Noncanonical target-strand cytosine base editing via engineered Un1Cas12f1 platform
CRISPR/Cas-derived base editors harness various deaminase or glycosylase activities to target bases within non-target strand (NTS) of the R-loop, catalyzing base conversions independent of double-strand break formation. To develop miniature BEs compatible with therapeutic viral vectors, we explore the compact Cas12f system. Through computational modeling and mutagenesis, we establish a highly active enUn1Cas12f1 protein, and subsequently construct the derivative cytosine BE (CBE). Remarkably, the engineered CBE exhibits an unexpected activity to also edit the target strand (TS), indicating its substantially expanded editable space. We refine this activity via a focused alanine scan, establishing a nickase-CBE that preferentially install TS edits (TSminiCBE). Further engineering with a non-specific DNA binding domain yields an optimized TS-editing BE that enables in vivo base edits in mice (male). Overall, through extensive engineering of the Cas12f platform and repurposing its intrinsic dynamics, our work establishes a strand-selectable miniature CBE toolkit with strong potential for diverse applications.
Authors
- Ziguo Song
- Yinghui Wei (ORCID: https://orcid.org/0000-0002-6642-1559)
- Shuhong Huang (ORCID: https://orcid.org/0000-0003-2302-4200)
- Lina Wu (ORCID: https://orcid.org/0000-0001-8640-6279)
- Bingchun Chen (ORCID: https://orcid.org/0009-0008-0908-3056)
- Xingxu Huang (ORCID: https://orcid.org/0000-0001-8934-1247)
- Zhanqing Fan (ORCID: https://orcid.org/0000-0003-2701-8115)
- Shisheng Huang (ORCID: https://orcid.org/0000-0003-4673-8388)
- Junfan Guo
- Jianghuai Liu (ORCID: https://orcid.org/0000-0002-2382-9140)
- Yulin Chen (ORCID: https://orcid.org/0000-0001-5679-4055)
- Xiaolong Wang (ORCID: https://orcid.org/0000-0003-1620-1344)
- Guanglei Li (ORCID: https://orcid.org/0000-0002-0979-288X)
- Wenxin Zheng (ORCID: https://orcid.org/0000-0002-6525-2212)
- Zhou-shen Zhao (ORCID: https://orcid.org/0009-0004-7083-2360)
Institutions
- Nanjing Normal University (CN)
- China Pharmaceutical University (CN)
- ShanghaiTech University (CN)
- North West Agriculture and Forestry University (CN)
- Xinjiang Academy of Animal Science (CN)
- Sanya University (CN)
- First Affiliated Hospital Zhejiang University (CN)
- Nanjing University (CN)
- Northwest A&F University (CN)
Publication Details
- Journal
- Nature Communications
- Published
- 2025-10-28
- DOI
- https://doi.org/10.1038/s41467-025-64562-0
- Citations
- 3
- Primary Topic
- CRISPR and Genetic Engineering
- Type
- article
- Field-Weighted Citation Impact
- 2.42
Funders
- National Natural Science Foundation of China
- Northwest A and F University
- China Agricultural Research System
- ShanghaiTech University