Detect-seq reveals out-of-protospacer editing and target-strand editing by cytosine base editors

Zhixin Lei1,2, Haowei Meng3, Zhicong Lv3, Menghao Liu1,2, Huanan Zhao4,5, Hao Wu1,2, Xiaoxue Zhang1,2, Lulu Liu3, Yuan Zhuang3, Kailin Yin3, Yongchang Yan3, Chengqi Yi6,7,8,9

  1. Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China.
  2. Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China.
  3. State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, China.
  4. School of Life Sciences, Tsinghua University, Beijing, China.
  5. Peking University-Tsinghua University-National Institute of Biological Sciences Joint Graduate Program, School of Life Sciences, Tsinghua University, Beijing, China.
  6. Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China. chengqi.yi@pku.edu.cn.
  7. State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, China. chengqi.yi@pku.edu.cn.
  8. Department of Chemical Biology and Synthetic and Functional Biomolecules Center, College of Chemistry and Molecular Engineering, Peking University, Beijing, China. chengqi.yi@pku.edu.cn.
  9. Peking University Genome Editing Research Center, Peking University, Beijing, China. chengqi.yi@pku.edu.cn.

Abstract

Cytosine base editors (CBEs) have the potential to correct human pathogenic point mutations. However, their genome-wide specificity remains poorly understood. Here we report Detect-seq for the evaluation of CBE specificity. It enables sensitive detection of CBE-induced off-target sites at the genome-wide level. Detect-seq leverages chemical labeling and biotin pulldown to trace the editing intermediate deoxyuridine, thereby revealing the editome of CBE. In addition to Cas9-independent and typical Cas9-dependent off-target sites, we discovered edits outside the protospacer sequence (that is, out-of-protospacer) and on the target strand (which pairs with the single-guide RNA). Such unexpected off-target edits are prevalent and can exhibit a high editing ratio, while their occurrences exhibit cell-type dependency and cannot be predicted based on the sgRNA sequence. Moreover, we found out-of-protospacer and target-strand edits nearby the on-target sites tested, challenging the general knowledge that CBEs do not induce proximal off-target mutations. Collectively, our approaches allow unbiased analysis of the CBE editome and provide a widely applicable tool for specificity evaluation of various emerging genome editing tools.

Presented By Zhixin Lei | ORCID iD

Genetics & Genome Biology, BiotechnologyNicholas Liao2021-06 (Jun)