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Published on: January 20, 2018
A Reproducible Electroporation Strategy for CRISPR-Cas9 RNP and mRNA Delivery in Fish Embryos
Yuanri Hu1,2,3, Fei Fang2,3,4, Zhongkai Cui5,6
1National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai, 201306, China.
Marine Biotechnology (New York, N.Y.)
|August 3, 2026
Summary
We developed an improved electroporation method for delivering molecules into zebrafish embryos. This technique enhances gene editing efficiency and protein expression, offering new possibilities for aquatic biotechnology.
Area of Science:
- Developmental Biology
- Molecular Biology
- Biotechnology
Background:
- Efficient macromolecular delivery into zebrafish embryos is crucial for genetic studies.
- The zebrafish chorion presents a significant barrier to delivery.
- Existing electroporation methods require optimization for improved efficiency and embryo survival.
Purpose of the Study:
- To develop and optimize a streamlined electroporation protocol for efficient macromolecular delivery into zebrafish embryos.
- To assess the efficacy of this method for gene knockout using Cas9 ribonucleoproteins (RNPs).
- To investigate the impact of polyglutamic acid (PGA) on RNP complex performance.
Main Methods:
- Characterization of the chorion barrier using Transmission Electron Microscopy (TEM).
- Systematic optimization of electroporation parameters (voltage, duration) for dechorionated zebrafish embryos.
- Delivery of enhanced green fluorescent protein (eGFP) mRNA for transfection efficiency assessment.
- Electroporation-mediated delivery of Cas9 RNPs targeting specific genes (tyr, slc24a5, ddx19b).
- Modification of RNP complexes with polyglutamic acid (PGA) to improve stability and efficacy.
Main Results:
- Optimal electroporation parameters were identified, ensuring high embryo survival and robust eGFP mRNA transfection.
- Targeted gene knockout achieved for tyr, slc24a5, and ddx19b loci, with phenotypic rates ranging from 38.33% to 44.45%.
- PGA-modified RNP complexes demonstrated enhanced gene editing efficiency compared to a commercial system.
- Sequencing confirmed successful introduction of indels at target sites.
Conclusions:
- A highly effective electroporation strategy for zebrafish embryos was established, overcoming chorion barriers.
- The method enables high-efficiency mRNA and RNP delivery for protein expression and gene editing.
- Nanotechnology-augmented RNP delivery shows significant potential for applications in aquatic biotechnology and genetic research.
