Fabien Nogué

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.

Key message : CRISPR-Cas multiplex editing generated a broad range of early-flowering soybean lines, advancing flowering by up to four weeks under controlled long-day conditions and by approximately one to two weeks in a field trial. Soybean is a promising crop for European agriculture, but its cultivation area remains smaller than its potential. A major limitation to broader adoption in Europe is the restricted number of elite lines adapted to local environmental conditions, particularly the photoperiod encountered at higher latitude. At these latitudes, soybean must flower early to complete its growth cycle before the onset of harsh autumn conditions. In this project, we employed a CRISPR-Cas multiplex knockout strategy, to generate allelic diversity in the gene network controlling flowering time, with the aim of developing earlier flowering soybeans lines better suited to European high latitudes. This approach successfully produced earlier flowering varieties, with flowering advances of up to four weeks compared to the controls. Starting from maturity group (MG) I and MG III lines, we developed, within two generations, gene-edited early flowering soybean lines with estimated maturity ranges between MG 000 and MG 0. Greenhouse observations were supported by a field trial, in an MG II environment, where edited lines flowered approximately one to two weeks earlier than their respective parental controls. These results demonstrate that multiplex editing of flowering-time genes can rapidly generate targeted allelic diversity and a broad spectrum of flowering phenotypes within elite soybean backgrounds. These results provide valuable breeding material and a genotype/phenotype resource for subsequent evaluation of flowering-time adaptation across environments.

The sexual life cycle of bryophytes and vascular plants, the two extant lineages of land plants, is dominated by a gametophytic and a sporophytic phase, respectively. Body plan diversification in these two phases has followed separate evolutionary trajectories in both lineages. However, how evolutionary conserved gene families have accompanied body plan diversification remains poorly understood. WIP transcription factors are conserved in land plants, and their function has been associated with root and flower development in angiosperms. Here, we dissect the function of Pp WIP1 and Pp WIP2 genes in a model bryophyte, the moss Physcomitrium patens . We demonstrate that Pp WIP s are expressed during both phases of the life cycle and regulate specific developmental processes. In the gametophytic phase, Pp WIP s promote the chloronema-to-caulonema transition, but restrict gametophore development. In the sporophytic phase, Pp WIPs prevent polysety, the formation of multiple sporogonia. RNA-seq, DAP-seq, and hormone analysis revealed that both P. patens and A. thaliana proteins act by controlling auxin homeostasis. Interspecies complementation experiments revealed that Pp WIP s are partially able to substitute At WIP molecular function in Arabidopsis root and seeds. Our findings demonstrate that WIPs control developmental progression in both phases of the land plant life cycle through an evolutionarily conserved molecular function.

BRCA2 is crucial for mediating homology-directed DNA repair (HDR) through its binding to single-stranded DNA (ssDNA) and the recombinases RAD51 and DMC1. Most BRCA2 orthologs have a canonical DNA-binding domain (DBD) with the exception of Drosophila melanogaster. It remains unclear whether such a noncanonical BRCA2 variant without DBD possesses a DNA-binding activity. Here, we identify a new noncanonical BRCA2 in the model plant Physcomitrium patens (PpBRCA2). We establish that PpBRCA2 is essential for genome integrity maintenance, somatic DNA double-strand break (DSB) repair, HDR-mediated gene targeting, and RAD51 foci recruitment at DNA break sites. PpBRCA2 is also critical for DSB repair during meiosis. Interestingly, PpBRCA2 interacts strongly with RAD51 but weakly with DMC1, suggesting a distinct meiotic function compared to other BRCA2 homologs. Despite lacking the canonical DBD, PpBRCA2 binds ssDNA through its disordered N-terminal region and efficiently promotes HDR. Our work highlights that the ssDNA binding capacity of BRCA2 homologs is conserved regardless of the presence of a canonical DBD and provides a deeper understanding of BRCA2’s functional diversity across species.

BRCA2 interacts with RAD51 and DMC1 recombinases and binds to single-stranded DNA (ssDNA), through its canonical DNA binding domain (DBD) to mediate homology-directed DNA repair (HDR). While the well-folded DBD is widely conserved in diverse eukaryotes, a non-canonical BRCA2 variant lacking this domain is found in Drosophila melanogaster . Whether such a non-canonical BRCA2 variant exists in other species is unknown. Additionally, the DNA-binding activity of a BRCA2 variant lacking DBD remains unclear. Here, we identify a new non-canonical BRCA2 in the model plant Physcomitrium patens (PpBRCA2). We establish that PpBRCA2 is essential for genome integrity maintenance, somatic DNA repair, HDR-mediated gene targeting, and RAD51 foci recruitment at DNA break sites. PpBRCA2 is also critical for DNA repair during meiosis, but interacts only weakly with DMC1, suggesting a distinct meiotic function compared to other BRCA2 homologs. Despite lacking the canonical DBD, PpBRCA2 binds ssDNA through its disordered N-terminal region and efficiently promotes HDR. Our work highlights that the ssDNA binding capacity of BRCA2 homologs is conserved regardless of the presence of canonical DBD and provides a deeper understanding of BRCA2’s functional diversity across species. Three Key messages: Physcomitrium patens BRCA2 (PpBRCA2) lacking canonical DBD mediates homologous recombination and promotes genome stability PpBRCA2 differentially interact with RAD51 and DMC1 PpBRCA2 binds single-stranded DNA via its N-terminal to compensate for the loss of canonical DBD

SUMMARY: Soybean (Glycine max Merr.) is the world's most important oilseed crop and its ability to fix atmospheric nitrogen makes it a cornerstone of sustainable agriculture. Despite its importance, Europe relies heavily on imports, leading to environmental and economic vulnerabilities. To address these challenges, the European Union has implemented policies to boost local soybean production, emphasizing sustainable practices and reduced dependency on imports. However, conventional breeding methods are time‐consuming and may not keep pace with the rapid environmental and consumer habit changes. Genome‐editing technologies, such as CRISPR‐Cas, offer precise and efficient tools for developing soybean varieties tailored to European conditions. These technologies can enhance traits related to precocity, stress responses, yield and quality that are essential for adapting to climate change and promoting ecological sustainability. This review explores the integration of genome editing (GE) in soybean breeding, highlighting its potential in advancing the agroecological transition in Europe. By having a clear regulation and enhancing breeding efforts, GE can significantly contribute to developing resilient and sustainable soybean varieties, fostering a competitive and environmentally friendly European agriculture.

SUMMARY: The development of CRISPR technologies provides a powerful tool for understanding the evolution and functionality of essential biological processes. Here we demonstrate successful CRISPR‐Cas9 genome editing in the dioecious moss species, Ceratodon purpureus. Using an existing selection system from the distantly related hermaphroditic moss, Physcomitrium patens, we generated knock‐outs of the APT reporter gene by employing CRISPR‐targeted mutagenesis under expression of native U6 snRNA promoters. Next, we used the native homology‐directed repair (HDR) pathway, combined with CRISPR‐Cas9, to knock in two reporter genes under expression of an endogenous RPS5A promoter in a newly developed landing site in C. purpureus. Our results show that the molecular tools developed in P. patens can be extended to other mosses across this ecologically important and developmentally variable group. These findings pave the way for precise and powerful experiments aimed at identifying the genetic basis of key functional variation within the bryophytes and between the bryophytes and other land plants.

Genome editing via CRISPR/Cas has enabled targeted genetic modifications in various species, including plants. The requirement for specific protospacer-adjacent motifs (PAMs) near the target gene, as seen with Cas nucleases like SpCas9, limits its application. PAMless SpCas9 variants, designed with a relaxed PAM requirement, have widened targeting options. However, these so-call PAMless SpCas9 still show variation of editing efficiency depending on the PAM and their efficiency lags behind the native SpCas9. Here we assess the potential of a PAMless SpCas9 variant for genome editing in the model plant Physcomitrium patens. For this purpose, we developed a SpRYCas9i variant, where expression was optimized, and tested its editing efficiency using the APT as a reporter gene. We show that the near PAMless SpRYCas9i effectively recognizes specific PAMs in P. patens that are not or poorly recognized by the native SpCas9. Pattern of mutations found using the SpRYCas9i are similar to the ones found with the SpCas9 and we could not detect off-target activity for the sgRNAs tested in this study. These findings contribute to advancing versatile genome editing techniques in plants.