In vivo targeted single-nucleotide editing in zebrafish

Area of Science:

• Molecular Biology
• Genetics
• Developmental Biology

Background:

• Current genome editing technologies often rely on nucleases to induce DNA double-strand breaks (DSBs).
• DSBs pose risks such as cell death and oncogenic transformation, limiting therapeutic applications.
• A safer alternative for in vivo genome editing is needed.

Purpose of the Study:

• To demonstrate the efficacy of the Target-AID system for in vivo targeted single-nucleotide editing in zebrafish.
• To assess the heritability and phenotypic effects of Target-AID-induced mutations.
• To establish a programmable genome editing tool in a vertebrate model organism.

Main Methods:

• Utilized the Target-AID system for targeted cytidine deamination in zebrafish.
• Applied the system to the chordin (chd) and one-eyed pinhead (oep) genes.
• Analyzed resulting nucleotide substitutions and their heritability.

Main Results:

• Successfully introduced targeted cytosine to thymine nucleotide substitutions in zebrafish genes.
• Achieved programmable introduction of premature stop codons (TAG or TAA).
• Confirmed heritable modifications that phenocopied known homozygous mutants.

Conclusions:

• The Target-AID system enables programmable, heritable nucleotide substitutions in vivo in vertebrates.
• This nuclease-free approach offers a safer alternative for genome editing applications.
• Demonstrated the utility of Target-AID in zebrafish for genetic studies and potential therapeutics.