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      Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9-mediated double-stranded DNA cleavage

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          Abstract

          Background

          Precise genome editing via homology-directed repair (HDR) after double-stranded DNA (dsDNA) cleavage facilitates functional genomic research and holds promise for gene therapy. However, HDR efficiency remains low in some cell types, including some of great research and clinical interest, such as human induced pluripotent stem cells (iPSCs).

          Results

          Here, we show that a double cut HDR donor, which is flanked by single guide RNA (sgRNA)-PAM sequences and is released after CRISPR/Cas9 cleavage, increases HDR efficiency by twofold to fivefold relative to circular plasmid donors at one genomic locus in 293 T cells and two distinct genomic loci in iPSCs. We find that a 600 bp homology in both arms leads to high-level genome knockin, with 97–100% of the donor insertion events being mediated by HDR. The combined use of CCND1, a cyclin that functions in G1/S transition, and nocodazole, a G2/M phase synchronizer, doubles HDR efficiency to up to 30% in iPSCs.

          Conclusions

          Taken together, these findings provide guidance for the design of HDR donor vectors and the selection of HDR-enhancing factors for applications in genome research and precision medicine.

          Electronic supplementary material

          The online version of this article (doi:10.1186/s13059-017-1164-8) contains supplementary material, which is available to authorized users.

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          Most cited references29

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          A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum

          Despite the existence of fluorescent proteins spanning the entire visual spectrum, the bulk of modern imaging experiments continue to rely on variants of the green fluorescent protein derived from Aequorea victoria. Meanwhile, a great deal of recent effort has been devoted to engineering and improving red fluorescent proteins, and relatively little attention has been given to green and yellow variants. Here we report a novel monomeric yellow-green fluorescent protein, mNeonGreen, which is derived from a tetrameric fluorescent protein from the cephalochordate Branchiostoma lanceolatum. This fluorescent protein is the brightest monomeric green or yellow fluorescent protein yet described, performs exceptionally well as a fusion tag for traditional imaging as well as stochastic single-molecule superresolution imaging, and is an excellent FRET acceptor for the newest generation of cyan fluorescent proteins.
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            Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells.

            CRISPR-Cas-mediated genome editing relies on guide RNAs that direct site-specific DNA cleavage facilitated by the Cas endonuclease. Here we report that chemical alterations to synthesized single guide RNAs (sgRNAs) enhance genome editing efficiency in human primary T cells and CD34(+) hematopoietic stem and progenitor cells. Co-delivering chemically modified sgRNAs with Cas9 mRNA or protein is an efficient RNA- or ribonucleoprotein (RNP)-based delivery method for the CRISPR-Cas system, without the toxicity associated with DNA delivery. This approach is a simple and effective way to streamline the development of genome editing with the potential to accelerate a wide array of biotechnological and therapeutic applications of the CRISPR-Cas technology.
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              • Article: not found

              Pathways of DNA double-strand break repair during the mammalian cell cycle.

              Little is known about the quantitative contributions of nonhomologous end joining (NHEJ) and homologous recombination (HR) to DNA double-strand break (DSB) repair in different cell cycle phases after physiologically relevant doses of ionizing radiation. Using immunofluorescence detection of gamma-H2AX nuclear foci as a novel approach for monitoring the repair of DSBs, we show here that NHEJ-defective hamster cells (CHO mutant V3 cells) have strongly reduced repair in all cell cycle phases after 1 Gy of irradiation. In contrast, HR-defective CHO irs1SF cells have a minor repair defect in G(1), greater impairment in S, and a substantial defect in late S/G(2). Furthermore, the radiosensitivity of irs1SF cells is slight in G(1) but dramatically higher in late S/G(2), while V3 cells show high sensitivity throughout the cell cycle. These findings show that NHEJ is important in all cell cycle phases, while HR is particularly important in late S/G(2), where both pathways contribute to repair and radioresistance. In contrast to DSBs produced by ionizing radiation, DSBs produced by the replication inhibitor aphidicolin are repaired entirely by HR. irs1SF, but not V3, cells show hypersensitivity to aphidicolin treatment. These data provide the first evaluation of the cell cycle-specific contributions of NHEJ and HR to the repair of radiation-induced versus replication-associated DSBs.
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                Author and article information

                Contributors
                chengtao@ihcams.ac.cn
                909-651-5886 , xzhang@llu.edu
                Journal
                Genome Biol
                Genome Biol
                Genome Biology
                BioMed Central (London )
                1474-7596
                1474-760X
                20 February 2017
                20 February 2017
                2017
                : 18
                : 35
                Affiliations
                [1 ]State Key Laboratory of Experimental Hematology, Tianjin, China
                [2 ]GRID grid.461843.c, , Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, ; Tianjin, China
                [3 ]Center for Stem Cell Medicine, Chinese Academy of Medical Sciences, Tianjin, China
                [4 ]ISNI 0000 0001 0662 3178, GRID grid.12527.33, Department of Stem Cell & Regenerative Medicine, , Peking Union Medical College, ; Tianjin, China
                [5 ]Collaborative Innovation Center for Cancer Medicine, Tianjin, China
                [6 ]Tianjin Key Laboratory of Blood Cell Therapy and Technology, Tianjin, China
                [7 ]ISNI 0000 0000 9852 649X, GRID grid.43582.38, Division of Regenerative Medicine MC1528B, Department of Medicine, , Loma Linda University, ; 11234 Anderson Street, Loma Linda, CA 92354 USA
                [8 ]ISNI 0000 0000 9852 649X, GRID grid.43582.38, Department of Orthopaedic Surgery, , Loma Linda University, ; Loma Linda, CA USA
                Article
                1164
                10.1186/s13059-017-1164-8
                5319046
                28219395
                ed0ad327-af11-4476-abe9-c63bb075c217
                © The Author(s). 2017

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided 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 Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 27 October 2016
                : 27 January 2017
                Funding
                Funded by: Ministry of Science and Technology of the People's Republic of China (CN)
                Award ID: 2015CB964902, 2013CB966902 and 2012CB966601
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/501100001809, National Natural Science Foundation of China ;
                Award ID: 81500148, 81570164 and 81421002
                Award Recipient :
                Funded by: the Loma Linda University School of Medicine GCAT grant (2015)
                Funded by: FundRef http://dx.doi.org/10.13039/100006965, Telemedicine and Advanced Technology Research Center;
                Award ID: W81XWH-08-1-0697
                Categories
                Research
                Custom metadata
                © The Author(s) 2017

                Genetics
                crispr,genome editing,knockin,homology-directed repair (hdr),non-homologous end joining (nhej),donor design,293 t,human induced pluripotent stem cells

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