A scalable CRISPR/Cas9-based fluorescent reporter assay to study DNA double-strand break repair choice

Paris Roidos, Stephanie Sungalee, Salvatore Benfatto, Özdemirhan Serçin, Adrian M. Stütz, Amir Abdollahi, Jan Mauer, Frank T. Zenke, Jan O. Korbel and Balca R. Mardin ()
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Paris Roidos: BioMed X Institute (GmbH), Im Neuenheimer Feld 583
Stephanie Sungalee: European Molecular Biology Laboratory, Genome Biology Unit
Salvatore Benfatto: BioMed X Institute (GmbH), Im Neuenheimer Feld 583
Özdemirhan Serçin: BioMed X Institute (GmbH), Im Neuenheimer Feld 583
Adrian M. Stütz: European Molecular Biology Laboratory, Genome Biology Unit
Amir Abdollahi: Division of Molecular and Translational Radiation Oncology, National Centre for Tumour Diseases (NCT), Heidelberg University Hospital
Jan Mauer: BioMed X Institute (GmbH), Im Neuenheimer Feld 583
Frank T. Zenke: Translational Innovation Platform Oncology, Merck KGaA
Jan O. Korbel: European Molecular Biology Laboratory, Genome Biology Unit
Balca R. Mardin: BioMed X Institute (GmbH), Im Neuenheimer Feld 583

Nature Communications, 2020, vol. 11, issue 1, 1-15

Abstract: Abstract Double-strand breaks (DSBs) are the most toxic type of DNA lesions. Cells repair these lesions using either end protection- or end resection-coupled mechanisms. To study DSB repair choice, we present the Color Assay Tracing-Repair (CAT-R) to simultaneously quantify DSB repair via end protection and end resection pathways. CAT-R introduces DSBs using CRISPR/Cas9 in a tandem fluorescent reporter, whose repair distinguishes small insertions/deletions from large deletions. We demonstrate CAT-R applications in chemical and genetic screens. First, we evaluate 21 compounds currently in clinical trials which target the DNA damage response. Second, we examine how 417 factors involved in DNA damage response influence the choice between end protection and end resection. Finally, we show that impairing nucleotide excision repair favors error-free repair, providing an alternative way for improving CRISPR/Cas9-based knock-ins. CAT-R is a high-throughput, versatile assay to assess DSB repair choice, which facilitates comprehensive studies of DNA repair and drug efficiency testing.

Date: 2020
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DOI: 10.1038/s41467-020-17962-3

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