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      Occurrence and Nature of Off-Target Modifications by CRISPR-Cas Genome Editing in Plants

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          Abstract

          CRISPR-Cas-based genome editing allows for precise and targeted genetic modification of plants. Nevertheless, unintended off-target edits can arise that might confer risks when present in gene-edited food crops. Through an extensive literature review we gathered information on CRISPR-Cas off-target edits in plants. Most observed off-target changes were small insertions or deletions (1–22 bp) or nucleotide substitutions, and large deletions (>100 bp) were rare. One study detected the insertion of vector-derived DNA sequences, which is important considering the risk assessment of gene-edited plants. Off-target sites had few mismatches (1–3 nt) with the target sequence and were mainly located in protein-coding regions, often in target gene homologues. Off-targets edits were predominantly detected via biased analysis of predicted off-target sites instead of unbiased genome-wide analysis. CRISPR-Cas-edited plants showed lower off-target mutation frequencies than conventionally bred plants. This Review can aid discussions on the relevance of evaluating off-target modifications for risk assessment of CRISPR-Cas-edited plants.

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          Most cited references56

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          Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements

          CRISPR-Cas9 is poised to become the gene editing tool of choice in clinical contexts. Thus far, exploration of Cas9-induced genetic alterations has been limited to the immediate vicinity of the target site and distal off-target sequences, leading to the conclusion that CRISPR-Cas9 was reasonably specific. Here we report significant on-target mutagenesis, such as large deletions and more complex genomic rearrangements at the targeted sites in mouse embryonic stem cells, mouse hematopoietic progenitors and a human differentiated cell line. Using long-read sequencing and long-range PCR genotyping, we show that DNA breaks introduced by single-guide RNA/Cas9 frequently resolved into deletions extending over many kilobases. Furthermore, lesions distal to the cut site and crossover events were identified. The observed genomic damage in mitotically active cells caused by CRISPR-Cas9 editing may have pathogenic consequences.
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            Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases

            Summary: The Type II clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system is an adaptive immune response in prokaryotes, protecting host cells against invading phages or plasmids by cleaving these foreign DNA species in a targeted manner. CRISPR/Cas-derived RNA-guided engineered nucleases (RGENs) enable genome editing in cultured cells, animals and plants, but are limited by off-target mutations. Here, we present a novel algorithm termed Cas-OFFinder that searches for potential off-target sites in a given genome or user-defined sequences. Unlike other algorithms currently available for identification of RGEN off-target sites, Cas-OFFinder is not limited by the number of mismatches and allows variations in protospacer-adjacent motif sequences recognized by Cas9, the essential protein component in RGENs. Cas-OFFinder is available as a command-line program or accessible via our website. Availability and implementation: Cas-OFFinder free access at http://www.rgenome.net/cas-offinder. Contact: baesau@snu.ac.kr or jskim01@snu.ac.kr
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              Efficient and transgene-free genome editing in wheat through transient expression of CRISPR/Cas9 DNA or RNA

              Editing plant genomes is technically challenging in hard-to-transform plants and usually involves transgenic intermediates, which causes regulatory concerns. Here we report two simple and efficient genome-editing methods in which plants are regenerated from callus cells transiently expressing CRISPR/Cas9 introduced as DNA or RNA. This transient expression-based genome-editing system is highly efficient and specific for producing transgene-free and homozygous wheat mutants in the T0 generation. We demonstrate our protocol to edit genes in hexaploid bread wheat and tetraploid durum wheat, and show that we are able to generate mutants with no detectable transgenes. Our methods may be applicable to other plant species, thus offering the potential to accelerate basic and applied plant genome-engineering research.
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                Author and article information

                Journal
                ACS Agric Sci Technol
                ACS Agric Sci Technol
                as
                aastgj
                ACS Agricultural Science & Technology
                American Chemical Society
                2692-1952
                03 March 2022
                18 April 2022
                : 2
                : 2 , Advances in Genome Editing for Sustainable Agriculture
                : 192-201
                Affiliations
                []Wageningen Food Safety Research, Wageningen University and Research , P.O. Box 230, 6700 AE Wageningen, The Netherlands
                []Sciensano , Rue Juliette Wytsmanstraat 14, 1050 Brussels, Belgium
                [§ ]Wageningen Plant Research, Wageningen University and Research , P.O. Box 16, 6700 AA Wageningen, The Netherlands
                Author notes
                Author information
                https://orcid.org/0000-0001-9073-2675
                Article
                10.1021/acsagscitech.1c00270
                9075866
                35548699
                5e939713-446a-4806-95a5-176a3d7e6d78
                © 2022 The Authors. Published by American Chemical Society

                Permits the broadest form of re-use including for commercial purposes, provided that author attribution and integrity are maintained ( https://creativecommons.org/licenses/by/4.0/).

                History
                : 02 December 2021
                : 18 February 2022
                : 18 February 2022
                Funding
                Funded by: Nederlandse Organisatie voor Wetenschappelijk Onderzoek, doi 10.13039/501100003246;
                Award ID: 15792
                Funded by: Ministerie van Landbouw, Natuur en Voedselkwaliteit, doi 10.13039/501100013890;
                Award ID: KB-037-002-013
                Categories
                Review
                Custom metadata
                as1c00270
                as1c00270

                crispr-cas,genome editing,plants,off-target modifications

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