The African turquoise killifishNothobranchius furzerihas recently gained interest as an emerging vertebrate model system for the study of aging, owing to its naturally short life span and generation time. Here, we provide a step-by-step guide for effective genome engineering using the CRISPR–Cas9 system to generate loss-of-function (i.e., knockout) alleles and for precise editing (i.e., knock-in) of short sequences into the genome. Using this approach, a new stable line can be created within several months. The killifish's tough chorion, rapid growth, and short life span are considered in this protocol and account for the key deviations from similar protocols in other fish models.

Using lineage tracing and fate mapping strategies to study vertebrate aging has lagged behind developmental studies, primarily due to of the relatively long lifespans of classical models. Here, we introduce theKillibow, an inducible transgenic model forin-vivomulticolor lineage tracing in the naturally short-lived turquoise killifish (N. furzeri). We demonstrate that Cre-mediated recombination in transgenic fish can generate robust and stochastic labeling that remains stable during aging and regeneration. In addition, to achieve inducible control of recombination, we either utilizein-vivoCre electroporation or use the tamoxifen system inKillibow-derived cells. To further enable transplantation assays, we establish the first immunocompromised killifish model by mutatingrag2. RNA sequencing reveals thatrag2mutants exhibit severely compromised expression of V(D)J recombination products, including immunoglobulins. Accordingly, we demonstrate that clearance of transplantedKillibow-derived cells is delayed inrag2recipients, and present a proof-of-principle for a KRASG12Dcancer model that is compatible with lineage tracing. Our platform provides the opportunity to examine tissue homeostasis, stem cell function, cancer dynamics, and tissue regeneration at unprecedented temporal resolution during vertebrate aging and disease.