CRISPR knockouts of pmela and pmelb engineered a golden tilapia by regulating relative pigment cell abundance

Abstract

AbstractPremelanosome protein (pmel) is a key gene for melanogenesis in vertebrates. Mutations in this gene are responsible for white plumage in chicken, but its role in pigmentation of fish remains to be demonstrated. In this study we found that most fishes have two pmel genes arising from the teleost-specific whole genome duplication. Both pmela and pmelb were expressed at high levels in the eyes and skin of Nile tilapia. We mutated both genes in tilapia using CRISPR/Cas9 gene editing. Homozygous mutation of pmela resulted in yellowish body color with weak vertical bars and a hypo-pigmented retinal pigment epithelium (RPE) due to significantly reduced number and size of melanophores. In contrast, we observed an increased number and size of xanthophores in mutants compared to wild-type fish. Homozygous mutation of pmelb resulted in a similar, but milder phenotype than pmela-/- mutants, without effects on RPE pigmentation. Double mutation of pmela and pmelb resulted in loss of additional melanophores compared to the pmela-/- mutants, and also an increase in the number and size of xanthophores, producing a strong golden body color without bars in the trunk. The RPE pigmentation of pmela-/-;pmelb-/- was similar to pmela-/- mutants, with much less pigmentation than pmelb-/- mutants and wild-type fish. Taken together, our results indicate that, while both pmel genes are important for the formation of body color in tilapia, pmela plays a more important role than pmelb. To our knowledge, this is the first report on mutation of pmelb or both pmela;pmelb in fish. Studies on these mutants suggest new strategies for breeding golden tilapia, and also provide a new model for studies of pmel function in vertebrates.Author SummaryMelanophores, the most common pigment cell type, have been studied for nearly 150 years. Many genes are involved in melanoblast migration, melanophore differentiation, and melanin biosynthesis. Pmel is fundamental for melanosome development by directing melanin biosynthesis and melanosome phase transition. Specifically, PMEL can form a fibrillar structure within the melanosome upon which melanin is deposited. We identified two pmel genes in Nile tilapia arising from the teleost-specific whole genome duplication. Disruption of either pmela or pmelb in tilapia leads to significant hypo-pigmentation. PMEL disrupted fish showed not only a reduction in melanin and tiny melanophores, but also a significant increase in the number of xanthophores, and even guanine-filled melanophores, which led to a golden tilapia with hypo-pigmented RPE. Our study confirmed the role of pmel in melanin biosynthesis and maturation, and also highlighted its effects on melanophore number and size. These results provide new insights into pigment cell biology and will help us better understand the mechanisms of color patterning in teleosts. Knockout of both pmela and pmelb provide a new strategy for engineering a golden tilapia, which might provide a foundation for developing new strains in the tilapia industry.