Functional knockout of FUT8 in Chinese hamster ovary cells using CRISPR/Cas9 to produce a defucosylated antibody
Functional knockout of FUT8 in Chinese hamster ovary cells using CRISPR/Cas9 to produce a defucosylated antibody
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DOI：
10.1002/elsc.201400218
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发表时间：
2015-09-01
影响因子：
2.7
通讯作者：
Zhu, Jianwei
中科院分区：
文献类型：
--
作者：
Sun, Tao;Li, Chaodong;Zhu, Jianwei
我们报道了新的CRISPR/Cas9（聚集规律间隔短回文重复/CRISPR相关蛋白9）系统的适应性，以破坏编码聚焦转移酶8 （FUT8）的基因，FUT8是一种1,6-聚焦转移酶，指导聚焦添加到衍生抗体Fc区天冬酰胺297，在中国仓鼠卵巢（CHO）细胞中。与先前报道的同源重组或锌指核酸酶（ZFNs）在CHO细胞中的应用相比，CRISPR/Cas9表现出更高的靶向效率和更容易定制。在3周内获得FUT8分裂克隆（FUT8(-/-)）， indel频率为9 ~ 25%，通过选择Lens culinaris凝集素（LCA）可将其提高到52%。基于凝集素印迹法，衍生的FUT8（-/-）克隆能够产生去聚焦的治疗性单抗，对细胞生长、活力或产品质量没有不利影响。该克隆具有用于治疗性抗体制造的工业应用潜力。我们已经从功能上证明了使用CRISPR/Cas9技术可以突变与产物合成相关的基因，从而改变表达的单抗的聚糖谱。我们相信，凭借其稳健性和有效性，CRISPR/Cas9可以广泛应用于细胞系发育，从而提高单克隆抗体和其他生物治疗药物的生产力和质量。
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 1,6-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.