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      Base Editing: The Ever Expanding Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Tool Kit for Precise Genome Editing in Plants

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          Abstract

          Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9), a newly developed genome-editing tool, has revolutionized animal and plant genetics by facilitating modification of target genes. This simple, convenient base-editing technology was developed to improve the precision of genome editing. Base editors generate precise point mutations by permanent base conversion at a specific point, with very low levels of insertions and deletions. Different plant base editors have been established by fusing various nucleobase deaminases with Cas9, Cas13, or Cas12a (Cpf1), proteins. Adenine base editors can efficiently convert adenine (A) to guanine (G), whereas cytosine base editors can convert cytosine (C) to thymine (T) in the target region. RNA base editors can induce a base substitution of A to inosine (I) or C to uracil (U). In this review, we describe the precision of base editing systems and their revolutionary applications in plant science; we also discuss the limitations and future perspectives of this approach.

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          CRISPR/Cas Genome Editing and Precision Plant Breeding in Agriculture

          Enhanced agricultural production through innovative breeding technology is urgently needed to increase access to nutritious foods worldwide. Recent advances in CRISPR/Cas genome editing enable efficient targeted modification in most crops, thus promising to accelerate crop improvement. Here, we review advances in CRISPR/Cas9 and its variants and examine their applications in plant genome editing and related manipulations. We highlight base-editing tools that enable targeted nucleotide substitutions and describe the various delivery systems, particularly DNA-free methods, that have linked genome editing with crop breeding. We summarize the applications of genome editing for trait improvement, development of techniques for fine-tuning gene regulation, strategies for breeding virus resistance, and the use of high-throughput mutant libraries. We outline future perspectives for genome editing in plant synthetic biology and domestication, advances in delivery systems, editing specificity, homology-directed repair, and gene drives. Finally, we discuss the challenges and opportunities for precision plant breeding and its bright future in agriculture.
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            Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions

            Base editing is a recently developed approach to genome editing that uses a fusion protein containing a catalytically defective Streptococcus pyogenes Cas9, a cytidine deaminase, and an inhibitor of base excision repair to induce programmable, single-nucleotide changes in the DNA of living cells without generating double-strand DNA breaks, without requiring a donor DNA template, and without inducing an excess of stochastic insertions and deletions 1 . Here we report the development of five new C→T (or G→A) base editors that use natural and engineered Cas9 variants with different protospacer-adjacent motif (PAM) specificities to expand the number of sites that can be targeted by base editing by 2.5-fold. Additionally, we engineered new base editors containing mutated cytidine deaminase domains that narrow the width of the apparent editing window from approximately 5 nucleotides to as little as 1 to 2 nucleotides, enabling the discrimination of neighboring C nucleotides that would previously be edited with comparable efficiency, thereby doubling the number of disease-associated target Cs that can be corrected preferentially over nearby non-target Cs. Collectively, these developments substantially increase the targeting scope of base editing and establish the modular nature of base editors.
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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.
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                Author and article information

                Journal
                Genes (Basel)
                Genes (Basel)
                genes
                Genes
                MDPI
                2073-4425
                24 April 2020
                April 2020
                : 11
                : 4
                : 466
                Affiliations
                State Key Laboratory of Rice Biology and China National Center for Rice Improvement, China National Rice Research Institute, Hangzhou 310006, China; mahmudamumu0@ 123456gmail.com (M.B.M.); shaogaoneng@ 123456caas.cn (G.S.); lvyusong0907@ 123456163.com (Y.L.); shakeelpbg@ 123456gmail.com (S.A.); weixiangjin@ 123456caas.cn (X.W.); hupeisong@ 123456caas.cn (P.H.)
                Author notes
                [* ]Correspondence: tangshaoqing@ 123456caas.cn
                [†]

                These authors contributed equally to this work.

                Author information
                https://orcid.org/0000-0001-6651-9399
                Article
                genes-11-00466
                10.3390/genes11040466
                7231171
                32344599
                a03f04a7-ea7a-4f91-800a-69e52b570b2e
                © 2020 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
                : 11 February 2020
                : 21 April 2020
                Categories
                Review

                clustered regularly interspaced short palindromic repeat,base editor,point mutation,genome editing,base conversion

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