Delivery of dCas9 Activator System Using Magnetic Nanoparticles Technology as a Vector Delivery Method for Human Skin Fibroblast

Author:

Mohammadi Ghanbarlou Mahdi12,Abdoli Shahriyar13,Omid Hamed1ORCID,Qazizadeh Leila1,Bamehr Hadi1,Raigani Mozhgan4,Shahsavarani Hosein52,Karimipour Morteza4,Shokrgozar Mohammad Ali12

Affiliation:

1. National Cell Bank of Iran, Pasteur Institute of Iran, Tehran 1316943551, Iran

2. Laboratory of Regenerative Medicine and Biomedical Innovations, Pasteur Institute of Iran, Tehran 1316943551, Iran

3. Biotechnology Research Center, Golestan University of Medical Sciences, Golestan 1575949138, Iran

4. Biotechnology Research Center, Pasteur Institute of Iran, Tehran 1316943551, Iran

5. Department of Cell and Mol Biology, Shahid Beheshti University, Tehran 1983969411, Iran

Abstract

The overexpression of stem cell-related genes such as octamer-binding transcription factor 4 (OCT4) and (sex determining region Y)-box 2 (SOX2) has been indicated to play several critical roles in stem cell self-renewal; moreover, the elevation of the self-renewal of cancer cells with stem cell-like properties has been suggested. The clustered and regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) protein fused to transactivation domains can be used to activate gene expression in human cells. CRISPR-mediated activation (CRISPRa) systems represent an effective genome editing tool for highly specific gene activation in which a nuclease-deficient Cas9 (dCas9) is utilized to target a transcriptional activator to the gene’s regulatory element, such as a promoter and enhancer. The main drawback of typical delivery methods for CRISPR/Cas9 components is their low transfection efficiency or toxic effects on cells; thus, we generated superparamagnetic iron oxide nanoparticles (SPIONs) coated with polyethylenimine (PEI) to improve the delivery of CRISPR/Cas9 constructs into human foreskin fibroblast cells. The delivery system with magnetic PEI-coated nanoparticles complex was applied to constitute plasmid DNA lipoplexes. CRISPRa systems were used to overexpress the endogenous OCT4 and SOX2 in fibroblast cells. The quantitative polymerase chain reaction (QPCR) assessment exhibited a three-times higher expression of OCT4 and SOX2 transfected by CRISPRa using MNPs. Moreover, no additional cytotoxicity was observed with the application of magnetic nanoparticles (MNPs) compared to lipofectamine. Our results demonstrate that MNPs enable the effective delivery of the CRISPR/Cas9 construct into human foreskin fibroblasts with low cell toxicity and a consequential overexpression of endogenous OCT4 and SOX2.

Funder

Pasteur Institute of Iran

National Institute for Medical Research Development

Iran Biotechnology Development Council

Publisher

MDPI AG

Subject

Materials Chemistry,Chemistry (miscellaneous),Electronic, Optical and Magnetic Materials

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