2. MATERIALS AND METHODS

2.1. Plasmid construction

The plasmids expressing Cas9 and sgRNAs targeted to ovalbumin distal promoter (px459-14 & px459-15) and that targeted to ovalbumin exon 2 (px459-6) were designed using the CRISPR design tool (http://crispr.mit.edu/) and then generated by routine subcloning techniques (Table 1). The donor vector for CRISPR HDR (homology-directed repair) (pHD_4520) was generated by ligating a PCR-amplified, 556 bp DNA ovalbumin fragment (beginning of the exon 2) as the 5′ homology arm, and a PCR-amplified, 526 bp DNA ovalbumin fragment as the 3′ homology arm (Table 1).

2.2.Targeted deletion of ovalbumin promoter in cultured DF1 cells

DF1 cells were received as a generous gift from Dr. Bertrand Pain (The Stem cell and Brain Research Institute, Lyon, France), and cultured as recommended by the ATCC. In order to perform CRISPR excision of ovalbumin promoter with dual sgRNAs, DF1 cells were transfected with px459-14 and px459-15 (Table 1) using Lipofectamine 3000 (Invitrogen, USA), as described previously (Abu-Bonsrah, Zhang, & Newgreen, 2016). Briefly, 0.5 μg from each of px459-14 and px459-15 plasmids were diluted with 50 μl OPTI-MEM + GlutaMax (Thermo Fisher Scientific, USA) and was mixed with 50 μl OPTI-MEM + GlutaMax containing 1 μl Lipofectamine 3000 reagent and then incubated with 0.1–0.15 × 106 DF1 cells for 4 hours. Subsequently, the cells were cultured in 500 μl of an antibiotic-free DMEM-F12 culture medium (Thermo Fisher Scientific) and incubated for 24 h at 38°C in a 7.5% CO2 environment. The transfectd DF1 cells were exposed to puromycin dihydrochloride (2.5 μg/ml; Sigma-Aldrich, USA) for 3 days. DF1 cells after antibiotic exposure were expanded for 2 to 3 weeks. A mixed population of these cells was subjected to genomic PCR to confirm the deletion of ovalbumin distal promoter in a fraction of cells. After single-cell isolation and clonal expansion, 3 clones of distal ovalbumin promoter knockout DF1 cells (DF1 +/OVA Pro ∆) were acquired that showed correct deletion in the ovalbumin distal promoter by PCR. These 3 clones were analyzed for the expression of ovalbumin by RT-qPCR (see below).

2.3.Confirmation of the ovalbumin distal promoter deletion

Genomic DNA was extracted from wild-type and distal ovalbumin promoter knockout DF1 cells (DF1 +/OVA Pro ∆) using the Genomic DNA Extraction Kit (DENAzist Asia Co., Iran). Gene-targeting events were detected by a single-round or nested PCR using the designed primers (Table 2) and Taq DNA polymerase master mix RED (Ampliqon, Denmark), and Sanger sequencing of the amplicons (Genomin Co., Iran).

2.4.Analysis of Ovalbumin expression in DF1 cells with the deletion of distal ovalbumin promoter

Total RNA was isolated from magnum tissue (from a 35-week old laying hen), wild-type, and distal ovalbumin promoter knockout DF1 cells (DF1+/OVA Pro ∆) using the Total RNA Isolation Kit (DENAzist Asia, Iran). The quality and quantity of the extracted RNA were evaluated using gel electrophoresis and a 2000 Nanodrop spectrophotometer (Thermo Scientific, USA). One μg of total RNA was reverse transcribed using random hexamer primers and MMLV reverse transcriptase (Thermo Fisher Scientific, USA). To quantify the level of transcripts for Ovalbumin and Gapdh genes, quantitative RT-PCR reactions containing 1x SYBR Green Real-time PCR Master Mix (Thermo Scientific, USA), 2 μl cDNA template and each primer (Table 2, Supplementary Figure 3) at 250 nM in a 20 μl reaction volume, were carried out in a Rotor-Gene Q real-time PCR cycler (Qiagen, USA). Amplification steps were: 95°C for 15 min, followed by 35 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 30 s. To derive the melting curve, the temperature was increased in steps of 0.2°C for 5 s from 55°C to 95°C. To confirm the identity of products, PCR-amplified bands after clean-up and reaction recovery (DENAzist Asia Co., Iran), were subjected to Sanger sequencing (Genomin Co., Iran).
To adjust the reaction temperature, to find the best concentration of primers, and to optimize the amplification and melting curves (Supplementary Figure 1A), qPCR reactions were repeated. The identity of qPCR products (Supplementary Figure 1B) was confirmed by Sanger sequencing (Genomin Co., Iran). Complementary DNA from the magnum of the 35-week-old hen was serially diluted and subjected to qPCR to make standard curves (Supplementary Figure 2). Each dilution was subjected to the real-time readings in triplicate. Then, the log10 of cDNA concentration for the Ovalbumin and Gapdh genes were plotted against the cycle threshold (Ct) numbers to make a standard curve (Supplementary Figure 2). To calculate the reaction efficiency, the slope of standard curves was used in the equation of E = (10–1/slope-1) × 100%. Both standard curves were linear in the analyzed range with an acceptable correlation coefficient (R2) (Supplementary Figure 2). Gene expression ratio for the Ovalbumingene over Gapdh was calculated for the magnum, wild-type DF1, and DF1 cell with deletion of distal ovalbumin promoter using the Pfaffl method of relative quantification (Pfaffl, 2001).

2.5.Targeted reporter knockin in DF1 cells with the deletion of distal ovalbumin promoter

DF1 +/OVA Pro ∆ cells were transfected with px4596 and pHD_4520 (donor reporter vector) using Lipofectamine 3000 (Invitrogen, USA), as described above. The cells 48 hr after transfection were subjected to antibiotic selection with puromycin dihydrochloride (2.5 μg/ml; Sigma-Aldrich, USA). To confirm knockin of the reporter construct (DsRed2-CMV-Puro-IRES-EGFP), genomic PCR and Sanger sequencing (Genomin Co., Iran) were performed. Cells with the inserted reporter and deleted ovalbumin promoter (DF1 +/OVA Pro ∆-Tg (promoterless dsRed)) were observed and photographed by fluorescence microscopy (Nikon Eclipse Ts2R, Japan) 2 weeks after transfection.