Sequence Context‐Agnostic TadA‐Derived Cytosine Base Editors for Genome‐Wide Editing in Zebrafish

Abstract Single‐nucleotide variants (SNVs) represent a significant form of genetic variation linked to various diseases. CRISPR‐mediated base editing has emerged as a powerful method for modeling diseases caused by SNVs, particularly in zebrafish, which serve as an excellent platform for investigating disease mechanisms and conducting drug screenings. However, existing cytosine base editors (CBEs) for zebrafish often have broad editing windows and strong sequence preferences, limiting their effectiveness. In this study, zebrafish (z) TadA‐derived cytosine base editors, termed zTadA‐CBEs, are developed by introducing key mutations into the TadA8e enzyme. These novel editors demonstrate improved efficiency and precision in cytosine base editing. Specifically, zTadA‐BE4max and zTadA‐BEmv offer complementary editing windows, while zTadA‐SpRY‐BE4max allows for PAM‐flexible editing. Using zTadA‐CBEs, a precise disease model for Axenfeld‐Rieger syndrome is established, and created two new models for Hermansky‐Pudlak syndrome. Additionally, a novel albinism model carrying two pathogenic SNVs in the F0 generation is developed. By employing specifically designed sgRNA, the fms ^ts± missense mutation is corrected back to the wild‐type nucleotide (C > T), successfully restoring macrophage levels to normal. These findings underscore the potential of zTadA‐CBEs to enhance genome editing techniques and their applications in developing therapies for SNV‐related disorders.