Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

作者： Ahmed Elaswad*1,2, Karim Khalil*1,3, David Cline1, Patrick Page-McCaw4, Wenbiao Chen4, Maximilian Michel5, Roger Cone5, Rex Dunham1 1School of Fisheries, Aquaculture and Aquatic Sciences,Auburn University, 2Department of Animal Wealth Development, Faculty of Veterinary Medicine,Suez Canal University, 3Anatomy and Embryology Department, Faculty of Veterinary Medicine,Cairo University, 4Department of Molecular Physiology and Biophysics,Vanderbilt University, 5Life Science Institute,University of Michigan

简介：

Overview

This article presents a detailed protocol for microinjecting CRISPR/Cas9 components into channel catfish embryos for gene editing. The method has been validated by targeting two immune-related genes, enhancing the understanding of functional genomics in this species.

Key Study Components

Area of Science

• Gene editing
• Functional genomics
• Embryology

Background

• The channel catfish genome has been sequenced, providing valuable genomic resources.
• Targeted gene editing is essential for studying gene function.
• CRISPR/Cas9 is a widely used tool for gene editing in various organisms.
• Efficient protocols are necessary for successful applications in aquaculture research.

Purpose of Study

• To develop a reliable microinjection protocol for CRISPR/Cas9 in channel catfish embryos.
• To validate the protocol by knocking out specific immune-related genes.
• To facilitate further research in functional genomics of channel catfish.

Methods Used

• Microinjection of guide RNAs and Cas9 protein into one-cell embryos.
• Targeting of TICAM1 and RBL genes for knockout validation.
• Assessment of gene editing efficiency through phenotypic analysis.
• Utilization of genomic resources for protocol development.

Main Results

• Successful microinjection of CRISPR/Cas9 components into embryos.
• Knockout of TICAM1 and RBL genes confirmed.
• Protocol demonstrated efficiency and reliability for gene editing.
• Enhanced understanding of immune-related gene functions in channel catfish.

Conclusions

• The developed protocol is a valuable tool for gene editing in channel catfish.
• It opens avenues for further research in functional genomics.
• Future studies can leverage this method for various genetic investigations.

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