Biotechnology Letters

, Volume 40, Issue 11–12, pp 1495–1506 | Cite as

Establishment of a HEK293 cell line by CRISPR/Cas9-mediated luciferase knock-in to study transcriptional regulation of the human SREBP1 gene

  • Zihang Li
  • Junli Zhao
  • Niaz Muhammad
  • Dongyang Wang
  • Qinwen Mao
  • Haibin XiaEmail author
Original Research Paper



To establish a HEK293 cell line with a luciferase knock-in reporter controlled by the endogenous SREBP1 promoter for investigating transcriptional regulation of the SREBP1 gene.


PCR confirmed the site-specific integration of a single copy of the exogenous luciferase gene into one allele of the genome and a 14 bp deletion of the targeted sequence in the other. Luciferase activity was directly correlated with the promoter activity of the endogenous SREBP1 gene in the HEK293-SREBP1-T2A-luciferase-KI cell line cell line.


We successfully generated a novel luciferase knock-in reporter system, which will be very useful for studying transcriptional regulation of the SREBP1 gene and for screening drugs or chemical molecules that regulate SREBP1 gene expression.


SREBP1 CRISPR/Cas9 Transcriptional regulation Knock-in Cell line 



This work was supported by the Fundamental Research Funds for the Central Universities, Shaanxi Normal University (No. 2016CSY015), and research grants to H.X. from the National Natural Science Foundation of China (No. 81471772 and No. 81773265) and the Key Research and Development Plan of Shaanxi Province (No. 2018SF-106).

Supporting information

Supplemental Fig. 1—Assay of the pGL3-SREBP1p-mCherry vector in HEK293 cells. Control vector pGL3-mCherry and experimental vector pGL3-SREBP1p-mCherry were transfected into HEK293 cells for 48 hours, respectively. Then, mCherry expression was detected under fluorescence microscope in the control vector (A and B) and experimental vector (C and D).

Supplemental Fig. 2—Illustration of the position of the sgRNA target sequence in the genome. The target regions of sgRNA were graphed according to their location, between − 1470 bp and + 1 bp of the SREBP1 transcription start site (TSS).

Supplemental Table 1—Oligonucleotide sequences for SREBP1 sgRNAs.

Supplemental Table 2—PCR primers used in this study.

Supplemental Table 3—Oligonucleotide sequences for SREBP1 promoter sgRNAs.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

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  1. Adams CM, Reitz J, De Brabander JK, Feramisco JD, Li L et al (2004) Cholesterol and 25-hydroxycholesterol inhibit activation of SREBPs by different mechanisms, both involving SCAP and Insigs. J Biol Chem 279:52772–52780CrossRefGoogle Scholar
  2. Brown MS, Goldstein JL (1997) The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell 89:331–340CrossRefGoogle Scholar
  3. Dif N, Euthine V, Gonnet E, Laville M, Vidal H et al (2006) Insulin activates human sterol-regulatory-element-binding protein-1c (SREBP-1c) promoter through SRE motifs. Biochem J 400:179–188CrossRefGoogle Scholar
  4. Guillet-Deniau I, Mieulet V, Le Lay S, Achouri Y, Carre D et al (2002) Sterol regulatory element binding protein-1c expression and action in rat muscles: insulin-like effects on the control of glycolytic and lipogenic enzymes and UCP3 gene expression. Diabetes 51:1722–1728CrossRefGoogle Scholar
  5. Jeon BN, Kim YS, Choi WI, Koh DI, Kim MK et al (2012) Kr-pok increases FASN expression by modulating the DNA binding of SREBP-1c and Sp1 at the proximal promoter. J Lipid Res 53:755–766CrossRefGoogle Scholar
  6. Konermann S, Brigham MD, Trevino AE, Joung J, Abudayyeh OO et al (2015) Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature 517:583–588CrossRefGoogle Scholar
  7. Millatt LJ, Bocher V, Fruchart JC, Staels B (2003) Liver X receptors and the control of cholesterol homeostasis: potential therapeutic targets for the treatment of atherosclerosis. Biochim Biophys Acta 1631:107–118CrossRefGoogle Scholar
  8. Nakakuki M, Shimano H, Inoue N, Tamura M, Matsuzaka T et al (2007) A transcription factor of lipid synthesis, sterol regulatory element-binding protein (SREBP)-1a causes G(1) cell-cycle arrest after accumulation of cyclin-dependent kinase (cdk) inhibitors. FEBS J 274:4440–4452CrossRefGoogle Scholar
  9. Oh GS, Kim G, Yoon J, Kim GH, Kim SW (2015) The E-box-like sterol regulatory element mediates the insulin-stimulated expression of hepatic clusterin. Biochem Biophys Res Commun 465:501–506CrossRefGoogle Scholar
  10. Saito K, Ishizaka N, Hara M, Matsuzaki G, Sata M et al (2005) Lipid accumulation and transforming growth factor-beta upregulation in the kidneys of rats administered angiotensin II. Hypertension 46:1180–1185CrossRefGoogle Scholar
  11. Shao F, Ford DA (2014) Elaidic acid increases hepatic lipogenesis by mediating sterol regulatory element binding protein-1c activity in HuH-7 cells. Lipids 49:403–413CrossRefGoogle Scholar
  12. Shibata C, Kishikawa T, Otsuka M, Ohno M, Yoshikawa T et al (2013) Inhibition of microRNA122 decreases SREBP1 expression by modulating suppressor of cytokine signaling 3 expression. Biochem Biophys Res Commun 438:230–235CrossRefGoogle Scholar
  13. Taskinen MR (2003) Diabetic dyslipidaemia: from basic research to clinical practice. Diabetologia 46:733–749CrossRefGoogle Scholar
  14. Thakore PI, D’Ippolito AM, Song L, Safi A, Shivakumar NK et al (2015) Highly specific epigenome editing by CRISPR-Cas9 repressors for silencing of distal regulatory elements. Nat Methods 12:1143–1149CrossRefGoogle Scholar
  15. Xiao D, Zhang W, Li Y, Liu K, Zhao J et al (2016) A novel luciferase knock-in reporter system for studying transcriptional regulation of the human Sox2 gene. J Biotechnol 219:110–116CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  1. 1.Laboratory of Gene Therapy, Department of Biochemistry, College of Life SciencesShaanxi Normal UniversityXi’anPeople’s Republic of China
  2. 2.Department of PathologyNorthwestern University Feinberg School of MedicineChicagoUSA

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