Construction Of Smad1-HA-Tag Mouse And SMAD1-HA-Tag Human Dental Mesenchymal Cells By CRISPR/Cas9-mediated Genome Editing

The BMP signaling pathways play an important role in organ formation through epithelial and mesenchymal interactions.The BMP signaling pathway exist in a new regulatory approach,unlike the BMP/Smad classical signaling pathway and the BMP nonclassical signaling pathway,which is known as atypical BMP classical signaling pathways in tooth development.In this pathway,the pSmadl/5/8 complex does not depend on Smad4 and that regulates the expression of the tooth development-related genes by binding to the unknown interacting protein.To analysis of pSmadl/5/8 of the interaction protein using affinity purification technology that is the main technical means to isolate and purify the identification of the interaction protein.And affinity purification technology need specificity of the antibody to efficiently isolate the interacting protein with high purity,we selected affinity label HA protein which is the target protein spatial structure is small and easy to be detected.And Smadl is the main entry gene in the pSmadl/5/8 complex.Therefore,we used CRISPR/Cas9 technique to knock the HA tag protein gene sequence into the 3' region of the Smadl and the 5' region of the SMAD1,without damaging the expression of Smadl protein.Through this method,we achieved the co-expression of HA protein and pSmad 1/5/8 protein and provided experimental material for isolation and purification of interacting proteins in the future.Cas9 protein,sgRNA,and the homologous recombination fragment donor with the exogenous gene constitute a foreign gene knock-in system.To verify sgRNA and donor,we perform validation experiments at the cell level.We designed 2 sgRNAs targeting the 3'region of the Smadl gene and transfect the sgRNAs plasmids into NIH3T3 cells with lipofection.Next,we analysised the efficiency of sgRNA targeting.As a result,2 sgRNAs(sgRNAl and sgRNA2)which can effiently edited the 3' region of the Smadl gene.Then,we co-transfected sgRNAs and DNA donor which designed the HA tag protein gene sequence with homolgy sequences on the 3' region of the Smadl into the NIH3T3 cells.The results demonstrate that HA sequence was correctly inserted into the 3' region of the Smadl and co-expression with pSmadl/5/8 protein.Finally,the Cas9 protein,sgRNAs and DNA donor were microinjected into mouse zygotes and Smadl-HA-Tag mice were successfully generated.At the same time,we successfully established SMAD1-HA-Tag human dental mesenchymal cell using CRISPR/Cas9 technology.Three sgRNAs were designed near the 5' region of the human SMAD1 gene and the sgRNAs expressing plasmid was constructed.We designed 3 sgRNAs targeting the 5' region of the SMAD1 gene and transfect the sgRNAs plasmids into HEK293T cells with lipofection,to analysised the efficiency of sgRNA targeting and off-target.As a result,low level of off-target sgRNAs(sgRNAl and sgRNA3)which can effiently edited the 5' region of the human SMAD1 gene.Then,we co-transfected sgRNAs and DNA donor which designed the HA tag protein gene sequence with homolgy sequences on the 5' region of the SMAD1 into human dental mesenchymal cells.The results of PCR,western blot and immunofluorescence demonstrate that HA sequence was correctly inserted into the 5' region of the SMAD1 gene and co-expression with pSmad 1/5/8 protein.In conclusion,we successfully constructe Smadl-HA-Tag mice.In the other hand,we established SMAD1-HA-Tag human mesenchymal cell.Our reslut provide a basis for studying the interaction of pSmadl/5/8 interacting proteins,laying the foundation for elucidating the signal transmission mechanism of the newly discovered atypical BMP classical signaling pathways.