文献类型：期刊文献

英文题名：A pre-integrated dCas9-VPR CRISPRa platform for high-level recombinant protein production in CHO cells

作者：Tian, Yuan[1,2];Zhou, Ya-Nan[1,2];Hu, Wan-Shun[1,2];Xu, Jiao[1,2];Ye, Qian[1,2];Tan, Wen-Song[1,2,3]

机构：[1]East China Univ Sci & Technol, State Key Lab Bioreactor Engn, 130 Meilong Rd,POB 309, Shanghai 200237, Peoples R China;[2]Shanghai Collaborat Innovat Ctr Biomfg Technol SCI, Shanghai 200237, Peoples R China;[3]Shanghai BioEngine Sci Tech Co LTD, Shanghai 201203, Peoples R China

年份：2026

卷号：232

外文期刊名：BIOCHEMICAL ENGINEERING JOURNAL

收录：;EI(收录号：20261720583074);WOS:【SCI-EXPANDED(收录号:WOS:001758593500001)】；

语种：英文

外文关键词：Chinese hamster ovary cells; CRISPR activation; Synthetic transcription platform; Engineered chassis cell line; Recombinant protein production

摘要：The use of strong promoters is essential for enhancing recombinant therapeutic protein (RTP) production, yet conventional engineering strategies struggle to exceed the strength of the human cytomegalovirus (hCMV) promoter. While CRISPR activation (CRISPRa) systems provide precise, tunable regulation and can be designed to surpass hCMV. Thus, we constructed a dCas9-VPR-based synthetic transcriptional activation platform, which demonstrated a 1.7-fold higher activation level than the hCMV promoter upon transient transfection. Nevertheless, the multicomponent nature of CRISPRa systems complicates the generation of stable producer cell lines. Therefore, we developed an innovative chassis cell line with pre-integrated dCas9-VPR. This design streamlines the workflow by reducing subsequent activation to transfection with a single gene-of-interest plasmid. The chassis cells exhibited robust performance, delivering up to 1.5-fold higher transient activation than the hCMV promoter while maintaining stability over 60 days. Compared to the hCMV promoter, this platform significantly enhanced the expression levels of multiple recombinant proteins. In fed-batch culture, the system achieved a maximum specific productivity (qP) of 44.38 pcd and a peak antibody titer of 5.92 g/L. During perfusion culture, the cumulative titer reached 17.15 g/L, with average and peak qp values of 62.28 pcd and 96.17 pcd, respectively, approaching the reported upper limit for CHO cells. This study provides a promising strategy for highyield recombinant therapeutic protein production.