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. 2022 Apr;29(3-4):157-170.
doi: 10.1038/s41434-021-00282-6. Epub 2021 Aug 6.

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

Jonathan O'Keeffe Ahern #  1 , Irene Lara-Sáez #  2 , Dezhong Zhou  1 , Rodolfo Murillas  3   4   5 , Jose Bonafont  3   4   5 , Ángeles Mencía  3 , Marta García  4   5   6 , Darío Manzanares  1 , Jennifer Lynch  1 , Ruth Foley  1 , Qian Xu  1 , A Sigen  1 , Fernando Larcher  3   4   5   6 , Wenxin Wang  7
Affiliations

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

Jonathan O'Keeffe Ahern et al. Gene Ther. 2022 Apr.

Abstract

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.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Agarose gel retardation assay to assess the CRISPR-C7 plasmid DNA complexation by the HPAE-EB polymer.
The cationic HPAE-EB polymer exhibits significant DNA condensation ability and can retard the movement of anionic DNA through the agarose gel at polymer:DNA w/w ratios ranging from 5:1 to 30:1. At w/w ratios below 5:1, incomplete retarding of DNA is evident as some DNA has migrated down through the gel, indicating insufficient complexation. Naked CRISPR-C7 plasmid alone (without polymer) was used as the control for this experiment. Data are representative of three independent experiments with each w/w ratio assessed in duplicate (n = 3).
Fig. 2
Fig. 2. DNA encapsulation efficiency assessment with the HPAE-EB polymer and CRISPR-C7 plasmid complexes.
Experiments were performed at polymer:DNA w/w ratios from 0.25:1 to 30:1 using 1 μg of DNA. At low polymer:DNA ratios of 0.25:1 and 0.5:1, the HPAE-EB polymer is incapable of adequately encapsulating the DNA from the PicoGreen® reagent. However, upon reaching w/w ratios of 5:1 and greater, an encapsulation efficiency of ~70% is achieved and maintained, indicating sufficient binding to protect the DNA. Samples were performed in triplicate and presented as average ± standard deviations (SD) for three independent experiments (n = 3).
Fig. 3
Fig. 3. Transfection process and physical characterisation of HPAE-EB polymer alone and complexed to CRISPR-C7 plasmid.
a Non-viral transfection process using HPAE-EB and CRISPR-C7 plasmid DNA to mediate gene editing. b Endosomal buffering capacity of the HPAE-EB polymer. Acid–base titration curve of HPAE-EB, PEI (25 kDa) and NaCl across a wide pH range was performed. PEI (25 kDa) was used as a commercial standard for comparison and NaCl was used as a negative control. With addition of HCl, NaCl shows sharp decline in pH, whereas PEI and HPAE-EB exhibit gradual decrease owing to higher proton buffering capacity (n = 3). c Zeta potential of HPAE-EB polyplexes with CRISPR-C7 plasmid DNA, complexed at different w/w ratios. DNA alone (0:1 w/w ratio) was used to demonstrate the negative charge of DNA, and from w/w ratios of 10:1 and higher, polyplex surface charge is positive for all DNA quantities. d Hydrodynamic size and PDI distribution of the HPAE-EB polymer and CRISPR-C7 plasmid DNA polyplexes at varying DNA quantities and polymer:DNA w/w ratios. As polymer:DNA ratio is increased, there is a sharp decrease in polyplex size across all DNA quantities. PDI distribution is heterogenous throughout all conditions tested. Data are presented as mean ± SD of three replicates from three independent experiments (n = 3).
Fig. 4
Fig. 4. Transfection capability and cytotoxicity of HPAE-EB polymer complexed to CRISPR-C7 plasmid in HEK293 cells.
a GFP within the CRISPR-C7 plasmid enabled visual transfection in cells. (I) Untreated cells were used as the negative control. (II) Lipofectamine 3000 was used as the commercial reagent for comparison, and treatments with different polymer:DNA w/w ratios and amounts of DNA (III–VIII) were performed. Scale bar 100 µm. Representative images from six replicates of three independent experiments (n = 3). b Cell viability 72 h post transfection in HEK293 cells by alamarBlue™, where preservation of cell metabolic health post transfections using HPAE-EB polymer and Lipofectamine 3000 was observed. Data were collected from six replicates of three independent experiments and presented as means ± SD (n = 3). *p < 0.05, as compared to control values.
Fig. 5
Fig. 5. Mean fluorescence intensity (MFI) and cell viability of transfected HEK293 cells.
MFI of GFP fluorescence for each transfection condition was grouped into polymer:DNA w/w ratios used and cell viability. In addition, one-plot histogram overlay of GFP fluorescence comparing untreated control and HPAE-EB transfections under different w/w ratios was performed. Only HPAE-EB at w/w ratios of 20:1 and 30:1 yields a statistically significant increase in MFI. All results are shown as mean ± SD, n = 3. ****p < 0.0001, as compared to control values.
Fig. 6
Fig. 6. Cas9 production and localisation in transfected HEK293 cells using plasmid CRISPR-C7.
a Untreated HEK293 cells acted as a negative control and Lipofectamine 3000 was used as the commercial reagent comparison. Samples were fixed 72 h post transfection and stained with Cas9 antibody in situ (red), and with DAPI as the nuclear stain. Magnification at 40×, scale bar 20 µm. b Representative western immunoblot for intracellular levels of Cas9 at the optimal conditions from transfections with HPAE-EB compared with the commercial reagent Lipofectamine 3000. Substantial Cas9 production was achieved in HEK293 cells following transfections with HPAE-EB at w/w ratios of 20:1 and 30:1. No expression of Cas9 was present in untreated or pDNA-only treated cells. GAPDH was used as the loading control. Data are representative of three independent experiments (n = 3).
Fig. 7
Fig. 7. CRISPR–Cas9 gene editing strategy for targeted exonal excision of mutation-containing exons in COL7A1 gene.
a CRISPR-C7 plasmid schematic detailing construct design containing dual-guide RNA sequences and Cas9 coupled with GFP reporter for transfection efficiency evaluation. b Dual-guide RNA strategy for targeted genomic deletion of pathogenic mutation-containing exon 80 in COL7A1 gene.
Fig. 8
Fig. 8. PCR analysis of genomic DNA from HEK293 cells transfected with CRISPR-C7 plasmid under different conditions.
HEK293 cells were treated with a 0.5 µg and b 1 µg of CRISPR-C7 plasmid. Upper arrow at 320 bp represents unedited DNA, while lower arrow at 263 bp represents DNA lacking exon 80. Lipofectamine 3000 was used as the commercial reagent control. Excision efficiency (%) estimated by densitometry analysis showed a maximum editing of 20.3%, achieved using a HPAE-EB:pDNA w/w ratio of 30:1. Data are representative of three independent experiments (n = 3).
Fig. 9
Fig. 9. Chromatograms of Sanger sequenced PCR amplicons of COL7A1 gene.
Guide RNAs denoting cut sites (dashed lines) are indicated highlighting the dual-guide strategy employed. a Sharp single peaks representative of no editing are seen in the control sample. b In contrast, overlapping peaks denoting the mixed pool population of cells are seen directly at cut sites confirming indel generation by HPAE-EB:pDNA (w/w) 20:1 using 0.5 µg DNA treated cells. Data are representative of three replicates from two independent experiments (n = 2).
Fig. 10
Fig. 10. RDEB keratinocytes transfections with the HPAE-EB polymer and CRISPR-C7 plasmid.
a Efficient transfection was evaluated by GFP expression. Cells were treated with different polymer:DNA ratios and DNA amounts (VII–XII). Furthermore, Lipofectamine 3000 was used as the commercial reagent for comparison (V, VI), untreated cells were used as a negative control (I, II) and plasmid DNA as the vector control (III, IV). Scale bar 100 µm. Representative images from six replicates of three independent experiments (n = 3). b alamarBlue™ test showed high preservation of RDEB keratinocytes viability 72 h post transfection using different HPAE-EB polymer conditions complexed to DNA, and Lipofectamine 3000 as a control. Data were collected from six replicates of three independent experiments and presented as mean ± SD (n = 3).
Fig. 11
Fig. 11. FACS analysis of GFP-positive RDEB keratinocytes.
Gating strategy stratified cells into three groups, namely, GFP negative, GFP “dim” and GFP “bright”, based on fluorescence intensity. Optimal transfection conditions using HPAE-EB and CRISPR-C7 plasmid resulted in 11.5% RDEB keratinocytes that were highly fluorescent. Data are representative of three replicates from three independent experiments (n = 3).
Fig. 12
Fig. 12. PCR analysis of genomic DNA from RDEB keratinocytes treated under different transfection conditions with 0.5 µg CRISPR-C7 plasmid.
Upper arrow of 320 bp band indicates unedited DNA, while lower arrow of 262 bp band corresponds to DNA lacking exon 80. Maximum excision efficiency of 8.2% was estimated by densitometry analysis. Data are representative of three independent experiments (n = 3).
Fig. 13
Fig. 13. COL7A1 gene exon 80 excision efficiency in RDEB keratinocytes transfected with CRISPR–Cas9 RNPs complexed to the HPAE-EB polymer.
a PCR amplification from genomic DNA of a fragment spanning over the sgRNA target sites confirmed removal of exon 80 by the presence of a smaller molecular band (440 bp) in line with the distance between both cut sites (55 bp), marked with a lower arrow (red). Upper arrow (blue) indicates unedited DNA. Densitometry analysis from three independent experiments estimated an exon 80 excision of 43.2% using 4 µg of CRISPR–Cas9 RNP complex at 20:1 w/w ratio with HPAE-EB polymer (n = 3). b Collagen VII expression (red) was restored in RDEB keratinocytes after CRISPR–Cas9 RNP treatment, assessed by immunofluorescence staining, using DAPI (blue) as a nuclear stain. Magnification at 40×, scale bar 50 µm. Data were collected from two repeats of two independent experiments (n = 2).
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