CRISPR-Cas9-Mediated Gene Knockout in a Non-Model Sea Urchin, .

Sea urchins have been used as model organisms in developmental biology research and the genomes of several sea urchin species have been sequenced. Recently, genome editing technologies have become available for sea urchins, and methods for gene knockout using the CRISPRCas9 system have been established. is an important marine fishery resource with edible gonads. Although has been used as a biological research material, its genome has not yet been published, and it is a non-model sea urchin for molecular biology research. However, as recent advances in genome editing technology have facilitated genome modification in non-model organisms, we applied genome editing using the CRISPR-Cas9 system to . In this study, we targeted genes encoding ETS transcription factor () and pigmentation-related polyketide synthase (). Gene fragments were isolated using primers designed by inter-specific sequence comparisons within Echinoidea. When gene was targeted using two sgRNAs, one successfully introduced mutations and impaired skeletogenesis. In the gene knockout, when two sgRNAs targeting the close vicinity of the site corresponding to the target site that showed 100% mutagenesis efficiency of the gene in , mutagenesis was not observed. However, two other sgRNAs targeting distant sites efficiently introduced mutations. In addition, knockout exhibited an albino phenotype in the pluteus larvae and adult sea urchins after metamorphosis. This indicates that the CRISPRCas9 system can be used to modify the genome of the non-model sea urchin .