Fig. 4: Design and characterization of frugal low-tech dual-color CRISPRkit.

a Schematic illustrating replacement of laboratory apparatus with frugal alternatives: pipettes with inoculation loops, incubator with room temperature setup, fluorescence plate reader with a smartphone, and professional analytical software with CRISPectra. b Performance characterization of the single-color CRISPRkit using eforRed at five different temperatures (4 °C to 42 °C). Reactions are incubated for 24 h. Red bars represent non-targeting sgRNA, and gray bars represent targeting sgRNA. A representative phone image of CRISPRkit reactions on parafilm is shown. Three biological replicates are plotted for each group, with mean fluorescence indicated by bars and standard deviation by error bars. Multiple two-tailed Student’s t-tests with Holm-Sidak correction were performed; multiplicity-adjusted p values are indicated above each compared pair. c Experimental data showing low-tech dual-color CRISPRkit reactions measured by a fluorescence plate reader. From left to right, each group of bars (red – eforRed, yellow – fwYellow) show eforRed positive control, fwYellow positive control, eforRed + fwYellow dual-color positive control, dual-color with empty sgRNA plasmid (NG), dual-color with eforRed-targeting sgRNA (sgRed), dual-color with fwYellow-targeting sgRNA (sgYellow), dual-color with both eforRed- and fwYellow-targeting sgRNAs, and negative control (water only). d Experimental data showing low-tech dual-color CRISPRkit reactions analyzed by CRISPectra. The order of the reactions mirrors c. For c and d, three biological replicates are plotted for each group, with biological replicate values being calculated from the mean of two technical replicates for d. The bars represent the mean of biological replicates, and the error bars represent the standard deviation. Two one-way ANOVA were performed; p values generated by comparison of chosen conditions to no-guide control via Dunnett’s multiple comparisons test are indicated above the chosen condition, above the fold of repression. e A representative smartphone image of the CRISPRkit reactions on parafilm after 24 h at room temperature, following the same order as in (d). f 2D scatter plots showing comparison of fluorescence plate reader data (x-axis) and phone image data (y-axis) for eforRed (left) and fwYellow (right). Individual dots represent biological replicates. Black lines represent the linear fit. Dotted lines represent 95% confidence interval. R² values are shown for the linear regression. Source data are provided as a Source Data file.