tRNA-based polycistronic CRISPR/Cas9 system boosts efficiency of multi-gene deletion in the moss Physcomitrella

CRISPR/Cas9-based genome editing in the model bryophyte Physcomitrium patens (commonly known as Physcomitrella) is widely used for gene knockout via small insertions or deletions (indels). In this study, we developed an efficient dual-gRNA system capable of producing large, targeted deletions across multiple genes, enabling straightforward detection by gel electrophoresis and simultaneous multi-gene knockout. We first compared the efficiency of polycistronic tRNA-gRNA arrays to conventional gRNA constructs expressed under individual promoters, using the checkpoint protein gene MAD2 as a target. We found that a polycistronic construct doubled the frequency of large gene deletions compared to a conventional design. We then demonstrated that simultaneous deletion of two or four genes, targeting the katanin and TPX2 gene families, respectively, can be achieved in a single transformation event. The polycistronic system also increased deletion frequencies in the multiplex context, with up to 42% efficiency for individual genes and successful recovery of quadruple mutants. As a drawback, we confirmed that deletion efficiency varied substantially among individual gRNA pairs, indicating that gRNA design remains a critical factor in multiplex editing. This study establishes a fast and efficient framework for simultaneous removal of multiple genes in Physcomitrella, providing a practical alternative to homologous recombination-based methods for functional and applied studies.