Fig. 6

Verification of the regulatory effect of rs559943616 by reporter gene assays and CRISPR-Cas9-mediated genome editing. a,b SNP rs559943616 disrupts POLR2A and CTCF binding. c The 1 kb sequence surrounding SNP rs559943616 is marked with DNase-Seq, ChIP-Seq, and histone modification signals, indicating that rs559943616 is located in an actively transcribed genomic region in neuronal cells. d Reporter gene assays validated the regulatory effect of rs559943616. The G allele of rs559943616 conferred significantly higher luciferase activity than the GGA allele in SH-SY5Y and U251 cells. e–h CTCF knockdown resulted in significant downregulation of CRHR1-IT1, DND1P1, and LRRC37A4P, indicating that these genes are regulated by the CTCF. i–l CRISPR-Cas9-mediated genome editing revealed that deletion of the genomic region containing rs559943616 led to significant expression changes of LRRC37A4P, DND1P1, CRHR1-IT1. i Electrophoresis showed that the given genomic region containing rs559943616 was deleted. WT indicates that the length of the DNA fragments containing rs559943616 is 987 bp in wild-type cells. KO indicates that the length of the DNA fragments containing rs559943616 is 437 bp in edited cells. N = 8 for the control group, n = 16 per experimental group for SH-SY5Y and U251 cells, n = 3 per group in e–h, j–l. Two-tailed Student’s t test was used for statistical analyses. *P < 0.05, **P < 0.01, ***P < 0.001