FIGURE 7.

Deletion of +22.6 kb cis-regulatory element (CRE) does not alter cystic fibrosis transmembrane conductance regulator (CFTR) expression in definitive endoderm (DE) or anterior foregut endoderm (AFE) cells. A, UCSC Genome Browser view of +22.6 kb CRE CRISPR/Cas9-mediated deletion schematic and sequencing alignments of two homozygous deletion clones. B, C, Reverse transcription quantitative PCR (RT-qPCR) of B., orthodenticle homeobox 2 (OTX2), GATA4, SOX17, SOX9, and PAX6; C, CFTR and cortactin binding protein 2 (CTTNBP2) in WT and +22.6 kb deleted DE and AFE cells. WT data combine CWRU205 WT, and two nontargeted clones; +22.6 kb deletion data combine two homozygous deletion clones. Expression is shown relative to DE WT, normalized to PGK1, n = 3. D, RNA-seq DEseq2 normalized counts in ND2.0 and CWRU205 in DE and AFE stages. **** denotes P < .0001, ** denotes P < .01, and * denotes P < .05 using a two-way analysis of variance (ANOVA) and Sidak’s multiple comparisons test. Not significant is not shown