Efficient Gene Editing in Fish Primary Germline Stem Cells

Genome editing by the CRISPR/Cas9 system is widely used for production of gene-modified animals, including fish. However, efficient gene editing in fish cultured cells, in particular germline stem cells (GSCs), is challenging, likely due to the difficulty in transfecting these cells. The ricefield eel (Monopterus albus), a sequential hermaphroditic species, is a freshwater fish of significant economic value in China. In this work, we report a simple method that can achieve high gene editing efficiency in primary GSCs of fish species, including ricefield eel. High transfection efficiency (50-95%) is achieved in fish GSCs by using a microchannel-based cell transfection system. Gene editing efficiency of up to 60% in primary ricefield eel GSCs is achieved using an integrated CRISPR/Cas9 vector strategy. High gene editing efficiency is also achieved in gibel carp (Carassius gibelio) GSCs and the medaka spermatogonial stem cell line SG3, demonstrating the general applicability of our method. Our data suggest that efficient transfection is key to high gene editing efficiency in fish cultured GSCs. This study establishes an efficient and reliable gene editing system for fish GSCs, which may facilitate the creation of new germplasm of genetically difficult-to-breed fish by combining GSC transplantation with gene editing techniques.