Fig. 6. Testing the frugal dual-color CRISPRkit by high school students in a classroom setting.

a Experimental setup of the frugal dual-color CRISPRkit in a frugal setting. The reagents are provided to the students, together with a detailed protocol. The students transfer the reagents including chromoprotein plasmids, dCas9, and sgRNA plasmids into reaction tubes preloaded with the cell-free system. The reactions are left at room temperature for 24 h. Smartphones are used to image the reactions and uploaded to CRISPectra for image analysis. b Illustration of the components of the frugal CRISPRkit. A, Reagent tubes (5), containing the chromoprotein plasmid for eforRed, fwYellow, dCas9 protein, and sgRNA targeting eforRed or fwYellow. B, Reaction tubes (7, preloaded with the cell-free system). C, Imaging tubes (7), for the phone image purpose. D, Inoculation loops (17 loops for each kit, and 3 spare ones). E, User manual and protocol. c Experimental data from 8 independent CRISPRkit experiments performed by one high school student in the frugal setting. d Experimental data from 15 independent CRISPRkit experiments performed by 15 groups of high school students in the classroom setting. For c, d, the bars represent the mean of biological replicates, and the error bars represent the standard deviation. Two one-way ANOVA were performed for each figure; 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 2D scatter plot showing the calculated efficiency and specificity for each reaction for eforRed (red) and fwYellow (yellow). The top right quadrant shows reactions with high efficiency and high specificity. Individual dots represent biological replicates. Data points with both specificity and efficiency of 100% were jittered by <5% to facilitate visualization of many overlapping data points.