Delivery of dCas9 Activator System Using Magnetic NanoparticlesTechnology as a Vector Delivery Method for Human Skin Fibroblast
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.
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 super-
paramagnetic 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.
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 super-
paramagnetic 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.