Fig. 3: Design and characterization of dual-color CRISPRkit using the high-tech setup.

a Experimental setup for high-tech dual-color CRISPRkit reactions. The CFS is mixed with one or two chromoprotein plasmids with one or two sgRNA plasmids, incubated at 28 °C for 24 h, and the fluorescence is quantified using a fluorescence plate reader. b Experimental data showing the performance of high-tech dual-color CRISPRkit reactions measured by a fluorescence plate reader. Each group of bars show eforRed or fwYellow single-color positive controls, eforRed+fwYellow dual-color positive control, with an empty sgRNA plasmid (NG), eforRed-targeting sgRNA (sgRed), fwYellow-targeting sgRNA (sgYellow), and both eforRed- and fwYellow-targeting sgRNAs, and negative control (water only). c Smartphone images of the high-tech dual-color CRISPR kit reactions on a parafilm (top) or PCR tubes (bottom), after incubation at 28 °C for 24 h. Fold of repression is labeled for each group. d Comparison of chromoprotein expression between high-tech single-color and dual-color CRISPRkit reactions. The graphs show the normalized fluorescence for eforRed (left) and fwYellow (right) in single-color or dual-color reactions to their positive controls (CFS + chromoprotein plasmid). For b–d, three biological replicates are plotted for each group, with biological replicate values being calculated from the mean of two technical replicates for the dual-color data. The bars represent the mean of biological replicates, and the error bars represent the standard deviation. For b, 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. For d, multiple two-tailed Student’s t-test were performed with the Holm-Sidak correction; multiplicity-adjusted p values are indicated above each compared pair. Source data are provided as a Source Data file.