The Establishment Of A Rabbit Model Of Spontaneous Hypercholesterolemia Mediated By CRISPR/Cas9

Objective:To generate novel rabbit models with deficiency of either LDL receptor(LDLR)and/or Apolipoprotein(ApoE)genes for the study of hyperlipidemic and atherosclerosis.Method:The CRISPR/Cas9 system was used in this research and a pair of sgRNAs(sgRNA1 sgRNA2)anchored 7th exon and a pair of sgRNAs(sgRNA3 sgRNA4)for the second exon in LDLR gene of rabbit were designed.For the first exon in ApoE gene,a pair of sgRNAs(sgRNA5 sgRNA6)were also designed,then all of them were ligated into CRISPR/Cas9 recombinant plasmid respectively.Subsequently,the Cas9 mRNA and different sgRNAs were transcripted in vitro Then Cas9 mRNA and sgRNAs in different groups were microinjected into the cytoplasm of multiple fertilized eggs,the grouping as follows:①Cas9 mRNA and sgRNA1,sgRNA3,sgRNA6;②Cas9 mRNA and sgRNA2,sgRNA4,sgRNA5;③Cas9 mRNA and sgRNA1,sgRNA2,sgRNA3,sgRNA4;④Cas9 mRNA and sgRNA5,sgRNA6.The microinjected fertilized eggs were transplanted into the oviduct of recipient,and the young were born after one mouth pregnancy.For these founders,the gene sequence,the analysis of amino acid arrangement,the simulation of protein structure and the test of off-target were performed to determine their genotypes.Then the founder rabbits with gene mutations were placed in a normal diet and their plasma lipids were tested at 12 weeks of age,and also plasma Apolipoprotein B(ApoB),ApoE and Apolipoprotein AI(Apo AI)were detected by Western blot.1%agarose gel electrophoresis and fast liquid chromatography(FPLC)were followed to analyse their plasma lipoprotein profile to determine whether spontaneous hyperlipidemia occur.After 36 weeks on normal diet,the aorta was dissected and opened out,then staining for lesions to observe whether atherosclerosis show up.After all the above analysis,it was determined whether the rabbit model of spontaneous hypercholesterolemia was established.Results:Thirteen founder mutant rabbits were finally lived,including single LDLR gene knockout(LDLR-/-)rabbits,the rabbits with LDLR gene large fragment deletion,single ApoE gene knockout(ApoE-/-)rabbits and LDLR/ApoE double knockout(LDLR-/-/ApoE-/-)rabbits.There are vary mutations from insertion,deletion and substitution to frameshift mutations,etc.And there was no off-target phenomenon in the predicted range.The protein structure simulation predicted that the proteins encoded by the mutant genes had varying structural or functional abnormalities.The plasma lipid concentrations in LDLR-/-and LDLR/ApoE double knockout rabbits were detected and the results showed that their plasma cholesterol levels were elevated significantly under normal diet.Plasma total cholesterol concentration was up to 18-fold higher than normal,and low-density lipoprotein cholesterol concentration was up to 40 times higher.Western blot results showed the ApoB and ApoE in mutant rabbit plasma(ApoE gene knockout was excluded)also increased,however the ApoAI decreased,which indicating that residual lipids were elevated in plasma of founders.Aorta Sudan IV staining showed the surface was covered with a mass of atherosclerotic plaques,and the coverage rate was as high as 92%.Followed by HE,immunohistochemical staining and Masson staining showed that typical atherosclerosis occured:intima thickening,neointimal,accumulation of numberous foam cells derived from macrophage and partially derived from smooth muscle in the intima,proliferation and migration of smooth muscle cells that migrated from media to the intima,the formation of fibrous plaques which containing abundant foam cells,calcium deposits and thin fibrous caps covering the surface.Plasma lipid analysis of all mutant rabbits revealed that the degree of elevated in blood lipids was not related to whether the LDLR gene or the ApoE gene were double-knocked or single-knocked,whereas it is mainly relevant to the alteration of the encoded protein caused by mutations in the LDLR gene and the ApoE gene.Conclusion:The spontaneous hypercholesterolemia-LDLR-/-rabbit-model was successfully estabilished through the CRISPR/Cas9 gene editing tool,and the LDLR/ApoE double knockout and the ApoE-/-rabbit model were also prepared.They will provide experimental models for the mechanisms and therapeutic studies of cardiovascular disease and related diseases.