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Cpf1 Is A Versatile Tool for CRISPR Genome Editing Across Diverse Species of Cyanobacteria

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Scientific Reports

Nature Publishing Group

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      Abstract

      Cyanobacteria are the ideal organisms for the production of a wide range of bioproducts as they can convert CO2 directly into the desired end product using solar energy. Unfortunately, the engineering of cyanobacteria to create efficient cell factories has been impaired by the cumbersome genetic tools that are currently available for these organisms; especially when trying to accumulate multiple modifications. We sought to construct an efficient and precise tool for generating numerous markerless modifications in cyanobacteria using CRISPR technology and the alternative nuclease, Cpf1. In this study we demonstrate rapid engineering of markerless knock-ins, knock-outs and point mutations in each of three model cyanobacteria; Synechococcus, Synechocystis and Anabaena. The markerless nature of cpf1 genome editing will allow for complex genome modification that was not possible with previously existing technology while facilitating the development of cyanobacteria as highly modified biofactories.

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      Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system.

      The microbial adaptive immune system CRISPR mediates defense against foreign genetic elements through two classes of RNA-guided nuclease effectors. Class 1 effectors utilize multi-protein complexes, whereas class 2 effectors rely on single-component effector proteins such as the well-characterized Cas9. Here, we report characterization of Cpf1, a putative class 2 CRISPR effector. We demonstrate that Cpf1 mediates robust DNA interference with features distinct from Cas9. Cpf1 is a single RNA-guided endonuclease lacking tracrRNA, and it utilizes a T-rich protospacer-adjacent motif. Moreover, Cpf1 cleaves DNA via a staggered DNA double-stranded break. Out of 16 Cpf1-family proteins, we identified two candidate enzymes from Acidaminococcus and Lachnospiraceae, with efficient genome-editing activity in human cells. Identifying this mechanism of interference broadens our understanding of CRISPR-Cas systems and advances their genome editing applications.
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        The CRISPR/Cas9 system for plant genome editing and beyond.

        Targeted genome editing using artificial nucleases has the potential to accelerate basic research as well as plant breeding by providing the means to modify genomes rapidly in a precise and predictable manner. Here we describe the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system, a recently developed tool for the introduction of site-specific double-stranded DNA breaks. We highlight the strengths and weaknesses of this technology compared with two well-established genome editing platforms: zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). We summarize recent results obtained in plants using CRISPR/Cas9 technology, discuss possible applications in plant breeding and consider potential future developments.
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          Purification and properties of unicellular blue-green algae (order Chroococcales).

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            Author and article information

            Affiliations
            [1 ]Department of Biology, Washington University , St. Louis, MO 63130 USA
            Author notes
            Journal
            Sci Rep
            Sci Rep
            Scientific Reports
            Nature Publishing Group
            2045-2322
            21 December 2016
            2016
            : 6
            28000776
            5175191
            srep39681
            10.1038/srep39681
            Copyright © 2016, 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/

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