Figure 4: circHIPK3 is derived from HIPK3 exon2 due to long flanking introns.

(a) Schematics showing that the genomic regions of HIPK3 Exon2 contain flanking Alu repeats and long introns. Alu elements and long introns were deleted using CRISPR/Cas9 systems. Primers flanking the gRNA recognition were used to detect deletions. (b) Schematic diagram of circHIPK3 expression vectors with various genomic sequences for circHIPK3 recapitulation (#1–4). The genomic region of circHIPK3 was cloned into the pcDNA3.0 vector with the upstream and downstream intron sequences, which included Alu elements (#1). Deletions were introduced into the HIPK3 expression plasmid. (#2–4). (c,d) Northern blot and qRT–PCR for circHIPK3 in cells transfected with the expression plasmids (#1–4). (e,f) Normal PCR and qRT–PCR data for CRISPR/Cas9-mediated genomic deletions in HEK-293 T cells. Two pairs of gRNAs (gRNA1+gRNA2, gRNA3+gRNA4) were used to mediate the deletion of the proximal Alu elements. M: 100 bp DNA ladder. (g) Normal PCR and qRT–PCR data for large deletion mediated by four sets of paired gRNAs. The primer site was designed outside the deleted region. For the control gRNA, the expected product was not shown because of too large PCR product. For the loading control, a genomic region from Actin gene loci was amplifed in e–g. Data in d–g are the means±s.e.m. of three experiments. *P<0.05, **P<0.01 (Student’s t-test).