Extended Data Fig. 1: Design and test of singular and binary CellREADR vectors.
From: Programmable RNA sensing for cell monitoring and manipulation
a, Schematic of a singular CellREADR vector. Left, PGK-tdT (top) expresses the tdTomato target RNA from a PGK promoter. READRtdT-GFP (bottom) expresses a READR RNA consisting of sesRNAtdT and efRNAGFP, driven by a CAG promoter. Vertical dashed lines indicate the complementary base-pairing region between tdT mRNA and sesRNAtdT, with sequence surrounding the editable STOP codon shown on the right. At the editing site, the editable adenine in sesRNAtdT (cyan) is mismatched to a cytosine in the tdT mRNA. TdTomato is a tandem repeat of two dTomato genes, thus a tdT RNA contains two copies of target sequence for sesRNAtdT base-pairing. b, Validation of the READRtdT-GFP vector. In 293T cells co-transfected with READRtdT-GFP and PGK-tdT, many cells switched on GFP translation and fluorescence (arrows in top row). In cells co-transfected with control empty vector, few cells showed GFP expression (bottom right). c, GFP expression was further assayed by Western blotting. d, Left, a binary vector design for CellREADR luciferase assay. READRtdT-tTA2 expresses a readrRNA consisting of sesRNAtdT and efRNAtTA2, and TRE3g-ffLuc expresses the luciferase RNA upon tTA2 activation. Right, luciferase activity dramatically increased only in cells transfected with three vectors. Co-transfection of TRE3g-ffLuc with CAG-tTA2, which constitutively expresses tTA2, served as a positive control. e, Schematic of READRtdT-GFP vector in which a spacer sequence is inserted before sesRNAtdT coding region (top). 293T cells were transfected with READRtdT-GFP vector encoding viable length of spacers without (gray) or with (pink) tdT target RNA expression, respectively. Quantification of conversion ratio calculated as percentage of GFP+ cells among RFP+ cells (bottom). f, A binary vector design for CellREADR assay (left). Right, representative images of GFP conversion with binary vectors. In cells co-transfected with sesRNACtrl vector, few GFP+ cells were observed. Conversion percentages are shown on the right. Error bars in d are mean values ± s.e.m. n = 3 independent experiments performed. The bars in e are mean values, n = 2 independent experiments performed. For gel source data in c, see Supplementary Fig. 2.
