Genome editing reveals reproductive and developmental dependencies on specific types of vitellogenin in zebrafish (Danio rerio)

Oviparous vertebrates produce multiple forms of vitellogenin (Vtg), the major source of yolk nutrients, but little is known about their individual contributions to reproduction and development. This study employed a CRISPR/Cas9 genome editing to assess essentiality and functionality of zebrafish (Danio rerio) type-I and -III Vtgs. The multiple CRISPR approach employed to knock out (KO) all genes encoding type-Ivtgs(vtg1, 4, 5, 6,and7) simultaneously (vtg1-KO), and the type-IIIvtg(vtg3) individually (vtg3-KO). Results of PCR genotyping and sequencing, qPCR, LC-MS/MS and Western blotting showed that onlyvtg6andvtg7escaped Cas9 editing. In fish whose remaining type-Ivtgswere incapacitated (vtg1-KO), and invtg3-KO fish, significant increases in Vtg7 transcript and protein levels occurred in liver and eggs, a heretofore-unknown mechanism of genetic compensation to regulate Vtg homeostasis. Fecundity was more than doubled invtg1-KO females, and fertility was ~halved invtg3-KO females. Substantial mortality was evident invtg3-KO eggs/embryos after only 8 h of incubation and invtg1-KO embryos after 5 d. Hatching rate and timing were markedly impaired invtgmutant embryos and pericardial and yolk sac/abdominal edema and spinal lordosis were evident in the larvae, with feeding and motor activities also being absent invtg1-KO larvae. By late larval stages,vtgmutations were either completely lethal (vtg1-KO) or nearly so (vtg3-KO). These novel findings offer the first experimental evidence that different types of vertebrate Vtg are essential and have disparate requisite functions at different times during both reproduction and development.