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      Development of Improved Fruit, Vegetable, and Ornamental Crops Using the CRISPR/Cas9 Genome Editing Technique

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          Abstract

          Horticultural crops, including fruit, vegetable, and ornamental plants are an important component of the agriculture production systems and play an important role in sustaining human life. With a steady growth in the world’s population and the consequent need for more food, sustainable and increased fruit and vegetable crop production is a major challenge to guarantee future food security. Although conventional breeding techniques have significantly contributed to the development of important varieties, new approaches are required to further improve horticultural crop production. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) has emerged as a valuable genome-editing tool able to change DNA sequences at precisely chosen loci. The CRISPR/Cas9 system was developed based on the bacterial adaptive immune system and comprises of an endonuclease guided by one or more single-guide RNAs to generate double-strand breaks. These breaks can then be repaired by the natural cellular repair mechanisms, during which genetic mutations are introduced. In a short time, the CRISPR/Cas9 system has become a popular genome-editing technique, with numerous examples of gene mutation and transcriptional regulation control in both model and crop plants. In this review, various aspects of the CRISPR/Cas9 system are explored, including a general presentation of the function of the CRISPR/Cas9 system in bacteria and its practical application as a biotechnological tool for editing plant genomes, particularly in horticultural crops.

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          Most cited references130

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          Targeting DNA double-strand breaks with TAL effector nucleases.

          Engineered nucleases that cleave specific DNA sequences in vivo are valuable reagents for targeted mutagenesis. Here we report a new class of sequence-specific nucleases created by fusing transcription activator-like effectors (TALEs) to the catalytic domain of the FokI endonuclease. Both native and custom TALE-nuclease fusions direct DNA double-strand breaks to specific, targeted sites.
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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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              Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice

              The type II CRISPR/Cas system from Streptococcus pyogenes and its simplified derivative, the Cas9/single guide RNA (sgRNA) system, have emerged as potent new tools for targeted gene knockout in bacteria, yeast, fruit fly, zebrafish and human cells. Here, we describe adaptations of these systems leading to successful expression of the Cas9/sgRNA system in two dicot plant species, Arabidopsis and tobacco, and two monocot crop species, rice and sorghum. Agrobacterium tumefaciens was used for delivery of genes encoding Cas9, sgRNA and a non-fuctional, mutant green fluorescence protein (GFP) to Arabidopsis and tobacco. The mutant GFP gene contained target sites in its 5′ coding regions that were successfully cleaved by a CAS9/sgRNA complex that, along with error-prone DNA repair, resulted in creation of functional GFP genes. DNA sequencing confirmed Cas9/sgRNA-mediated mutagenesis at the target site. Rice protoplast cells transformed with Cas9/sgRNA constructs targeting the promoter region of the bacterial blight susceptibility genes, OsSWEET14 and OsSWEET11, were confirmed by DNA sequencing to contain mutated DNA sequences at the target sites. Successful demonstration of the Cas9/sgRNA system in model plant and crop species bodes well for its near-term use as a facile and powerful means of plant genetic engineering for scientific and agricultural applications.
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                Author and article information

                Journal
                Plants (Basel)
                Plants (Basel)
                plants
                Plants
                MDPI
                2223-7747
                13 December 2019
                December 2019
                : 8
                : 12
                : 601
                Affiliations
                [1 ]Department of Agronomy, Universidade Estadual de Londrina, Londrina 86057-970, PR, Brazil; ligiacorte@ 123456uel.br
                [2 ]Pomology Department, Faculty of Agriculture, Mansoura University, 35516 Mansoura, Egypt; lamiaa.mahmoud@ 123456ufl.edu
                [3 ]Citrus Research and Education Center, University of Florida, Lake Alfred, FL 33850, USA; jgrosser@ 123456ufl.edu
                [4 ]Centro de Energia Nuclear na Agricultura, Universidade de São Paulo, Piracicaba 13416-000, SP, Brazil; t.moraes@ 123456usp.br
                [5 ]Department of Microbiology and Cell Sciences, University of Florida, Gainesville, FL 32603, USA; zhlmou@ 123456ufl.edu
                Author notes
                [* ]Correspondence: manjul@ 123456ufl.edu
                Author information
                https://orcid.org/0000-0002-0226-3520
                https://orcid.org/0000-0002-8027-7012
                Article
                plants-08-00601
                10.3390/plants8120601
                6963220
                31847196
                fb2101a6-a48f-4a2e-9599-d34a977370aa
                © 2019 by the authors.

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license ( http://creativecommons.org/licenses/by/4.0/).

                History
                : 17 November 2019
                : 11 December 2019
                Categories
                Review

                crispr/cas9,genome editing,gene knockout,horticultural plants,precision editing

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