PAM-flexible Cas9-mediated base editing of a hemophilia B mutation in induced pluripotent stem cells

Peripheral blood samples from a healthy donor and a patient with severe hemophilia B were collected after obtaining written informed consent, and experiments were approved by the ethical committee of Jichi Medical University (approved number: 18-18). iPSCs were generated as previously described with minor modifications²². Peripheral blood-derived mononuclear cells were cultured in StemFitAK02N (Ajinomoto Healthy Supply Co., Inc. Tokyo, Japan) without Supplement C, containing 50 ng/mL of human interleukin (IL)–6 (BioLegend, San Diego, CA, USA), 100 ng/mL of human stem cell factor (BioLegend), 10 ng/mL of human thrombopoietin (BioLegend), and 100 ng/mL human Fms-related tyrosine kinase ligand (BioLegend) for 3 days. Mononuclear cells were then transduced with Sendai virus vectors expressing reprogramming genes (CytoTune-iPS 2.0, ID Pharma Co., Tokyo, Japan). Four days after transduction, cells were harvested and seeded on plates coated with Corning Matrigel hESC-Qualified Matrix (Corning Inc., Corning, NY, USA) and cultured in StemFitAK02N. The medium was changed every other day until colonies appeared. Colonies were mechanically dissociated and seeded onto Matrigel-coated dishes. Established iPSCs were maintained in StemFitAK02N on plates coated with Matrigel or iMatrix-511 (Matrixome Inc., Osaka, Japan).

HEK293 cells were maintained in DMEM (Merck SA., Darmstadt, Germany) containing 10% fetal bovine serum (FBS; ThermoFisher Scientific, Waltham, MA, USA), GlutaMAX^TM Supplement (ThermoFisher Scientific), and penicillin–streptomycin solution (FUJIFILM Wako Pure Chemical, Osaka, Japan).

cDNAs for SpCas9, SpCas9-NG, and SpCas9 (D10A) or SpCas9 (D10A) conjugated with the cytidine base editor PmCDA1 from sea lamprey (TAID) were provided by Prof. Nishimasu and Prof. Nureki (The University of Tokyo)²¹. Cytidine base editor APOBEC1 (BE4)-GAM²³ plasmid (#100806) was obtained from Addgene (Watertown, MA, USA). Plasmids were constructed using routine molecular cloning methods and confirmed by DNA sequencing.

Transduction to iPSCs was performed using P3 Primary Cell 4D-Nucleofector X Kit S (Lonza, Basel, Switzerland) as per the manufacturer's instructions. Briefly, 4 × 10⁵ cells were suspended in a Nucleofector solution mix with 7 µg of plasmids. Cell suspension mixtures were then transferred into cuvettes, and the nucleofector process was initiated using a 4D-Nucleofector core unit (Lonza, Basel, Switzerland). After electroporation, cells were seeded on iMatrix-511-coated wells and cultured in StemFitAK02N with 10 µM Y-27632 (FUJIFILM Wako).

To establish HEK293 cells stably expressing a human F9 cDNA, the linearized plasmid (pBApo-Neo-EF1α, Takara Bio, Shiga, Japan) expressing F9 cDNA (R338L, R338L+I316T patient mutation) was transfected into the cells with Lipofectamine 3000 Reagent (ThermoFisher Scientific), according to the manufacturer's instructions. G418 (400 µg/mL) (Nacalai Tesque Inc., Kyoto, Japan) was added to the culture medium to obtain stably expressed clones.

Genomic DNA was extracted using the DNeasy Blood & Tissue kit (QIAGEN, Venlo, Netherlands). PCR fragments were amplified using ExTaq DNA polymerase (Takara Bio), then denatured and re-annealed using a thermal cycler. Genomic mutations were detected using the Surveyor Mutation Detection Kit (Integrated DNA Technologies, Skokie, IL). DNA fragments were analyzed by agarose gel electrophoresis. The oligonucleotide primer sequences used to detect mutations are described in Supplementary Table . 1

Genomic DNA fragments were amplified using Prime Star MAX polymerase (Takara Bio) and purified using AMPure XP beads (Beckman Coulter, Brea, CA). A library was prepared to add adapters and a barcode sequence to the amplicon target. PCR amplicons were subjected to 250 or 300 pair-end read sequencing using Illumina MiSeq (100,000 reads) at Research Institute for Microbial Diseases at Osaka University (Osaka, Japan) or Hokkaido System Science (Hokkaido, Japan). The primer sequences are described in Supplementary Table . 1

The immunohistochemical staining of iPSCs was performed to confirm the undifferentiated state²⁴. Human iPSCs cultured in 12-well plates were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) at 4 °C for 30 min and permeabilized with PBS containing 0.1% Triton X-100. Sections were blocked with 5% skim milk in PBS containing 0.1% Tween-20 (PBS-T) for 30 min at 4 °C. Cells were stained with anti-NANOG mouse monoclonal antibody (10 µg/mL, clone 23D2-3C6; BioLegend) and anti-OCT4 mouse monoclonal antibody (2.5 µg/mL, clone 3A2A20; BioLegend) at 4 °C overnight. Antibody binding was detected using anti-mouse IgG conjugated with Alexa Fluor 594 (2 µg/mL, ThermoFisher Scientific). Sections were mounted in VECTASHIELD Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA). Immunofluorescence staining was observed by using an all-in-one microscope (BIOREVO BZ-9000, KEYENCE, Tokyo, Japan).

For the immunohistochemistry of HEK293 cells, the cells were fixed with 4% paraformaldehyde for 20 min at room temperature and were washed with PBS containing 0.1% Tween-20. The cells were permeabilized with PBS containing 0.3% Triton X-100 and 2% goat serum for 1 h, and they were subsequently incubated with goat anti-human FIX polyclonal antibody conjugated with biotin (10 µg/mL, GAFIX-AP, Affinity Biologicals, Ancaster, ON, Canada) and rabbit anti-Giantin polyclonal antibody (1 µg/mL, Poly19087; BioLegend) overnight at 4 °C. Next, the binding antibodies were visualized using streptavidin conjugated with Alexa Fluor 594 and anti-rabbit antibody conjugated with Alexa Fluor 488 (10 µg/mL, ThermoFisher Scientific) for 1 h at room temperature. Slides were mounted with VECTASHIELD mounting medium with DAPI. Immunofluorescence staining was observed and photographed using a confocal microscope (Leica TCS SP8; Leica Microsystems, Wetzlar, Germany).

Cells were suspended in PBS containing 0.5% bovine serum albumin and 2 mM EDTA and incubated with R-Phycoerythirin-conjugated anti-human SSEA-4 monoclonal antibody (2 µg/10⁶ cells, clone MC-813-70; BioLegend), and Alexa Fluor 488-conjugated anti-human Tra-1-60-R monoclonal antibody (2 µg/10⁶ cells, clone TRA-1-60-R; BioLegend). Cells were resuspended using the BD Cell Viability kit (2 µg/10⁶ cells, BD Bioscience, Franklin Lakes, NJ). The surface expression of target proteins in living cells was analyzed on an LSRFortessa X-20 (BD Bioscience). FCS files were obtained using Diva software and re-analyzed using FlowJo software (BD Bioscience).

NOG (NOD.Cg-Prkdcscid II2rgtm1Sug/Jic) and TK-NOG [NOD.Cg-Prkdcscid Il2rgtm1SugTg (Alb-UL23)7-2/ShiJic] mice were purchased from In-Vivo Science Inc. (Tokyo, Japan). Knock-in (KI) mice expressing human F9 cDNA without or with the hemophilia B mutation (I316T) [C57BL/6-F9^{tm1.1(hF9_R338L)} and C57BL/6-F9^{tm1.2(hF9_R338L_I316T)}]were developed by UNITECH (Chiba, Japan). Briefly, a linearized targeting vector containing a human F9 minigene (exon 1, truncated intron 1, and exon 2–8) (R338L mutation or R338L+I316T mutations), SV40 polyA sequence, and PGK-Neo cassette with 5′ arm (4.3 kb) and 3′ arm (2.3 kb) was transduced into mouse embryonic stem cells (ESGRO Complete™ Adapted C57/BL6 mouse embryonic stem cell line, Merck, Darmstadt, Germany) by electroporation. The embryonic stem cells were then injected into mouse blastocysts after confirming gene targeting by Southern blotting. Chimeric mice were bred with C57BL/6 mice to produce F1 mice and then bred with CAG-FLPe transgenic mice to delete the PGK-Neo cassette. All animals were housed in groups of a maximum of five mice in individually ventilated cages under specific pathogen-free animal facilities. Dark/light cycles were 12 h/12 h with a temperature of 23 °C and humidity of 50%. All experiments were performed in compliance with the Institutional Animal Care and Concern Committee at Jichi Medical University, and animal care was carried out in accordance with the committee's guidelines.

iPSCs (1 × 10⁶ cells) were injected into the testis of immunodeficient NOG male mice (6-week-old). At 12–17 weeks post-injection, mice were anesthetized with isoflurane and perfused with PBS. Tumor tissues were then fixed with 10% formalin. Paraffin-embedded tissue sections were dewaxed using xylene and rehydrated using ethanol and water. Sections were processed for hematoxylin and eosin staining and then visualized using an all-in-one microscope (BIOREVO BZ-9000, KEYENCE).

Hepatic cell lineage differentiation was performed in vitro as previously described with minor modification^25,26. iPSCs were dissociated into single cells using TrypLE-Select (ThermoFisher Scientific) and plated onto Matrigel Growth Factor Reduced Basement Membrane Matrix (Corning). Cells were maintained in StemFitAK02N for a few days until 80% confluent. Endoderm differentiation was initiated using RPMI1640 medium supplemented with 50 ng/mL of human WNT3A (R&D systems, Minneapolis, MN), 100 ng/mL of human Activin A (BioLegend), and B-27 Supplement (ThermoFisher Scientific) for 4 days. Induction of hepatocyte differentiation was performed by culturing the endoderm cells in RPMI1640 supplemented with 20 ng/mL of fibroblast growth factor 4 (BioLegend), 20 ng/mL of bone morphogenetic protein 4 (BioLegend), and B-27 Supplement for 5 days, followed by culture in RPMI1640 supplemented with 20 ng/mL of human hepatocyte growth factor and B-27 Supplement in hypoxic conditions using Hypoxia Chamber (STEMCELL Technologies Inc., Vancouver, BC, Canada). Cells were further cultured for 11 days in Hepatocyte Culture Media (Hepatocyte Culture Media Bullet kit, Lonza, Basel, Switzerland) supplemented with 20 ng/mL of human Oncostatin M (FUJIFILM Wako Pure Chemical).

Engraftment of differentiated iPSCs was performed using TK-NOG mice²⁶. Eight-week-old TK-NOG male mice were injected intraperitoneally with 6 mg/kg ganciclovir at 8 and 6 days prior to transplantation. Differentiated iPSC-derived cells (2 × 10⁶) were suspended in ice-cold Hepatocyte Culture Media (Lonza) and Matrigel Growth Factor Reduced Basement Membrane Matrix and then transplanted in the subrenal capsule of TK-NOG mice anesthetized with isoflurane. Mouse kidneys were harvested 12 weeks after transplantation, and mRNA expression of F9 was analyzed. For the detection of the FIX antigen in engrafted tissue via immunohistochemistry, tissue samples were fixed with 4% paraformaldehyde, incubated with PBS containing sucrose, and then frozen in Tissue-Tek O.C.T. Compound (Sakura Fintek Japan, Tokyo, Japan). The sections were incubated with 2% goat serum and 0.3% Triton X-100 and further treated with Ready Probes Streptavidin/Biotin Blocking Solution (Invitrogen, USA). The sections were then incubated with goat-human FIX polyclonal antibody conjugated with biotin (10 µg/mL, GAFIX-AP, Affinity Biologicals) overnight at 4 °C. The antibody binding was detected with streptavidin conjugated with Alexa Fluor 594 (10 µg/mL, ThermoFisher Scientific). Slides were mounted with VECTASHIELD mounting medium with DAPI. Immunofluorescence staining was observed with a Leica SP8 confocal microscope (Leica Microsystems).

Total RNA was isolated using the RNeasy Mini Kit (QIAGEN). Human liver RNA was purchased from Takara Bio. cDNA was synthesized using the PrimeScript RT-PCR Kit (Takara Bio). Quantitative polymerase chain reaction (PCR) was performed using THUNDERBIRD Probe qPCR Mix (Toyobo, Osaka, Japan) or THUNDERBIRD SYBR qPCR Mix (TOYOBO) on QuantStudio 12K Flex (ThermoFisher Scientific). Reactions were analyzed in duplicate and expression levels were normalized to GAPDH mRNA levels. Predesigned primer and probe sets were used for the experiments (TaqMan Gene Expression Assays, ThermoFisher Scientific, AFP: Hs00173490, ALB: Hs00910225, CYP3A4: Hs00604506, HNF4A: Hs00230853, F9: Hs01592597, GAPDH: Hs02758991). The predesigned primers for SYBR qPCR were purchased from Takara Bio (F9: HA166488 GAPDH: HA067812).

The medium of confluent cells was changed into a fresh medium containing 5 μg/mL of menatetrenone (Eisai, Tokyo, Japan), and the culture was continued for the following 48 h. Blood sample was drawn from the jugular vein of anesthetized mice with a 29G micro-syringe (TERUMO Corp., Tokyo, Japan) containing 1/10 (volume/volume) sodium citrate. FIX activity (FIX:C) was measured with a one-stage clotting-time assay using an automated coagulation analyzer (Sysmex CA-1600 analyzer; Sysmex, Kobe, Japan). The FIX antigen (FIX:Ag) was measured as follows: microtiter plates were incubated with PBS containing anti-human FIX antibody (CL20039AP; CEDARLANE, Ontario, Canada) (1 μg/mL) overnight at 4 °C. After blocking with 5% casein in PBS for 1 h at room temperature, samples were incubated in PBS containing 1% casein and 0.1% Triton X-100 for 1 h at 37 °C. After washing with PBS containing 0.1% Triton X-100, the bound FIX antigen was detected with the anti-human FIX antibody conjugated with horseradish peroxidase (Affinity Biologicals) using ABTS microwell peroxidase substrate (Seracare, Milford, MA, USA).

The SpCas9 cannot be packaged in one AAV vector; thus, we employed an intein-mediated split-Cas9 system²⁷. The SpCas9-base editor was expressed by two AAV vectors. To express the target gene specifically in hepatocytes, we created a plasmid that contains a chimeric promoter (HCRhAAT; an enhancer element of the hepatic control region of the Apo E/C1 gene and the human anti-trypsin promoter), cDNAs, and the SV40 polyadenylation signal. The AAV genes were packaged by triple plasmid transfection of AAVpro293T cells (Takara Bio) to generate the AAV vector⁴. We used the AAV8 serotype, as it efficiently expresses the target gene in the mouse liver in vivo. The AAV vector was quantified by qPCR measuring the SV40 polyadenylation signal. The primer pairs and probe were as follows:

5'-AGCAATAGCATCACAAATTTCACAA-3' (sense)

5'-CCAGACATGATAAGATACATTGATGAGTT-3' (anti-sense)

5'-AGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTC-3' (FAM probe)

For the in vivo base-editing experiments, we administered two AAV vectors expressing the N- and C terminal of SpCas9-base editor intraperitoneally (10 µL) in neonatal mice (1 × 10¹¹ viral genomes).

GUIDE-Seq was carried out as previously described, with minor modifications²⁸. HEK293 cells were transfected with a plasmid expressing SpCas9 or SpCas9-NG and gRNA3 for 7 days together with dsODN. Genomic DNA was isolated, and then the libraries were prepared from 400 ng of genomic DNA using the NEBNext Ultra II FS DNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA) according to the manufacturer's instructions. Next, they were subjected to 150 pair-end read sequencing using the Illumina NovaSeq platform (50,000,000 reads) at the Genome Information Research Center at Osaka University. Data analysis was performed using the GUIDE-Seq analysis pipeline following standard procedures²⁹. The primer sequences are described in Supplementary Table 1, and the in silico predicted off-target sites are listed in Supplementary Data 1.

Statistical analyses were performed using Prism 9 software (Graph Pad Software, San Diego, CA, USA). All data are presented as mean ± standard error of the mean (SEM). Each data was obtained from biologically independent experiments (Supplementary Data 2). The sample number of the experiment was indicated in each figure legend. When indicated, statistical significance was determined using a two-tailed Student's t-test or one-way ANOVA with post hoc Tukey's multiple comparison test. All P-values <0.05 were considered statistically significant.

Further information on research design is available in the linked to this article. Nature Portfolio Reporting Summary

We found three possible PAM sites at the 3′ end of the mutation and designed three guide RNAs (gRNAs) to repair the mutation by a base-editing approach (Fig. 1b). We then inserted each gRNA downstream of the U6 promoter in the plasmid harboring wild-type SpCas9 or SpCas9-NG driven by CAG promoter (Fig. 1c). Using Surveyor endonuclease assay, we assessed a DSB near the mutation site induced by the expression of each gRNA and Cas9 after transduction of patient-derived iPSCs with each plasmid. SpCas9 induced DSBs with gRNA2 (TGG PAM) but not with gRNA1 (TGG PAM) or gRNA3 (CGA PAM). (Fig. 1d and Supplementary Fig. 1). In contrast, SpCas9-NG induced DSBs with all three gRNAs, albeit a low efficiency with gRNA1(Fig. 1d and Supplementary Fig. 1). We quantified the frequency of DSBs induced by Cas9 with gRNA2 and gRNA3 by next-generation sequencing. SpCas9 induced DSBs at the TGG PAM site with gRNA2 more efficiently than SpCas9-NG (SpCas9, 8.72 ± 1.29%; SpCas9-NG, 3.16 ± 0.71%) (Fig. 1e). Notably, SpCas9-NG, but not SpCas9, induced DSBs at the CGA PAM site with gRNA3 efficiently (SpCas9, 0.21 ± 0.04%; SpCas9-NG, 6.55 ± 2.13%) (Fig. 1e). These results confirmed the broad PAM flexibility of SpCas9-NG in iPSCs, as observed in HEK293 cells. To evaluate off-target events in the whole genome, we performed GUIDE-Seq in HEK293 cells transducing the plasmid expressing SpCas9 or SpCas9-NG and gRNA3. The peak score was significantly higher for SpCas9-NG at one guide RNA mismatch, which corresponds to the patient's point mutation (Supplementary Fig. 2a,b and Supplementary Data 1). We could not detect any difference between the other scores and influenced genes of SpCas9 and those of SpCas9-NG (Supplementary Fig. 2a, b and Supplementary Data 1).

We next cloned iPSC colonies after the transduction of the patient-derived iPSCs with plasmids harboring SpCas9-NG-TAID with gRNA3. We detected 84 colonies and selected one that had the gene correction at the mutation (Fig. 2c). The isolated gene-corrected iPSCs could be maintained in an undifferentiated status since the cells expressed pluripotency markers (NANOG, OCT4, SSEA-4, and Tra-1-60) (Fig. 2d). Furthermore, the gene-corrected iPSCs developed teratomas, representing differentiation into three germ layers, in NOG mice (Fig. 2e).

Supplementary Materials Description of Additional Supplementary Files Supplementary Data 1 Supplementary Data 2 Peer Review File Reporting Summary