Fig 1: Efficient one-step organoid population editing by cRNP. (A) (Top) Schematic describing the R26LSL-ERG allele. LSL, LoxP-STOP-LoxP cassette. (Bottom) ERG detection in organoid cells by intracellular flow cytometry. (B) Cas9 protein detection in bulk EPC organoid cells after Cas9 lentiviral transduction. (C) ERG loss in mCherry-labeled sgRNA-infected R26LSL-ERG/LSL-ERG;Ptenfl/fl;Pb-Cre (EPC) organoids as measured by intracellular flow cytometry. EV, empty vector control. (D) sgERG–cRNP dosage titration in EPC organoids with corresponding editing outcomes as measured by ERG intracellular flow cytometry (n = 1). Unt, untreated. (E) Western blot comparing efficiency of editing the Ptenfl/fl allele by Adeno-Cre and sgPten–cRNP in R26LSL-ERG/LSL-ERG;Ptenfl/fl organoids. (F) Pten loss in wild-type (WT) organoids by cRNP as measured by intracellular flow cytometry. (G) Representative images showing a hyperplastic morphology displayed by sgPten–cRNP-treated organoids. (Scale bar, 100 µm.)
Fig 2: cRNP generates large chromosomal deletions in mouse prostate organoids. (A) (Left) Schematic showing a paired-cRNP strategy to induce ERG expression by disrupting the preceding LSL cassette. (Right) ERG expression from the cRNP-treated R26LSL-ERG/LSL-ERG organoid population was measured by intracellular flow cytometry. (B) (Top) Schematic showing Tmprss2-Erg fusion by paired cRNPs with different sgRNA combinations in WT organoids. (Bottom) Genomic fusion detection from the electroporated organoid populations by fusion-specific genomic PCRs. (C) ERG expression of the paired cRNP-treated WT organoid population from B was measured by intracellular flow cytometry. (D) Fusion-specific genomic PCRs of organoid clones treated with indicated cRNP pairs. DNA electrophoresis results of representative clones are shown. (E) Paired CRISPR-mediated deletion of the Foxp1-Shq1 region (schematic shown on the Left). WT organoids were either electroporated with cRNP or infected with lentiGuide (with preengineered Cas9 expression). Fusion-specific genomic PCR of resulting organoid clones and quantification of deletion-positive clones are shown on the Right.
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