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

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          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.

          Abstract

          Cells employ different repair pathways to repair DNA double strand breaks. Here, the authors develop a CRISPR/Cas9-dependent method to study choices in DNA repair called the Color Assay Tracing-Repair (CAT-R) which simultaneously measure outcomes of DSB repair via end-protection and end-resection pathways.

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          Most cited references 44

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          Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements

          CRISPR-Cas9 is poised to become the gene editing tool of choice in clinical contexts. Thus far, exploration of Cas9-induced genetic alterations has been limited to the immediate vicinity of the target site and distal off-target sequences, leading to the conclusion that CRISPR-Cas9 was reasonably specific. Here we report significant on-target mutagenesis, such as large deletions and more complex genomic rearrangements at the targeted sites in mouse embryonic stem cells, mouse hematopoietic progenitors and a human differentiated cell line. Using long-read sequencing and long-range PCR genotyping, we show that DNA breaks introduced by single-guide RNA/Cas9 frequently resolved into deletions extending over many kilobases. Furthermore, lesions distal to the cut site and crossover events were identified. The observed genomic damage in mitotically active cells caused by CRISPR-Cas9 editing may have pathogenic consequences.
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            DNA double strand break repair via non-homologous end-joining.

             S. Chen,  J. Davis (2013)
            DNA double-stranded breaks (DSB) are among the most dangerous forms of DNA damage. Unrepaired DSBs results in cells undergoing apoptosis or senescence whereas mis-processing of DSBs can lead to genomic instability and carcinogenesis. One important pathway in eukaryotic cells responsible for the repair of DSBs is non-homologous end-joining (NHEJ). In this review we will discuss the interesting new insights into the mechanism of the NHEJ pathway and the proteins which mediate this repair process. Furthermore, the general role of NHEJ in promoting genomic stability will be discussed.
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              Tracking genome engineering outcome at individual DNA breakpoints

              Site-specific genome engineering technologies are increasingly important tools in the post-genomic era, where biotechnological objectives often require organisms with precisely modified genomes. Rare-cutting endonucleases, through their capacity to create a targeted DNA strand break, are one of the most promising of these technologies. However, realizing the full potential of nuclease-induced genome engineering requires a detailed understanding of the variables that influence resolution of nuclease-induced DNA breaks. Here we present a genome engineering reporter system, designated Traffic Light, that supports rapid flow cytometric analysis of repair pathway choice at individual DNA breaks, quantitative tracking of nuclease expression and donor template delivery, and high throughput screens for factors that bias the engineering outcome. We applied the Traffic Light system to evaluate the efficiency and outcome of nuclease-induced genome engineering in human cell lines and identified strategies to facilitate isolation of cells in which a desired engineering outcome has occurred.
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                Author and article information

                Contributors
                mardin@bio.mx
                Journal
                Nat Commun
                Nat Commun
                Nature Communications
                Nature Publishing Group UK (London )
                2041-1723
                14 August 2020
                14 August 2020
                2020
                : 11
                Affiliations
                [1 ]BioMed X Institute (GmbH), Im Neuenheimer Feld 583, Heidelberg, 69120 Germany
                [2 ]GRID grid.4709.a, ISNI 0000 0004 0495 846X, European Molecular Biology Laboratory, Genome Biology Unit, ; Heidelberg, Germany
                [3 ]GRID grid.5253.1, ISNI 0000 0001 0328 4908, Division of Molecular and Translational Radiation Oncology, National Centre for Tumour Diseases (NCT), Heidelberg University Hospital, ; Heidelberg, Germany
                [4 ]GRID grid.39009.33, ISNI 0000 0001 0672 7022, Translational Innovation Platform Oncology, Merck KGaA, ; Darmstadt, Germany
                [5 ]GRID grid.5333.6, ISNI 0000000121839049, Present Address: Swiss Institute for Experimental Cancer Research (ISREC), School of Life Science, École Polytechnique Fédérale de Lausanne (EPFL), ; Lausanne, Switzerland
                Article
                17962
                10.1038/s41467-020-17962-3
                7429917
                32796846
                © The Author(s) 2020

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                Funding
                Funded by: European Research Council (ERC) starting grant, #336045
                Funded by: FundRef https://doi.org/10.13039/100009945, Merck KGaA;
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                © The Author(s) 2020

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                drug screening, genetic techniques, mechanisms of disease

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