Gene Editing of Luteinizing Hormone, Follicle-stimulating Hormone and Gonadotropin-releasing Hormone Genes to Sterilize Channel Catfish, Ictalurus punctatus, using Zinc Finger Nuclease, Transcription Activator-like Effector Nuclease and Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 Technologies

Abstract

Channel catfish (Ictalurus punctatus) is the leading freshwater aquaculture species in the US, but the catfish industry has been in crisis during the past few years. Genetically enhanced fish can greatly contribute to production efficiency and profit, but could pose environmental and ecological risk upon escapement. Targeted gene editing technologies, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 were applied to induce mutagenesis of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and catfish type gonadotropin-releasing hormone (cfGnRH) genes, and to sterilize channel catfish. Three sets of ZFNs targeting LH gene were electroporated into one-cell embryos, and different concentrations were tested. The overall mutation rate was 19.7% and the best treatment was ZFN set 1 at a concentration of 25 μg/ml. Channel catfish carrying the mutated LH gene were sterile, as confirmed by mating experiments. The plasmids were eventually degraded without integration as they were not detectable in mutated individuals. Carp pituitary extract failed to induce spawning and restoration of fertility indicating the need for developing other hormone therapies to achieve reversal of sterility upon demand. TALENs were electroporated into one-cell embryos targeting LH, FSH and cfGnRH genes. Mutations were produced and confirmed with Cel-I assay and DNA sequencing. The mutation rates of LH, FSH and cfGnRH genes were 44.7%, 63.2% and 52.9% respectively. Embryo hatch rates and fry survival rates were not different among either TALEN groups (p=0.387, p=0.182) or among the TALEN and control groups (p=0.340, p=0.107), indicating low cell toxicity of TALENs. CRISPR/Cas9 plasmids were electroporated into embryos to mutate LH and cfGnRH genes, and CRISPR/Cas9 RNAs were microinjected to target cfGnRH gene. Both methods successfully produced mutations. The mutation rates for the electroporation were 37.5% and 38.5%, and 100% for microinjection. However, the egg hatch rate was only 9.0% using microinjection and significant lower than that for electroporation (23.0% and 21.0%, p=0.0004). Low cell toxicity was also confirmed. These results and future research will help in understanding the roles of reproductive genes, sterilization of teleost fish and is a step towards control of domestic, hybrid, exotic, invasive and transgenic fish.