Deciphering the DSB repair mechanisms involved in CRISPR-induced mutagenesis and gene targeting in the model plant, Physcomitrella patens.

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