We previously generated an iPSC line (HA-iPSCs) derived from the urine cells of a HA patient with F8 Inv1 (Hu et al., 2015). Here the HA-iPSCs were identified via immunofluorescence. The HA-iPSCs maintained pluripotency according to immunofluorescence. The HA-iPSCs expressed Oct4, Nanog and SSEA-4, while SSEA-1 wasn’t expressed (Supplementary Figure S1A). To further evaluate the pluripotency in vivo, HE staining of teratomas was performed and the results showed that teratomas contained ectoderm, endoderm and mesoderm (Supplementary Figure S1B). Meanwhile, the HA-iPSCs maintained a normal karyotype (Supplementary Figure S1C).

Inv1 of F8 splits the F8 gene into two parts and disrupts the F8 transcription, resulting in no FVIII protein production. The part which contains exon 2-exon 26 covers 98% of F8 coding region. So the Inv1 mutation could be rescued by gene addition of a promoter and the coding sequences of exon 1 (Figure 1). We then designed six single-guide RNAs (sgRNAs) F8-sg1, F8-sg2, F8-sg3, F8-sg4, F8-sg5, and F8-sg6 mapping to target sites in intron 1 (Figure 2A) and constructed and verified the cleavage activity via T7 Endonuclease I (T7E1) (Figure 2B). The cleavage frequency of F8-sg4 was 56.53% and was used for targeted addition. The donor plasmids were designed (Figure 2C), constructed and verified with Sanger sequencing (Figure 2D).

The HA-iPSCs were nucleofected with the plasmids expressing the CRISPR/Cas9 complex and F8-sg4 along with the donor plasmid F8-NHEJ, F8-HDR, F8-HMEJ, respectively. The single-cell clone was screened using primers across homology arms 5F/R (Figure 3A) and 3F/R (Figure 3B), and the sequences were verified via Sanger sequencing (Figure 3C). The targeting efficiency was 10.19% with the donor plasmid F8-HMEJ, higher than that in the F8-HDR group (6.25%) and the F8-NHEJ group (0.99%) (Table 1). Two targeted addition clones (T-26 and T-73) generated from the HA-iPSCs were then used for further research. The primers 5F and 3R were used to detect the purity of the single-cell clone (Figure 3D). The immunofluorescence showed that T-26 and T-73 maintained pluripotency (Figure 3E). The HE staining of teratomas further confirmed the pluripotency in vivo (Figure 3F), and T-26 and T-73 maintained a normal karyotype (Figure 3G). To evaluate the off-target effect of CRISPR/Cas9, the potential off-target sites (≤4 mismatches) of F8-sg4 predicted using CHOPCHOP (http://chopchop.cbu.uib.no/) were amplified and sequenced. No off-target indels in the potential off-target sites were observed comparing the sequences in HA-iPSCs with that in T-26 and T-73 (Supplementary Figure S2).

Considering a modified hAAT promoter was used in the gene addition, we detected the transcription of the hAAT gene and found that the hAAT gene was transcribed in the iPSCs (Figure 4A). Then the F8 transcription was detected in T-26 and T-73 via reverse transcription PCR (RT-PCR), while no F8 transcript was detected in HA-iPSCs (Figure 4B). The sequencing results revealed that the promoter and the exon one were successfully inserted into F8 in T-26 and T-73 with the F8 transcription restored (Figure 4C), demonstrating the F8 expression was rescued in the gene targeting group.

To evaluate the F8 expression in hepatocyte, we differentiated the HA-iPSCs, the gene-corrected iPSCs T-26 and T-73, the normal hiPSCs (N-iPSCs) into the hepatocyte-like cells (HA-iHPLCs, T-26-iHPLCs, T-73-iHPLCs, and N-iHPLCs) as the diagram in Figure 5A. During the differentiation, the cells went through four stages. The cell morphology was gradually changed from iPSC clone to epithelioid cell morphology (Figure 5B) and identified the cell marker with immunofluorescence. All cells in the first stage expressed the definitive endoderm cell markers SOX17 and FOXA2; 5 days later, the AFP signal was positive in hepatoblast-like cells; after 5 days of culture, followed by 11 days of culture in Hepatocyte Culture Medium (HCM) contained 20 ng/mL oncostatin M (OsM), the hepatocyte-like cells expressed ALB (Figure 5C). In addition, differentiated HPLCs on Day 25 with characteristic functions of mature hepatocytes could store glycogen and metabolize indocyanine green (ICG) (Figure 5D, E).

RT-PCR results indicated that the F8 transcripts were detected in T-26-iHPLCs and T-73-iHPLCs (Figure 6A). The supernatant of HA-iHPLCs, T-26-iHPLCs, T-73-iHPLCs, and N-iHPLCs were collected for FVIII antigen detecting using ELISA. Results showed that human FVIII antigen secreted by T-26-iHPLCs was higher than that in HA-iHPLCs (Figure 6B). And the FVIII coagulation activities in the supernatants from the T-26-iHPLCs were detectable by FVIII activity assay (Figure 6C). More importantly, the FVIII was expressed in T-26-iHPLCs, T-73-iHPLCs via immunofluorescence staining, but no FVIII was detected in HA-iHPLCs due to the interrupted F8 gene (Figure 6D). These results suggest that FVIII expression was restored in gene-corrected iPSCs derived HPLCs.

The expression of iPSC surface markers were detected using immunofluorescence staining. iPS cells were fixed with 4% paraformaldehyde for 15 min, then permeabilized with DPBS (Thermo Fisher Scientific #C14190500BT, Waltham, MA, United States) contained 0.1% Triton-X100 for 15 min, followed by blocking with 5% bovine serum albumin (BSA, Geneview #FA016, St. Galveston, TX, United States) in DPBS for 30 min, then the cells were incubated with primary antibody diluted 1:100 in 5% bovine serum albumin in DPBS (OCT4 (Abcam #ab181557, Cambridge, United Kingdom), NANOG (Abcam #ab109250), stage-specific embryonic antigen (SSEA)-1 and SSEA-4 (Merck Millipore #SCR001, Billerica, MA, United States) at room temperature for 1 h. After washing with DPBS appropriately, cells were blocked again for 30 min and treated with secondary antibodies for 1 h in the dark. The nuclear staining was performed using 4′, 6′-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific #D1306). Then the cells were photographed via fluorescence microscope (Leica DM IRB, Wetzlar, Germany).

For teratoma formation, iPSCs plated on 60 mm dish were digested and resuspended in 140 μL mTeSR1 (STEMCELL Technologies #85850, Vancouver, BC, Canada) with 70 μL Matrigel (Corning #354277, NY, United States). The iPSCs were transplanted into the groins of NSG mice subcutaneously. Eight weeks later, the formed teratoma was harvested, fixed, paraffin embedded, sectioned, stained with hematoxylin and eosin, photographed and analyzed. The care and use of the animals are in accordance with the guidelines of the Ethics Committee of the School of Life Sciences of Central South University. All operations involving animal experiments were approved by the Institutional Animal Care and Use Committee of School of Life Sciences of Central South University (No. 2021-2–12, Date: 12 March 2021).

G-banding analysis of chromosomes was performed. The cells were incubated with 0.1 μg/mL colcemid (Sigma-Aldrich #D7385, St. Louis, MO, United States) for 4 h, followed by trypsinization, and hypotonic treatment with 0.075 M KCl for 10 min at 37°C. Then the cells were fixed with Carnoy fixative, and the metaphase chromosomes were spreaded using an air-drying method, then treated with Giemsa (Sigma-Aldrich #48900) and analyzed.

All short guide RNA oligos which were designed via CRISPOR (http://crispor.tefor.net/) were synthesized by Sangon Biotech. The plasmid pX601 (pX601-AAV-CMV:NLS-SaCas9-NLS-3xHA-bGHpA;U6:BsaI-sgRNA) was a gift from Feng Zhang (Addgene plasmid #61591; http://n2t.net/addgene:61591; RRID:Addgene_61591, Watertown, MA, United States) (Ran et al., 2015). The annealed complementary sgRNA oligos were ligated with the pX601 digested with BbsI (New England Biolabs #R3539, Ipswich, MA, United States) using T4 DNA ligase (Thermo Fisher Scientific #EL0011). To evaluate the activity, HEK293T cells were transfected with CRISPR/SaCas9 expression plasmid using Lipofectamine 2000 (Invitrogen #11668–019, Carlsbad, CA, United States). After 2 days, genomic DNA was extracted. The PCR products encompassing the targeted locus were purified and treated with mismatch-sensitive T7E1 (Vazyme #EN303-01, Nanjing, China). After electrophoresis, gene disruption was evaluated via a gel imaging system.

The donor plasmids NHEJ, HDR, HMEJ were synthesized by Sangon Biotech and were confirmed by Sanger sequencing.

The iPSCs cultured on Matrigel were transfected with the human stem cell Nucleofector kit 2 (Lonza #VPH-5022, Alpharetta, GA, United States) using Nucleofector II (Lonza) set at program B016. 2.5 µg of CRISPR/Cas9 plasmid and the donor plasmid were used to transfect 1×10⁶ cells (HA-iPSCs). After 2 days, 1,000 single transfected cells were seeded on Matrigel coated 6-cm dish in CloneR medium (STEMCELL Technologies #1000691). Approximately 12 days later, clones were picked up and identified by PCR and Sanger sequencing. PCR was performed using two pairs of primers: F8-5F, 5′- CAA​AAT​GAT​ACA​GAA​AGT​AGA​ATG​G-3′ and F8-5R, 5′- CAG​GGA​GGG​CTG​TGT​GTT​T-3'; and F8-3F, 5′- TTT​CTG​AGC​CAG​GTA​CAA​TGA-3′ and F8-3R, 5′- GCT​GTA​ATT​CAG​AAT​CAG​TCC​TAC-3'. The PCR products were sequenced by Sanger sequencing. The N-iPSCs (DYR0100) purchased from ATCC were used as normal control.

The potential off-target sites of sgRNA4 were searched using CHOPCHOP (http://chopchop.cbu.uib.no/) (Montague et al., 2014), seven potential sites were predicted for mismatches of up to four nucleotides. Then the regions encompassing the seven potential sites in gene-edited clones were PCR amplified, followed by Sanger sequencing (Supplementary Table S1). Different indels in HA-iPSCs and genetically edited iPSCs were used to evaluate off-target effects. Primer sequences are shown in Supplementary Table S2.

Total RNA isolated with TRIzol reagent (Sigma-Aldrich #T9424) was digested with DNase for 30 min, followed by reverse transcribed via HiScript II 1st Strand cDNA Synthesis Kit (Vazyme #R212). The primers were based on exons one and four to detect the F8 transcripts. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified to represent an endogenous control. Primer sequences are shown in Supplementary Table S3.

Derivation of hepatocyte from human iPSCs was performed based on Yukiko Toba’s protocol with some modifications (Labun et al., 2019). Briefly, for induction of the definitive endoderm cells, iPSCs were cultured in Differ1 medium (RPMI1640 medium, Hyclone #SH30027, South Logan, UT, United States; 100 ng/mL Activin A, Peprotech #120–14P, Rocky, Hill, NJ, United States; 1 × GlutaMAX, Thermo Fisher Scientific #35050061; 0.2% fetal bovine serum, Thermo Fisher Scientific #16000044, 1 × B27 Supplement Minus Vitamin A, Thermo Fisher Scientific #12587010) for 4 days.

For the hepatoblast-like cells differentiation, the definitive endoderm cells were cultured in Differ2 medium (RPMI1640 medium, 20 ng/mL recombinant human BMP4 (Biolegend #595202, San Diego, CA, United States); 1 × GlutaMAX, and 1 × B27 Supplement Minus Vitamin A) for 5 days.

To perform the hepatocyte differentiation, the medium was replaced with Diff3 medium (RPMI1640 medium, 20 ng/mL HGF, Peprotech #100-39H; 1 × GlutaMAX, and 1 × B27 Supplement Minus Vitamin A) and cultured for 5 days. Then the cells were cultured for 11 days in Hepatocyte Culture Medium (HCM, Lonza #cc3198) containing 20 ng/mL Recombinant Human Oncostatin M (OsM, Peprotech #300–10).

Immunofluorescence staining of hepatocyte was performed as that described for iPSC characterization. The primary antibodies used were anti-SOX17 (R&D SYSTERM #AF 1924, Minneapolis, MN, United States), anti-FOXA2 (Merck Millipore #07–633), anti-FVIII N-terminus antibody (Santa Cruz Biotechnology #sc27649, Dallas, TX, United States), and anti-AFP (Sigma-Aldrich #A8452), anti-ALB (R&D SYSTERM #MAB1455).

For periodic acid schiff stain, the hepatocytes on Day 25 were stained with the Periodic Acid Schiff Stain Kit (Solarbio #G1280, Beijing, China) according to the manufacturer’s instructions. The periodic acid schiff stain was detected by microscopy.

For indocyanine green (ICG) uptake assay, hepatocytes on Day 25 were treated with 1 mg/mL ICG (Sigma-Aldrich #1340009) for 30 min, then washed with DPBS thoroughly and cultured in fresh Hepatocyte Culture Medium. The cells were detected by microscopy. After 12 h, the cells were observed using microscopy.

Culture supernatants of mature hepatocytes from 12-well plates were harvested in triplicate after medium replacement for 24 h. ELISA was performed with paired antibodies for ELISA-Factor VIII:C (Cedarlane #CL20035K, Burlington, ON, Canada) according to manufacturer instructions. The standard curves were constructed using serial dilutions of normal pooled plasma, with a correlation coefficient (R2) greater than 0.990 using a semilog fit.

For the FVIII activity assay, 24-hour-old culture supernatants of mature hepatocytes were collected. The activated partial thromboplastin time (aPTT) was detected using a Destiny Max hemostasis analyzer (Tcoag, Lemgo, Germany) according to the manufacturer’s instructions.

GraphPad Prism 8.0 was used for data analysis. Data were analyzed using ANOVA for more than two groups.