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Microinjection of CRISPR/Cas9 protein into Channel Catfish, Ictalurus punctatus, Embryos to Knockout Genes

Karim Khalil*, Ahmed Elaswad, Patrick Page-McCaw, Wenbiao Chen, Roger Cone and Rex Dunham
School of Fisheries, Aquaculture and Aquatic Sciences,
Auburn University, Auburn, AL, 36849

Channel catfish is the most popular catfish species in the United States. The genome of channel catfish has been sequenced, however, there are still many genes in which gene function is not clearly understood. There is an emerging importance to study gene function in channel catfish. Understanding gene function can assist in selecting the best fish for commercial production as well as sport activities. Channel catfish also could be used as a model for research since there is extensive information on the biology of channel catfish. Gene knockout has been used to study gene functions in vivo. Clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system is a powerful and versatile tool used to edit genomic DNA sequences including gene knockout. Microinjection is the first and widely used method to transfer genes and various constructs in aquaculture species.  

In this work, we describe a detailed protocol for microinjection of channel catfish embryos with CRISPR/Cas9 protein as an example for large aquaculture species. Briefly, eggs and sperm are collected then artificial fertilization is performed. Fertilized eggs are transferred to a petri dish with Holtfreter's solution. A microinjection needle is loaded with the injection solution then connected to a microinjector. Injection volume is determined with a hemocytometer by injecting into a drop of mineral oil. Once the needle is adjusted to inject the required volume, fertilized eggs are injected within the first cell stage of embryonic development. The needle is introduced into the yolk and injection material is expelled. Embryos are then incubated in Holtfreter's solution with antibiotic treatment until hatch. To demonstrate the efficiency of the microinjection protocol, sgRNAs were designed and injected to target some channel catfish genes.

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