Rapid generation of endogenously driven transcriptional reporters in cells through CRISPR/Cas9.
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.
Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dow Street, Dundee, DD1 5EH, Scotland, United Kingdom.
Alejandro
A
Rojas-Fernandez
MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee, DD1 5EH, Scotland, United Kingdom.
Lina
L
Herhaus
MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee, DD1 5EH, Scotland, United Kingdom.
Thomas
T
Macartney
MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee, DD1 5EH, Scotland, United Kingdom.
Christophe
C
Lachaud
Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dow Street, Dundee, DD1 5EH, Scotland, United Kingdom.
Ronald T
RT
Hay
MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dow Street, Dundee, DD1 5EH, Scotland, United Kingdom.
Gopal P
GP
Sapkota
D064113:CRISPR-Cas Systems / Q000235:genetics*; D045744:Cell Line, Tumor; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics*; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics; D006801:Humans; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics*; D016212:Transforming Growth Factor beta / Q000235:genetics; D019076:Transgenes / Q000235:genetics; D020663:ras Guanine Nucleotide Exchange Factors
D020662:Guanine Nucleotide Exchange Factors; D017395:Plasminogen Activator Inhibitor 1; C417242:RAPGEF5 protein, human; D016212:Transforming Growth Factor beta; D020663:ras Guanine Nucleotide Exchange Factors