Journal: Nature

Article Title: Programmable RNA sensing for cell monitoring and manipulation

doi: 10.1038/s41586-022-05280-1

Figure Lengend Snippet: a, Schematic showing the design of CellREADR. CellREADR is a modular readrRNA molecule, consisting of a 5′ sensor domain (sesRNA) and 3′ effector domain (efRNA), separated by a T2A coding region. sesRNA is complementary to a cellular RNA and contains a stop codon that prevents efRNA translation. Base-pairing between sesRNA and target RNA recruits ADARs, which mediate A-to-I editing and convert the UAG stop to a UGG Trp codon, switching on translation of effector protein. b, CAG-tdT encodes tdTomato target RNA and READRtdT-GFP encodes a readrRNA consisting of a BFP sequence followed by sesRNAtdT and efRNAGFP. WPRE and 2a are sequences for a virus post-transcriptional regulatory element and a self-cleaving peptide coding sequence, respectively. pA, polyadenylated tail. c, Cells transfected with both CAG-tdT (tdT) and READRtdT-GFP exhibited robust GFP expression that co-localized with BFP and RFP (bottom). Cells transduced with READRtdT-GFP only (top) or CAG-tdT and READRctrl encoding sesRNActrl (middle) exhibited very low GFP expression. Right, FACS analysis of GFP and RFP expression. The percentage of GFP+ cells is indicated. d, Quantification of CellREADR efficiency by FACS analysis. e–g, The effect of target RNA expression levels on CellREADR. e, rtTA-TRE3g-ChETA (top) is designed so that BFP-ChETA target RNA is transcribed from TRE3g, driven by constitutively expressed rtTA in a tetracycline-concentration-dependent manner. READRChETA-GFP (bottom) is designed to express readrRNA comprising mCherry (mCh) followed by sesRNAChETA and efRNAGFP. f, Increasing tetracycline concentrations resulted in increasing numbers of BFP+ cells, expressed here as a percentage of RFP+ cells (conversion ratio). g, The conversion ratio increased with increasing tetracycline concentration. CAG-ChETA, which results in constitutive expression of BFP-ChETA, served as positive control. READRctrl served as negative control. h,i, ADAR1 is required for CellREADR function. FACS analysis (h) and quantification (i) of wild-type (WT) or ADAR1-KO cells with sesRNAtdT only, sesRNActrl and CAG-tdT, sesRNAtdT and CAG-tdT, or sesRNAtdT and CAG-tdT with overexpression of ADAR2. Arrows indicate GFP+RFP+ populations. j,k, Electropherograms of Sanger sequencing (j) and quantification (k), showing A to G conversion at the intended editing site in different samples. Data in d,f,g,i,k are mean ± s.e.m.; n = 3 independent experiments.

Article Snippet: 39 , Extended Data Fig 1 g , ​ ,h h , pCAG-spacer-sesRNA-2a-eGFP , Spacer sequence was from CasRx coding regions of Addgene# 109049, different length was used from ATG start as indicated.

Techniques: Sequencing, Transfection, Expressing, Transduction, RNA Expression, Concentration Assay, Positive Control, Negative Control, Over Expression

Journal: Nature

Article Title: Programmable RNA sensing for cell monitoring and manipulation

doi: 10.1038/s41586-022-05280-1

Figure Lengend Snippet: Plasmids used in this study.

Article Snippet: 39 , Extended Data Fig 1 g , ​ ,h h , pCAG-spacer-sesRNA-2a-eGFP , Spacer sequence was from CasRx coding regions of Addgene# 109049, different length was used from ATG start as indicated.

Techniques: Construct, Sequencing, Amplification

Journal: Nature

Article Title: Programmable RNA sensing for cell monitoring and manipulation

doi: 10.1038/s41586-022-05280-1

Figure Lengend Snippet: SesRNA used in this study.

Article Snippet: 39 , Extended Data Fig 1 g , ​ ,h h , pCAG-spacer-sesRNA-2a-eGFP , Spacer sequence was from CasRx coding regions of Addgene# 109049, different length was used from ATG start as indicated.

Techniques: Sequencing