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). However, this approach may leave residual gene activity and typically requires sequencing-based validation. In this study, we established an efficient strategy for generating large, targeted deletions across multiple genes using dual-gRNA targeting. 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 versatile and scalable framework for generating multi-gene deletion mutants in P. patens , facilitating functional genomics and biotechnological applications requiring precise gene removal.