Sun T.,Shanghai JiaoTong University |
Li C.,Shanghai JiaoTong University |
Han L.,Shanghai JiaoTong University |
Jiang H.,Jecho Laboratories Inc. |
And 6 more authors.
Engineering in Life Sciences | Year: 2015
We report the adaptation of the new CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9) system to disrupt the gene encoding fucosyltransferase 8 (FUT8), an α1,6-fucosyltransferase that directs fucose addition to derived antibody Fc region asparagine 297, in Chinese hamster ovary (CHO) cells. Compared to previously reported homologous recombination or zinc-finger nucleases (ZFNs) applications in CHO cells, CRISPR/Cas9 demonstrated higher targeting efficiency and easier customization. FUT8 disruptive clones (FUT8-/-) were obtained within 3 weeks at indel frequencies ranging from 9 to 25%, which could be enhanced to 52% with Lens culinaris agglutinin (LCA) selection. Based on the lectin blot method, the derived FUT8-/- clone had the ability to produce defucosylated therapeutic mAb with no detrimental effects on cell growth, viability, or product quality. The clone had the potential of industrial application for therapeutic antibodies manufacturing. We have demonstrated functionally that a gene related to product synthesis could be mutated using CRISPR/Cas9 technology, and consequently the glycan profile of expressed mAb was alternated. We believe that with its robustness and effectiveness, CRISPR/Cas9 can be widely applicable in cell line development leading to higher productivity and better quality of mAbs and other biological therapeutics. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source