Fig. 6

Validation of the regulatory effect of rs2027349 by reporter gene assays, TF knockdown and CRISPR/Cas9-mediated genome editing. a, b The SNP rs2027349 disrupts CTCF and TAF1 binding. c The 1 kb sequence near the SNP rs2027349 is marked with a strong DNase-Seq (light blue), TF ChIP-Seq (green), and histone modification (purple) signals. d Reporter gene assays showed that the G allele of rs2027349 produced significantly higher luciferase activity than the A allele in all three tested cell lines. e–k CTCF knockdown led to downregulation of ANP32E, TARS2, and RPRD2. However, knockdown of TAF1 resulted in downregulation of TARS2 and upregulation of VPS45. l–o A genomic sequence containing rs2027349 was deleted by CRISPR/Cas9-mediated genome editing, which resulted in downregulation of ANP32E, TARS2, and VPS45 expression. l Electrophoresis showed that the genomic region containing rs2027349 was deleted by the sgRNAs. WT, genomic DNA containing rs2027349 (996 bp) in wild-type cells. KO, genomic DNA containing rs2027349 (473 bp) in edited cells. N = 8 per group for HEK293T cells, n = 8 for the control group, n = 16 per experimental group for SH-SY5Y and U251 cells, n = 3 for each group in (e–k) and (l–o). Two-tailed Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001