CRISPR/Cas9 Ribonucleoprotein Delivery Enhanced by Lipo-Xenopeptide Carriers and Homology-Directed Repair Modulators: Insights from Reporter Cell Lines

Abstract

CRISPR-Cas9 genome editing is a versatile platform for studying and treating various diseases. Homology-directed repair (HDR) with DNA donor templates serves as the primary pathway for gene correction in therapeutic applications, but its efficiency remains a significant challenge. This study investigates strategies to enhance gene correction efficiency using a T-shaped lipo-xenopeptide (XP)-based Cas9 RNP/ssDNA delivery system combined with various HDR enhancers. Nu7441, a known DNA-PKcs inhibitor, was found to be most effective in enhancing HDR-mediated gene correction. An over 10-fold increase in HDR efficiency was achieved by Nu7441 in HeLa-eGFPd2 cells, with a peak HDR efficiency of 53% at a 5 nM RNP concentration and up to 61% efficiency confirmed by Sanger sequencing. Surprisingly, the total gene editing efficiency including non-homologous end joining (NHEJ) was also improved. For example, Nu7441 boosted exon skipping via NHEJ-mediated splice site destruction by 30-fold in a DMD reporter cell model. Nu7441 modulated the cell cycle by reducing cells in the G1 phase and extending the S and G2/M phases without compromising cellular uptake or endosomal escape. The enhancement in genome editing by Nu7441 was widely applicable across several cell lines, several Cas9 RNP/ssDNA carriers (LAF-XPs), and also Cas9 mRNA/sgRNA/ssDNA polyplexes. These findings highlight a novel and counterintuitive role for Nu7441 as an enhancer of both HDR and total gene editing efficiency, presenting a promising strategy for Cas9 RNP-based gene therapy.

Keywords: CRISPR Cas9; Cas9 mRNA/sgRNA; Cas9 ribonucleoprotein; cell cycle; enhancer; homology-directed repair.

Figure 1

Characterization of Cas9 RNP complexes and selection of gene correction enhancers. (A) The chemical structure of T-shaped lipo-XP 1738 (dGtp). (B) Schematic illustration depicting T-shaped lipo-XP-based Cas9 RNP complexes prepared by mixing Cas9 protein with sgRNA, ssDNA, and lipo-XP 1738. Dynamic light scattering (DLS) intensity size distribution (C) and zeta potential (D) of 1738-based Cas9 RNP/ssDNA complexes at an RNP/ssDNA ratio of 1:1 and N/P = 12. (E) Transmission electron microscopy (TEM) image of 1738-based Cas9 RNP/ssDNA complexes at an RNP/ssDNA ratio of 1:1 and N/P = 12 (scale bar, 80 nm). (F) Schematic illustration of eGFP-to-BFP conversion in eGFPd2 cells. GFP expression can be eliminated via NHEJ, or the 66th amino acid, tyrosine (TAC), can be changed to histidine (CAC) through HDR to produce BFP expression. (G) HDR efficiency, (H) total gene editing efficiency (both HDR and NHEJ events), and (I) cell viability of HeLa-eGFPd2 cells treated with 1738-based Cas9 RNP/ssDNA complexes (18.75 nM, RNP) with various gene correction enhancers, including Pevonedistat (0.5, 1, 2 nM), Rs-1 (1, 2.5, 10 nM), SCR7 (1, 5, 10 nM), M3814 (5, 10, 20 nM), KU-0060648 (0.1, 0.25, 1 nM), and Nu7441 (2.5, 5, 7.5 nM). “1738” refers to treatment with 1738 Cas9 RNP/ssDNA complexes without enhancer. Enhancer abbreviations represent the enhancer name followed by its concentration in nM. *** p < 0.001 vs. 1738; ns denotes no significant difference. Data are shown as means ± SD (n = 3), with statistical significance determined by unpaired Student’s t-test.

Figure 2

HDR and gene editing efficiency of Cas9 RNP/ssDNA complexes formed with nine different lipo-XP carriers and tested without or with enhancers in HeLa-GFPd2 cells. (A) HDR efficiency and total gene editing efficiency of T-shaped oligomer-derived Cas9 RNP/ssDNA complexes (18.75 nM RNP), transfected with or without 5 nM Nu7441. (B) HDR efficiency and total gene editing efficiency of 1738 Cas9 RNP/ssDNA complexes (18.75 nM RNP), in combination with enhancer mixtures, added for 24 h transfection. Enhancer mixtures include Nu7441 (2.5, 5, 7.5 nM), Rs1 (1, 2.5 nM), KU-0060648 (0.1, 0.25 nM), or M3814 (10, 20 nM). “1738” refers to treatment with 1738 Cas9 RNP/ssDNA complexes without enhancer. Enhancer abbreviations represent the enhancer name followed by its concentration in nM. **** p < 0.0001 vs. 1738; #### p < 0.0001 vs. group Nu2.5; ns denotes no significant difference. Data are shown as means ± SD (n = 3), with statistical significance determined by unpaired Student’s t-test.

Figure 3

Mechanistic investigation of the Nu7441 effect. (A) Cellular uptake of 1738 Cas9 RNP/ssDNA complexes (37.5 nM) containing 20% ATTO647N-labeled Cas9 protein, with or without 5 nM Nu7441, in HeLa cells, analyzed by flow cytometry after 2 h of incubation. (B) Quantification and (C) confocal laser scanning microscopy (CLSM) imaging of gal8 puncta in HeLa gal8-mRuby3 cells treated with HBG or Cas9 RNP/ssDNA nanoparticles (37.5 nM), with or without 5 nM Nu7441 for 4 h. Nuclei were stained with DAPI (blue), while red punctate gal8-mRuby3 fluorescence indicates endosomal membrane disruption. Gal8 puncta were quantified using ImageJ analysis. (D) Experimental design for cell cycle analysis. (E) PI-based cell cycle analysis of HeLa cells treated with various 1738-based nanocarriers (18.75 nM Cas9 RNP), analyzed on day 3 after treatment. Data are shown as means ± SD (n = 3), with statistical significance determined by unpaired Student’s t-test. * p < 0.05; ** p < 0.01; ns denotes no significant difference.

Figure 4

Exon skipping efficiency of Cas9 RNP complexes with NU7441 in a DMD reporter cell model. (A) Schematic representation of the exon 23 skipping mechanism using Cas9 RNP complexes in the absence or presence of 5 nM Nu7441. (B) Comparison of exon skipping efficiency between single 1738 Cas9 RNP complexes and the group supplemented with 5 nM Nu7441. Cas9 RNP concentrations ranged from 0.05 to 50 nM. The column chart illustrates significant improvements in exon skipping efficiency with Nu7441. (C) CLSM images of HeLa mCherry-DMD_Ex23 cells treated with 1738 Cas9 RNP complexes at 10 nM RNP, either with or without 5 nM Nu7441. Cell nuclei stained with DAPI (blue), the cytoskeleton with Alexa Fluor^TM 488 phalloidin (green), and mCherry fluorescence (red). The scale bar represents 50 μm. All complexes were prepared at a N/P ratio of 24 and transfected for 24 h. Data are shown as means ± SD (n = 3). Statistical significance is indicated as **** p < 0.0001.

Figure 5

Gene correction and editing efficiency of Cas9 RNP/ssDNA complexes with NU7441 under various conditions. Gene editing was evaluated by flow cytometry in C2C12-eGFPd2 (A) and 16HBE14o-eGFPd2 (B) cell lines following transfection with 1738 Cas9 RNP complexes without a DNA template (referred to as 1738 no ssDNA, 18.75 nM) or 1738 Cas9 RNP/ssDNA complexes (18.75 nM) in the presence or absence of 5 nM Nu7441. The HDR efficiency (C) and total gene editing efficiency (D) of LAF Cas9 RNP/ssDNA complexes (1611 and 1719) with or without 5 nM Nu7441 in HeLa-eGFPd2. All complexes were prepared at a N/P ratio of 12 and transfected for 24 h. Data are shown as means ± SD (n = 3). Statistical significance is indicated as * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 6

Enhanced HDR-mediated genome editing of HeLa-eGFPd2 applying the optimized HDR T-shaped XP (1636)-based Cas9 RNP/ssDNA complexes with Nu7441. (A) HDR efficiency, (B) total gene editing performance, and (C) cellular viability were evaluated after treatment with 1636-based Cas9 RNP/ssDNA complexes at varying sgRNA/ssDNA ratios (1/2, 1/3, 1/4, 1/5, 1/8) and RNP concentrations ranging from 0.1 nM to 75 nM. Cytotoxicity was assessed using the MTT assay. All formulations maintained a N/P ratio of 12. Data are presented as the mean (n = 3). Numeric percentages are listed in Figure S6.

Figure 7

Gene sequence analysis of HeLa-eGFPd2 cells treated with Cas9 RNP/ssDNA complexes and Nu7441. (A) Schematic representation of the HDR evaluation process using flow cytometry and Sanger sequencing. (B) Flow cytometry gating strategy used to differentiate between eGFP-positive (non-edited), eGFP-negative (knock-out), and BFP-positive (knock-in) populations in samples treated with 1636 RNP/ssDNA complexes and 5 nM Nu7441. (C) Gel electrophoresis assay of PCR products (target gene sequence bands for GFP and BFP at 769 bp) from a sample exhibiting 50.7% HDR prior to Sanger sequencing. (D) Distribution of indel sizes. (E) Alignment of Sanger sequencing; control group is untreated HeLa-GFPd2 cells. (F) Contribution of each sequence after GFP-to-BFP conversion. Sanger sequencing results analyzed using the Synthego ICE tool (https://ice.synthego.com/#/, accessed on 18 October 2023).

Figure 8

Schematic illustration of the gene editing process utilizing the lipo-xenopeptide-based RNP complex and the HDR enhancer Nu7441, with an additional possible impact on the cell cycle and the breakdown of the nuclear envelope.