One-step generation of multiple gene knock-outs in the diatom Phaeodactylum tricornutum by DNA-free genome editing
| Autor: | Manuel Serif, Denis Jallet, Marie-Ange Teste, Anne-Laure Finoux, Gwendoline Dubois, Fayza Daboussi |
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| Přispěvatelé: | Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Gordon and Betty Moore Foundation [GBMF 4966], Region Midi-Pyrenees [15058490], 3BCAR Carnot Institute, ANR-16-CE05-0006,SynDia,Conception d'une plateforme d'ingénierie génomique pour les microalgues(2016), TESTE, Marie Ange, Conception d'une plateforme d'ingénierie génomique pour les microalgues - - SynDia2016 - ANR-16-CE05-0006 - AAPG2016 - VALID, Institut National des Sciences Appliquées (INSA)-Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées (INSA)-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS) |
| Jazyk: | angličtina |
| Rok vydání: | 2018 |
| Předmět: |
0106 biological sciences
0301 basic medicine [SDV.BIO]Life Sciences [q-bio]/Biotechnology Adenine Phosphoribosyltransferase gène codant General Physics and Astronomy diatomée 01 natural sciences Genome chemistry.chemical_compound Gene Knockout Techniques Genome editing Microalgae Multiplex lcsh:Science Gene Editing Multidisciplinary Vegetal Biology Ribonucleoproteins Orotate Phosphoribosyltransferase Transgene Science Orotidine-5'-Phosphate Decarboxylase Computational biology Biotechnologies Biology Transfection General Biochemistry Genetics and Molecular Biology 03 medical and health sciences Multienzyme Complexes Phaeodactylum tricornutum Amino Acid Sequence Gene photorécepteur Diatoms micro-algue Base Sequence Sequence Homology Amino Acid transgénique Algal Proteins édition de gènes Reproducibility of Results General Chemistry biology.organism_classification [SDV.BIO] Life Sciences [q-bio]/Biotechnology 030104 developmental biology chemistry Exogenous DNA lcsh:Q CRISPR-Cas Systems DNA Biologie végétale 010606 plant biology & botany |
| Zdroj: | Nature Communications Nature Communications, Nature Publishing Group, 2018, 9, pp.3924. ⟨10.1038/s41467-018-06378-9⟩ Nature Communications 1 (9), 10 p.. (2018) Nature Communications, 2018, 9 (1), pp.3924. ⟨10.1038/s41467-018-06378-9⟩ Nature Communications, Nature Publishing Group, 2018, 9 (1), pp.3924. ⟨10.1038/s41467-018-06378-9⟩ Nature Communications, Vol 9, Iss 1, Pp 1-10 (2018) |
| ISSN: | 2041-1723 |
| DOI: | 10.1038/s41467-018-06378-9⟩ |
| Popis: | Recently developed transgenic techniques to explore and exploit the metabolic potential of microalgae present several drawbacks associated with the delivery of exogenous DNA into the cells and its subsequent integration at random sites within the genome. Here, we report a highly efficient multiplex genome-editing method in the diatom Phaeodactylum tricornutum, relying on the biolistic delivery of CRISPR-Cas9 ribonucleoproteins coupled with the identification of two endogenous counter-selectable markers, PtUMPS and PtAPT. First, we demonstrate the functionality of RNP delivery by positively selecting the disruption of each of these genes. Then, we illustrate the potential of the approach for multiplexing by generating double-gene knock-out strains, with 65% to 100% efficiency, using RNPs targeting one of these markers and PtAureo1a, a photoreceptor-encoding gene. Finally, we created triple knock-out strains in one step by delivering six RNP complexes into Phaeodactylum cells. This approach could readily be applied to other hard-to-transfect organisms of biotechnological interest. |
| Databáze: | OpenAIRE |
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