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Targeted CRISPR‐Cas9‐based gene knockouts in the model brown alga Ectocarpus
Author(s) -
Badis Yacine,
Scornet Delphine,
Harada Minori,
Caillard Céline,
Godfroy Olivier,
Raphalen Morgane,
Gachon Claire M. M.,
Coelho Susana M.,
Motomura Taizo,
Nagasato Chikako,
Cock J. Mark
Publication year - 2021
Publication title -
new phytologist
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.742
H-Index - 244
eISSN - 1469-8137
pISSN - 0028-646X
DOI - 10.1111/nph.17525
Subject(s) - biology , crispr , model organism , organism , reverse genetics , gene , multicellular organism , genetics , cas9 , computational biology , mutant , gene knockout , forward genetics , genome editing
Summary Brown algae are an important group of multicellular eukaryotes, phylogenetically distinct from both the animal and land plant lineages. Ectocarpus has emerged as a model organism to study diverse aspects of brown algal biology, but this system currently lacks an effective reverse genetics methodology to analyse the functions of selected target genes. Here, we report that mutations at specific target sites are generated following the introduction of CRISPR‐Cas9 ribonucleoproteins into Ectocarpus cells, using either biolistics or microinjection as the delivery method. Individuals with mutations affecting the ADENINE PHOSPHORIBOSYL TRANSFERASE ( APT ) gene were isolated following treatment with 2‐fluoroadenine, and this selection system was used to isolate individuals in which mutations had been introduced simultaneously at APT and at a second gene. This double mutation approach could potentially be used to isolate mutants affecting any Ectocarpus gene, providing an effective reverse genetics tool for this model organism. The availability of this tool will significantly enhance the utility of Ectocarpus as a model organism for this ecologically and economically important group of marine organisms. Moreover, the methodology described here should be readily transferable to other brown algal species.