CRISPR/Cas9 targeted genetic screening in Physcomitrella identifies novel cell division genes

Although plants share core cell division mechanisms with other eukaryotes, their unique features—such as acentrosomal spindle formation and cytokinesis via the phragmoplast—suggest the existence of plant-specific genes. This study used the model bryophyte Physcomitrium patens to uncover such genes and employed CRISPR-based screening to identify novel cell division genes in plants.

Co-expression data from known mitotic genes were used to create a pool of 216 candidate genes, which were then targeted in CRISPR/Cas9 screening. Frameshift mutants with division defects were characterized using high-resolution imaging of mitosis and gene tagging with fluorophores for localization analysis.

Three novel gene families—CYR (Cytokinesis-Related), LACH (Lagging Chromosome), and SpinMi (Spindle and Phragmoplast Midzone)—were identified. CYR genes were linked to cytokinesis defects, LACH was essential for chromosome segregation, and SpinMi localized to the spindle and phragmoplast midzone. Notably, none of these gene families had homologs in algae, suggesting their emergence during land colonization.

Our findings provide a framework for combining co-expression analysis with targeted screening to identify genes associated with specific cellular processes, in this case, cell division. Beyond characterizing three novel gene families, this study also offers insights into evolutionary changes in the plant cell division machinery.