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Abstract CRISPR and TALENs are efficient systems for gene editing in many organisms including plants. In many cases the CRISPR–Cas or TALEN modules are expressed in the plant cell only transiently. Theoretically, transient expression of the editing modules should limit unexpected effects compared to stable transformation. However, very few studies have measured the off-target and unpredicted effects of editing strategies on the plant genome, and none of them have compared these two major editing systems. We conducted, in Physcomitrium patens, a comprehensive genome-wide investigation of off-target mutations using either a CRISPR–Cas9 or a TALEN strategy. We observed a similar number of differences for the two editing strategies compared to control non-transfected plants, with an average of 8.25 SNVs and 19.5 InDels for the CRISPR-edited plants, and an average of 17.5 SNVs and 32 InDels for the TALEN-edited plants. Interestingly, a comparable number of SNVs and InDels could be detected in the PEG-treated control plants. This shows that except for the on-target modifications, the gene editing tools used in this study did not show a significant off-target activity nor unpredicted effects on the genome, and did not lead to transgene integration. The PEG treatment, a well-established biotechnological method, in itself, was the main source of mutations found in the edited plants.

Adapting plants to sustainable cropping systems is a major challenge for facing climate change and promoting agroecological transition. Camelina sativa is an emerging oilseed crop species with climate-resilient properties that could be used in double-cropping systems, in particular as a summer catch crop. The availability of early-flowering camelina is essential in such cropping systems to allow full completion of the growth cycle during summer. Targeted induced gene variation (TIGV) was used in camelina on several flowering repressor genes identified in Arabidopsis to obtain early-flowering lines. Multiplex editing of 15 target genes representing FLOWERING LOCUS C, SHORT VEGETATIVE PHASE, LIKE HETEROCHROMATIN PROTEIN 1, TERMINAL FLOWER 1 and EARLY FLOWERING LOCUS 3 induced combinatorial mutations that were screened for early-flowering phenotypes. Certain mutants showing a stable early-flowering trait after five generations also presented additional phenotypes: determinate flowering, shorter stature and/or basal branching. Different combinations of mutations had a positive or negative impact on yield. This work demonstrates that efficient multiplex CRISPR is achievable in hexaploid plants like camelina, providing valuable genetic diversity for better selecting lines adapted to new cropping systems.