Abstract：
Manipulation of the lactate metabolism is an efficient way for cancer treatment given its involvement in cancer development, metastasis, and immune escape. However, most of the inhibitors of lactate transport carriers suffer from poor specificity. Herein, we use the CRISPR/Cas9 system to precisely downregulate the monocarboxylate carrier 1 (MCT1) expression. To avoid the self-repairing during the gene editing process, a dual-Cas9 ribonucleoproteins (duRNPs) system is generated using the biological fermentation method and delivered into cells by the zeolitic imidazolate framework-8 (ZIF-8) nanoparticles, enabling precise removal of a specific DNA fragment from the genome. For efficient cancer therapy, a specific glucose transporter 1 inhibitor (BAY-876) is co-delivered with the duRNPs, forming BAY/duRNPs@ZIF-8 nanoparticle. ZIF-8 nanoparticles can deliver the duRNPs into cells within 1 h, which efficiently downregulates the MCT1 expression, and prohibits lactate influx. Through simultaneous inhibition of the lactate and glucose influx, BAY/duRNPs@ZIF-8 prohibits ATP generation, arrests cell cycle, inhibits cell proliferation, and finally induces cellular apoptosis both in vitro and in vivo. Consequently, we demonstrate that the biologically produced duRNPs delivered into cells by the nonviral ZIF-8 carrier have expanded the CRISPR/Cas gene editing toolbox and elevated the gene editing efficiency, which will promote biological studies and clinical applications. STATEMENT OF SIGNIFICANCE: The CRISPR/Cas9 system, widely used as an efficient gene editing tool, faces a challenge due to cells\' ability to self-repair. To address this issue, a strategy involving dual-cutting of the genome DNA has been designed and implemented. This strategy utilizes biologically produced dual-ribonucleoproteins delivered by a metal-organic framework. The effectiveness of this dual-cut CRISPR-Cas9 system has been demonstrated through a therapeutic approach targeting the simultaneous inhibition of lactate and glucose influx in cancer cells. The utilization of the dual-cut gene editing strategy has provided valuable insights into gene editing and expanded the toolbox of the CRISPR/Cas-based gene editing system. It has the potential to enable more efficient and precise manipulation of specific protein expression in the future.
摘要：
由于乳酸代谢参与癌症发展，因此操纵乳酸代谢是癌症治疗的有效方法，转移,免疫逃逸。然而,大多数乳酸转运载体的抑制剂特异性差。在这里，我们使用CRISPR/Cas9系统精确下调单羧酸载体1（MCT1）的表达。为了避免基因编辑过程中的自我修复,使用生物发酵方法产生双Cas9核糖核蛋白（duRNPs）系统，并通过沸石咪唑酯框架-8（ZIF-8）纳米颗粒递送到细胞中，能够从基因组中精确去除特定的DNA片段。为了有效的癌症治疗，特定的葡萄糖转运蛋白1抑制剂（BAY-876）与duRNP共同递送，形成BAY/duRNPs@ZIF-8纳米颗粒。ZIF-8纳米颗粒可以在1小时内将duRNPs递送到细胞中，有效下调MCT1表达，并禁止乳酸流入。通过同时抑制乳酸和葡萄糖流入，BAY/duRNP@ZIF-8禁止产生ATP，阻止细胞周期，抑制细胞增殖，并最终在体外和体内诱导细胞凋亡。因此,我们证明了由非病毒ZIF-8载体递送到细胞中的生物产生的duRNPs扩展了CRISPR/Cas基因编辑工具箱并提高了基因编辑效率，这将促进生物学研究和临床应用。重要声明：CRISPR/Cas9系统，作为一种高效的基因编辑工具，由于细胞的自我修复能力而面临挑战。为了解决这个问题，已经设计并实施了涉及基因组DNA双重切割的策略。该策略利用由金属-有机框架递送的生物产生的双核糖核蛋白。这种双切CRISPR-Cas9系统的有效性已通过靶向同时抑制癌细胞中乳酸和葡萄糖流入的治疗方法得到证明。双切基因编辑策略的利用为基因编辑提供了有价值的见解，并扩展了基于CRISPR/Cas的基因编辑系统的工具箱。它有可能在未来更有效和精确地操作特定的蛋白质表达。
