精确调控癌细胞中的关键蛋白，如抗凋亡蛋白，是治疗癌症和发现相关分子机制的关键策略之一。然而，在实际研究和实践中，这也是具有挑战性的。广泛使用的CRISPR/Cas9基因编辑技术和蛋白质降解靶向融合物（PROTACs）在细胞中调节基因表达和蛋白功能方面发挥了关键作用。然而，它们的靶向精确性和可控性仍然是必要的。使用睡莲系统构建UMUC-3-EGFP稳定转基因细胞系，通过流式细胞术、定量实时PCR、免疫印迹、荧光微板读取器和荧光倒置显微镜分析EGFP强度。通过使用Annexin V-FITC/PI凋亡、calcein/PI/DAPI细胞存活/细胞毒性试验、克隆形成试验和划痕试验对Survinin抑制进行表征。采用细胞源性异种移植模型（CDX）评估降低Survinin表达的体内效果。在此，我们建立了一个协同控制平台，以光激活的分裂Cas9靶向基因编辑和光诱导蛋白降解为基础，在该平台上，核内的Survivin基因可通过蓝光照射进行可控编辑（命名为paCas9-Survivin），同时，细胞质中的Survivin蛋白可通过纳米抗体介导的靶向精确降解（命名为paProtacL-Survivin）。同时，在体外实验中证明，降低Survivin表达能有效促进凋亡，减少膀胱癌细胞的增殖和迁移。此外，使用UMUC-3细胞株建立了CDX模型，动物研究结果表明，paCas9-Survivin系统和paProtacL-Survivin都显著抑制肿瘤生长，当两者联合使用时抑制率更高。简而言之，协同调控策略和可组合技术平台在可控性和靶向性方面具有明显优势，在通过多级调控关键细胞因子的命运控制癌细胞的过程中具有优良的参考价值和普适性。© 2023 The Authors. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.

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.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.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.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.© 2023 The Authors. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.