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      Easy quantification of template-directed CRISPR/Cas9 editing

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          Abstract

          Template-directed CRISPR/Cas9 editing is a powerful tool for introducing subtle mutations in genomes. However, the success rate of incorporation of the desired mutations at the target site is difficult to predict and therefore must be empirically determined. Here, we adapted the widely used TIDE method for quantification of templated editing events, including point mutations. The resulting TIDER method is a rapid, cheap and accessible tool for testing and optimization of template-directed genome editing strategies. A free web tool for TIDER data analysis is available at http://tide.nki.nl.

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          High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases.

          Zinc-finger nucleases (ZFNs) have enabled highly efficient gene targeting in multiple cell types and organisms. Here we describe methods for using simple ssDNA oligonucleotides in tandem with ZFNs to efficiently produce human cell lines with three distinct genetic outcomes: (i) targeted point mutation, (ii) targeted genomic deletion of up to 100 kb and (iii) targeted insertion of small genetic elements concomitant with large genomic deletions.
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            Optimization of scarless human stem cell genome editing

            Efficient strategies for precise genome editing in human-induced pluripotent cells (hiPSCs) will enable sophisticated genome engineering for research and clinical purposes. The development of programmable sequence-specific nucleases such as Transcription Activator-Like Effectors Nucleases (TALENs) and Cas9-gRNA allows genetic modifications to be made more efficiently at targeted sites of interest. However, many opportunities remain to optimize these tools and to enlarge their spheres of application. We present several improvements: First, we developed functional re-coded TALEs (reTALEs), which not only enable simple one-pot TALE synthesis but also allow TALE-based applications to be performed using lentiviral vectors. We then compared genome-editing efficiencies in hiPSCs mediated by 15 pairs of reTALENs and Cas9-gRNA targeting CCR5 and optimized ssODN design in conjunction with both methods for introducing specific mutations. We found Cas9-gRNA achieved 7–8× higher non-homologous end joining efficiencies (3%) than reTALENs (0.4%) and moderately superior homology-directed repair efficiencies (1.0 versus 0.6%) when combined with ssODN donors in hiPSCs. Using the optimal design, we demonstrated a streamlined process to generated seamlessly genome corrected hiPSCs within 3 weeks.
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              Nucleosomes impede Cas9 access to DNA in vivo and in vitro

              The prokaryotic CRISPR (clustered regularly interspaced palindromic repeats)-associated protein, Cas9, has been widely adopted as a tool for editing, imaging, and regulating eukaryotic genomes. However, our understanding of how to select single-guide RNAs (sgRNAs) that mediate efficient Cas9 activity is incomplete, as we lack insight into how chromatin impacts Cas9 targeting. To address this gap, we analyzed large-scale genetic screens performed in human cell lines using either nuclease-active or nuclease-dead Cas9 (dCas9). We observed that highly active sgRNAs for Cas9 and dCas9 were found almost exclusively in regions of low nucleosome occupancy. In vitro experiments demonstrated that nucleosomes in fact directly impede Cas9 binding and cleavage, while chromatin remodeling can restore Cas9 access. Our results reveal a critical role of eukaryotic chromatin in dictating the targeting specificity of this transplanted bacterial enzyme, and provide rules for selecting Cas9 target sites distinct from and complementary to those based on sequence properties. DOI: http://dx.doi.org/10.7554/eLife.12677.001
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                Author and article information

                Journal
                Nucleic Acids Res
                Nucleic Acids Res
                nar
                Nucleic Acids Research
                Oxford University Press
                0305-1048
                1362-4962
                01 June 2018
                10 March 2018
                10 March 2018
                : 46
                : 10
                : e58
                Affiliations
                [1 ]Division of Gene Regulation, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands
                [2 ]Division of Molecular Pathology, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands
                [3 ]Division of Tumor Biology & Immunology, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands
                [4 ]Oncode Institute, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands
                Author notes
                To whom correspondence should be addressed. Tel: +31 20 5122040; Email: b.v.steensel@ 123456nki.nl
                Article
                gky164
                10.1093/nar/gky164
                6007333
                29538768
                918898a7-8dcf-4bc9-836f-cd03f5d56188
                © The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@ 123456oup.com

                History
                : 22 February 2018
                : 13 February 2018
                : 06 December 2017
                Page count
                Pages: 9
                Funding
                Funded by: Netherlands Organization for Scientific Research ZonMW-TOP 10.13039/501100001718
                Award ID: 91210033
                Award ID: 91211061
                Funded by: European Research Council 10.13039/501100000781
                Award ID: 694466
                Funded by: Danish Council for Independent Research 10.13039/501100004836
                Award ID: DFF-4183-00599
                Funded by: Danish Cancer Society 10.13039/100008363
                Award ID: R91-A7351-14-S9
                Categories
                Methods Online

                Genetics
                Genetics

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