Delivery of dCas9 CRISPR System Into the Hard Transfection Cells by Magnetofection Approach

: Background: The CRISPR-Cas9 system, a powerful tool, has revolutionized genome engineering in eukaryotic cells and living organisms. However, this approach poses unique concerns and limitations when used by conventional transfection methods, including limited packaging size and low delivery efficiency. Here, we aim at assessing the transfection efficiency of DNA encoding for the CRISPR-Cas9 system by PEI coated Magnetic NanoParticle (MNPs) to improve the delivery of CRISPR/Cas9 constructs into eukaryotic cells. Results: Superparamagnetic iron oxide nanoparticles (SPIONs) coated with polyethylenimine (PEI) and then complexed with pCXLE-dCas9VPH-T2A-GFP-shP53 plasmid DNA. We used HEK-293 (human embryonic kidney) and Human foreskin fibroblasts (HFF) cells to express GFP after transfection to evaluate delivery efficiency with MNPs and Lipofection methods. PEI-coated nanoparticles with magnetic iron oxide core were synthesized by co-precipitation technique resulting in an average size of ~ 20 nm in diameter. Characterization of Magnetic Nano Particle (MNPs) revealed that particles have narrow size distribution sufficient colloidal stability. The result showed that the magnetofection method with an efficiency around 85.7% for HEK-293 and 28.2% for HFF . Also, transfection efficiency by lipofection method was 83.2% and 7.89% for HEK-293 and HFF respectively. Conclusion: The magnetofection was revealed to be more efficient than classic Lipofectamine transfection as measured by GFP expression. We show that PEI-MNPs enable effective delivery and improved safety of plasmids encoding CRISPR/Cas9 into eukaryote cells.


Background
In biomedicine, from 2012 clustered regularly interspaced short palindromic repeats-associated proteins (CRISPR-Cas9) technology has emerged as a promising and highly efficient approach employed for gene editing in various living organisms and contexts. the CRISPR/Cas9 system allows insertions and deletions at a specific genomic locus through DNA double-strand breaks (DSBs), which are repaired using error-prone non-homologous end joining (NHEJ)-mediated mechanisms (1,2). Furthermore, this system represents the most promising strategy in biomedical applications such as diseases pathology, gene function, gene therapy, and generation of transgenic animal models (3,4).
To date, a wide variety of possible delivery methods have been developed for delivery of CRISPR/Cas9 machinery as form of a ribonucleotide protein (RNP) complex, DNA, viral vector, or mRNA which can be cross cellular barriers of target cells (5). CRISPR/Cas9 delivery systems can be classified into three general types including: 1-physical delivery such as microinjection and electroporation are useful for in vitro delivery (6). Microinjection has been defined as gold standard of CRISPR delivery system by Yang et al 2013 (7). Also, it's no limited to molecular weight of CRISPR/Cas9 component (6). This approach faces critical challenges for in vivo applications (8). 2viral vectors known as the most common delivery vectors and wide range of viral delivery systems are available such as retrovirus, adenovirus (types 2 and 5), adeno-associated virus(AAV), herpes virus, pox virus, human foamy virus (HFV), and lentivirus (9). viral vectors have established advantages such as high transduction efficiency and long term transgene expression ,but their application concern with safety issues and limited packing capacities (10) increased risk of unwanted immunogenicity, expensive large scale production ,and risk of integrating viral sequences into the host chromosome (11).3-Non-viral vector such as human serum albumin nanoparticles (NPs), lipid nanoparticles, cell-penetrating peptides, and gold nanoparticles. Represent some advantages over previous approaches include: safety, efficiency and customizability (12). NP based delivery systems can be engineered to target the interest cell or tissues. Also, this approach represents both higher packaging capacity for CRISPR/Cas9 components and protective effect for the loaded cargo against degradation until to reach the host cell. NPs generally are coast effective for scale-up production and very acceptable safety profiles ,improved colloidal stability , biocompatibility and low risk of mutagenicity in contrast to viral vectors (13). Despite its advantages, non-viral delivery systems application has been limited due to Low delivery efficiency therefor investigation for development of new polymer or material with optimal delivery efficiency are needed (5). Magnetic nanoparticles (MNPs) primarily has found as a bio-magnetic compasses in bacteria, insects, and larger animals (14).
Due to their unique properties such as high magnetization values and ability to cross cellular barriers, MNPs has received considerable critical attention and it has shed the light the road to develop of new generation of gene delivery tool (15).
Polyethylenimine (PEI) is a stable, easy to handle, cationic polymer Known as an effective transfection reagent (16). PEI acts by forming a positively charged complex with DNA which led to effective condensation of DNA into compact particles and subsequently binding to anionic residues on the target cell surface (17,18) and cross the cell membrane via endocytosis (19). To date, various surface modified nanoparticles such as graphene oxide-polyethylene glycol-PEI cationic arginine gold NPs, CRISPR-PAsp gold NPs ,have been previously assessed for delivering CRISPR/Cas9 components into different cell line (20). In this study, we aimed to compare the efficiency of the delivery of dCas9 CRISPR system into human fibroblast and HEK-293 cell line. Two different method were evaluated for each cell. Also, in each case, magnetofection method in presence of magnetic nanoparticles was used for the comparison with lipofection. So, two different methods and cells were tested to investigation of transfection efficiency. Our study provides a foundation for improvement of transfection method selection.

Fluorescent microscopy
Fluorescent microscopy images showed that HEK293 and HFF cells morphology 48h post-transfection has no significant differences between magnetofected and lipofected cells. A low number of floating dead cells were observed, as shown in Fig.5. approximately is 20 pg. Therefore; this concentration was used for further analyze.

Cell viability assay
The survival rate of the HEK-293cell line and HFF cell were measured by MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell toxicity assay. Mean value of viable cells was calculated by determining absorbance at 570/600 nm. Therefore, SPIONs are biocompatible toward HEK-293 and HFF cells. But viability for both transfection methods represent a dose-dependent manner and was reduced in magnetofection to 76%; and in lipofection to 82.0%. Also, DMSO was used as a positive control as shown in Figure 6. The result showed that transfection with CRISPR/Cas9-PEI-SPIONS has not significant cytotoxicity associated with the SPIONs compared with the lipofected cell (p-value = 0.12).

Effect of magnetic field on the magnetofection
The magnetic fields (SMFs) application to the accumulation of magneto complexes on the cell surface before magnetofection has been investigated and demonstrated to be efficient (8,21). We conducted a transfection experiment with and without a magnetic field to assess whether it can influence the biological pathway such as endocytosis and apoptosis. Also this transfection was done with Lipofectamine. The transfection efficiency was determined as EGF intensity to be around 28.2% for magnetofection by CRISPR/Cas9-PEI-SPIONs magneto-complexes and 7.89% for transfection with CRISPR/Cas9-PEI, as shown in Figure 7.

Cellular uptake of magneto complexes and GFP expression
To further establish the effect of magneto complexes on cellular uptake, the fluorescence intensity was  negatively charged heparin sulfate proteoglycans on cells' surfaces (22). Common entry pathway of cargo into a target cell is through clathrin-dependent endocytosis, a size-dependent path (23,24). One advantage of the DNA/PEI complexes is that they represent property such as escaping from endosomes and preventing fusion of endo-lysosome, which are associated with the proton sponge effect (25,26).
Therefore it prevents lysis (or burst) of the DNA by DNase or low pH by lysosome (26). MNPs via electrostatic adsorption combined with CRISPR/Cas9-PEI complexes and then transfected into HEK-293 and HFF cell line to improve the transfection efficiency. Also, transfection using lipofectamine reagent was conducted, and the result compares to magnetofection. This process is illustrated as Fig 9. Overall, this result proved that Magnetofection has successfully increased the penetration and subsequent dissociation of the CRISPR/Cas9-PEI-SPIONs magnetocomplexes into the nucleus.

Conclusion
The CRISPR/Cas9 system has emerged as a promising tool for genome editing of genetic disorders and infectious diseases. Its programmability and ease of use have gained attention, but the efficient delivery CRISPR/Cas9 system remains a significant challenge (35).

Synthesis of PEI coated MNPs and complexed with CRISPR-Cas9 plasmid
Preparation of magnetic complexes was done by conjugating the aqueous solution of PEI and DNA with a nitrogen in PEI-coated MNPs to phosphorus in DNA (N/P) conjugation ratio of 10/1 as reported by Zhang et al. 2014. Briefly, Magnetic complexes were prepared by mixing 50 μl of PEI (40 μg/mL) and 2 μg of plasmid DNA, and the mixture was incubated with MNPs for 20 min at room temperature (37,38). After that, the CRISPR/Cas9-PEI-MNPs complex, which has MNPs to CRISPR-Cas9 plasmid weight ratio of 10 used for further experiment.

Physicochemical characterization.
The size distribution and zeta potential of CRISPR/Cas9-PEI -SPION was determined using the DLS device (Sympatech, NANOPHOX Model, Germany). Samples sonicated for 1-2 minutes in injectable distilled water. He-Ne laser beam measurements were conducted by detecting at a scattering angle 90° at 633 nm at 25 °C. The zeta potential determine using a universal zeta dip cell. The morphology and the particle size of the MNPs were analyzed by FESEM microscopy. The magnetic properties of the synthesised Fe3O4 was evaluated by vibrating sampling magnetometer (VSM) at room temperature.

Magnetofection
One day before magnetofection/transfection, the cells were seeded at cell density (2× 10

Evaluation of transfection efficiency by fluorescent microscopy
Following transfection, plates were incubated for 24 h with these magnetic complexes. Cell growth and morphology were assessed for control, lipofection, and magnetofection groups using fluorescence microscopy.

Toxicity assay
The cytotoxicity of the magnetic complexes was evaluated using MTT cell toxicity assay. The cells were seeded at a density of 1× 10 3 cells/well on a 96-well plate (TPP, Sigma Aldrich) in 100 µL of supplemented DMEM and incubated for 24h until reached to confluency of 70%. The cells were transfected with 20 pg of CRISPR/Cas9-PEI-SPIONs using Mega Magnetic Plate. Also, other plates transfected with Lipofectamine and then incubated for 24h. Following that, the supernatant was replaced with fresh DMEM and then 25 μL of a 5.5 mg/mL MTT reagent was added to the cells. The plates were incubated for 3 h in the dark, and finally, the supernatant was removed and the remaining purple formazan was lysed with DMSO100 μl/l for 40 min. The assay was performed in triplicate.
Absorbance was measured using ELISA Reader (Bio Tek Instruments, Inc., VT, USA) at 570nm. The cell viability was calculated from the absorbance versus concentration curve.

Statistical analysis
The experiments were done in triplicate, and The Results are expressed as means ± standard deviation.
A paired t-test, one-way analysis of variance with Bonferroni's post hoc test, or two-way analysis of variance with post hoc test were used for statistical analysis. The P-values of <0.05 were considered significant. Statistical analysis was performed using commercially available software IBM ® SPSS ® Statistics software (IBM Corp., Armonk, NY, USA).

Ethics approval and consent to participate
All the participants were thoroughly informed about the study and procedures before signing consent forms. Participants were assured of anonymity and confidentiality. The Research Ethics Committee of the Pasteur Institute of Iran, Tehran, approved this study (IR.NIMAD.REC.1398.051).

Consent for publication
All authors have participated in conception and design, or analysis and interpretation of the data; drafting the article or revising it critically for important intellectual content; and approval of the final version.

Availability of data and materials
All data generated or analyzed in this study are included in the present article.

Funding
Data of this research was obtained from PhD dissertation of MMG and financially supported by Pasteur Institute of Iran (Thesis No. BD-9473).

Authors' contributions
MK and MS conceived and designed the study. MMG performed experiments, data analysis and writing initial draft of the manuscript. HI, HB, LG, HO, SA and HS checking data analysis.