CRISPR/Cas9-mediated knock-in of masu salmon (Oncorhyncus masou) elongase gene in the melanocortin-4 (mc4r) coding region of channel catfish (Ictalurus punctatus) genome.

Channel catfish, Ictalurus punctatus, have limited ability to synthesize Ω-3 fatty acids. The ccβA-msElovl2 transgene containing masu salmon, Oncorhynchus masou, elongase gene driven by the common carp, Cyprinus carpio, β-actin promoter was inserted into the channel catfish melanocortin-4 receptor (mc4r) gene site using the two-hit two-oligo with plasmid (2H2OP) method. The best performing sgRNA resulted in a knockout mutation rate of 92%, a knock-in rate of 54% and a simultaneous knockout/knock-in rate of 49%.

Gene Editing of the Follicle-Stimulating Hormone Gene to Sterilize Channel Catfish, , Using a Modified Transcription Activator-like Effector Nuclease Technology with Electroporation.

Follicle-stimulating hormone () plays an important role in sexual maturation in catfish. Knocking out the gene in the fish zygote should suppress the reproduction of channel catfish (). In this study, transcription activator-like effector nuclease (TALEN) plasmids targeting the gene were electroporated into fertilized eggs with the standard double electroporation technique.

A New Strategy for Increasing Knock-in Efficiency: Multiple Elongase and Desaturase Transgenes Knock-in by Targeting Long Repeated Sequences.

CRISPR/Cas9-mediated knock-in (KI) has a wide application in gene therapy, gene function study, and transgenic breeding programs. Unlike gene therapy, which requires accurate KI to correct gene mutation, transgenic breeding programs can accept robust KI as long as integration does not interrupt normal gene functions and result in any negative pleiotropic effects. High KI efficiency is required to reduce the breeding cost and shorten the breeding period, especially in transferring multiple foreign genes to a single individual.

Improved Growth and High Inheritance of Melanocortin-4 Receptor (mc4r) Mutation in CRISPR/Cas-9 Gene-Edited Channel Catfish, Ictalurus punctatus.

Effects of CRISPR/Cas9 knockout of the melanocortin-4 receptor (mc4r) gene in channel catfish, Ictalurus punctatus, were investigated. Three sgRNAs targeting the channel catfish mc4r gene in conjunction with Cas9 protein were microinjected in embryos and mutation rate, inheritance, and growth were studied. Efficient mutagenesis was achieved as demonstrated by PCR, Surveyor® assay, and DNA sequencing.

Gene Editing of the Catfish Gonadotropin-Releasing Hormone Gene and Hormone Therapy to Control the Reproduction in Channel Catfish, .

Transcription activator-like effector nuclease (TALEN) plasmids targeting the channel catfish gonadotropin-releasing hormone (cfGnRH) gene were delivered into fertilized eggs with double electroporation to sterilize channel catfish (Ictalurus punctatus). Targeted cfGnRH fish were sequenced and base deletion, substitution, and insertion were detected. The gene mutagenesis was achieved in 52.

CRISPR/Cas9-Mediated Transgenesis of the Masu Salmon (Oncorhynchus masou) elovl2 Gene Improves n-3 Fatty Acid Content in Channel Catfish (Ictalurus punctatus).

Omega-3 polyunsaturated fatty acids (n-3 PUFAs), particularly eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), play a very important role in human health. Channel catfish (Ictalurus punctatus) is one of the leading freshwater aquaculture species in the USA, but has low levels of EPA and DHA compared to some fish such as salmon. To improve EPA and DHA content, a modification of the n-3 PUFA biosynthetic pathway was achieved through the insertion of an elovl2 transgene isolated from masu salmon (Oncorhynchus masou) driven by a carp β-actin promoter using a two-hit by gRNA and two oligos with a targeting plasmid (2H2OP) CRISPR/Cas9 approach.

Direct and pleiotropic effects of the Masou Salmon Delta-5 Desaturase transgene in F1 channel catfish (Ictalurus punctatus).

Channel catfish (Ictalurus punctatus) is the primary culture species in the US along with its hybrid made with male blue catfish, I. furcatus. In an effort to improve the nutritional value of channel catfish, the masou salmon Δ5-desaturase like gene (D5D) driven by the common carp beta-actin promoter (βactin) was inserted into channel catfish.

CRISPR/Cas9-mediated knock-in of alligator cathelicidin gene in a non-coding region of channel catfish genome.

CRISPR/Cas9-based gene knockout in animal cells, particularly in teleosts, has proven to be very efficient with regards to mutation rates, but the precise insertion of exogenous DNA or gene knock-in via the homology-directed repair (HDR) pathway has seldom been achieved outside of the model organisms. Here, we succeeded in integrating with high efficiency an exogenous alligator cathelicidin gene into a targeted non-coding region of channel catfish (Ictalurus punctatus) chromosome 1 using two different donor templates (synthesized linear dsDNA and cloned plasmid DNA constructs). We also tested two different promoters for driving the gene, zebrafish ubiquitin promoter and common carp β-actin promoter, harboring a 250-bp homologous region flanking both sides of the genomic target locus.

TNF-α is involved in apoptosis triggered by grass carp reovirus infection in vitro.

Grass carp reovirus (GCRV) infection causes apoptosis in Ctenopharyngodon idella kidney cells (CIK). However, the cause of GCRV-induced apoptosis and its signaling pathways remain unknown. This study investigated the role of TNF-α-induced capase-8 pathways in mediating GCRV-induced apoptosis in the grass carp (Ctenopharyngodon idella).