Fig. 1

CRISPRi-mediated activation of the Ovalbumin promoter in DF1 cells. A The schematic representation of the promoter and coding region of the OVA gene in DF1 cells. Two regulatory elements of SDRE and NRE are shown in the distal promoter. The bottom panel shows binding sites for two guide RNAs (Silencer-gRNA and CAR-gRNA) that bind the silencer and CAR regions in the NRE element, respectively. 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; dCas9, Catalytically dead Cas9. The enlarged inset in the lower section of panel A shows the location and orientation of PAM regions and protospacers for the two regulatory regions of ‘silencer’ and ‘CAR’. B The left panel shows agarose gel electrophoresis for analysis of the RT-PCR products which were amplified by primers P8 and P9 (for OVA, Fig. 2A and Table 1). The right panel shows agarose gel electrophoresis for analysis of the RT-PCR products which were amplified by P10 and P11 (for GAPDH, Table 1). RNA was extracted from DF1 cells which were transfected with CRISPRi vectors that target the NRE element at CAR, Silencer, both CAR and silencer sequences, and pdCas9-X as the negative control. The expected amplicon size for OVA was 179 bp, and for GAPDH was 187 bp. WT, wild-type; Magnum, hormonally-activated tissue of magnum from  a 35-week-old laying hen; M, DNA size marker; NTC, no template control. C Upregulation of the OVA mRNA in CRISPRi-modified DF1 cells was assessed by RT-qPCR. Upon transfection with CRISPRi vectors that target the NRE element at CAR, Silencer, and both CAR and silencer sequences, an increment in the OVA gene expression level was determined. The transcript levels for OVA in the hormonally-activated tissue of the magnum (from a 35-week-old laying hen) show the highest level of expression. The gene expression ratio for the OVA over GAPDH was calculated by the Pfaffl method of relative quantification [38]. For each group of CRISPRi-DF1 cells, three biological replicates were used. Each biological replicate was read as three technical replicates. The Mann–Whitney assay was used to analyze significant statistical differences between groups. The asterisk (*) indicates statistical differences with p values < 0.05