GENE EDITING WITH CRISPR/CAS9 IN THE EASTERN OYSTER Crassostrea virginica

Clustered Regularly Interspersed Short Palindromic Repeats-Cas9 (CRISPR/Cas9) is a powerful technology for targeted gene editing. It can be used to knockout or knockin genes for studying gene function and for genetic improvement of aquaculture species. Genetic improvement of oysters has so far relied on selective breeding, polyploidization or marker-assisted selection. As candidate genes underlying economically important traits are being identified through functional genomics, gene editing by CRISPR/Cas9 may provide an effective approach to genetic improvement. Gene editing by CRISPR/Cas9 requires efficient plasmid transfection, which remains a challenge in many invertebrates. We developed an effective transfection protocol for oysters using the transfection reagent Superfect (Sigma) and obtained successful transfection with a green fluorescence protein (GFP) plasmid. Green fluorescence was observed at 48h after fertilization suggesting that the plasmid was incorporated and expressed. The observation that dead larvae exhibited the strongest fluorescence signals indicates that over-expression of GFP may be toxic to oyster larvae. CRISPR/Cas9 plasmids targeting four genes, Foxl2, Sox9, GDF8 and Dynein Block 2 of the eastern oyster, were constructed with mCherry as the reporter protein. Red mCherry fluorescence was detected in oyster embryos at 40h after fertilization suggesting that transfection was successful and CRISPR/Cas9 plasmids were expressed. Cas9 DNA sequences were detected at day 2 and 7 after fertilization, indicating that CRISPR/Cas9 plasmid can persist in oyster larvae for at least 7 days. Spat from the experiments have been sampled for genetic confirmation of gene editing.