Gene Editing and Transgenic Reporter Assays in African Killifish

Abstract

As an emerging genetic model, the short-lived African killifish Nothobranchius furzeri has displayed unique advantages for vertebrate aging and regeneration studies. Gene editing and transgenesis are critical strategies widely used for discovering and dissecting underlying mechanisms of specific biological questions. In popular teleost models, such genetic manipulations are time-consuming due to the relatively long-generation time. Remarkably, the turnover time of genetic manipulation in the African killifish is much shorter because of its rapid sexual maturation. The fast genetic manipulation makes African killifish very powerful in addressing many biological questions. Here, we discuss a detailed and efficient pipeline step by step for generating loss-of-function mutants using the CRISPR/Cas9 approach and performing transgenic reporter assays to dissect gene regulatory elements in African killifish. We cover the sgRNA design and in vitro transcription, the assembly of reporter constructs, embryo collection, microinjection, African killifish hatching and maintenance, and the identification of mutants or transgenic animals.