CRISPR/Cas9 system characterization and genome editing improvement for genetically modified human iPS cells and rat embryos generation

CRISPR/Cas9 a révolutionné l’édition de génome en l’élargissant à des espèces animales et des types cellulaires variés. Les nombreuses études sur CRISPR permettent une bonne compréhension générale du système (études cristallographiques, enzymatiques et mécanistiques…). Toutefois, il reste encore de nombreux aspects à étudier pour une meilleure maîtrise de l’édition de génome et une meilleure efficacité de génération de modèles génétiquement modifiés, même pour les plus complexes. Ces derniers nécessitent l’insertion d’une large séquence ADN (cassette d’expression, gène rapporteur, système conditionnel…) ou plusieurs séquences contenant une structure particulièrement complexe (double insertion de sites loxP flanquant un exon…) qui diminuent fortement l’efficacité de recombinaison homologue requise pour leur génération. J’ai donc caractérisé la base moléculaire du système CRISPR/Cas9. En particulier, j’ai étudié la formation du complexe RNP par différentes techniques notamment la « Nano Differential Scanning Fluorimetry » et sa fonctionnalité clivage in vitro. Ces études m’ont permises de démontrer que, dans des conditions optimales de formation de ce complexe (tampon, Cas9, guide ARN…), un ratio Cas9 protéine /dual guide ARN 1/1 est suffisant. J’ai donc appliqué ce principe aux cellules iPS humaines et embryons de rats pour confirmer ces résultats et déterminer in vivo les concentrations optimales de Cas9 et court donneur ADN afin d’obtenir les meilleurs taux d’insertion d’une courte séquence simples. Cette preuve de concept nous a permis d’obtenir plus de 50% de succès dans les deux modèles utilisés. En parallèle, nous avons tenté deux approches d’import d’un donneur ADN via le guide ARN qui ne nous ont pas permis d’améliorer la génération de nos modèles mais d’autres stratégies de ce type sont en cours. L’ensemble de ces résultats permettra un emploi de CRISPR/Cas9 plus maîtrisée et ouvre la voie à une réflexion sur l’optimisation de l’utilisation des longs donneurs ADN pour la génération de modèles complexes de façons plus efficaces et maîtrisé.CRISPR/Cas9 has revolutionized genome editing by spreading human modeling to a large variety of species and types of cells. The numerous studies of this system have accelerated its understanding, in particular crystallography, enzymatic and mechanistic studies. Nevertheless, a large number of aspects remained to be studied for better control of genome editing and for improved genetically modified model’s generation efficacy, especially for complex models. To generate those ones, insertion of large DNA sequence (expression cassette, reporter, conditional system…) or multiple sequences with complex structure (double insertion of loxP sites flanking exons…) is required. Those particularities reduce homologous recombination used for their generation. Thus, I have characterized the molecular basis of CRISPR/Cas9 system. In particular, I have studied RNP complex formation by different technics, in particular “Nano Differential Scanning Fluorimetry”, and its functionality by in vitro cleavage. I have demonstrated that using optimal RNP complex formation conditions (buffer, Cas9, guide RNA...) Cas9 protein/dual guide RNA ratio 1/1 is enough. We have next applied those settings to human iPS cells and rat embryos to confirm them in vivo. We also determine optimal Cas9 and short DNA donnor concentrations to improve insertion efficiency. This proof of concept allowed us to reach more than 50% of succes for both models. In parrallel, we tested two unsuccessful strategies to import the DNA donor to the nucleus via the guide RNA. Other strategies are ongoing. All these results gives new insight into CRISPR/Cas9 usage to better master this tool. Moreover, it opened new reflexion for optimal large DNA donor usage for complex models generation with high efficacy and better control

Advances in transgenic animal models and techniques

International audienceOn May 11th and 12th 2017 was held in Nantes, France, the international meeting "Advances in transgenic animal models and techniques" ( http://www.trm.univ-nantes.fr/ ). This biennial meeting is the fifth one of its kind to be organized by the Transgenic Rats ImmunoPhenomic (TRIP) Nantes facility ( http://www.tgr.nantes.inserm.fr/ ). The meeting was supported by private companies (SONIDEL, Scionics computer innovation, New England Biolabs, MERCK, genOway, Journal Disease Models and Mechanisms) and by public institutions (International Society for Transgenic Technology, University of Nantes, INSERM UMR 1064, SFR François Bonamy, CNRS, Région Pays de la Loire, Biogenouest, TEFOR infrastructure, ITUN, IHU-CESTI and DHU-Oncogeffe and Labex IGO). Around 100 participants, from France but also from different European countries, Japan and USA, attended the meeting

Immunological characterization of dystrophin-deficient Dmd[mdx] rats

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Transgenic animals and genetic engineering techniques. Nantes, France, 2–3 July, 2015

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A Rapid and Cost-Effective Method for Genotyping Genome-Edited Animals: A Heteroduplex Mobility Assay Using Microfluidic Capillary Electrophoresis

International audienceThe recent emergence and application of engineered endonucleases have led to the development of genome editing tools capable of rapidly implementing various targeted genome editions in a wide range of species. Moreover, these novel tools have become easier to use and have resulted in a great increase of applications. Whilst gene knockout (KO) or knockin (KI) animal models are relatively easy to achieve, there is a bottleneck in the detection and analysis of these mutations. Although several methods exist to detect these targeted mutations, we developed a heteroduplex mobility assay on an automated microfluidic capillary electrophoresis system named HMA-CE in order to accelerate the genotyping process. The HMA-CE method uses a simple PCR amplification of genomic DNA (gDNA) followed by an automated capillary electrophoresis step which reveals a heteroduplexes (HD) signature for each mutation. This allows efficient discrimination of wild-type and genome-edited animals down to the single base pair level

Immunophenotype of a Rat Model of Duchenne's Disease and Demonstration of Improved Muscle Strength After Anti-CD45RC Antibody Treatment

International audienceCorticosteroids (CS) are standard therapy for the treatment of Duchenne's muscular dystrophy (DMD). Even though they decrease inflammation, they have limited efficacy and are associated with significant side effects. There is therefore the need for new protolerogenic treatments to replace CS. Dystrophin-deficient rats (Dmd mdx ) closely resemble the pathological phenotype of DMD patients. We performed the first Immunophenotyping of Dmd mdx rats and showed leukocyte infiltration in skeletal and cardiac muscles, which consisted mostly of macrophages and T cells including CD45RChigh T cells. Muscles of DMD patients also contain elevated CD45RChigh T cells. We treated Dmd mdx rats with an anti-CD45RC MAb used in previous studies to deplete CD45RChigh T cells and induce immune tolerance in models of organ transplantation. Treatment of young Dmd mdx rats with anti-CD45RC MAb corrected skeletal muscle strength and was associated with depletion of CD45RChigh T cells with no side effects. Treatment of young Dmd mdx rats with prednisolone resulted in increase in skeletal muscle strength but also severe growth retardation. In conclusion, anti-CD45RC MAb treatment has potential in the treatment of DMD and might eventually result in reduction or elimination of CS use

In vivo analysis of human immune responses in immunodeficient rats.

International audienceBACKGROUND:Humanized immune system immunodeficient mice have been extremely useful for the in vivo analyses of immune responses in a variety of models, including organ transplantation and GVHD but they have limitations. Rat models are interesting complementary alternatives presenting advantages over mice, such as their size and their active complement compartment. Immunodeficient rats have been generated but human immune responses have not yet been described.METHODS:We generated immunodeficient RRGS rats (for Rat Rag-/- Gamma chain-/- hSIRPa-positive) combining Rag1 and Il2rg deficiency with the expression of human SIRPalpha, a negative regulator of macrophage phagocytosis allowing repression of rat macrophages by human CD47-positive cells. We then immune humanized RRGS animals with human PBMCs to set up a human acute GVHD model. Treatment of GVHD was done with a new porcine anti-human lymphocyte serum (LIS1) active through complement-dependent cytotoxicity. We also established a tumor xenograft rejection model in these human PBMCs immune system RRGS animals by subcutaneous implantation of a human tumor cell line.RESULTS:RRGS animals receiving human PBMCs showed robust and reproducible reconstitution, mainly by T and B cells. A dose-dependent acute GVHD process was observed with progressive weight loss, tissue damage and death censoring. LIS1 antibody completely prevented acute GVHD. In the human tumor xenograft model, detectable tumors were rejected upon hPBMCs injection.CONCLUSIONS:Human PBMC can be implanted in RRGS animals and elicit acute GVHD or rejection of human tumor cells and these are useful models to test new immunotherapies

Excess of guide RNA reduces knockin efficiency and drastically increases on-target large deletions

YC and VC were genOway employees. The other authors declare no conflict of interest.International audienceCRISPR-Cas9 cleavage efficacy and accuracy are the main challenges gene editing faces, and they are particularly affected by the optimal formation of the ribonucleoprotein (RNP) complex. We used nano differential scanning fluorimetry, a label and immobilization-free assay, to demonstrate that an equimolar ratio of Cas9 and guide RNA (gRNA) is optimal for RNP complex formation. We almost achieved 50% of green fluorescent protein (GFP) to blue fluorescent protein (BFP) conversion using a biallelic homozygous GFP human induced pluripotent stem cell line, when 0.4 μM of Cas9, equimolar Cas9/gRNA ratio and 2 μM of single-stranded oligonucleotide, were used and showed that increasing Cas9/gRNA ratio did not further improve KI efficiency. Additionally, excess gRNA decreased point mutation KI efficiency in rat embryos and drastically increased the occurrence of on-target large deletions. These findings highlight the importance of CRISPR/Cas9 stoichiometric optimization to ensure efficient and accurate KI generation, which will be applicable to other in vitro as well as in vivo models

Comparative Analysis of piggyBac, CRISPR/Cas9 and TALEN Mediated BAC Transgenesis in the Zygote for the Generation of Humanized SIRPA Rats

International audienceBAC transgenic mammalian systems offer an important platform for recapitulating human gene expression and disease modeling. While the larger body mass, and greater genetic and physiologic similarity to humans render rats well suited for reproducing human immune diseases and evaluating therapeutic strategies, difficulties of generating BAC transgenic rats have hindered progress. Thus, an efficient method for BAC transgenesis in rats would be valuable. Immunodeficient mice carrying a human SIRPA transgene have previously been shown to support improved human cell hematopoiesis. Here, we have generated for the first time, human SIRPA BAC transgenic rats, for which the gene is faithfully expressed, functionally active, and germline transmissible. To do this, human SIRPA BAC was modified with elements to work in coordination with genome engineering technologies-piggyBac, CRISPR/Cas9 or TALEN. Our findings show that piggyBac transposition is a more efficient approach than the classical BAC transgenesis, resulting in complete BAC integration with predictable end sequences, thereby permitting precise assessment of the integration site. Neither CRISPR/Cas9 nor TALEN increased BAC transgenesis. Therefore, an efficient generation of human SIRPA transgenic rats using piggyBac opens opportunities for expansion of humanized transgenic rat models in the future to advance biomedical research and therapeutic applications

Immunological characterization of a rat model of Duchenne’s disease and increase in muscle strength after anti-CD45RC antibody treatment

International audienceIn this study, we phenotyped by flow cytometry and immunohistochemistry (data not shown) the immune cell subsets infiltrating Dmd mdx rat skeletal and cardiac muscles. Leukocyte infiltrates were absent or very low at 2 weeks of age, peaked at 4 and 8 weeks and decreased at 12 weeks. M2 macrophages represented >90% of infiltrating immune cells and Teff cells were the majority of the remaining ones. We also analyzed muscle enzymes and cytokines in sera. Creatin kinase was increased at weeks 4 and 8 and decreased at week 12 and thereafter (data not shown). This results are consistent with those observed in mdx mice model. Anti-CD45RC MAb treatment of young Dmd mdx rats normalized skeletal muscle strength associated to a depletion of effectors CD45RC high cells and no obvious side-effects. As a control prednisolone treatment of Dmd mdx rats similarly increased skeletal muscle strength and was also associated to a depletion of effectors CD45RC high cells but resulted in severe weight loss. Conclusion Duchenne Muscular Dystrophy (DMD) is a severe genetic muscle-wasting disorder due to the lack of dystrophin characterized by a progressive muscle weakness and a cardiomyopathy leading to premature death. The dystrophin-deficient Dmd mdx rats were generated using TALENs and offer a more reliable representation of human DMD, with marked muscle strength reduction, cardiomyopathy and muscle fibrosis that are higher that those observed in the mdx mouse model (1). A role for inflammation and autoimmune responses in muscle damages was shown both in DMD patients and the mdx mouse model (2).In this study, we assessed by flow cytometry and immunohistochemistry the immune cell subsets infiltrating Dmd mdx rat skeletal and cardiac muscles especially immunoregulatory and pro-inflammatory subsets (M1 and M2 macrophages, CD4 + and CD8 + Teff or Tregs…).Then, we investigated the possibility of reducing disease in Dmd mdx rats by administrating immunomodulatory treatments. The standard therapy for DMD patients is corticoids that decrease inflammation and immune responses but with variable responses, limited efficacy and important and numerous side effects. Therefore, there is need for new anti-inflammatory and pro-tolerogenic treatments that could replace or decrease the doses of corticoids. Anti-CD45RC monoclonal antibody (MAb) treatment has induced immune tolerance in models of organ transplantation and GVHD