Using Genome-Editing Tools to Develop a Novel In Situ Coincidence Reporter Assay for Screening ATAD3A Transcriptional Inhibitors

  • Liwei Lang
  • Yong TengEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2138)


Transgene-based reporter gene assays have been used for discovery of inhibitors targeting vital gene transcription. In traditional assays, the reporter gene is commonly fused with a cloned promoter and integrated into a random genomic location. This has been widely applied but significantly dampened by disadvantages, including incomplete cis-acting elements, the influence of foreign epigenetic environments, and generation of false hits that disrupt the luciferase reporter activity. Therefore, there is a need to develop novel strategies for developing in situ reporter assays closely mimicking endogenous gene expression without disrupting its function. By employing the CRISPR-Cas9 system, we developed an effective in situ coincidence reporter system with a selection marker in the endogenous locus of ATAD3A, which provides a means of screening for transcription-targeted lead compounds with high confidence.

Key words

ATAD3A CRISPR-Cas9 Coincidence reporter In situ Transcriptional inhibition 



This research was supported by NIH grant R03DE028387 and R01DE028351 (to Y.T.).


  1. 1.
    Chau N-M, Rogers P, Aherne W, Carroll V, Collins I, McDonald E et al (2005) Identification of novel small molecule inhibitors of hypoxia-inducible factor-1 that differentially block hypoxia-inducible factor-1 activity and hypoxia-inducible factor-1α induction in response to hypoxic stress and growth factors. Cancer Res 65(11):4918–4928CrossRefGoogle Scholar
  2. 2.
    Lang L, Ding H-F, Chen X, Sun S-Y, Liu G, Yan C (2015) Internal ribosome entry site-based bicistronic in situ reporter assays for discovery of transcription-targeted lead compounds. Chem Biol 22(7):957–964CrossRefGoogle Scholar
  3. 3.
    Dansithong W, Paul S, Scoles DR, Pulst SM, Huynh DP (2015) Generation of SNCA cell models using zinc finger nuclease (ZFN) technology for efficient high-throughput drug screening. PLoS One 10(8):e0136930. Scholar
  4. 4.
    Hasson SA, Fogel AI, Wang C, MacArthur R, Guha R, Heman-Ackah S et al (2015) Chemogenomic profiling of endogenous PARK2 expression using a genome-edited coincidence reporter. ACS Chem Biol 10(5):1188–1197CrossRefGoogle Scholar
  5. 5.
    Yang W, Zhang S, Zhang Y, Huang X (2017) A novel strategy to dissect endogenous gene transcriptional regulation in live cells. Biochem Biophys Res Commun 487(3):573–579CrossRefGoogle Scholar
  6. 6.
    Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F (2013) Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8(11):2281–2308CrossRefGoogle Scholar
  7. 7.
    Auld DS, Southall NT, Jadhav A, Johnson RL, Diller DJ, Simeonov A et al (2008) Characterization of chemical libraries for luciferase inhibitory activity. J Med Chem 51(8):2372–2386CrossRefGoogle Scholar
  8. 8.
    Cheng KC, Inglese J (2012) A coincidence reporter-gene system for high-throughput screening. Nat Methods 9(10):937. Scholar
  9. 9.
    Schuck BW, MacArthur R, Inglese J (2017) Quantitative high-throughput screening using a coincidence reporter biocircuit. Curr Protoc Neurosci 79:5.32.1–5.32.27. Scholar
  10. 10.
    Teng Y, Ren X, Li H, Shull A, Kim J, Cowell JK (2016) Mitochondrial ATAD3A combines with GRP78 to regulate the WASF3 metastasis-promoting protein. Oncogene 35(3):333–343CrossRefGoogle Scholar
  11. 11.
    Dickerson T, Jauregui CE, Teng Y (2017) Friend or foe? Mitochondria as a pharmacological target in cancer treatment. Future Med Chem 9(18):2197–2210CrossRefGoogle Scholar
  12. 12.
    Fang H-Y, Chang C-L, Hsu S-H, Huang C-Y, Chiang S-F, Chiou S-H et al (2010) ATPase family AAA domain-containing 3A is a novel anti-apoptotic factor in lung adenocarcinoma cells. J Cell Sci 123(Pt 7):1171–1180CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Department of Oral Biology and Diagnostic Sciences, Dental College of GeorgiaAugusta UniversityAugustaUSA
  2. 2.Georgia Cancer Center, Department of Biochemistry and Molecular Biology, Medical College of GeorgiaAugusta UniversityAugustaUSA
  3. 3.Department of Medical Laboratory, Imaging and Radiologic Sciences, College of Allied HealthAugusta UniversityAugustaUSA

Personalised recommendations