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