Optimizing linker length of base editors for precise crop breeding and gene therapy

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.