Generation of Stable knock-out lines

A ribonucleoprotein (RNP) complex, conformed by Cas9 and the sgRNA(s) designed against the selected gene, is injected into wild-type one-cell stage embryos (F0)

Once they reach sexual maturity, injected larvae (F0) are screened to identify a founder carrying a loss-of-function mutation in the germline

The selected founder is then crossed to a wild-type fish, thus generating a population of heterozygous and wild-type offspring (F1).

The inbreeding of adult heterozygous fish (F1) gives rise to homozygous mutant, heterozygous and wild type larvae (F2), in which the phenotypes deriving from gene inactivation can be analyzed.