Extended Data Fig. 2: NCREs regulate their surrounding genes to exert their functions. | Nature Biomedical Engineering

Extended Data Fig. 2: NCREs regulate their surrounding genes to exert their functions.

From: Genome-wide Cas9-mediated screening of essential non-coding regulatory elements via libraries of paired single-guide RNAs

Extended Data Fig. 2

a, TADs identified by Hi-C surrounding PAX6_Ta are shown, and the location of PAX6_Ta is indicated by the vertical blue bar. Horizontal yellow and blue bars indicate distinct TADs. Transcription of three nearby genes within the same TAD as PAX6_Ta was quantified by qPCR (n = 3 biological independent samples; bars show mean value ± s.d.; ****P < 0.0001, calculated using unpaired t-test). b, RNA-seq was performed to identify differentially expressed genes around NCRE PAX6_Ta from PAX6_Ta_KO cells versus K562 WT cells. The x axis represents the coordinates of genes surrounding PAX6_Ta ranging from - 5 Mb to + 5 Mb. The y axis shows the -log10(FDR) of nearby genes by DESeq2. The dashed horizontal line indicates the FDR cutoff of 0.05, and the vertical line indicates the location of PAX6_Ta. Each dot represents one gene, the red dots represent significantly up-regulated genes, blue dots represent significantly down-regulated genes, and the gray ones are either non-significant genes or genes with |log2FC | <1. c, CRISPR knockout was used to silence the PTPRD gene in de_novo_1_KO#1 cells. Sanger sequencing of gRNA targeting region within the exon region of PTPRD gene (chr9:8314246-8733946, hg19) showed a 4-nucleotide-deletion leading to a frameshift mutation, compared to the original de_novo_1_KO#1. d, Growth effect of PTPRD gene knockout in de_novo_1 KO clone (de_novo_1_KO#1 + PTPRD_KO) compared to the de_novo_1 KO clone (de_novo_1_KO#1). 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. K562 cells served as control. 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%. (n = 3 independent biological samples; values are shown as the mean ± s.d.; *P = 0.0472, **P = 0.0061, ***P = 0.0008, calculated using two-way ANOVA).

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