title: CRISPR-Cas9-mediated efficient directed mutagenesis and RAD51-dependent and RAD51-independent gene targeting in the moss Physcomitrella patens.
abstract: 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.
authors.affiliation: INRA Centre de Versailles-Grignon, IJPB (UMR1318), Versailles Cedex, France.
authors.forename: Cécile
authors.initials: C
authors.lastname: Collonnier
authors.affiliation: INRA Centre de Versailles-Grignon, IJPB (UMR1318), Versailles Cedex, France.
authors.forename: Aline
authors.initials: A
authors.lastname: Epert
authors.affiliation: INRA Centre de Versailles-Grignon, IJPB (UMR1318), Versailles Cedex, France.
authors.forename: Kostlend
authors.initials: K
authors.lastname: Mara
authors.affiliation: INRA Centre de Versailles-Grignon, IJPB (UMR1318), Versailles Cedex, France.
authors.forename: François
authors.initials: F
authors.lastname: Maclot
authors.affiliation: INRA Centre de Versailles-Grignon, IJPB (UMR1318), Versailles Cedex, France.
authors.forename: Anouchka
authors.initials: A
authors.lastname: Guyon-Debast
authors.affiliation: INRA Centre de Versailles-Grignon, IJPB (UMR1318), Versailles Cedex, France.
authors.forename: Florence
authors.initials: F
authors.lastname: Charlot
authors.affiliation: Génétique, Reproduction et Développement, UMR CNRS 6293, Clermont Université, INSERM U1103, Université Blaise Pascal, Clermont Ferrand, France.
authors.forename: Charles
authors.initials: C
authors.lastname: White
authors.affiliation: Laboratoire de Biologie Moléculaire et Cellulaire, Institut de Biologie, Université de Neuchâtel, Neuchâtel, Switzerland.
authors.forename: Didier G
authors.initials: DG
authors.lastname: Schaefer
authors.affiliation: INRA Centre de Versailles-Grignon, IJPB (UMR1318), Versailles Cedex, France.
authors.forename: Fabien
authors.initials: F
authors.lastname: Nogué
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D029681:Arabidopsis Proteins / Q000235:genetics*; D019068:Bryopsida / Q000201:enzymology* / Q000235:genetics*; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D059766:DNA End-Joining Repair; D004720:Endonucleases; D018390:Gene Targeting / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods; D018745:Genome, Plant; D059765:Homologous Recombination; D016296:Mutagenesis; D030821:Plants, Genetically Modified; D011523:Protoplasts; D051135:Rad51 Recombinase / Q000235:genetics* / Q000378:metabolism; D017384:Sequence Deletion; D017385:Sequence Homology; D013297:Streptococcus pyogenes / Q000235:genetics; D014170:Transformation, Genetic
chemical_list: D029681:Arabidopsis Proteins; C038208:2-fluoroadenine; C495146:ATRAD51 protein, Arabidopsis; D051135:Rad51 Recombinase; D004720:Endonucleases; D000225:Adenine
keywords: 
  Physcomitrella patens
  ; CRISPR-Cas9; RAD51; alt-EJ; gene targeting; genome editing