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

Belshaw, Nigel, Grouneva, Irina, Aram, Lior, Gal, Assaf, Hopes, Amanda and Mock, Thomas ORCID: https://orcid.org/0000-0001-9604-0362 (2023) Efficient gene replacement by CRISPR/Cas-mediated homologous recombination in the model diatom Thalassiosira pseudonana. New Phytologist, 238 (1). pp. 438-452. ISSN 0028-646X

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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.

Item Type:	Article
Additional Information:	Funding Information: TM acknowledges support from the Gordon and Betty Moore Foundation (grant no. 4961) and support received from the Natural Environment Research Council (NERC) (NE/K013734/1). AH acknowledges funding from NERC for her PhD studentship. Publisher Copyright: © 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.
Uncontrolled Keywords:	algae,crispr,diatom,genome editing,homology-directed repair,recombination,synthetic biology,thalassiosira pseudonana,physiology,plant science ,/dk/atira/pure/subjectarea/asjc/1300/1314
Faculty \ School:	Faculty of Science > School of Environmental Sciences University of East Anglia Research Groups/Centres > Theme - ClimateUEA
UEA Research Groups:	Faculty of Science > Research Centres > Centre for Ecology, Evolution and Conservation Faculty of Science > Research Groups > Centre for Ocean and Atmospheric Sciences Faculty of Science > Research Groups > Environmental Biology
Related URLs:	http://www.scopus.com/inward/record.url?...
Depositing User:	LivePure Connector
Date Deposited:	11 Mar 2024 10:31
Last Modified:	11 Mar 2024 10:31
URI:	https://ueaeprints.uea.ac.uk/id/eprint/94624
DOI:	10.1111/nph.18587

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