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      Tracking genome engineering outcome at individual DNA breakpoints

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          Abstract

          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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          Most cited references29

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          XRCC3 promotes homology-directed repair of DNA damage in mammalian cells.

          Homology-directed repair of DNA damage has recently emerged as a major mechanism for the maintenance of genomic integrity in mammalian cells. The highly conserved strand transferase, Rad51, is expected to be critical for this process. XRCC3 possesses a limited sequence similarity to Rad51 and interacts with it. Using a novel fluorescence-based assay, we demonstrate here that error-free homology-directed repair of DNA double-strand breaks is decreased 25-fold in an XRCC3-deficient hamster cell line and can be restored to wild-type levels through XRCC3 expression. These results establish that XRCC3-mediated homologous recombination can reverse DNA damage that would otherwise be mutagenic or lethal.
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            Regulation of DNA double-strand break repair pathway choice.

            DNA double-strand breaks (DSBs) are critical lesions that can result in cell death or a wide variety of genetic alterations including large- or small-scale deletions, loss of heterozygosity, translocations, and chromosome loss. DSBs are repaired by non-homologous end-joining (NHEJ) and homologous recombination (HR), and defects in these pathways cause genome instability and promote tumorigenesis. DSBs arise from endogenous sources including reactive oxygen species generated during cellular metabolism, collapsed replication forks, and nucleases, and from exogenous sources including ionizing radiation and chemicals that directly or indirectly damage DNA and are commonly used in cancer therapy. The DSB repair pathways appear to compete for DSBs, but the balance between them differs widely among species, between different cell types of a single species, and during different cell cycle phases of a single cell type. Here we review the regulatory factors that regulate DSB repair by NHEJ and HR in yeast and higher eukaryotes. These factors include regulated expression and phosphorylation of repair proteins, chromatin modulation of repair factor accessibility, and the availability of homologous repair templates. While most DSB repair proteins appear to function exclusively in NHEJ or HR, a number of proteins influence both pathways, including the MRE11/RAD50/NBS1(XRS2) complex, BRCA1, histone H2AX, PARP-1, RAD18, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), and ATM. DNA-PKcs plays a role in mammalian NHEJ, but it also influences HR through a complex regulatory network that may involve crosstalk with ATM, and the regulation of at least 12 proteins involved in HR that are phosphorylated by DNA-PKcs and/or ATM.
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              Regulation of DNA repair throughout the cell cycle.

              The repair of DNA lesions that occur endogenously or in response to diverse genotoxic stresses is indispensable for genome integrity. DNA lesions activate checkpoint pathways that regulate specific DNA-repair mechanisms in the different phases of the cell cycle. Checkpoint-arrested cells resume cell-cycle progression once damage has been repaired, whereas cells with unrepairable DNA lesions undergo permanent cell-cycle arrest or apoptosis. Recent studies have provided insights into the mechanisms that contribute to DNA repair in specific cell-cycle phases and have highlighted the mechanisms that ensure cell-cycle progression or arrest in normal and cancerous cells.
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                Author and article information

                Journal
                101215604
                32338
                Nat Methods
                Nat. Methods
                Nature methods
                1548-7091
                1548-7105
                1 August 2011
                10 July 2011
                09 August 2012
                : 8
                : 8
                : 671-676
                Affiliations
                [1 ]Program in Molecular and Cellular Biology, University of Washington, Seattle, Washington
                [2 ]Center of Immunity and Immunotherapies, Seattle Children's Research Institute, Seattle, Washington
                [3 ]Quellos High Throughput Core, Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA
                [4 ]Department of Immunology, University of Washington, Seattle, Washington
                Author notes
                [* ]Corresponding author, andrewms@ 123456u.washington.edu
                Article
                nihpa305598
                10.1038/nmeth.1648
                3415300
                21743461
                f7e7ffa8-d9fc-4151-9c13-66aa12a393c8

                Users may view, print, copy, download and text and data- mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms

                History
                Funding
                Funded by: National Center for Research Resources : NCRR
                Award ID: UL1 RR024921-01 || RR
                Funded by: National Institute of Dental and Craniofacial Research : NIDCR
                Award ID: UL1 DE019582-05 || DE
                Funded by: National Institute of Dental and Craniofacial Research : NIDCR
                Award ID: UL1 DE019582-04 || DE
                Funded by: National Institute of Dental and Craniofacial Research : NIDCR
                Award ID: UL1 DE019582-03 || DE
                Funded by: National Institute of Dental and Craniofacial Research : NIDCR
                Award ID: UL1 DE019582-02 || DE
                Funded by: National Cancer Institute : NCI
                Award ID: RL1 CA133832-05 || CA
                Funded by: National Cancer Institute : NCI
                Award ID: RL1 CA133832-04 || CA
                Funded by: National Cancer Institute : NCI
                Award ID: RL1 CA133832-03 || CA
                Funded by: National Cancer Institute : NCI
                Award ID: RL1 CA133832-02 || CA
                Funded by: National Cancer Institute : NCI
                Award ID: RL1 CA133832-01 || CA
                Funded by: National Institute of Allergy and Infectious Diseases Extramural Activities : NIAID
                Award ID: R21 AI064581-02 || AI
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                Article

                Life sciences
                Life sciences

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