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      Generation of virus‐resistant potato plants by RNA genome targeting

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          Summary

          CRISPR/Cas systems provide bacteria and archaea with molecular immunity against invading phages and foreign plasmids. The class 2 type VI CRISPR/Cas effector Cas13a is an RNA‐targeting CRISPR effector that provides protection against RNA phages. Here we report the repurposing of CRISPR/Cas13a to protect potato plants from a eukaryotic virus, Potato virus Y ( PVY). Transgenic potato lines expressing Cas13a/sg RNA (small guide RNA) constructs showed suppressed PVY accumulation and disease symptoms. The levels of viral resistance correlated with the expression levels of the Cas13a/sg RNA construct in the plants. Our data further demonstrate that appropriately designed sg RNAs can specifically interfere with multiple PVY strains, while having no effect on unrelated viruses such as PVA or Potato virus S. Our findings provide a novel and highly efficient strategy for engineering crops with resistances to viral diseases.

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          Most cited references41

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          Diversity and evolution of class 2 CRISPR–Cas systems

          Class 2 CRISPR–Cas systems are characterized by effector modules that consist of a single multidomain protein. In this Analysis, using a computational pipeline, the authors discover three novel families of class 2 effectors that correspond to three new CRISPR–Cas subtypes and present a comprehensive census of class 2 systems that are encoded in complete and draft bacterial and archaeal genomes.
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            Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing.

            CRISPR/Cas9 uses a guide RNA (gRNA) molecule to execute sequence-specific DNA cleavage and it has been widely used for genome editing in many organisms. Modifications at either end of the gRNAs often render Cas9/gRNA inactive. So far, production of gRNA in vivo has only been achieved by using the U6 and U3 snRNA promoters. However, the U6 and U3 promoters have major limitations such as a lack of cell specificity and unsuitability for in vitro transcription. Here, we present a versatile method for efficiently producing gRNAs both in vitro and in vivo. We design an artificial gene named RGR that, once transcribed, generates an RNA molecule with ribozyme sequences at both ends of the designed gRNA. We show that the primary transcripts of RGR undergo self-catalyzed cleavage to generate the desired gRNA, which can efficiently guide sequence-specific cleavage of DNA targets both in vitro and in yeast. RGR can be transcribed from any promoters and thus allows for cell- and tissue-specific genome editing if appropriate promoters are chosen. Detecting mutations generated by CRISPR is often achieved by enzyme digestions, which are not very compatible with high-throughput analysis. Our system allows for the use of universal primers to produce any gRNAs in vitro, which can then be used with Cas9 protein to detect mutations caused by the gRNAs/CRISPR. In conclusion, we provide a versatile method for generating targeted mutations in specific cells and tissues, and for efficiently detecting the mutations generated. © 2013 Institute of Botany, Chinese Academy of Sciences.
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              A CRISPR–Cpf1 system for efficient genome editing and transcriptional repression in plants

              Clustered regularly interspaced short palindromic repeats (CRISPR)-Cpf1 has emerged as an effective genome editing tool in animals. Here we compare the activity of Cpf1 from Acidaminococcus sp. BV3L6 (As) and Lachnospiraceae bacterium ND2006 (Lb) in plants, using a dual RNA polymerase II promoter expression system. LbCpf1 generated biallelic mutations at nearly 100% efficiency at four independent sites in rice T0 transgenic plants. Moreover, we repurposed AsCpf1 and LbCpf1 for efficient transcriptional repression in Arabidopsis, and demonstrated a more than tenfold reduction in miR159b transcription. Our data suggest promising applications of CRISPR-Cpf1 for editing plant genomes and modulating the plant transcriptome.
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                Author and article information

                Contributors
                nbihua@mail.hzau.edu.cn
                zhangjiang@hubu.edu.cn
                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
                08 March 2019
                September 2019
                : 17
                : 9 ( doiID: 10.1111/pbi.v17.9 )
                : 1814-1822
                Affiliations
                [ 1 ] State Key Laboratory of Biocatalysis and Enzyme Engineering School of Life Sciences Hubei University Wuhan China
                [ 2 ] Key Laboratory of Potato Biology and Biotechnology Ministry of Agriculture and Rural Affairs National Center for Vegetable Improvement (Central China) Huazhong Agricultural University Wuhan China
                [ 3 ] Max‐Planck‐Institut für Molekulare Pflanzenphysiologie Potsdam‐Golm Germany
                Author notes
                [*] [* ] Correspondence (Tel +86 27 88663882 8064; fax +86 27 88663882 8064; emails nbihua@ 123456mail.hzau.edu.cn (B.N.); zhangjiang@ 123456hubu.edu.cn (J.Z.))
                [†]

                These authors contribute equally to this work.

                Author information
                http://orcid.org/0000-0001-7502-6940
                http://orcid.org/0000-0003-3949-3765
                Article
                PBI13102
                10.1111/pbi.13102
                6686122
                30803101
                800c46d7-6a54-43c5-a1b6-a6d7c67c537a
                © 2019 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

                This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

                History
                : 18 December 2018
                : 20 February 2019
                : 21 February 2019
                Page count
                Figures: 5, Tables: 0, Pages: 9, Words: 6146
                Funding
                Funded by: National Key Research and Development Program of China
                Award ID: 2017YFD0600101
                Funded by: National Natural Science Foundation of China
                Award ID: 31572071
                Funded by: Science and Technology Department of Hubei Province of China
                Award ID: 2016CFA052
                Funded by: Recruitment Program of Global Experts (China)
                Categories
                Research Article
                Research Articles
                Custom metadata
                2.0
                pbi13102
                September 2019
                Converter:WILEY_ML3GV2_TO_NLMPMC version:5.6.7 mode:remove_FC converted:08.08.2019

                Biotechnology
                crispr/cas13a,rna targeting,potato virus y,virus interference,molecular immunity,virus resistance

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