Study On Generation Of Rabbit Models Of Cardiovascular Disease Caused By Point Mutation In PCSK9 And CBS Using CRISPR/Cas9

ObjectiveCardiovascular disease is a kind of common disease that seriously threatens the health of Chinese residents.In this study,CRISPR/Cas9 gene editing technique was used to construct rabbit models of S386A-PCSK9 and G307S-CBS point mutation to further clarify the effects of mutated PCSK9/CBS gene on lipid metabolism and cardiovascular disease.Methods1.After reading the literature,the point mutation of PCSK9/CBS gene was selected.The protein functional regions of human and rabbit PCSK9/CBS and were analyzed by Blast according to the protein data of Pub Med line gene2.SgRNAs and ssODN were designed according to the base substitution position and sequence analysis results corresponding to the point mutation in rabbits.3.SgRNAs,Cas9-mRNA and ssODN transcribed in vitro were co-injected into the cytoplasm of rabbit fertilized eggs and the embryos were transferred into the pregnant rabbits.4.PCR,TA cloning and miss detection were performed on FO rabbits to identify whether the mutation was successfully completed.5.According to the amino acid sequence obtained from gene sequencing,the protein structure was simulated to predict the effect of mutation on the structure of protein.6.The lipid metabolism of mutant rabbits was analyzed by the detection of blood lipid and the lipoprotein agarose gel electrophoresis7.At the age of 12 weeks,rabbits were given a high cholesterol diet for 10 weeks,and then the aorta was dissected for oil red O staining,H&E staining,Masson pine staining and immunohistochemical staining of smooth muscle cells,macrophages and TNF-α.Liver tissue was taken for immunohistochemical staining of TNF-α.8.CBS mutant rabbits were fed with vitamin B and betaine complex,and the effects of drugs on blood lipids,plasma homocysteine and liver lipids in mutant rabbits were observedResults1.Fifteen S386A-PCSK9 and six G307S-CBS F0 generation rabbits were obtained2.The sequencing results showed that one rabbit was homozygous for S386A-PCSK9 and two were heterozygotes for S386A-PCSK9.The three designed sgRNAs did not miss within the predicted range.The results of protein structure simulation showed that the PCSK9 proteins of mutant rabbits had different degrees of structural changes.The results of blood lipid and lipoprotein detection under normal diet showed that,compared with WT rabbits,the plasma levels of low-density lipoprotein cholesterol,total cholesterol and triglyceride decreased and the level of high-density lipoprotein cholesterol increased in one S386A-PCSK9-/-rabbit and one S386A-PCSK9+/-rabbit.The result of lipoprotein detection is consistent with this.The results of special staining and immunohistochemical staining after 10 weeks of high-fat diet showed that compared with WT rabbits,the intimal thickening was not obvious,the migration level of smooth muscle cells decreased,the infiltration of macrophages in intima and the inflammatory level of liver decreased in S386A-PCSK9 rabbits.The levels of blood lipids,lipoproteins and atherosclerotic plaques in the other S386A-PCSK9 rabbit were not significantly different from those in WT rabbits,which may be related to its mutation mode.The S386A-PCSK9 mutation can be stably inherited.3.Mutation in CBS caused significant growth retardation and high mortality rates within 6 weeks after birth.The 6-week old CBS-KO rabbits showed higher plasma levels of homocysteine,triglycerides total cholesterol and low-density lipoprotein cholesterol compared to the age-matched wild-type controls.Histological analysis of the mutants showed accumulation of micro-vesicular cytoplasmic lipid droplets in the hepatocytes.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.Conclusion1.The rabbit models of S386A-PCSK9 and G307S-CBS point mutation were successfully constructed by CRISPR/Cas9 technique。2.S386A-PCSK9 mutation has a protective effect on atherosclerosis.The construction of models provide a good animal model for exploring the molecular mechanism of impaired PCSK9 function and developing reliable,effective diagnosis and treatment measures3.G307S-CBS rabbit models were 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.