Extended Data Fig. 9: CRISPR-Cas9 deletion of the rs17713054 enhancer.
From: Identification of LZTFL1 as a candidate effector gene at a COVID-19 risk locus
a, ENCODE DNase I-seq in HUVEC and IMR-90 cells and ATAC-seq in Blood Outgrowth Endothelial Cells (BOECs) and H441 epithelial cells showing the rs17713054 containing enhancer with schematic of generated deletions and short guide RNA (sgRNA) binding sites. b, Example D1000 trace of genotyping PCR product amplified from cells transfected with Cas9 protein only, Cas9 protein with sgRNA1 + 2 (∆108), or Cas9 protein with sgRNA1 + 3 (∆191). c, Example Sanger sequencing trace following ICE analysis over the sgRNA1 and sgRNA2 binding sites in unedited cells, and the double strand break repair site in cells containing the 108 bp deletions. sgRNA sequence shown by black boxes, protospacer adjacent motif sites shown with red letters. d, Calculated deletion efficiency for each sgRNA pair and cell type. Transfections failing to achieve >70% deletion (blue circles) were excluded from expression analyses. n shown are for independent transfections e, Expression of LZTFL1 normalized to RPS18 and expressed as relative to the mean expression in Cas9 only treated cells for each cell type. Corrected P values from an ordinary one-way ANOVA with Dunnett’s multiple comparisons test. n shown are for independent samples from at least 3 independent transfections. For d,e bars show mean and one standard deviation. f, ChIP-seq for the active transcription marker (H3K27ac) was performed in umbilical vein endothelial cells (HUVECs), blood outgrowth endothelial cells (BOECs), H441 lung epithelial cells, and IMR-90 lung fibroblast cells. The rs17713054 enhancer (grey box, g) lacks strong modification under standard growth conditions in these cells.
