Fig. 2

Design and validation of the targeted deletion of Ovalbumin distal promoter elements in DF1 cells. A The schematic representation of CRISPR/Cas9 mediated deletion strategy of the OVA promoter in DF1 cells. The top diagram shows the wild-type (WT) chicken OVA locus. The two guide RNA (SDRE-gRNA and NRE-gRNA) binding sites are shown. The NRE- and SDRE- gRNAs target two positions downstream of NRE (downstream of CAR) and upstream of SDRE, respectively. The bottom diagram shows the locus after CRISPR-mediated deletion of the distal OVA promoter in DF1 cells (DF1^{+/OVA Pro ∆ cell}). The PCR primers used for the assessment of deletion (P5 to P7), and the OVA gene expression (P8 and P9, used in Figs. 1 and 3) are shown as small red arrows. B Two-step genomic PCR to confirm the deletion of the distal promoter of the OVA gene. In the first PCR (using P5 and P7 primers, Table 1), an amplicon of 1310 bp was amplified from the wild-type (WT) allele (In the first PCR, amplicon of ~ 370 bp were not detected from the promoter-deleted (DF1^∆) alleles). In a hemi-nested PCR (using P5 and P6 primers), amplicons of 1256 bp and ~ 316 bp were amplified from the wild-type and promoter-deleted (DF1^∆) alleles, respectively. C Alignment of the representative sequences of the wild-type (WT DF1) and promoter-deleted (DF1^∆) sequences determined by Sanger sequencing. The gRNA-binding sites are shown in blue, and the PAM regions are shown in green letters. WT, wild-type; DF1 ^∆, DF1 cells knockout for the distal OVA promoter (DF1 ^{+/OVA Pro ∆}); NHEJ, non-homologous end-joining; ERE, estrogen-responsive enhancer element; TSSL, tissue-specific silencer-like element; SDRE, steroid-dependent regulatory element; NRE, negative regulatory element; CAR, COUP-adjacent repressor site; COUP, Chicken OVA upstream promoter; TATA, TATA box; TSS, transcription start site; P, primer. M, DNA size marker; NTC, no template control