Format

Send to

Choose Destination
Proc Natl Acad Sci U S A. 2018 May 8;115(19):4903-4908. doi: 10.1073/pnas.1712963115. Epub 2018 Apr 23.

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

Author information

1
Department of Biomedical Engineering, Columbia University, New York, NY 10027.
2
Department of Materials Science and Engineering, University of Illinois at Urbana-Champaign, Champaign, IL 61801.
3
Institute of Functional Nano & Soft Materials (FUNSOM), Soochow University, Suzhou 215123, China.
4
Jiangsu Key Laboratory for Carbon-Based Functional Materials & Devices, Soochow University, Suzhou 215123, China.
5
Collaborative Innovation Center of Suzhou Nano Science and Technology, Soochow University, Suzhou 215123, China.
6
Joint International Research Laboratory of Carbon-Based Functional Materials and Devices, Soochow University, Suzhou 215123, China.
7
Key Laboratory for Polymeric Composite and Functional Materials of Ministry of Education, School of Materials Science and Engineering, Sun Yat-Sen University, Guangzhou 510275, China.
8
Institute of Functional Nano & Soft Materials (FUNSOM), Soochow University, Suzhou 215123, China; lcyin@suda.edu.cn jianjunc@illinois.edu kam.leong@columbia.edu.
9
Department of Materials Science and Engineering, University of Illinois at Urbana-Champaign, Champaign, IL 61801; lcyin@suda.edu.cn jianjunc@illinois.edu kam.leong@columbia.edu.
10
Department of Biomedical Engineering, Columbia University, New York, NY 10027; lcyin@suda.edu.cn jianjunc@illinois.edu kam.leong@columbia.edu.

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-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.

KEYWORDS:

CRISPR/Cas9; cell-penetrating peptide; genome editing; helical polypeptide; nanomedicine

PMID:
29686087
PMCID:
PMC5948953
DOI:
10.1073/pnas.1712963115
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for HighWire Icon for PubMed Central
Loading ...
Support Center