[en] Site-directed nucleases make it very easy now to mutate (knock-out) any genomic sequence and to direct the insertion of a template DNA at a specific target (knock-in). The DNA repair mechanisms that are presumed to be mainly involved in these two types of events are respectively canonical non-homologous end-joining (c-NHEJ) and, if the template shares homology to the target, homologous recombination (HR). By using, the model plant Physcomitrella patens, where efficient gene editing (knock-out or knock-in) via SDN can be obtained, we demonstrated that it may not be as simple. By applying the CRISPR/Cas9 system to a series of mutants impacted in different DNA repair pathways, we progressed in the understanding of the different mechanisms that could be involved in CRISPR-induced mutagenesis and gene targeting in plants. Without template DNA, we showed that the mutation efficiency was not decreased in absence of key factors of c-NHEJ, which could indicate that CRISPR-induced mutations may not only be due to c-NHEJ but also to alternative end-joining like micro-homology mediated end joining (MMEJ) and other types of non-canonical end joining. Targeted insertion of a circular template DNA presenting homology to the target appeared to be mainly dependent on the RAD51-dependent HR pathway but not entirely, and other pathways, including single-strand annealing (SSA), could potentially be involved.
Research Center/Unit :
INRA Versailles, ULiège-GxABT
Disciplines :
Biotechnology
Author, co-author :
Collonnier, Cécile; 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
Epert, Aline; INRA Centre de Versailles-Grignon, Versailles Cedex, France > Institut Jean-Pierre Bourgin (UMR1318) > Méiose et Recombinaison
Mara, Kostlend; INRA Centre de Versailles-Grignon, Versailles Cedex, France > Institut Jean-Pierre Bourgin (UMR1318) > Méiose et Recombinaison
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
Nogué, Fabien; INRA Centre de Versailles-Grignon, Versailles Cedex, France > Institut Jean-Pierre Bourgin (UMR1318) > Méiose et Recombinaison
Language :
English
Title :
Deciphering the DSB repair mechanisms involved in CRISPR-induced mutagenesis and gene targeting in the model plant, Physcomitrella patens.
Publication date :
28 January 2016
Event name :
2nd edition of the VIB Congress, Genome engineering and synthetic biology: tools and technologies
Event organizer :
Vlaams Interuniversitair Instituut Voor Biotechnologie (Flanders Interuniversity Institute for Biotechnology)
Event place :
Gent, Belgium
Event date :
January 28-29 2016
Funders :
French programme “Investissements d’Avenir” and French National Research Agency through the research project GENIUS ANR‐11‐BTBR‐0001