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

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

Abstract

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 

Notes

Acknowledgments

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

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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

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