Gene Knockout in a Cephalopod

Research using squid as a model led to seminal breakthroughs in diverse areas of biology and biophysics. Despite their biological novelties, such as possessing by far the largest brains among invertebrates, highly plastic systems for camouflage, and an advanced system for recoding genetic information as it passes through mRNA, squid and other cephalopods are not widely used in research today. This is mostly because at present they are genetically intractable. In this work we demonstrate the first gene knockout in a cephalopod. The pigments in squid chromatophores and retinas are ommochromes. In many organisms, the first step in ommochrome biosynthesis is catalyzed by Tryptophan 2,3 Dioxygenase (TDO). We show that in the squid Doryteuthis pealeii TDO message is highly expressed in chromatophores and eyes, and that pigmentation can be blocked in developing embryos using TDO-specific antagonists. Likewise, TDO knockouts in developing embryos using the CRISPR-Cas9 system efficiently eliminated pigmentation. By fertilizing oocytes in vitro and precisely timing CRISPR-Cas9 delivery, the degree of pigmentation could be finely controlled. Genotyping of hatchlings through deep sequencing revealed highly efficient gene knockouts (routinely greater than 90%) in the G0 generation. These results open a pathway for exploring gene function in cephalopods and the associated biological innovations.