Extended Data Fig. 2: CellREADR enables RNA sensing dependent gene editing and cell ablation, and Cre or Flp as an effort shows leaky activities.
From: Programmable RNA sensing for cell monitoring and manipulation
a, left, vector design for CellREADR-mediated and target RNA-dependent gene editing. In READR tdT-Cas9/GFP, a CAG promoter drives expression of BFP followed by sequences coding for sesRNAtdT, Cas9, and eGFP effectors. In another vector, EF1a promoter drives tdT expression and U6b promoter drives the expression of a guide RNA (gRNA) targeting the DYRK1A gene in 293T cells. Right, quantification of READRtdT-Cas9/GFP efficiency as percent of GFP among RFP and BFP expressing cells with or without tdT target RNA. b, Cells transfected with the both U6b-gRNADYRK1A-CAG-tdT and READRtdT-Cas9/GFP showed robust GFP expression co-localized with BFP and RFP (bottom). Cells transfected with READR tdT-Cas9/GFP only (top) showed almost no GFP expression. c, SURVEYOR assay showed Cas9-mediated cleavage in the human DYRK1A locus. DNA cleavage was observed in cell lysates transfected with U6b-gRNADYRK1A-CAG-tdT and READR tdT-Cas9/GFP, but not in U6b-gRNADYRK1A and READRtdT-Cas9/GFP that lacked tdT target RNA. CAG-Cas9 with U6b-gRNADYRK1A cell lysate and 293T cell lysate without plasmid transfection were used as positive control and negative control, respectively. Arrows indicate cleavage products. d, left, vector design for CellREADR-mediated and target RNA-dependent cell death induction. In READR tdT-taCasp3-TEVp, a CAG promoter drives expression of BFP followed by sequences coding for sesRNAtdT and taCasp3-TEVp as effector to induce cell death. Right, cell apoptosis level measured by luminescence was increased in the cells transfected READR tdT-taCasp3-TEVp and EF1a-tdT compared with cells with no tdT RNA. e, Schematic READR vector with Cre coding sequence as effector RNA (left). Representative images of GFP conversion in CellREADR (right). In READRtdT-Cre vector without tdTom RNA, numerous cells showed GFP expression resulting from leaky CRE translation and recombination (right top). Cells transfected with READRtdT-Cre and tdTom RNA showed robust and strong GFP expression (right bottom). Cbh-DIO-eGFP was used as reporter vector for Cre. f, Schematic READR vector with Flp coding sequence as effector RNA (left). Representative images of GFP conversion in CellREADR (right). In READRtdT-Flp vector without tdTom RNA, numerous cells showed GFP expression from leaky FLP translation and recombination (right top). Cells transfected with READRtdT-Flp and tdTom RNA showed robust and strong GFP expression (right bottom). Cbh-fDIO-eGFP was used as reporter vector for Flp. Error bars in a and d are mean values ± s.e.m. n = 3 independent experiments performed.
