[en] The ability to address the CRISPR-Cas9 nuclease complex to any target DNA using customizable
single-guide RNAs has now permitted genome engineering in many species. Here, we report its
first successful use in a nonvascular plant, the moss Physcomitrella patens. Single-guide RNAs
(sgRNAs) were designed to target an endogenous reporter gene, PpAPT, whose inactivation
confers resistance to 2-fluoroadenine. Transformation of moss protoplasts with these sgRNAs
and the Cas9 coding sequence from Streptococcus pyogenes triggered mutagenesis at the
PpAPT target in about 2% of the regenerated plants. Mainly, deletions were observed, most of
them resulting from alternative end-joining (alt-EJ)-driven repair. We further demonstrate that, in
the presence of a donor DNA sharing sequence homology with the PpAPT gene, most transgene
integration events occur by homology-driven repair (HDR) at the target locus but also that Cas9-
induced double-strand breaks are repaired with almost equal frequencies by mutagenic
illegitimate recombination. Finally, we establish that a significant fraction of HDR-mediated gene
targeting events (30%) is still possible in the absence of PpRAD51 protein, indicating that
CRISPR-induced HDR is only partially mediated by the classical homologous recombination
pathway.
Disciplines :
Biotechnologie
Auteur, co-auteur :
Collonnier, Cécile; INRA Centre de Versailles-Grignon, Versailles Cedex, France > Institut Jean-Pierre Bourgin (UMR1318) > Méiose et Recombinaison
Epert, Aline; INRA Centre de Versailles-Grignon, Versailles Cedex, France > Institut Jean-Pierre Bourgin > Méiose et Recombinaison
Mara, Kostlend; INRA Centre de Versailles-Grignon, Versailles Cedex, France > Institut Jean-Pierre Bourgin (UMR1318) > Méiose et Recombinaison
Maclot, François ; Université de Liège > Agronomie, Bio-ingénierie et Chimie (AgroBioChem) > Gestion durable des bio-agresseurs
Guyon-Debast, Anouchkla; INRA Centre de Versailles-Grignon, Versailles Cedex, France > Institut Jean-Pierre Bourgin (UMR1318) > Méiose et Recombinaison
Charlot, Florence; INRA Centre de Versailles-Grignon, Versailles Cedex, France > Institut Jean-Pierre Bourgin (UMR1318) > Méiose et Recombinaison
White, Charles; Institut Blaise Pascal, Clermont Ferran, France > Génétique, Reproduction et Développement, UMR CNRS 6293
Schaefer, Didier G.; Université de Neuchâtel, Neuchâtel, Suisse > Institut de Biologie > Laboratoire de Biologie Moléculaire et Cellulaire
Nogué, Fabien; INRA Centre de Versailles-Grignon, Versailles Cedex, France > Institut Jean-Pierre Bourgin (UMR1318) > Méiose et Recombinaison
Langue du document :
Anglais
Titre :
CRISPR-Cas9-mediated efficient directed mutagenesis and RAD51-dependent and RAD51-independent gene targeting in the moss Physcomitrella patens
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