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J. Chen et al.
not completely clear.14,15 No single animal model responds to all the requirements for studying every aspect of a dis- ease. Classical hemophilic mice and dogs have greatly accelerated the exploration of the pathophysiology and treatment of hemophilia.14-16 The advantages of hemophilic mice are availability, fast reproduction, and easy handling. They have proved to be instrumental in screening the phar- macokinetic and pharmacodynamic properties of potential drugs and gene therapeutics prior to other animal models of hemophilia.17,18 However, studies with hemophilic mice allow only small-volume blood samples and a limited fre- quency of blood sampling. Moreover, spontaneous bleed- ing rarely occurs in hemophilic mice, and intervention is necessary in certain studies.16,19,20 For decades, therapeutic efficacy in hemophilic dogs has proven to be an excellent predictor of human clinical efficacy.16 In addition, hemo- philic dogs are frequently used to validate the effects of gene therapy.21-23 They show the spontaneous bleeding phe- notype; however, the relatively infrequent occurrence of clinically recognizable spontaneous joint bleeding currently hampers any in-depth study of hemophilic arthropathy.16 Pigs are an excellent animal model for understanding the pathogenesis of human disease and developing therapeutic strategies.24-26 They are similar to humans in anatomy, phys- iology, and genome. Therefore, we decided to generate HB pigs that potentially provide their own unique study oppor- tunities. Moreover, CRISPR/Cas9-mediated homology- directed gene targeting was used to determine whether it could ameliorate the bleeding phenotype by site-specific gene insertion.
Methods
Animal studies and ethics statements
All animal studies and the breeding process were carried out in accordance with guidelines approved by the Animal Welfare and Research Ethics Committee of Jilin University. All invasive proce- dures were performed under inhalation anesthesia with 1.5% isoflurane. The wild-type (WT) controls used in our study were age- and sex-matched.
Plasmid construction
pX330-U6-Chimeric_BB-CBh-hSpCas9 was a gift from Feng Zhang (Addgene plasmid #42230).27 Two paired oligonucleotides, which were designed on the basis of porcine F9 sequences, were ligated to the pX330 plasmid to form single guide RNA (sgRNA)- expressing vectors. The coding sequences (CDS) of human F9, two homologous DNA arms of porcine F9 sequences and the pLB vec- tor (TIANGEN, VT205) were assembled together to generate the donor vector. Details of the methods used are available in the Online Supplementary Methods.
Cell culture and selection
The constructed sgRNA-expressing vectors and the donor vec- tor were transfected via electroporation into cultured porcine fetal fibroblasts (PFF). Fibroblasts were isolated from 33-day old fetuses of Large White pigs. The transfected cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco, USA) supplemented with 15% fetal bovine serum (Gibco) at 39°C in an incubator with 5% CO2. The positive PFF were selected by the limiting dilution method. Details are given in the Online Supplementary Methods.
Somatic cell nuclear transfer and embryo transfer
The positive cells were pooled as donor cells for somatic cell
nuclear transfer (SCNT).28 Briefly, a single donor cell was microin- jected into an enucleated pig oocyte. Then, the reconstructed embryos were activated and transferred into the synchronized recipient pigs. We performed ultrasonography 30 days post trans- fer and obtained cloned pigs by eutocia.
Quantitative real-time polymerase chain reaction and western blot analysis
Standard protocols were used for RNA and protein isolation, quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis. Details are given in the Online Supplementary Methods.
Clinical observations
An 8-week clinical observational recording process was initiat- ed after the piglets were born. The pigs were examined daily, and the number and type of bleeding episodes were observed, with a particular focus on joints.
Blood sampling and blood analysis
A detailed description of the blood sampling and blood analysis is provided in the Online Supplementary Methods.
Radiography
Radiographs of the pig limbs were obtained using a digital X- ray imaging system (PaxScan 4343R, Varian; Palo Alto, CA, USA).
Histological assessment
The ankle joints and livers from 2-month old pigs were placed in 4% formaldehyde for fixation. The joints were decalcified for 3 weeks before tissue processing and paraffin embedding. The liver sections were stained with Hematoxylin & Eosin (HE). The ankle joints were stained with HE and Safranin O. After examination by light microscopy, HE-stained sections were scored for the pres- ence of synovitis, and Safranin O stains were scored for cartilage degradation. The score was based on a modified grading system of hemophilic synovitis;29 scoring criteria are shown in Online Supplementary Table S1.
Statistical analysis
The data from the experiments were analyzed with GraphPad Prism software (t-test). P<0.05 was considered statistically signifi- cant.
Results
Generation of porcine F9 knockout pigs and human F9 knockin pigs
Two sgRNAs were specifically designed to target the porcine F9 gene using CRISPR/Cas9 (Figure 1A), consider- ing the following: (i) a computational BLAST tool was used to analyze the selected sgRNAs to ensure that they are unique in the porcine genome, reducing the risk of off- target gene editing; (ii) to produce a non-functional coagu- lation factor IX protein, the target sites were selected at the 5’ end of the F9 gene; and (iii) the target sites con- tained mutations known to cause HB, based on the human FIX database (http://www.factorix.org/). We trans- fected male and female fetal pig fibroblasts with the two Cas9/sgRNA plasmids separately, 118 fetal pig fibroblasts (88 males; 30 females) were screened by PCR and we identified 13 positive cell clones that contained 11 male clones and two female clones (Online Supplementary Figure S1). We selected some positive clones for SCNT, and the
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