Rapid generation of endogenously driven transcriptional reporters in cells through CRISPR/Cas9.

Alejandro Rojas-Fernandez,Ronald T Hay,Gopal P Sapkota,Christophe Lachaud,Thomas Macartney,Lina Herhaus

doi:10.1038/srep09811

Alejandro Rojas-Fernandez, Ronald T Hay + Show 4 more

Open Access

https://doi.org/10.1038/srep09811

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Abstract

CRISPR/Cas9 technologies have been employed for genome editing to achieve gene knockouts and knock-ins in somatic cells. Similarly, certain endogenous genes have been tagged with fluorescent proteins. Often, the detection of tagged proteins requires high expression and sophisticated tools such as confocal microscopy and flow cytometry. Therefore, a simple, sensitive and robust transcriptional reporter system driven by endogenous promoter for studies into transcriptional regulation is desirable. We report a CRISPR/Cas9-based methodology for rapidly integrating a firefly luciferase gene in somatic cells under the control of endogenous promoter, using the TGFβ-responsive gene PAI-1. Our strategy employed a polycistronic cassette containing a non-fused GFP protein to ensure the detection of transgene delivery and rapid isolation of positive clones. We demonstrate that firefly luciferase cDNA can be efficiently delivered downstream of the promoter of the TGFβ-responsive gene PAI-1. Using chemical and genetic regulators of TGFβ signalling, we show that it mimics the transcriptional regulation of endogenous PAI-1 expression. Our unique approach has the potential to expedite studies on transcription of any gene in the context of its native chromatin landscape in somatic cells, allowing for robust high-throughput chemical and genetic screens.

Highlights

CRISPR/Cas[9] technologies have been employed for genome editing to achieve gene knockouts and knock-ins in somatic cells
We demonstrate that firefly luciferase cDNA can be efficiently delivered downstream of the promoter of the transforming growth factor-beta (TGFb)-responsive gene PAI-1
The CRISPR/Cas[9] methodology has been used for the generation of gene knockouts and knock-ins in cells derived from a multitude of species including human, rats, mice, zebrafish, drosophila, nematodes and the parasite Plasmodium yoelii . 18-30 In some cases, fluorescent proteins such as green fluorescent protein (GFP) have been used for the characterisation and quantification of homologous recombination events as well as for endogenous gene-tagging and determination of subcellular localisation of expressed proteins.[31]

Summary

Introduction

CRISPR/Cas[9] technologies have been employed for genome editing to achieve gene knockouts and knock-ins in somatic cells. Conventional reporters were constructed on a cDNA expression vector carrying either a promoter fragment or repeats of specific nucleotide sequences In both cases, the DNA sequences are designed to recruit their cognate transcription factors followed by a minimal promoter and a gene, such as luciferase, beta lactamase or a fluorescent protein, which is used as a functional readout. A reporter system should engage all components of an endogenous gene regulation, including the native chromatin architecture We have achieved this by using CRISPR/Cas[9] genome engineering technology[8,9,10,11,12] to integrate a luciferase and GFP reporter cassette directly downstream of the endogenous promoter of a transforming growth factor-beta (TGFb) responsive gene PAI-1 (plasminogen activator inhibitor-1)[13]. As the detection of any fluorescent protein relies on the levels of expression, low to very low levels of fluorescent tagged proteins driven by endogenous promoters can be challenging to detect even with sophisticated tools such as confocal microscopy or flow cytometry

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