Ceratopteris richardii U6 promoters and plants expressing Cas9 endonuclease as tools for efficient genome editing in a fern

Ferns and their allies (monilophytes) represent the second most species-rich group of land plants and are of considerable ecological importance. As a sister group of seed plants (including flowering plants) they are also of great evolutionary interest. Compared to flowering plants, however, much less is known about the developmental and molecular biology of ferns. Among the most important reasons have been the huge genome sizes of ferns and technical obstacles such as the lack of an efficient transformation system. In recent years the situation has improved considerably, however. For the fern model system Ceratopteris richardii a whole genome sequence has been published, and an efficient transformation system has been developed. To further facilitate studies on fern biology we aim at simplifying genome engineering of C. richardii with the CRISPR-Cas9 system. We report C. richardii plants that express Cas9 nuclease under control of the strong CaMV 35S promoter. For efficient expression of single guide RNA (sgRNA) by RNA polymerase III we identified C. richardii U6 promoters. These technical improvements may foster many fields of fern physiology, development and evolution.