The Establishment Of CRISPR/Cas9-mediated Transcriptional Activation System And Its In Vitro Experimental Study

CRISPR/Cas9 system is a new type of genome targeted editing technique.In this system,the target gene is digested with the guidance of a small RNA.Compared with earlier ZFN and TALEN systems,the new genome editing technology has the advantages of simple operation,low cost,high efficiency and targeting multiple genes at the same time etc.In CRISPR/Cas9 system,the nucleic acid enzyme region of Cas9 protein,which has the function of identifying and cutting,is composed of two parts(HNH and RuvC).RuvC and HNH were able to digest the corresponding DNA complementary and non-complementary chains of gRNA respectively.D10A and H840A mutations in Cas9 protein can lead to the inactivation of RuvC and HNH domains respectively,which generated Cas9 protein with loss cleavage activity(dCas9).Existing researches show that compared with dCas9 protein,Cas9m4 has better genomic binding activity and lower off-target effect,which was generated by two additional amino acid mutations of D839A and N863A on the basis of dCas9.Cas9m4 is fused with TET1-CD with the function of DNA demethylation or VP64 with the function of transcriptional activation by a flexible connecting peptide.These fusion protein were then used to generate the transcription factor activation system based on CRISPR/Cas9 or CRISPR/Cas9 mediated DNA demethylation activation system combined with the designed sgRNA.iPS cell technology is significant importance in biology and medicine.Although great progress has been made in the study of iPS cell technology in the past studies,many problems still await to be solved.Many scientists are always improving strategies of inducing the iPS cell,for example,the application of vectors from the integration vectors to the non-integration vectors,and then to the application of the plasmid or protein,their securities have been continuously increased.In addition,the application of small molecule compounds,miRNA,hpoxia condition and so on greatly improved the efficiency of iPS induction.But so far,the lack of efficient and safe technology of iPS cell induction limited the safe application of iPS in clinical practice.In this project,we tried to construct the specific transcriptional activation system and the specific demethylation system by CRISPR/Cas9 system,then they would be applied for improving the endogenous expression of the four genes,Oct4,Sox2,Klf4 and c-Myc,which would provide a new method for the induction of iPS cell.The research results of this paper are as follows.(1)Two fusion protein expression vectors of pCMV-Cas9m4-TET1-CD-Flag and pCMV-Cas9m4-VP64-Flag were successfully constructed,and the expression of the fusion protein in eukaryotic cells was verified.(2)The transcriptional activation sgRNAs of Oct4,Sox2,Klf4 and c-Myc were designed,and sgRNA activity was analysing by T7E1 enzyme digestion assay.(3)Taking Oct4 gene as an example,the results of bisulfite sequencing showed that CRISPR/Cas9 mediated DNA demethylation activation system can effectively reduce the DNA methylation status of the target site.(4)The effect of two kinds of activation system on the expression level of the target gene was detected by quantitative real-time PCR.The results indicated that CRISPR/Cas9 mediated transcription factor activation system can significantly increase the expressin level of the four target genes.The effect of CRISPR/Cas9 mediated DNA demethylation activation system on target gene expression is weaker than that of CRISPR/Cas9 mediated transcription factor activation system on target gene expression,and the transcriptional expression of four target gene regulated by CRISPR/Cas9 mediated DNA demethylation activation system was varied.In summary,we successfully established two kinds of CRISPR/Cas9 mediated transcriptional activation system,one is transcription factor activation system based on CRISPR/Cas9 and another is CRISPR/Cas9 mediated DNA demethylation activation system,which could improve the expression of endogenous genes including Oct4,Sox2,Klf4 and c-Myc in different extent.This study provides a new way of thinking for exploring the new method of iPS,and provides a convenient and inexpensive research tool for the study of transcriptional regulation of other genes.