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      DNA-free two-gene knockout in Chlamydomonas reinhardtii via CRISPR-Cas9 ribonucleoproteins

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          Abstract

          Microalgae are versatile organisms capable of converting CO 2, H 2O, and sunlight into fuel and chemicals for domestic and industrial consumption. Thus, genetic modifications of microalgae for enhancing photosynthetic productivity, and biomass and bio-products generation are crucial for both academic and industrial applications. However, targeted mutagenesis in microalgae with CRISPR-Cas9 is limited. Here we report, a one-step transformation of Chlamydomonas reinhardtii by the DNA-free CRISPR-Cas9 method rather than plasmids that encode Cas9 and guide RNAs. Outcome was the sequential CpFTSY and ZEP two-gene knockout and the generation of a strain constitutively producing zeaxanthin and showing improved photosynthetic productivity.

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          COPPER ENZYMES IN ISOLATED CHLOROPLASTS. POLYPHENOLOXIDASE IN BETA VULGARIS.

          D ARNON (1949)
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            Efficient Delivery of Genome-Editing Proteins In Vitro and In Vivo

            John Zuris, David Thompson, Yilai Shu … (2014)
            Efficient intracellular delivery of proteins is needed to fully realize the potential of protein therapeutics. Current methods of protein delivery commonly suffer from low tolerance for serum, poor endosomal escape, and limited in vivo efficacy. Here we report that common cationic lipid nucleic acid transfection reagents can potently deliver proteins that are fused to negatively supercharged proteins, that contain natural anionic domains, or that natively bind to anionic nucleic acids. This approach mediates the potent delivery of nM concentrations of Cre recombinase, TALE- and Cas9-based transcriptional activators, and Cas9:sgRNA nuclease complexes into cultured human cells in media containing 10% serum. Delivery of Cas9:sgRNA complexes resulted in up to 80% genome modification with substantially higher specificity compared to DNA transfection. This approach also mediated efficient delivery of Cre recombinase and Cas9:sgRNA complexes into the mouse inner ear in vivo, achieving 90% Cre-mediated recombination and 20% Cas9-mediated genome modification in hair cells.
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              An outlook on microalgal biofuels.

              R. H. Wijffels, M Barbosa (2010)
              Microalgae are considered one of the most promising feedstocks for biofuels. The productivity of these photosynthetic microorganisms in converting carbon dioxide into carbon-rich lipids, only a step or two away from biodiesel, greatly exceeds that of agricultural oleaginous crops, without competing for arable land. Worldwide, research and demonstration programs are being carried out to develop the technology needed to expand algal lipid production from a craft to a major industrial process. Although microalgae are not yet produced at large scale for bulk applications, recent advances-particularly in the methods of systems biology, genetic engineering, and biorefining-present opportunities to develop this process in a sustainable and economical way within the next 10 to 15 years.
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                Author and article information

                Journal
                Journal ID (nlm-ta): Sci Rep
                Journal ID (iso-abbrev): Sci Rep
                Title: Scientific Reports
                Publisher: Nature Publishing Group
                ISSN (Electronic): 2045-2322
                Publication date (Electronic): 28 July 2016
                Publication date Collection: 2016
                Volume: 6
                Electronic Location Identifier: 30620
                Affiliations
                [1 ]Department of Life Science, Hanyang University , Seoul, South Korea
                [2 ]Department of Chemistry, Hanyang University , Seoul, South Korea
                [3 ]Department of Chemical and Biological Engineering, Korea University , Seoul, South Korea
                [4 ]Department of Plant and Microbial Biology, University of California , Berkeley, CA 94720-3102, USA
                [5 ]Center for Genome Engineering, Institute for Basic Science , Seoul, South Korea
                [6 ]Department of Chemistry, Seoul National University , Seoul, South Korea
                Author notes
                [*]

                These authors contributed equally to this work.

                Article
                Publisher Item ID: srep30620
                DOI: 10.1038/srep30620
                PMC ID: 4964356
                PubMed ID: 27466170
                SO-VID: fa8537f4-23ac-461f-9a81-b6de81f7b427
                Copyright statement: Copyright © 2016, Macmillan Publishers Limited
                License:

                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
                Date received : 30 April 2016
                Date accepted : 06 July 2016
                Categories
                Subject: Article

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