Polyethylenimine based magnetic nanoparticles mediated non-viral CRISPR/Cas9 system for genome editing

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.

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“…CRISPR is a gene editing technique with high precision and potential to be used in gene therapy. Mainly, CRISPR/Cas9 are inserted into the cells in the form of plasmids, mRNA or ribonucleotide proteins, which can be used by nanocarrier systems to increase the transfection of cells [164,165].…”

Tailoring Iron Oxide Nanoparticles for Efficient Cellular Internalization and Endosomal Escape

“…In consequence, PEI has been extensively used to modify the surface of IONs, primarily for gene editing applications where endosomal escape is a fundamental step for an effective transfection and electrostatic interactions can be exploited to form complexes with nucleic acids [ 98 , 249 , 250 ]. In this regard, Rohiwal and colleagues developed CRISPR/Cas9-PEI-IONs capable of disrupting endosomes and effectively inserting the genes of both the blue fluorescent protein (BFP) and the green fluorescent protein (GFP) in vitro [ 251 ]. Similarly, pluronic/PEI shell crosslinked nanocapsules with embedded iron oxide nanocrystals (PPMCs) were developed by Lee and colleagues for the delivery of siRNA to cancerous cells.…”

“…Hybrid ION/UCNP systems have been widely applied in cancer therapy, MRI and diagnosis, gene therapy, and drug delivery [ 308 ]. In general, the most prevalent host matrices for iron oxide core-shell nanoparticles include Y 2 O 3 , Y 2 O 2 S, LaF 3 , BaYF 5 , NaYF 4 , and NaGdF 4 , which have been doped with Yb 3+ /Tm 3+ and Yb 3+ /Er 3+ ions [ 230 , 231 , 232 , 233 , 234 , 235 , 236 , 237 , 238 , 239 , 240 , 241 , 242 , 243 , 244 , 245 , 246 , 247 , 248 , 249 , 250 , 251 , 252 , 253 , 254 , 255 , 256 , 257 , 258 , 259 , 260 , 261 , 262 , 263 , 264 , 265 , 266 , 267 , 268 , 269 , …”

“…Among cationic polymers, perhaps PEI shows the highest protective effects over plasmid DNA (pDNA). In this regard, Rohiwal and colleagues demonstrated that after exposure to 10% (v/v) fetal bovine serum, naked pDNA degraded within 24 h while the polyplex PEI-pDNA remained stable beyond the same timespan [ 251 ]. For this reason, IONs have been modified with PEI to improve transfection efficiency, as demonstrated by Kami and colleagues, who achieved an 8-fold increase for episomal vectors after exposure to a magnetic field [ 351 ].…”

One-step synthesis of polyethyleneimine-coated magnetite nanoparticles and their structural, magnetic and power absorption study

“…They have been applied for the synthesis at high temperatures, 20 during long periods of synthesis 6 using several reaction steps, 21,22 such as rst core synthesis, and aer coated with PEI. [23][24][25][26][27] PEI molecule is considered a good candidate for the functionalization of other molecules, the biocompatibility and stability of MNPs, that are important in the elds of biomedicine. 28 In order to improve and simplify the synthesis for the formation of PEI-coated MNPs, we propose an effective procedure that combines co-precipitation steps in a one-step procedure in an aqueous medium.…”