Efficient gene replacement by CRISPR/Cas-mediated homologous recombination in the model diatom Thalassiosira pseudonana

Nigel Belshaw, Irina Grouneva, Lior Aram, Assaf Gal, Amanda Hopes, Thomas Mock

Research output: Contribution to journalArticlepeer-review

10 Citations (Scopus)

Abstract

CRISPR/Cas enables targeted genome editing in many different plant and algal species including the model diatom Thalassiosira pseudonana. However, efficient gene targeting by homologous recombination (HR) to date is only reported for photosynthetic organisms in their haploid life-cycle phase. Here, a CRISPR/Cas construct, assembled using Golden Gate cloning, enabled highly efficient HR in a diploid photosynthetic organism. Homologous recombination was induced in T. pseudonana using sequence-specific CRISPR/Cas, paired with a dsDNA donor matrix, generating substitution of the silacidin, nitrate reductase and urease genes by a resistance cassette (FCP:NAT). Up to c. 85% of NAT-resistant T. pseudonana colonies screened positive for HR by nested PCR. Precise integration of FCP:NAT at each locus was confirmed using an inverse PCR approach. The knockout of the nitrate reductase and urease genes impacted growth on nitrate and urea, respectively, while the knockout of the silacidin gene in T. pseudonana caused a significant increase in cell size, confirming the role of this gene for cell-size regulation in centric diatoms. Highly efficient gene targeting by HR makes T. pseudonana as genetically tractable as Nannochloropsis and Physcomitrella, hence rapidly advancing functional diatom biology, bionanotechnology and biotechnological applications targeted on harnessing the metabolic potential of diatoms.

Original languageEnglish
Pages (from-to)438-452
Number of pages15
JournalNew Phytologist
Volume238
Issue number1
Early online date28 Oct 2022
DOIs
Publication statusPublished - Apr 2023

Keywords

  • algae
  • CRISPR
  • diatom
  • genome editing
  • homology-directed repair
  • recombination
  • synthetic biology
  • Thalassiosira pseudonana

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