Diosgenin enhances liposome-enabled nucleic acid delivery and CRISPR/Cas9-mediated gene editing by modulating endocytic pathways

Abstract

The CRISPR/Cas9 system holds great promise in treating genetic diseases, owing to its safe and precise genome editing. However, the major challenges to implementing the technology in clinics lie in transiently limiting the expression of genome editing factors and achieving therapeutically relevant frequencies with fidelity. Recent findings revealed that non-viral vectors could be a potential alternative delivery system to overcome these limitations. In our previous research, we demonstrated that liposomal formulations with amide linker-based cationic lipids and cholesterol were found to be effective in delivering a variety of nucleic acids. In the current study, we screened steroidal sapogenins as an alternative co-lipid to cholesterol in cationic liposomal formulations and found that liposomes with diosgenin (AD, Amide lipid: Diosgenin) further improved nucleic acid delivery efficacy, in particular, delivering Cas9 pDNA and mRNA for efficient genome editing at multiple loci, including AAVS1 and HBB, when compared to amide cholesterol. Mechanistic insights into the endocytosis of lipoplexes revealed that diosgenin facilitated the lipoplexes' cholesterol-independent and clathrin-mediated endocytosis, which in turn leads to increased intracellular delivery. Our study identifies diosgenin-doped liposomes as an efficient tool to deliver CRISPR/Cas9 system.

Keywords: CRISPR/Cas9; cationic lipid; diosgenin; genome editing; transfections.

FIGURE 1

Liposomal physicochemical characterizations. Schematic representation of the lipids used in the liposomal formulations (A), Hydrodynamic diameters of liposomes (B), and Surface potentials of the liposomes (C).

FIGURE 2

Transfection studies with pDNA and mRNA of different liposomes. Screening of sapogenin liposomes using the eGFP pDNA reporter gene, Representative images in epifluorescence microscope (A), Flow cytometry analysis of eGFP pDNA expression (B), Screening of sapogenin liposomes using eGFP mRNA, Representative images in epifluorescence microscope (C), Flow cytometry analysis of eGFP mRNA expression (D).

FIGURE 3

Transfections in the presence of endocytosis blockers. Normalized eGFP expression by endocytosis inhibitors. HEK-293 cells were pretreated with Dynosore (80 μM), M-β-CD (2 mM), Nystatin (80 μM), Chlorpromazine (15 μM), and Sucrose (450 mM) in serum-free media for 1 hour before exposure to the 1:1 lipid/DNA charge ratio of AC and AD lipoplexes, respectively (A) Imipramine was added in the different concentrations of 10, 20, and 40 μM after 4 h of seeding of HEK293 cells, and then the cells were treated with AC and AD lipoplexes with a 1:1 lipid-to-DNA charge ratio (n = 3; **p < 0.001 compared with AC and LF3000) (B).

FIGURE 4

Delivery of varied sizes of plasmids. AD, AC, and LF3000 liposomes delivered a 4.7 kb eGFP-N1 plasmid (A) and larger size plasmids, i.e., 11.7 kb pL-CRISPR. EFS.GFP (B) and 13.7 kb pL-CRISPR. SFFV.GFP plasmid construct respectively (compacted with P3000 DNA-condensing agent) (C) (n = 3; **p < 0.001 compared with AC and LF3000).

FIGURE 5

Delivery of different forms of CRISPR/Cas9 in HEK-293T cell lines: Agarose gel electrophoresis showing T7 activity. The cleaved product is highlighted in red. The editing efficiencies are indicated at the bottom of the gel. CRISPR-Cas9 plasmid DNA-mediated gene editing. Transfection of lenti-plasmid CRISPR-AAVS1 sgRNA, which expresses human codon-optimized Cas9 protein and an AAVS1-targeting sgRNA element from the U6 promoter, using transfection reagents AD, AC, and LF3000 (A). sgRNA mediated gene editing in Cas9-expressing cells. sgRNA targeting the SCD locus was transfected with AD and AC (B). Cas9 mRNA mediated gene editing. Transfection of SCD sgRNA and Cas9 mRNA targeting the Hbb locus using Mmax, AC (C).