Engineering single-cycle MeV vector for CRISPR-Cas9 gene editing

Abstract

CRISPR-Cas9-mediated gene editing has vast applications in basic and clinical research and is a promising tool for several disorders. Our lab previously developed a non-integrating RNA virus, measles virus (MeV), as a single-cycle reprogramming vector by replacing the viral attachment protein with the reprogramming factors for induced pluripotent stem cell generation. Encouraged by the MeV reprogramming vector efficiency, in this study, we develop a single-cycle MeV vector to deliver the gRNA(s) and Cas9 nuclease to human cells for efficient gene editing. We show that the MeV vector achieved on-target gene editing of the reporter (mCherry) and endogenous genes (HBB and FANCD1) in human cells. Additionally, the MeV vector achieved precise knock-in via homology-directed repair using a single-stranded oligonucleotide donor. The MeV vector is a new and flexible platform for gene knock-out and knock-in modifications in human cells, capable of incorporating new technologies as they are developed.

Keywords: CRISPR; Cas9; HDR; NHEJ; gene editing; iPSC; measles; viral vector.

Graphical abstract

Figure 1

Production and characterization of MVCas9 and MVgRNA^mch to cause mCherry mutagenesis in a reporter cell line (A) Schematic of MeV virus and MeV gene editing vectors. (B) Western blot analysis of Cas9 expression in 293T cells after transduction with MVCas9 or LVCas9. Uninfected 293T (negative control), beta-actin (loading control), and MeV N (infection control). (C) Representative confocal images of Cas9 nuclear expression in transduced neonatal human fibroblasts (BJ) and Vero cells (Vero) with MVCas9 vector. Scale bars, 100 μm. (D) Titers of cell-associated and released virus produced upon infection of Vero-H2 cells with MVCas9 (gray) and MVgRNA (black) vectors compared to control vector MVVac2(GFP)H (white) at 24, 48, and 72 h after infection. Error bars represent SD from three individual experiments. A two-way ANOVA was used, followed by Tukey’s multiple comparison test (∗∗∗∗p ≤ 0.0001, all non-significant values are not labeled). (E) Indel (light gray) and KO (gray) efficiency analyzed by ICE-Synthego software created by transfection or/and infection of specified vectors. A two-way ANOVA was used followed by Tukey’s multiple comparison test (∗∗∗∗p ≤ 0.0001, ∗∗∗p ≤ 0.001, all non-significant values are not labeled). n = 3–6.

Figure 2

Characterization of all-in-one MeV vectors expressing Cas9 and gRNA^mch to cause mCherry KO in a reporter cell line (A) Schematic of all-in-one MeV gene editing vectors expressing Cas9 and gRNA in a single genome. (B) Representative microscopy images of mCherry and GFP expression in 293Tmch cells after transduction with each MeV gene editing vectors. Scale bar, 100 mm. (C) ICE-Synthego analysis of NHEJ mediated indel and KO percentages on mCherry gene in 293T^mch cells when infected with MV(gRNA^mch)^PVac(Cas9) (light gray), MV(gRNA^mch)^PWT(Cas9) (gray), MV^PWT(Cas9) (gRNA^mch)^H (dark gray), and pgRNA^mchCas9 (stripe) at an MOI of 0.5 or 1; n = 6. Error bars represent mean ± SD. A two-way ANOVA was used, followed by Tukey’s multiple comparison test (∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001, not significant values are not labeled).

Figure 3

MeV vector mediated gene editing on endogenous HBB and FANCD1 genes (A) ICE Synthego analysis of NHEJ-mediated indel (white) and KO (black) percentages on HBB gene of 293T cells when transduced with MV(gRNA^HBB)^PVac(Cas9) at an MOI of 0.5 or 1.0. Error bars represent mean ± SD of five independent experiments with each containing a biological replicate. A two-way ANOVA was used followed by Sidak post hoc multiple comparisons test (∗∗p ≤ 0.01, not significant values not labeled). (B) ICE Synthego analysis of NHEJ-mediated indel (white) and KO (black) efficiencies on HBB gene of four different primary AHFs (#1, #2, #3, and #4) transduced with MV(gRNA^HBB)^PVac(Cas9) at an MOI of 0.5 or 1.0. Error bars represent the mean ± SD of three independent experiments with each containing a biological replicate. A two-way ANOVA was used followed by Tukey’s multiple comparisons test (∗∗p ≤ 0.01, not significant values are not labeled). (C) ICE Synthego analysis of NHEJ-mediated indel (white) and KO (black) GE efficiencies on FANCD1 mutagenesis of 293T cells when infected with MV(gRNA^FANCD1)^PVac(Cas9) at MOI of 0.5 or 1.0. Error bars represent mean ± SD of three independent experiments with each containing a biological replicate. A two-way ANOVA was used followed by Sidak post hoc multiple comparisons test (∗∗p ≤ 0.01, not significant values not labeled). (D) ICE Synthego analysis of NHEJ-mediated indel (white) and KO (black) efficiencies on FANCD1 gene of four different primary AHFs (#1, #2, #3, and #4) when infected with MV(gRNA^FANC)^PVac(Cas9) at an MOI of 0.5 or 1.0. Error bars represent mean ± SD of three independent experiments with each containing a biological replicate. A two-way ANOVA was used followed by Tukey’s multiple comparisons test (not significant values are not labeled). (E) ICE Synthego analysis of NHEJ-mediated indel (white) and KO (black) efficiencies on HBB gene of iPSC when infected with MV(gRNA^HBB)^PVac(Cas9) at an MOI of 0.25, 0.5, or 1.0 and collected at days 2, 3 and 5. Error bars represent mean ± SD of three independent experiments with each containing a biological replicate. A two-way ANOVA was used followed by Tukey’s multiple comparisons test (∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001, not significant values are not labeled).

Figure 4

MeV editing vectors can promote efficient HDR mediated knock-in mCherry and HBB genes (A and B) ICE-Synthego GE analysis of mCherry (A) or HBB (B) gene in 293T^mch cells demonstrating the knock-in (KI) (A) in presence of 10 pM or 20 pM of the indicated ssODN. AS, antisense to target region; S, sense. Error bars represent mean ± SD of three independent experiments with each containing a biological replicate. A two-way ANOVA was used followed by Sidak post hoc multiple comparisons test (∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001, not significant values aerw not labeled). (C and D) ICE-Synthego GE analysis of mCherry (A) or HBB (B) gene in 293T^mch cells demonstrating the KO contribution in the HDR experiment for each sample presented in (A) and (B). Error bars represent the mean ± SD of three independent experiments, with each containing a biological replicate. A two-way ANOVA was used followed by Sidak post hoc multiple comparisons test (∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001, not significant values not labeled). (E and F) Optimization of HDR-mediated knock-in HBB gene of 293T using timing and amount of HBB 80_3S ssODN. Cells were transfected with 10 pM (white triangle), 20 pM (white circle), 30 pM (black triangle), 50 (black square), 75 pM (black circle) and 100 pM (white square). Eight hours later, the cells were transduced with MV(gRNA^HBB)^PVac(Cas9). Cells were collected at 24, 36, 48, 72, 96 h, or 5 days after transfection, n = 4. The percentage of KI was determined after DNA amplification, Sanger sequencing, and ICE-Synthego analysis.