Extended Data Fig. 2: CRISPR-Cas9 editing of the chr21q22 locus and ETS2 in monocytes.
From: A disease-associated gene desert directs macrophage inflammation through ETS2
a. Cas9 gRNAs were designed to flank the chr21q22 enhancer region at the indicated sites. b. Representative bioanalyzer trace of PCR-amplified target region following monocyte CRISPR/Cas9 editing with an equimolar mix of RNPs containing 5′ and 3′ chr21q22 gRNAs. Example editing efficiency calculation shown. c. Editing efficiency at the chr21q22 locus. Mean enhancer deletion: 42.4% (n = 11). d. Location and sequence of gRNAs used to disrupt ETS2. e. ETS2 editing efficiency. gRNA1 (mean), 89.7% (n = 31); gRNA2 (mean), 78.6% (n = 14). f. ETS2 expression (relative to NTC) following CRISPR/Cas9 editing, measured by qPCR (housekeeping gene PPIA; equivalent results with other housekeeping genes; n = 10). g. Viability following monocyte nucleofection with Cas9 RNPs and macrophage differentiation. Mean values: NTC, 97.9%; gRNA1: 98.3%; gRNA2, 98.6% (n = 6). h. Expression of myeloid lineage markers following ETS2 editing and TPP differentiation (n = 5). Gating strategy shown in Supplementary Information Fig. 2. i. GSVA enrichment scores for 67 different monocyte/macrophage activation conditions to identify stimuli that phenocopy CD14+ monocytes/macrophages from IBD patients. j. Chromatin accessibility in ETS2-edited versus unedited inflammatory macrophages (n = 3). k. Enhancer activity (H3K27ac) in ETS2-edited versus unedited inflammatory macrophages (n = 3). P values calculated using edgeR (two-sided) in j, k. Red points denote adjusted P-value (Padj) < 0.1, grey points NS. Error bars are mean±SEM in c, e-h. * P < 0.05. NTC: non-targeting control.
