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Plastids pp 367-378 | Cite as

Generation of Mutants of Nuclear-Encoded Plastid Proteins Using CRISPR/Cas9 in the Diatom Phaeodactylum tricornutum

  • Guillaume AllorentEmail author
  • Erika Guglielmino
  • Cécile Giustini
  • Florence Courtois
Part of the Methods in Molecular Biology book series (MIMB, volume 1829)

Abstract

Genome modifications in microalgae are becoming a widespread and mandatory tool for research in both fundamental and applied biology. Among genome editing methods in these photosynthetic organisms, CRISPR/Cas9 offers a specific, powerful and efficient tool for genome engineering by inducing mutations in targeted regions of the genome. Here we described a protocol that allows the generation of knockout mutants by CRISPR/Cas9 in the diatom Phaeodactylum tricornutum using biolistic transformation.

Key words

Phaeodactylum tricornutum CRISPR/Cas9 Genome editing Biolistic transformation 

Notes

Acknowledgments

This work was supported by the French National Research Agency (ANR-10-LABEX-04 GRAL Labex, Grenoble Alliance for Integrated Structural Cell Biology) and by a grant from the HFSP (to G.A).

References

  1. 1.
    Chisti Y (2007) Biodiesel from microalgae. Biotechnol Adv 25(3):294–306CrossRefPubMedGoogle Scholar
  2. 2.
    Ambrust EV (2009) The life of diatoms in the world’s ocean. Nature 459(7244):185–192CrossRefGoogle Scholar
  3. 3.
    Bowler C, Allen AE, Badger JH et al (2008) The Phaeodactylum genome reveals the evolutionary history of diatom genomes. Nature 456(7219):239–244CrossRefGoogle Scholar
  4. 4.
    Daboussi F, Leduc S, Maréchal A et al (2014) Genome engineering empowers the diatom Phaeodactylum tricornutum for biotechnology. Nat Commun 5:3831CrossRefPubMedGoogle Scholar
  5. 5.
    Weyman PD, Beeri K, Lefebvre SC et al (2015) Inactivation of Phaeodactylum tricornutum urease gene using transcription activator-like effector nuclease-based targeted mutagenesis. Plant Biotechnol J 13(4):460–470CrossRefPubMedGoogle Scholar
  6. 6.
    Nymark M, Sharma AK, Sparstad T et al (2016) A CRISPR/Cas9 system adapted for gene editing in marine algae. Sci Rep 6:24951CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Jinek M, Chylinski K, Fonfara I et al (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337(6096):816–821CrossRefGoogle Scholar
  8. 8.
    Sander JD, Joung JK (2014) CRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotechnol 32(4):347–355CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Wang H, La Russa M, Qi LS (2016) CRISPR/Cas9 in genome editing and beyond. Annu Rev Biochem 85:227–264CrossRefPubMedGoogle Scholar
  10. 10.
    Villanova V, Fortunato AE, Singh D et al (2017) Investigating mixotrophic metabolism in the model diatom Phaeodactylum tricornutum. Philos Trans R Soc Lond Ser B Biol Sci 372(1728). https://doi.org/10.1098/rstb.2016.0404
  11. 11.
    Falciatore A, Casotti R, Leblanc C et al (1999) Transformation of nonselectable reporter genes in marine diatoms. Mar Biotechnol (NY) 1(3):239–251CrossRefGoogle Scholar
  12. 12.
    Rastogi A, Murik O, Bowler C et al (2016) PhytoCRISP-ex : a web-based and stand-alone application to find specific target sequences for CRISPR/CAS editing. BMC Bioinformatics 17(1):261CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Guillaume Allorent
    • 1
    Email author
  • Erika Guglielmino
    • 1
  • Cécile Giustini
    • 1
  • Florence Courtois
    • 1
  1. 1.Laboratoire de Physiologie Cellulaire et Végétale, Centre National de la Recherche Scientifique, Institut National Recherche Agronomique, Commissariat à l’Energie Atomique et aux Energies Alternatives, CEA GrenobleUMR5168, Université Grenoble AlpesGrenobleFrance

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