Fig. 4: Identifying essential cluster of enhancers with redundant functions.
a, Illustration of a cluster of enhancers regulating gene transcription. Created with BioRender.com. b, Top: Venn diagram indicating the overlapping clusters identified by GSEA and RRA. Bottom: plot showing the ranking of the individual enhancers from the top 3 clusters in the original screening analysis. The y axis represents the MAGeCK RRA score. The x axis represents the ranking of the individual enhancers based on the RRA score. R1, R2 and R3 represent the three individual potential enhancers on the respective chromosomes (chr) targeted by the dual-CRISPR libraries c, Enhancer activities were measured by luciferase assay. Individual putative enhancer elements from the top 3 enhancer clusters were cloned by PCR into the enhancer reporter plasmid pGL4.23 with a minimal promoter. The empty pGL4.23 plasmid was used as the control for the baseline luciferase activities. The y axis represents the relative unit of luciferase activity compared to that of pGL4.23 empty plasmid (n = 3 independent biological samples; bars show mean ± s.d.; chr6R1 NSP = 0.6800, chr6R2 NSP = 0.1147, chr6R3 NSP = 0.4200, chr10R1 NSP = 0.3176, chr10R2 NSP = 0.1312, chr10R3 NSP = 0.0721, chr22R1 ***P = 0.0006, chr22R2 **P = 0.0017, chr22R3 *P = 0.0122, calculated using two-tailed unpaired t-test). d–f, Top: illustration of one-enhancer (d), two-enhancer (e) and three-enhancer (f) removal from the cluster. Bottom: cell proliferation assay was performed by mixing the KO cell lines with cells expressing GFP at a 1:1 ratio. The changes in GFP percentage were monitored at indicated time points by FACS. Cells with dual-CRISPR guide RNAs targeting GFP sequences served as negative controls (Ctrl_KO). The y axis represents the relative ratio of the GFP-negative cells to the positive cells. The ratio of cells in the initial mixture was set as 100%. R1, R2 and R3 represent the three individual potential enhancers on chr22 targeted by the dual-CRISPR libraries (n = 3 independent biological samples; values are shown as mean ± s.d.; R1_KO NSP = 0.7047, R3_KO NSP = 0.3443, R2_KO *P = 0.0348 and R1 + R3_KO, R1 + R2_KO, R2 + R3_KO, All_KO ****P < 0.0001, calculated using two-way ANOVA). g, Top: epigenetic signatures surrounding the chr22 enhancer cluster. The RNA Pol II ChIA-PET loops are shown. The red arrow indicates the location of gene SLC25A1. Bottom: transcription of SLC25A1 in different enhancer KO clones was quantified by qPCR (n = 3 independent biological samples; values are shown as mean ± s.d. for each bar; R1_KO **P = 0.0066, R2_KO **P = 0.0075, R3_KO ***P = 0.0001, R1 + R2_KO *P = 0.0195, R2 + R3_KO **P = 0.0015, R1 + R3_KO **P = 0.0062 and All_KO **P = 0.0025, calculated using two-tailed unpaired t-test).
