High-efficiency TadA cytosine base editors for precise modelling of human disease variants

Many missense mutations identified in genetic testing are variants of uncertain significance (VUS), not yet classified as either benign or pathogenic. Systematic determination of their functional relevance is a pressing clinical need. CRISPR-mediated base editing can precisely introduce precise variants into model organisms for functional testing, but current editors face efficiency and targeting constraints. We developed TCBE-Umax, a family of TadA-derived cytosine base editors optimized for zebrafish. Engineering the TadA deaminase domain improved editing efficiency and reduced sequence-context bias, expanded PAM compatibility, and minimized bystander edits and indel formation. Our editors achieved efficient biallelic editing, enabling rapid functional assessment of genetic variants in the F0 (founding) zebrafish. As a proof of concept, we evaluated 15 VUS linked to hereditary hearing loss, determining pathogenicity through phenotypic analysis. With high efficiency and versatility, TCBE-Umax base editors provide a powerful platform for studying genetic variants and disease in vivo. A suite of TadA-derived cytosine base editors was optimized to improve efficiency, reduce sequence bias, expand PAM compatibility and minimize artefacts, enabling precise genetic interrogation of variants of uncertain significance (VUS) in zebrafish.