Establishment Of OSBPL2-knockout Bama Miniature Pig Phenotyping With Hearing Loss Via CRISPR/Cas99 Method

Background: Deafness is one of the most common perceptual disorders.It is estimated that 360 million people worldwide are suffering from mild to profound deafness.About 1 of every 1000 newborns are detected with hearing loss,and at least half of these cases are attributable to genetic factors.Among them,non-syndromic deafness(NSHL)accounts for about 70% of inherited deafness.In our previous study,OSBPL2 was firstly mapped and cloned as a novel causal gene for autosomal dominant NSHL(DFNA67)in a large affected Chinese family.Oxysterol binding protein like-2(OSBPL2)is highly homologous to oxysterol-binding protein(OSBP)and belongs to the OSBP-related protein(ORP)family.The ORP family constitutes numerous homologues as high-affinity oxysterol receptors,and plays an important role in many cellular processes,including lipid metabolism,vesicular trafficking and cell signaling.This study will establish an OSBPL2-knockout Bama miniature pig model via CRISPR/Cas9 method.And the pathogenic mechanism of OSBPL2 mutation implicated in NSHL will be explored.Objective: This study is devoted to establishing OSBPL2-knockout porcine fetal fibroblasts(PFFs)cell line as the donor of somatic cell nuclear transplantation using CRISPR/Cas9 approach.And OSBPL2-knockout piglets are generated via embryo transfer.Based on mentioned above,it is expected to verify the correlation of genotype-phenotype in pig model,probe the effect of OSBPL2-defects as well as high-fat diet on blood-lipid levels,hearing threshold and inner ear morphology.Methods:(1)Bioinformatic analysis: Bioinformatics methods were applied to analyze the collinearity and homology of OSBPL2 between human and pig.The secondary and tertiary structures of OSBPL2 protein of human and pig were predicted and simulated using the Chou-Fasman algorithm and Swiss Model online tool.(2)Construction of OSBPL2-knockout PPFs cell line: Single-guide RNAs(sg RNAs)were designed and synthesized that targeting exon 5 and exon 6 in porcine OSBPL2 gene.Two CRISPR/Cas9 targeting vectors were constructed and co-transfected with neomycin-expression plasmid p CMV-td-Tomato into the PPFs.G418 was used to screen monoclonal cells.The monoclonal cell genome was used as a template for PCR amplification.TA cloning was performed and sequenced to verify the genotypes of monoclonal cell line.(3)Establishment of OSBPL2-knockout Bama miniature pig model: The OSBPL2-knockout Bama miniature pig model was constructed using somatic cell nuclear transfer and embryo transfer approaches.The genomic DNA of the isolated pig ear tissue was extracted and used in PCR amplification.The genotypes of piglets were verified by Sanger sequencing after TA cloning.The OSBPL2 expression was assayed by Western blotting.The location and expression of OSBPL2 in cochleae were investigated by the immunohistochemistry method.(4)Analysis of auditory and inner ear morphology: Auditory brainstem response(ABR)was used to detect hearing thresholds;and the cochleae were isolated,paraffin-embedded and sectioned.HE staining method was used to observe the morphology of spiral ganglion,stria vascularis and basement membrane in the pig cochlea.(5)Biochemical analysis for blood lipid: OSBPL2-knockout Bama piglets were weaned at 1 month old and then fed with normal diet.At 2 months old,4 pigs were still fed with normal diet(KO-C group),and another 4 pigs were fed with high-fat diet instead of normal diet(KO-H group).5 wild-type Bama piglets of 2 months old were all fed with high-fat diet(WT-H group).Blood biochemical analysis for blood lipid were conducted,including total cholesterol(TC),triglyceride(TG),low-density lipoprotein(LDL),high-density lipoprotein(HDL)levels.The formation of atherosclerotic plaques in the aorta and coronary arteries was examined using Sudan ? staining.Results:(1)The OSBPL2 gene and its flanking sequences of pigs and humans are highly collinear.The amino acid sequence identity of them is up to 88% and the protein homology is high.Their secondary structures have approximately equal amounts of ?-helix,?-sheet and ?-turn structure.Three-dimensional modeling structure of the protein has high similarity and both contain strictly conserved OSBP fingerprinting motif “EQVSHHPP”.(2)Two pairs of sg RNAs targeting OSBPL2 exon 5 and exon 6 were designed and synthesized,respectively.The recombined vectors were successfully transfected into primary PFFs.The cleavage efficiencies were 11.4%(exon 5)and 0%(exon 6).35 individual cell clones were screened using the vector targeting exon 5,and 19 clones were identified with biallelic mutations(mutation efficiency: 54.29%).(3)OSBPL2-knockout cell pools were used as donors and transferred into oocyte by somatic cell nuclear transfer.The reconstructed embryos were transplanted into the uterus of 8 surrogate pigs and 3 of them were pregnant.At 37 days of pregnancy,14 pig fetuses were separated to prepare primary PPFs from a surrogate.All genotypes of the fetuses were identified as biallelic OSBPL2-knockout.The other two surrogate pigs were pregnant for the full term and naturally produced 4 and 6 piglets,respectively.One of the piglets showed limb and lip malformation and was killed at birth.The identified genotypes of all piglets were consistent with that of donor cells.OSBPL2 is mainly expressed in the spiral ganglion,stria vascularis,and basement membrane in the cochlea of Bama miniature pigs.Meanwhile,OSBPL2 is not detected in brain and liver tissues of cloned piglets.(4)Audiological test was conducted every one month after weaning.Compared with the hearing threshold(40~60 d B SPL)of wild-type Bama miniature pigs at the same age,both the KO-C and KO-H piglets showed progressive hearing loss.It was noteworthy that the KO-H group showed profound deafness(hearing threshold: ?100 d B SPL)early at the age of 4 months.Cochlear HE staining showed no obvious changes in histological morphology of the inner ear.(5)Obesity and abnormal lipid parameters were detected in both KO-C and KO-H groups,especially in the KO-H group.The plaques were significantly visible in the thoracic aorta and abdominal aorta of KO-H group by Sudan ? staining,whereas no obvious lesions were detected in corresponding arteries in the WT-H and KO-C groups.Conclusion:The OSBPL2-knockout Bama pig model was successfully constructed and showed dual phenotypes of abnormal blood lipid parameters and progressive hearing loss.In addition,high-fat diet could promote the development of hearing loss caused by OSBPL2-defects.This study will contribute to the elucidation of pathogenic mechanism of OSBPL2 mutation implicated in NSHL and provide experimental evidence for the association between lipid metabolism disorders and hearing loss,which would lay a theoretical foundation for the prevention and treatment of inherited deafness.