a. Department of Radiology, The Affiliated Panyu Central Hospital, GMU-GIBH Joint School of Life Sciences, Guangdong Provincial Key Laboratory of Protein Modification and Disease, The Guangdong-Hong Kong-Macao Joint Laboratory for Cell Fate Regulation and Diseases, Guangzhou Medical University, Guangzhou, Guangdong 511436, China;
b. Guangdong Basic Research Center of Excellence for Precise Breeding of Future Crops, College of Agriculture, South China Agricultural University, Guangzhou, Guangdong 510642, China;
c. State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangdong Provincial Key Laboratory of Plant Molecular Breeding, South China Agricultural University, Guangzhou, Guangdong 510642, China;
d. Key Laboratory for Enhancing Resource Use Efficiency of Crops in South China, Ministry of Agriculture and Rural Affairs, South China Agricultural University, Guangzhou, Guangdong 510642, China;
e. College of Fisheries, Southwest University, Chongqing 402460, China;
f. Institute of BioFoundry, College of Life Sciences, South China Agricultural University, Guangzhou, Guangdong 510642, China
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Thanks for the instrument analysis and measurement provided by the Scientific Research Center of Guangzhou Medical University and Center of Genomics and Bioinformatics of South China Agricultural University. We thank Kewei Xu for his insightful comments on the manuscript. Parts of the figures were created with BioRender.
Base editing enables efficient nucleotide conversions without inducing DNA double-strand breaks (DSBs) or requiring exogenous donor DNA templates. However, its broader editing window often causes bystander editing, increasing the risk of unintended mutations. In this study, we find that linker length significantly influences the editing window, and base editors with a 7-amino-acid linker reduce bystander editing by an average of 54.4% across 13 endogenous genomic sites in both rice and human cell lines. We further develop an optimized strategy by modulating the linker length between various deaminases and Cas9 nickases, which effectively reduces bystander editing across multiple applications, including functional studies, precise crop breeding, and correction of pathogenic variants. Our work reveals that shortening the linker enhances the specificity of base editing, addressing a key safety concern for its agricultural and therapeutic applications.
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