Programmable base editing of zebrafish genome using a modified CRISPR-Cas9 system.

Area of Science:

• Genetics and Genomics
• Molecular Biology
• Biomedical Research

Background:

• Precise genetic modifications in model animals are crucial for advancing biomedical research.
• Existing genome editing technologies require further refinement for high-efficiency, site-specific base conversions.

Purpose of the Study:

• To develop and demonstrate a programmable base editing system for precise genetic modifications in zebrafish.
• To assess the efficiency of base editing using cytidine deaminase fused to Cas9 nickase and a Cas9-VQR variant.

Main Methods:

• Utilized a cytidine deaminase fused to a Cas9 nickase for targeted base conversion.
• Employed an engineered Cas9-VQR variant with altered 5'-NGA protospacer adjacent motif (PAM) specificities.
• Applied the base editing system to introduce site-specific single-base mutations in multiple zebrafish gene loci.

Main Results:

• Achieved up to 28% efficiency in site-specific single-base mutations in zebrafish.
• Demonstrated successful base conversion using both the standard and the engineered Cas9-VQR variant.
• Validated the programmability and effectiveness of the base editing system across different gene loci.

Conclusions:

• The developed base editing system offers a precise and efficient strategy for genome editing in zebrafish.
• Cas9 variants, such as Cas9-VQR, can expand the utility and target range of base editing technology.
• This advancement facilitates sophisticated genetic studies in zebrafish, a key model organism for biomedical research.