We searched for the target site of CRISPR/Cas9 on porcine platelet-derived growth factor receptor alpha (PDGFRA, Gene ID: 100627123) using the online tool CHOPCHOP (https://chopchop.cbu.uib.no/) [20]. Four target sequences were selected for two genomic sites on PDGFRA (two target sequences for each genomic site; Table 1). The genomic sites were inserted into the cloning site of the pCAG-EGxxFP vector. Oligos for the target sequences were dimerized by slow cooling from 95°C to 25°C, followed by insertion into the guide RNA sequence of the pX330 vector (oligo sequences in Table 2). All vectors were verified by nucleotide sequencing.

Basic cell culture and lipofection were carried out following procedures in our previous report [14]. Briefly, EGxxFP and px330 vectors were introduced into porcine fetal fibroblasts at a 1:1 ratio using Lipofectamine 3000 Reagent (Thermo Fisher Scientific, Waltham, MA, USA). We replaced culture media with fresh modified Dulbecco’s modified eagle’s medium (DMEM) with 10% fetal bovine serum 48 hours after lipofection, followed by culture for 2 days. To identify the efficiency of the target sequences, the enhanced green fluorescent protein signal was measured in each sample.

The ovaries of prepubertal gilts were obtained from a local slaughterhouse and transferred to the laboratory within warmed saline. Cumulus-oocyte complexes (COCs) were collected by aspirating 3- to 7-mm follicles of prepubertal gilts with a 10-mL syringe and an 18-gauge needle. COCs with compact multiple layers of cumulus cells and fine cytoplasm were collected from aspirated porcine follicular fluid (pFF) and allowed to mature for 44 hours in tissue culture medium 199 (TCM 199) (Gibco, Grand Island, NY, USA) supplemented with 10% pFF, L-cysteine (0.1 mg/mL), sodium pyruvate (44 ng/mL), epidermal growth factor (10 ng/mL), insulin (1 mg/mL), and kanamycin (75 μg/mL) at 39°C. The COCs were matured with 10 IU/mL gonadotropin hormones, pregnant mare serum gonadotropin (Lee Biosolutions, Maryland Heights, MO, USA) and human chorionic gonadotropin only for the first 22 hours. After maturation, cumulus cells were removed from the oocytes with hyaluronidase. Sperm cells were washed twice with Dulbecco’s phosphate buffered saline (DPBS) supplemented with 0.1% bovine serum albumin (BSA) at 1,400 rpm for 3 minutes. Washed sperm (4×10⁴/mL in final concentration) were coincubated with the matured oocytes in 500 μL of modified tris-buffered medium (mTBM) for 4 hours [21]. mTBM consisted of 113.1 mM sodium chloride, 3 mM potassium chloride, 7.5 mM calcium chloride, 20 mM Trizma base, 11 mM glucose, 5 mM pyruvate, 1 mM caffeine, and 0.8% BSA. After this process, eggs were incubated in 5% CO₂ and 5% O₂ at 39°C in 20 μL of porcine zygote medium 3 (PZM3) [22]. The cleavage rate was measured on day 2 after insemination. AG1296 (146535-11-7; Sigma, USA) was dissolved with DMSO and diluted in PZM3.

We used commercial Cas9 mRNA (Thermo, USA) and custom synthesized gRNAs. The microinjection procedure was conducted with a microscope (Eclipse TE2000; Nikon, Tokyo, Japan) and micromanipulator (Narishige, Tokyo, Japan) with holding and injection pipettes. We used Femtotip II (Eppendorf, Hamburg, Germany) as an injection pipette. The concentrations of Cas9 RNA and each gRNA were 20 ng/μL and 10 ng/μL, respectively, following a previous report [23]. RNA was injected into fertilized eggs at 2 hours after IVF.

Embryos were washed twice with DPBS supplemented with 0.1% BSA and fixed with 4% paraformaldehyde in DPBS at room temperature (RT) for 15 minutes. Fixed embryos were permeabilized using 0.2% Tween-20 and 0.2% Triton X-100 in DPBS at RT for 15 minutes, followed by blocking with 10% donkey serum in DPBS at RT for 1 hour. Samples were stained with anti-SOX2 (5 μg/mL) and anti-SOX17 (1 μg/mL) in DPBS containing 10% donkey serum at 4°C overnight. After washing 3 times in washing solution (DPBS with 0.2% Tween-20 and 1% BSA for 10 minutes), embryos were incubated with donkey anti-rabbit Alexa594 or donkey anti-goat Alexa488 (Invitrogen, Waltham, MA, USA; 1:5,000) in DPBS with 10% donkey serum at RT for 1 hour. For double staining, samples were stained again with primary and secondary antibodies. The steps were the same, but primary antibody treatment was conducted at RT for 2 hours. All samples were washed 3 times with washing solution after secondary antibody treatment. Immunostained embryos were mounted on a glass slide with Prolong Gold and 4′, 6-diamidino-2-phenylindole (DAPI) (Invitrogen, USA) and cured for more than 24 hours. We described the list of antibodies in Table 3. The digital imaging system for microscope (DS-L1; Nikon, Japan) was used to obtain fluorescence and bright-field images. We used the ImageJ program for images.

Embryonic RNA was extracted by an Arcturus PicoPure RNA Isolation Kit (Applied Biosystem, Waltham, MA, USA) following the standard manual. cDNA was synthesized from the total RNA of a single embryo with a High-Capacity RNA-to-cDNA Kit (Applied Biosystems, USA) following the standard protocol. Power SYBR Green PCR Master Mix (Applied Biosystems, USA) was used to run quantitative PCR of cDNA samples following the standard manual of the vender. The levels of the transcripts were normalized to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression level by 2^−ΔΔCT method [24]. The list of primers is described in Table 2.

Statistical analysis of the data was performed using GraphPad Prism Software (version 5.01; San Diego, CA, USA). Significant differences among experimental groups were determined by one-way analysis of variance followed by Tukey’s multiple comparison test, and unpaired t test was used for the binomial data. A p-value <0.05 was considered significant. Data are presented as the mean±the standard error. The number of embryos used for each experiment is described in Supplementary Table S1.

We designed four gRNA sequences to target two different gDNA sites of PDGFRA (Figure 1A). To evaluate the activity of sgRNAs, modified pCAG-EGxxFP vectors were transfected with px330 vectors containing sgRNA sequences. sgRNAs showed their activity via expression of EGFP (Figure 1B). From the four sequences, we chose sgRNAs #1-2 and #2-1, which are predicted to have higher efficiency and lower off-target effects than #1-1 and #2-2 (Table 1). mRNA of Cas9 and sgRNA #1-2 and #2-1 were injected into IVF embryos. Injection of RNAs showed no significant effect on the developmental competency of embryos (Figure 1C). To evaluate SOX17- and SOX2-positive populations in embryos, ICC and DAPI staining were conducted with day 7 blastocysts (Figure 1D). The total cell number did not show a significant difference between control and RNA-injected embryos (Figure 1E). The numbers of SOX2-positive cells and SOX2-&SOX17-positive cells were reduced, but there was no significant difference in the number of SOX17-positive cells (Figure 1F). In the scatter plot of SOX17-positive cell numbers, 9 of 12 RNA-injected embryos (Group A) showed significantly fewer populations than control embryos (Figure 1G). In view of the ratio of total cells, cells that were positive for both SOX17 and SOX2 were present in lower numbers than the control (Figure 1H). However, when we counted the ratio only within the ICM, only the SOX2 ratio showed a significant difference (Figure 1I).

AG1296 was applied to parthenogenetic activated (PA) embryos. Treatment with AG1296 did not affect cleavage, morula, blastocyst formation, or hatched blastocyst rate. Within embryos that developed to blastocysts stage, hatching rate was reduced with AG1296 (Figure 2A). At concentrations of 0 to 20 μM, no significant difference was observed in the total number of embryonic cells (Figure 2B). In the ICC, SOX17- and SOX2-positive cell populations were identified (Figure 2C). In the case of cell number, SOX17-positive cells were significantly decreased with 5 and 10 μM AG1296 (Figure 2D). In total cells, the ratio of cells expressing SOX17 was reduced with 5, 10, and 20 μM AG1296 (Figure 2E). Within ICM cells, AG1296 suppressed SOX17-positive cells and number of SOX2-positive cells was increased (Figure 2F).

We also investigated the effect of AG1296 on IVF embryos. Similar to PA embryos, the hatching rate within blastocysts was decreased with AG1296 (Figure 3A). The number of total cells was not affected by AG1296 treatment (Figure 3B, 3C). In the ICC experiment, AG1296 significantly suppressed the number of SOX17-positive cells (Figure 3D and 3E). However, the ratio of marker-positive cell populations was not significantly different with AG1296 treatment (Figure 3F), and ratio of SOX17 and SOX2-positive cells in ICM did not show any significant difference (Figure 3G). In the qPCR analysis of PDGF downstream genes, Txnip was upregulated in 4-cell embryos of the treatment group (Figure 3H), but the other genes (Zfnad5, Myo1e, Sgpl1, Tiparp, Csrnp1, Plekha1) did not show any difference. However, no significant difference was observed in late blastocysts (Figure 3I). Furthermore, the expression level of Txnip increased 20-fold in the morula and decreased in early blastocysts treated with AG1296 (Figure 3J).