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      Efficient inhibition of duck hepatitis B virus DNA by the CRISPR/Cas9 system

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          Abstract

          Current therapeutic strategies cannot eradicate hepatitis B virus covalently closed circular DNA (HBV cccDNA), which accounts for the persistence of HBV infection. Very recently, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been used as an efficient and powerful tool for viral genome editing. Given that the primary duck hepatocyte (PDH) infected with duck hepatitis B virus (DHBV) has been widely used to study human HBV infection in vitro, the present study aimed to demonstrate the targeted inhibition of DHBV DNA, especially cccDNA, by the CRISPR/Cas9 system using this model. We designed six single-guide RNAs (sgRNA1-6) targeting the DHBV genome. The sgRNA/Cas9 plasmid was transfected into DHBV-infected PDHs, and then DHBV total DNA (in culture medium and PDHs) and cccDNA were quantified by reverse transcription-quantitative polymerase chain reaction. The combined inhibition of CRISPR/Cas9 system and entecavir (ETV) was also assessed. Two sgRNAs, sgRNA4 and sgRNA6, exhibited efficient inhibition on DHBV total DNA (77.23 and 86.51%, respectively), cccDNA (75.67 and 85.34%, respectively) in PDHs, as well as DHBV total DNA in the culture medium (62.17 and 59.52%, respectively). The inhibition remained or enhanced from day 5 to day 9 following transfection. The combination of the CRISPR/Cas9 system and ETV further increased the inhibitory effect on DHBV total DNA in PDHs and culture medium, but not cccDNA. The CRISPR/Cas9 system has the potential to be a useful tool for the suppression of DHBV DNA.

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          A TALE nuclease architecture for efficient genome editing.

          Nucleases that cleave unique genomic sequences in living cells can be used for targeted gene editing and mutagenesis. Here we develop a strategy for generating such reagents based on transcription activator-like effector (TALE) proteins from Xanthomonas. We identify TALE truncation variants that efficiently cleave DNA when linked to the catalytic domain of FokI and use these nucleases to generate discrete edits or small deletions within endogenous human NTF3 and CCR5 genes at efficiencies of up to 25%. We further show that designed TALEs can regulate endogenous mammalian genes. These studies demonstrate the effective application of designed TALE transcription factors and nucleases for the targeted regulation and modification of endogenous genes.
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            Genome Engineering of Drosophila with the CRISPR RNA-Guided Cas9 Nuclease

            We have adapted a bacterial CRISPR RNA/Cas9 system to precisely engineer the Drosophila genome and report that Cas9-mediated genomic modifications are efficiently transmitted through the germline. This RNA-guided Cas9 system can be rapidly programmed to generate targeted alleles for probing gene function in Drosophila.
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              The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats

              Background In Archeae and Bacteria, the repeated elements called CRISPRs for "clustered regularly interspaced short palindromic repeats" are believed to participate in the defence against viruses. Short sequences called spacers are stored in-between repeated elements. In the current model, motifs comprising spacers and repeats may target an invading DNA and lead to its degradation through a proposed mechanism similar to RNA interference. Analysis of intra-species polymorphism shows that new motifs (one spacer and one repeated element) are added in a polarised fashion. Although their principal characteristics have been described, a lot remains to be discovered on the way CRISPRs are created and evolve. As new genome sequences become available it appears necessary to develop automated scanning tools to make available CRISPRs related information and to facilitate additional investigations. Description We have produced a program, CRISPRFinder, which identifies CRISPRs and extracts the repeated and unique sequences. Using this software, a database is constructed which is automatically updated monthly from newly released genome sequences. Additional tools were created to allow the alignment of flanking sequences in search for similarities between different loci and to build dictionaries of unique sequences. To date, almost six hundred CRISPRs have been identified in 475 published genomes. Two Archeae out of thirty-seven and about half of Bacteria do not possess a CRISPR. Fine analysis of repeated sequences strongly supports the current view that new motifs are added at one end of the CRISPR adjacent to the putative promoter. Conclusion It is hoped that availability of a public database, regularly updated and which can be queried on the web will help in further dissecting and understanding CRISPR structure and flanking sequences evolution. Subsequent analyses of the intra-species CRISPR polymorphism will be facilitated by CRISPRFinder and the dictionary creator. CRISPRdb is accessible at
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                Author and article information

                Journal
                Mol Med Rep
                Mol Med Rep
                Molecular Medicine Reports
                D.A. Spandidos
                1791-2997
                1791-3004
                November 2017
                19 September 2017
                19 September 2017
                : 16
                : 5
                : 7199-7204
                Affiliations
                [1 ]Artificial Liver Center, Beijing Youan Hospital, Capital Medical University, Beijing 100069, P.R. China
                [2 ]Department of Gastroenterology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan 450052, P.R. China
                [3 ]Department of Gastroenterology, The First Affiliated Hospital of Xinxiang Medical College, Weihui, Henan 410781, P.R. China
                [4 ]Department of Gastroenterology, Pennsylvania Hospital, University of Pennsylvania, Philadelphia, PA 19104, USA
                Author notes
                Correspondence to: Professor Zhongping Duan, Artificial Liver Center, Beijing Youan Hospital, Capital Medical University, 8 Xitoutiao Road, Fengtai, Beijing 100069, P.R. China, E-mail: duan2517@ 123456163.com
                Dr Jiansheng Li, Department of Gastroenterology, The First Affiliated Hospital of Zhengzhou University, 1 Jianshe East Road, Zhengzhou, Henan 450052, P.R. China, E-mail: lijiansheng@ 123456medmail.com.cn
                [*]

                Contributed equally

                Article
                mmr-16-05-7199
                10.3892/mmr.2017.7518
                5865846
                28944845
                9b2fa1dd-6632-45d7-bb55-52ad73245c43
                Copyright: © Zheng et al.

                This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 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
                : 01 May 2016
                : 28 March 2017
                Categories
                Articles

                crispr/cas9,duck hepatitis b virus,hepatitis b virus,covalently closed circular dna,antivirus

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