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      CRISPR/Cas9 targeting events cause complex deletions and insertions at 17 sites in the mouse genome

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          Abstract

          Although CRISPR/Cas9 genome editing has provided numerous opportunities to interrogate the functional significance of any given genomic site, there is a paucity of data on the extent of molecular scars inflicted on the mouse genome. Here we interrogate the molecular consequences of CRISPR/Cas9-mediated deletions at 17 sites in four loci of the mouse genome. We sequence targeted sites in 632 founder mice and analyse 54 established lines. While the median deletion size using single sgRNAs is 9 bp, we also obtain large deletions of up to 600 bp. Furthermore, we show unreported asymmetric deletions and large insertions of middle repetitive sequences. Simultaneous targeting of distant loci results in the removal of the intervening sequences. Reliable deletion of juxtaposed sites is only achieved through two-step targeting. Our findings also demonstrate that an extended analysis of F1 genotypes is required to obtain conclusive information on the exact molecular consequences of targeting events.

          Abstract

          CRISPR/Cas9 gene editing has been used to generate mutations in several mouse genes. Here, the authors show that targeting events using single guide RNAs cause large deletions at 17 sites in the mouse genome, suggesting that careful genotyping is needed and sequential targeting may avoid such deletions.

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          Most cited references46

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          Genome Engineering of Drosophila with the CRISPR RNA-Guided Cas9 Nuclease

          We have adapted a bacterial CRISPR RNA/Cas9 system to precisely engineer the Drosophila genome and report that Cas9-mediated genomic modifications are efficiently transmitted through the germline. This RNA-guided Cas9 system can be rapidly programmed to generate targeted alleles for probing gene function in Drosophila.
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            CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes.

            The CRISPR-Cas9 RNA-guided DNA endonuclease has contributed to an explosion of advances in the life sciences that have grown from the ability to edit genomes within living cells. In this Review, we summarize CRISPR-based technologies that enable mammalian genome editing and their various applications. We describe recent developments that extend the generality, DNA specificity, product selectivity, and fundamental capabilities of natural CRISPR systems, and we highlight some of the remarkable advancements in basic research, biotechnology, and therapeutics science that these developments have facilitated.
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              Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence.

              Prokaryotic clustered regularly interspaced short palindromic repeat (CRISPR)/Cas (CRISPR-associated sequences) systems provide adaptive immunity against viruses when a spacer sequence of small CRISPR RNA (crRNA) matches a protospacer sequence in the viral genome. Viruses that escape CRISPR/Cas resistance carry point mutations in protospacers, though not all protospacer mutations lead to escape. Here, we show that in the case of Escherichia coli subtype CRISPR/Cas system, the requirements for crRNA matching are strict only for a seven-nucleotide seed region of a protospacer immediately following the essential protospacer-adjacent motif. Mutations in the seed region abolish CRISPR/Cas mediated immunity by reducing the binding affinity of the crRNA-guided Cascade complex to protospacer DNA. We propose that the crRNA seed sequence plays a role in the initial scanning of invader DNA for a match, before base pairing of the full-length spacer occurs, which may enhance the protospacer locating efficiency of the E. coli Cascade complex. In agreement with this proposal, single or multiple mutations within the protospacer but outside the seed region do not lead to escape. The relaxed specificity of the CRISPR/Cas system limits escape possibilities and allows a single crRNA to effectively target numerous related viruses.
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                Author and article information

                Journal
                Nat Commun
                Nat Commun
                Nature Communications
                Nature Publishing Group
                2041-1723
                31 May 2017
                2017
                : 8
                : 15464
                Affiliations
                [1 ]Laboratory of Genetics and Physiology, National Institute of Diabetes and Digestive and Kidney Diseases, US National Institutes of Health , Bethesda, Maryland 20892, USA
                [2 ]Department of Biomedical Science and Engineering, Konkuk University , Seoul 05029, Republic of Korea
                [3 ]Department of Cell and Developmental Biology & Dental Research Institute, Seoul National University , Seoul 110-749, Republic of Korea
                [4 ]Department of Life Systems, Sookmyung Women's University , Seoul 140-742, Republic of Korea
                [5 ]Transgenic Core, National Heart, Lung, and Blood Institute, US National Institutes of Health , Bethesda, Maryland 20892, USA
                Author notes
                [*]

                These authors contributed equally to this work.

                [†]

                These authors jointly supervised this work.

                Author information
                http://orcid.org/0000-0003-2346-2884
                Article
                ncomms15464
                10.1038/ncomms15464
                5460021
                28561021
                c881b34e-18b7-402f-a8e0-eae7f16fb28b
                Copyright © 2017, The Author(s)

                This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

                History
                : 27 August 2016
                : 31 March 2017
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