Hyperhomocysteinemia and dyslipidemia in point mutation G307S of cystathionine β-synthase-deficient rabbit generated using CRISPR/Cas9

Congenital hyper-homocysteinemia (HHcy) is caused by a defective cystathionine β-synthase (CBS) gene, and is frequently associated with dyslipdemia. The aim of this study was to further elucidate the effect of mutated CBS gene on circulating lipids using a rabbit model harboring a homozygous G307S point mutation in CBS. CRISPR/Cas9 system was used to edit the CBS gene in rabbit embryos. The founder rabbits were sequenced, and their plasma homocysteine (Hcy) and lipid profile were analyzed. Six CBS-knockout (CBS-KO) founder lines with biallelic modifications were obtained. Mutation in CBS caused significant growth retardation and high mortality rates within 6 weeks after birth. In addition, the 6-week old CBS-KO rabbits showed higher plasma levels of Hcy, triglycerides (TG), total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) compared to the age-matched wild-type (WT) controls. Histological analysis of the mutants showed accumulation of micro-vesicular cytoplasmic lipid droplets in the hepatocytes. However, gastric infusion of vitamin B and betaine complex significantly decreased the plasma levels of TG, TC and LDL-C in the CBS-KO rabbits, and alleviated hepatic steatosis compared to the untreated animals. A CBSG307S rabbit model was generated that exhibited severe dyslipidemia when fed on a normal diet, indicating that G307S mutation in the CBS gene is a causative factor for dyslipidemia.

Hcy is re-methylated to methionine by 5methyltetrahydrofolate-homocysteine methyltransferase (MHMT) and the cofactor folate-cobalamin, and the methyl group is donated by betaine via betainehomocysteine methyl transferase (BHMT). In addition, CBS metabolizes Hcy to cystathionine in the presence of vitamin B6 [25][26][27][28][29][30]. Interestingly, there is considerable clinical heterogeneity among individuals with homocystinuria based on their responsiveness to pyridoxine (vitamin B6) [31]. While patients harboring the G307S mutation are completely non-responsive, those with I278T partially respond to B6. In addition, pyridoxine resistance is associated with a more severe clinical phenotype [20]. Although the precise mechanism through which G307S affects CBS function is unclear, there is evidence that it restricts the ability of tyrosine at position 308 to assume the proper conformational state required for the pyridoxial-cystathionine intermediate, eventually inhibiting the catalytic performance [32]. In addition, the pyridoxine non-responsive patients may benefit from betaine supplementation [33], either alone or in combination with vitamin B12, folic acid or a methionine-restricted diet.
HHcy is routinely accompanied by life-threatening vascular complications, and individuals with high circulating levels of Hcy are at a higher risk of CVD and increased 5-year mortality [34]. However, most animal models currently used for cardiovascular research do not accurately simulate the human system [35]. For instance, most disease-causing mutations in humans do not replicate the symptoms in rodents, and their short life-span precludes any investigation of long-term effects. Furthermore, rats, mice, tree shrews and dogs are resistant to atherosclerosis and hypercholesterolemia, unlike primates, hamsters and rabbits [36][37][38][39]. The rabbit (Oryctolagus cuniculus) model offers several advantages over rodents, such as greater phylogenetic similarity to primates, adequate amount of blood for plasma biochemical analysis, suitable heart size for studying atherosclerosis in both aorta and coronary arteries [35,40], and a more diverse genetic background which is conducive to modelling complex diseases and simulating the effects of genetic diversity in the human population [35].
Since most CBS mutations in humans are of the missense type caused by base pair substitutions, HHcy modeling require gene knock-in as opposed to knock-out. To this end, we generated CBS gene mutant rabbits including point mutation G307S using the CRISPR/Cas9 system in order to gain new insights into the HHcy pathogenesis. In addition, the CBS-KO rabbits were fed with vitamin B and betaine complex supplements to devise possible therapeutic approaches.
Zygote injection with Cas9/sgRNA and 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 Female rabbits were superovulated by intramuscular injection of follicle-stimulating hormone (Sansheng Pharmaceutical Co., Ltd., 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 h post-coitus, the female rabbits were anesthetized with pentobarbital (2%, 20 mg/kg, i.p.), and the pronuclear zygotes were extracted and transferred into M2 medium (Sigma-Aldrich, St. Luis, MO, USA) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, 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 (Sigma-Aldrich, St. Luis, MO, USA) and incubated at 38°C under 5% CO 2 for 30-60 min. Approximately 15-20 embryos were transferred to one recipient female.

Genotypic analysis
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 (Transgene Biotechnology Co., Ltd., Beijing, China). The sequences were then cloned into pGEM-T vector (Promega, Madison, WI, USA), and sequenced by Shanghai Sangon Biotechnology Co. Ltd. Sequences were analyzed by the Lasergene DNA analysis package (DNAStar Inc., Madison, WI, USA).
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 then 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, Table S2).

Drug test
Two weeks-old CBS-KO and WT rabbits were infused daily with 2.5 mg/kg vitamin B6, 25 μg/kg vitamin B12, 45 μg/kg folate and 25 mg/kg betaine via the gastric route. The animals were acclimatized to gentle restraint and handling, and infused daily from 10 am to 12 noon for 4 weeks by the same technician.

Western blotting
The

Histomorphological assessment
The CBS-KO and WT littermates were maintained under similar conditions, and fed according to their growth stage. The body weight of the rabbits was 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 Leica light microscope.

Statistical analysis
Data were expressed as mean ± SD and compared by Student's t test. GraphPad Prism was used for all statistical analyses, and P < 0.05 was considered statistically significant.

Generation of CBS-KO rabbits and genotypic analysis
As shown in Table 2, we injected 217 zygotes with the CRISPR/Cas9 and sgRA constructs, and transferred 181 into 9 surrogate females. Eighteen pups harbored a mutant CBS (Table 2), of which only 6 survived and were numbered C2♀, C8♀, C9♀, C14♂, C15♀ and C16♂. C8♀ was homozygous for G307S, C2♀ harbored a frameshift mutation in CBS due to deletion or substitution, and C9♀, C14♂ and one allele of C16♂ harbored deletions/ substitutions in the CBS gene without a frameshift in the amino acid sequence. The second CBS allele of C16♂ had deletions/insertions resulting in a frameshift mutation. One allele of C15♀ harbored the G307S and the other showed deletions/insertions resulting in frameshift mutation (Fig. 2). In addition, 15 potential off-target sites (OTs, five for each sgRNA) were also amplified and sequenced, and no overlapping peaks were detected near the OTs.

Mutations in CBS induced hyperlipidemia which was reversed by betaine
Since HHcy is closely associated with dyslipidemia, we next analyzed the levels of blood lipids in the WT and mutant rabbits. As shown in Table 3, the C2♀ and C8♀ rabbits fed with normal chow had significantly higher serum levels of Hcy, TG, TC and LDL-C compared to the age-matched WT rabbits. The Hcy levels in C2♀ (48.80 μmol/L) and C8♀ (52.66 μmol/L) were almost twice as high as that in WT controls, whereas the TG levels (3614.93 mg/dL and 3878.47 mg/dL respectively) showed a 50-54 fold increase. The TC levels in C2♀ (738.98 mg/dL) and C8♀ (342.62 mg/dL) were respectively 6-and 3-fold higher, and that of LDL-C levels in both (67.24 mg/dL and 76.80 mg/dL respectively) were 2-fold higher compared to the WT. In contrast, the HDL-C level in C8♀ (16.64 mg/dL) was significantly lower compared to the WT. Consistent with the high levels of circulating lipids in C8♀, the color of its plasma was milky white (Fig. 4b).
To evaluate a potential hypolipidemic effect of betaine, we infused both WT and mutant animals with vitamin B and betaine complex for 4 weeks. Betaine supplementation reduced plasma Hcy, TG, TC and LDL-C by 30 39.33% (17.14 ± 0.56 mg/dL vs 28.25 ± 3.54 mg/dL) respectively in the WT rabbits compared to non-supplemented littermates. In contrast, no significant changes were seen in plasma HDL-C levels (Fig. 3a). The similarly fed C9♀, C14♂, C15♀ and C16♂ mutants showed a significant decrease in plasma levels of TG, TC, LDL-C and Hcy compared to that in C2♀ and C8♀. In addition, the TG levels in C14♂ (76.14 mg/dL) and C15♀ (35.95 mg/dL), and the LDL-C levels in C16♂ (24.64 mg/dL) dropped to near normal range after betaine supplementation (Tables 3 and 4). Consistent with these observations, the C2♀ and C8♀ rabbits showed a significant increase in ApoE and ApoB compared to the WT, whereas betaine supplementation in C9♀, C14♂, C15♀ and C16♂ reversed these trends (Fig. 3c).

The morphology of CBS-KO rabbits
The body weights of the WT and CBS-KO rabbits were similar on postnatal days 1 and 7. At 3 weeks however, the CBS-KO rabbits weighed significantly less compared to the WT animals (Table 5), and some failed to gain weight even after 6 weeks. In addition, CBS-KO rabbits were overall smaller, and had sparser fur and pale mucous membranes (Fig. 4c-e), all of which are indicative of growth retardation. The eye-lens were also dislocated in the CBS-KO rabbits (Fig. 4a). Finally, most of the mutant animals did not survive beyond 5~7 weeks after birth ( Table 6).

Histological analysis
The 6-week-old WT and CBS-KO rabbits receiving normal and vitamin B/betaine complex-supplemented feed were sacrificed for histological examination. The livers of CBS-KO rabbits were brick red compared to the reddish-brown color of the WT livers (Fig. 5a).  Values are expressed as means ± SD. a The data does not contain vLDL-C Histologically, no cytoplasmic lipid droplets were observed in the liver of WT rabbits even in the absence of vitamin B and betaine complex supplementation. However, the CBS-KO rabbits had significant accumulation of macro cytoplasmic lipid droplets in their liver, which decreased following vitamin B and betaine complex supplementation (Fig. 5b). However, Sudan IV staining did not reveal any atheromatous plaques in the aorta and other vascular segments of the CBS-KO rabbits (Fig. 5c).

Discussion
Homocysteinemia is observed in 5-30% of the human population, and leads to vascular complications in onefourth of the patients [43][44][45]. We obtained 6 CBS-KO rabbits including one harboring the G307S point mutation, using the CRISPR/Cas9 gene editing technology. All mutants (C2♀ and C8♀) lacked the glycine 307 residue in CBS protein, and displayed high serum levels of Hcy and lipids. Supplementing the normal chow with vitamin B and betaine alleviated the pathological symptoms of HHcy. Although there was some research reported on the G307S point mutation, it was mostly based on the heterologous gene expression system such as E. coli, S. cerevisiae, and Chinese hamster ovary cells. But it was unfortunate that the heterologous CBS G307S protein lacked enzyme activity [46][47][48]. At present, there was no CBS-deficient animal model of G307S point mutation or deletion of glycine at position 307. The CBS-KO rabbit showed HHcy, dyslipidemia, a short life span, hepatic steatosis and other lesions. This indicates that HHcy and hyperlipidemia are likely the result of 307G deletion as well as G → S mutation. Compared with CBS-deficient mice in the previous research [49,50], the results of this study may be more precise and accurate in explaining the molecular mechanism of hereditary hyperhomocysteinemia etiology. Therefore, the CBS-KO rabbit model is suitable for studying the pathogenesis and therapeutic strategies of HHcy. Studies show that HHcy is an independent risk factor for CVDs [3], which correlates positively with atherosclerotic symptoms induced by the high levels of TG, TC and LDL-C, and normal plasma HDL-C level [51,52]. In addition, HHcy increases the risk of hypertriglyceridemia [53,54]. Consistent with this, the serum triglyceride levels were high in the CBS-KO founder rabbits, and decreased significantly following betaine and vitamin B6 supplementation.
Dyslipidemia is a major factor underlying CVDs, and normalizing blood lipid levels can lower the morbidity and mortality in patients with heart disease [55]. The quaternary amine betaine and B vitamins are efficient methyl donors that produce a large amount of free carnitine in the liver, which promotes long-chain ester acyl-CoA entry into the mitochondria and accelerates fatty acid oxidation, eventually reducing the blood lipid levels [56]. Betaine may inhibit hepatic biosynthesis of LDL-C Values are expressed as means ± SD. *P < 0.05, **P < 0.01 and ****P < 0.0001 Values are expressed as means ± SD. a The data does not contain vLDL-C or accelerate the conversion of LDL to HDL [57]. In addition, oral betaine and B vitamins can reduce serum Hcy levels in healthy individuals by increasing their metabolism, and have therapeutic effects on patients with hyperlipidemia, fatty liver and atherosclerosis [58][59][60]. Consistent with this, vitamin B and betaine complex supplementation significantly improved the blood lipid profile of both WT and CBS-KO rabbits. Interestingly, the normal rabbits also showed Hcy levels higher than 15 μmol/L, which may explain the rapid induction of hyperlipidemia via high-fat diet consumption. Thus, the species and nutrient intake should be taken into account when assessing the effect of Hcy metabolism on dyslipidemia.
The fatty liver parenchyma of CBS-KO rabbits was filled with microvesicular cytoplasmic lipid droplets due to the influence of Hcy on the expression level of Smethyltransferase (BHMT). CBS-KO rabbits showed signs of growth retardation and short survival. Betaine and vitamin B supplementation reduced the fatty liver symptoms and prolonged survival of the mutants. Maclean [61] et al. reported that transgenic expression of CBS alleviated liver steatosis and prolonged survival in a mouse model. We showed for the first time that the addition of betaine and B vitamins partly compensated for the lack of CBS. This is significant since the previously reported hypolipidemic effects of betaine were seen animal models with high-fat diet-induced hyperlipidemia.
HHcy is an important factor in hyperlipidemia and cardiovascular disease. In this study, animal models of HHcy showed hyperlipidemia and cardiovascular disease symptoms. Although the lipoprotein metabolism and cardiovascular pathophysiology of rabbits are similar to that of humans [62], the inherently higher Hcy levels in rabbits may have resulted in premature death in the absence of CBS.
Compared to the untreated animals, the CBS-KO rabbits with betaine and vitamin B supplementation had lower ApoB100, ApoB48 and ApoE levels in their plasma. ApoB is the major component of very low density lipoprotein (vLDL) and LDL particles. Namekata et al. [49] detected elevated ApoB in CBS −/− mouse serum, and Salahi et al. [63] found that betaine supplementation decreased LDL concentration and increased that of HDL in a rat model. ApoB is abundant in human and rabbit but not murine plasma [64]. In addition, the chemical composition and cholesterol ester transfer subunits of human and rabbit ApoB-related lipoproteins are similar, which correlates with the role of rabbit ApoB in atherosclerosis [65].
In conclusion, CBS-KO rabbits have impaired growth and metabolism, although it remains to elucidated whether these changes are the direct effect of the mutation, or an indirect effect of hyperlipidemia or high homocysteine.

Study strengths and limitations
We have generated CBS-KO rabbits with G307S mutation for the first time using the CRISPR/Cas9 system, and the model exhibits hyperhomocysteinemia and  dyslipidemia on a normal chow diet. It is also the first report on the effect of betaine on the serum lipids of CBS-KO rabbits. However, the short lifespan of these rabbits obviated the control data of C9♀, C14♂, C15♀ and C16♂ without betaine and vitamin B complex supplementation. Nevertheless, Hcy, TG, TC, LDL-C and other indices of most CBS-KO rabbits were higher compared to that of the normal controls even after betaine and vitamin B complex supplementation.