Improvement Of The Cardiac Repairing Capacity Of MSCs Via CRISPR/Cas9 Based Wnt Signal Pathway Regulation

Objective:To fine-tune the Wnt pathway based on CRISPR/Cas9 gene editing system,and to explore its implication in regulation of MSCs towards multi-lineages,such as,fibroblastic,endothelial,and myogenic differentiation,shedding light on improving its effects on myocardial fibrosis in mouse?Methods(1)Construction and verification of the plasmids fine-tuning Wnt pathway based on CRISPR/Cas9 gene editing system.The negative regulators of Wnt pathway were selected in this study.Guide RNAs targeting the promoter region of these genes were designed and corresponding DNA oligos were synthesized for cloning into lenti-sgRNA-Ms2-zeo.The plasmid construction was done by annealing the two complementary DNA sequences before insertion into lenti-sgRNA-Ms2-zeo vectors with the enzyme BsmBI.The acquired clones are confirmed by sequencing.After confirmation,the plasmids were co-transfected with lenti-Ms2-P65-HSF1-Hygro and lenti-dcas9-VP64-blastine into 293 T cells.Expression of the target genes were analyzed by qPCR assay.(2)Effects on the differentiation of MSCs via CRISPR/Cas9 based Wnt pathway lenti-virous regulation.293TCells were transfected with 4 kinds of plasmids,psPAX2 and pMD2.G by Lipofectamine2000.Virus supernatant was harvested and filtered at 48 h post-transfection.Virus supernatant of gRNA,MS2-p65 and dcas9-vp 64(1:1:1)were co-infected to MSCsvia polybrene,expansion in vitro.Either kind of inducing medium was added to the corresponding well.QRT-PCR was performed to verify the expression of genes,7 days post-infected.Results:(1)Construction and verification of the plasmids fine-tuning Wnt pathway based on CRISPR/Cas9 gene editing system.Four genes were selected in this study,all of which were well-established negative regulators of Wnt pathway.Two guide RNAs recognizing the promoter of each gene were designed and corresponding plasmids expressing the gRNA fused with a MS2 scaffold were constructed.Sequencing results indicated that all the clones were correctly done.4 kinds of plasmids were randomly selected for further gene activation efficiency analysis.As expected,the gRNA expression vectors could significantly increase the target genes when co-transfected with lenti-Ms2-P65-HSF1-Hygro and lenti-dcas9-VP64-blastine.(2)Effects on the differentiation of MSCs via CRISPR/Cas9 based Wnt pathway lenti-virous regulation..At 3 days post-infection,selection agents were added to each well.QPCR showed the expression of VEGF,?-SMA,Col1a1 in experimental group were all lower than control in different levels(p<0.05).Western blot were performed at 7day,compared with?-actin,the expression of ?-SMA and Col1 agen were lower.Conclusion:1.The plasmids that expressed gRNAs targeting the known Wnt negative regulators were successfully constructed,which could fine-tune the Wnt pathway by increase the target gene expression.2.The differentiation capacity of MSCs towards different lineages can be regulated by Wnt pathway modulators based on CRISPR/Cas9 gene editing system.