Fig. 1: Identification of the KLF5 enhancer region by in vitro enChIP-seq.

a List of regions that bind to the KLF5 promoter by in vitro enChIP-seq analysis. The regions of the top five highest P scores on chromosome 13 are shown. First line shows the KLF5 promoter region including the target region of two gRNAs. The obtained reads from NGS were mapped to the human hg19 reference genome using the COBWeb algorithm. Peak calling was performed using the MACS peak detection algorithm at default settings on Strand NGS software version 3.4 (Agilent Technologies, Santa Clara, CA, USA). The peaks were determined using gRNA-A and gRNA-B as biological duplicates against gRNA-NC (negative control) filtered for P score (−log10(P values)) and fold change ≥2.0. b Integrative genome viewer (IGV) tracks of in vitro enChIP-seq peaks (gRNA-A, gRNA-B, gRNA-NC) and ChIP-seq peaks of H3K27ac and DNase-seq peaks from the ChIP-Atlas database in HT29 cells. The region surrounded by a dotted square is the rank #3rd peak listed in (a). Scissors indicate the position of the gRNAs to create the deletion mutants. c Schematic illustration of the KLF5 enhancer candidate region deleted by the CRISPR/Cas9 system. The positions of the primers for validation are indicated. All candidate clones of the deletion mutant were validated by PCR. Gel images show the PCR product amplified by the indicated primers (Enh-F1 and Enh-R2 or Enh-F1 and Enh-R1) in parental cells and five heterodeletion mutants. d Sequencing result of PCR products that are amplified by Enh-F1 and Enh-R2 primers in five heterodeletion mutants. The deleted region is surrounded by a square and the predicted sequence after deletion is shown as ‘Predicted’. e Expression level of KLF5 mRNA in five deletion mutants. The relative value is calculated by the expression level of the parental cells. The average value of five heterodeletion mutants is also shown. *P < 0.01.