Advancing knock-in approaches for robust genome editing in zebrafish

Area of Science:

• Molecular Biology
• Genomics
• Zebrafish Model Systems

Background:

• Precise genome editing, especially for knock-in (KI) alleles, is crucial for functional genomics in model organisms.
• Existing methods face challenges in efficiency and specificity.

Purpose of the Study:

• To introduce a versatile Golden Gate-based platform, the mini-golden system, for rapid assembly of donor constructs for genome editing.
• To develop strategies for enhanced precision in genome editing, including a synthetic exon-based donor template approach.

Main Methods:

• Utilized the mini-golden system for Golden Gate assembly of donor constructs with homology arms and genes of interest.
• Generated donor minicircles for KI applications, including a foxd3CreER KI zebrafish line.
• Employed a synthetic exon-based donor template strategy with fluorescence screening for precise gene editing.

Main Results:

• Successfully generated a foxd3CreER KI zebrafish line for conditional recombination in neural crest cells.
• Engineered a specific isoleucine-to-valine substitution in the hbaa1.2 gene in zebrafish, demonstrating high editing precision.
• The synthetic exon strategy minimized off-target recombination and improved identification of edited lines, even with highly similar paralogs.

Conclusions:

• The mini-golden system provides a robust toolkit for efficient and precise genome engineering in zebrafish.
• The synthetic exon approach enhances editing specificity and simplifies screening for successful KI events.
• This methodology has broad applicability for genome engineering in various model systems.