Constructing A Syria Hamster MYBPC3 Gene Knock Out Inheritec Cardiomyopathy Model Via CRISPR-Cas9

Background and Objective:Inherited cardiomyopathies,one of the major reasons for the worldwide public health problem of Heart failure(HF),have a high prevalent rate in the general population and is also the leading cause of sudden cardiac death(SCD)among young people.It is regarded as the main cause of sudden cardiac death among young people.Treatment of inherited cardiomyopathies nowadays like drug therapy,device help service,surgical procedures which only have a symptom relief effects can hardly reverse or even slow down the process of disease pathophysiology advancing.Inherited cardiomyopathies is known to have a close relation with mutations in sarcomere component encoding genes.Among the pathogenic mutation genes,most located in myosin heavy chain encoding gene(MYH7)and cardiac myosin binding protein encoding gene(MYBPC3).The commonly variant type in MYBPC3 gene related inherited cardiomyopathies is frameshift mutation.Animal models have an unreplaceable value in the study of inherited disease molecule mechanism.The existing animal models for inherited cardiomyopathies available confined in mice,rat,rabbit which have a disparity in both genetic and phenotype aspects compared to human.It is urgently needed to develop new animal models to provide tools for the research of inherited cardiomyopathies pathogenesis.With the improvement and maturity of new gene editing tool of CRISPR-Cas9(clustered regularly interspaced short palindromic repeats),gene editing field have witnessed tremendous change.The CRISPR-Cas9 system,which are more cheaper,convenient and efficient than previous gen editing tools,have been widely applied in scientific study.Syria hamster,also named golden hamster,which have many advantages like proper body size,short periods of gestation,more pupils every birth,is one of the mostly used animals in lab across the world.However,rare reports of gene editing inherited cardiomyopathies models in hamster have been reported.Thus,we aimed to creating a Syria hamster MYBPC3 gene knock out inherited cardiomyopathies models by using the CRISPR-Cas9 system to provide tool support for the deeper research of inherited cardiomyopathies.Methods:The gene targeting site and sgRNA was first selected.Then after the successfully construction of CRISPR-Cas9 targeting system,the editing efficiency of sgRNA is tested in vitro.The highest efficiency one was picked up for zygotes injection to get MYBPC3 gene knock out hamster.Genotype of F0 was identified through PCR-RELP analysis and off-target effects was also carried out.The founder knock out hamster was selected if the pupil genotype have frameshift effect and then breed for the stable MYBPC3 knock out hamster generations.Phenotype detecting were applied in 6-months-old homozygous,heterozygous and wild littermates(control group).Echocardiography were done before the slaughter of experiment hamster.After body weighting,the hamsters were killed to harvest heart.The heart was weighted before spliced to different part for various experiment purpose.Transcription-quantitative polymerase chain reaction(RT-qPCR)were used to for the analysis of ANP,BNP,MYBPC3,β-MYH,TGF-β mRNA expression.cMyBP-C protein analysis was carried out through western blot.Myocardial histochemistry were measured by Masson trichrome and HE staining.Result:The sixteen exon of hamster MYBPC3 gene was selected as the targeting site.SgRNA1 and sgRNA2 was designed,which displayed an efficiency of 30.22%and 0%separately in vitro study after successfully constructed to CRISPR-Cas9 targeting system.The use of sgRNA1 binding CRISPR-Cas9 system to zygotes injection managed to received 8 F0 hamster,five of which produced gene editing effect.Off-target analysis didn’t test any off target effect.The F0#2,4 base pair deleted,resulting frame shift mutation,was breed for experiment usage.Phenotype analysis using the F2 generation 4bp deletion hamster at 6 months old age among homozygous,heterozygous and wild type group showed great disparity.Heart size in homozygous group is larger than the other two group.Ratio of heart to body weight is higher in homozygous and heterozygous group compared to wild type littermates.ANP,BNP mRNA expression detected via RT-qPCR revealed a relative higher increase in homozygous and heterozygous group,most prominent in homozygous group.(P<0.05).MYBPC3 mRNA expression is lower in gene knock out hamster than in control group,while the cMyBP-C protein is increased in heterozygous group and not detected in homozygous group.Expression of cardiac fibrosis marker TGF-β and cardiac hypertrophic markerβ-MYH have no difference among three groups.HE and Masson stain observed cardiomyocyte disarray in homozygous hamster.Echocardiography indicated left ventricular enlargement and the reduction of ejection fraction and fraction shortening in homozygous group.Conclusions:With highl efficiency and safety,the CRISPR-Cas9 system managed to get MYBPC3 gene knocked out in vivo of Syria hamster.The homozygous 4bp MYBPC3 deleted hamster displayed phenotype of dilated cardiomyopathy which indicated the successfully constructing of MYBPC3 knock out Syria hamster inherited cardiomyopathies animal model.Phenotype of heterozygous hamster need to extend observation time.