Editing of the Luteinizing Hormone Gene to Sterilize Channel Catfish, Ictalurus punctatus, Using a Modified Zinc Finger Nuclease Technology with Electroporation

Abstract

Channel catfish (Ictalurus punctatus) is the most important freshwater aquaculture species in the USA. Genetically enhanced fish that are sterile could both profit the catfish industry and reduce potential environmental and ecological risks. As the first step to generate sterile channel catfish, three sets of zinc finger nuclease (ZFN) plasmids targeting the luteinizing hormone (LH) gene were designed and electroporated into one-cell embryos, different concentrations were introduced, and the Cel-I assay was conducted to detect mutations. Channel catfish carrying the mutated LH gene were sterile, as confirmed by DNA sequencing and mating experiments. The overall mutation rate was 19.7 % for 66 channel catfish, and the best treatment was ZFN set 1 at the concentration 25 μg/ml. To our knowledge, this is the first instance of gene editing of fish via plasmid introduction instead of mRNA microinjection. The introduction of the ZFN plasmids may have reduced mosaicism, as mutated individuals were gene edited in every tissue evaluated. Apparently, the plasmids were eventually degraded without integration, as they were not detectable in mutated individuals using PCR. 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. This is the first sterilization achieved using ZFN technology in an aquaculture species and the first successful gene editing of channel catfish. Our results will help understand the roles of the LH gene, purposeful sterilization of teleost fishes, and is a step towards control of domestic, hybrid, exotic, invasive, and transgenic fishes.

Efficient Gene Transfer and Gene Editing in Sterlet (Acipenser ruthenus)

“…Reverse genetics research on animals and plants was largely pushed ahead by zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR) ( Gaj et al, 2013 ), which showed great potential to modify genes of interest ( Ding et al, 2013 ). Until now, targeted gene editing has been reported in zebrafish, medaka, Nile tilapia, rainbow trout, common carp, channel catfish, rohu ( Labeo rohita ), Chinese tongue sole ( Cynoglossus semilaevis ), rice field eel ( Monopterus albus ), and so on ( Doyon et al, 2008 ; Ansai et al, 2014 ; Li et al, 2014 ; Yano et al, 2014 ; Chakrapani et al, 2016 ; Qin et al, 2016 ; Zhong et al, 2016 ; Cui et al, 2017 ; Feng et al, 2017 ).…”

Biotechnological innovation in genetic breeding and sustainable green development in Chinese aquaculture

“…自此, 基因组编辑在多种养殖鱼类中得到了应用, 如虹 鳟 [151] 、罗非鱼 [152] 、鲤 [153] 、大西洋鲑 [154] 、斑点叉尾 鮰 [155] 、黄鳝 [156] 、半滑舌鳎 [157] 和小体鲟 [158] 等. 基因 组编辑技术将在水产养殖动物基因功能解析和经济性 状精准设计育种中发挥重要作用 [102,145,149] .…”

Disease reduction in aquaculture with genetic and genomic technology: current and future approaches

“…Zinc fingers have gene-targeting frequencies of up to 20% success (Porteus & Carroll 2005). In channel catfish, the overall mutation rate for three ZFN sets was 19.7%, with one ZFN set inducing average mutation rate of 38.5% in two of the treatments (Qin et al 2016). Because of greater costs, complexity of design, off-target effects, cytotoxicity and lower mutation rate, research on ZFNs is decreasing, especially once TALENs became available.…”