CRISPR/Cas9 knock-in of GST-tagged human Noggin in the β-casein gene locus of bovine ear fibroblast cells

Abstract

We developed knock-in vector system of human Noggin mature sequence with glutathione S-transferase (GST) containing factor Xa protease linker to facilitate the subsequent purification of recombinant protein. To achieve this, bovine ear fibroblast cells were isolated and transfection conditions were optimized by electroporation. To generate knock-in vector, human Noggin lacking its native signal peptide is fused to GST and foot and month disease virus 2A (F2A), and then inserted into bovine β-casein gene exon 3. We also generated enhanced green fluorescent protein (EGFP) expression vector of GST-human Noggin mature fused to β-casein signal peptide and F2A, and successfully detected recombinant human Noggin protein secreted into culture media, followed by factor Xa cleavage. Then, we co-transfected human Noggin knock-in vector with single-guided RNA and Cas9 expression vectors into bovine ear fibroblasts and obtained the stably-integrated colonies by antibiotic selection. PCR screening analysis revealed that 26 out of 35 colonies positively integrated human Noggin knock-in vector into bovine β-casein locus. One of positive clones was subjected to chromosome analysis, presenting normal karyotypes. Our data may provide the additional purification guideline of recombinant proteins by tagging GST with a protease linker sequence in the upstream of target genes and a high efficiency of integration ratio into bovine β-casein locus.