Methods Methods A blueprint for gene function analysis through Base Editing in the model plant Physcomitrium (Physcomitrella) patens Anouchka Guyon-Debast1 , Alessandro Alboresi2 , Zoé Terret3 , Florence Charlot1 , Floriane Berthier1 , Pol Vendrell-Mir4 , Josep M. Casacuberta4 , Florian Veillet5 , Tomas Morosinotto2 , Jean-Luc Gallois3 and Fabien Nogué1 1 Institut Jean-Pierre Bourgin, INRAE, AgroParisTech, Université Paris-Saclay, Versailles 78000, France; 2Department of Biology, University of Padova, Padova 35121, Italy; 3INRAE, GAFL, Montfavet 84143, France; 4Centre for Research in Agricultural Genomics CSIC-IRTA-UAB-UB, Campus UAB, Edifici CRAG, Bellaterra, Barcelona 08193, Spain; 5IGEPP, INRAE, Institut Agro, Université de Rennes, Ploudaniel 29260, France Summary Author for correspondence:  CRISPR-Cas9 has proven to be highly valuable for genome editing in plants, including the Fabien Nogué model plant Physcomitrium patens. However, the fact that most of the editing events pro- Email: Fabien.nogue@inrae.fr duced using the native Cas9 nuclease correspond to small insertions and deletions is a limita- tion. Received: 5 October 2020  CRISPR-Cas9 base editors enable targeted mutation of single nucleotides in eukaryotic Accepted: 21 December 2020 genomes and therefore overcome this limitation. Here, we report two programmable base- editing systems to induce precise cytosine or adenine conversions in P. patens. New Phytologist (2021) 230: 1258–1272  Using cytosine or adenine base editors, site-specific single-base mutations can be achieved doi: 10.1111/nph.17171 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 Key words: adenine deaminase, APRT, base for the production of protein variants with multiple amino-acid changes. Finally, we set up a editing, Cas9, CRISPR, cytosine deaminase, co-editing selection system, named selecting modification of APRT to report gene targeting Physcomitrella patens, Physcomitrium (SMART), allowing up to 90% efficiency site-specific base editing in P. patens. 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. elimination of background mutations that could interfere with Introduction the functional analysis of the modified protein. Modification of the sequence of a protein is a powerful approach For a few years now, the CRISPR-Cas9 system has been effi- to decipher the protein’s sequence–function relationship and ciently applied to induce targeted mutagenesis in plant genes. understand its biological roles. In addition, production of differ- Guided by an sgRNA, the Cas9 nuclease produces a targeted ent variants for a given gene and its corresponding protein also DNA double-stranded break (DSB) by site-specific cleavage, has practical applications and is a way to go beyond what evolu- which typically results in insertion or deletion mutations after tion has shaped in terms of function for this protein. For a long error-prone NHEJ DNA repair (Jiang & Doudna, 2017). In time, intracellular protein engineering through substitution, many cases these mutations correspond to loss of function due to insertion or deletion of nucleotides in its corresponding gene, out-of-frame mutations, premature terminations or aberrant was restricted to unicellular eukaryotes. One possible way to splicing variants. By contrast, gene function analysis and the modify a gene in planta is the TILLING strategy, based on EMS- development of new traits of interest in crops need a more precise induced mutations in the genome (Jacob et al., 2018). However, mutation system delivering more predictable mutations, includ- such in vivo mutagenesis is not targeted to a specific locus and, ing modification of one or more bases in a given gene. Such for this reason, needs first a thorough selection system to find the mutations that facilitate basic research can be achieved through desired mutation in the target gene. In addition, TILLING will different CRISPR-derived tools, as recently reviewed (Manghwar only very rarely allow the selection of simultaneous mutations in et al., 2019; Veillet et al., 2020a), but also accelerate plant breed- a given gene. Finally, such a strategy will necessitate the ing (Zhang et al., 2019; Gaillochet et al., 2020). While 1258 New Phytologist (2021) 230: 1258–1272 Ó 2021 The Authors www.newphytologist.com New Phytologist Ó 2021 New Phytologist Foundation This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. New Phytologist Research 1259 modification of one or more bases via CRISPR-mediated gene efficient tools for base editing, including multiplex editing, in targeting constitutes an elegant strategy, its efficiency remains P. patens. We characterised the respective efficiencies and deami- low in flowering plants (Huang & Puchta, 2019). nation windows for CBE and ABE and demonstrated that they Recently, a strategy named Prime editing, has been developed can be useful tools for gene function analysis. In addition, data in human cells (Anzalone et al., 2019) and then adapted to wheat gained from this study can be translated to drive directed in and rice (Lin et al., 2020). Prime editing is based on the fusion of planta evolution of other targets. Finally, we propose here an a Cas9 nickase to a reverse transcriptase that, guided by a Prime original co-editing selection strategy, named selecting modifica- editing guide RNAs (pegRNAs), enables point mutations, inser- tion of APRT to report gene targeting (SMART), based on the tions and deletions. This strategy is highly promising but still restoration of the APT gene function, for efficient and easy-to- needs refinement to achieve editing that is efficient enough for screen base editing of any gene of interest in P. patens. routine use (Marzec & Hensel, 2020). As an alternative to gene targeting, and before the development of the promising Prime Materials and Methods editing strategy, CRISPR-mediated base editors (BEs) have been developed, to induce targeted base modification, first in human Molecular cloning and murine cell lines (Komor et al., 2016) and then in rice (Li et al., 2017; Lu & Zhu, 2017). Like Prime editing, base editing is Guide RNA (sgRNA) sequences specific to the APT a gene editing strategy free of donor DNA and double-stranded (Pp3c8_16590), Pp3c3_13220, Pp3c14_9040 and breaks. Two types of BEs exist, cytosine base editors (CBEs) and Pp3c17_3870 genes were chosen using the webtool CRISPOR adenine base editors (ABEs) that correspond to a Cas9 nickase 4.97 (Concordet & Haeussler, 2018). Expression cassettes (nCas9) or a catalytically inactive Cas12a (dCas12a) (Komor sgRNA#5, sgRNA#7, sgRNA#21, sgRNA#23, sgRNA#24 and et al., 2016; Li et al., 2018a) that is fused to either a cytosine- or sgRNA#25, comprising the promoter of the P. patens U6 snRNA an adenine-deaminase domain, respectively. BEs generate tar- (Collonnier et al., 2017a), the 50 -G-N(19)-30 guide sequences geted nucleotide substitutions on ssDNA in a small editing win- targeting the APT gene and the tracrRNA scaffold were synthe- dow that is accessible during the CRISPR-mediated R-loop sised by Twist Bioscience (San Francisco, California, USA; Sup- formation. The action of the cytidine deaminase of the CBE porting Information Table S1). The sgRNA expression cassette complex on a cytosine can generate transition (C-to-T) and sgRNAPp3c14, based on the same backbone but targeting the transversion (C-to-A and C-to-G) substitutions, however addi- Pp3c14_9040 gene was synthesised by Twist Bioscience. tion of a uracil DNA glycosylase inhibitor (UGI) to the CBE will sgRNA#5 and sgRNA#7 were subcloned into the pDONR207 permit the production mostly of transition (C-to-T) substitutions vector by GatewayTM BP reaction (Invitrogen) to give (Evanoff & Komor, 2019). ABEs almost exclusively result in A- psgRNA#5 and psgRNA#7. sgRNA#21, sgRNA#23, to-G substitutions (Evanoff & Komor, 2019). sgRNA#24, sgRNA#25 and sgRNAPp3c14 were cloned into the BEs using nCas9 fusions have been used in different model pDONR207-neomycin resistance (NeoR) vector using a Gate- and crop plants including rice, wheat, maize, potato and tomato wayTM BP reaction (Invitrogen) to give psgRNA#21, (Lu & Zhu, 2017; Shimatani et al., 2017; Zong et al., 2017; Li psgRNA#23, psgRNA#24, psgRNA#25 and psgRNAPp3c14. et al., 2018b; Bastet et al., 2019; Veillet et al., 2019a,b). These pDONR207-NeoR was obtained by cloning the 35S::neoR frag- studies have shown that CBE or ABE activities can vary between ment (SmaI–ApaI, 1824 pb) from pBNRF (Schaefer et al., 2010) plants, but also between target sites in term of editing efficiency into pDONR207. The sgRNAPp3c3, sgRNAPp3c17 and of an effective deamination window and of occurrence of byprod- sgRNArestor expression cassettes, targeting the Pp3c3_13220 ucts (insertions, deletions or unpredicted substitutions). In the gene, the Pp3c17_3870 gene and a mutated version of the APT model plant bryophyte Physcomitrium (Physcomitrella) patens, the gene respectively, were synthesised and cloned in the pTwist use of CRISPR-Cas9 or CRISPR-Cas12a strategies has permitted Amp vector by Twist Bioscience to give psgRNAPp3c3 efficient gene knock-out (Nomura et al., 2016; Lopez-Obando spgRNAPp3c17and psgRNArestor. psgRNA#2 containing the et al., 2016; Pu et al., 2019; Mallett et al., 2019) or gene knock- expression cassette sgRNA#2 has been described previously (Col- in (Collonnier et al., 2017a; Yi & Goshima, 2020), but no base- lonnier et al., 2017a). All the expression cassettes used in this editing strategies have been reported so far. study are described in Table S1. Physcomitrium patens is a well recognised model to study evo- For the CBE system we used a CRISPR-nCas9 cytosine deam- lutionary developmental biology questions, stem cell reprogram- inase consisting of a fusion of nCas9 (D10A) to the Petromyzon ming, and the biology of nonvascular plants (Rensing et al., marinus cytosine deaminase (PmCDA1). The pnCas9-CBE1 vec- 2020). In order to expand the toolbox for gene function analysis tor expressing this fusion enzyme is based on the in P. patens, we explored the possibility of editing specific bases pDicAID_nCas9-PmCDA_NptII_Della vector (Shimatani et al., of the genome through CBE and ABE in this model plant. For 2017), from which the sgRNA expression cassette targeting the this purpose, we used the APT gene that we and other groups tomato DELLA gene was dropped out (AanI digest). For The had previously used as a reporter of gene modification or modu- ABE system we used a CRISPR-nCas9-adenine deaminase con- lation in P. patens (Trouiller et al., 2006; Holá et al., 2013; Orr sisting of a heterodimer of a wild-type bacterial tRNA adenosine et al., 2020). We demonstrate here for the first time that the deaminase (TadA) and a mutated version (TadA*), fused to CRISPR-Cas9 deaminase systems CBE and ABE are very nCas9 (Gaudelli et al., 2017). For this purpose, using Invitrogen © 2021 The Authors New Phytologist (2021) 230: 1258–1272 New Phytologist © 2021 New Phytologist Foundation www.newphytologist.com New 1260 Research Phytologist Platinum SuperFi DNA polymerase (Thermo Fisher Scientific), primers PpAPT#8 and PpAPT#10 primers for the sgRNA#7 we PCR amplified the ABE7.10-nCas9 gene fusion from pCMV- locus, primers PpAPT#60 and PpAPT#61 for the sgRNA#5 and ABE7.10 (Addgene plasmid #102919) with AttB1 and AttB2 primers PpAPT#25 and PpAPT#5 for sgRNA#2 and sgRNA#21 flanking sequences using the ABE7.10-AttB1 and ABE7.10- loci. PCR primers used in this study are listed in Table S3. AttB2 primers. The PCR fragment was cloned into pDONR207 using the GatewayTM BP reaction and then subcloned into pBS- Structural analysis of mutations in P. patens APRT TPp-B (Thévenin et al., 2012) using the GatewayTM LR reaction (Invitrogen) to give the pnCas9-ABE1 plasmid, in which Nine APRT templates were selected with Modeller 9.18 (Webb & ABE7.10 is flanked by the rice Actin 1 promoter and CaMVter Sali, 2017), based on sequence identity (>20%) from: Escherichia terminator. The pAct-Cas9 plasmid used in this study has been coli (PDB:2DY0), Saccharomyces cerevisiae (PDB:1G2Q), Giardia described previously (Collonnier et al., 2017a). Sequences of the intestinalis (PDB:1L1Q), Thermoanaerobacter pseudethanolicus plasmids used in this study are listed in Table S2. (PDB:4LZA), Rhodothermus marinus (PDB:4M0K), Yersinia pseudotuberculosis (PDB:4MB6) and Homo sapiens (PDB:4X45). Multiple alignment was used to develop high quality models and Moss culture and transformation the best model was chosen using the discrete optimised protein P. patens wild-type Gransden strain was propagated vegetatively as energy (DOPE) method (Shen & Sali, 2006) and/or the GA341 previously described (Cove et al., 2009). Plants were grown on method (John & Sali, 2003; Melo et al., 2009). Optimisation of PpNH4 medium (PpNO3 medium supplemented with 2.7 mM the model was achieved using energy minimisation protocols NH4-tartrate) in growth chambers set at 60% humidity with 16 h available at YASARA software (Elmar et al., 2010). The visualisation of light (quantum irradiance of 80 μmol m−2 s−1) at 24°C and 8 h of the multiple alignment with the secondary structure was made of dark at 22°C. Moss protoplast isolation and transfection were using the ESPript3 server (Robert & Gouet, 2014). performed as previously described (Schaefer & Zryd, 1997). Protoplasts were transfected with a total of 20 µg of circular In vivo measurements of chlorophyll fluorescence DNA divided as follow: 8 µg of the pAct-Cas9, pDIC-AID- APTgRNA#21, pnCas9-CBE1 or pnCas9-ABE1 plasmids and a Here, 10-d-old plants grown on PpNO3 medium were probed mix of 12 µg of sgRNA plasmids. Regenerating protoplasts were for chlorophyll fluorescence with the modular version of the Dual spread on cellophane disks on PpNH4 medium supplemented with PAM-100 fluorometer (Walz). Plants were dark adapted for 40 0.33 M mannitol for 1 wk. Plants on cellophane disks were then min and then induction curve analyses were performed using 850 selected either on PpNH4 supplemented with 50 mg l−1 G418 µmol of photons m−2 s−1 red actinic light for 8 min followed by (Duchefa) to select clones that were transiently transfected (Lopez- 8 min of dark recovery. Chlorophyll fluorescence was measured Obando et al., 2016) or directly on PpNH4 supplemented with in plants dark adapted for 30 min that were then exposed to 10 μM 2-FA (Fluorochem) to select clones that were mutated at actinic light of 800 μmol of photons m−2 s−1 for 8 min. Fm and the APT locus (Collonnier et al., 2017a). For the experiment con- Fm0 are the fluorescence values after exposure to saturating pulses sisting of the restoration of the APRT activity of the apt mutant (6000 μmol of photons m−2 s−1, duration 600 ms) in, respec- ABEv#1, protoplasts isolated from this mutant were transfected tively, dark adapted plants and plants exposed to actinic light. with 10 µg of each of the pnCas9-CBE1 and psgRNArestor plas- Non-photochemical quenching (NPQ) was calculated as Fm/ mids. Regenerating protoplasts were spread on cellophane disks on Fm0 − 1 (Klughammer & Schreiber, 1994). PpNH4 medium supplemented with 0.33 M mannitol and 1.75 mM adenine (Sigma A8626) for 1 wk and then transferred onto Results PpNH4 supplemented with 1.75 mM adenine for 3 wk for selec- tion of clones in which APRT function was restored. Efficient base editing in P. patens via RNA-guided cytosine or adenine deaminases PCR and sequence analysis of the edited plants The APRT enzyme is a member of the Type I phosporibosyltrans- For PCR analysis, genomic DNA was extracted from 50 mg of ferase family and is involved in the nucleotide salvage pathway, by fresh tissue as previously described (Lopez-Obando et al., 2016). which organisms, including plants (Ashihara et al., 2018), convert The quality of the DNA samples was controlled using primers tar- adenine to adenosine monophosphate (AMP). APRT enzyme, geting the P. patens RAD51-1 gene, PpRAD51-1#6 and encoded by the APT gene, is also able to convert some adenine PpRAD51-1#7. Molecular analysis was based on Sanger sequenc- analogues into toxic compounds and this has been used for effi- ing (Genoscreen, Lille, France) of PCR fragments using primers cient selection of APRT-deficient mutants in many organisms surrounding the targeted locus. For edited plants obtained using a (Taylor et al., 1985). For this reason, we hypothesised that base single sgRNA, PpAPT#25 and PpAPT#5 were used for APT, modifications of the P. patens APT gene (Fig. 1a) could be easily Pp3c3#1 and Pp3c3#2 for the Pp3c3_13220 locus, Pp3c14#1 monitored by selection on 2-FA in order to quickly assess the effi- and Pp3c14#2 for the Pp3c14_9040 locus, Pp3c17#1 and ciency of base editing (Fig. S1). To determine whether pro- Pp3c17#2 for the Pp3c1783870 locus. For edited plants obtained grammable BEs could catalyse site-specific base editing in the using multiple sgRNAs, molecular analysis was carried out using genome of P. patens, we used both a CBE based on the cytosine New Phytologist (2021) 230: 1258–1272 © 2021 The Authors www.newphytologist.com New Phytologist © 2021 New Phytologist Foundation New Phytologist Research 1261 (a) 200 bp (b) pAct-Cas9 NLS OsAct1 Pro SpCAS9 35S Ter pnCas9-ABE1 NLS OsAct1 Pro ABE7.10 SpnCAS9 (D10A) 35S Ter pnCas9-CBE1 Linker NLS NLS pcUbi4-2 Pro SpnCAS9 (D10A) PmCDA1 Pea3A Ter Linker (c) (d) 11% 3.5 pnCas9-CBE1 Mutaon efficiency (% of regenerants) 3 n=46 2.5 89% 2 Predicted eding By-products 1.5 1 pnCas9-ABE1 0.5 n=34 0 pnCas9-CBE1 + Cas9 + sgRNA#2 pnCas9-ABE1 + Cas9 + sgRNA#1 100% sgRNA#2 sgRNA#1 (e) T T G A G C G T T A C C G G G A C C A G A A G G T G G G G G A A G A G T A T A G T C T A G A G T A T G G T A Wild type Wild type (target sgRNA#2) (target sgRNA#1) T T G A G T G T T A C C G G G A C C A G A A G G T G G A G G G A A G A G T G T A G T C T A G A G T A T G G T A CBE1-v#1 ABE1-v#1 (target sgRNA#2) (target sgRNA#1) Fig. 1 Relative efficiency of mutagenesis and the nature of editing by CBE and ABE in Physcomitrium patens. (a) Structure of the PpAPT gene and sgRNAs positions. Boxes in white represent the exons and black lines represent the introns. The eight sgRNAs positions are indicated in orange, at the top for sgRNAs that target forward strand, and at the bottom for sgRNA that target reverse strand. Green arrows represent the primers used for PCR and sequencing. (b) Schematic representation of the native Cas9 and the two base editors. Linker sequences are in light blue, NLS sequences are in red. (c) Mutation rates using pnCas9-CBE1 or pnCas9-ABE1 (in black) vs active Cas9 (in grey). (d) Nature of the base editing was characterised by sequence analysis of 2FA-resistant plants. (e) Sequence chromatograms from wild-type and CBE or ABE edited clones. Target sequence (in red) and PAM (in blue) are highlighted in the wild-type (WT) sequence; red arrows point to the positions with edited base. Primers used for amplification and Sanger sequencing can be found in the Materials and Methods section. Number of analysed plants is indicated. © 2021 The Authors New Phytologist (2021) 230: 1258–1272 New Phytologist © 2021 New Phytologist Foundation www.newphytologist.com New 1262 Research Phytologist deaminase from P. marinus (PmCDA1) and an ABE based on the different targets in a given plant showed that precise multi- tRNA adenosine deaminase from E. coli (TadA) (Fig. 1b). plex base editing could be achieved, as 40% of the precisely We selected eight different sgRNAs targeting the APT reporter edited plants (C substitutions with no byproducts and no guide- gene that contained cytosine or adenine residues in the predicted to-guide deletions, n = 30) were modified at the four targeted editing windows (Fig. 1a). loci (Fig. 2b,c). For ABE multiplexing, the predictability of base First, in order to validate the CBE and ABE constructs, we editing was comparable with the simplex strategy, as 98% (100% compared their respective editing efficiencies to that of the native for the simplex) of the mutations corresponded with the precise Cas9 (pAct-Cas9). psgRNA#1 was transfected with pnCas9- editing of an adenine (Figs 2a, S2). In addition, 14% of the pre- ABE1 (for ABE) or pAct-Cas9 (for native Cas9) and psgRNA#2 cisely edited plants (A substitutions with no byproduct) (n = 43) was transfected with pnCas9-CBE1 (for CBE) or pAct-Cas9 (for showed concomitant mutations at the three targeted loci (Fig. native Cas9) respectively, in P. patens protoplasts. Regenerating 2b,d). These data showed that, when a cell is subjected to the protoplasts were transferred onto a medium containing 2-FA in CBE or ABE systems with multiple sgRNAs, concomitant edit- order to detect plants that had been mutated at the APT locus. ing of the targeted loci is possible, making multiplexing a power- The relative efficiencies of APT mutagenesis, estimated by divid- ful tool in P. patens. ing the number of 2-FA-resistant plants by the number of ini- tially regenerating plants, were 0.8% for ABE using sgRNA#1 Characteristics of BE and nature of the substitutions and 1.6% for CBE using sgRNA#2 (Table S4; Fig. 1c). Using the same two guides, these values were, respectively, 2.7 and First, in order to check that selection of the edited plants on 2- 2.9% when mutagenesis was performed with the native Cas9 sys- FA did not create a bias in the type of mutations that could be tem (Fig. 1c). Analysis of the type of mutations obtained with observed using BE, we aimed at analysing plants that were trans- the two different BE strategies showed that a majority of mutant fected with the CBE system without the a priori knowledge that plants obtained with the CBE strategy corresponded to precise editing would result in an alteration of APRT activity. For this base-editing events (89%), and the remaining mutants corre- purpose, protoplasts were transfected with the CBE system and sponded to short insertions or deletions (hereafter called byprod- sgRNA#2 or sgRNA#21. The regenerating protoplasts were ucts) (Fig. 1d,e). For the ABE strategy, 100% of the mutant transferred onto a medium containing the antibiotic G418 in plants corresponded to precise base editing (solely A-to-G substi- order to select clones that expressed the transfected plasmids that tution, Fig. 1d,e). This showed that CBE and ABE can be used contained a NeoR gene cassette (Table S5). We observed that to precisely modify cytosine or adenine bases in P. patens. 34.8% for sgRNA2 and 52.3% for sgRNA#21 of the G418 resis- tant transfected clones were mutated at the APT locus (Fig. 3). Of these mutated clones, 32.6% for sgRNA#2 and 20.9% for Multiplex base editing is efficient in P. patens sgRNA#21 corresponded to precise base-edited plants, the other In order to evaluate the possibility of multiplex editing using the mutants corresponded to deletions (Fig. 3). Interestingly, a small BE systems we co-transfected P. patens protoplasts with plasmids fraction of the G418-resistant clones (2.2% for sgRNA#2 and psgRNA#2, psgRNA#5, psgRNA#7, psgRNA#21 (Fig. 1) and 4.7% for sgRNA#21, Fig. 3) corresponded to chimeric clones pnCas9-CBE1 plasmids (for the CBE system), and with plasmids composed of a mixture of wild-type and mutated cells (Fig. S3). psgRNA#1, psgRNA#21, psgRNA#23 and pnCas9-ABE1 plas- Such chimeric clones were not observed using direct 2-FA selec- mids (for the ABE system) (Fig. 1). As before, regenerating pro- tion. This suggests that, even if the BE system is preferentially toplasts were grown on medium containing 2-FA, and plants that active very early after protoplast transfection, it can still be active survived (apt mutants) were analysed. Sequence analysis of the after multiple divisions of the originally transfected cell thanks to apt mutants showed that, under these conditions, the relative effi- episomal replication of the vector (Muren et al., 2009). Even if ciencies of mutagenesis (2.8% for CBE and 0.7% for ABE, illegitimate integration of a nonhomologous supercoiled plasmid Table S4) were comparable with those observed with the simplex is low in P. patens (Schaefer & Zryd, 1997), episomal persistence strategy (1.6% for CBE and 0.8% for ABE, Table S4). For CBE, of the vector in the clones in the presence of G418 could, in the- predictability of base editing was decreased, compared with the ory, lead to stable integration in the genome. In order to check simplex strategy, as 47% (89% for the simplex) of the mutations for the level of unexpected integration of the ABE or CBE vectors corresponded to precise editing of a cytosine (Figs 2a, S2). Analy- in the selected plants, we tested the sensitivity of 288 ABE- or sis of the mutations showed that the decrease observed was due to CBE-transfected clones to G418. We could observe that sensitiv- byproducts corresponding to small indels (Fig. S2), but also to ity to G418 was restored in most of the clones as only one clone the occurrence of deletions between the sgRNAs used for multi- out of 480 (0.21%) showed resistance to G418 (Fig. S4). All the plexing. As expected, these deletions always involved sgRNAs edited clones presented in this study were G418 sensitive. Finally, that were in opposite orientation on the DNA (Figs 1a, S2), as in order to test whether the different mutations observed in the nicking of both DNA strands by a pair of Cas9 nickases is known APT locus would have an effect on the activity of the APRT to lead to site-specific DSBs and NHEJ, and by contrast nicks on enzyme we sequenced the APT gene in the 132 G418-resistant the same DNA strand were predominantly repaired through the (transfected) clones obtained with sgRNA#2 or sgRNA#21 and high-fidelity base excision repair pathway (BER) (Ran et al., placed them on a medium containing 2-FA. Under these condi- 2013). Analysis of the mutations generated by the CBE system at tions, 100% of the clones containing a mutation (77 out of the New Phytologist (2021) 230: 1258–1272 © 2021 The Authors www.newphytologist.com New Phytologist © 2021 New Phytologist Foundation New Phytologist Research 1263 (a) 4% 21% CBE 47% ABE n=63 n=45 32% 96% Predicted eding By-products Guide to guide deleons (b) 17% 14% 35% 40% CBE ABE n=30 n=43 27% 4 sgRNA targets 51% 3 sgRNA targets 3 sgRNA targets 2 sgRNA targets 17% 2 sgRNA targets 1 sgRNA target 1 sgRNA target (c) target sgRNA#2 target sgRNA#5 target sgRNA#7 target sgRNA#21 G T T G A G C G T T A C C G G G A C C A G A A G G T G AT C CA G TA T A T C T C G G AC A G C A T C C G T T C C C G C T T C C G C A A T T T T G T T G C A G T G G C C A C C C A T T G C T C T T G C CA T C G G T G Wild type 280 290 G T T G A G T G T T A C C G G G A C C A G A A G G T G AT C C A G T A T AT C T C G G A CA C C A T C C G T T C C C G C T T C C G C A A T T T T A T T A C A G T G G T T A T A C A T T G C T C T T G C C A T C G G T G CBE1-v#30 (d) target sgRNA#1 target sgRNA#21 target sgRNA#23 G G G A A G A G T A T A G T C T A G A G T A T G G T A G G C C A C C C A T T G C T C T T G C CA T C G G T G G A C CA G A A G G T G G A C G T C A T T G T G G G T Wild type G G G A A G A G T G T A G T C T A G A G T A T G G T A G AC C A G A A G G T G G A C G T C A C T G T G G G T G G G C C A C C C G T T G C T C T T G C CA T C G G T G ABE1-v#7 620 630 6 0 20 30 40 Fig. 2 Predictability and efficiency of multiplex editing by CBE and ABE in Physcomitrium patens. (a) Frequencies of predicted base editing, byproducts and guide-to-guide deletions using pnCas9-CBE1 with four sgRNAs or pnCas9-ABE1 with three sgRNAs. (b) Percentage of plants where the APT gene has been modified precisely (no byproducts or guide-to-guide deletions) at multiple sgRNA targets concomitantly. Primers used for amplification and Sanger sequencing can be found in the Materials and Methods section. Number of analysed plants is indicated. (c, d) Sequence chromatograms from wild-type and CBE or ABE edited clones. Target sequence (in red) and PAM (in blue) are highlighted in the WT sequence; red arrows point to the positions with edited base. © 2021 The Authors New Phytologist (2021) 230: 1258–1272 New Phytologist © 2021 New Phytologist Foundation www.newphytologist.com New 1264 Research Phytologist (n=46) (n=86) existence of multiple Cs in the editing window, as is the case for 100.0% sgRNA#21, favoured the occurrence of deletion byproducts at 90.0% the targeted locus, that in this case corresponded to 31.4% of the mutations (Fig. 3a). For ABE, analysis of the nature of the 80.0% 43.0% adenine modifications (n = 156 modified adenines) showed that, 70.0% as observed in other organisms, all the modifications corre- 63.0% Ratio of each class (%) sponded to A-to-G substitutions (Figs S5b, S7). It has been 60.0% shown recently in animal cells that, in addition to converting 4.7% 50.0% adenine to guanine, ABEs could also convert cytosines that are in a narrow editing window (positions −16 to −14) and in a con- 40.0% 2.2% fined ‘TCN’ nucleic-acid sequence context, into guanine or 31.4% 30.0% 2.2% thymine (Kim et al., 2019). In order to check whether this unpre- dicted activity was present in P. patens, we designed two sgRNAs, 20.0% sgRNA#24 and sgRNA#25 containing a ‘TCN’ sequence context 32.6% 10.0% 20.9% with the C present in the narrow editing window (Fig. S7). Anal- ysis of 75 plants that were modified by the ABE editor at the 0.0% sgRNA#24 or #25 targets showed efficient substitution of the A CBE + sgRNA#2 CBE + sgRNA#21 in the editing window with no co-substitution of the C in the C subs on Del on Chimeric WT ‘TCN’ sequence context (Fig. S7), suggesting that the phe- Fig. 3 Efficiency of mutagenesis and nature of editing by CBE in nomenon observed in animal cells could not be generalised. Con- Physcomitrium patens. Efficiency of mutagenesis using pnCas9-CBE1 for cerning the positions of the adenines that could be substituted, sgRNA#2 and sgRNA#21 observed among transient transformants (G418 selection). Efficiency and nature of the mutagenesis were assessed by analysis in five different sgRNAs targets showed that adenines sequencing analysis using PpAPT#25/PpAPT#5 primers on G418-resistant present in the 4-bp editing window (positions −17 to −14) previ- plants. Number of analysed plants is indicated. ously reported (Eid et al., 2018), were efficiently edited also in P. patens. Interestingly, exclusive substitution of an A in position −13 could also be observed (sgRNA#1 and sgRNA#24, Figs 132 G418-resistant clones) were resistant to 2-FA (Table S6). S5b,S7). Finally, for target sequences presenting more than one This confirmed our hypothesis that the APRT enzyme is very A in the editing window, as was the case for sgRNA#23 constrained in terms of amino-acid changes and is therefore very (Fig. S5b), at least two As could be modified simultaneously. sensitive to mutations. Therefore 2-FA screening is very effective in identifying any modification in the APT gene. Predicted potential off-target sites are not affected by Based on the fact that a majority, if not all, of the mutations base-editing activity in P. patens induced by CBE or ABE lead to a nonfunctional APRT, we decided to pool the different events of editing, obtained through The sgRNAs used in this study were designed to minimise poten- selection on G418 or directly on 2-FA, in order to analyse the tial off-target cleavage in the P. patens genome (Phytozome 3.1) nature of the CBE or ABE editing products. For CBE, the analy- using the CRISPOR software package (Concordet & Haeussler, sis of the nature of the cytosines modifications (n = 693 modi- 2018). We focused our analysis on two sgRNAs (sgRNA#1 and fied cytosines of 2005 analysed) showed that a majority sgRNA#2) for which no perfect 20-bp matches were found, but corresponded to C-to-T substitutions (75.8%), but some C-to-G potential off-target sequences presenting three to six mismatches (18.6%) or C-to-A (5.8%) substitutions were also observed (Fig. were identified: nine for sgRNA#1 and four for sgRNA#2 4a). Analysis of the positions of the cytosines that could be substi- (Table S8). All these potential off-target loci were amplified with tuted in eight different sgRNAs targets showed that cytosines pre- surrounding primers and sequenced in 39 clones transformed sent in the 5-bp editing window (positions −19 to −15 from the with pnCas9-ABE1 and psgRNA#1, and 39 clones transformed PAM) previously described (Nishida et al., 2016), are efficiently with pnCas9-CBE1 and psgRNA#2 that were all previously iden- edited also in P. patens. However, a significant number of C sub- tified as mutated at the APT locus. No mutation could be stitutions (17%) could also be observed outside this editing win- detected in the potential off-target sequences for any of the tested dow, including a cytosine in position −21 from the PAM that clones, suggesting that Cas9-dependent predicted off-target activ- was outside the 20-bp target sequence of sgRNA (Fig. 4c; ity may be low in P. patens. Table S7). For target sequences presenting more than one C in the editing window, as is the case for sgRNA#21, at least two Cs BE allows the generation of multiple variants of the APRT and up to five Cs could be modified simultaneously (Fig. S3). In enzyme addition, important variation of the efficiency and nature of the substitutions could be observed for a cytosine in a given position Altogether, use of CBE and ABE strategies with multiple sgRNAs from one targeted locus to another (Figs 4b, S5a, S5), confirming allowed the selection of 38 plants containing different variants of the influence of the environment of the cytosine on the efficiency the APRT enzyme and showing combinations of one to four and nature of its modification. Finally, we observed that the amino-acid modifications (Table S9; Fig. S8). To understand New Phytologist (2021) 230: 1258–1272 © 2021 The Authors www.newphytologist.com New Phytologist © 2021 New Phytologist Foundation New Phytologist Research 1265 (a) (b) 5.8 C to T substuon C to G substuon C to A substuon 100 18,6% 90 n=693 80 Nature of C modificaon (%) 75,8% 70 60 50 (c) 40 30 20 10 0 -20 -19 -17 -16 -15 -10 -8 -4 -3 C posion to the PAM 5’ nd nd CC CC -21 -20 -19 -18 -17 -16 -15 -14 -13 -12 -11 -10 -9 -8 C nd C C C C C -7 -6 -5 -4 -3 -2 -1 3’ C to T C to G C to A By-products C Fig. 4 Type of cytosine substitutions and importance of the position of the cytosine. (a) Nature of cytosine editing using pnCas9-CBE1 with the different sgRNAs used in this study was characterised (693 cytosine substitutions analysed). (b) Nature of cytosine editing using CBE with gRNA#21 for each cytosines present in this 20-bp target (n = 100 Physcomitrium patens plants). (c) Frequency of substitution for cytosines at each position of the eight sgRNAs used in this study (2005 cytosines analysed, 693 substituted), only cytosines in positions −13, −12 and −6 were not present (for number of cytosines at each position see Supporting Information Table S7). Primers used for amplification and Sanger sequencing can be found in the Materials and Methods section. how CBE- and ABE-induced substitutions were associated with helix H4, are all located in the core subdomain, and tyrosine 103 APRT loss of function, we focused our analysis on apt mutants (Tyr103) is at the end of β-strand S5. Arg54 is surrounded by showing one amino-acid change, and investigated the role of residues implicated in intraside-chain polar contact (Tyr55–- these single mutations on the APRT 3D structure. Because of the Glu154–Gln58/Asp6–Tyr176) and the R54C mutation could high level of identity of the P. patens APRT to the APRT pro- impair the formation of this interaction, and therefore the correct teins from different kingdoms (45% and 42% to E. coli and folding of the protein. It is highly likely that the mutation at posi- human APRT proteins, respectively) and the availability of sev- tion 75 could disturb the helix H4 and lead to abnormal protein eral APRT 3D structures, we used homology modelling to build folding. Tyr103 is highly conserved in the eight APRT enzymes of a P. patens APRT 3D model. All the sequences were aligned the different kingdoms, and is thought to be involved in the con- using Modeller 9.18 (Fig. S8). Based on multiple alignments of formation of the flexible loop that is proposed to close the active the APRT sequences, a model was created for the P. patens APRT site during catalysis (Shi et al., 2002). (Fig. 5). The P. patens APRT monomer model is a single-do- main structure composed of eight α-helices and nine β-strands Efficient base editing of a gene of interest in P. patens using that can be divided further into the ‘hood’ (residues 1–39) CBE and ABE responsible primarily for base recognition and the definition of substrate specificity, the ‘flexible loop’ (residues 98–115) and the We have shown before that APT-base-edited plants could be iso- core (residues 40–184) (Fig. S8) that corresponds to the con- lated after selection on G418 of CBE-transfected protoplasts. In served type I PRTase fold (Phillips et al., 1999; Shi et al., 2001). order to test the portability of this BE strategy based on selection The four mutated residues (single mutations) associated with on G418, for genes for which the mutations cannot be positively APRT loss of function were reported on the APRT 3D model, selected we decided to target three genes located on three differ- where they were all in important structural or functional domains ent chromosomes in the P. patens genome (Fig. S10) and encod- (Figs 5,S9). Arginine 54 (Arg54) at the end of the helix H3, ing respectively, violaxanthin de-epoxidase (VDE) protein isoleucine 63 (Ile63) in β-strand S3, proline 75 (Pro75) in the (Pp3c3_13220), a C2H2 zinc finger transcription factor © 2021 The Authors New Phytologist (2021) 230: 1258–1272 New Phytologist © 2021 New Phytologist Foundation www.newphytologist.com New 1266 Research Phytologist 180° Arg54 Tyr103 Pro75 Iso63 Fig. 5 Single amino-acid modifications by CBE or ABE in the Physcomitrium patens APRT. View of the Physcomitrium patens APRT 3D model. The APRT protein consists of eight α-helices (in red) and nine β-strands (in yellow) which can be divided further into the hood (residues 1–39), the flexible loop (residues 98–115; β5 and β6) and the core (residues 40–184). Amino acids that could be modified as single substitutions using CBE or ABE are indicated. (Pp3c14_9040), and the tetratricopeptide-repeat protein 39C similar to vde KO plants (Fig. 6c,d). These results confirmed on (TTC39C) protein (Pp3c17_3870). Protoplasts were transfected one side the essential in vivo role of the targeted amino acids in with the pnCas9-CBE1 plasmid and either of the sgRNAs expres- VDE activity in P. patens, and more importantly in this context, sion vectors targeting the three genes. The regenerating proto- provided an in vivo demonstration of the specific alteration of plasts were transferred onto a medium containing the antibiotic protein activity using gene editing of selected amino acids (Fig. G418 in order to select clones expressing the transfected plas- 6). mids. Sequence analysis of the targeted genes showed that 55% These data showed that selection of transfected clones on for sgRNAPp3c3, 23% for sgRNAPp3c14 and 51% for G418 can be used to detect base-editing events in the three tar- sgRNAPp3c17 of the transfected (G418 resistant) clones showed geted genes of this study leading to efficiencies ranging from a cytosine edited at the targeted locus (Fig. 6a). As observed for 23% to 55%. However, it must be noted that 12–16% of the the sgRNAs targeting the APT gene, the ratio of C-to-T, C-to-A edited clones were chimeric (Fig. 6a). and C-to-G varied from one sgRNA to the other, but with a majority of C-to-T substitutions (Figs 6b, S6). Analysis of the The SMART strategy for efficient base co-editing of a gene mutations induced at the three different loci showed that editing of interest in P. patens of the cytosines present in the editing window led to the produc- tion of different amino-acid changes and premature stop codons In order to increase the efficiency of selection for base-edited for the three different proteins. clones and reduce the risk of chimerism we used the APT gene as The genomic region targeted in the P. patens VDE gene has a a reporter of editing efficiency. Because the apt mutants show a key role in regulation of photosynthesis. When plants are exposed developmental phenotype (decreased number of gametophores, to excess light, VDE catalyses the conversion of the carotenoid see Fig. S8b) that could potentially interfere with phenotyping of violaxanthin into another one, zeaxanthin (Baroli et al., 2000). a mutant in a given gene of interest, we could not use a strategy The VDE-dependent zeaxanthin synthesis contributes to the dis- based on co-editing of the wild-type APT gene and the gene of sipation of excess energy through a mechanism called NPQ. The interest. For this reason, we based our strategy on the reversion of assessment of NPQ levels, calculated from the measurement of an existing apt mutation toward the wild-type allele of the APT chlorophyll a fluorescence, therefore allows inferring VDE activ- gene. Co-editing of this apt mutation and of a gene of interest ity in vivo (Pinnola et al., 2013). The availability of structural should permit the selection of individuals mutated in the gene of data on VDE allows the identification of key residues for its activ- interest but wild-type for the rest of their genome. For this pur- ity (Arnoux et al., 2009) such as amino acids involved in violax- pose, we took advantage of the hypersensitivity of the apt anthin binding that have been shown to be essential for protein mutants, compared with the wild-type, to the substrate of APRT, activity in Arabidopsis VDE thanks to in vitro assays (Saga et al., the adenine (Figs S1, S8b). We selected the apt mutant ABEv#1 2010) (Fig. S11). Base editing of the three cytosines present in obtained in this study (Fig. S8b) and tried to suppress the sensi- this catalytic region led to the production of six variants. Variant tivity to adenine in this mutant by reverting the initial mutation. vde#20 contains a premature stop codon leading to a truncated In ABEv#1, the Y103C substitution that confers resistance to 2- VDE, the other variants are modified on one or two of amino FA and hypersensitivity to adenine, can be restored to the wild- acids D177, W178 and Y179 (Table S10; Fig. S11). All vde-edited type sequence via a C103Y substitution. We designed an sgRNA, plants showed an impaired NPQ response, with a phenotype sgRNArestor, that through a C-to-T base editing with the CBE New Phytologist (2021) 230: 1258–1272 © 2021 The Authors www.newphytologist.com New Phytologist © 2021 New Phytologist Foundation New Phytologist Research 1267 (a) Selecon on (b) Selecon on G418 Selecon on G418 adenine (n=44) (n=44) (n=43) (n=42) Pp3c3 Pp3c14 Pp3c17 100% 2% 7% 4% 5% 90% 2% 30% 30% 27% 19% 80% n=68 n=22 43% n=49 53% Ratio of each class (%) 70% 74% 73% 16% 12% 60% 77% 7% 50% 90% Pp3c3 40% 7% 4% 30% C to T substuon 55% 51% C to G substuon 20% C to A substuon n=107 10% 23% 89% 0% Pp3C3 Pp3c3 Pp3c14 Pp3c17 Pp3c14 Pp3c17 Pp3c3 Pp3c3 Selecon on (VDE) (C2H2 ZF) (TTC39C) (VDE) adenine C substuon Deleon Chimeric WT (c) (d) LIGHT DARK 4 WT vde#12 vde#20 vde#15 vde#12 3 vde#20 vde#22 vde#34 NPQ vde#37 2 vde#37 vde#15 WT vde KO vde#34 1 vde#22 0 0 200 400 600 800 1000 Time (s) Fig. 6 Efficiency and nature of editing by cytidine deaminase on Physcomitrium patens genes of interest and consequences on NPQ activity for the VDE (Pp3c3_13220) variants. (a) Efficiency of CBE editing of Pp3c3_13220 (VDE), Pp3c14_9040 (C2H2 ZF) and Pp3c17_3870 (TTC39C) via transfection of wild-type (WT) protoplasts and selection of transient transformants on G418 and, for Pp3c3_13220 via transfection of ABEv#1 protoplasts and co- selection on adenine. Editing efficiency was assessed by sequence analysis performed on G418-resistant plants and also on adenine-resistant plants for Pp3c3_13220. Primers used for amplification and Sanger sequencing can be found in the Materials and Methods section. Number of analysed plants is indicated. (b) Nature of editing for Pp3c3_13220, Pp3c17_3870 and Pp3c14_9040 loci was assessed on 68, 22 and 49 C substitutions for selection on G418, respectively, and 107 for selection on adenine for locus Pp3c3_13220. (c) Phenotype of 20-d-old wild-type and VDE variant plants. (d) NPQ induction and relaxation kinetics of the wild-type and VDE base-edited variants. The wild-type is compared with clones mutated for the VDE gene by cytosine base editing (Supporting Information Table S10). A null mutant of VDE (vde KO; Pinnola et al, 2013) was used as a control for VDE inactivation. Data are mean values of plants grown on three independent plates  SD. © 2021 The Authors New Phytologist (2021) 230: 1258–1272 New Phytologist © 2021 New Phytologist Foundation www.newphytologist.com New 1268 Research Phytologist GOI not edited GOI edited Base-eding GOI PpCBE1 + Selecon GOI goi sgRNArestor + on adenine apt sgRNA-GOI APT APT ABEv#1(2-FAR AdeS) Restored APT (2-FAS AdeR) Wild type GOI Fig. 7 The SMART strategy for efficient base co-editing of a gene of interest in Physcomitrium patens. Principle of selection of base-edited events using adenine selection. The ABEv#1 mutant is co-transfected with the CBE system and two different sgRNAs, sgRNArestor and a sgRNA targeting a gene of interest (GOI). Base editing at the apt locus with sgRNArestor can restore APRT function and confer resistance to adenine. Adenine-resistant clones can be co-edited or not at the GOI locus. system could cause this mutation. This reversion should lead to The results presented here show that, in P. patens, the clones that are sensitive to 2-FA but resistant to adenine (Figs 7, PmCDA1-based CBE strategy allows substitution of one or more S12). cytosines in an editing window slightly larger than the one previ- Based on this, protoplasts from the ABEv#1 mutant were co- ously described (Nishida et al., 2016) including nucleotides −20 transfected with the CBE system and two different sgRNAs, to −14 from the PAM. The majority of the modifications corre- sgRNArestor and sgRNAPp3c3 used previously to target the VDE sponded to C-to-T substitutions but, depending on the sgRNA gene. The regenerating protoplasts were transferred onto medium used, the ratio of edited plants showing indel mutations at the tar- containing adenine in order to select clones of the ABEv#1 mutant geted site could reach almost 50%. This is in line with what can be that were edited and in which function of the APRT enzyme was observed in rice, for which the different CBE strategies gave rise to restored. Sequence analysis of the APT gene in these clones 22% to 71% of plants showing indel mutations (Li et al., 2017; (Table S11) showed that sgRNArestor can efficiently restore a Ren et al., 2018). The observed byproducts occurred in the editing wild-type APRT activity by reverting the Y103C mutation to window, and were more frequent when the editing window con- C103Y. This reversion was sufficient to restore a perfect wild-type tained more than one cytosine. These are likely to be due to the APRT sequence (38% of the clones), or it could be accompanied unfaithful BER of the uracil through nonhomologous end joining. by the replacement of the glutamic acid in position 102 by an As previously observed in other plants or animal cells, the TadA- aspartic acid, a lysine or a glutamine. Interestingly, restoration of a based ABE strategy allows efficient substitution of one or more wild-type APRT activity could also be obtained by replacement of adenines present in the predicted editing window by guanines the cysteine in position 103 by a phenylalanine (C103F) accompa- with very few byproducts, which were observed only when multi- nied or not by the replacement of the glutamic acid in position ple nearby sgRNAs were used at the same time. These byproducts 102 by an aspartic acid. Sequence analysis of the VDE gene show are close to the predicted cutting site of the nickase, and are proba- that its concomitant editing with the APT gene was very efficient bly due to Cas9 nickase activity. For ABE, only very few byprod- (Table S11; Fig. 6a). Using this strategy of selection on adenine ucts could be found with more than 98% of the modifications for co-editing of the gene of interest and of the APT reporter gene, corresponding to A-to-G substitutions. In animal cells, ABE has the efficiency of base editing of the VDE gene reached 90%, com- been shown to be able to convert cytosines that are in a confined pared with the 55% efficiency observed using selection on G418, ‘TCN’ nucleic-acid sequence context into guanine or thymine and furthermore increased the number of VDE variants. In addi- (Lee et al., 2018; Kim et al., 2019). We could not detect this addi- tion, the number of chimeric clones was decreased more than tional activity in P. patens, reinforcing the high level of precision three-fold (Fig. 6a), making this system of selection, that we of the ABE strategy in this organism. named SMART, a very powerful tool for base editing of genes of Because BE strategies are based on dead or nickase Cas9, and interest in P. patens. as such do not produce double-stranded breaks, a theoretical major advantage of BE over classical editing via Cas9 is the recov- ery of a more precise edited product with few or no off-targets. Discussion Low levels of gRNA-dependent off-target DNA base-editing A wide range of gene function analysis tools is available for the activity for CBE or ABE editors have been shown in animals (for model plant P. patens, such as gene replacement through homol- a review see Molla & Yang, 2019) and in plants such as rice or ogous recombination, gene knock-down through RNA silencing tomato (Shimatani et al., 2017; Hua et al., 2018). However, or gene knock-out through CRISPR-Cas (Rensing et al., 2020). unbiased whole genome analyses (WGA) in rice, oilseed rape and Here, we adapted the plant CBE and ABE systems to efficiently mouse embryos (Jin et al., 2019; Zuo et al., 2019; Cheng et al., and specifically achieve targeted modification of single or multi- 2021), have shown that CBEs derived from the rat APOBEC1 ple cytosines and adenines in P. patens. We showed that CBE deaminase can induce substantial genome-wide Cas9-indepen- and ABE strategies are efficient in P. patens and deciphered their dent off-target mutations. Interestingly, this gRNA-independent different characteristics and outputs. off-target DNA base-editing activity was not observed for the New Phytologist (2021) 230: 1258–1272 © 2021 The Authors www.newphytologist.com New Phytologist © 2021 New Phytologist Foundation New Phytologist Research 1269 ABE editor in rice (Jin et al., 2019). Such unpredicted off-target substrate adenine, we could both diminish the proportion of chimeric activity of CBEs was also demonstrated for alternate cytosine clones and increase the efficiency of precise cytosine base editing sig- deaminase domains in E. coli, and motivated the production of nificantly, reaching an efficiency of editing of 90%. This strategy is engineered CBEs with low levels of Cas9-independent off-target also possible for ABE starting from an apt mutant obtained via the activity (Doman et al., 2020). We have shown in this study that CBE strategy. Furthermore, as multiplex base editing has been the PmCDA1-based CBE and TadA-based ABE editors used in reported in dicot and monocot plants (Shimatani et al., 2017, 2018; this study have a low or null predicted gRNA-dependent off-tar- Hua et al., 2018) and because the APRT function is a very conserved get activity. Concerning a possible unpredicted off-target activity enzymatic function in all kingdoms, the SMART strategy presented of these BEs, it must be noticed that no such activity could be here should be applicable to different flowering plants. It would detected in rice for the ABE used in our study (Jin et al., 2019). potentially be an additional tool for transgene free editing in crops, For CBE, if the PmCDA1-based CBE showed only low levels of already obtained using the ALS gene for example (Veillet et al., Cas9-independent deamination in E. coli compared with other 2019b), but would present the advantage of not including a selection cytosine deaminases, this activity was significantly higher in step on herbicides or antibiotics. mammalian cells (Doman et al., 2020). Only an unbiased WGS In conclusion, we demonstrated here that CBE and ABE edi- analysis would address the possible unpredicted off-target activity tors can be very useful tools for in-depth gene function analysis of these BEs in P. patens and permit a conclusion on the preci- in P. patens. We provide information on the nature of the edited sion of these BEs in this plant. products, windows of editing, simplex versus multiplex systems Using selection on G418 for CBE- or ABE-transfected proto- and selection strategies, which should facilitate their use in this plasts we could demonstrate efficient editing of genes of interest. model plant. BE editors extend the already imposing tool box for This allowed us to produce multiple variants of the APRT precise genome editing in P. patens, such as gene replacement enzyme. In humans, alterations of APRT activity can lead to kid- through homologous recombination (Schaefer, 2001), that could ney stone disease (nephrolithiasis), and more than 40 mutations be made more efficient and precise using a CRISPR-Cas9 strat- have been described to date leading to this metabolic defect egy (Collonnier et al., 2017b), or via an elegant recent strategy (Rumsby, 2016). Some of these mutations are present in domains also based on CRISPR-Cas9 but using oligonucleotide templates that were affected in our P. patens APRT variants. For instance, (Yi & Goshima, 2020). In theory, all these strategies could bene- the common D65V mutation found in British, Icelandic and fit from the SMART co-editing selection system described here. Spanish patients affected by nephrolithiasis modifies APRT helix Finally, in addition to being an easy-to-implement alternative to H4, as do our variants P75I, P75L and P75R. Mimicking in the these base modification strategies, the BE system described here P. patens the APRT mutations causing human nephrolithiasis makes possible the in vivo random mutagenesis of a given gene, a could be potentially informative in terms of conservation powerful new tool for gene function analysis in P. patens that between kingdoms of APRT functions. Taking into considera- should reinforce the status of P. patens as a powerful platform for tion the potential sgRNAs (using an NGG PAM) present in the functional analysis of plant genes. APT gene, 40% of the APRT amino acids could theoretically be modified using a combination of the two ABE and CBE editors Acknowledgements used in this study. Recently, we have shown that the SpCas9 vari- ant, SpCas9-NG, is active in P. patens for CRISPR-mediated We are grateful to Akihiko Kondo who provided the Target-AID gene knock-out applications (Veillet et al., 2020b). Because this plasmid. We thank Mark Tepfer and Pierre-François Perroud for variant recognises NGN PAMs, setting up a BE strategy based on their critical revision of the manuscript. The work, including a SpnCas9-NG variant should theoretically permit the modifica- study design, data collection, analysis and interpretation and tion of 100% of the amino acids encoded by the APT gene, rein- manuscript writing, was supported by the French National forcing the usefulness of these ABE and CBE strategies. The Research Agency (ANR11-BTBR-0001-GENIUS). The IJPB great potential of these BE strategies for functional analysis was benefits from the support of the LabEx Saclay Plant Sciences-SPS confirmed for three other genes, including the VDE gene. The (ANR-10-LABX-0040-SPS). The authors declare no conflict of latter allowed us to demonstrate in vivo the essential role of the interest. targeted amino acids in VDE activity in P. patens. Nevertheless, analysis of the edited clones obtained after selection on G418 Author contributions showed that significant numbers of edited clones were chimeric. This should be taken into consideration when P. patens proto- FN and AG-D designed the research; AG-D performed the plasts clones were selected for transient transfection of BE editors research with the help of AA, ZT, FC, FV, FB, PV-M, JMC, using antibiotics. The possibility of the existence of such chimeric TM and J-LG; FN and AG-D wrote the manuscript with contri- clones in other protoplast-based BE editor transfection systems butions from all the authors. (e.g. potato or tomato) would probably deserve attention. Finally, by using APT as a reporter gene, we propose the SMART ORCID approach to efficiently select base editing in target genes. Using the SMART selection system, based on co-editing of a gene of interest Alessandro Alboresi https://orcid.org/0000-0003-4818-7778 and of a mutated version of the APT, and selection on the APRT Floriane Berthier https://orcid.org/0000-0003-3368-8078 © 2021 The Authors New Phytologist (2021) 230: 1258–1272 New Phytologist © 2021 New Phytologist Foundation www.newphytologist.com New 1270 Research Phytologist Josep M. Casacuberta https://orcid.org/0000-0002-5609- Gaudelli NM, Komor AC, Rees HA, Packer MS, Badran AH, Bryson DI, Liu 4152 DR. 2017. Programmable base editing of A T to G C in genomic DNA • • without DNA cleavage. Nature 551: 464–471. Florence Charlot https://orcid.org/0000-0003-1218-951X Holá M, Kozák J, Vágnerová R, Angelis KJ. 2013. Genotoxin induced Jean-Luc Gallois https://orcid.org/0000-0003-0451-1740 mutagenesis in the model plant Physcomitrella patens. BioMed Research Anouchka Guyon-Debast https://orcid.org/0000-0003-3489- International 2013: 1–7. 1035 Hua K, Tao X, Yuan F, Wang D, Zhu JK. 2018. Precise AT to GC base editing Tomas Morosinotto https://orcid.org/0000-0002-0803-7591 in the rice genome. Molecular Plant 11: 627–630. Huang TK, Puchta H. 2019. CRISPR/Cas-mediated gene targeting in plants: Fabien Nogué https://orcid.org/0000-0003-0619-4638 finally a turn for the better for homologous recombination. Plant Cell Reports Zoé Terret https://orcid.org/0000-0002-8057-755X 38: 443–453. Florian Veillet https://orcid.org/0000-0002-6892-6825 Jacob P, Avni A, Bendahmane A. 2018. Translational research: exploring and Pol Vendrell-Mir https://orcid.org/0000-0002-5695-5880 creating genetic diversity. Trends in Plant Science 23: 42–52. Jiang F, Doudna JA. 2017. CRISPR–Cas9 structures and mechanisms. Annual Review of Biophysics 46: 505–529. Jin S, Zong Y, Gao Q, Zhu Z, Wang Y, Qin P, Liang C, Wang D, Qiu JL, References Zhang F et al. 2019. Cytosine, but not adenine, base editors induce genome- wide off-target mutations in rice. Science 364: 292–295. Anzalone AV, Randolph PB, Davis JR, Sousa AA, Koblan LW, Levy JM, John B, Sali A. 2003. Comparative protein structure modeling by iterative Chen PJ, Wilson C, Newby GA, Raguram A et al. 2019. Search-and- alignment, model building and model assessment. Nucleic Acids Research 31: replace genome editing without double-strand breaks or donor DNA. 3982–3992. Nature 576: 149–157. Kim HS, Jeong YK, Hur JK, Kim JS, Bae S. 2019. Adenine base editors catalyze Arnoux P, Morosinotto T, Saga G, Bassi R, Pignol D. 2009. A structural basis cytosine conversions in human cells. Nature Biotechnology 37: 1145–1148. for the ph-dependent xanthophyll cycle in Arabidopsis thaliana. The Plant Cell Klughammer C, Schreiber U. 1994. An improved method, using saturating light 21: 2036–2044. pulses, for the determination of photosystem I quantum yield via P700+- Ashihara H, Stasolla C, Fujimura T, Crozier A. 2018. Purine salvage in plants. absorbance changes at 830 nm. Planta 192: 261–268. Phytochemistry 147: 89–124. Komor AC, Kim YB, Packer MS, Zuris JA, Liu DR. 2016. Programmable Baroli I, Niyogi KK, Barber J, Heifetz P. 2000. Molecular genetics of editing of a target base in genomic DNA without double-stranded DNA xanthophyll-dependent photoprotection in green algae and plants. Philosophical cleavage. Nature 533: 420–424. Transactions of the Royal Society B: Biological Sciences 355: 1385–1394. Lee HK, Willi M, Miller SM, Kim S, Liu C, Liu DR, Hennighausen L. 2018. Bastet A, Zafirov D, Giovinazzo N, Guyon-Debast A, Nogué F, Robaglia C, Targeting fidelity of adenine and cytosine base editors in mouse embryos. Gallois J-L. 2019. Mimicking natural polymorphism in eIF4E by CRISPR- Nature Communications 9: 7–12. Cas9 base editing is associated with resistance to potyviruses. Plant Li J, Sun Y, Du J, Zhao Y, Xia L. 2017. Generation of targeted point mutations Biotechnology Journal 17: 1736–1750. in rice by a modified CRISPR/Cas9 system. Molecular Plant 10: 526–529. Cheng H, Hao M, Ding B, Mei D, Wang W, Wang H, Zhou R, Liu J, Li C, Hu Li X, Wang Y, Liu Y, Yang B, Wang X, Wei J, Lu Z, Zhang Y, Wu J, Huang X Q. 2021. Base editing with high efficiency in allotetraploid oilseed rape by et al. 2018a. Base editing with a Cpf1-cytidine deaminase fusion. Nature A3A-PBE system. Plant Biotechnology Journal 19: 87–97. Biotechnology 36: 324–327. Collonnier C, Epert A, Mara K, Maclot F, Guyon-Debast A, Charlot F, White Li ZY, Wang Y, Jin S, Zhang D, Song Q, Zhang R, Gao C, Li C, Zong Y et al. C, Schaefer DG, Nogué F. 2017a. CRISPR-Cas9-mediated efficient directed 2018b. Expanded base editing in rice and wheat using a Cas9-adenosine mutagenesis and RAD51-dependent and RAD51-independent gene targeting deaminase fusion. Genome Biology 19: 59. in the moss Physcomitrella patens. Plant Biotechnology Journal 15: 122–131. Lin Q, Zong Y, Xue C, Wang S, Jin S, Zhu Z, Wang Y, Anzalone AV, Raguram Collonnier C, Guyon-Debast A, Maclot F, Mara K, Charlot F, Nogué F. 2017b. A, Doman JL et al. 2020. Prime genome editing in rice and wheat. Nature Towards mastering CRISPR-induced gene knock-in in plants: survey of key Biotechnology 38: 582–585. features and focus on the model Physcomitrella patens. Methods 121–122: Lopez-Obando M, Hoffmann B, Géry C, Guyon-Debast A, Téoulé E, Rameau 103–117. C, Bonhomme S, Nogué F. 2016. Simple and efficient targeting of multiple Concordet J-P, Haeussler M. 2018. CRISPOR: intuitive guide selection for genes through CRISPR-Cas9 in Physcomitrella patens. G3: Genes, Genomes, CRISPR/Cas9 genome editing experiments and screens. Nucleic Acids Research Genetics 6: 3647–3653. 46: W242–W245. Lu Y, Zhu J-K. 2017. Precise editing of a target base in the rice genome using a Cove DJ, Perroud P-F, Charron AJ, McDaniel SF, Khandelwal A, Quatrano modified CRISPR/Cas9 system. Molecular Plant 10: 523–525. RS. 2009. Culturing the moss Physcomitrella patens. In: Emerging model Mallett DR, Chang M, Cheng X, Bezanilla M. 2019. Efficient and organisms: a laboratory manual, Vol. 1. New York, NY, USA: Cold Spring modular CRISPR-Cas9 vector system for Physcomitrella patens. Plant Direct Harbor Laboratory Press. 3: 1–15. Doman JL, Raguram A, Newby GA, Liu DR. 2020. Evaluation and Manghwar H, Lindsey K, Zhang X, Jin S. 2019. CRISPR/Cas system: recent minimization of Cas9-independent off-target DNA editing by cytosine base advances and future prospects for genome editing. Trends in Plant Science 24: editors. Nature Biotechnology 38: 620–628. 1102–1125. Eid A, Alshareef S, Mahfouz MM. 2018. CRISPR base editors: genome editing Marzec M, Hensel G. 2020. Prime editing: game changer for modifying plant without double-stranded breaks. Biochemical Journal 475: 1955–1964. genomes. Trends in Plant Science 25: 722–724. Elmar K, Keehyoung J, Jinwoo L, Jooyoung L, Srivatsan R, James T, Mike T, Melo F, Sánchez R, Sali A. 2009. Statistical potentials for fold assessment. Protein David B, Kevin K. 2010. Improving physical realism, stereochemistry and Science 11: 430–448. side-chain accuracy in homology modeling: four approaches that performed Molla KA, Yang Y. 2019. CRISPR/Cas-mediated base editing: technical well in CASP8 Elmar. Proteins 77: 114–122. considerations and practical applications. Trends in Biotechnology 37: Evanoff M, Komor AC. 2019. Base Editors: Modular Tools for the Introduction 1121–1142. of Point Mutations in Living Cells. Emerging topics in life sciences 3: 483–491. Muren E, Nilsson A, Ulfstedt M, Johansson M, Ronne H. 2009. Rescue and Gaillochet C, Develtere W, Jacobs TB. 2020. CRISPR screens in plants: characterization of episomally replicating DNA from the moss approaches, guidelines, and future prospects. The Plant Cell tpc.00463.2020. Physcomitrella. Proceedings of the National Academy of Sciences, USA 106: doi: https://doi.org/10.1105/tpc.20.00463 19444–19449. New Phytologist (2021) 230: 1258–1272 © 2021 The Authors www.newphytologist.com New Phytologist © 2021 New Phytologist Foundation New Phytologist Research 1271 Nishida K, Arazoe T, Yachie N, Banno S, Kakimoto M, Tabata M, Mochizuki Taylor MW, Simon AE, Kothari RM. 1985. The APRT system. In Gottesman M, Miyabe A, Araki M, Hara KY et al. 2016. Targeted nucleotide editing MM, ed. Molecular cell genetics. New York, NY, USA: John Wiley & Sons: using hybrid prokaryotic and vertebrate adaptive immune systems. Science 102: 311–332. 553–563. Thévenin J, Dubos C, Xu W, Le Gourrierec J, Kelemen Z, Charlot F, Nogué F, Nomura T, Sakurai T, Osakabe Y, Osakabe K, Sakakibara H. 2016. Efficient Lepiniec L, Dubreucq B. 2012. A new system for fast and quantitative analysis and heritable targeted mutagenesis in mosses using the CRISPR/Cas9 system. of heterologous gene expression in plants. New Phytologist 193: 504–512. Plant and Cell Physiology 57: 2600–2610. Trouiller B, Schaefer DG, Charlot F, Nogué F. 2006. MSH2 is essential for the Orr RG, Foley SJ, Sherman C, Abreu I, Galotto G, Liu B, González-Guerrero preservation of genome integrity and prevents homeologous recombination in M, Vidali L. 2020. Robust survival-based RNA interference of gene families the moss Physcomitrella patens. Nucleic Acids Research 34: 232–242. using in tandem silencing of adenine phosphoribosyltransferase. Plant Veillet F, Chauvin L, Kermarrec MP, Sevestre F, Merrer M, Terret Z, Physiology 184: 607–619. Szydlowski N, Devaux P, Gallois JL, Chauvin JE. 2019a. The Solanum Phillips CL, Ullman B, Brennan RG, Hill CP. 1999. Crystal structures of tuberosum GBSSI gene: a target for assessing gene and base editing in tetraploid adenine phosphoribosyltransferase from Leishmania donovani. EMBO Journal potato. Plant Cell Reports 38: 1065–1080. 18: 3533–3545. Veillet F, Durand M, Kroj T, Cesari S, Gallois J-L. 2020a. Precision breeding Pinnola A, Dall’Osto L, Gerotto C, Morosinotto T, Bassi R, Alboresi A. 2013. made real with CRISPR: illustration through genetic resistance to pathogens. Zeaxanthin binds to light-harvesting complex stress-related protein to enhance Plant Communications 1: 100102. nonphotochemical quenching in Physcomitrella patens. The Plant Cell 25: Veillet F, Perrot L, Chauvin L, Kermarrec M-P, Guyon-Debast A, Chauvin 3519–3534. J-E, Nogué F, Mazier M. 2019b. Transgene-free genome editing in Pu X, Liu L, Li P, Huo H, Dong X, Xie K, Yang H, Liu L. 2019. A CRISPR/ tomato and potato plants using Agrobacterium-mediated delivery of a LbCas12a-based method for highly efficient multiplex gene editing in CRISPR/Cas9 cytidine base editor. International Journal of Molecular Physcomitrella patens. The Plant Journal 100: 863–872. Sciences 20: 402. Ran FA, Hsu PD, Lin C-Y, Gootenberg JS, Konermann S, Trevino AE, Scott Veillet F, Perrot L, Guyon-Debast A, Kermarrec M, Chauvin L, Chauvin J, DA, Inoue A, Matoba S, Zhang Y, et al. 2013. Double nicking by RNA- Gallois J, Mazier M, Nogué F. 2020b. Expanding the CRISPR toolbox in P. guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154: patens using SpCas9-NG variant and application for gene and base editing in 1380–1389. solanaceae crops. International Journal of Molecular Sciences 21: 1024. Ren B, Yan F, Kuang Y, Li N, Zhang D, Zhou X, Lin H, Zhou H. 2018. Webb B, Sali A. 2017. Protein structure modeling with MODELLER. Methods Improved base editor for efficiently inducing genetic variations in rice with in Molecular Biology 1654: 39–54. CRISPR/Cas9-guided hyperactive hAID mutant. Molecular Plant 11: Yi P, Goshima G. 2020. Transient cotransformation of CRISPR/Cas9 and 623–626. oligonucleotide templates enables efficient editing of target loci in Rensing SA, Goffinet B, Meyberg R, Wu SZ, Bezanilla M. 2020. The moss Physcomitrella patens. Plant Biotechnology Journal 18: 599–601. physcomitrium (Physcomitrella) patens: A model organism for non-seed plants. Zhang Y, Malzahn AA, Sretenovic S, Qi Y. 2019. The emerging and The Plant Cell 32: 1361–1376. uncultivated potential of CRISPR technology in plant science. Nature Plants 5: Robert X, Gouet P. 2014. Deciphering key features in protein structures with the 778–794. new ENDscript server. Nucleic Acids Research 42: 320–324. Zong Y, Wang Y, Li C, Zhang R, Chen K, Ran Y, Qiu JL, Wang D, Gao C. Rumsby G. 2016. Genetic defects underlying renal stone disease. International 2017. Precise base editing in rice, wheat and maize with a Cas9-cytidine Journal of Surgery 36: 590–595. deaminase fusion. Nature Biotechnology 35: 438–440. Saga G, Giorgetti A, Fufezan C, Giacometti GM, Bassi R, Morosinotto T. Zuo E, Sun Y, Wei W, Yuan T, Ying W, Sun H, Yuan L, Steinmetz LM, Li Y, 2010. Mutation analysis of violaxanthin de-epoxidase identifies substrate- Yang H. 2019. Cytosine base editor generates substantial off-target single- binding sites and residues involved in catalysis. Journal of Biological Chemistry nucleotide variants in mouse embryos. Science 364: 289–292. 285: 23763–23770. Schaefer D. 2001. Gene targeting in Physcomitrella patens. Current Opinion in Plant Biology 4: 143–150. Supporting Information Schaefer DG, Delacote F, Charlot F, Vrielynck N, Guyon-Debast A, Le Guin S, Neuhaus JM, Doutriaux MP, Nogué F. 2010. RAD51 loss of function Additional Supporting Information may be found online in the abolishes gene targeting and de-represses illegitimate integration in the moss Supporting Information section at the end of the article. Physcomitrella patens. DNA Repair 9: 526–533. Schaefer DG, Zryd J-P. 1997. Efficient gene targeting in the moss Physcomitrella Fig. S1 Schematic description of the APT reporter gene and patens. The Plant Journal 11: 1195–1206. Shen M, Sali A. 2006. Statistical potential for assessment and prediction of APRT function. protein structures. Protein Science 15: 2507–2524. Shi W, Sarver AE, Wang CC, Tanaka KSE, Almo SC, Schramm VL. 2002. Fig. S2 Examples of deletions observed during BE multiplexing. Closed site complexes of adenine phosphoribosyltransferase from Giardia lamblia reveal a mechanism of ribosyl migration. Journal of Biological Chemistry Fig. S3 Examples of multiple cytosines editing or chimerism 277: 39981–39988. Shi W, Tanaka KSE, Crother TR, Taylor MW, Almo SC, Schramm VL. 2001. observed in some clones. Structural analysis of adenine phosphoribosyltransferase from Saccharomyces cerevisiae. Biochemistry 40: 10800–10809. Fig. S4 G418 sensitivity of ABE and CBE single and multiplex Shimatani Z, Fujikura U, Ishii H, Matsui Y, Suzuki M, Ueke Y, Taoka KI, edited clones after relaxing of the antibiotic selection pressure. Terada R, Nishida K, Kondo A. 2018. Inheritance of co-edited genes by CRISPR-based targeted nucleotide substitutions in rice. Plant Physiology and Biochemistry 131: 78–83. Fig. S5 Nature of editing using CBE or ABE for each cytosine or Shimatani Z, Kashojiya S, Takayama M, Terada R, Arazoe T, Ishii H, adenine present in the target locus. Teramura H, Yamamoto T, Komatsu H, Miura K et al. 2017. Targeted base editing in rice and tomato using a CRISPR-Cas9 cytidine deaminase fusion. Fig. S6 Nature of editing using CBE on genes of interest for each Nature Biotechnology 35: 441–443. cytosine in the target locus. © 2021 The Authors New Phytologist (2021) 230: 1258–1272 New Phytologist © 2021 New Phytologist Foundation www.newphytologist.com New 1272 Research Phytologist Fig. S7 Sequence of two sgRNAs containing cytosines potentially Table S6 Mutation rates of the CBE system after preselection on target of ABE activity and nature of ABE editing using these G418. sgRNAs. Table S7 Frequency of substitution for cytosines at each position Fig. S8 Alignment of APRT sequences from different species and of the eight sgRNAs used in this study. phenotype of the apt P. patens mutants. Table S8 Sequences and positions of possible off-target sites for Fig. S9 View of the P. patens APRT 3D model with amino acids sgRNA1 and sgRNA2. (in blue) that could be modified as single substitutions using CBE or ABE. Table S9 List of amino acids modified in the APT gene using the CBE or ABE strategy. Fig. S10 Structure of the Pp3c3_13220, Pp3c14_9040 and Pp3c17_3870 targeted genes. Table S10 Consequence of CBE editing in the different edited clones for the three genes of interest. Fig. S11 Sequence alignment of VDE from Arabidopsis and Physcomitrella. Table S11 Sequence analysis of the APT and Pp3c3_13220 locus in adenine-resistant clones obtained after co-transfection of the Fig. S12 Use of the APT gene as a marker of base-editing effi- ABEv#1 mutant with the CBE system and the two sgRNAs, ciency. sgRNArestor and sgRNAPp3c3. Table S1 List of sgRNAs expression cassettes used in this study. Table S2 Sequences of plasmids used in this study. Please note: Wiley Blackwell are not responsible for the content or functionality of any Supporting Information supplied by the Table S3 List of PCR primers used in this study. authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office. 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. New Phytologist is an electronic (online-only) journal owned by the New Phytologist Foundation, a not-for-profit organization dedicated to the promotion of plant science, facilitating projects from symposia to free access for our Tansley reviews and Tansley insights. Regular papers, Letters, Viewpoints, Research reviews, Rapid reports and both Modelling/Theory and Methods papers are encouraged. We are committed to rapid processing, from online submission through to publication ‘as ready’ via Early View – our average time to decision is <26 days. There are no page or colour charges and a PDF version will be provided for each article. The journal is available online at Wiley Online Library. 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