Figure 1: Gene editing using γPNAs in mouse bone marrow.
(a) Strategy for targeted correction of a β-globin gene IVS2-654 (C->T) mutation in β-globin/GFP transgenic mice using triplex-forming tail-clamp PNAs and donor DNAs. (b) tcPNA and γtcPNAs designed to bind to homopurine regions within intron 2 of the human β-globin gene near IVS2-654 (C->T). (c) DNA, unmodified PNA and MPγPNA. (d) tcPNAs and γtcPNAs to bind to the positions indicated in B. γtcPNA4-Scr is a scrambled version of γtcPNA4. Bold/underline indicates γPNA residues. K indicates lysine; J, pseudoisocytosine (for c) for pH-independent triplex formation. O, 8-amino-2,6,10-trioxaoctanoic acid linkers connecting the Hoogsteen and Watson–Crick domains of the tcPNAs. (e) Scanning electron microscope images of nanoparticles. Scale bar, 2.0 μm. (f) Gene correction of the IVS2-654 (C->T) mutation within the β−globin/GFP fusion gene in mouse BM cells treated ex vivo with NPs containing the indicated tcPNAs and donor DNA. %GFP+ was determined by flow cytometry and indicates successful gene editing. Data are mean±s.e., n=3; statistical analysis by Student’s t-test, **P<0.005. (g) %GFP+ cells in mouse BM after ex vivo treatment with NPs containing tcPNA1, γtcPNA4 or γtcPNA4-Scr, plus donor DNAs. Data are mean±s.e.m., n=3; analysis by Student’s t-test, *P<0.05. (h,i) Quantification of γH2AX foci by immune fluorescence microscopy, indicative of DNA DSBs in primary fibroblasts (from the β-globin/GFP transgenic mice) either untreated or treated with 5 Gy of IR, blank NPs, NPs containing γtcPNA4 and donor DNA, lipofectamine alone, lipofectamine transfection of a Cas9 expression vector, lipofectamine transfection of a Cas9 vector and a separate guide RNA expression vector (targeting the same site in β-globin sequences as γtcPNA4; Cas9+gRNA), or transfection of a vector containing both Cas9 and guide RNA (Cas9 and gRNA). Quantification as per cent of cells with 15 or more foci (h) or average number of foci per cell (i). 100 cells were counted per condition, data are mean±s.e.m, n=3; analysis by Student’s t-test, *P<0.05.
