A blueprint for gene function analysis through Base Editing in the model plant Physcomitrium (Physcomitrella) patens

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

Capitalisation des données géologiques, structurales et métallogéniques du Craton Ouest Africain : vers une meilleure compréhension de la distribution spatiale de l'or dans le craton

International audienceLe Craton Ouest Africain(COA) est actuellement le réservoir majeur d’or protérozoïque, avec environ 10 000 t d’or de réserves estimées en 2017 et 9 gisements de classe mondiale, le plaçant parmi les plus grandes provinces aurifères mondiales. La très grande majorité des gisements connus sont de type orogénique, encaissés dans les volcano-sédiments et sédiments déformés et métamorphisés formant les ceintures de roches vertes d’âge Paléoprotérozoïque inférieur (Rhyacien). Cependant, la mise au jour de nouveaux types de gisement dans les autres domaines litho-tectoniques ainsi que la description de minéralisations complexes, polyphasées, conduisent à compiler et intégrer les connaissances géologiques et métallogéniques actuelles dans un schéma pétro-structural multi-échelle.L’engouement pour l’or du COA est relativement récent (années 1990) et son exploration minière est donc encore partielle. La première synthèse géologique et métallogénique est publiée en 1989 par le BRGM. Son exploration s’accélère dans les années 2000 et de nombreux acteurs privés et entités de recherche, notamment via le programme international « West African eXploration Initiative », ont produit une importante quantité de données permettant de réviser les concepts géologiques,métallogéniques et géodynamiques aussi bien à l’échelle du gisement qu’à l’échelle de la province. L'intégration de ces nouveaux concepts aux cartes géologiques publiées à toutes échelles et à toutes époques,aboutit, après un exercice d'harmonisation et de réinterprétation,à une définition géodynamique des unités lithologiques et à une délimitation précise des grands linéaments structuraux et domaines litho-tectoniques associés à l'échelle du COA. La comparaison de cette carte harmonisée avec une base de données exhaustive des indices minéraux, aussi bien mines, projets d’explorations avancés et occurrences, permet de discuter du potentiel métallogénique de ces différents domaines plus clairement définis et délimité

Patronus is the elusive plant securin, preventing chromosome separation by antagonizing separase.

PNAS August 6, 2019 116 (32) 16018-16027; first published July 19, 2019Accurate chromosome segregation at mitosis and meiosis is crucial to prevent genome instability, birth defect, and cancer. Accordingly, separase, the protease that triggers chromosome distribution, is tightly regulated by a direct inhibitor, the securin. However, securin has not been identified, neither functionnally nor by sequence similarity, in other clades that fungi and animals. This raised doubts about the conservation of this mechanism in other branches of eukaryotes. Here, we identify and characterize the securin in plants. Despite extreme sequence divergence, the securin kept the same core function and is likely a universal regulator of cell division in eukaryotes

Patronus is the elusive plant securin, preventing chromosome separation by antagonizing separase

International audienceChromosome distribution at anaphase of mitosis and meiosis is triggered by separase, an evolutionarily conserved protease. Separase must be tightly regulated to prevent the untimely release of chromatid cohesion and disastrous chromosome distribution defects. Securin is the key inhibitor of separase in animals and fungi, but has not been identified in other eukaryotic lineages. Here, we identified PATRONUS1 and PATRONUS2 (PANS1 and PANS2) as the Arabidopsis homologs of securin. Disruption of PANS1 is known to lead to the premature separation of chromosomes at meiosis, and the simultaneous disruption of PANS1 and PANS2 is lethal. Here, we show that PANS1 targeting by the anaphase-promoting complex is required to trigger chromosome separation, mirroring the regulation of securin. We showed that PANS1 acts independently from Shugosins. In a genetic screen for pans1 suppressors, we identified SEPARASE mutants, showing that PANS1 and SEPARASE have antagonistic functions in vivo. Finally, we showed that the PANS1 and PANS2 proteins interact directly with SEPARASE. Altogether, our results show that PANS1 and PANS2 act as a plant securin. Remote sequence similarity was identified between the plant patronus family and animal securins, suggesting that they indeed derive from a common ancestor. Identification of patronus as the elusive plant securin illustrates the extreme sequence divergence of this central regulator of mitosis and meiosis

Rare Coding Variants in ANGPTL6 Are Associated with Familial Forms of Intracranial Aneurysm

International audienceIntracranial aneurysms (IAs) are acquired cerebrovascular abnormalities characterized by localized dilation and wall thinning in intracranial arteries, possibly leading to subarachnoid hemorrhage and severe outcome in case of rupture. Here, we identified one rare nonsense variant (c.1378A>T) in the last exon of ANGPTL6 (Angiopoietin-Like 6)—which encodes a circulating pro-angiogenic factor mainly secreted from the liver—shared by the four tested affected members of a large pedigree with multiple IA-affected case subjects. We showed a 50% reduction of ANGPTL6 serum concentration in individuals heterozygous for the c.1378A>T allele (p.Lys460Ter) compared to relatives homozygous for the normal allele, probably due to the non-secretion of the truncated protein produced by the c.1378A>T transcripts. Sequencing ANGPTL6 in a series of 94 additional index case subjects with familial IA identified three other rare coding variants in five case subjects. Overall, we detected a significant enrichment (p = 0.023) in rare coding variants within this gene among the 95 index case subjects with familial IA, compared to a reference population of 404 individuals with French ancestry. Among the 6 recruited families, 12 out of 13 (92%) individuals carrying IA also carry such variants in ANGPTL6, versus 15 out of 41 (37%) unaffected ones. We observed a higher rate of individuals with a history of high blood pressure among affected versus healthy individuals carrying ANGPTL6 variants, suggesting that ANGPTL6 could trigger cerebrovascular lesions when combined with other risk factors such as hypertension. Altogether, our results indicate that rare coding variants in ANGPTL6 are causally related to familial forms of IA