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

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

Research output: Contribution to journalArticle

Abstract

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-glu-tamate) 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.

Original languageEnglish (US)
Pages (from-to)4903-4908
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume115
Issue number19
DOIs
StatePublished - May 8 2018

Keywords

  • CRISPR/Cas9
  • Cell-penetrating peptide
  • Genome editing
  • Helical polypeptide
  • Nanomedicine

ASJC Scopus subject areas

  • General

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