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. 2020:2138:159-166.
doi: 10.1007/978-1-0716-0471-7_8.

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

Liwei Lang  1 , Yong Teng  2   3   4
Affiliations

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

Liwei Lang et al. Methods Mol Biol. 2020.

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.

Keywords: ATAD3A; CRISPR-Cas9; Coincidence reporter; In situ; Transcriptional inhibition.

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Figures

Fig. 1
Fig. 1
The in situ coincidence reporter gene assay in searching for the transcriptional inhibitors of ATAD3A. (a) Schematic of pCI9.4-ATAD3A. The coincidence reporter genes and pac gene are adjacent to homology arms of ATAD3A. LA homology left arm, RA homology right arm, pac puromycin resistance gene, FLuc firefly luciferase, NLuc nanoluciferase. (b) Schematic figure showing insertion of a cassette into in the native ATAD3A locus on Chromosome 1. The cassette contains coincidence reporter genes (FLuc-P2A-NLuc) and the pac gene. TSS transcriptional start site
See this image and copyright information in PMC

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