Zhang Lingmin, Yang Langyu, Huang Jionghua, Chen Sheng, Huang Chuangjia, Lin Yinshan, Shen Ao, Zheng ZhouYikang, Zheng Wenfu, Tang Shunqing
Key Laboratory of Molecular Target & Clinical Pharmacology and the State & NMPA Key Laboratory of Respiratory Disease, School of Pharmaceutical Sciences & The Third and Fifth Affiliated Hospital, Guangzhou Medical University, Guangzhou 511436, China.
Department of Biomedical Engineering, Jinan University, Guangzhou 510632, China.
Asian J Pharm Sci. 2022 Aug;17(5):666-678. doi: 10.1016/j.ajps.2022.08.001. Epub 2022 Sep 20.
The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR/Cas9) adaptive immune system is a cutting-edge genome-editing toolbox. However, its applications are still limited by its inefficient transduction. Herein, we present a novel gene vector, the zwitterionic polymer-inspired material with branched structure (ZEBRA) for efficient CRISPR/Cas9 delivery. Polo-like kinase 1 (PLK1) acts as a master regulator of mitosis and overexpresses in multiple tumor cells. The Cas9 and single guide sgRNA (sgRNA)-encoded plasmid was transduced to knockout , which was expected to inhibit the expression of PLK1. Our studies demonstrated that ZEBRA enabled to transduce the CRISPR/Cas9 system with large size into the cells efficiently. The transduction with ZEBRA was cell line dependent, which showed ∼10-fold higher in CD44-positive cancer cell lines compared with CD44-negative ones. Furthermore, ZEBRA induced high-level expression of Cas9 proteins by the delivery of CRISPR/Cas9 and efficient gene editing of gene, and inhibited the tumor cell growth significantly. This zwitterionic polymer-inspired material is an effective and targeted gene delivery vector and further studies are required to explore its potential in gene delivery applications.
II型原核CRISPR（成簇规律间隔短回文重复序列）/Cas9（CRISPR/Cas9）适应性免疫系统是一种前沿的基因组编辑工具箱。然而，其应用仍受限于转导效率低下。在此，我们提出了一种新型基因载体，即具有分支结构的两性离子聚合物启发材料（ZEBRA），用于高效递送CRISPR/Cas9。Polo样激酶1（PLK1）作为有丝分裂的主要调节因子，在多种肿瘤细胞中过表达。将编码Cas9和单向导sgRNA（sgRNA）的质粒转导以敲除，预期可抑制PLK1的表达。我们的研究表明，ZEBRA能够将大尺寸的CRISPR/Cas9系统高效转导入细胞。ZEBRA介导的转导具有细胞系依赖性，与CD44阴性癌细胞系相比，在CD44阳性癌细胞系中显示出约10倍的更高转导率。此外，ZEBRA通过递送CRISPR/Cas9诱导Cas9蛋白的高水平表达以及对基因的高效基因编辑，并显著抑制肿瘤细胞生长。这种两性离子聚合物启发材料是一种有效且靶向的基因递送载体，需要进一步研究以探索其在基因递送应用中的潜力。
