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4 results on '"Jean-Baptiste Renaud"'

1. Tagging Proteins with Fluorescent Reporters Using the CRISPR/Cas9 System and Double-Stranded DNA Donors

Author

Jean-Paul Concordet, Sophie Jacquemin, Sylvain Geny, Arnaud Poterszman, Simon Pichard, Alice Brion, Jean-Baptiste Renaud, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Structure et Instabilité des Génomes (STRING), Muséum national d'Histoire naturelle (MNHN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), and POTERSZMAN, Arnaud

Subjects
0301 basic medicine, Models, Molecular, Protein Conformation, Recombinant Fusion Proteins, Green Fluorescent Proteins, Gene Expression, Context (language use), Computational biology, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, 03 medical and health sciences, Structure-Activity Relationship, 0302 clinical medicine, Genome editing, CRISPR, Humans, Cloning, Molecular, Gene, CRISPR/Cas9, ComputingMilieux_MISCELLANEOUS, Double-stranded DNA donors, Gene Editing, Base Sequence, Drug discovery, Cas9, Chemistry, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, DNA, Flow Cytometry, Fluorescent protein, Protein subcellular localization prediction, 030104 developmental biology, HEK293 Cells, Microscopy, Fluorescence, 030220 oncology & carcinogenesis, Gene Targeting, Target protein, CRISPR-Cas Systems, Plasmids, RNA, Guide, Kinetoplastida
Abstract

International audience; Macromolecular complexes govern the majority of biological processes and are of great biomedical relevance as factors that perturb interaction networks underlie a number of diseases, and inhibition of protein-protein interactions is a common strategy in drug discovery. Genome editing technologies enable precise modifications in protein coding genes in mammalian cells, offering the possibility to introduce affinity tags or fluorescent reporters for proteomic or imaging applications in the bona fide cellular context. Here we describe a streamlined procedure which uses the CRISPR/Cas9 system and a double-stranded donor plasmid for efficient generation of homozygous endogenously GFP-tagged human cell lines. Establishing cellular models that preserve native genomic regulation of the target protein is instrumental to investigate protein localization and dynamics using fluorescence imaging but also to affinity purify associated protein complexes using anti-GFP antibodies or nanobodies.

Published
2021

2. Chromosomal Translocations in Human Cells Are Generated by Canonical Nonhomologous End-Joining

Author

Hind Ghezraoui, Jean Baptiste Renaud, Eric A. Hendrickson, Erika Brunet, Benjamin Renouf, Brian L. Ruis, Carine Giovannangeli, Alan E. Tomkinson, Marion Piganeau, Sehyun Oh, Maria Jasin, and Annahita Sallmyr

Subjects
DNA End-Joining Repair, DNA Ligases, Chromosomal translocation, Biology, LIG4, DNA-binding protein, Article, Translocation, Genetic, DNA Ligase ATP, Mice, Species Specificity, Tumor Cells, Cultured, CRISPR, Animals, Chromosomes, Human, Humans, Molecular Biology, Sequence Deletion, Genetics, chemistry.chemical_classification, Transcription activator-like effector nuclease, DNA ligase, Deoxyribonucleases, Breakpoint, fungi, Cell Biology, DNA repair protein XRCC4, DNA-Binding Proteins, enzymes and coenzymes (carbohydrates), chemistry, embryonic structures
Abstract

Breakpoint junctions of the chromosomal translocations that occur in human cancers display hallmarks of nonhomologous end-joining (NHEJ). In mouse cells, translocations are suppressed by canonical NHEJ (c-NHEJ) components, which include DNA ligase IV (LIG4), and instead arise from alternative NHEJ (alt-NHEJ). Here we used designer nucleases (ZFNs, TALENs, and CRISPR/Cas9) to introduce DSBs on two chromosomes to study translocation joining mechanisms in human cells. Remarkably, translocations were altered in cells deficient for LIG4 or its interacting protein XRCC4. Translocation junctions had significantly longer deletions and more microhomology, indicative of alt-NHEJ. Thus, unlike mouse cells, translocations in human cells are generated by c-NHEJ. Human cancer translocations induced by paired Cas9 nicks also showed a dependence on c-NHEJ, despite having distinct joining characteristics. These results demonstrate an unexpected and striking species-specific difference for common genomic rearrangements associated with tumorigenesis.

Published
2014
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3. Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR

Author

Jim Kent, Kai Schönig, Jean-Baptiste Renaud, Joffrey Mianné, Helene Eckert, Sylvie Schneider-Maunoury, Lydia Teboul, Jean-Stéphane Joly, Maximilian Haeussler, Alena Shkumatava, Alexis Eschstruth, Jean-Paul Concordet, Santa Cruz Genomics Institute, University of California [Santa Cruz] (UCSC), University of California-University of California, Central Institute of Mental Health, Medical Faculty Mannheim, Génétique et Biologie du Développement, Centre National de la Recherche Scientifique (CNRS)-Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC), Laboratoire de Biologie du Développement (LBD), Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Paris Seine (IBPS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Mary Lyon Centre, MRC, Institut des Neurosciences Paris-Saclay (NeuroPSI), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Morphogénèse du Cerveau des Vertébrés = Morphogenesis of the vertebrate brain (LBD-E10), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Paris Seine (IBPS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Structure et Instabilité des Génomes (STRING), Muséum national d'Histoire naturelle (MNHN)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), PERIGNON, Alain, University of California [Santa Cruz] (UC Santa Cruz), University of California (UC)-University of California (UC), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Biologie Paris Seine (IBPS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Biologie Paris Seine (IBPS), and Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
0301 basic medicine, Mutation rate, Bioinformatics, [SDV.NEU.NB]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology, [SDV.NEU.PC] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Psychology and behavior, Bioengineering, Biology, Genome, Promoter Regions, arn, 03 medical and health sciences, 0302 clinical medicine, Small Nuclear, Genetic, Genome editing, Information and Computing Sciences, RNA, Small Nuclear, Genetics, False positive paradox, CRISPR, Guide RNA, Kinetoplastida, Promoter Regions, Genetic, Selection (genetic algorithm), Gene Editing, Internet, [SDV.NEU.PC]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Psychology and behavior, Cas9, génome, Research, Human Genome, [SDV.NEU.NB] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology, [SDV.NEU.SC]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Cognitive Sciences, Biological Sciences, 030104 developmental biology, RNA, CRISPR-Cas Systems, Algorithm, Guide, Environmental Sciences, algorithme, 030217 neurology & neurosurgery, Algorithms, Software, [SDV.NEU.SC] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Cognitive Sciences, Biotechnology, RNA, Guide, Kinetoplastida
Abstract

Background The success of the CRISPR/Cas9 genome editing technique depends on the choice of the guide RNA sequence, which is facilitated by various websites. Despite the importance and popularity of these algorithms, it is unclear to which extent their predictions are in agreement with actual measurements. Results We conduct the first independent evaluation of CRISPR/Cas9 predictions. To this end, we collect data from eight SpCas9 off-target studies and compare them with the sites predicted by popular algorithms. We identify problems in one implementation but found that sequence-based off-target predictions are very reliable, identifying most off-targets with mutation rates superior to 0.1 %, while the number of false positives can be largely reduced with a cutoff on the off-target score. We also evaluate on-target efficiency prediction algorithms against available datasets. The correlation between the predictions and the guide activity varied considerably, especially for zebrafish. Together with novel data from our labs, we find that the optimal on-target efficiency prediction model strongly depends on whether the guide RNA is expressed from a U6 promoter or transcribed in vitro. We further demonstrate that the best predictions can significantly reduce the time spent on guide screening. Conclusions To make these guidelines easily accessible to anyone planning a CRISPR genome editing experiment, we built a new website (http://crispor.org) that predicts off-targets and helps select and clone efficient guide sequences for more than 120 genomes using different Cas9 proteins and the eight efficiency scoring systems evaluated here.

Published
2016

4. Efficient gene targeting by homology-directed repair in rat zygotes using TALE nucleases

Author

Jean-Baptiste Renaud, Laurent Tesson, Virginie Thepenier, Alexandre Fraichard, Gregory J. Cost, Marine Charpentier, Séverine Ménoret, Anne De Cian, Roland Buelow, Carine Giovannangeli, Claire Usal, Ignacio Anegon, Daniel Baron, Reynald Thinard, Jean-Paul Concordet, Séverine Remy, Centre de Recherche en Transplantation et Immunologie (U1064 Inserm - CRTI), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), Institut de transplantation urologie-néphrologie (ITUN), Université de Nantes (UN)-Centre hospitalier universitaire de Nantes (CHU Nantes), plate-forme Rat Transgenesis Immunophenomic (Inserm/CHU Nantes-IFR Santé François Bonamy), Structure fédérative de recherche François Bonamy (SFR François Bonamy), Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche en Santé de l'Université de Nantes (IRS-UN)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche en Santé de l'Université de Nantes (IRS-UN), Régulation et dynamique des génomes, Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Open Monoclonal Technology, Inc., Ligand Pharmaceuticals, Sangamo BioSciences [Richmond, CA, USA], Plate-forme genOway [Lyon], Region Pays de la Loire through Biogenouest, IBiSA program, Fondation Progreffe and TEFOR (Infrastructures d’Avenir of the French government)., and Le Bihan, Sylvie

Subjects
Male, Hypoxanthine Phosphoribosyltransferase, Microinjections, Zygote, Transgene, Method, Biology, Rats, Sprague-Dawley, Homology directed repair, chemistry.chemical_compound, Genetics, Animals, CRISPR, Cells, Cultured, Genetics (clinical), [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, Base Sequence, Cas9, Recombinational DNA Repair, Gene targeting, DNA Restriction Enzymes, Molecular biology, Recombinant Proteins, Genetically modified organism, chemistry, Gene Targeting, Female, Expression cassette, Rats, Transgenic, Genetic Engineering, DNA, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology
Abstract

The generation of genetically modified animals is important for both research and commercial purposes. The rat is an important model organism that until recently lacked efficient genetic engineering tools. Sequence-specific nucleases, such as ZFNs, TALE nucleases, and CRISPR/Cas9 have allowed the creation of rat knockout models. Genetic engineering by homology-directed repair (HDR) is utilized to create animals expressing transgenes in a controlled way and to introduce precise genetic modifications. We applied TALE nucleases and donor DNA microinjection into zygotes to generate HDR-modified rats with large new sequences introduced into three different loci with high efficiency (0.62%–5.13% of microinjected zygotes). Two of these loci (Rosa26 and Hprt1) are known to allow robust and reproducible transgene expression and were targeted for integration of a GFP expression cassette driven by the CAG promoter. GFP-expressing embryos and four Rosa26 GFP rat lines analyzed showed strong and widespread GFP expression in most cells of all analyzed tissues. The third targeted locus was Ighm, where we performed successful exon exchange of rat exon 2 for the human one. At all three loci we observed HDR only when using linear and not circular donor DNA. Mild hypothermic (30°C) culture of zygotes after microinjection increased HDR efficiency for some loci. Our study demonstrates that TALE nuclease and donor DNA microinjection into rat zygotes results in efficient and reproducible targeted donor integration by HDR. This allowed creation of genetically modified rats in a work-, cost-, and time-effective manner.

Published
2014

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