Open AccessCRISPR ‐Cas9‐mediated efficient directed mutagenesis and RAD 51‐dependent and RAD 51‐independent gene targeting in the moss Physcomitrella patens
Author(s) -
Collonnier Cécile,
Epert Aline,
Mara Kostlend,
Maclot François,
GuyonDebast Anouchka,
Charlot Florence,
White Charles,
Schaefer Didier G.,
Nogué Fabien
Publication year - 2017
Publication title -
plant biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.525
H-Index - 115
eISSN - 1467-7652
pISSN - 1467-7644
DOI - 10.1111/pbi.12596
Subject(s) - biology , physcomitrella patens , crispr , cas9 , homologous recombination , gene , genetics , gene targeting , genome editing , guide rna , mutagenesis , genome engineering , homology (biology) , dna , nuclease , homology directed repair , computational biology , dna repair , mutant , nucleotide excision repair
Summary The ability to address the CRISPR ‐Cas9 nuclease complex to any target DNA using customizable single‐guide RNA s 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 RNA s (sg RNA s) were designed to target an endogenous reporter gene, Pp APT , whose inactivation confers resistance to 2‐fluoroadenine. Transformation of moss protoplasts with these sg RNA s and the Cas9 coding sequence from Streptococcus pyogenes triggered mutagenesis at the Pp APT 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 Pp APT 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 Pp RAD 51 protein, indicating that CRISPR ‐induced HDR is only partially mediated by the classical homologous recombination pathway.