The KGN human ovarian granulosa cells were supplied by Professor Yiming Mu of the Chinese PLA General Hospital (Beijing, China) and human embryonic kidney (293T and 293A) cells were obtained from the American Type Culture Collection (Manassas, VA, USA). The KGN cells were incubated in Dulbecco’s modified Eagle’s medium: Nutrient Mixture F-12 (DMEM/F-12; Hyclone; GE Healthcare Life Sciences, Logan, UT, USA), and the 293T and 293A cells were incubated in DMEM (Hyclone; GE Healthcare Life Sciences) supplemented with 10% fetal bovine serum (FBS; Hyclone; GE Healthcare Life Sciences) and antibiotics at 37°C in a humidified atmosphere (Thermo Fisher Scientific, Inc.; Waltham, MA, USA) with 5% CO₂.

Three G (N19) NGG sequences, where N stands for any A, T, G, or C nucleotide, downstream of the FOXL2 translation start site (ATG) were selected as potential target sites with the assistance of the Optimized CRISPR Design website (http://crispr.mit.edu/). The resulting three target sequences were as follows: F404: GCG CGC CGG CTT TGT CAT GAT GG; F425: GGC CAG CTA CCC CGA GCC CGA GG; and F446: GGA CGC GGC GGG GGC CCT GCT GG.

The following primers were then synthesized: F404F, 5′-GAA AGG ACG AAA CAC CGC GCG CCG GCT TTG TCA TG AGTT TTA GAG CTA GAA AT-3′ and F404R, 5′-ATT TCT AGC TCT AAA ACT CAT GAC AAA GCC GGC GCG CGG TGT TTC GTC CTT TC-3′; F425F, 5′-GAA AGG ACG AAA CAC CG G CCA GCT ACC CCG AGC CCG GTT TTA GAG CTA GAA AT-3′ and F425R, 5′-ATT TCT AGC TCT AAA ACC GGG CTC GGG GTA GCT GGC CGG TGT TTC GTC CTT TC-3′; F446F, 5′- GAA AGG ACG AAA C ACC GGA CGC GGC GGG GGC CCTGCG TT T TAG AGC TAG AAA T-3′ and F446R, 5′-ATT TCT AGC TC TAAA ACG CAG GGC CCC CGC CGC GTC CGG TGT TTC GT C CTT TC-3′; CRIF, 5′-GTA TTT CGA TTT CTT GGC TTT ATA TAT CTT GTG GAA AGG ACG AAA CAC CG-3′ and CRIR, 5′-GTT GAT AAC GGA CTA GCC TTA TTT TAA CTT GCT ATT TCT AGC TCT AAA AC-3′.

To construct the F404 gRNA lentivirus plasmid, F404F and F404R were denatured and annealed to act as the template for CRIF and CRIR polymerase chain reaction (PCR) amplification. PCR was conducted using the following parameters: An initial denaturation at 94°C for 3 min, followed by 20 cycles of amplification (94°C for 20 sec, 53°C for 20 sec, and 72°C for 20 sec) with a final extension step at 72°C for 10 min. The PCR products were then inserted into the pLKO.1 plasmid using Gibson master mix according to the manufacturer’s protocol (New England Biolabs, Beverly, MA, USA). The F425 and F446 gRNA plasmids were also constructed in a similar manner. A flag-tagged Cas9 gene was cloned into the pShuttle-CMV plasmid using the BglII and XhoI restriction sites. Following linearizing with PmeI, the pShuttle-CMV-Cas9 was co-transformed with the pAdEasy-1 plasmid into Escherichia coli strain BJ5183.(Stratagene, La Jolla, CA, USA) by electroporation using a Bio-Rad Gene Pulser electroporator (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

To package the gRNA lentivirus, the gRNA plasmid and the packaging plasmids pMD and SPA were co-transfected into 293T cells. At 48 h post-transfection, the medium was harvested and used to infect KGN cells. The recombinant Ad plasmid was linearized with PacI and transfected into AD-293 cells (Stratagene) to produce the adenovirus.

The genomic DNA of KGN cells derived from single colony was extracted using a kit (Tiangen Biotech Co., Ltd., Beijing, China) following the manufacturer’s protocol. The sequence including the target sites was amplified with F296F and F818R primers and purified with a kit (Tiangen Biotech Co., Ltd.). The resulting PCR product was 523 bp. The sequences of the primers were as follows: F296F: 5′-CAT CCG CAG TCT CCA GAA GTT TGA-3′; F818R: 5′-AGG CCG GGT CCA GCG TCC AGT AGT-3′.

Prior to T7 EI digestion, the PCR product was denatured, by placing the reaction tube in a 500-ml beaker of 94°C water, and annealed, by allowing it to cool to room temperature. Following cooling, the PCR product was digested with T7 EI at 37°C for 2 h and then assayed with agarose gel electrophoresis.

Total RNA was extracted and purified with the Takara MiniBEST Universal RNA Extraction kit (Takara Bio, Inc., Tokyo, Japan) according to the manufacturer’s protocol. For cDNA synthesis, 2 μg of RNA was reverse transcribed with PrimeScript RT reagent kit (Takara Bio, Inc.) according to the manufacturer’s protocol. The real-time PCR was standardised using SYBR FAST qPCR mix (Takara Bio, Inc.). The primers were as follows: β-actin, forward 5′-GTC CCT CAC CCT CCC AAA AG-3′ and reverse 5′-GCT GCC TCA ACA CCT CAA CCC-3′; StAR, forward 5′-TGG GCA TCC TTA GCA ACC A-3′ and reverse 5′-GGG ACC ACT TTA CTC ATC ACT TTG T-3′; and CYP19A, forward 5′-TGA GGA TCC CTT TGG ACG AA-3′ and reverse 5′-AAT AAC CTT GGA TTT TAA CCA CGA TAG-3′. qPCR was conducted using the following parameters: An initial denaturation at 95°C for 5 min, followed by 25 cycles of amplification (95°C for 30 sec, 60°C for 40 sec, and 72°C for 1 min) with a final extension step at 72°C for 10 min. The PCR results were quantified using the Eppendorf realplex 4S RTqPCR system. The qPCR data were analyzed with the comparative 2^-ΔΔCq method (25) using β-actin to normalize the expression data. Values are presented as changes relative to the KGN wt values, which were set to 1.

The cells were collected and lysed with buffer [50 mM Tris-HCl, 150 mM NaCl, 1% NP40, 0.1% SDS, 1 mM DTT (pH 7.5)] and protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) and sonicated briefly. Following centrifugation (12,000 × g) at 4°C for 15 min, the supernatant was collected, and protein concentrations were determined using the BCA protein quantification assay kit (Thermo Fisher Scientific, Inc.). The supernatant was boiled in Laemmli buffer and analyzed by western blot analysis; 40 μg of protein homogenates were loaded onto a denaturing 10% sodium dodecyl sulfate-polyacrylamide gel using a Mini Protean apparatus (Bio-Rad Laboratories, Inc.) and transferred onto polyvinylidene difluoride membranes (Merck KGaA, Darmstadt, Germany). The membrane was blocked with Tris-buffered saline containing 5% non-fat dry-milk at room temperature for 2 h, then exposed to primary antibodies overnight at 4°C. Based on the described reactivity, anti-FOXL2 antibody (1:1,000; cat. no. ab188584, Abcam, Cambridge, MA, USA) and anti-flag antibody (1:2,000; cat. no. F2555, Sigma-Aldrich; Merck KGaA) were used for detection of the transfection effectiveness, and anti-StAR antibody (1:1,000; cat. no. sc-166821, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), and anti-CYP19A antibody (1:1,000; cat. no. sc-374176, Santa Cruz Biotechnology, Inc.) were used to evaluate the disruption of FOXL2. Anti-cyclin D1 antibody (1:1,000; cat. no. ab134175, Abcam), anti-cyclin-dependent kinase (CDK)4 antibody (1:1,000; cat. no. ab108357, Abcam), and anti-CDK6 antibody (1:1,000; cat. no. ab124821, Abcam) were used to detect knockout of the cell cycle. Anti-β-actin antibody (1:5,000; cat. no. ab8227, Abcam) was used as a protein loading control. The secondary antibody (anti-rabbit or anti-mouse peroxidase-conjugated IgG, 1:5,000; cat. nos. ab6721 and ab6789, Abcam) was incubated with the membrane for 1 h at room temperature. The reaction products were revealed by enhanced chemiluminescence (ECL Plus; Thermo Fisher Scientific, Inc.). The intensities of the digitally detected bands were evaluated by densitometry using Image-Pro Plus software (Version X; Media Cybernetics, Inc., Silver Springs, MD, USA).

The cells (1×10⁶) were collected following trypsinization and washed twice with PBS. The cells were then stained using the cycle staining kit and Annexin V-FITC/PI apoptosis kit (Multi Sciences Biotech Co., Ltd., Hangzhou, China) according to the manufacturer’s protocol, respectively. In the cell cycle assay, 2×10⁵ cells for each sample were measured with a Becton Dickinson FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed with ModFit software version 3.0 (Verity Software House; Topsham, ME, USA). Cell cycle status was analyzed using propidium iodide (PI) as a specific fluorescent dye probe. For the apoptosis assay, 1×10⁵ cells were measured and analyzed with CellQuest software version 2.0 (BD Biosciences). Annexin V(+)/PI(-) and Annexin V(+)/PI(+) represent the cells in early apoptosis and late apoptosis/necrosis respectively and the proportion of these cells out of the total number of cells analyzed were determined.

Cell proliferation experiments were performed using the xCELLigence Real Time Cell Analyzer (RTCA) DP instrument (ACEA Biosciences, San Diego, CA, USA). The cells were seeded in 16-well plates (E-plate 16 ACEA Biosciences, Inc.) at a density 2×10³ and 4×10³ cells in 150 μl medium per well, and impedance measurements were monitored at regular time intervals. The cell growth curves were automatically recorded with the xCELLigence system in real time. The area of growth covered in an E-plate due to cell adhesion was recorded as the cell index (CI). A high CI indicates more cell adhesion and vice versa. The experiment was allowed to run for 3 days and each experiment was performed in triplicate. Data were analyzed using RTCA software version 1.2.

The cellular viabilities of the FOXL2-knockout and wt KGN cells were determined using two methods: BrdU labelling and a CCK-8 assay. The BrdU labelling assays were performed using the APC-BrdU Flow kit (BD Biosciences, cat. no. 552598) according to the manufacturer’s protocol. Briefly, during the final 1 h of culture, the cells were pulsed with 10 μM of BrdU APC and 7-AAD. The stained cells were analyzed on a flow cytometer using a low flow rate. For the CCK-8 assay, the cells were seeded in 96-well plates at a density of 1×10⁴ cells per well and incubated for 24 h. The cells were then treated with different concentrations of FBS (0, 2, 5 and 10%) for 24 h. Following treatment, CCK-8 (10 μl/well) was added to the wells and the cells were incubated at 37°C for 1 h. Subsequently, the OD value was we detected at 450 nm (A450) using a microplate spectrophotometer.

The experimental data are expressed as the mean ± standard deviation. For statistical analysis, Student’s t-test for quantitative data was processed with Excel version 2007 (Microsoft Corporation, Redmond, WA, USA). P<0.05 was considered to indicate a statistically significant difference. Error bars represent the standard deviation of at least three independent experiments.

Three potential G (N19) NGG target sites were screened just downstream of the translation start site. All three gRNA sequences were cloned into the pLKO.1 lentivirus vector, and were confirmed by sequencing (Fig. 1A). In addition, as shown in Fig. 1B, gRNA lentivirus infection did not alter the expression of FOXL2. The gRNA lentivirus-infected KGN cells were further infected with flag-Cas9 adenovirus and the expression of Cas9 was monitored by anti-flag western blot analysis (Fig. 1C).

To assess the efficacy of Cas9-mediated genome editing, the T7 EI digestion of PCR products from the genomic DNA of control and infected KGN cells was analyzed by agarose gel electrophoresis. As shown in Fig. 2A, the PCR products derived from the double infected cells were cut to produce bands of the predicted size, whereas the PCR products of control cells remained almost intact. Quantitative analysis revealed that the cells infected with F425 gRNA were most efficiently edited by Cas9 (Fig. 2B). The luciferase reporter assay results also confirmed that F425 had the highest efficiency of the three targets (Fig. 2C).

A total of 16 F425 single cell clones were then screened and the genomic DNA of the cells was extracted to perform T7 EI digestion following PCR amplification. As shown in Fig. 3A, the majority of the PCR products from different clones were digested by T7 EI, which included clones 1, 3, 9, 10, 11, 12, 13, 14, and 16. The results of the western blot analysis further indicated that clone 3 had been fully disrupted at the FOXL2 locus (Fig. 3B). To further investigate whether FOXL2 has been completely disrupted in clone 3, the sequence that included the target sites was cloned into the T vector and 50 positive clones were sequenced. The sequencing results showed only two genotypes of the target sites, one of which lacked five base pairs and the other lacked four base pairs (Fig. 3C and D).

To further rule out the potential disruption of FOXL2, StAR and CYP-19A, two major reported downstream modulators of FOXL2, were measured. Previous reports have identified that FOXL2 positively regulates StAR and negatively regulates CYP19A (10). As expected, the results showed that StAR was markedly elevated and CYP19A was markedly decreased in clone 3 at the mRNA (Fig. 3E) and protein (Fig. 3F and G) levels. Therefore, the results confirmed that FOXL2 had been successfully knocked out in clone 3 using the CRISPR/Cas9 method.

To further investigate whether the physiological activities of KGN cells were influenced by FOXL2 knockout, proliferation, cell cycle and apoptosis assays were been performed. The viability assay results using CCK-8 detection showed that, in the presence of different concentrations of FBS (0, 2, 5 and 10%), the proliferation rate of clone 3 was significantly inhibited, compared with that in the wt cells (Fig. 4A). In addition, the proliferation rate inhibition in clone 3 was confirmed by the xCELLigence RTCA assay (Fig. 4B). However, no significant changes were observed in total apoptosis following the knockout of FOXL2 (Fig. 4C and D). Therefore, the results indicated that FOXL2 knockout mainly attenuated the proliferation of KCN cells.

The present study then measured the effects of FOXL2 knockout on cell cycle. The results showed that the percentage of cells in the G0/G1 phases in clone 3 cells, which lacked FOXL2, was significantly enhanced, whereas the percentage in the S phase was decreased; this suggested that proliferation was inhibited through cell cycle arrest at G0/G1 following FOXL2 knockout (Fig. 5A and B). The BrdU labelling assays with flow cytometry also confirmed that the clone 3 cells had enhanced G0/G1 phases (Fig. 5C and D). In addition, the results of the western blot analysis demonstrated that the expression of cyclin D1 and CDK4 were upregulated whereas that of CDK6 was downregulated (Fig. 5E and F). This suggested that the knockout of FOXL2 arrested cell cycle progression at the G0/G1 phase. Therefore, the results indicated that knocking out FOXL2 in KGN cells inhibited the proliferation of cells and promoted cell cycle arrest at the G0/G1 phase.