Journal: Nature Communications
Article Title: A combinatorial synthetic strategy for developing genome-editing protein-delivery agents targeting mouse retina
doi: 10.1038/s41467-026-69077-w
Figure Lengend Snippet: a Schematic diagram of the color-switching construct of the rd12 reporter-cell line. An intervening sequence from the Rpe65 - rd12 genomic sequence containing the mutation (c.130 C > T; p.R44X) was inserted into a gene expressing both mCherry and eGFP. Expression of eGFP in the reporter cells is prevented by a STOP codon, which can be restored via proper action of ABE. b Representative fluorescence-microscopic images of rd12 -reporter cells 48 h after adding the RNP-CBB complex. Scale bar, 400 μm. c , d Quantitative assessment of ABE-RNP delivery into rd12 -reporter cells, mediated by lipidoid complexes of ABE8e with CBB compounds ( c ) by flow cytometry; or CBBZ compounds ( d ) by fluorescence-microscopic imaging only. [ABE8e]/[CBB(Z)] = 1:8. A, ABE8e-RNP only. NE, no enzyme. Quantification of ABE8e RNP delivery mediated by Lipofectamine 3000 (LF) is included as a positive control. The ratio (eGFP/mCherry) reflects the relative editing efficiency. The CBB compounds were considered positive hits if the ratio of green-to-red (eGFP/mCherry) was more than twice that of A (the dashed line). Data are presented as mean ± SD, n = 4 replicates ( c ) and 3 replicates ( d ). e Schematic diagram of the ABE-activity assay. EndoV = endonuclease V. Blue, SpCas9 PAM; red, target base; orange, bystander base; arrowhead, nick site; FAM, fluorescein. f Representative urea-PAGE-gel image of products of ABE deamination cleaved by EndoV. The gel was imaged with a fluorescein filter. N, no enzyme; P, positive control: 60 bp DNA with inosine in the middle; A, ABE-RNP in TAS; D, ABE-RNP in TAS with 10% DMSO. Numbers indicate molar excess of the CBB compound versus ABE. g Fluorescence quantification of substrate and products of deamination and EndoV cleavage. Two independent samples per data point were assayed in parallel. Source data are provided as a Source Data file.
Article Snippet: The cleavage products were analyzed by denaturing polyacrylamide gel electrophoresis with urea (Urea-PAGE) in Bio-Rad MiniProtean continuous hand cast 15% acrylamide gels in Tris-borate-EDTA (TBE; Bio-Rad, 1610770) with 7 M urea and 5% crosslinker (acrylamide:bis-acrylamide ratio of 19:1; Bio-Rad,1610144).
Techniques: Construct, Sequencing, Mutagenesis, Expressing, Fluorescence, Flow Cytometry, Imaging, Positive Control, Activity Assay
Bio-Rad is a verified supplier 
![a Schematic diagram of the color-switching construct of the rd12 reporter-cell line. An intervening sequence from the Rpe65 - rd12 genomic sequence containing the mutation (c.130 C > T; p.R44X) was inserted into a gene expressing both mCherry and eGFP. Expression of eGFP in the reporter cells is prevented by a STOP codon, which can be restored via proper action of ABE. b Representative fluorescence-microscopic images of rd12 -reporter cells 48 h after adding the RNP-CBB complex. Scale bar, 400 μm. c , d Quantitative assessment of ABE-RNP delivery into rd12 -reporter cells, mediated by lipidoid complexes of ABE8e with CBB compounds ( c ) by flow cytometry; or CBBZ compounds ( d ) by fluorescence-microscopic imaging only. [ABE8e]/[CBB(Z)] = 1:8. A, ABE8e-RNP only. NE, no enzyme. Quantification of ABE8e RNP delivery mediated by Lipofectamine 3000 (LF) is included as a positive control. The ratio (eGFP/mCherry) reflects the relative editing efficiency. The CBB compounds were considered positive hits if the ratio of green-to-red (eGFP/mCherry) was more than twice that of A (the dashed line). Data are presented as mean ± SD, n = 4 replicates ( c ) and 3 replicates ( d ). e Schematic diagram of the ABE-activity assay. EndoV = endonuclease V. Blue, SpCas9 PAM; red, target base; orange, bystander base; arrowhead, nick site; FAM, fluorescein. f Representative <t>urea-PAGE-gel</t> image of products of ABE deamination cleaved by EndoV. The gel was imaged with a fluorescein filter. N, no enzyme; P, positive control: 60 bp DNA with inosine in the middle; A, ABE-RNP in TAS; D, ABE-RNP in TAS with 10% DMSO. Numbers indicate molar excess of the CBB compound versus ABE. g Fluorescence quantification of substrate and products of deamination and EndoV cleavage. Two independent samples per data point were assayed in parallel. Source data are provided as a Source Data file.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_2809/pmc12992809/pmc12992809__41467_2026_69077_Fig4_HTML.jpg)