Efficient homologous recombination-mediated genome engineering in zebrafish using TALE nucleases

Area of Science:

• Molecular Biology
• Genetics
• Developmental Biology

Background:

• Custom-designed nucleases enable gene disruption and genome modification.
• Homologous recombination (HR) is valuable for inserting DNA fragments (e.g., GFP) into specific genomic loci.
• Precise HR-mediated genome editing in zebrafish remains technically challenging.

Purpose of the Study:

• To establish a GFP reporter system for measuring HR event frequency in live zebrafish embryos.
• To optimize HR-mediated genome editing efficiency in zebrafish by defining optimal homology arm lengths and construct configurations.

Main Methods:

• Co-injection of TALE nucleases and GFP reporter targeting constructs with varying homology arm lengths into zebrafish embryos.
• Utilizing a GFP reporter system to quantify HR event frequency.
• Analysis of targeting construct configuration impact on HR efficiency.

Main Results:

• Defined optimal homology arm lengths for increased HR efficiency.
• Identified targeting construct configuration as a crucial parameter for HR efficiency.
• Achieved over 10% germline transmission of gene-targeting events in F0 zebrafish.
• Generated two HR-mediated insertion alleles (sox2 and gfap) expressing sfGFP or tdTomato.

Conclusions:

• Developed an efficient strategy for HR-mediated knock-in generation in zebrafish.
• The optimized method significantly improves the efficiency of generating gene-targeted zebrafish.
• This approach facilitates sophisticated genetic manipulations and in vivo monitoring of gene expression and cell types in zebrafish.