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 F(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. 0
CRISPR Gene Editing