RESEARCH OVERVIEW

Eukaryotic cells have developed diverse repair pathways to resolve DNA damage and the faithful repair is crucial to maintaining genome integrity.  On the other hand, genome editing with custom nucleases take advantage of these pathways to produce permanent genetic changes and is now a major strategy for investigating genome organization and gene function, creating animal models of disease and improving gene therapy. Our team is interested in studying DSB repair pathways, 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 three major objectives:

• improvement of precise genome editing strategies by modulation of DNA repair
• study of the mechanisms and functions of Microhomology-Mediated End-Joining
• characterization of adaptation of DNA repair to extreme environmental conditions in tardigrades

​People involved

• Carine Giovannangeli, DR CNRS
• Jean-Paul Concordet, CR Inserm
• Danièle Praseuth, MC Museum
• Quentin Helleu, MC Museum
• Anne De Cian, IR Inserm
• Alice Brion, IE Museum
• Khadija Lamribet, IE Inserm
• Marie As, CDD IE Inserm
• Mathieu Carrara, Post-Doc Inserm
• José-Roberto Alvarez Vargas, PhD Student
• Gaia Gentile, CDD IE

Former Team Members

• Lauréline Roger, MC Museum
• Mathieu Carrara, Doctorant (Co-direction G. Pezeron UMR 7221, MNHN)
• Marwan Anoud, Doctorant
• Fatemeh Rabbani, Doctorante (Université Tarbiat Modares, Téhéran, Iran)
• Sarah Hirèche-Proteau M2 Univ Paris
• Juliette Bonillo, M1
• Loïc Perrouault, IE Museum
• Ahmed Kheder, PhD student
• Sylvain Geny, Post-Doc
• 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 DNA DOUBLE-STRAND BREAK REPAIR

Cells have deployed the capacity to deal with DSBs and genome editing, first introducing a targeted lesion, further relies on cellular DNA repair to generate genome modification. The ultimate goal of genome editing is to produce efficiently a precise edit and this remains difficult in many biological systems including clinically relevant ones. Indeed 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) are generally efficient but result in diverse outcomes; in contrast homology-dependent repair (HDR) that is involved if an homologous donor DNA is provided as a repair template, in addition to the nuclease, can generate precise edits but is often inefficient. A central goal of our team is to increase the efficiency of precise 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 biological systems, including various animal models for functional studies ^(1,2,3),and human cells for gene therapy approaches ⁽⁴⁾. This expertise has been 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 ^{(5,6,7,8,9, 15)}

Figure 1 : Programmed sequence modifications with CRISPR-Cas9 systems

Credits

©C-Giovannangeli

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 mitochondrial DNA deletions ⁽¹⁰⁾ and in chromosomal translocations ⁽¹¹⁾ and to characterize associated cellular responses ⁽¹²⁾. 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 ⁽¹³⁾.

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 ⁽¹⁴⁾ and is shown to be essential for some physiological functions, such as the repair of double strand breaks during embryogenesis. 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 further characterize MMEJ components, regulations and physiological functions, by investigating its role in DSB repair at specific genomic sites and in model organisms.

Figure 2 : DSB repair pathways choice

Credits

©L.Roger

ADAPTATION OF DNA REPAIR TO EXTREME ENVIRONMENTAL CONDITIONS IN TARDIGRADES

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 in different tardigrades species, using functional and comparative genomics approaches.

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

Credits

©A.De-Cian

Main collaborations

• Maximilian Haeussler, University of California at Santa Cruz (CRISPOR website)
• Bernard Lopez, Institut Cochin/CNRS-Inserm, Paris (DNA repair)
• Anna Buj-Bello/Mario Amendola, Genethon/Inserm, Evry (gene therapy)
• Annarita Miccio, Imagine/Inserm-CNRS, paris
• Tsuyoshi Momose, SU-CNRS, Villefranche s/ Mer (Genome editing and repair in Clitya jellyfish)
• Arnaud Poterszman, IGBMC/CNRS-Inserm, Strasbourg (NER repair)
• Jean-Michel Itier, Sanofi, Vitry (genome editing in iPS cells)
• Antoine Coulon, Institut Curie/CNRS-Inserm (DNA imaging with CRISPR)
• Ignacio Anegon, TRIP/Inserm, Nantes (Genome editing improvement in rat)
• Filippo Del Bene, Institut de la Vision/Inserm, Paris (Genome editing improvement in zebrafish)
• Erika Brunet, Imagine/Inserm-CNRS, Paris (Chromosomal translocations)
• Hervé Tostivint, Guillaume Pezeron, MNHN/CNRS (Genome editing and MMEJ in zebrafish)
• Saman Hosseinkhani, Tarbiat Modares University, Iran (Apoptosome reporter cells)
• Roberto Guidetti, University of Modena, Italie (tardigrade biology)

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. Weber L., Frati G., Felix T., Hardouin G., Casini A., Wollenschlaeger C., Meneghini V., Masson C., De Cian A., Chalumeau A., Mavilio F., Amendola M., Andre-Schmutz I., Cereseto A., El Nemer W., Concordet, J.-P., Giovannangeli, C., Cavazzana, M. & Miccio, A. (2020) Editing a γ-globin repressor binding site restores fetal hemoglobin synthesis and corrects the sickle cell disease phenotype. Sci Adv. 2020 Feb 12;6(7):eaay9392. doi: 10.1126/sciadv.aay9392.

5. 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.

6. 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. 

7. 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.

8. Charpentier M., Khedher A.H.Y., Menoret S., Brion A., Lamribet K., Dardillac E., Boix C., Perrouault L., Tesson L., Geny S., De Cian A., Itier J.-M., Anegon I., Lopez B., Giovannangeli C. & Concordet J.-P. (2018) CtIP fusion to Cas9 enhances transgene integration by homology-dependent repair. Nat Commun. 9, 1133. 

9. Elaheh Sadat Hosseini, Maryam Nikkhah, Amir Ali Hamidieh, Howard O Fearnhead, Jean-Paul Concordet, and Saman Hosseinkhani (2020)Lumiptosome, an Engineered Luminescent Form of Apoptosome Reports Cell Death Through the Same Apaf-1 Dependent Pathway. J Cell Sci, 133(10):jcs242636.  doi:10.1242/jcs.242636.

10. 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.

11. 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.

12. Babin L, Piganeau M, Renouf B, Lamribet K, Thirant C, Deriano L, Mercher T, Giovannangeli C & Brunet EC. (2018) Chromosomal Translocation Formation Is Sufficient to Produce Fusion Circular RNAs Specific to Patient Tumor Cells. iScience. 2018 Jul 27;5:19-29. doi: 10.1016/j.isci.2018.06.007.

13. 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.

14. Momose T, De Cian A, Shiba K, Inaba K, Giovannangeli C & Concordet J-P. (2018) High doses of CRISPR/Cas9 ribonucleoprotein efficiently induce gene knockout with low mosaicism in the hydrozoan Clytia hemisphaerica through microhomology-mediated deletion.Sci Rep. 2018 Aug 6;8(1):11734. doi: 10.1038/s41598-018-30188-0.

15. Geny S, Pichard S, Brion A, Renaud JB, Jacquemin S, Concordet JP, Poterszman A. Tagging Proteins with Fluorescent Reporters Using the CRISPR/Cas9 System and Double-Stranded DNA Donors. Methods Mol Biol. 2021;2247:39-57. doi: 10.1007/978-1-0716-1126-5_3.