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      Induced Genetic Variations in Fruit Trees Using New Breeding Tools: Food Security and Climate Resilience

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          Abstract

          Fruit trees provide essential nutrients to humans by contributing to major agricultural outputs and economic growth globally. However, major constraints to sustainable agricultural productivity are the uncontrolled proliferation of the population, and biotic and abiotic stresses. Tree mutation breeding has been substantially improved using different physical and chemical mutagens. Nonetheless, tree plant breeding has certain crucial bottlenecks including a long life cycle, ploidy level, occurrence of sequence polymorphisms, nature of parthenocarpic fruit development and linkage. Genetic engineering of trees has focused on boosting quality traits such as productivity, wood quality, and resistance to biotic and abiotic stresses. Recent technological advances in genome editing provide a unique opportunity for the genetic improvement of woody plants. This review examines application of the CRISPR-Cas system to reduce disease susceptibility, alter plant architecture, enhance fruit quality, and improve yields. Examples are discussed of the contemporary CRISPR-Cas system to engineer easily scorable PDS genes, modify lignin, and to alter the flowering onset, fertility, tree architecture and certain biotic stresses.

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          Most cited references269

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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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            Multiplex genome engineering using CRISPR/Cas systems.

            Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.
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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
                Role: Academic Editor
                Journal
                Plants (Basel)
                Plants (Basel)
                plants
                Plants
                MDPI
                2223-7747
                01 July 2021
                July 2021
                : 10
                : 7
                : 1347
                Affiliations
                [1 ]Central Laboratories, King Faisal University, Al-Ahsa 31982, Saudi Arabia; mnsattar@ 123456kfu.edu.sa (M.N.S.); zafar@ 123456kfu.edu.sa (Z.I.)
                [2 ]Department of Agricultural Biotechnology, College of Agriculture and Food Sciences, King Faisal University, Al-Ahsa 31982, Saudi Arabia
                [3 ]Department of Agricultural Sciences, PL-27, University of Helsinki, 00014 Helsinki, Finland; mohan.jain@ 123456helsinki.fi
                Author notes
                [* ]Correspondence: jkhayri@ 123456kfu.edu.sa
                Author information
                https://orcid.org/0000-0001-9507-0201
                Article
                plants-10-01347
                10.3390/plants10071347
                8309169
                0bab2872-1217-46a1-957d-4b45d4372eb9
                © 2021 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 ( https://creativecommons.org/licenses/by/4.0/).

                History
                : 22 May 2021
                : 28 June 2021
                Categories
                Review

                mutagenesis,tilling,genome editing,targeted mutation,crispr-cas,transgene-free,fruit trees

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