海洋軟体動物で標的遺伝子変異を持つゲノム編集個体群の作出に成功（Researchers Establish G0 Founder Population Carrying Targeted Mutations in Marine Mollusks）

Genome editing-based breeding has progressed rapidly in aquaculture species but remains challenging in mollusks. The limited number of mutant larvae and frequent deaths during metamorphosis, together with the potential deficiencies caused by gene mutation, make the long-term culture of mutant individuals difficult. Currently, there are no reports on the development of G0 populations in aquaculture mollusks. Here, we established a G0 founder population carrying mutations targeted a tyrosinase gene in the Pacific abalone Haliotis discus hannai. By addressing various issues in generating and culturing the small number of manipulated larvae (50–100), we developed a reliable workflow encompassing mutagenesis through genome editing, larval/juvenile culture, metamorphosis induction, and assessing mutation-carrying juveniles. After obtaining mutant larvae by microinjecting Cas9/sgRNA to the oocyte, frequent mass mortality of them was proved to be the major obstacle for subsequent culture. We addressed this issue by culturing each larva individually, which ensured high survival rates. By determining the optimal induction time point for each larval batch, an efficient metamorphosis-induction strategy was developed with the metamorphosis rates of over 90%. Pure diatom cultures were also prepared to ensure rapid juvenile growth. Using this workflow, we successfully generated a G0 founder population containing 98 juveniles with shell lengths of 4–10 mm (3–5mpf). Genotyping confirmed that mutant individuals were cultured to the juvenile stage. This work established a G0 founder population in an economic mollusk, representing an essential step toward completing the technical chain of molluscan genome editing-based genetic breeding.