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      Use of CRISPR systems in plant genome editing: toward new opportunities in agriculture

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          Initially discovered in bacteria and archaea, CRISPR–Cas9 is an adaptive immune system found in prokaryotes. In 2012, scientists found a way to use it as a genome editing tool. In 2013, its application in plants was successfully achieved. This breakthrough has opened up many new opportunities for researchers, including the opportunity to gain a better understanding of plant biological systems more quickly. The present study reviews agricultural applications related to the use of CRISPR systems in plants from 52 peer-reviewed articles published since 2014. Based on this literature review, the main use of CRISPR systems is to achieve improved yield performance, biofortification, biotic and abiotic stress tolerance, with rice ( Oryza sativa) being the most studied crop.

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          Most cited references 54

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          Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis.

          The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) system has emerged as a powerful tool for targeted gene editing in many organisms, including plants. However, all of the reported studies in plants focused on either transient systems or the first generation after the CRISPR/Cas system was stably transformed into plants. In this study we examined several plant generations with seven genes at 12 different target sites to determine the patterns, efficiency, specificity, and heritability of CRISPR/Cas-induced gene mutations or corrections in Arabidopsis. The proportion of plants bearing any mutations (chimeric, heterozygous, biallelic, or homozygous) was 71.2% at T1, 58.3% at T2, and 79.4% at T3 generations. CRISPR/Cas-induced mutations were predominantly 1 bp insertion and short deletions. Gene modifications detected in T1 plants occurred mostly in somatic cells, and consequently there were no T1 plants that were homozygous for a gene modification event. In contrast, ∼22% of T2 plants were found to be homozygous for a modified gene. All homozygotes were stable to the next generation, without any new modifications at the target sites. There was no indication of any off-target mutations by examining the target sites and sequences highly homologous to the target sites and by in-depth whole-genome sequencing. Together our results show that the CRISPR/Cas system is a useful tool for generating versatile and heritable modifications specifically at target genes in plants.
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            Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana.

            Engineered nucleases can be used to induce site-specific double-strand breaks (DSBs) in plant genomes. Thus, homologous recombination (HR) can be enhanced and targeted mutagenesis can be achieved by error-prone non-homologous end-joining (NHEJ). Recently, the bacterial CRISPR/Cas9 system was used for DSB induction in plants to promote HR and NHEJ. Cas9 can also be engineered to work as a nickase inducing single-strand breaks (SSBs). Here we show that only the nuclease but not the nickase is an efficient tool for NHEJ-mediated mutagenesis in plants. We demonstrate the stable inheritance of nuclease-induced targeted mutagenesis events in the ADH1 and TT4 genes of Arabidopsis thaliana at frequencies from 2.5 up to 70.0%. Deep sequencing analysis revealed NHEJ-mediated DSB repair in about a third of all reads in T1 plants. In contrast, applying the nickase resulted in the reduction of mutation frequency by at least 740-fold. Nevertheless, the nickase is able to induce HR at similar efficiencies as the nuclease or the homing endonuclease I-SceI. Two different types of somatic HR mechanisms, recombination between tandemly arranged direct repeats as well as gene conversion using the information on an inverted repeat could be enhanced by the nickase to a similar extent as by DSB-inducing enzymes. Thus, the Cas9 nickase has the potential to become an important tool for genome engineering in plants. It should not only be applicable for HR-mediated gene targeting systems but also by the combined action of two nickases as DSB-inducing agents excluding off-target effects in homologous genomic regions. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.
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              Precise base editing in rice, wheat and maize with a Cas9- cytidine deaminase fusion

              Single DNA base pairs are edited in wheat, rice and maize using a Cas9 nickase fusion protein.

                Author and article information

                Emerg Top Life Sci
                Emerg Top Life Sci
                Emerging Topics in Life Sciences
                Portland Press Ltd.
                10 November 2017
                10 November 2017
                : 1
                : 2 , Gene Editing in Agriculture: Biotechnology and Biosafety
                : 169-182
                [1 ]AgroParisTech, 16 rue Claude Bernard, Paris F-75231, France
                [2 ]Université de Paris-Sud, Faculté Jean-Monnet, Collège d'Etudes Interdisciplinaires, 54, Boulevard Desgranges, Sceaux F-92330, France
                [3 ]John Innes Centre, Norwich Research Park, Norwich NR4 7UH, U.K.
                Author notes
                Correspondence: Agnès Ricroch ( agnes.ricroch@ )
                © 2017 The Author(s)

                This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and the Royal Society of Biology and distributed under the Creative Commons Attribution License 4.0 (CC BY).

                Plant Biology
                Agricultural & Industrial Bioscience
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                plant genome editing, grna, crispr, agriculture


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