TadA-Derived Cytosine Base Editor for Precise Genome Editing in Zebrafish

CRISPR base editors are crucial for precise genome manipulation. Existing APOBEC-based cytosine base editors (CBEs), while powerful, exhibit indels and sequence context limitations, where TC-context preferences restrict effective editing of CC and GC motifs. To address these challenges, we evaluated various tRNA adenine deaminase (TadA)-derived CBEs, ultimately engineering zTadCBE that demonstrates high editing efficiency, minimized off-target effects, and an expanded targeting range. Our approach integrates beneficial mutations from TadA-based adenine base editors (ABEs) with SpRYCas9n-enhanced protospacer-adjacent motif (PAM) compatibility. Additionally, we engineered expanded-window zTadCBE variants, zTadCBE-ex1 and zTadCBE-ex2, to target wider nucleotide ranges, further increasing the versatility of this tool. To demonstrate the utility of zTadCBE variants in the functional assessment of genetic mutants, we generated a model for CDH23-associated hearing loss to validate the pathogenicity of a patient-specific variant in zebrafish. Furthermore, we induced a premature stop codon in the mediator complex gene med12 to inactivate its function using a CRISPR-STOP strategy and recapitulated patient-specific phenotypes in the founding (F0) generation. zTadCBE variants thus offer a robust set of CBEs for precise and efficient C-to-T editing in zebrafish, promising to advance the rapid functional assessment of genetic variants in vivo.