Validation of a Modular Base Editing Strategy, Free-BE, in Physcomitrium patens, Maize, and Wheat

Base editing (BE) technologies enable precise genome modification but often generate undesired editing outcomes, including indels, bystander edits, and editing outside the optimal activity windows. Here, we report a modular BE strategy, termed Free-BE, where the cytidine deaminase and Cas9 nickase are delivered as separate components rather than as a single fusion protein. In the moss Physcomitrium patens, Free-BE showed lower editing efficiency than the conventional fused system (Fused-BE). However, it generated fewer indels, a higher proportion of single-nucleotide edits, and reduced out-of-window editing. Free-BE also altered the spectrum of base substitutions, producing a higher proportion of C-to-G and C-to-A transversions than Fused-BE in P. patens. No mutations were detected at four predicted off-target loci examined in P. patens. We further validated Free-BE in crop systems. In a transient maize protoplast reporter assay, Free-BE achieved targeted base conversions, whereas stable transformation of wheat demonstrated successful editing of endogenous loci, although with lower efficiency than Fused-BE. In wheat, Free-BE similarly reduced multicytidine editing and by-products. Together, these results establish Free-BE as a functional modular BE strategy that broadens the plant genome-editing toolbox and may be advantageous for applications requiring greater control over editing outcomes, despite lower overall editing efficiency.