Safe harbor landing sites as community resource for next-generation transgenesis in zebrafish

PROJECT SUMMARY Transgenesis to generate reporters or genetic modifiers is a key tool for biomedical research organisms. Zebrafish are a critical model to study early vertebrate development, regeneration, and congenital disease, with transgenic zebrafish as uniquely powerful tools to visualize and perturb biological processes. However, routine generation of transgenic zebrafish using random genomic integration of linearized DNA vectors or Tol2 transposons remains a major challenge in the field: beyond highly variable transgene activity due to chromatin position effects, the required number of animals, resources, and time to select high-quality, single-integration transgenes remain prohibitive to numerous labs. Site-directed transgenesis into so-called safe-harbor landing sites, genomic locations with faithful transgene activity, is critically missing in zebrafish. In Drosophila and mouse, phiC31 integrase-mediated transgenesis of DNA vectors into engineered, carefully selected attP landing sites has revolutionized the field. Access to several attP landing sites enable predictable, reproducible, and economical transgene generation for numerous biomedical research applications, including quantitative assessment of disease variant genes, predictable expression of genetic modifiers, and routine generation of fluorescent reporters. Building on our pioneering work on phiC31 integrase transgenesis, we will establish and expand community-accessible resources for site-directed transgenesis in zebrafish. Replacing two previously established Tol2-based transgenes with attP sites through CRISPR-Cas9, we generated the first possibly universal landing site strains for zebrafish as phiC31 Integrase Genomic Loci Engineered for Transgenesis (pIGLET). We documented reproducible and exceedingly high transgenesis efficiencies upon injection and germline transmission for diverse transgenes. Guided by community feedback to expand on our first two landing sites, in Aim 1 we will generate next-generation landing sites for reporter and enhancer testing applications in our validated loci and will establish previously challenging new Cre/lox tools. However, our current landing sites on chromosomes 14 and 24 limit combinatorial transgene experiments and strain health through inbreeding; additional landing sites with different properties (expression strength, maternal contribution, etc.) on other chromosomes are critically needed akin to Drosophila and mice. In Aim 2, we will streamline mapping of established, faithful transgenes using innovative DNA capture and long-read sequencing to identify suitable additional safe-harbor sites and to generate additional landing site strains and quasi-enhancer traps for diverse biomedical research applications. Our reagents and protocols will be openly accessible through the Zebrafish International Resource Center (ZIRC), Addgene, and direct distribution. Together, our work aims to advance zebrafish transgenesis applicable across NIH institute interests, greatly reducing the required resources, animals, and time to establish transgenic zebrafish strains.