Systematic Vertebrate Functional Genomics

Abstract With the generation of more than 100 sequenced vertebrate genomes, the current key question is how to determine the role(s) of uncharacterized gene products in specific biological and pathological processes. For example, genes associated with human disease are being discovered at a rapid rate, thanks in part to the development of next generation sequencing technologies. However, the biological functions underlying this linkage are often unclear, in part because the majority of published work investigates genes encoded by ~10% of the genome. The overall purpose of this long-standing and collaborative research program is to develop innovative ways to address this key gap of functional annotation in genome science. For example, mitochondria have integral functions in metabolism, organ homeostasis, apoptosis and aging. They also play important but still largely perplexing roles in human pathophysiology, as demonstrated by the enormous biological variation and diverse disorders in patients with mitochondrial disease that can compromise nearly every organ system. Over 1100 known nuclear proteins reside in vertebrate mitochondria, with the majority of unknown biological function or no known role in pathogenesis. Deploying loss of function approaches in model systems has been essential to the annotation of the genome to date from the discovery of novel processes to the biological mechanisms underlying disease. Among vertebrates, Danio rerio (zebrafish) has emerged as an outstanding and pioneering vertebrate amenable to both forward and reverse genetic approaches. This funding period will deploy at scale recently developed genome engineering technologies designed to address these gaps in the field. We will use Predominant Microhomology-mediated end-joining Allele (PreMA) generation for rapid functional screening using reproducible allele generation in F0 animals. For those loci with accessible phenotypes, second phase analyses will follow via GeneWeld large-insert targeted knock-in technology in conjunction with gene-breaking protein traps for detailed functional annotation of the genome using targeted protein trapping in F1+ stable lines. Mitochondria are the products of two genomes – the nucleus and from mtDNA. The conservation between zebrafish and human in the mitochondrial genomes includes nearly identical size and perfect synteny of all 37 protein-coding and RNA genes. We will also deploy our recently developed zebrafish mitochondrial base editor to make the first majority heteroplasmy animal models with targeted mutations in mtDNA-encoded proteins for in vivo functional annotation. We will use this pioneering model organism for mitochondrial genomic annotation focusing initially on the mitochondrially encoded proteome and at a targeted panel of vertebrate-specific, nuclearly encoded conserved mitochondrial proteins of unknown function. We will annotate the functional role of these genes using a rich array of biological, phenotypic and biochemical tests in this highly collaborative research program. This work will yield 1) Novel tools for rapid PreMA deployment and new methods for targeted integration of Gene-breaking Protein Trap alleles will be generated and disseminated 2) New biological and genomic annotation of vertebrate mitochondrial innovations in development and regeneration including mechanisms underlying genetic compensation 3) Longitudinal knowledge of mtDNA SNP heteroplasmy maintenance and 4) Complete a unique functional annotation of the mtDNA-encoded proteome. This combination of novel technologies, annotation and genome science will establish important new functional information on the role of mitochondria in biology that, when compromised, underlies disease.