Department of Clinical Laboratory, Guangdong Provincial Key Laboratory of Major Obstetric Diseases; Guangdong Provincial Clinical Research Center for Obstetrics and Gynecology; The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, China.
Department of Urology, Carson International Cancer Centre, Shenzhen University General Hospital, Shenzhen, China.
Cell Mol Biol Lett. 2024 May 9;29(1):66. doi: 10.1186/s11658-024-00569-7.
The development of compact CRISPR systems has facilitated delivery but has concurrently reduced gene editing efficiency, thereby limiting the further utilization of CRISPR systems. Enhancing the efficiency of CRISPR systems poses a challenging task and holds significant implications for the advancement of biotechnology. In our work, we report a synthetic dual-antibody system that can stably exist in the intracellular environment, specifically inhibiting the functions of NF-κB and β-catenin. This not only elevates the transgenic expression of the CRISPR system by suppressing the innate immune response within cells to enhance the gene editing efficiency but also demonstrates a notable tumor inhibitory effect. Based on the specific output expression regulation of CRISPR-CasΦ, we constructed a CRISPR-based gene expression platform, which includes sensor modules for detecting intracellular β-catenin and NF-κB, as well as an SDA module to enhance overall efficiency. In vitro experiments revealed that the CRISPR-based gene expression platform exhibited superior CDK5 expression inhibition efficiency and specific cytotoxicity towards tumor cells. In vitro experiments, we found that CRISPR-based gene expression platforms can selectively kill bladder cancer cells through T cell-mediated cytotoxicity. Our design holds significant assistant potential of transgene therapy and may offer the capability to treat other diseases requiring transgene therapy.
紧凑型 CRISPR 系统的发展促进了递送，但同时降低了基因编辑效率，从而限制了 CRISPR 系统的进一步利用。提高 CRISPR 系统的效率是一项具有挑战性的任务，对生物技术的发展具有重要意义。在我们的工作中，我们报告了一种合成的双抗体系统，它可以在细胞内环境中稳定存在，专门抑制 NF-κB 和 β-catenin 的功能。这不仅通过抑制细胞内的固有免疫反应来提高 CRISPR 系统的转基因表达，从而提高基因编辑效率，而且还表现出显著的肿瘤抑制作用。基于 CRISPR-CasΦ 的特定输出表达调控，我们构建了一个基于 CRISPR 的基因表达平台，其中包括用于检测细胞内 β-catenin 和 NF-κB 的传感器模块，以及用于增强整体效率的 SDA 模块。体外实验表明，基于 CRISPR 的基因表达平台表现出优异的 CDK5 表达抑制效率和针对肿瘤细胞的特异性细胞毒性。在体外实验中，我们发现基于 CRISPR 的基因表达平台可以通过 T 细胞介导的细胞毒性选择性地杀死膀胱癌细胞。我们的设计具有转基因治疗的重要辅助潜力，并可能提供治疗其他需要转基因治疗的疾病的能力。
