Fig. 3: Reprogrammable PAIRing (RePAIR) enables a multiplexed output system.

a Schematic of RePAIR, a CRISPR induced DNA reorganization process for multiplexed outputs in a cell-free protein expression system (CFS). b Validation of RePAIR, using a crRNA. Here, reactions contained a truncated LacZα coding sequence and were supplemented with all the components needed for RePAIR (orange) or, alternatively, selected components were removed (Δcomponent). The product of RePAIR was added to a CFS reaction. Absorbance was monitored at 570 nm in a plate reader to reflect functional RePAIRed LacZα. c Validation of RePAIR using a synthetic DNAcrRNA. Truncated LacZα and all the components needed for RePAIR were added to the reactions, with (orange) or without (neg-H₂O, gray) the addition of the DNAcrRNA. The product of RePAIR was added to a CFS reaction. Absorbance was monitored at 570 nm in a plate reader to reflect functional RePAIRed LacZα. Insert shows the initial rate of absorbance within 1 h. d Various concentrations of DNAcrRNAtrc3a, or H₂O (neg) were added to the RePAIR reaction to test sensitivity. Products of RePAIR were added to a CFS and absorbance was monitored at 570 nm in a plate reader. *p = 0.0188. e RePAIR reactions containing truncated blue fluorescent protein (BFP, lipET15trcBFP), truncated green fluorescent protein (GFP, lipBRtrcGFP), or truncated red fluorescent protein (RFP, lipET15trcRFP) were supplemented with H₂O (neg) or with their respective DNAcrRNA. Products of RePAIR were added to a CFS reaction. Fluorescence was monitored at 399/456 nm for BFP, 485/530 nm for GFP and 584/607 nm for RFP. f RePAIR reactions containing truncated trehalase (lipET15trcTre) coding sequence were supplemented with H₂O (neg) or with the matching DNAcrRNA. Products of RePAIR were added to a CFS. Production of glucose was monitored at 1 h and 2 h on a glucose meter. ****p < 0.0001. Error bars: mean ± SD. Experiments displayed are representative of independent biological triplicates (n = 3). Statistical test used for data analysis was a two-tailed unpaired t tests. Source data are provided as a Source Data file.