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      Conventional and Molecular Techniques from Simple Breeding to Speed Breeding in Crop Plants: Recent Advances and Future Outlook

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          Abstract

          In most crop breeding programs, the rate of yield increment is insufficient to cope with the increased food demand caused by a rapidly expanding global population. In plant breeding, the development of improved crop varieties is limited by the very long crop duration. Given the many phases of crossing, selection, and testing involved in the production of new plant varieties, it can take one or two decades to create a new cultivar. One possible way of alleviating food scarcity problems and increasing food security is to develop improved plant varieties rapidly. Traditional farming methods practiced since quite some time have decreased the genetic variability of crops. To improve agronomic traits associated with yield, quality, and resistance to biotic and abiotic stresses in crop plants, several conventional and molecular approaches have been used, including genetic selection, mutagenic breeding, somaclonal variations, whole-genome sequence-based approaches, physical maps, and functional genomic tools. However, recent advances in genome editing technology using programmable nucleases, clustered regularly interspaced short palindromic repeats (CRISPR), and CRISPR-associated (Cas) proteins have opened the door to a new plant breeding era. Therefore, to increase the efficiency of crop breeding, plant breeders and researchers around the world are using novel strategies such as speed breeding, genome editing tools, and high-throughput phenotyping. In this review, we summarize recent findings on several aspects of crop breeding to describe the evolution of plant breeding practices, from traditional to modern speed breeding combined with genome editing tools, which aim to produce crop generations with desired traits annually.

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          Most cited references203

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          A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.

          Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand. The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.
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            Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system.

            The microbial adaptive immune system CRISPR mediates defense against foreign genetic elements through two classes of RNA-guided nuclease effectors. Class 1 effectors utilize multi-protein complexes, whereas class 2 effectors rely on single-component effector proteins such as the well-characterized Cas9. Here, we report characterization of Cpf1, a putative class 2 CRISPR effector. We demonstrate that Cpf1 mediates robust DNA interference with features distinct from Cas9. Cpf1 is a single RNA-guided endonuclease lacking tracrRNA, and it utilizes a T-rich protospacer-adjacent motif. Moreover, Cpf1 cleaves DNA via a staggered DNA double-stranded break. Out of 16 Cpf1-family proteins, we identified two candidate enzymes from Acidaminococcus and Lachnospiraceae, with efficient genome-editing activity in human cells. Identifying this mechanism of interference broadens our understanding of CRISPR-Cas systems and advances their genome editing applications.
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              DNA targeting specificity of RNA-guided Cas9 nucleases.

              The Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of single-guide RNAs (sgRNAs) to enable genome editing. Here, we characterize SpCas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target effects. Our study evaluates >700 guide RNA variants and SpCas9-induced indel mutation levels at >100 predicted genomic off-target loci in 293T and 293FT cells. We find that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches. We also show that SpCas9-mediated cleavage is unaffected by DNA methylation and that the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modification. To facilitate mammalian genome engineering applications, we provide a web-based software tool to guide the selection and validation of target sequences as well as off-target analyses.
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                Author and article information

                Journal
                Int J Mol Sci
                Int J Mol Sci
                ijms
                International Journal of Molecular Sciences
                MDPI
                1422-0067
                08 April 2020
                April 2020
                : 21
                : 7
                : 2590
                Affiliations
                [1 ]National Key Laboratory of Crop Genetic Improvement, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, Hubei, China; sunny.ahmar@ 123456yahoo.com (S.A.); uzairqasim1149@ 123456yahoo.com (M.U.Q.)
                [2 ]Oil Crops Research Institute, Chinese Academy of Agriculture Sciences, Wuhan 430070, China; drragill@ 123456caas.cn
                [3 ]Graduate School of Biotechnology & Crop Biotech Institute, Kyung Hee University, Yongin 17104, Korea
                [4 ]State Key Laboratory of Agricultural Microbiology and State Key Laboratory of Microbial Biosensor, College of Life Sciences Huazhong Agriculture University, Wuhan 430070, China
                [5 ]State Key Laboratory of Agricultural Microbiology and Provincial Key Laboratory of Plant Pathology of Hubei Province, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, China
                [6 ]Institute of Crop Science and Zhejiang Key Laboratory of Crop Germplasm, Zhejiang University, Hangzhou 310058, China
                Author notes
                [* ]Correspondence: khjung2010@ 123456khu.ac.kr (K.-H.J.); wjzhou@ 123456zju.edu.cn (W.Z.)
                [†]

                These authors contributed equally to this work.

                Author information
                https://orcid.org/0000-0003-0427-5901
                Article
                ijms-21-02590
                10.3390/ijms21072590
                7177917
                32276445
                fe6926cf-3d40-4047-b4ae-c548a8b4a31c
                © 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
                : 08 February 2020
                : 05 April 2020
                Categories
                Review

                Molecular biology
                food security,food scarcity,conventional breeding,crispr/cas9,crispr/cpf1,high-throughput phenotyping,speed breeding

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