An Efficient Vector-Based CRISPR/Cas9 System in Zebrafish Cell Line.

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• The CRISPR/Cas9 system is a powerful genome editing tool but faces challenges in fish cell lines due to inefficient promoter systems.
• Existing vectors often require separate components for sgRNA and Cas9 expression, hindering efficient delivery in fish cells.

Purpose of the Study:

• To develop an optimized all-in-one CRISPR/Cas9 vector for efficient gene editing in zebrafish cell lines.
• To establish a robust in vitro platform for high-throughput gene function analysis in fish.

Main Methods:

• Utilized the zebrafish U6 RNA polymerase III (ZFU6) promoter to drive sgRNA and Cas9 expression within a single lentiviral vector.
• Constructed an optimized vector based on the lentiCRISPRV2 system, replacing the human U6 promoter with ZFU6.
• Tested the vector's efficacy in zebrafish fibroblast (PAC2) cells targeting the ctgfa gene and in mammalian 293T cells targeting the EMX1 gene.

Main Results:

• The ZFU6 promoter efficiently induced tyrosinase (tyr) gene editing in zebrafish embryos, leading to loss of retinal pigments.
• The optimized all-in-one vector successfully targeted the ctgfa gene in PAC2 cells, demonstrating its functionality.
• The vector also demonstrated efficacy in editing the EMX1 gene in mammalian 293T cells, indicating broad applicability.

Conclusions:

• An efficient CRISPR/Cas9 gene editing tool was established for zebrafish cell lines using the ZFU6 promoter.
• This novel vector system facilitates high-throughput gene function analysis in fish cell lines.
• The tool shows potential for wide application across different cell types, including mammalian cells.