ALTERING MELANOCORTIN-4 RECEPTOR ACTIVITY IN CHANNEL CATFISH (Ictalurus punctatus) USING CRISPR/Cas9 SYSTEM

Melanocortin receptors (MCRs) are membrane proteins and classified as G protein-coupled receptors. One of these five MCRs is melanocortin-4 receptor (MC4R) which has a critical role in the regulation of body weight. As a consequence of its importance in feeding regulation, MC4R has been identified in many species of mammals, birds and teleost fish including zebrafish, goldfish, see bass and cyprinid carps. Polymorphisms in MC4R gene have been observed and linked to growth, carcass composition and meat quality traits, thus knocking out the melanocortin-4 receptor (MC4R) gene could increase size and growth rate in the livestock, including fish.

CRISPR was used to edit the MC4R gene of the channel catfish (Ictalurus punctatus) to determine the effects of knockout.

Female catfish were artificially spawned, eggs collected and fertilized with electroporated sperm followed by post fertilization electroporation in the buffer solution containing a mix of Cas9 and sgRNA, which was designed to target the MC4R gene. Embryos were reared in the modified Holtfreter`s solution till hatching.  DNA was obtained from barbel tissue of fingerlings, and PCR was performed using Expand High Fidelity^PLUS PCR System (Roche). PCR product length (932 bp) was verified on 1% agarose gel. Surveyor® mutation detection kit (Transgenomic) was used to detect mutations according to manufacturer instructions. Digestion products were separated in 2% agarose gel and compared to a non-edited channel catfish (Figure 1). To confirm and identify the mutations, PCR products will be cloned and sequenced.