One-Vector System for Multiplexed CRISPR/Cas9 against Hepatitis B Virus cccDNA Utilizing High-Capacity Adenoviral Vectors – ScienceOpen
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      One-Vector System for Multiplexed CRISPR/Cas9 against Hepatitis B Virus cccDNA Utilizing High-Capacity Adenoviral Vectors

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          Abstract

          High-capacity adenoviral vectors (HCAdVs) devoid of all coding genes are powerful tools to deliver large DNA cargos into cells. Here HCAdVs were designed to deliver a multiplexed complete CRISPR/Cas9 nuclease system or a complete pair of transcription activator-like effector nucleases (TALENs) directed against the hepatitis B virus (HBV) genome. HBV, which remains a serious global health burden, forms covalently closed circular DNA (cccDNA) as a persistent DNA species in infected cells. This cccDNA promotes the chronic carrier status, and it represents a major hurdle in the treatment of chronic HBV infection. To date, only one study demonstrated viral delivery of a CRISPR/Cas9 system and a single guide RNA (gRNA) directed against HBV by adeno-associated viral (AAV) vectors. The advancement of this study is the co-delivery of multiple gRNA expression cassettes along with the Cas9 expression cassette in one HCAdV. Treatment of HBV infection models resulted in a significant reduction of HBV antigen production and the introduction of mutations into the HBV genome. In the transduction experiments, the HBV genome, including the HBV cccDNA, was degraded by the CRISPR/Cas9 system. In contrast, the combination of two parts of a TALEN pair in one vector could not be proven to yield an active system. In conclusion, we successfully delivered the CRISPR/Cas9 system containing three gRNAs using HCAdV, and we demonstrated its antiviral effect.

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          Multiplex genome engineering using CRISPR/Cas systems.

          Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.
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            Molecular Cloning : A Laboratory Manual

            <p>The first two editions of this manual have been mainstays of molecular biology for nearly twenty years, with an unrivalled reputation for reliability, accuracy, and clarity.<br>In this new edition, authors Joseph Sambrook and David Russell have completely updated the book, revising every protocol and adding a mass of new material, to broaden its scope and maintain its unbeatable value for studies in genetics, molecular cell biology, developmental biology, microbiology, neuroscience, and immunology.<br>Handsomely redesigned and presented in new bindings of proven durability, this three–volume work is essential for everyone using today’s biomolecular techniques.<br>The opening chapters describe essential techniques, some well–established, some new, that are used every day in the best laboratories for isolating, analyzing and cloning DNA molecules, both large and small.<br>These are followed by chapters on cDNA cloning and exon trapping, amplification of DNA, generation and use of nucleic acid probes, mutagenesis, and DNA sequencing.<br>The concluding chapters deal with methods to screen expression libraries, express cloned genes in both prokaryotes and eukaryotic cells, analyze transcripts and proteins, and detect protein–protein interactions.<br>The Appendix is a compendium of reagents, vectors, media, technical suppliers, kits, electronic resources and other essential information.<br>As in earlier editions, this is the only manual that explains how to achieve success in cloning and provides a wealth of information about why techniques work, how they were first developed, and how they have evolved. </p>
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              Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting

              TALENs are important new tools for genome engineering. Fusions of transcription activator-like (TAL) effectors of plant pathogenic Xanthomonas spp. to the FokI nuclease, TALENs bind and cleave DNA in pairs. Binding specificity is determined by customizable arrays of polymorphic amino acid repeats in the TAL effectors. We present a method and reagents for efficiently assembling TALEN constructs with custom repeat arrays. We also describe design guidelines based on naturally occurring TAL effectors and their binding sites. Using software that applies these guidelines, in nine genes from plants, animals and protists, we found candidate cleavage sites on average every 35 bp. Each of 15 sites selected from this set was cleaved in a yeast-based assay with TALEN pairs constructed with our reagents. We used two of the TALEN pairs to mutate HPRT1 in human cells and ADH1 in Arabidopsis thaliana protoplasts. Our reagents include a plasmid construct for making custom TAL effectors and one for TAL effector fusions to additional proteins of interest. Using the former, we constructed de novo a functional analog of AvrHah1 of Xanthomonas gardneri. The complete plasmid set is available through the non-profit repository AddGene and a web-based version of our software is freely accessible online.
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                Author and article information

                Contributors
                Journal
                Mol Ther Nucleic Acids
                Mol Ther Nucleic Acids
                Molecular Therapy. Nucleic Acids
                American Society of Gene & Cell Therapy
                2162-2531
                07 June 2018
                07 September 2018
                07 June 2018
                : 12
                : 242-253
                Affiliations
                [1 ]Center of Biomedical Education and Research (ZBAF), Department of Human Medicine, Faculty of Health, Witten/Herdecke University, Witten, Germany
                [2 ]Institute of Virology, Technical University of Munich/Helmholtz Zentrum München, Munich, Germany
                [3 ]German Center for Infection Research (DZIF), partner site Munich, Munich, Germany
                Author notes
                []Corresponding author: Anja Ehrhardt, Center of Biomedical Education and Research (ZBAF), Department of Human Medicine, Faculty of Health, Witten/Herdecke University Stockumerstr. 10, 58453 Witten, Germany. anja.ehrhardt@ 123456uni-wh.de
                Article
                S2162-2531(18)30103-3
                10.1016/j.omtn.2018.05.006
                6023846
                30195763
                d328fd85-9fd6-4449-a8ab-d726aeab3e2c
                © 2018 The Authors

                This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

                History
                : 28 March 2018
                : 10 May 2018
                Categories
                Article

                Molecular medicine
                chronic hbv infection,multiplexed crispr/cas9,high-capacity adenovirus,cccdna degradation

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