Abstract
Clustered regularly interspaced short palindromic repeats-associated protein (CRISPR/Cas9) system has become a revolutionary tool for gene editing. Since viral delivery systems have significant side effects, and naked DNA delivery is not an option, the nontoxic, non-viral delivery of CRISPR/Cas9 components would significantly improve future therapeutic delivery. In this study, we aim at characterizing nanoparticles to deliver plasmid DNA encoding for the CRISPR-Cas system in eukaryotic cells in vitro. CRISPR/Cas9 complexed polyethylenimine (PEI) magnetic nanoparticles (MNPs) were generated. We used a stable HEK293 cell line expressing the traffic light reporter (TLR-3) system to evaluate efficient homology- directed repair (HDR) and non-homologous end joining (NHEJ) events following transfection with NPs. MNPs have been synthesized by co-precipitation with the average particle size around 20 nm in diameter. The dynamic light scattering and zeta potential measurements showed that NPs exhibited narrow size distribution and sufficient colloidal stability. Genome editing events were as efficient as compared to standard lipofectamine transfection. Our approach tested non-viral delivery of CRISPR/Cas9 and DNA template to perform HDR and NHEJ in the same assay. We demonstrated that PEI-MNPs is a promising delivery system for plasmids encoding CRISPR/Cas9 and template DNA and thus can improve safety and utility of gene editing.
Highlights
There is a new trend in the development of gene therapies for monogenic diseases investigating the application of the CRISPR/Cas[9] system for editing the genome in a precise, sequence-dependent manner, resulting in a permanent change of the genomic information at the target site
Plasmid DNA ensures a temporary burst of transgene expression, limiting the activity of the CRISPR/Cas[9] system and reducing off target toxicity when comparing it to long-term viral vector mediated transgene expression[9]
This CRISPR/Cas9-PEI-magnetic nanoparticles (MNPs) complex was transfected into a stable TLR-HEK293 cell line previously generated by us to test the efficacy of genome editing[4]
Summary
There is a new trend in the development of gene therapies for monogenic diseases investigating the application of the CRISPR/Cas[9] system for editing the genome in a precise, sequence-dependent manner, resulting in a permanent change of the genomic information at the target site. Even PEI–MNPs were shown to improve efficiency of commonly used transfection reagent (DreamFect Gold) to deliver CRISPR/Cas[9] for gene editing in porcine fibroblast for generating genetically modified pig models[24]. This transfection combination does not have the potential to be applicable in therapeutic in vivo genome editing. The long term goal of our study is to develop MNPs that allow efficient therapeutic genome editing applications This requires a robust chemical characterization, prove of stability and colloidal dispersion, as well as efficient internalization into human cells, first in in vitro study, followed by in vivo studies. We generated and characterized MNPs with regard to their physical and chemical properties and their potential to deliver CRISPR/ Cas[9] plasmids into cells without the help of Lipofectamine like transfection reagents
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