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      Advanced domestication: harnessing the precision of gene editing in crop breeding

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          Summary

          Human population growth has increased the demand for food crops, animal feed, biofuel and biomaterials, all the while climate change is impacting environmental growth conditions. There is an urgent need to develop crop varieties which tolerate adverse growth conditions while requiring fewer inputs. Plant breeding is critical to global food security and, while it has benefited from modern technologies, it remains constrained by a lack of valuable genetic diversity, linkage drag, and an effective way to combine multiple favourable alleles for complex traits. CRISPR/Cas technology has transformed genome editing across biological systems and promises to transform agriculture with its high precision, ease of design, multiplexing ability and low cost. We discuss the integration of CRISPR/Cas‐based gene editing into crop breeding to advance domestication and refine inbred crop varieties for various applications and growth environments. We highlight the use of CRISPR/Cas‐based gene editing to fix desirable allelic variants, generate novel alleles, break deleterious genetic linkages, support pre‐breeding and for introgression of favourable loci into elite lines.

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          Most cited references126

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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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            Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage

            Current genome-editing technologies introduce double-stranded (ds) DNA breaks at a target locus as the first step to gene correction. 1,2 Although most genetic diseases arise from point mutations, current approaches to point mutation correction are inefficient and typically induce an abundance of random insertions and deletions (indels) at the target locus from the cellular response to dsDNA breaks. 1,2 Here we report the development of base editing, a new approach to genome editing that enables the direct, irreversible conversion of one target DNA base into another in a programmable manner, without requiring dsDNA backbone cleavage or a donor template. We engineered fusions of CRISPR/Cas9 and a cytidine deaminase enzyme that retain the ability to be programmed with a guide RNA, do not induce dsDNA breaks, and mediate the direct conversion of cytidine to uridine, thereby effecting a C→T (or G→A) substitution. The resulting “base editors” convert cytidines within a window of approximately five nucleotides (nt), and can efficiently correct a variety of point mutations relevant to human disease. In four transformed human and murine cell lines, second- and third-generation base editors that fuse uracil glycosylase inhibitor (UGI), and that use a Cas9 nickase targeting the non-edited strand, manipulate the cellular DNA repair response to favor desired base-editing outcomes, resulting in permanent correction of ∼15-75% of total cellular DNA with minimal (typically ≤ 1%) indel formation. Base editing expands the scope and efficiency of genome editing of point mutations.
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              Search-and-replace genome editing without double-strand breaks or donor DNA

              Summary Most genetic variants that contribute to disease 1 are challenging to correct efficiently and without excess byproducts 2–5 . Here we describe prime editing, a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a catalytically impaired Cas9 fused to an engineered reverse transcriptase, programmed with a prime editing guide RNA (pegRNA) that both specifies the target site and encodes the desired edit. We performed >175 edits in human cells including targeted insertions, deletions, and all 12 types of point mutations without requiring double-strand breaks or donor DNA templates. We applied prime editing in human cells to correct efficiently and with few byproducts the primary genetic causes of sickle cell disease (requiring a transversion in HBB) and Tay-Sachs disease (requiring a deletion in HEXA), to install a protective transversion in PRNP, and to precisely insert various tags and epitopes into target loci. Four human cell lines and primary post-mitotic mouse cortical neurons support prime editing with varying efficiencies. Prime editing shows higher or similar efficiency and fewer byproducts than homology-directed repair, complementary strengths and weaknesses compared to base editing, and much lower off-target editing than Cas9 nuclease at known Cas9 off-target sites. Prime editing substantially expands the scope and capabilities of genome editing, and in principle can correct up to 89% of known genetic variants associated with human diseases.
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                Author and article information

                Contributors
                Sateesh.Kagale@nrc-cnrc.gc.ca
                Journal
                Plant Biotechnol J
                Plant Biotechnol J
                10.1111/(ISSN)1467-7652
                PBI
                Plant Biotechnology Journal
                John Wiley and Sons Inc. (Hoboken )
                1467-7644
                1467-7652
                25 March 2021
                April 2021
                : 19
                : 4 ( doiID: 10.1111/pbi.v19.4 )
                : 660-670
                Affiliations
                [ 1 ] Aquatic and Crop Resource Development National Research Council Canada Saskatoon SK Canada
                [ 2 ] Global Institute for Food Security University of Saskatchewan Saskatoon SK Canada
                [ 3 ] Crop Development Centre University of Saskatchewan Saskatoon SK Canada
                Author notes
                [*] [* ] Correspondence (Tel +1 306 975 4194; fax +1 306 975 6503; email Sateesh.Kagale@ 123456nrc-cnrc.gc.ca )

                Author information
                https://orcid.org/0000-0002-7213-1590
                Article
                PBI13576
                10.1111/pbi.13576
                8051614
                33657682
                6036d771-10f1-4095-9aab-9d6ba26c14b8
                © 2021 Her Majesty the Queen in Right of Canada. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd. Reproduced with the permission of the Minister of National Research Council Canada.

                This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

                History
                : 19 February 2021
                : 20 December 2020
                : 26 February 2021
                Page count
                Figures: 5, Tables: 2, Pages: 0, Words: 10771
                Funding
                Funded by: Genome Canada LSARP
                Award ID: 4D Wheat
                Funded by: Agriculture and Agri‐Food Canada , open-funder-registry 10.13039/501100000040;
                Funded by: Western Grains Research Foundation , open-funder-registry 10.13039/100009370;
                Funded by: Saskatchewan Ministry of Agriculture
                Funded by: Saskatchewan Wheat Development Commission
                Funded by: Alberta Wheat Commission
                Funded by: Manitoba Crop Alliance
                Funded by: Ontario Research Fund
                Funded by: Viterra, Canadian Agricultural Partnership, and Illumina
                Categories
                Review Article
                Review Article
                Custom metadata
                2.0
                April 2021
                Converter:WILEY_ML3GV2_TO_JATSPMC version:6.0.2 mode:remove_FC converted:16.04.2021

                Biotechnology
                gene editing,crispr/cas,domestication,precision breeding,recombination,novel alleles
                Biotechnology
                gene editing, crispr/cas, domestication, precision breeding, recombination, novel alleles

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