文献类型：期刊文献

英文题名：Enhanced Biosynthesis Performance of Heterologous Proteins in CHO-K1 Cells Using CRISPR-Cas9

作者：Wang, Wenpeng[1];Zheng, Wenyun[2];Hu, Fengzhi[2];He, Xiujuan[2];Wu, Dong[1];Zhang, Wenliang[3];Liu, Haipeng[4];Ma, Xingyuan[1]

机构：[1]East China Univ Sci & Technol, Sch Biotechnol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China;[2]East China Univ Sci & Technol, Sch Pharm, Shanghai Key Lab New Drug Design, Shanghai 200237, Peoples R China;[3]Univ North Carolina Greensboro, Ctr Translat Biomed Res, Greensboro, NC 27310 USA;[4]Xiamen Univ, State Key Lab Marine Environm Sci, Xiamen 361102, Fujian, Peoples R China

年份：2018

卷号：7

期号：5

起止页码：1259

外文期刊名：ACS SYNTHETIC BIOLOGY

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000432901300011)】；

基金：This study was supported by the National Natural Science Foundation (31670944, 81673345), Science and Technology Innovation Action Plan of Shanghai (14431904300, 17431904600), Shanghai Pujiang Program (13PJD012), and supported by the National Science Research Project "Significant New Drugs Created" of Eleventh Five-Year Plan (2009ZX09103-693).

语种：英文

外文关键词：CHO-K1 mammalian cell; ER microenvironment; antiapoptotic ability; genome editing by CRISPR-Cas9; high quality protein production

摘要：Chinese hamster ovary (CHO) cells are the famous expression system for industrial production of recombinant proteins, such as therapeutic antibodies. However, there still remain bottlenecks in protein quality and weakness in expression efficiency because of the intrinsic genetic properties of the cell. Here we have enhanced biosynthesis performance of heterologous proteins in CHO-K1 cells using CRISPR-Cas9 by editing the genome precisely with two genes for improving ER microenvironment and reinforcing antiapoptotic ability. A linear donor plasmid harboring eGFP-HsQSOX1b and Survivin genes was knocked in specific locus in CHO-K1 genome by the CRISPR-Cas9 RNA guided nucleases via NHEJ with efficiencies of up to 3.85% in the CHO-K1 cell pools following FACS, and the hQSOX1 and hSurvivin genes were integrated into expected genome locus successfully. Compared with control, the antiapoptotic viability of edited CHO-K1 cells was increased by 6.40 times, and the yield has been raised by 5.55 times with GLuc as model protein. The possible molecular mechanisms and pathways of remarkable antiapoptotic ability and protein biosynthesis in modified CHO-K1 cells have been elucidated reasonably. In conclusion, the novel ideas and reliable techniques for obtaining foreign proteins more efficiently in engineered animal cells were very valuable to meet large clinical needs.