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Abstract Until recently, precise genome editing has been limited to a few organisms. The ability of Cas9 to generate double stranded DNA breaks at specific genomic sites has greatly expanded molecular toolkits in many organisms and cell types. Before CRISPR‐Cas9 mediated genome editing,P. patenswas unique among plants in its ability to integrate DNA via homologous recombination. However, selection for homologous recombination events was required to obtain edited plants, limiting the types of editing that were possible. Now with CRISPR‐Cas9, molecular manipulations inP. patenshave greatly expanded. This protocol describes a method to generate a variety of different genome edits. The protocol describes a streamlined method to generate the Cas9/sgRNA expression constructs, design homology templates, transform, and quickly genotype plants. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Constructing the Cas9/sgRNA transient expression vector Alternate Protocol 1: Shortcut to generating single and pooled Cas9/sgRNA expression vectors Basic Protocol 2: Designing the oligonucleotide‐based homology‐directed repair (HDR) template Alternate Protocol 2: Designing the plasmid‐based HDR template Basic Protocol 3: Inducing genome editing by transforming CRISPR vector intoP. patensprotoplasts Basic Protocol 4: Identifying edited plants. 

Polarized growth drives the morphogenesis of elongated cellular structures. In plants, polarized growth depends on actin and a tip focused ionic calcium gradient. How the calcium gradient is maintained remains unclear. We discovered that autoinhibitory calcium ATPases (ACAs) redundantly contribute to the steepness of the calcium gradient. ACA1 and ACA2 localize to the subapical plasma membrane and ACA5 to the vacuole membrane, providing spatial regulation of calcium efflux. Tip-growing plant cells also exhibit apical calcium fluctuations. Even though Δaca1/2/5 cells have a diminished calcium gradient, they exhibit normal fluctuations and actin but have significantly reduced apical secretion. Furthermore, cells lacking apical actin retain a strong calcium gradient but have reduced apical secretion. Suppression of both the calcium gradient and apical actin dramatically impairs growth, supporting a model where two independent and parallel processes, the calcium gradient and apical actin, promote rapid polarized growth. 

Similar to cellulose synthases (CESAs), cellulose synthase–like D (CSLD) proteins synthesize β-1,4-glucan in plants. CSLDs are important for tip growth and cytokinesis, but it was unknown whether they form membrane complexes in vivo or produce microfibrillar cellulose. We produced viable CESA-deficient mutants of the mossPhyscomitrium patensto investigate CSLD function without interfering CESA activity. Microscopy and spectroscopy showed that CESA-deficient mutants synthesize cellulose microfibrils that are indistinguishable from those in vascular plants. Correspondingly, freeze-fracture electron microscopy revealed rosette-shaped particle assemblies in the plasma membrane that are indistinguishable from CESA-containing rosette cellulose synthesis complexes (CSCs). Our data show that proteins other than CESAs, most likely CSLDs, produce cellulose microfibrils inP. patensprotonemal filaments. The data suggest that the specialized roles of CSLDs in cytokinesis and tip growth are based on differential expression and different interactions with microtubules and possibly Ca²⁺, rather than structural differences in the microfibrils they produce.