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      CRISPR/dCas9 platforms in plants: strategies and applications beyond genome editing

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          Summary

          Clustered regularly interspaced short palindromic repeat ( CRISPR) and Cas9‐associated protein systems provide a powerful genetic manipulation tool that can drive plant research forward. Nuclease‐dead Cas9 ( dCas9) is an enzymatically inactive mutant of Cas9 in which its endonuclease activity is non‐functional. The applications of CRISPR/ dCas9 have expanded and diversified in recent years. Originally, dCas9 was used as a CRISPR/Cas9 re‐engineering tool that enables targeted expression of any gene or multiple genes through recruitment of transcriptional effector domains without introducing irreversible DNA‐damaging mutations. Subsequent applications have made use of its ability to recruit modifying enzymes and reporter proteins to DNA target sites. In this paper, the most recent progress in the applications of CRISPR/ dCas9 in plants, which include gene activation and repression, epigenome editing, modulation of chromatin topology, live‐cell chromatin imaging and DNA‐free genetic modification, will be reviewed. The associated strategies for exploiting the CRISPR/ dCas9 system for crop improvement with a dimer of the future of the CRISPR/ dCas9 system in the functional genomics of crops and the development of traits will be briefly discussed.

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          Epigenome editing by a CRISPR/Cas9-based acetyltransferase activates genes from promoters and enhancers

          Technologies that facilitate the targeted manipulation of epigenetic marks could be used to precisely control cell phenotype or interrogate the relationship between the epigenome and transcriptional control. Here we have generated a programmable acetyltransferase based on the CRISPR/Cas9 gene regulation system, consisting of the nuclease-null dCas9 protein fused to the catalytic core of the human acetyltransferase p300. This fusion protein catalyzes acetylation of histone H3 lysine 27 at its target sites, corresponding with robust transcriptional activation of target genes from promoters, proximal enhancers, and distal enhancers. Gene activation by the targeted acetyltransferase is highly specific across the genome. In contrast to conventional dCas9-based activators, the acetyltransferase effectively activates genes from enhancer regions and with individual guide RNAs. The core p300 domain is also portable to other programmable DNA-binding proteins. These results support targeted acetylation as a causal mechanism of transactivation and provide a new robust tool for manipulating gene regulation.
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            Central dogma of molecular biology.

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              CRISPR interference (CRISPRi) for sequence-specific control of gene expression.

              Sequence-specific control of gene expression on a genome-wide scale is an important approach for understanding gene functions and for engineering genetic regulatory systems. We have recently described an RNA-based method, CRISPR interference (CRISPRi), for targeted silencing of transcription in bacteria and human cells. The CRISPRi system is derived from the Streptococcus pyogenes CRISPR (clustered regularly interspaced palindromic repeats) pathway, requiring only the coexpression of a catalytically inactive Cas9 protein and a customizable single guide RNA (sgRNA). The Cas9-sgRNA complex binds to DNA elements complementary to the sgRNA and causes a steric block that halts transcript elongation by RNA polymerase, resulting in the repression of the target gene. Here we provide a protocol for the design, construction and expression of customized sgRNAs for transcriptional repression of any gene of interest. We also provide details for testing the repression activity of CRISPRi using quantitative fluorescence assays and native elongating transcript sequencing. CRISPRi provides a simplified approach for rapid gene repression within 1-2 weeks. The method can also be adapted for high-throughput interrogation of genome-wide gene functions and genetic interactions, thus providing a complementary approach to RNA interference, which can be used in a wider variety of organisms.
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                Author and article information

                Contributors
                snaa@upm.edu.my
                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
                03 September 2019
                January 2020
                : 18
                : 1 ( doiID: 10.1111/pbi.v18.1 )
                : 32-44
                Affiliations
                [ 1 ] Laboratory of Science and Technology Institute of Plantation Studies Universiti Putra Malaysia Serdang Selangor Malaysia
                [ 2 ] Department of Agricultural Technology Faculty of Agriculture Universiti Putra Malaysia Serdang Selangor Malaysia
                Author notes
                [*] [* ]Correspondence (Tel +603-9769 1044; fax +603-9769 1044; email snaa@ 123456upm.edu.my )
                Author information
                https://orcid.org/0000-0002-3611-6094
                Article
                PBI13232
                10.1111/pbi.13232
                6920162
                31392820
                3bf10221-e003-40a1-9b9c-0ec9fc6823e8
                © 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-nc-nd/4.0/ License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.

                History
                : 10 May 2019
                : 27 July 2019
                : 30 July 2019
                Page count
                Figures: 3, Tables: 3, Pages: 13, Words: 9869
                Categories
                Review
                Reviews
                Custom metadata
                2.0
                January 2020
                Converter:WILEY_ML3GV2_TO_JATSPMC version:5.7.3 mode:remove_FC converted:18.12.2019

                Biotechnology
                crispr/dcas9,sgrnas,transcriptional regulation,transcriptional activation,epigenome editing,chromatin topology,chromatin imaging,crispr/dcas9 ribonucleoproteins

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