Noncanonical target-strand cytosine base editing via engineered Un1Cas12f1 platform

Ziguo Song, Junfan Guo, Zhanqing Fan, Shuhong Huang, Guanglei Li, Zichang Zhao, Bingchun Chen, Shisheng Huang, Wenxin Zheng, Yinghui Wei, Yulin Chen, Xingxu Huang, Jianghuai Liu (), Lina Wu () and Xiaolong Wang ()
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Ziguo Song: Hainan Institute of Northwest A&F University
Junfan Guo: ShanghaiTech University
Zhanqing Fan: Hainan Institute of Northwest A&F University
Shuhong Huang: Hainan Institute of Northwest A&F University
Guanglei Li: ShanghaiTech University
Zichang Zhao: Nanjing Normal University
Bingchun Chen: Hainan Institute of Northwest A&F University
Shisheng Huang: ShanghaiTech University
Wenxin Zheng: Xinjiang Academy of Animal Sciences
Yinghui Wei: Hainan Institute of Northwest A&F University
Yulin Chen: Hainan Institute of Northwest A&F University
Xingxu Huang: ShanghaiTech University
Jianghuai Liu: Medical School of Nanjing University
Lina Wu: Nanjing Normal University
Xiaolong Wang: Hainan Institute of Northwest A&F University

Nature Communications, 2025, vol. 16, issue 1, 1-19

Abstract: Abstract 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.

Date: 2025
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DOI: 10.1038/s41467-025-64562-0

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