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      Prime Editing Technology and Its Prospects for Future Applications in Plant Biology Research

      review-article
      1 , 2 , 3 , 1 , 2 , 1 , 2 , 1 , 2 , 1 , 2 ,
      Biodesign Research
      AAAS

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          Abstract

          Many applications in plant biology requires editing genomes accurately including correcting point mutations, incorporation of single-nucleotide polymorphisms (SNPs), and introduction of multinucleotide insertion/deletions (indels) into a predetermined position in the genome. These types of modifications are possible using existing genome-editing technologies such as the CRISPR-Cas systems, which require induction of double-stranded breaks in the target DNA site and the supply of a donor DNA molecule that contains the desired edit sequence. However, low frequency of homologous recombination in plants and difficulty of delivering the donor DNA molecules make this process extremely inefficient. Another kind of technology known as base editing can perform precise editing; however, only certain types of modifications can be obtained, e.g., C/G-to-T/A and A/T-to-G/C. Recently, a new type of genome-editing technology, referred to as “prime editing,” has been developed, which can achieve various types of editing such as any base-to-base conversion, including both transitions (C→T, G→A, A→G, and T→C) and transversion mutations (C→A, C→G, G→C, G→T, A→C, A→T, T→A, and T→G), as well as small indels without the requirement for inducing double-stranded break in the DNA. Because prime editing has wide flexibility to achieve different types of edits in the genome, it holds a great potential for developing superior crops for various purposes, such as increasing yield, providing resistance to various abiotic and biotic stresses, and improving quality of plant product. In this review, we describe the prime editing technology and discuss its limitations and potential applications in plant biology research.

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          Most cited references104

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          The plant immune system.

          Many plant-associated microbes are pathogens that impair plant growth and reproduction. Plants respond to infection using a two-branched innate immune system. The first branch recognizes and responds to molecules common to many classes of microbes, including non-pathogens. The second responds to pathogen virulence factors, either directly or through their effects on host targets. These plant immune systems, and the pathogen molecules to which they respond, provide extraordinary insights into molecular recognition, cell biology and evolution across biological kingdoms. A detailed understanding of plant immune function will underpin crop improvement for food, fibre and biofuels production.
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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
                Journal
                Biodes Res
                Biodes Res
                bdr
                Biodesign Research
                AAAS
                2693-1257
                26 June 2020
                2020
                : 2020
                : 9350905
                Affiliations
                1Biosciences Division, Oak Ridge National Laboratory, Oak Ridge TN 37831, USA
                2Center for Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA
                3Department of Genetics and Plant Breeding, Patuakhali Science and Technology University, Dumki, Patuakhali 8602, Bangladesh
                Author notes

                The authors declare that they have no conflicts of interest regarding the publication of this article.

                Author information
                https://orcid.org/0000-0002-1752-4201
                https://orcid.org/0000-0001-5207-4210
                Article
                9350905
                10.34133/2020/9350905
                10530660
                37849904
                183d2c0f-4929-45e3-a173-9935554fa052
                Copyright © 2020 Md. Mahmudul Hassan et al.

                Exclusive Licensee Nanjing Agricultural University. Distributed under a Creative Commons Attribution License (CC BY 4.0).

                History
                : 15 April 2020
                : 19 May 2020
                Page count
                Figures: 2, Tables: 2, References: 109, Pages: 14
                Funding
                Funded by: Genomic Science Program
                Funded by: Oak Ridge National Laboratory
                Funded by: Laboratory Directed Research and Development
                Funded by: Office of Biological and Environmental Research
                Funded by: Office of Science
                Funded by: Center for Bioenergy Innovation
                Funded by: U.S. Department of Energy
                Award ID: DE-AC05-00OR22725
                Categories
                Review Article
                Review Article

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