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

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.

Fig. 1.

Schematic illustration showing the formation of P-HNPs and the intracellular activity of Cas9 expression plasmid/sgRNA in performing genome editing or gene activation.

Fig. 2.

Transfection of Cas9-GFP plasmid px458 in a variety of cells. The HNPs were formed by PPABLG/px458 at the wt/wt ratio of 30:1. The P-HNPs were formed by PPABLG/px458/PEG_2k-T₄₀ at the wt/wt/wt ratio of 30:1:1. Free, treatment with naked plasmids; UT, untreated group.

Fig. 3.

In vitro gene disruption. (A) EGFP disruption assay of Cas9 expression plasmid and sgRNA expression plasmid delivery by HNPs or P-HNPs in U2OS.EGFP reporter cells. The ratios in the figure are weight ratios for Cas9 expression plasmid:sgRNA expression plasmid. (B and C) EGFP disruption assay of Cas9 expression plasmid and in vitro-synthesized sgRNA delivery in U2OS.EGFP reporter cells. Short dash under the bars in B indicates the group treated by only Cas9 expression plasmids. (D–F) GCD assay of indels in the EGFP gene in U2OS.EGFP cells (D), the AAVS1 gene in HEK293T cells (E), and the HPRT1 gene in HEK293T cells (F). “Multi” is the group treated by Cas9 expression plasmid delivery and subsequent codelivery of AAVS1- and HPRT1-targeting sgRNAs. Arrows indicate the cutting products. Free, treatment with naked nucleic acids; UT, untreated group.

Fig. 4.

In vitro gene insertion and gene activation. (A) Donor architecture for HDR of the AAVS1 gene. The arrows show the locations of PCR primers for detecting successful HDR events. (B) PCR assay to detect HDR events 3 d after transfection. (C) Microscopic images of two GFP+ clones after treatment by P-HNPs loaded with Cas9 expression plasmids and the synthetic sgRNAs and HDR donor after selection with puromycin for 2 wk. (Scale bar, 100 μm.) (D) Sanger sequencing of the HDR events in the selected GFP+ clones in C. (E) The transfection efficiency of P-HNPs for pMax-^VP64dCas9-BFP^VP64 plasmid by detecting the BFP-positive cells. (F) The qRT-PCR evaluation of relative Myod1 mRNA level to detect the Myod1 activation. The results were shown as the ratio relative to the mRNA expression of the untreated cells. Free, treatment with naked nucleic acids; UT, untreated group.

Fig. 5.

In vivo gene disruption and tumor growth suppression. (A) Indels of the Plk1 gene in HeLa cells treated with P-HNPs containing px165 only (P-HNP_PCas9) or P-HNPs containing px165 and Plk1-targeting sgRNAs (P-HNP_PCas9+sgPlk1). (B) Viability of HeLa cells at 72 h after transfection with P-HNP_PCas9 or P-HNP_PCas9+sgPlk1. Free, cells treated with free px165 and free Plk1-targeting sgRNAs; UT, untreated cells. (C) The inhibition of tumor growth in HeLa xenograft tumor-bearing mice after intratumoral injection of P-HNP_PCas9 or P-HNP_PCas9+sgPlk1 (n = 7). Black and red arrows indicate the injection day of P-HNP_PCas9 or P-HNPs containing Plk1-targeting sgRNAs (P-HNP_sgPlk1), respectively. The injection dose was 20 μg for PCas9 or sgPlk1. (D) Survival of HeLa xenograft mice in C. Euthanasia was performed when tumor volumes reached 1,000 mm³. (E) Expression level of Plk1 protein in tumor tissues in C, analyzed at 48 h post last injection.