Figure 3.

CRISPR−Cas9 genome editing of enhancer region 2 on 20q13.33. (a) Chromosome view of the section of putative enhancer region 2 targeted by CRISPR−Cas9 genome editing technique in the UCSC Genome Browser. The total region (∼2 kb) represents a part of putative enhancer region 2, containing the candidate functional SNP rs3761124, amplified by PCR (using Forward and Reverse primers). The region highlighted in gray represents region (∼0.5 kb) targeted by CRISPR gRNAs (gRNA1 and gRNA2). The 1.5‐kb band in DNA gel electrophoresis demonstrates the targeted deletion of putative enhancer region 2, containing SNP rs3761124, in LN‐229 (b) and U‐87 MG cells (c). Cas9, cells transfected with Cas9 only (no guide RNAs); Cas9+ control, cells transfected with Cas9 vector and gRNA empty vector; Cas9+ target, cells transfected with Cas9 vector and guide RNA target vectors; NC, mock‐transfected parental cells; PCR, polymerase chain reaction; SNP, single‐nucleotide polymorphism; UCSC, University of California Santa Cruz