12Oviparous vertebrates produce multiple forms of vitellogenin (Vtg), the major source of yolk 13 nutrients, but little is known about their individual contributions to reproduction and development. This14 study employed a CRISPR/Cas9 genome editing to assess essentiality and functionality of zebrafish 15 (Danio rerio) type-I and -III Vtgs. The multiple CRISPR approach employed to knock out (KO) all genes 16 encoding type-I vtgs (vtg1, 4, 5, 6, and 7) simultaneously (vtg1-KO), and the type-III vtg (vtg3) 17 individually (vtg3-KO). Results of PCR genotyping and sequencing, qPCR, LC-MS/MS and Western 18blotting showed that only vtg6 and vtg7 escaped Cas9 editing. In fish whose remaining type-I vtgs were 19 incapacitated (vtg1-KO), and in vtg3-KO fish, significant increases in Vtg7 transcript and protein levels 20 occurred in liver and eggs, a heretofore-unknown mechanism of genetic compensation to regulate Vtg 21 homeostasis. Fecundity was more than doubled in vtg1-KO females, and fertility was ~halved in vtg3-KO 22 females. Substantial mortality was evident in vtg3-KO eggs/embryos after only 8 h of incubation and in 23 vtg1-KO embryos after 5 d. Hatching rate and timing were markedly impaired in vtg mutant embryos and 24 pericardial and yolk sac/abdominal edema and spinal lordosis were evident in the larvae, with feeding and 25 motor activities also being absent in vtg1-KO larvae. By late larval stages, vtg mutations were either 26 2 completely lethal (vtg1-KO) or nearly so (vtg3-KO). These novel findings offer the first experimental 27 evidence that different types of vertebrate Vtg are essential and have disparate requisite functions at 28 different times during both reproduction and development. 29 30