Keywords: adenine deaminase; APRT; base editing; Cas9; CRISPR; cytosine deaminase; Physcomitrella patens; Physcomitrium patens Summary CRISPR-Cas9 has proven to be highly valuable for genome editing in plants, including the model plant Physcomitrium patens. However, the fact that most of the editing events produced using the native Cas9 nuclease correspond to small insertions and deletions is a limitation. CRISPR-Cas9 base editors enable targeted mutation of single nucleotides in eukaryotic genomes and therefore overcome this limitation. Here, we report two programmable base-editing systems to induce precise cytosine or adenine conversions in P. patens. Using cytosine or adenine base editors, site-specific single-base mutations can be achieved with an efficiency up to 55%, without off-target mutations. Using the APT gene as a reporter of editing, we could show that both base editors can be used in simplex or multiplex, allowing for the production of protein variants with multiple amino-acid changes. Finally, we set up a co-editing selection system, named selecting modification of APRT to report gene targeting (SMART), allowing up to 90% efficiency site-specific base editing in P. patens. These two base editors will facilitate gene functional analysis in P. patens, allowing for site-specific editing of a given base through single sgRNA base editing or for in planta evolution of a given gene through the production of randomly mutagenised variants using multiple sgRNA base editing. CAPTION(S): Fig. S1 Schematic description of the APT reporter gene and APRT function. Fig. S2 Examples of deletions observed during BE multiplexing. Fig. S3 Examples of multiple cytosines editing or chimerism observed in some clones. Fig. S4 G418 sensitivity of ABE and CBE single and multiplex edited clones after relaxing of the antibiotic selection pressure. Fig. S5 Nature of editing using CBE or ABE for each cytosine or adenine present in the target locus. Fig. S6 Nature of editing using CBE on genes of interest for each cytosine in the target locus. Fig. S7 Sequence of two sgRNAs containing cytosines potentially target of ABE activity and nature of ABE editing using these sgRNAs. Fig. S8 Alignment of APRT sequences from different species and phenotype of the apt P. patens mutants. Fig. S9 View of the P. patens APRT 3D model with amino acids (in blue) that could be modified as single substitutions using CBE or ABE. Fig. S10 Structure of the Pp3c3_13220, Pp3c14_9040 and Pp3c17_3870 targeted genes. Fig. S11 Sequence alignment of VDE from Arabidopsis and Physcomitrella. Fig. S12 Use of the APT gene as a marker of base-editing efficiency. Table S1 List of sgRNAs expression cassettes used in this study. Table S2 Sequences of plasmids used in this study. Table S3 List of PCR primers used in this study. Table S4 Mutation rates of the CBE and ABE systems tested (2-FA direct selection). Table S5 Transfection efficiency of the CBE and ABE systems. Table S6 Mutation rates of the CBE system after preselection on G418. Table S7 Frequency of substitution for cytosines at each position of the eight sgRNAs used in this study. Table S8 Sequences and positions of possible off-target sites for sgRNA1 and sgRNA2. Table S9 List of amino acids modified in the APT gene using the CBE or ABE strategy. Table S10 Consequence of CBE editing in the different edited clones for the three genes of interest. Table S11 Sequence analysis of the APT and Pp3c3_13220 locus in adenine-resistant clones obtained after co-transfection of the ABEv#1 mutant with the CBE system and the two sgRNAs, sgRNArestor and sgRNAPp3c3. Please note: Wiley Blackwell are not responsible for the content or functionality of any Supporting Information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office. Byline: Anouchka Guyon-Debast, Alessandro Alboresi, Zoe Terret, Florence Charlot, Floriane Berthier, Pol Vendrell-Mir, Josep M. Casacuberta, Florian Veillet, Tomas Morosinotto, Jean-Luc Gallois, Fabien Nogue