Efficient Gene Editing in Fish Primary Germline Stem Cells

Area of Science:

• Aquaculture
• Genetics
• Molecular Biology

Background:

• CRISPR/Cas9 gene editing is crucial for creating gene-modified animals, but its efficiency in fish germline stem cells (GSCs) is limited by transfection challenges.
• The ricefield eel (Monopterus albus) is an economically important freshwater fish species in China, yet efficient genetic modification remains difficult.

Purpose of the Study:

• To establish a highly efficient gene editing system for fish GSCs, overcoming current transfection limitations.
• To demonstrate the broad applicability of the developed method across different fish species.

Main Methods:

• Utilized a microchannel-based cell transfection system to achieve high transfection efficiency in fish GSCs.
• Employed an integrated CRISPR/Cas9 vector strategy for precise gene editing.
• Applied the method to primary GSCs of ricefield eel, gibel carp, and the medaka SG3 cell line.

Main Results:

• Achieved high transfection efficiency (50-95%) in fish GSCs using the microchannel system.
• Reached up to 60% gene editing efficiency in primary ricefield eel GSCs.
• Demonstrated successful high gene editing efficiency in gibel carp GSCs and medaka SG3 cells, confirming method's general applicability.

Conclusions:

• Efficient transfection is the critical factor for achieving high gene editing efficiency in fish GSCs.
• The developed CRISPR/Cas9 gene editing system provides a reliable method for fish GSC modification.
• This system facilitates the creation of novel germplasm in genetically challenging fish species through GSC transplantation and gene editing.