for a universal method to analyze their off-target effects. Here we describe a versatile method, called Tracking-seq, for in situ identification of off-target effects that is broadly applicable to common genome-editing tools, including Cas9, base editors and prime editors. Through tracking replication protein A (RPA)-bound single-stranded DNA followed by strand-specific library construction, Tracking-seq requires a low cell input and is suitable for in vitro, ex vivo and in vivo genome editing, providing a sensitive and practical genome-wide approach for off-target detection in various scenarios. We show, using the same guide RNA, that Tracking-seq detects heterogeneity in off-target effects between different editor modalities and between different cell types, underscoring the necessity of direct measurement in the original system.