Fig. 2: Design, implementation, and characterization of a single-color CRISPRkit under high-tech setup.

a Schematic of plasmids encoding chromoproteins (top) and sgRNAs (bottom). Three primary plasmids encode eforRed, fwYellow, and aeBlue, each containing a strong bacterial σ₇₀ promoter J23119, a strong ribosome binding site (RBS), codon-optimized coding sequence, and a transcriptional terminator. Each sgRNA plasmid targets a specific chromoprotein gene, also containing a J23119 promoter, the sgRNA-coding sequence, and a transcriptional terminator. b High-tech single-color experiment setup using CRISPRkit. The cell-free system is mixed with one chromoprotein plasmid and one sgRNA plasmid per tube. After incubation at 28 °C for 24 h, results are quantified using a fluorescence plate reader. c Data showing performance of single-color CRISPRkit reactions using Cas9, measured by a fluorescence plate reader. Four conditions are compared for each chromoprotein: negative control (NC) with water only, positive control (PC) with CFS and chromoprotein plasmid, no-guide condition (NG) with CFS, chromoprotein plasmid, Cas9 protein, and empty sgRNA plasmid, and targeting sgRNA condition (G) with CFS, chromoprotein plasmid, Cas9 protein, and cognate sgRNA. d Data showing performance of single-color CRISPRkit reactions using nuclease-dead dCas9 with a similar setup to Fig. 2c. For c, d, three biological replicates calculated from the mean of two technical replicates each are plotted for each group. The bars represent the mean and the error bars represent the standard deviation. Unpaired two-sided Student’s t-test was performed; p values are indicated above the no guide and guide condition bars. e Heatmap showing the orthogonality of sgRNAs for repressing each chromoprotein expression. Normalized fluorescence (for eforRed and fwYellow) and absorbance (for aeBlue) to positive controls (PC) are shown. f, g Smartphone images of the single-color CRISPRkit reactions transferred to a parafilm (f) or 0.6 mL PCR tubes (g), after incubation at 28 °C for 24 h. From left to right, each group represent negative control (NC), positive control (PC, CFS + chromoprotein plasmid), no-guide (NG, CFS + chromoprotein plasmid + dCas9 protein + empty sgRNA plasmid), and targeting sgRNA for eforRed (sgRed), fwYellow (sgYellow), and aeBlue (sgBlue). Source data are provided as a Source Data file.