The Regulation Of PGRN Gene Expression By A CRISPR/dCas9-based Tet-on-induced Transcriptional Repression System

CRISPR/Cas9 system is a new efficient site-directed technology for genome editing after the zinc-finger nucleases and transcription activator-like effector nucleases technology.CRISPR/Cas9 system is widely used in gene function research because of its convenience for construction and high efficiency.The result of gene editing by the CRISPR/Cas9 under the guidance of sgRNA leads to gene function lost.Based on this system,the dCas9(dead Cas9)with the double mutation of D10A and H840A sites was obtained,which can bind to the DNA,but has no ability of cleaving DNA.Under the guidance of sgRNA(single guide RNA),the dCas9 can interfere the transcription of target gene by the steric hindrance.The system was also named CRISPRi(CRISPR interference).Subsequently,dCas9-KRAB system generated by fusing dCas9 with the KRAB produces a more efficient transcriptional interference.Its suppression efficiency has been greatly improved.The efficiency of the gene transcriptional interference can achieve 90%?95%by the system and it had a very low off-target effect.Therefore,CRISPR/dCas9 system plays an important role in the regulation of gene expression.However,the continuous interference of some genes which have very important physiological functions may lead to abnormal function of the cell.In order to control the expression of the target gene more efficiently and accurately,the Tet-on regulatory system in combination with the CRISPRi system was used to induce the expression of the target gene.The system can not only silence the target gene accurately and efficiently,but also flexibly regulate the inhibition level of target gene expression,so it has important application value in the research of gene function.Granular protein precursor(progranulin,PGRN)as a growth factor protein has a variety of functions,such as the physiological functions of cell proliferation,wound repair,anti-inflammatory and so on.PGRN is highly expressed in many tumor cell lines,such as breast cancer cell line MDA-MB-231,malignant glioma cell line U87 and neuroblastoma cell line SH-SY5Y and so on.Over-expression of PGRN can promote the occurrence of tumor,and the low expression level of PGRN can significantly reduce the formation of tumor,suggesting that PGRN is one of the important molecules in tumor development and can be used as a good target molecule for tumor diagnosis and treatment.However,the molecular mechanisms involving biological function of PGRN in tumor cells is not clear,and the study on the transcriptional regulation of PGRN will lay a foundation for clarifying the molecular mechanism involving its biological function in tumor cells.The aim of this study was to establish an efficient and inducible transcriptional regulatory system.Then,on the basis of the transcriptional regulatory system,SH-SY5Y cell line with transcription inhibition targeting PGRN in Tet-on-induction manner was generated,which provides a new strategy for the study of the role of PGRN in neuroblastoma.This project has been studied from three aspects,and achieved the following results.1.Construction and in vitro study of the transcriptional suppression system targeting PGRN gene by Tet-on induced on the basis of CRISPR/dCas9 system:First,a lentiviral expression vector carrying the Tet-on system was constructed and its inducibility was detected by luciferase reporter gene,the results showed that the induction ratio was 180 times.Next,the expression vector carrying dCas9-KRAB fusion protein was constructed.The sgRNAs(1-4)targeting the promoter region of the human PGRN gene was designed and screened.Then sgRNA expressing cassette was cloned into the vector carrying dCas9-KRAB induced by Tet on system,resulted in the generation of a Tet-on-induced transcriptional inhibition system targeting human PGRN.In order to detect the inhibitory efficiency of the system,we constructed a test vector,pGL3 basic-PGRN promoter-Luciferase.Then the test vector and Tet-on-induced transcriptional inhibition system were co-transfected into HEK293,and the Luciferase expression was detected.The results showed that Tet-on-induced transcriptional inhibition system guided by sgRNA3 could inhibit the exogenous PGRN promoter by 10-fold compared with the control group in the presence of Dox.2.Establishment and studies in vitro of HEK293 and HepG2 cell lines with the knock-in of PGRN-T2A-Luciferase:In order to test the expression of the endogenous PGRN inhibited by the induced transcriptional inhibition system.First,the sgRNAs(1-4)targeting the downstream of PGRN promoter was designed.Then the expression vectors of sgRNAs were constructed.The sgRNA expression vector and the Cas9 expression vector were co-transfected into HEK293 to test the efficiency of sgRNA targeting.The results showed that the targeting efficiency of sgRNA2 was higher than that of others.Next,a donor vector with the homologous up and down arms and T2A-Luciferase exogenous gene for homologous recombination in the targeting region was constructed.The donor vector and the target vector containing the Cas9 and sgRNA2 were co-transfected into HEK293 or HepG2 cell lines.The HEK293 cell line or HepG2 cell line with knock-in of exogenous gene in single allele were obtained and confirmed by cell screening,cell cloning,PCR and sequencing.On the basis of this,the lentiviral vector carrying Tet-on induced transcriptional inhibition system targeting human PGRN was transfected into HEK293 cell line or HepG2 cell line with the knock-in of PGRN-T2A-Luciferase.The results showed that different sgRNA-mediated transcriptional suppression systems targeting the PGRN promoter under the Dox induction conditions had no inhibitory effect on the expression of endogenous PGRN in PGRN-T2A-Luciferase HEK293 cell line.Therefore,the system had a weak inhibitory effect on the expression of endogenous PGRN in PGRN-T2A-Luciferase HepG2 cell line,in which the system guided by sgRNA3 had a better inhibitory effect on the expression of endogenous PGRN under Dox induction in hPGRN-T2A-Luciferase knock-in HepG2 cell lines.3.Establishment and in vitro study of SH-SY5Y cell line with Tet-on-induced transcriptional inhibition system targeting PGRN gene:Through testing of the transcriptional inhibitory activity,the sgRNA3 showed a higher inhibitory activity than others.Therefore,the lentiviral vector carrying sgRNA3 guided Tet-on induced transcriptional inhibition system was packaged and purified.Then the human neuroblastoma cell line SH-SY5Y was infected with the lentivirus to establish a cell line with Tet-on induced transcriptional inhibition system.The expression level of human PGRN mRNA and protein in the established cell lines was detected by Real-time PCR and Western Blot.The results showed that the expression level of PGRN mRNA and protein in SH-SY5Y cell line were significantly decreased under Dox induction.The result indicated that the system could efficiently inhibit the PGRN expression in SH-SY5Y cell line in a Tet-on induction manner,which would lay a foundation for the study of PGRN functions.