Nonviral gene editing via CRISPR/Cas9 delivery by membrane-disruptive and endosomolytic helical polypeptide.
Nonviral gene editing via CRISPR/Cas9 delivery by membrane-disruptive and endosomolytic helical polypeptide.
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通过膜破坏性和内体溶解螺旋多肽通过 CRISPR/Cas9 传递进行非病毒基因编辑
DOI：
10.1073/pnas.1712963115
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发表时间：
2018-05-08
影响因子：
11.1
通讯作者：
Leong KW
中科院分区：
文献类型：
--
作者：
Wang HX;Song Z;Lao YH;Xu X;Gong J;Cheng D;Chakraborty S;Park JS;Li M;Huang D;Yin L;Cheng J;Leong KW
对于CRISPR/Cas9的稳健实施，递送仍然是一个重大挑战。我们报道了一种高效的CRISPR/Cas9递送系统，该系统包含基于α-螺旋多肽PPABLG的聚乙二醇化纳米颗粒。在多肽高膜穿透能力的帮助下，P-HNPs实现了高效的细胞内化和内体逃逸。CRISPR/Cas9传递系统在细胞内的基因编辑率可达47.3%，在体内的基因缺失率可达35%，HeLa肿瘤生长抑制率可达71%，与现有的常规多阳离子转染试剂相比具有优势。报道的CRISPR/Cas9传递系统在敲入和基因激活方面也很有效，有助于推进体外和体内的基因编辑。有效和安全地递送CRISPR/Cas9基因编辑元件仍然是一个挑战。在这里，我们报道了基于阳离子α-螺旋多肽聚（γ-4-（（2-（胡椒苷-1-酰基）乙基）氨基甲基）苄基-谷氨酸）的聚乙二醇化纳米粒子（命名为P-HNPs）的开发，用于将Cas9表达质粒和sgRNA递送到各种细胞类型和基因编辑场景。穿透细胞的α-螺旋多肽增强细胞摄取，促进pCas9和/或sgRNA从核内体逃逸并转运到细胞核。胶体稳定的P-HNPs实现了高达60%的Cas9转染效率和67.4%的sgRNA摄取效率，比现有的基于多阳离子的基因传递系统有了改进。在体外以47.3%的效率进行单次或多重基因编辑后，我们发现，携带靶向Plk1基因的Cas9质粒/sgRNA的P-HNPs在HeLa肿瘤组织中实现了35%的基因缺失，使Plk1蛋白水平降低66.7%，从而在60天内抑制肿瘤生长，使动物存活率提高到60%。P-HNP系统能够将Cas9质粒传递到各种细胞类型，实现体外和体内多重基因敲除、敲入和基因激活，为生物学研究和治疗应用提供了一个多功能的基因编辑平台。
Delivery remains a significant challenge for robust implementation of CRISPR/Cas9. We report an efficient CRISPR/Cas9 delivery system comprising PEGylated nanoparticles based on the α-helical polypeptide PPABLG. Assisted by the high membrane-penetrating ability of the polypeptide, P-HNPs achieved efficient cellular internalization and endosomal escape. The CRISPR/Cas9 delivery system could reach 47.3% gene editing in cells, 35% gene deletion in vivo, and HeLa tumor growth suppression >71%, demonstrating an advantage over the existing conventional polycationic transfection reagents. Efficient also in knock-in and gene activation, the reported CRISPR/Cas9 delivery system serves to advance gene editing in vitro and in vivo. Effective and safe delivery of the CRISPR/Cas9 gene-editing elements remains a challenge. Here we report the development of PEGylated nanoparticles (named P-HNPs) based on the cationic α-helical polypeptide poly(γ-4-((2-(piperidin-1-yl)ethyl)aminomethyl)benzyl-l-glutamate) for the delivery of Cas9 expression plasmid and sgRNA to various cell types and gene-editing scenarios. The cell-penetrating α-helical polypeptide enhanced cellular uptake and promoted escape of pCas9 and/or sgRNA from the endosome and transport into the nucleus. The colloidally stable P-HNPs achieved a Cas9 transfection efficiency up to 60% and sgRNA uptake efficiency of 67.4%, representing an improvement over existing polycation-based gene delivery systems. After performing single or multiplex gene editing with an efficiency up to 47.3% in vitro, we demonstrated that P-HNPs delivering Cas9 plasmid/sgRNA targeting the polo-like kinase 1 (Plk1) gene achieved 35% gene deletion in HeLa tumor tissue to reduce the Plk1 protein level by 66.7%, thereby suppressing the tumor growth by >71% and prolonging the animal survival rate to 60% within 60 days. Capable of delivering Cas9 plasmids to various cell types to achieve multiplex gene knock-out, gene knock-in, and gene activation in vitro and in vivo, the P-HNP system offers a versatile gene-editing platform for biological research and therapeutic applications.
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用于癌症治疗的基因组编辑：通过金纳米簇/脂质核壳纳米载体递送 Cas9 蛋白/sgRNA 质粒
影响因子：
15.1
作者：
Wang, Peng;Zhang, Lingmin;Jiang, Xingyu
通讯作者：
Jiang, Xingyu
影响因子：
64.5
作者：
Hsu PD;Lander ES;Zhang F
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影响因子：
16.6
作者：
Gabrielson, Nathan P.;Lu, Hua;Yin, Lichen;Li, Dong;Wang, Fei;Cheng, Jianjun
通讯作者：
Cheng, Jianjun
影响因子：
12.4
作者：
Wu, Zhijian;Yang, Hongyan;Colosi, Peter
通讯作者：
Colosi, Peter
影响因子：
17.1
作者：
Li, Ling;Song, Linjiang;Wei, Yuquan
通讯作者：
Wei, Yuquan