Endogenous Activating Primordial Germ Cell (PGC) Specialization Genes

Primordial germ cells(PGCs)are progenitors of germ cells,which have the ability to differentiate into sperm cells and egg cells.The combination of somatic cell reprogramming(SCR)and in vitro induced differentiation(IVD)to obtain primordial germ cells(PGCs)has become an important research strategy for clinical treatment of infertility.Recent years,CRISPR/Cas9 technology has been found not only to be used for gene knockout,but also to activate gene transcription.In this study,CRISPR-Cas9-mediated targeting gene activation was used to compare the different regions of sgRNA targeting gene,sgRNA combinations and different activation systems to study the endogenous activation of primordial germ cell specific related genes.The results are as follows:(1)The transcriptional activation system Cas9-p300 was used for endogenous activation of germ cell specific related genes.sgRNAs were designed in the promoter regions of Blimp1,Tfap2 c,Dazl,Sox17,Nanos2,Pax5,Boll,Ddx4.Then,the expression of above target gene was detected in 293 T cells that co-transfected with the vector carrying Cas9-p300 system and the sgRNA-bearing vector.Results showed that the Cas9-p300 system can activate the expression of germ cell specific genes,but the activation efficiency of different genes is different.Simultaneous activation of target gene by multiple sgRNAs is more efficient than that by a single sgRNA.To further explore whether there are other potential sites that can activate the target gene,sgRNA was designed to activate the target gene through the potential enhancer regions of Nanos2 and Ddx4,and the results showed that the potential enhancer regions did not show significant activation efficiency.(2)Activation of germ cell-specific genes such as Blimp1,Tfap2 c,and Nanos2 was performed using two different "second generation" transcriptional activation systems,the Cas9-p300 system and the CRISPR/dCas9-VPR system,and the activation efficiencies of the two transcriptional activation systems were compared.The results showed that the Cas9-p300 system has higher activation efficiency inactivating the Blimp1,while the dCas9-VPR system has higher activation efficiency in activating the Nanos2,Dazl,and Sox17.Both systems have similar activation efficiency in activating Tfap2 c and Ddx4,but neither system can activate Ddx4 effectively.Immunofluorescence staining showed that the expression of Tfap2 c could be detected by CRISPR/dCas9-VPR system at protein level.The above results indicate that both Cas9-p300 and CRISPR/dCas9-VPR systems can be used for transcriptional activation regulation and exhibit different activation trends when different genes are activated.