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      Advances in genomics and genome editing for improving strawberry ( Fragaria ×ananassa)

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          Abstract

          The cultivated strawberry, Fragaria ×ananassa, is a recently domesticated fruit species of economic interest worldwide. As such, there is significant interest in continuous varietal improvement. Genomics-assisted improvement, including the use of DNA markers and genomic selection have facilitated significant improvements of numerous key traits during strawberry breeding. CRISPR/Cas-mediated genome editing allows targeted mutations and precision nucleotide substitutions in the target genome, revolutionizing functional genomics and crop improvement. Genome editing is beginning to gain traction in the more challenging polyploid crops, including allo-octoploid strawberry. The release of high-quality reference genomes and comprehensive subgenome-specific genotyping and gene expression profiling data in octoploid strawberry will lead to a surge in trait discovery and modification by using CRISPR/Cas. Genome editing has already been successfully applied for modification of several strawberry genes, including anthocyanin content, fruit firmness and tolerance to post-harvest disease. However, reports on many other important breeding characteristics associated with fruit quality and production are still lacking, indicating a need for streamlined genome editing approaches and tools in Fragaria ×ananassa. In this review, we present an overview of the latest advancements in knowledge and breeding efforts involving CRISPR/Cas genome editing for the enhancement of strawberry varieties. Furthermore, we explore potential applications of this technology for improving other Rosaceous plant species.

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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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            RNA-guided human genome engineering via Cas9.

            Bacteria and archaea have evolved adaptive immune defenses, termed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems, that use short RNA to direct degradation of foreign nucleic acids. Here, we engineer the type II bacterial CRISPR system to function with custom guide RNA (gRNA) in human cells. For the endogenous AAVS1 locus, we obtained targeting rates of 10 to 25% in 293T cells, 13 to 8% in K562 cells, and 2 to 4% in induced pluripotent stem cells. We show that this process relies on CRISPR components; is sequence-specific; and, upon simultaneous introduction of multiple gRNAs, can effect multiplex editing of target loci. We also compute a genome-wide resource of ~190 K unique gRNAs targeting ~40.5% of human exons. Our results establish an RNA-guided editing tool for facile, robust, and multiplexable human genome engineering.
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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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                Author and article information

                Contributors
                URI : https://loop.frontiersin.org/people/2647715/overviewRole: Role: Role: Role:
                URI : https://loop.frontiersin.org/people/1136032/overviewRole:
                URI : https://loop.frontiersin.org/people/688545/overviewRole:
                URI : https://loop.frontiersin.org/people/222988/overviewRole: Role: Role: Role:
                Journal
                Front Genet
                Front Genet
                Front. Genet.
                Frontiers in Genetics
                Frontiers Media S.A.
                1664-8021
                19 April 2024
                2024
                : 15
                : 1382445
                Affiliations
                [1] 1 Gulf Coast Research and Education Center , Institute of Food and Agricultural Sciences , University of Florida , Wimauma, FL, United States
                [2] 2 University of Florida , Horticultural Sciences Department , Institute of Food and Agricultural Sciences , Gainesville, FL, United States
                [3] 3 University of Florida , Agronomy Department , Institute of Food and Agricultural Sciences , Gainesville, FL, United States
                Author notes

                Edited by: Rongbin Hu, University of California, Riverside, United States

                Reviewed by: Aftab Ahmad, University of Agriculture, Pakistan

                Thomas M. Davis, University of New Hampshire, United States

                *Correspondence: Seonghee Lee, seonghee105@ 123456ufl.edu
                Article
                1382445
                10.3389/fgene.2024.1382445
                11066249
                38706796
                3fa3833c-f6fa-4407-8b0d-7e818d7157cd
                Copyright © 2024 Vondracek, Altpeter, Liu and Lee.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 05 February 2024
                : 04 April 2024
                Funding
                The author(s) declare that financial support was received for the research, authorship, and/or publication of this article. This research is supported by grants from the United Stated Department of Agriculture National Institute of Food and Agriculture (NIFA) Specialty Crops Research Initiative (#2022–51181-38328).
                Categories
                Genetics
                Review
                Custom metadata
                Genomics of Plants and the Phytoecosystem

                Genetics
                crispr/cas genome editing,octoploid strawberry,genome sequencing,transformation,polyploid
                Genetics
                crispr/cas genome editing, octoploid strawberry, genome sequencing, transformation, polyploid

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