Fig. 6

Zero-passage organoid compatibility with lentiviral transduction and genetic modification. A Representative image of one organoid transduced with a mixture of lentiviruses encoding EGFP (green) and TdTomato (magenta). Scale bar is 50 µm. B Quantification of organoids containing cells expressing one or more fluorophores. Percent fluorescence positive ( +) is reported as the mean ± standard deviation from n = 4 independent luminal breast cancers. Differences in organoid sizes were assessed by two-way ANOVA (n = 11–27 organoids per group). C Map of the pDual_dsCas9_Venus lentiviral plasmid encoding destabilized Cas9 fused by a P2A sequence with Venus. Cloning sites for coexpressed dual-sgRNAs are indicated. D Harvesting single Venus-positive organoids for genotyping. Fluorescent organoids confirmed by epifluorescence (top right) are harvested by micropipette aspiration (before: bottom left; after: bottom right). Scale bars are 50 µm (top) and 250 µm (bottom). E and F Single-organoid genotyping of zero-passage cultures transduced with pDual_dsCas9_Venus targeting a noncoding region of human chromosome 1 (E) or chromosome 2 (F). The first lane is a PCR negative control (no genomic DNA), the second lane is a nontargeting control (single organoid transduced with empty pDual_dsCas9_Venus), and the subsequent three lanes are single organoids transduced with dual-sgRNAs. DNA markers are on the left and PCR band sizes for wild-type and knockout amplicons are on the right. Relative efficacy of targeting is indicated on the bottom of the gel