Conditional genetics and precision genome editing resources for Zebrafish

The overarching goal of this proposal is to create conditional gene animal resources for modeling human health and disease in zebrafish through improved genome editing tools. Zebrafish (Danio rerio) is second only to mouse as the most commonly published model system for human health. In 2023, NIH supports over 150 Principal Investigators in >200 Research and Resource Zebrafish projects (>$80 million) across 19 institutes. Zebrafish possess high fecundity for genetic studies and optically clear embryos for live imaging and visualization of developmental processes. Readily accessible methods have been established to modify gene expression or edit the genome by injection of reagents into the single cell embryo. Zebrafish is increasingly used for post-embryonic and adult models of disease and regeneration, requiring strategies for inducible genetic manipulation with tight spatial and temporal control across the lifespan. The ability to fully utilize this powerful animal model is limited by a lack of tools for tissue and cell-type specific gene functional studies, reproducible and accurate transgene expression, and reliable integration of nucleotide variants and short sequences into the nuclear genome. Epigenome regulation and energy metabolism are rapidly emerging areas impacting development, disease, and regeneration, yet few zebrafish genetic alleles are available to study either in zebrafish. Moreover, the ubiquitous expression of epigenetic and nuclear mitochondrial genes requires conditional alleles to model tissue and organ specific pathologies associated with these fundamental biological processes. For zebrafish to remain at the forefront of animal models that address the research mission of institutes across the NIH, innovative methods are needed for precision genome editing and expanding the conditional genetic toolbox to these understudies areas. Building on our pioneering strategies in zebrafish genome editing, the improvements and conditional genetics resources we propose to develop here will have a significant, lasting impact on zebrafish. We propose the following aims: Aim 1. Expand the zebrafish community resource for Cre recombinase conditional gene studies to established fields of zebrafish studies (embryonic development, major tissues and organ systems) and understudied, rapidly emerging areas (epigenetics, mitochondrial biology). Aim 2. Further develop and improve methods for precision targeted integration to increase the accessibility of our GeneWeld and DonorGuide methods for integration of long DNA fragments and short sequences. Aim 3. Continue development and distribution of targeted integration resources to the zebrafish community through seminars, conferences, social media, and virtual and hands on training. We expect by the end of the funding period to significantly expand our Cre and conditional allele resource. Our methods to improve GeneWeld and DonorGuide precision genome editing in zebrafish will be widely applicable, given the near universal application of CRISPR genome editing in model and non-model systems.