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 th

SUBMITTER: Collonnier C 

PROVIDER: S-EPMC5253467 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature