Multiple parameters drive the efficiency of CRISPR/Cas9-induced gene modifications in Yarrowia lipolytica

Yarrowia lipolytica is an oleaginous yeast of growing industrial interest for biotechnological applications. In the last few years, genome edition has become an easier and more accessible prospect with the world wild spread development of CRISPR/Cas9 technology. In this study, we focused our attention on the production of the two key elements of the CRISPR-Cas9 ribonucleic acid protein complex in this non-conventional yeast. The efficiency of NHEJ-induced knockout was measured by time-course monitoring using multiple parameters flow cytometry, as well as phenotypic and genotypic observations, and linked to nuclease production levels showing that its strong overexpression is unnecessary. Thus, the limiting factor for the generation of a functional ribonucleic acid protein complex clearly resides in guide expression, which was probed by testing different linker lengths between the transfer RNA promoter and the sgRNA. The results highlight a clear deleterious effect of mismatching bases at the 5' end of the target sequence. For the first time in yeast, an investigation of its maturation from the primary transcript was undertaken by sequencing multiple sgRNAs extracted from the host. These data provide insights into of the yeast small RNA processing, from synthesis to maturation, and suggests a pathway for their degradation in Y. lipolytica. Subsequently, a whole-genome sequencing of a modified strain detected no abnormal modification due to off-target effects, confirming CRISPR/Cas9 as a safe strategy for editing Y. lipolytica genome. Finally, the optimized system was used to promote in vivo directed mutagenesis via homology-directed repair with a ssDNA oligonucleotide

From azoospermia to macrozoospermia, a phenotypic continuum due to mutations in the ZMYND15 gene

International audienceThanks to tremendous advances in sequencing technologies and in particular to whole exome sequencing (WES), many genes have now been linked to severe sperm defects. A precise genetic diagnosis is obtained for a minority of patients and only for the most severe defects like azoospermia or macrozoospermia which is very often due to defects in the aurora kinase C (AURKC) gene. Here, we studied a subject with a severe oligozoospermia and a phenotypic diagnosis of macrozoospermia. AURKC analysis did not reveal any deleterious variant. WES was then initiated which permitted to identify a homozygous loss of function variant in the zinc finger MYND-type containing 15 (ZMYND15) gene. ZMYND15 has been described to serve as a switch for haploid gene expression, and mice devoid of ZMYND15 were shown to be sterile due to nonobstructive azoospermia (NOA). In man, ZMYND15 has been associated with NOA and severe oligozoospermia. We confirm here that the presence of a bi-allelic ZMYND15 variant induces a severe oligozoospermia. In addition, we show that severe oligozoospermia can be associated macrozoospermia, and that a phenotypic misdiagnosis is possible, potentially delaying the genetic diagnosis. In conclusion, genetic defects in ZMYND15 can induce complete NOA or severe oligozoospermia associated with a very severe teratozoospermia. In our experience, severe oligozoospermia is often associated with severe teratozoospermia and can sometimes be misinterpreted as macrozoospermia or globozoospermia. In these instances, specific AURKC or dpy-19 like 2 (DPY19L2) diagnosis is usually negative and we recommend the direct use of a pan-genomic techniques such as WES

Allemagne 2001

Confronté au double défi de l'unification et de la mondialisation, le « modèle rhénan » semblait s'être progressivement englué dans ses pesanteurs et ses rigidités. Pourtant, le gouvernement Schröder est parvenu à sortir l'Allemagne de sa torpeur. Réforme fiscale, privatisations, modernisation bancaire et financière, épargne-retraite, flexibilité du travail, gouvernance sociale,... - aucun domaine n'échappe à de profondes mutations. Une nouvelle dynamique collective de modernisation a saisi pouvoirs publics, acteurs économiques, partenaires sociaux ; elle se fonde sur une acceptation raisonnée du changement. Dans un contexte en pleine évolution, l'Allemagne réagit et tente de libérer sa compétitivité, ses forces d'innovation et de croissance. L'impératif réformateur est triple : la globalisation et l'intégration croissante de l'Union européenne exigent l'adaptation des structures et de la réglementation ; la « révolution technologique » force à la réorganisation des activités et des règles du jeu ; la normalité nouvelle de l'Allemagne réunifiée appelle la redéfinition des fondements mêmes du « modèle rhénan ». Le défi que relève actuellement la société allemande dépasse l'horizon mental national. C'est avant tout un choix européen assumé. Parce que le site Allemagne est au cœur de l'Europe, il ne peut échapper à la modernisation : de son dynamisme retrouvé dépendra la compétitivité de l'espace économique européen dans son ensemble, et donc celle de chacun des Etats membres. En révélant le haut degré d'interdépendance des économies nationales au sein de la zone euro, l'avènement de la monnaie unique en a forcé la prise de conscience collective. Tout autant que la globalisation ou la « nouvelle économie », le vrai levier des mutations que connaît l'économie allemande n'est autre que la réalité vécue et acceptée de l'Europe économique et monétaire. En cela, l'Allemagne est peut-être en train de reprendre une longueur d'avance. Cet ouvrage est le fruit d'un travail collectif issu de l'activité d'étude et d'observation suivies de l'équipe du CIRAC et de son réseau, rassemblés autour de la revue Regards sur l'économie allemande. Les auteurs des contributions sont des spécialistes réputés, de jeunes chercheurs et experts, ainsi que des journalistes spécialisés

Le modèle social allemand en mutation

Le modèle social allemand est en crise. L’hypertrophie de l’Etat social inhibe la dynamique de croissance de la première économie européenne. Mais sans une économie performante et prospère, comment continuer à assurer une protection sociale satisfaisante et équitable ? Depuis 2003, le gouvernement Schröder et les partenaires sociaux ont commencé à relever ce défi. Garants de l’Etat social, les pouvoirs publics ont entrepris d’en infléchir les normes, réformant le système d’indemnisation chômage (lois « Hartz IV ») et revalorisant l’apport individuel à l’assurance-maladie ou aux régimes de retraite. Responsables de la régulation du travail et co-gestionnaires de la protection sociale, les partenaires sociaux mènent une politique salariale dont la nouvelle approche globale cherche à concilier la compétitivité des entreprises et la solidarité collective. Mais ces inflexions ne suffisent pas. Les mutations induites par la globalisation des activités ont fragilisé les bases de ce libéralisme organisé qui est au cœur du modèle économique et social allemand. L’Allemagne ne doit pas seulement lever les « rigidités » de son marché de l’emploi et assurer le financement des régimes sociaux mis à mal par le vieillissement démographique. Elle doit aussi refonder son contrat social et trouver un nouvel équilibre entre le marché et la solidarité, entre la promotion de l’activité et la redistribution, entre les droits et devoirs respectifs de l’individu et de la collectivité. C’est là un processus collectif complexe qui associe les pouvoirs publics, les partenaires sociaux et l’opinion. Car les réformes en cours impliquent aussi un débat sur les valeurs qui fonderont les nouveaux choix de société allemands et assureront le maintien de la performance économique du pays dans le cadre d’une globalisation assumée. L’acceptation du changement se nourrit également de l’engagement européen réaffirmé. C’est dans la perspective d’une Europe compétitive dans l’économie du savoir que l’Allemagne inscrit la refondation de son modèle économique et social

Genetic causes of macrozoospermia and proposal for an optimized genetic diagnosis strategy based on sperm parameters

International audienc

Identification of a new recurrent Aurora kinase C mutation in both European and African men with macrozoospermia.

International audienceSTUDY QUESTION: Can we identify new sequence variants in the aurora kinase C gene (AURKC) of patients with macrozoospermia and establish a genotype-phenotype correlation? SUMMARY ANSWER: We identified a new non-sense mutation, p.Y248*, that represents 13% of all mutant alleles. There was no difference in the phenotype of individuals carrying this new mutation versus the initially described and main mutation c.144delC. WHAT IS KNOWN ALREADY: The absence of a functional AURKC gene causes primary infertility in men by blocking the first meiotic division and leading to the production of tetraploid large-headed spermatozoa. We previously demonstrated that most affected men were of North African origin and carried a homozygous truncating mutation (c.144delC). STUDY DESIGN, SIZE, DURATION: This is a retrospective study carried out on patients consulting for infertility and described as having >5% large-headed spermatozoa. A total of 87 patients are presented here, 43 patients were published previously and 44 are new patients recruited between January 2008 and December 2011. PARTICIPANTS/MATERIALS, SETTING, METHODS: All patients consulted for primary infertility in fertility clinics in France (n = 44), Tunisia (n = 30), Morocco (n = 9) or Algeria (n = 4). Sperm analysis was carried out in the recruiting fertility clinics and all molecular analyses were performed at Grenoble teaching hospital. DNA was extracted from blood or saliva and the seven AURKC exons were sequenced. RT-PCR was carried out on transcripts extracted from leukocytes from one patient homozygous for p.Y248*. Microsatellite analysis was performed on all p.Y248* patients to evaluate the age of this new mutation. MAIN RESULTS AND THE ROLE OF CHANCE: We identified a new non-sense mutation, p.Y248*, in 10 unrelated individuals of European (n = 4) and North African origin (n = 6). We show that this new variant represents 13% of all mutant alleles and that the initially described c.144delC variant accounts for almost all of the remaining mutated alleles (85.5%). No mutated transcripts could be detected by RT-PCR suggesting a specific degradation of the mutant transcripts by non-sense mediated mRNA decay. A rare variant located in the 3' untranslated region was found to strictly co-segregate with p.Y248*, demonstrating a founding effect. Microsatellite analysis confirmed this linkage and allowed us to estimate a mutational age of between 925 and 1325 years, predating the c.144delC variant predicted by the same method to have arisen 250-650 years ago. Patients with no identified AURKC mutation (n = 15) have significantly improved parameters in terms of vitality and concentration of normal spermatozoa, and a decreased rate of spermatozoa with a large head and multiple flagella (P < 0.001). LIMITATIONS, REASONS FOR CAUTION: Despite adherence to the World Health Organization guidelines, large variations in most characteristic sperm parameters were observed, even for patients with the same homozygous mutation. We believe that is mainly related to inter-laboratory variability in sperm parameter scoring. This prevented us from establishing clear-cut values to indicate a need for molecular analysis of patients with macrozoospermia. WIDER IMPLICATIONS OF THE FINDINGS: This study confirms yet again the importance of AURKC mutations in the aetiology of macrozoospermia. Although a large majority of patients are of North African origin, we have now identified European patients carrying a new non-sense mutation indicating that a diagnosis of absence of a functional AURKC gene should not be ruled out for non-Magrebian individuals. Indirect evidence indicates that AURKC might be playing a role in the meiotic spindle assembly checkpoint (SAC) during meiosis. We postulate that heterozygous men might have a more relaxed SAC leading to a more abundant sperm production and a reproductive advantage. This could be the reason for the rapid accumulation of the two AURKC mutations we observe in North African individuals. STUDY FUNDING/COMPETING INTEREST(S): None of the authors have any competing interest. This work is part of the project 'Identification and Characterization of Genes Involved in Infertility (ICG2I)' funded by the programme GENOPAT 2009 from the French Research Agency (ANR)