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      Multiplex gene regulation by CRISPR-ddCpf1

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          Abstract

          The clustered regularly interspaced short palindromic repeats (CRISPR)/dCas9 system has been widely applied in both transcriptional regulation and epigenetic studies. However, for multiple targets, independent expression of multiple single guide RNAs (sgRNAs) is needed, which is less convenient. To address the problem, we employed a DNase-dead Cpf1 mutant (ddCpf1) for multiplex gene regulation. We demonstrated that ddCpf1 alone could be employed for gene repression in Escherichia coli, and the repression was more effective with CRISPR RNAs (crRNAs) specifically targeting to the template strand of its target genes, which was different from that of dCas9. When targeting the promoter region, both strands showed effective repression by the ddCpf1/crRNA complex. The whole-transcriptome RNA-seq technique was further employed to demonstrate the high specificity of ddCpf1-mediated repression. Besides, we proved that the remaining RNase activity in ddCpf1 was capable of processing a precursor CRISPR array to simply generate multiple mature crRNAs in vivo, facilitating multiplex gene regulation. With the employment of this multiplex gene regulation strategy, we also showed how to quickly screen a library of candidate targets, that is, the two-component systems in E. coli. Therefore, based on our findings here, the CRISPR-ddCpf1 system may be further developed and widely applied in both biological research and clinical studies.

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          Most cited references31

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          Two-component signal transduction.

          Most prokaryotic signal-transduction systems and a few eukaryotic pathways use phosphotransfer schemes involving two conserved components, a histidine protein kinase and a response regulator protein. The histidine protein kinase, which is regulated by environmental stimuli, autophosphorylates at a histidine residue, creating a high-energy phosphoryl group that is subsequently transferred to an aspartate residue in the response regulator protein. Phosphorylation induces a conformational change in the regulatory domain that results in activation of an associated domain that effects the response. The basic scheme is highly adaptable, and numerous variations have provided optimization within specific signaling systems. The domains of two-component proteins are modular and can be integrated into proteins and pathways in a variety of ways, but the core structures and activities are maintained. Thus detailed analyses of a relatively small number of representative proteins provide a foundation for understanding this large family of signaling proteins.
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            Expression profiling reveals off-target gene regulation by RNAi.

            RNA interference is thought to require near-identity between the small interfering RNA (siRNA) and its cognate mRNA. Here, we used gene expression profiling to characterize the specificity of gene silencing by siRNAs in cultured human cells. Transcript profiles revealed siRNA-specific rather than target-specific signatures, including direct silencing of nontargeted genes containing as few as eleven contiguous nucleotides of identity to the siRNA. These results demonstrate that siRNAs may cross-react with targets of limited sequence similarity.
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              CRISPR/Cas9 in Genome Editing and Beyond

              The Cas9 protein (CRISPR-associated protein 9), derived from type II CRISPR (clustered regularly interspaced short palindromic repeats) bacterial immune systems, is emerging as a powerful tool for engineering the genome in diverse organisms. As an RNA-guided DNA endonuclease, Cas9 can be easily programmed to target new sites by altering its guide RNA sequence, and its development as a tool has made sequence-specific gene editing several magnitudes easier. The nuclease-deactivated form of Cas9 further provides a versatile RNA-guided DNA-targeting platform for regulating and imaging the genome, as well as for rewriting the epigenetic status, all in a sequence-specific manner. With all of these advances, we have just begun to explore the possible applications of Cas9 in biomedical research and therapeutics. In this review, we describe the current models of Cas9 function and the structural and biochemical studies that support it. We focus on the applications of Cas9 for genome editing, regulation, and imaging, discuss other possible applications and some technical considerations, and highlight the many advantages that CRISPR/Cas9 technology offers.
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                Author and article information

                Journal
                Cell Discov
                Cell Discov
                Cell Discovery
                Nature Publishing Group
                2056-5968
                06 June 2017
                2017
                : 3
                : 17018
                Affiliations
                [1 ]Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences , Shanghai, China
                [2 ]School of Life Science and Technology, Shanghai Tech University , Shanghai, China
                [3 ]University of Chinese Academy of Sciences , Beijing, China
                [4 ]State Engineering Laboratory of Medical Key Technologies Application of Synthetic Biology, Shenzhen Second People’s Hospital, The First Affiliated Hospital of Shenzhen University , Shenzhen, China
                [5 ]Sun Yat-sen University Cancer Center , Guangzhou, China
                [6 ]Shanghai Tolo Biotechnology Company Limited , Shanghai, China
                Author notes
                [* ] Tel: +86 21 54971125; Fax: +86 21 54971126; E-mail: wangj01@ 123456hotmail.com
                [7]

                These authors contributed equally to this work.

                JW and GZ designed the experiments. XZ, JW and QC conducted all experiments. XZ drew the illustration figure of the Cpf1-mediated repression of transcription elongation. JW wrote the manuscript, and all authors revised the manuscript.

                Article
                celldisc201718
                10.1038/celldisc.2017.18
                5460296
                28607761
                daa37217-423c-489f-943c-c0c2be9b5790
                Copyright © 2017 The Author(s)

                This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

                History
                : 23 March 2017
                : 24 April 2017
                Categories
                Article

                crispr,cpf1,dnase-dead cpf1 (ddcpf1),crispri,multiplex gene regulation

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