Rationally Designed TadA‐Derived Cytosine Editors Enable Context‐Independent Zebrafish Genome Editing

Abstract CRISPR base editors are crucial for precise genome manipulation. Existing APOBEC‐based cytosine base editors (CBEs), while powerful, exhibit indels and sequence context limitations, and editing CC and GC motifs is challenging and inefficient. To address these challenges, existing tRNA adenine deaminase (TadA)‐derived CBEs are evaluated in zebrafish, and a series of zTadCBE variants is developed that demonstrate high editing efficiency, minimized off‐target effects, and an expanded targeting range compared to existing tools. The approach integrates beneficial mutations from TadA‐based adenine base editors (ABEs) with SpRYCas9n‐enhanced protospacer‐adjacent motif (PAM) compatibility. The expanded window zTadCBE variants enable the targeting of cytosines at a broader range of nucleotide positions relative to the PAM sequence, further enhancing the versatility of this tool. Using zTadCBEs, four zebrafish disease models affecting the auditory, nervous, metabolic, and muscular systems are generated directly in the F0 generation—models that cannot be efficiently produced using earlier CBE tools. Together, zTadCBE variants provide a robust and flexible toolkit for efficient and precise C‐to‐T base editing in zebrafish, facilitating rapid in vivo functional assessment of genetic variants.