Nonviral gene editing via CRISPR/Cas9 delivery by membrane-disruptive and endosomolytic helical polypeptide

• Published in: Proceedings of the National Academy of Sciences - PNAS 2018-05, Vol.115 (19), p.4903-4908
• Main Authors: Wang, Hong-Xia, Song, Ziyuan, Lao, Yeh-Hsing, Xu, Xin, Gong, Jing, Cheng, Du, Chakraborty, Syandan, Park, Ji Sun, Li, Mingqiang, Huang, Dantong, Yin, Lichen, Cheng, Jianjun, Leong, Kam W.
• Format: Article
• Language: English
• Subjects: Animals; Biological research; Biological Sciences; Cell-Penetrating Peptides - chemistry; Cell-Penetrating Peptides - pharmacology; Clonal deletion; CRISPR; CRISPR-Cas Systems; Efficiency; Gene deletion; Gene Editing - methods; Gene expression; Gene transfer; Gene Transfer Techniques; Genetic modification; Glutamic acid; HEK293 Cells; HeLa Cells; Humans; K562 Cells; Membranes; Mice; Multiplexing; Nanoparticles; Nanoparticles - chemistry; NIH 3T3 Cells; Plasmids; Plasmids - chemistry; Plasmids - genetics; Plasmids - pharmacology; Plk1 protein; Polo-like kinase; Polo-like kinase 1; Polyelectrolytes; Polypeptides; Proteins; Therapeutic applications; Transfection; Tumors
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1712963115

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.