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 utilized clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) genome editing to assess essentiality and functionality of zebrafish (Danio rerio) type-I and type-III Vtgs. A multiple CRISPR approach was employed to knockout (KO) all genes encoding type-I vtgs (vtg1, 4, 5, 6, and 7) simultaneously (vtg1-KO), and the type-III vtg (vtg3) individually (vtg3-KO). Results of polymerase chain reaction (PCR) genotyping and sequencing, quantitative PCR, liquid chromatography-tandem mass spectrometry, and Western blot analysis showed that only vtg6 and vtg7 escaped Cas9 editing. In fish whose remaining type-I vtgs were incapacitated (vtg1-KO), and in vtg3-KO fish, significant increases in Vtg7 transcript and protein levels occurred in liver and eggs, revealing a heretofore-unknown mechanism of genetic compensation regulating Vtg homeostasis. Egg numbers per spawn were elevated more than 2-fold in vtg1-KO females, and egg fertility was approximately halved in vtg3-KO females. Substantial mortality was evident in vtg3-KO eggs/embryos after only 8 hr of incubation and in vtg1-KO embryos after 5 days. Hatching rate and timing were markedly impaired in embryos from vtg mutant mothers and pericardial and yolk sac/abdominal edema and spinal lordosis were evident in the larvae, with feeding and motor activities also being absent in vtg1-KO larvae. By late larval stages, vtg mutations 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