Advancing Knock-In Approaches for Robust Genome Editing in Zebrafish.

Area of Science:

• Genomics and Molecular Biology
• Developmental Biology
• Zebrafish as a Model Organism

Background:

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

Purpose of the Study:

• To introduce a versatile Golden Gate-based platform, the mini-golden system, for rapid assembly of donor constructs for KI applications.
• To develop and validate a synthetic exon-based donor template strategy for enhanced genome editing precision and specificity.

Main Methods:

• Utilized the mini-golden system for efficient assembly of donor constructs with homology arms and genes of interest.
• Generated a foxd3^CreER KI zebrafish line using the mini-golden system.
• Developed a synthetic exon strategy combined with fluorescence screening for precise gene editing, including a targeted Ile-to-Val substitution in hbaa1.2.

Main Results:

• Successfully generated a foxd3^CreER KI zebrafish line for conditional recombination in neural crest cells.
• Achieved precise, targeted editing of the hbaa1.2 gene, demonstrating specificity despite high sequence similarity with its paralog hbaa1.1.
• The synthetic exon approach minimized undesired recombination and improved the identification of edited lines.

Conclusions:

• The mini-golden system provides a robust toolkit for efficient genome engineering in zebrafish.
• The synthetic exon strategy enhances precision and specificity in genome editing, particularly for challenging targets.
• These advancements have broad applicability for genome engineering in various model systems.