CRISPR/Cas9-Induced Targeted Mutagenesis of the Moss

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a widely used programmable nuclease system for gene modification in many organisms, including Physcomitrium patens. P. patens is a model species of moss plants, a basal land plant group, which has been extensively studied from the viewpoint of evolution and diversity of green plant lineages. So far, gene modifications by CRISPR/Cas9 in P. patens have been carried out exclusively by the polyethylene glycol (PEG)-mediated DNA transfer method, in which a transgene (or transgenes) is introduced into protoplast cells prepared from protonemal tissues. However, this PEG-mediated method requires a relatively large amount of transgene DNA (typically 30 µg for a single transformation), consists of many steps, and is time-consuming. Additionally, this PEG-mediated method has only been established in a few species of moss. In the current protocol, we succeeded in CRISPR/Cas9-induced targeted mutagenesis of P. patens genes by making good use of the biolistic method, which i) requires amounts of transgene DNA as low as 5 μg for each vector, ii) consists of fewer steps and is time-saving, and iii) is known to be applicable to a wide variety of species of plants. Key features • In this protocol, particle bombardment-mediated gene transfer is used for CRISPR/Cas9-induced mutagenesis in the moss Physcomitrium patens. • By this application of particle bombardment, a gene can be modified by the CRISPR/Cas9 system much more conveniently with a smaller amount of transgene DNA. • This protocol is expected to be easily applicable to non-model moss species, some of which have noteworthy traits, such as tolerance to various stresses.