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      Variability in Genome Editing Outcomes: Challenges for Research Reproducibility and Clinical Safety

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          Abstract

          Genome editing tools have already revolutionized biomedical research and are also expected to have an important impact in the clinic. However, their extensive use in research has revealed much unpredictability, both off and on target, in the outcome of their application. We discuss the challenges associated with this unpredictability, both for research and in the clinic. For the former, an extensive validation of the model is essential. For the latter, potential unpredicted activity does not preclude the use of these tools but requires that molecular evidence to underpin the relevant risk:benefit evaluation is available. Safe and successful clinical application will also depend on the mode of delivery and the cellular context.

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          Abstract

          Genome editing tools have already revolutionized biomedical research and will have a major impact in the clinic. However, the unpredictability of genome editing outcomes raises the question of its safety for human therapy. This article reviews molecular evidences both in animals models and human and their implications for risk:benefit evaluation.

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          Most cited references 122

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          Progress and problems with the use of viral vectors for gene therapy.

          Gene therapy has a history of controversy. Encouraging results are starting to emerge from the clinic, but questions are still being asked about the safety of this new molecular medicine. With the development of a leukaemia-like syndrome in two of the small number of patients that have been cured of a disease by gene therapy, it is timely to contemplate how far this technology has come, and how far it still has to go.
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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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              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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                Author and article information

                Contributors
                Journal
                Mol Ther
                Mol. Ther
                Molecular Therapy
                American Society of Gene & Cell Therapy
                1525-0016
                1525-0024
                03 June 2020
                20 March 2020
                : 28
                : 6
                : 1422-1431
                Affiliations
                [1 ]The Mary Lyon Centre, Medical Research Council Harwell Institute, Harwell Campus, Didcot OX11 0RD, Oxon, UK
                [2 ]Université de Strasbourg, CNRS, INSERM, IGBMC, PHENOMIN-Institut Clinique de la Souris, Celphedia, Strasbourg 67404, France
                [3 ]Great Ormond Street Institute of Child Health, NIHR Biomedical Research Centre, London WC1N 1EH, UK
                Author notes
                []Corresponding author: Lydia Teboul, The Mary Lyon Centre, Medical Research Council Harwell Institute, Harwell Campus, Didcot OX11 0RD, Oxon, UK. l.teboul@ 123456har.mrc.ac.uk
                [∗∗ ]Corresponding author: Waseem Qasim, Great Ormond Street Institute of Child Health NIHR Biomedical Research Centre, London WC1N 1EH, UK. w.qasim@ 123456ucl.ac.uk
                [∗∗∗ ]Corresponding author: Guillaume Pavlovic, Université de Strasbourg, CNRS, INSERM, IGBMC, PHENOMIN-Institut Clinique de la Souris, Celphedia, Strasbourg 67404, France. pavlovic@ 123456igbmc.fr
                Article
                S1525-0016(20)30149-0
                10.1016/j.ymthe.2020.03.015
                7264426
                32243835
                © 2020 The Authors

                This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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