Establishment And Experimental Study Of SREBPs-T2A-luciferase Knock-in Cell Line Based On CRISPR/Cas9 Targeted Genome Modification Technology

Sterol regulatory element binding proteins(SREBPs)are key transcriptional factors that directly activate the transcription of more than 30 genes by binding to the sterol regulatory element sequence(SER)in the downstream gene promoter,which play important roles in the regulation of lipid metabolism signal network.SREBP has 3 isomers including SREBP-1a,SREBP-1c and SREBP-2.Recent studies have shown that lipid metabolic signal transduction pathway plays an important role in the development and progression of tumors.Therefore,the study of the transcriptional regulation of SREBP molecules involving lipid metabolism signal network is of great significance to explore the molecular mechanisms of related metabolic diseases and the study of drug screening for treatment of related diseases.The CRISPR/Cas9 system is a novel targeted genome editing technology after the zinc finger nucleases(ZFNs)and transcription-activator-like effects of nucleases(TALENs),which has been widely used in the study of gene function for its simple design time consuming and efficient.To investigate the transcriptional regulation of SREBP gene,a luciferase knock-in reporter system was established in HEK293 and HepG2 cells by inserting the T2A-Luciferase gene in the 3-terminal located right before the stop codon of the SREBPs gene using clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated system(Cas)mediated genome editing technique.PCR results confirmed the site-specific integration of a single copy of exogenous luciferase gene into the genome.To prove the reliability and sensitivity of T2A-Luciferase-KI cell line,a CRISPR/dCas transcription activation/repression system for the SREBP gene was constructed and applied to the cell line.The dCas9(dead Cas9)with the double mutation of D10A and H840A sites can bind to the DNA,but has no ability of cleaving DNA.dCas9 fused with the VP64 activator or Kruppel associated box inhibitor domain(KRAB)could be recruited to target gene promoter region under the guidance of sgRNA(single guide RNA,sgRNA),and implements transcription activation or repression of target gene.Further,the transcription factor and some drugs that were proven to regulate the expression of SREBP gene was tested in the generated cell line.The results showed thatluciferase activity was directly correlated with the SREBP endogenous promoter activity in T2A-Luciferase-KI cell line.This novel cell line would be a useful tool to study transcriptional regulation of SREBP gene,and has great potential in screening the drug or chemical molecules for regulating the expression of SREBP gene.The specific research contents of the proiect were as follows:(1)Designing of biological activity screening of sgRNAs targeting downstream region of 3-terminal of stop codon of SREBP gene.The human genome sequences of SREBP1&2 were obtained through NCBI,then the downstream region of 3 terminal of the stop codon of SREBP1&2 genes were selected as the target region.Four sgRNA binding sites with higher specificity were selected in this region by sequence comparison.The sgRNA expression vectors were constructed and detected by cell transfection and T7 El assay.The sgRNAs with higher biological cleavage activity were obtained,which laid a foundation for the establishment of subsequent cell lines.(2)Construction of the donor vector targeting SREBP gene.The targeting vector was designed and successfully constructed for the insertion of luciferase reporter gene into C-terminal of SREBPs gene in genome.The targeting vector contains a positive screening element,eGFP and Neomycin,between upstream and downstream homologous arm and a negative screening element,mCherry and TK,on the outer side of the homologous arm.Positive and negative screening expression elements in the target vector can directly distinguish homologous recombination from random integrated cells through different reporter genes,but also enrich homologous recombinant cells by the screening of Neo and TK suicide gene,which can efficiently increase the proportion of the positive cells with homologous recombination.(3)Establishment of SREBP1/SREBP2-T2A-luciferase-KI HEK293 and SREBP1/SREBP2-T2A-luciferase-KI HepG2 cell lines.The single cell was obtained by limited dilution cloning based on the screened cells,followed by the validation of PCR and sequencing.The positive cells named SREBP1/SREBP2-T2A-luciferase-KI HEK293 cell line and SREBP1/SREBP2-T2A-luciferase-KI HepG2 cell line,respectively.(4)Construction of sgRNA expression vector targeting SREBP1&2 promoters region.According to the literature,the promoter regions of SREBP1&2 genes were obtained and 8 sgRNAs recognition target sites were selected according to the sgRNA design principle.The corresponding primers were synthesized,respectively,annealed and ligated with sgRNA expression vector.Through the sequencing validation,sgRNA1-8 expression vectors targeting SREBP1&2 promoters were finally obtained.The construction of the sgRNA expression vectors provides the possibility for the study of transcriptional regulation of SREBP gene.(5)The study of SREBP1/SREBP2-T2A-luciferase-KI HEK293 and SREBP1/SREBP2-T2A-luciferase-KI HepG2 cell line in vitro.CRISPR/dCas-mediated transcriptional activation and repression system were constructed by fusing dCas9 with VP64 or KRAB.Then CRISPR/dCas-mediated transcriptional activation and repression system,the related transcription factors affecting the expression of SREBPs gene and the related drugs affecting the expression of SREBPs gene were applied to the cell lines for testing the transcriptional regulation of the target genes.The results showed that the expression level of luciferase reporter was correlated with the expression level of endogenous gene.In summary,.T2A-Luciferase reporter gene was for the first time specifically integrated into 3 terminal of SREBP gene in the genome by CRISPR/Cas9 technology in 3 terminal,resulted in the establishment of SREBP1/2-T2A-luciferase-KI HEK293 and SREBP1/2-T2A-luciferase-KI HepG2 cell lines.The exogenous gene integration in the genome was verified by sequencing.At the same time,CRISPR/dCas9 system mediated transcriptional regulation system,transcription factors and drugs were applied to the cells for testing the transcriptional regulation of target genes of the SREBP1-T2A-luciferase-KI and SREBP2-T2A-luciferase-KI cell lines.The results showed that the luciferase expression level of SREBP1-T2A-luciferase-KI and SREBP2-T2A-luciferase-KI cell lines can accurately reflect the expression level of the SREBP genes of the cells.The establishment of the cell lines provides a new solution strategy for the study of the molecular mechanisms involving the interaction of related lipid metabolism molecules and the screening of small chemical drugs which effect the expression of SREBPs.Meanwhile,the method that monitors the expression of target gene by integrating luciferase reporter in the downstream of target gene is able to be widely applied to other kinds of gene research.