Journal: bioRxiv
Article Title: Multiplexed CRISPR/Cas9 Editing of Tumor Suppressor Genes Recapitulates Molecular and Morphological Features of High-Risk Endometrial Cancer
doi: 10.1101/2025.11.28.691175
Figure Lengend Snippet: (A) Graphic representation of the spatial mRNA-based method used for validation of tumor heterogeneity generated with CRISPR/Cas9. Briefly, a padlock probe is hybridized in the mRNA of the specific gene, designed at the Cas9-cutting site. Then, SplintR ligase is used to ligate de padlock probe, but if there is not 100% homology between the padlock probe and the mRNA (edited sequence), ligation does not occur. Next step is Rolling Circle Amplification (RCA), where EquiPhi29 polymerase amplifies the padlock sequence. Finally, specific regions of different padlock probes are detected with fluorochrome-labelled probes. If the detection is individual for a single gene, generic probes are used. But if the detection is pooled, a fluorochrome code is used. (B) Individual detection of selected TSGs ( Fbxw7, Pten, Trp53, Ppp2r1a, Arhgap35, Arid1a, Pik3r1, Muc16, Kmt2d and Chd4 ) in wildtype epithelial endometrial cells detected with generic probes (green). Hoechst (blue) is used for nuclear staining. Scale bars: 50µm (40X magnification). (C) Pooled detection of mRNA from selected TSGs in wildtype epithelial endometrial cells detected with generic probes (green). Hoechst (blue) is used for nuclear staining. Scale bar: 50µm (40X magnification). (D) Individual detection of Actb mRNA with generic probes. Scale bar: 50µm (40X magnification). (E) Quantification of individual and pooled detections of selected TSG in wildtype cells, shown in (B) and (C) . (F) Representative image of pooled detection of selected TSG in non-electroporated (control) epithelial endometrial cells, detected with fluorochrome code. Images show detection with Atto488 (green), Cy3 (orange), Atto647 (red) and/or AlexaFluor750 (grey). Hoechst (blue) is used for nuclear staining. Scale bars: 50µm (60X magnification). (G) Representative image of pooled detection of selected TSG in electroporated with a pool of RNPs (EP: 10 TSG), detected with fluorochrome code. Images show detection with Atto488 (green), Cy3 (orange), Atto647 (red) and/or AlexaFluor750 (grey). Hoechst (blue) is used for nuclear staining. Scale bars: 50µm (60X magnification). (H) Quantification and co-localization of RNA spots according to the fluorochrome code at a single cell resolution. Binary heatmap represents the presence (green) or absence (yellow) of Fbxw7, Pten, Trp53, Ppp2r1a, Arhgap35, Arid1a, Pik3r1, Muc16, Kmt2d or Chd4 detection in each cell (rows).
Article Snippet: To wash the samples, they were rinsed twice with PBS-T. Rolling Circle Amplification (RCA) was performed for 2 hours at 42°C using 0.5U/μl EquiPhi29 DNA polymerase (A39391, Thermo Fisher Scientific) in a reaction containing 1X EquiPhi29 buffer, 1mM dithiothreitol (DTT) and 1mM dNTPs.
Techniques: Biomarker Discovery, Generated, CRISPR, Sequencing, Ligation, Amplification, Staining, Control
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