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      Genome Editing in Rice: Recent Advances, Challenges, and Future Implications

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          Abstract

          Rice ( Oryza sativa L.) is the major food source for more than three billion people of the world. In the last few decades, the classical, mutational, and molecular breeding approaches have brought about tremendous increase in rice productivity with the development of novel rice varieties. However, stagnation in rice yield has been reported in recent decade owing to several factors including the emergence of pests and phyto pathogens, climate change, and other environmental issues posing great threat to global food security. There is an urgent need to produce more rice and associated cereals to satisfy the mammoth task of feeding a still growing population expected to reach 9.7 billion by 2050. Advances in genomics and emergence of multiple genome-editing technologies through use of engineered site-specific nucleases (SSNs) have revolutionized the field of plant science and agriculture. Among them, the CRISPR/Cas9 system is the most advanced and widely accepted because of its simplicity, robustness, and high efficiency. The availability of huge genomic resources together with a small genome size makes rice more suitable and feasible for genetic manipulation. As such, rice has been increasingly used to test the efficiency of different types of genome editing technologies to study the functions of various genes and demonstrate their potential in genetic improvement. Recently developed approaches including CRISPR/Cpf1 system and base editors have evolved as more efficient and accurate genome editing tools which might accelerate the pace of crop improvement. In the present review, we focus on the genome editing strategies for rice improvement, thereby highlighting the applications and advancements of CRISPR/Cas9 system. This review also sheds light on the role of CRISPR/Cpf1 and base editors in the field of genome editing highlighting major challenges and future implications of these tools in rice improvement.

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          Most cited references92

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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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            Editing DNA Methylation in the Mammalian Genome.

            Mammalian DNA methylation is a critical epigenetic mechanism orchestrating gene expression networks in many biological processes. However, investigation of the functions of specific methylation events remains challenging. Here, we demonstrate that fusion of Tet1 or Dnmt3a with a catalytically inactive Cas9 (dCas9) enables targeted DNA methylation editing. Targeting of the dCas9-Tet1 or -Dnmt3a fusion protein to methylated or unmethylated promoter sequences caused activation or silencing, respectively, of an endogenous reporter. Targeted demethylation of the BDNF promoter IV or the MyoD distal enhancer by dCas9-Tet1 induced BDNF expression in post-mitotic neurons or activated MyoD facilitating reprogramming of fibroblasts into myoblasts, respectively. Targeted de novo methylation of a CTCF loop anchor site by dCas9-Dnmt3a blocked CTCF binding and interfered with DNA looping, causing altered gene expression in the neighboring loop. Finally, we show that these tools can edit DNA methylation in mice, demonstrating their wide utility for functional studies of epigenetic regulation.
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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

                Contributors
                Journal
                Front Plant Sci
                Front Plant Sci
                Front. Plant Sci.
                Frontiers in Plant Science
                Frontiers Media S.A.
                1664-462X
                19 September 2018
                2018
                : 9
                : 1361
                Affiliations
                [1] 1National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Science, Chinese Academy of Agricultural Sciences , Beijing, China
                [2] 2Department of Biotechnology, Rama Devi Women’s University , Bhubaneswar, India
                Author notes

                Edited by: Raúl Alvarez-Venegas, Centro de Investigación y de Estudios Avanzados (CINVESTAV), Mexico

                Reviewed by: Hemant Ritturaj Kushwaha, Jawaharlal Nehru University, India; Uener Kolukisaoglu, University of Tübingen, Germany

                *Correspondence: Kaijun Zhao, zhaokaijun@ 123456caas.cn

                This article was submitted to Plant Biotechnology, a section of the journal Frontiers in Plant Science

                Article
                10.3389/fpls.2018.01361
                6156261
                30283477
                36aefd11-da30-4969-9907-66a167bc57ce
                Copyright © 2018 Mishra, Joshi and Zhao.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 25 June 2018
                : 28 August 2018
                Page count
                Figures: 2, Tables: 1, Equations: 0, References: 110, Pages: 12, Words: 0
                Categories
                Plant Science
                Review

                Plant science & Botany
                base editors,crop improvement,crispr/cas9,crispr/cpf1,genome editing,rice,targeted mutagenesis

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