Editing of CSN2 (beta-casein) and LGB (beta-lactoglobulin) genes by CRISPR/Cas9 in Bos taurus cells as a model for recombinant protein production

Abstract

The production and commercialization of recombinant proteins is an ever-expanding multi-billion dollar market. Due to the limitations of production models in bacteria and yeast, alternative methods, such as those based on mammalian cells, have emerged. One of the most promising approaches in this sense is to produce recombinant proteins in the mammary glands of bovines taking advantage of all the molecular machinery of protein expression in milk of these animals and replacing the two main proteins present in it, beta-casein (CSN2) and beta-lactoglobulin (LGB) by other proteins of commercial interest. In this context, the present proposal has as the main objective the knockout of the CSN2 and LGB genes in a bovine model by CRISPR/Cas9 technology. With the new lineage established, it is intended to introduce a short non-coding sequence through knock-in in order to validate the technique and as a proof of concept. Initially, standardization of all steps of gene editing strategies (design of the guide RNAs, construction of the donor DNA, and gene cloning) will be performed. Subsequently, the cells will be transfected using the AMAXA NucleofectorTM 2B equipment. After two days in culture, the cells will be analyzed by flow cytometry to assess the transfection rate. Next, sorting of the cells will be performed to generate clones in culture. These clones will be cultured, selected, and evaluated by Sanger sequencing for subsequent indels frequency analysis by Synthego ICE program as knockout and knock-in validation. This proposal can potentially generate a new model of recombinant protein production by allowing the introduction of transgenes of commercial and therapeutic interest in bovine mammary gland.