A CRISPR/Cas9 toolkit for multiplex genome editing in plants – ScienceOpen
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      A CRISPR/Cas9 toolkit for multiplex genome editing in plants

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          Abstract

          Background

          To accelerate the application of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein 9) system to a variety of plant species, a toolkit with additional plant selectable markers, more gRNA modules, and easier methods for the assembly of one or more gRNA expression cassettes is required.

          Results

          We developed a CRISPR/Cas9 binary vector set based on the pGreen or pCAMBIA backbone, as well as a gRNA (guide RNA) module vector set, as a toolkit for multiplex genome editing in plants. This toolkit requires no restriction enzymes besides BsaI to generate final constructs harboring maize-codon optimized Cas9 and one or more gRNAs with high efficiency in as little as one cloning step. The toolkit was validated using maize protoplasts, transgenic maize lines, and transgenic Arabidopsis lines and was shown to exhibit high efficiency and specificity. More importantly, using this toolkit, targeted mutations of three Arabidopsis genes were detected in transgenic seedlings of the T1 generation. Moreover, the multiple-gene mutations could be inherited by the next generation.

          Conclusions

          We developed a toolkit that facilitates transient or stable expression of the CRISPR/Cas9 system in a variety of plant species, which will facilitate plant research, as it enables high efficiency generation of mutants bearing multiple gene mutations.

          Electronic supplementary material

          The online version of this article (doi:10.1186/s12870-014-0327-y) contains supplementary material, which is available to authorized users.

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          Most cited references26

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          Efficient In Vivo Genome Editing Using RNA-Guided Nucleases

          Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems have evolved in bacteria and archaea as a defense mechanism to silence foreign nucleic acids of viruses and plasmids. Recent work has shown that bacterial type II CRISPR systems can be adapted to create guide RNAs (gRNAs) capable of directing site-specific DNA cleavage by the Cas9 nuclease in vitro. Here we show that this system can function in vivo to induce targeted genetic modifications in zebrafish embryos with efficiencies comparable to those obtained using ZFNs and TALENs for the same genes. RNA-guided nucleases robustly enabled genome editing at 9 of 11 different sites tested, including two for which TALENs previously failed to induce alterations. These results demonstrate that programmable CRISPR/Cas systems provide a simple, rapid, and highly scalable method for altering genes in vivo, opening the door to using RNA-guided nucleases for genome editing in a wide range of organisms.
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            Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9.

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              Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system.

              A simple and robust method for targeted mutagenesis in zebrafish has long been sought. Previous methods generate monoallelic mutations in the germ line of F0 animals, usually delaying homozygosity for the mutation to the F2 generation. Generation of robust biallelic mutations in the F0 would allow for phenotypic analysis directly in injected animals. Recently the type II prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system has been adapted to serve as a targeted genome mutagenesis tool. Here we report an improved CRISPR/Cas system in zebrafish with custom guide RNAs and a zebrafish codon-optimized Cas9 protein that efficiently targeted a reporter transgene Tg(-5.1mnx1:egfp) and four endogenous loci (tyr, golden, mitfa, and ddx19). Mutagenesis rates reached 75-99%, indicating that most cells contained biallelic mutations. Recessive null-like phenotypes were observed in four of the five targeting cases, supporting high rates of biallelic gene disruption. We also observed efficient germ-line transmission of the Cas9-induced mutations. Finally, five genomic loci can be targeted simultaneously, resulting in multiple loss-of-function phenotypes in the same injected fish. This CRISPR/Cas9 system represents a highly effective and scalable gene knockout method in zebrafish and has the potential for applications in other model organisms.
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                Author and article information

                Contributors
                xinghuili1989@163.com
                li.dong127@gmail.com
                wangzp6119@163.com
                hzzhanghaiyan@163.com
                qy15652815165@163.com
                liubing.9024@163.com
                xcwang@cau.edu.cn
                qjchen@cau.edu.cn
                Journal
                BMC Plant Biol
                BMC Plant Biol
                BMC Plant Biology
                BioMed Central (London )
                1471-2229
                29 November 2014
                29 November 2014
                2014
                : 14
                : 1
                : 327
                Affiliations
                State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing, 100193 China
                Article
                327
                10.1186/s12870-014-0327-y
                4262988
                25432517
                6152dc38-dde8-4003-9b63-84033ec50d38
                © Xing et al.; licensee BioMed Central Ltd. 2014

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. 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
                : 9 October 2014
                : 6 November 2014
                Categories
                Methodology Article
                Custom metadata
                © The Author(s) 2014

                Plant science & Botany
                crispr/cas9,genome editing,multiple gene mutations,assembly of multiple grnas

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