文献类型：期刊文献

英文题名：Split-Cas9-based targeted gene editing and nanobody-mediated proteolysis-targeting chimeras optogenetically coordinated regulation of Survivin to control the fate of cancer cells

作者：Deng, Changping[1];Li, Shihui[1];Liu, Yuping[2];Bao, Wen[2];Xu, Chengnan[2];Zheng, Wenyun[2,6];Wang, Meiyan[3,4];Ma, Xingyuan[1,5]

机构：[1]East China Univ Sci & Technol, State Key Lab Bioreactor Engn, Shanghai, Peoples R China;[2]East China Univ Sci & Technol, Sch Pharm, Shanghai Key Lab New Drug Design, Shanghai, Peoples R China;[3]East China Normal Univ, Synthet Biol & Biomed Engn Lab, Biomed Synthet Biol Res Ctr, Shanghai Key Lab Regulatory Biol,Inst Biomed Sci, Shanghai, Peoples R China;[4]East China Normal Univ, Sch Life Sci, Shanghai, Peoples R China;[5]East China Univ Sci & Technol, State Key Lab Bioreactor Engn, 130 Meilong Rd, Shanghai 200237, Peoples R China;[6]East China Univ Sci & Technol, Shanghai Key Lab New Drug Design, 130 Meilong Rd, Shanghai 200237, Peoples R China

年份：2023

卷号：13

期号：8

外文期刊名：CLINICAL AND TRANSLATIONAL MEDICINE

收录：;WOS:【SCI-EXPANDED(收录号:WOS:001053983800001)】；

基金：We thank the Public Instrument Service Platform of State Key Laboratory of Bioreactor Engineering and the Research Centre of Analysis in this study.

语种：英文

外文关键词：Coordinated regulating to Survivin; Fate of cancer cells; Nanobody targeted degradation; Photoactivatable proteolysis and editing

摘要：Background Precise regulation of partial critical proteins in cancer cells, such as anti-apoptotic proteins, is one of the crucial strategies for treating cancer and discovering related molecular mechanisms. Still, it is also challenging in actual research and practice. The widely used CRISPR/Cas9-based gene editing technology and proteolysis-targeting chimeras (PROTACs) have played an essential role in regulating gene expression and protein function in cells. However, the accuracy and controllability of their targeting remain necessary.Methods Construction of UMUC-3-EGFP stable transgenic cell lines using the Sleeping Beauty system, Flow cytometry, quantitative real-time PCR, western blot, fluorescence microplate reader and fluorescence inverted microscope analysis of EGFP intensity. Characterization of Survivin inhibition was done by using Annexin V-FITC/PI apoptosis, calcein/PI/DAPI cell viability/cytotoxicity assay, cloning formation assay and scratch assay. The cell-derived xenograft (CDX) model was constructed to assess the in vivo effects of reducing Survivin expression.Results Herein, we established a synergistic control platform that coordinated photoactivatable split-Cas9 targeted gene editing and light-induced protein degradation, on which the Survivin gene in the nucleus was controllably edited by blue light irradiation (named paCas9-Survivin) and simultaneously the Survivin protein in the cytoplasm was degraded precisely by a nanobody-mediated target (named paProtacL-Survivin). Meanwhile, in vitro experiments demonstrated that reducing Survivin expression could effectively promote apoptosis and decrease the proliferation and migration of bladder cancerous cells. Furthermore, the CDX model was constructed using UMUC-3 cell lines, results from animal studies indicated that both the paCas9-Survivin system and paProtacL-Survivin significantly inhibited tumour growth, with higher inhibition rates when combined.Conclusions In short, the coordinated regulatory strategies and combinable technology platforms offer clear advantages in controllability and targeting, as well as an excellent reference value and universal applicability in controlling the fate of cancer cells through multi-level regulation of key intracellular factors.