Non-viral delivery of CRISPR–Cas9 complexes for targeted gene editing via a polymer delivery system

Recent advances in molecular biology have led to the CRISPR revolution, but the lack of an efficient and safe delivery system into cells and tissues continues to hinder clinical translation of CRISPR approaches. Polymeric vectors offer an attractive alternative to viruses as delivery vectors due to their large packaging capacity and safety profile. In this paper, we have demonstrated the potential use of a highly branched poly(β-amino ester) polymer, HPAE-EB, to enable genomic editing via CRISPRCas9-targeted genomic excision of exon 80 in the COL7A1 gene, through a dual-guide RNA sequence system. The biophysical properties of HPAE-EB were screened in a human embryonic 293 cell line (HEK293), to elucidate optimal conditions for efficient and cytocompatible delivery of a DNA construct encoding Cas9 along with two RNA guides, obtaining 15–20% target genomic excision. When translated to human recessive dystrophic epidermolysis bullosa (RDEB) keratinocytes, transfection efficiency and targeted genomic excision dropped. However, upon delivery of CRISPR–Cas9 as a ribonucleoprotein complex, targeted genomic deletion of exon 80 was increased to over 40%. Our study provides renewed perspective for the further development of polymer delivery systems for application in the gene editing field in general, and specifically for the treatment of RDEB.


Zeta potential and Zetasizer Measurements
Poylplex size, polydispersity index (PDI) and surface charge, were evaluated using a Malvern Zetasizer Nano ZS (Malvern Instrument) equipped with a scattering angle of 173°. Zeta potential samples were measured using a Malvern folded capillary cell (DTS1070), while polyplex size measurements were performed in a clear plastic disposable cuvette. Polyplexes were prepared as described in Materials and Methods section. All samples were further diluted with 980 μl of molecular water for measurement in appropriate cuvette.

Cell Viability Calculations
Untreated cells were used to normalize fluorescence values and plotted as 100% viable. Wells containing alamarBlue™ reagent only were subtracted as background prior to obtaining the % of cell viability, calculated as follows: (Absorbancetreatment /Absorbanceuntreated) x100%.

Immunocytochemistry
Cells were seeded in 24-well plates containing 12 mm coverslips. These coverslips were washed three times in ice cold phosphate buffer saline (PBS, Thermo Fisher, St. Louis, MO, USA) and then fixed with ice cold acetone:methanol (1:1, Sigma Aldrich) for 20 min at -20 °C.
Samples underwent three washes with PBS, then blocking in 3% bovine serum albumin (BSA, Sigma Aldrich) for 20 min at RT and incubated overnight at 4 °C with primary antibody. For probing Cas9, the rabbit monoclonal (ab203933) (Abcam, Cambridge, UK) antibody was used at 1:500 dilution in 0.1% BSA in PBS solution. For collagen VII, a monospecific polyclonal anti-C7 antibody (a generous gift from Dr. A Nystrom, University of Freiburg) was used at 1:5000 dilution in 0.1% BSA in PBS solution as well. Next day, coverslips were incubated for 1 hr in darkness at RT with AlexaFluor 568 nm secondary antibody (A-11031) (Thermo Fisher) at 1:2000 dilution in 0.1% BSA in PBS. Then, coverslips were mounted on microscope slides with Fluoroshield (Abcam) mounting medium and imaged using an Olympus IX83 microscope (Olympus, Tokyo, Japan).

Western Blotting
Cell lysates were obtained by directly adding 100 µl radioimmunoprecipitation assay (RIPA) buffer containing protease cocktail inhibitor (Sigma Aldrich) into well culture plates and collected in 1.5 mL eppendorfs. Lysates were centrifuged for 30 min at 13,000 rpm at 4 °C to pellet cell debris. Protein sample concentrations was determined by Bradford reagent following manufacturer guidelines and samples were boiled for 5 min at 95 °C in Laemmli buffer (4% SDS, 20% glycerol, 125 mM Tris-HCL, 10% β-mercaptoethanol [pH 6.8]) before loading onto 4-20% Mini-PROTEAN TGX gels (Bio-rad, Hercules, CA, USA). Following electrophoresis, samples were electro-transferred onto a nitrocellulose membrane for 2 hrs at 300 mA.
Membranes were then blocked on membrane blocking solution (MBS) (Sigma Aldrich) for 1 hr at RT before incubating overnight at 4 °C with target primary antibodies. Rabbit monoclonal (ab203933) antibody was again used, at a dilution of 1:5000. Next day, membranes were incubated with the secondary antibody, conjugated to HRP (anti-rabbit IgG) (ab6721) (Abcam) for 2 hr at 1:2000 dilution at RT and visualized using Pierce ECL western blotting substrate. In the case of the loading control protein GAPDH, a mouse monoclonal primary antibody was used (G8795) (Sigma) at 1:5000 dilution, and a goat anti-mouse (IgG) antibody conjugated to HRP was employed as secondary antibody (ab205719) (Abcam) at a dilution of 1:5000 as well.