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. 2022 Jun 9;26:119–131. doi: 10.1016/j.omtm.2022.06.002

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© 2022 The Authors

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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Allele specificity and excision efficiency of OMNI A1 V10 nuclease compositions

(A) Scheme depicting experimental workflow. HSCs from healthy donors and SCN patients were electroporated with RNPs or left non-treated followed by 3 days of recovery in CD34⁺ expansion medium. Cells were then subjected to differentiation by culturing for 7 days with IL-3, SCF, GM-CSF, and G-CSF for proliferation and myeloid progenitor differentiation, followed by a 7-day culture in G-CSF for neutrophil differentiation. (B) Bar graph representing percentages of un-edited reference (black) and alternative (gray) alleles at day 6 of differentiation in HSCs taken from either a healthy donor (HD-V3) or an SCN patient (SCN-P41) and treated with RNP(ref) composition or left non-treated (NT), as measured by ddPCR (n = 3 groups of cells from HD-V3 healthy/SCN-P41 patient donors). Statistical significance is indicated by ∗∗∗∗p < 0.0001; ns, not statistically significant. (C) Bar graph representing percentages of excision at days 6 and 14 of differentiation in HSCs taken from either a healthy donor (HD-V3) (non-treated [NT, black] or RNP(ref)-treated [gray]) or an SCN patient (SCN-P41) (NT [white] or RNP(ref)-treated [hatched]), as measured by ddPCR (n = 3 groups of cells from HD-V3 healthy/SCN-P41 patient donors). Statistical significance is indicated by ∗∗p < 0.01, ∗∗∗∗p < 0.0001. (D) Bar graph representing percentages of wild-type (black) and mutated (gray) alleles in cDNA taken from SCN-P41 patient HSCs that were either RNP(ref)-treated or left NT, as measured by NGS targeting the mutation site (n = 3 groups of cells from patient SCN-P41). Statistical significance is indicated by ∗∗∗p < 0.001. (E) Bar graph representing percentages of unedited reference (black) and alternative (gray) alleles at day 6 of differentiation in HSCs taken from an SCN patient (SCN-P55) treated with RNP(alt) composition or left NT, as measured by ddPCR (n = 3 groups of cells from patient donor SCN-P55). Statistical significance is indicated by ∗∗∗∗p < 0.0001; ns, not statistically significant. (F) Bar graph representing percentages of excision at days 6 and 14 of differentiation in HSCs taken from an SCN patient (SCN-P55): NT (white) or RNP(alt)-treated (hatched), as measured by ddPCR (n = 3 groups of cells from patient donor SCN-P55). Statistical significance is indicated by ∗∗∗p < 0.001,∗∗∗∗p < 0.0001. (G) Bar graph representing percentages of wild-type (black) and mutated (gray) alleles in cDNA taken from patient SCN-P55 HSCs that were either RNP(alt)-treated or left NT, as measured by NGS targeting the mutation site (n = 3 groups of cells from patient SCN-P55). Statistical significance is indicated by ∗∗∗p < 0.001. (H) Bar graph representing ELANE mRNA levels in day 6 differentiated HSCs of patients SCN-P41 and SCN-P55 that were either NT (black) or RNP(ref)/RNP(alt)-treated, respectively (excised, gray). Data are presented relative to the NT group (n = 3 groups of cells from patients SCN-P41 and SCN-P55). Statistical significance is indicated by ∗p < 0.05. Bars represent mean values with standard deviation.