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, Xiaolong Wang
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. CRISPR base editors directly modify the non-target strand (NTS). Here, authors reveal an activity by miniature Cas12f cytosine base editor (CBE) to also edit the TS, and next engineer a TS-preferential CBE. The work establishes a strand-selectable miniature CBE toolkit with therapeutic potential.