Skip to main content
. 2023 Jun 8;33:57–74. doi: 10.1016/j.omtn.2023.06.004

Figure 6.

Figure 6

Efficient NB-induced KO in human intestinal organoids

(A) Human rectal eGFP-expressing organoids 14 days after treatment or not with anti-GFP NBs or electroporation with Cas9 and anti-GFP gRNA encoding plasmid. Flow cytometry analysis of percent eGFP-expressing organoid cells (left panels, representative of n = 3). The viability (right panel) upon treatment with anti-GFP NBs or electroporation of Cas9 and anti-GFP gRNA encoding plasmids is shown after 72 h (means ± SD, n = 3, biological replicates, t test, Mann-Whitney test, non-parametric; ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗p < 0.05). (B and C) Validation of anti-hCFTR NBs in HEK293T cells. (B) One sgRNA was used for KO of human CFTR in HEK293T cells. Bulk gDNA sequencing analysis of the cells treated with NBs targeting hCFTR. Sanger sequencing was performed of the PCR band including the hCFTR target locus and was subjected to DECODR analysis, which revealed on-target INDEL frequencies at the gRNA CFTR target locus (means ± SD, n = 3, biological replicates, t test, Mann-Whitney test, non-parametric; ∗∗∗∗p < 0.0001). (C) Detection of hCFTR plasma membrane (PM) expression by FACS analysis for HEK293T cells, overexpressing 3HA-CFTR treated with anti-hCFTR NBs or control NBs (means ± SD, n = 3, biological replicates, t test, Mann-Whitney test, non-parametric; ∗∗∗∗p < 0.0001). (D) Toxicity of NBs in HEK293T cells by DAPI staining at 48 h after NB incubation (means ± SD, n = 3, biological replicates, unpaired t test, not significant). (E) Bulk gDNA sequencing analysis of human rectal organoids treated with NBs targeting hCFTR. Sanger sequencing was performed of the PCR band including the CFTR target locus and was subjected to DECODR analysis which revealed on-target INDEL frequencies at the gRNA hCFTR target locus. Sequence decomposition of INDEL events is shown (upper panel) and is summarized in the histogram (F) (means ± SD n = 4, technical replicates, t test, Mann-Whitney test, non-parametric; ∗∗∗p < 0.001). (G) Toxicity of NBs in organoids was evaluated 14 days after treatment by counting the number of organoids treated or not with NBs (means ± SD, n = 10, technical replicates). CTRL, no NBs.