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

Area of Science:

• Genetics
• Molecular Biology
• Biotechnology

Background:

• CRISPR base editors (CBEs) are vital for precise genome editing.
• APOBEC-based CBEs face limitations including indels and restricted targeting, especially for CC and GC motifs.
• TadA-derived CBEs offer an alternative but require optimization for efficiency and targeting range.

Purpose of the Study:

• To evaluate and enhance tRNA adenine deaminase (TadA)-derived cytosine base editors (CBEs) in zebrafish.
• To develop novel zTadCBE variants with improved efficiency, reduced off-target effects, and expanded targeting capabilities.
• To create zebrafish disease models using zTadCBEs for in vivo functional genetic assessment.

Main Methods:

• Evaluation of existing TadA-derived CBEs in zebrafish.
• Development of zTadCBE variants by integrating beneficial mutations from TadA-based adenine base editors (ABEs).
• Incorporation of SpRYCas9n for enhanced protospacer-adjacent motif (PAM) compatibility.
• Generation of four zebrafish disease models (auditory, nervous, metabolic, muscular) in the F0 generation.

Main Results:

• Developed zTadCBE variants demonstrate high C-to-T base editing efficiency in zebrafish.
• Minimized off-target mutations were observed compared to existing CBE tools.
• Expanded targeting window allows editing of cytosines at a broader range of positions relative to the PAM.
• Successfully generated four distinct zebrafish disease models in the F0 generation, which were difficult to produce with previous CBEs.

Conclusions:

• zTadCBE variants represent a robust and versatile toolkit for precise C-to-T base editing in zebrafish.
• These editors overcome limitations of APOBEC-based CBEs, offering enhanced efficiency and targeting range.
• The developed zTadCBEs facilitate rapid in vivo functional assessment of genetic variants and disease modeling.