|A microinjection protocol was developed and validated for CRISPR/Cas9 gene editing in channel catfish, Ictalurus punctatus. This protocol proved to be rapid, efficient and does not require elaborate equipment or a high level of technical skill. To demonstrate the efficiency of the protocol, two channel catfish immune-related genes were targeted, toll/interleukin 1 receptor domain-containing adapter molecule (TICAM 1) gene and rhamnose binding lectin (RBL) gene. Brood stock were artificially spawned and the eggs were fertilized. A microinjection needle was loaded with the injection solution then connected to a microinjector. Injection volume was determined with a hemocytometer by injecting into a drop of mineral oil. Microinjection was performed by introducing the needle into the yolk and expelling the injection material. Embryos were then incubated in Holtfreter’s solution until hatch. Indels in TICAM 1 and RBL genes were confirmed by DNA sequencing. Dramatic changes in the predicted protein sequence included frameshift and truncated protein due to a premature stop codon.
The effects of microinjection of different dosages of guide RNA (gRNA)/Cas9 protein on the mutation rate, embryo mortality, hatchability and early fry survival were investigated in channel catfish. Three dosages of gRNA/Cas9 protein (low, 2.5 ng gRNA/7.5 ng Cas9 protein, medium, 5 ng gRNA/15 ng Cas9 protein and high, 7.5 ng gRNA/22.5 ng Cas9 protein) targeting TICAM 1 and RBL genes, were compared. Microinjection increased the embryo mortality compared to non-injected controls. Injection of gRNA/Cas9 protein increased the embryo mortality when compared to the injected control embryos. Hatching percent was reduced with injection of higher dosages of gRNA/Cas9 protein. Increasing the dosage of gRNA/Cas9 protein increased the mutation rate. Mutation rate was higher in dead embryos than 4-month old fingerlings, suggesting that off-target effects caused some mortality or knockout of the target genes affected viability. These experiments lay the foundations for designing and conducting gene editing experiments in channel catfish and can be used as a guide for other fish species.
A third study evaluated the efficacy of interspecific hybridization of channel catfish females and blue catfish I. furcatus males (CB hybrid catfish), and the transfer of cecropin B gene in enhancing the resistance to Ichthyophthirius multifiliis in catfish fingerlings and food size fish using a communal challenge. At the fingerling stage, cecropin transgenic channel catfish, cecropin transgenic CB hybrid catfish and non-transgenic CB hybrid catfish had similar resistance to ich and the three genotypes had significantly improved resistance to ich when compared to non-transgenic channel catfish. The experiment at the food size stage was conducted on non-transgenic channel catfish and non-transgenic CB hybrid catfish. CB hybrid catfish had a lower infection rate and higher survival rate than channel catfish. The results suggest that genetic enhancement of resistance to ich in catfish can be achieved by either generation of cecropin transgenic channel catfish or production of non-transgenic CB hybrid catfish, but no additional benefit was obtained by applying both genetic improvement programs simultaneously.