Figure 5.

Interrogation of the 6 Myrf enhancer candidates by CRISPRi. (A) CRISPRi validation for the Myrf locus. The expression level of Myrf was estimated by the reporter activity of Rffl, a highly specific and sensitive Myrf luciferase reporter^25,37. The Rffl activity for each sgRNA was divided by the average Rffl activity for Scr1 and Scr2 to get the relative Rffl activity. For each sgRNA, the mean and standard error are shown. *p value < 1.4 × 10⁻⁴ by two-sided one sample Student’s t test corrected by the Bonferroni procedure (n = 4). (B) For each of the 6 Myrf enhancer candidates, 5 independent sgRNAs were used. For each sgRNA, the mean and standard error of the relative Rffl activity are shown. *p value < 3.3 × 10⁻² by two-sided one sample Student’s t test corrected by the Bonferroni procedure (n = 9). (C) The signal from each fluorescence channel was quantified for individual cells by CellProfiler⁴⁰. The number of cells analyzed is as follows: Scr1 (91), Pro (79), EC1 (42), EC2 (46), EC1&2 (82). Scale bar, 20 µm. AU: arbitrary unit. Targeting dCas9-KRAB to the Myrf promoter (Pro, by G5 in panel A), EC1 (by 5 in panel B), EC2 (by 4 in panel B), or EC1&2 (by 5 and 4 in panel B, respectively) led to a significant drop in Myrf expression. *p value < 4.6 × 10⁻¹⁰ by two-sided unpaired Student’s t test corrected by the Bonferroni procedure (comparison with Scr1). (D) Epigenome editing analysis was repeated for two negative control regions, NC1 and NC2. The mean and standard error for 8 and 7 sgRNAs that tile NC1 and NC2, respectively, are shown. *p value < 2.4 × 10⁻³ by two-sided one sample Student’s t test corrected by the Bonferroni procedure (n = 8).