Highly Efficient Knockout of a Squid Pigmentation Gene

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In this paper, we demonstrate efficient gene knockout in the squid Doryteuthis pealeii using CRISPR-Cas9. Ommochromes, the pigments found in squid retinas and chromatophores, are derivatives of tryptophan, and the first committed step in their synthesis is normally catalyzed by Tryptophan 2,3 Dioxygenase (TDO [18Williams T.L. DiBona C.W. Dinneen S.R. Labadie S.F.J. Chu F. Deravi L.F. Contributions of phenoxazone-based pigments to the structure and function of nanostructured granules in squid chromatophores.Langmuir. 2016; 32: 3754-3759Crossref PubMed Scopus (17) Google Scholar, 19Schwinck I. Über den Nachweis eines Redox-Pigmentes (Ommochrom) in der Haut von Sepia officinalis.Naturwissenschaften. 1953; 40: 365Crossref Scopus (5) Google Scholar, 20Aubourg S.P. Torres-Arreola W. Trigo M. Ezquerra-Brauer J.M. Partial characterization of jumbo squid skin pigment extract and its antioxidant potential in a marine oil system.Eur. J. Lipid Sci. Technol. 2016; 118: 1293-1304Crossref Scopus (14) Google Scholar]). Knocking out TDO in squid embryos efficiently eliminated pigmentation. By precisely timing CRISPR-Cas9 delivery during early development, the degree of pigmentation could be finely controlled. Genotyping revealed knockout efficiencies routinely greater than 90%. This study represents a critical advancement toward making squid genetically tractable.