Simple and Efficient Targeting of Multiple Genes Through CRISPR-Cas9 inPhyscomitrella patens.
Powerful genome editing technologies are needed for efficient gene function analysis. The CRISPR-Cas9 system has been adapted as an efficient gene-knock-out technology in a variety of species. However, in a number of situations, knocking out or modifying a single gene is not sufficient; this is particularly true for genes belonging to a common family, or for genes showing redundant functions. Like many plants, the model organismPhyscomitrella patenshas experienced multiple events of polyploidization during evolution that has resulted in a number of families of duplicated genes. Here, we report a robust CRISPR-Cas9 system, based on the codelivery of a CAS9 expressing cassette, multiple sgRNA vectors, and a cassette for transient transformation selection, for gene knock-out in multiple gene families. We demonstrate that CRISPR-Cas9-mediated targeting of five different genes allows the selection of a quintuple mutant, and all possible subcombinations of mutants, in one experiment, with no mutations detected in potential off-target sequences. Furthermore, we confirmed the observation that the presence of repeats in the vicinity of the cutting region favors deletion due to the alternative end joining pathway, for which induced frameshift mutations can be potentially predicted. Because the number of multiple gene families inPhyscomitrellais substantial, this tool opens new perspectives to study the role of expanded gene families in the colonization of land by plants.
Institut Jean-Pierre Bourgin, National Institute for Agricultural Research, AgroParisTech, Centre National de la Recherche Scientifique, Université Paris-Saclay, 78000 Versailles, France.
Mauricio
http://orcid.org/0000-0002-1380-0643
M
Lopez-Obando
Institut Jean-Pierre Bourgin, National Institute for Agricultural Research, AgroParisTech, Centre National de la Recherche Scientifique, Université Paris-Saclay, 78000 Versailles, France.
Beate
B
Hoffmann
Institut Jean-Pierre Bourgin, National Institute for Agricultural Research, AgroParisTech, Centre National de la Recherche Scientifique, Université Paris-Saclay, 78000 Versailles, France.
Carine
C
Géry
Institut Jean-Pierre Bourgin, National Institute for Agricultural Research, AgroParisTech, Centre National de la Recherche Scientifique, Université Paris-Saclay, 78000 Versailles, France.
Anouchka
A
Guyon-Debast
Institut Jean-Pierre Bourgin, National Institute for Agricultural Research, AgroParisTech, Centre National de la Recherche Scientifique, Université Paris-Saclay, 78000 Versailles, France.|Université Pierre et Marie Curie, Sorbonne Universités, 75005 Paris, France.
Evelyne
E
Téoulé
Institut Jean-Pierre Bourgin, National Institute for Agricultural Research, AgroParisTech, Centre National de la Recherche Scientifique, Université Paris-Saclay, 78000 Versailles, France.
Catherine
C
Rameau
Institut Jean-Pierre Bourgin, National Institute for Agricultural Research, AgroParisTech, Centre National de la Recherche Scientifique, Université Paris-Saclay, 78000 Versailles, France sandrine.bonhomme@versailles.inra.fr fabien.nogue@versailles.inra.fr.
Sandrine
S
Bonhomme
Institut Jean-Pierre Bourgin, National Institute for Agricultural Research, AgroParisTech, Centre National de la Recherche Scientifique, Université Paris-Saclay, 78000 Versailles, France sandrine.bonhomme@versailles.inra.fr fabien.nogue@versailles.inra.fr.
Fabien
http://orcid.org/0000-0003-0619-4638
F
Nogué
AP2/ERF transcription factor; CRISPR-Cas9; KAI2; butenolide receptor; moss; multiple gene targeting