Generation of a transparent killifish line through multiplex CRISPR/Cas9mediated gene inactivation

Abstract

Body pigmentation is a limitation for <i>in vivo</i> imaging and thus for the performance of longitudinal studies in biomedicine. A possibility to circumvent this obstacle is the employment of pigmentation mutants, which are used in fish species like zebrafish and medaka. To address the basis of aging, the short-lived African killifish <i>Nothobranchius furzeri</i> has recently been established as a model organism. Despite its short lifespan, <i>N. furzeri</i> shows typical signs of mammalian aging including telomere shortening, accumulation of senescent cells, and loss of regenerative capacity. Here, we report the generation of a transparent <i>N. furzeri</i> line by the simultaneous inactivation of three key loci responsible for pigmentation. We demonstrate that this stable line, named <i>klara</i>, can serve as a tool for different applications including behavioral experiments and the establishment of a senescence reporter by integration of a fluorophore into the <i>cdkn1a (p21</i>) locus and <i>in vivo</i> microscopy of the resulting line.