# Advancing knock-in approaches for robust genome editing in zebrafish

## TL;DR

The paper introduces a new system for precise genome editing in zebrafish, enabling efficient and accurate genetic modifications.

## Contribution

The mini-golden system and synthetic exon-based donor template strategy enable precise genome editing in zebrafish.

## Key findings

- The mini-golden system allows rapid assembly of donor constructs for knock-in applications.
- A synthetic exon-based strategy successfully edited a specific hemoglobin gene in zebrafish without affecting its paralog.
- The method improved the identification of edited zebrafish lines and minimized undesired recombination.

## Abstract

Precise genome editing remains a major challenge in functional genomics, particularly for generating knock-in (KI) alleles in model organisms. Here, we introduce the mini-golden system, a versatile Golden Gate-based subcloning platform that enables rapid assembly of donor constructs containing homology arms and a gene of interest. This system offers a library of middle entry vectors including diverse genes, enhancing the preparation of donor minicircles for KI applications. Using the mini-golden system, we efficiently generated a foxd3CreER KI zebrafish line, allowing conditional recombination in neural crest cells. To further improve genome editing precision, we developed a synthetic exon-based donor template strategy combined with fluorescence screening. Using this approach, we successfully engineered a targeted isoleucine-to-valine substitution (Ile-to-Val) in hbaa1.2, one of the two adult hemoglobin alpha genes in zebrafish. Importantly, despite the high sequence similarity between hbaa1.2 and its paralog hbaa1.1, our strategy specifically edited hbaa1.2, demonstrating the effectiveness of the synthetic exon approach. This method minimized undesired recombination and significantly improved the identification of lines carrying the edited genome. Together, we provide a robust toolkit for efficient and precise genome engineering in zebrafish, with broad applicability to other model systems.

Summary: This paper describes a robust toolkit for efficient and precise genome engineering in zebrafish, with broad applicability to other model systems.