The turquoise killifish is the shortest-lived vertebrate species bred in the lab

Aging is a complex process that affects multiple organs. Modeling aging and age-related diseases in the lab is challenging because classical vertebrate models have relatively long lifespans. Here, we develop the first platform for rapid exploration of age-dependent traits and diseases in vertebrates, using the naturally short-lived African turquoise killifish. We provide an integrative genomic and genome-editing toolkit in this organism using our de-novo-assembled genome and the CRISPR/Cas9 technology. We mutate many genes encompassing the hallmarks of aging, and for a subset, we produce stable lines within 2–3 months. As a proof of principle, we show that fish deficient for the protein subunit of telomerase exhibit the fastest onset of telomere-related pathologies among vertebrates. We further demonstrate the feasibility of creating specific genetic variants. This genome-to-phenotype platform represents a unique resource for studying vertebrate aging and disease in a high-throughput manner and for investigating candidates arising from human genome-wide studies.

Introduction

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Moore D.J. Genetic mouse models of neurodegenerative diseases. Aging is the number one risk factor for many human pathologies, including diabetes, cancer, cardiovascular, and neurodegenerative diseases (). Thus, delaying aging could help postpone the onset of these devastating ailments and increase healthspan. Because aging affects multiple organs and systems in humans (), it is one of the most challenging processes to model in the lab. So far, the study of aging has been dominated by non-vertebrate short-lived model organisms, such as yeast (C. cerevisiae), worm (C. elegans), and fly (D. melanogaster), which has allowed the identification of remarkably conserved aging-related pathways, such as the TOR and Insulin/IGF pathways (). However, some important aspects of human aging and disease phenotypes cannot be faithfully recapitulated in invertebrate models, as they lack specific organs and systems (e.g., blood, bones, and an adaptive immune system) that are crucial components of human aging and age-related pathologies. Vertebrate model systems, namely the mouse (M. musculus) and zebrafish (D. rerio), have also been exploited to probe genes involved in aging and age-related diseases. However, experimental studies have been hampered by the relatively long lifespan of mice and zebrafish (maximal lifespan of 3–4 and 5 years, respectively []) and high costs of maintenance, especially for mice. Mouse models with accelerated onset of age-associated disease (e.g., neurodegeneration) can partially address this issue (), but these models uncouple the disease phenotype from its main risk factor—aging—and they remain expensive to use. Thus, a new vertebrate model is needed to better understand the principles of vertebrate aging and to study age-related diseases in the context of aging.

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Cellerino A. Temperature affects longevity and age-related locomotor and cognitive decay in the short-lived fish Nothobranchius furzeri. The turquoise killifish has additional advantages as a model system. Contrary to many other fish, including zebrafish, the turquoise killifish has an XY-based sexual determination (). Furthermore, there exists a highly inbred strain of the turquoise killifish (the GRZ strain, used in this study), as well as a number of wild-derived strains (). The availability of multiple strains provides an important advantage for genetic studies and for mapping traits that are different between strains (e.g., color, maximal lifespan) (). Collectively, these characteristics of the turquoise killifish—coupled with the ease of rapidly generating many offspring and low maintenance costs—make this fish a promising vertebrate model, uniquely fit to address aging and age-related diseases ().

Here, we create the first platform for the rapid exploration of aging and aging-related diseases in vertebrates by developing new genomic and genome-editing tools in a promising vertebrate model, the naturally short-lived African turquoise killifish. As a proof of principle for the versatility of this platform, we generate a suite of mutated alleles for 13 genes encompassing the hallmarks of aging and report six stable lines to date. We characterize a loss-of-function mutation in the gene encoding the protein component of telomerase and show that telomerase-deficient turquoise killifish recapitulate characteristics of human pathologies. This platform should allow high-throughput studies on aging and longevity in vertebrates, as well as longitudinal modeling of human diseases. Our platform should also enable systematic examination of unexplored candidates identified in human genomic studies.