Journal Article

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

Gong, Jing, Song, Ziyuan, Xu, Xin, Chakraborty, Syandan, Cheng, Jianjun, Wang, Hong-Xia, Cheng, Du, Li, Mingqiang, Yin, Lichen, Lao, Yeh-Hsing, Park, Ji Sun, Huang, Dantong, Leong, Kam W.

2018

Overview

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.

• Publisher: National Academy of Sciences
• Subject: Animals / Applied Biological Sciences / 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 / Survival / Therapeutic applications / Transfection / Tumors