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GE2R        

GE2R

Genome Editing, DNA double-strand break Repair and cellular Responses


Research overview

Genome editing with custom nucleases is now a major strategy for investigating genome organization and gene function, creating animal models of disease and improving gene therapy. Genetic studies were now possible in virtually all forms of life accessible to experimental manipulation. Modification of the genome sequence takes place during DNA repair of the double-strand break (DSB) induced by the nuclease. Many pathways exist for repair of DSBs and our team is interested in studying their roles in genome editing as well as in different biological situations taking advantage of the novel opportunities raised by programmable nucleases.

Our projects are focused on the development of artificial nucleases with two major objectives:

  • improvement of genome editing strategies in different biological systems
  • and study of double-strand break (DSB) repair


People involved

  • Carine Giovannangeli, DR CNRS
  • Jean-Paul Concordet, CR Inserm
  • Danièle Praseuth, MC Museum
  • Laureline Roger, MC Museum
  • Anne De Cian, IR Inserm
  • Loïc Perrouault, IE Museum
  • Charlotte Boix, AI Inserm
  • Alice Brion, IE-MNHN
  • Khadija Lamribet, IE-INSERM
  • Ahmed Kheder, PhD student (CIFRE with Sanofi)
  • Sylvain Geny, Post-Doc
  • Mathieu Carrara, PhD student (Co-direction G. Pezzeron UMR 7221, MNHN)
  • Elaheh Hosseini, PhD student (University of Teheran)
  • Litchy Boueya, Master 1 student
  • Marwan Anoud, Master 2 student (January 2018)

 

Former Team Member

  • Erika Brunet, CR Inserm
  • Loelia Babin, PhD Student
  • Sonia Dubois, Post-Doc
  • Jean-Baptiste-Renaud, IE-CDD
  • Marine Charpentier, PhD Student
  • Armêl Millet, PhD student

Genome editing methods and double-strand break repair

Among the DSB repair pathways, canonical non-homologous end-joining (cNHEJ) and alternative end-joining pathways (altEJ) such as micro-homology-mediated end-joining (MMEJ) proceed by ligation of DNA ends and result in targeted and generally unpredictable indels, small insertions or deletions. In contrast, if homologous donor DNA is provided as a repair template, homology-dependent repair (HDR) can direct precise genome editing by sequence integration (also called, KI for knock-in). Depending on the type of sequence change wanted, for example correction of a gene mutation or integration of a transgene, different strategies are possible, with varying precision and efficiency. A central goal of our team is to increase the efficiency of genome editing in different experimental contexts and to better understand and control the mechanisms of DSB repair involved.

These last few years, we developed some gene editing tools and successfully used them for optimization of gene KOs in various animal models (1,2,3), this expertise being now transferred to the TACGENE facility. We also work on improvements of sequence integration following DSB induction with custom nucleases, mainly by exploiting and manipulating DSB repair pathways (4,5,6).

Image

Figure 1: Programmed sequence modifications with CRISPR-Cas9 systems

Mechanisms and functions of DSB repair

Using genome editing methods to mimic some genomic rearrangements, we succeeded to elucidate the DSB repair pathways involved in chromosomal translocations (7). and in mitochondrial DNA deletions (8). We also developed a biochemical analysis of reconstituted and functional DSB repair complexes in vitro and identified the DNA ends proteome by semi-quantitative mass spectrometry (9).

Our on-going projects focus on alternative end-joining mechanisms. Among repair pathways for double-strand breaks (DSBs), an alternative end-joining pathway called MMEJ, for microhomology-mediated end-joining, appears to play a predominant role in repair of DSBs inflicted by artificial nucleases. Its main characteristics are that repair involves annealing of short sequences flanking the break, called microhomologies, and that DNA polymerase θ is necessary. In order to gain novel insights into the MMEJ pathway, we want to identify novel DNA polymerase θ partners and characterize MMEJ regulation and physiological functions by investigating its role in DSB repair at specific genomic sites and in model organisms.

Image

Figure 2: DSB repair pathways choice

We have chosen to expand our studies of DNA repair to tardigrades, a novel in vivo model organism for tolerance to extreme environments. Tardigrades are resistant to a great variety of harsh environments, including conditions that induce high levels of DNA damage. The study of tardigrades may therefore pave the way to identification of new proteins and pathways that handle DNA damages. We propose to examine the molecular basis of tardigrade genome tolerance by identification and analysis of candidate genes of DNA repair pathways.

Image

Figure 3: Hypsibius dujardini tardigrade with oocytes and embryos at various cell stages (DIC and Dapi staining)

Main collaborations

  • Maximilian Haeussler, University of California at Santa Cruz (CRISPOR website)
  • Bernard Lopez, Paris (DNA repair)
  • Anna Buj-Bello, Genethon, Evry (gene therapy)
  • Tsuyoshi Momose, UMR7009, Villefranche s/ Mer (Genome editing and repair in Clitya jellyfish)
  • Arnaud Poterszman, IGBMC, Strasbourg (NER repair)
  • Jean-Michel Itier, Sanofi, Vitry (genome editing in iPS cells)
  • Maxime Dahan, ENS Paris (DNA imaging with CRISPR)
  • Ignacio Anegon, TRIP - UMR1064, Nantes (Genome editing improvement in rat)
  • Filippo Del Bene, Institut Curie, Paris (Genome editing improvement in zebrafish)
  • Erika Brunet, Imagine, Paris (Chromosomal translocations)
  • Hervé Tostivint, Guillaume Pezeron, UMR 7221, MNHN (Genome editing in zebrafish)
  • Filipo Rusconi, CNRS, Orsay (Mass spectrometry, proteomics)


Selected references


1. Haeussler M., Schönig K., Eckert H., Eschstruth A., Mianné J., Renaud J-B., Schneider-Maunoury S., Shkumatava A., Teboul L., Kent J., Joly J-S. & Concordet J-P. (2016) Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome Biol. 17(1):148.
2. Di Donato V., De Santis F., Auer T-O., Testa N., Sánchez-Iranzo H., Mercader N., Concordet J-P. & Del Bene F. (2016) 2C-Cas9: a versatile tool for clonal analysis of gene function. Genome Res. 26,5:681-92.
3. Auer, T-O., Duroure, K., Concordet, J-P. & Del Bene, F. (2014) CRISPR/Cas9-mediated conversion of eGFP- into Gal4-transgenic lines in zebrafish. Nat Protoc. 9,12: 2823-40.
4. Renaud J-B., Boix C., Charpentier M., De Cian A., Cochennec J., Duvernois-Berthet E., Perrouault L., Tesson L., Edouard J., Thinard R., Cherifi Y., Menoret S., Fontanière S., de Crozé N., Fraichard A., Sohm F., Anegon I., Concordet J-P. & Giovannangeli C. (2016) Improved Genome Editing Efficiency and Flexibility Using Modified Oligonucleotides with TALEN and CRISPR-Cas9 Nucleases. Cell Reports. 14,9:2263-72.
5. Ménoret, S., De Cian, A., Tesson, L., Remy, S., Usal, C., Boulé, J-B., Boix, C., Fontanière, S., Crénéguy, A., Nguyen, T-H., Brusselle, L., Thinard, R., Gauguier, D., Concordet, J-P., Cherifi, Y., Fraichard, A., Giovannangeli, C. & Anegon, I.  (2015) Homology-directed repair in rodent zygotes using Cas9 and TALEN engineered proteins. Sci Rep. 7(5):14410. 
6. Rémy, S., Tesson, L., Ménoret, S., Usal, C., De Cian, A., Thepenier, V., Thinard, R., Baron, D., Charpentier, M., Renaud, J-B., Buelow, R., Cost, G-J., Giovannangeli, C., Fraichard, A., Concordet, J-P. & Anegon, I. (2014) Efficient gene targeting by homology-directed repair in rat zygotes using TALE nucleases. Genome Res. 24,8: 1371-83.
7. Ghezraoui, H., Piganeau, M., Renouf, B., Renaud, J-B., Sallmyr, A., Ruis, B., Oh, S., Tomkinson, A-E., Hendrickson, E-A., Giovannangeli, C., Jasin, M. & Brunet, E. (2014) Chromosomal translocations in human cells are generated by canonical nonhomologous end-joining. Mol Cell. 55, 6: 829-42.
8. Phillips A-F., Millet A-R., Tigano M., Dubois S-M., Crimmins H., Babin L., Charpentier M., Piganeau M., Brunet E &, Sfeir A. (2017)Single-Molecule Analysis of mtDNA Replication Uncovers the Basis of the Common Deletion. Mol Cell. 65(3):527-538 e526.
9. Berthelot V., Mouta-Cardoso G., Hégarat N., Guillonneau F., François J-C., Giovannangeli C., Praseuth D. & Rusconi F. (2016) The human DNA ends proteome uncovers an unexpected entanglement of functional pathways. Nucleic Acids Res. 44,10:4721-33.

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