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 (sg RNAs) were designed to target an endogenous reporter gene, Pp APT, whose inactivation confers resistance to 2-fluoroadenine. Transformation of moss protoplasts with these sg RNAs 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 RAD51 protein, indicating that CRISPR-induced HDR is only partially mediated by the classical homologous recombination pathway. [ABSTRACT FROM AUTHOR]