Construction of the CRISPR/Cas9 system
The CRISPR/Cas9 single guide RNAs (sgRNAs) for rabbit CBS were designed using http://crispr.mit.edu based on its sequence (Genbank: NW_003160195.1; http://www.ncbi.nlm.nih.gov/). Three CBS-targeting sgRNAs were screened (sgRNA1 sited E8: g.6631-6650, sgRNA2 sited E8: g.6637-6656 and sgRNA3 sited E8: 6641-6660) (Figure 1). The detailed protocol has been described previously [41, 42]. Single-stranded oligodeoxynucleotide donor templates (ssODN) with silent mutations were designed and synthesized by Sangon as follows: GGCTCCATCCTGGCGGAGCCGGAGGAGCTGAACCAGACGGAGGTGACGGCCTAtGAaGTaGAaGGtATCtcCTACGACTTCATCCCCACCGTGCTCGACCGGACGGTGTGTGGGCCCCAG.
Zygote injection with Cas9/sgRNAand embryo transfer
New Zealand White rabbits (6-8 months old) were housed at the Animal Genetic Engineering Laboratory of Yangzhou University under a 12 h diurnal/nocturnal cycle, and fed twice a day with free access to water. All protocols were approved by the Care and Use of Laboratory Animals (Ministry of Science and Technology of the People’s Republic of China) and the Animal Care and Use Committee of Yangzhou University, Yangzhou, China (license number: SYXK(Su)2017-0044).
Female rabbits were superovulated by intramuscular injection of follicle-stimulating hormone (SANSHENG, NingBo, China) twice daily. The dosage was 15 IU for the first two injections, 10 IU for the next two and 5 IU for the last two injections. After the final injection, both the superovulated and recipient females were injected with 100 IU human chorionic gonadotropin (HCG), and the former were mated with male rabbits. Approximately 18-20 hours post-coitus, the female rabbits were anesthetized with pentobarbital (2%, 20mg/kg, i.p.), and the pronuclear zygotes were extracted and transferred into M2 medium (M7167, Sigma, USA) supplemented with 10% fetal bovine serum (FBS; SH30084.03, HyClone, USA). Following cytoplasmic injection of 40 ng/μl Cas9 mRNA, 10 ng/μl of an sgRNA and 25 ng/μl ssODN, the embryos were transferred to complete (with 10% FBS) M16 medium (M7292, Sigma, USA) and incubated at 38°C under 5% CO2 for 30-60 minutes. Approximately 15-20 embryos were transferred to one recipient female.
Genomic DNA was extracted from ear biopsies via phenol-chloroform extraction, and the CBS sequences were amplified using specific primers (Table 1). The amplified products were extracted and purified from the gel using a PCR Purification kit (EP101-01, TransGen, China). The sequences were then cloned into pGEM-T vector (A1360, Promega, USA), and sequenced by Lasergene (DNASTAR Inc., U.S.).
To determine any off-target effects, the CRISPR design tool (http://tools.genomeengineering.org) was used to predict potential sites homologous to the 23-bp sgRNA + PAM sequence across the rabbit genome. These sites were amplified in the genomic DNA of founder CBS-KO rabbits and sequenced. The off-target sites and primer pairs are listed in Supplementary Tables 1 and 2 (Table S1 and Table S2).
Two weeks-old CBS-KO and WT rabbits were infused daily with 2.5 mg/kg vitamin B6, 25μg/kg vitamin B12, 45μg/kg folate and 25 mg/kg betaine via the gastric route. The animals were acclimatized to the gentle restraint and handling, and infused daily from 10 am to 12 noon for 4 weeks by one technician.
Four milliliters peripheral blood was collected into EDTA-coated tubes from each animal after withholding food for 10–12h. Plasma was separated by centrifuging the blood at 3000 rpm at 4°C. The levels of triglycerides (TG), total cholesterol (TC), and high-density (HDL-C) and low-density lipoprotein cholesterol (LDL-C) were measured using specific kits (A110–1, A111–1, A112–1 and A113–1; Nanjing Jiancheng Bioengineering Institute, Nanjing, China). In addition, Hcy levels were measured using an ELISA kit (Laier Biotechnology Co. Ltd., Hefei, China).
The expression levels of the apolipoproteins (Apo) B, E and A-I in the plasma were analyzed by Western blotting as per standard protocols. The protein bands were probed with the goat anti-ApoE (600–101-197) and anti-ApoB (600–101-111; both from Rockland Inc., Limerick, PA, US), and sheep anti-ApoA-I (0650–0180, Bio-Rad AbD Serotec, Kidlington, UK) antibodies. The secondary antibodies were HRP-conjugated donkey anti-goat IgG (Jackson Immuno Research Laboratories, West Grove, PA, US) and donkey anti-sheep IgG (Chemicon, Temecula, CA, US).
The CBS-KO and WT littermates were maintained under similar conditions, and fed according to their growth stage. The body weight of the rabbits were monitored from birth till 6 weeks of age. After euthanizing the animals with sodium pentobarbital overdose, the liver lobes were harvested and fixed in 4% paraformaldehyde. For histological analysis, the fixed tissues were embedded in paraffin and cut into sections that were stained using hematoxylin and eosin (HE) using standard protocols. The stained sections were viewed under a light microscope (Leica, DM2000, Germany).
Data were expressed as mean ± SEM and compared by Student’s t test. GraphPad Prism was used for all statistical analyses, and p < 0.05 was considered statistically significant.