Impact of Microhomology Mediated End-Joining DNA repair pathway in vivo – i-MMEJ

In this project, we studied the importance of MMEJ/NHEJ in living animal context by creating Clytia and zebrafish mutants for genes involved in MMEJ or NHEJ. A few target genes known to be responsible for MMEJ pathway were destroyed by using CRISPR/Cas9 gene-editing technique. The effect of MMEJ-depletion was assessed in normal animals and under treatment by DSB-inducing drug calicheamicin (Clytia) or ionizing radiation (zebrafish). In parallel, we develop in vivo reporter systems, in order to understand cellular, developmental and physiological context favoring MMEJ or NHEJ. To do so, we established Tol2 transposon-mediated transgenesis method, which has been used in zebrafish, in Clytia (Weissbourd et al, 2021). Furthermore, we developed an innovative screen in cell culture to identify novel actors for MMEJ pathway. We also took advantage of single-cell transcriptome information to reveal the cell-type specific deployment of MMEJ pathway.

In the jellyfish Clytia hemisphaerica, we revealed that MMEJ pathway genes, as well as another DSB repair pathway components, are enriched in pluripotent stem cell interstitial cell i-cell lineages and germ line cells, from single cell RNAseq data. This observation is consistent with our existing observation that MMEJ pathway is dominant during early embryogenesis (Momose et al, 2018), which retains maternal proteins from eggs. Clytia embryo stage exhibited higher sensitivity to DSB DNA damage, than the jellyfish stage. With gene knockout of ChePOLQ gene (DNA polymerase ?) oogenesis was severely distributed, indicating its critical role during oocyte development and maintenance.
In zebrafish, we showed that genes involved in MMEJ are largely expressed during the first hours of development when embryonic cells divide very actively and that in 1 dpf embryos, ionising radiation induced an increase in the expression of both MMEJ and NHEJ genes.
We showed a pivotal role for Lig4 for normal growth, reminiscent of Lig4 syndrome in humans and found that both lig3 and polq mutant embryos were highly sensitive to DSB
whereas embryo mutants for lig4 were not. In summary, our results with polq, lig3 and lig4 mutants extend and further confirm the importance of MMEJ relative to cNHEJ.
In human cells we also showed the importance of MMEJ pathway after CRISPR cleavage (Weber et al, 2020). To identify new genes involved in MMEJ and NHEJ, we developed a novel DSB repair pathway reporter in human cells and performed a genome-wide CRISPR screen. A set of 250 candidate genes was selected and validation experiments are currently ongoing.

We successfully fulfilled a large part of the project goals. Some parts of the project need additional data acquisition for the final publication due to the delay caused by the public health crisis on animal maintenance. The scientific outcome of the i-MMEJ project is extremely crucial and will be published in future. Also, the project will be further developed, focusing on stem cell and germline cell maintenance and DNA repair pathways. Cnidarian is particularly well-known for its senescence and high regeneration capacity, in which the maintenance of the stem cell and the genomic integrity is one of the major questions to be answered.

A genetically tractable jellyfish model for systems and evolutionary neuroscience.
Weissbourd B, Momose T, Nair A, Kennedy A, Hunt B, Anderson DJ.
Cell. 2021 Nov 24;184(24):5854-5868.e20. doi: 10.1016/j.cell.2021.10.021. (Transgnic methods developed in this project.)

Role of MMEJ in oocyote development in Clytia hemisphaerica
Uveira J., Lechable M. and Momose M. in preparation.

Analysis of MMEJ and NHEJ activity in zebrafish.
Carrara M., Gaillard AL., Brion A., Duvernois-Berthet E., Giovannangeli C., Concordet JP. and Pézeron G. in preparation

High doses of CRISPR/Cas9 ribonucleoprotein efficiently induce gene knockout with low mosaicism in the hydrozoan Clytia hemisphaerica through microhomology-mediated deletion. Momose T, De Cian A, Shiba K, Inaba K, Giovannangeli C, Concordet JP. Sci Rep. 2018 Aug 6;8(1):11734. doi: 10.1038/s41598-018-30188-0. PMID: 30082705

DNA is continuously exposed to damages in cells. Among the varieties of DNA damages, double strand breaks (DSBs) are one of the most severe threats to genome integrity. Eukaryotes have multiple evolutionary conserved pathways to repair DSBs, namely homologous recombination (HR), non-homologous end-joining (NHEJ) and the lesser-known microhomology mediated end-joining (MMEJ) pathways. HR is a highly precise DSB repair mechanism by copying sequences from the homologous repair template i.e. sister chromatid. NHEJ is an end-tethering repair mechanism and can generate mutations; thus it is often qualified as “error-prone”. MMEJ has long been considered as a backup pathway for NHEJ and has been characterized by the implication, during DSB repair, of short homologous sequences called microhomologies (MH). In the last decade MMEJ has started to be recognized as an independent pathway, particularly after identification of proteins involved such as DNA polymerase?, but the mechanisms of MMEJ remain largely unclear. Importantly in cultured cells, MH-containing deletions often represent a majority of mutations generated during repair of a targeted DSB, supporting the importance of MMEJ in DSB repair. The different DSB repair pathways are mutually exclusive. How and in which conditions cells select one pathway, is still under investigation. In cultured cells, HR is favoured in S/G2 phases of the cell cycle, when a repair template is available; while NHEJ is active along the cell cycle and very little is known for MMEJ. In parallel, it is reasonable to speculate that another level of DSB repair regulation, depending on the stage of embryonic development and on cell type, is taking place in living animals, which consist of many differentiated or undifferentiated cells, dynamically changing and interacting. Recent observations suggested that MMEJ is critical for animal embryos. In zebrafish embryos, pol? mutant is highly sensitive to DSB. In C. elegans, pol?-mediated MMEJ is dominant in germline stem cells. In mouse embryos, high frequency of deletions between MH, characteristic of MMEJ, has been observed following TALEN or CRISPR/Cas9-mediated knockout. Moreover, our recent work has shown that MMEJ is acting almost exclusively in early embryos of a jellyfish, Clytia hemisphaerica.

In this project, we aim to address questions about roles and mechanisms of MMEJ. One key feature of our project is to address these questions in developing animals, using zebrafish and Clytia. Therefore our project includes three axes:

- Characterization of the role of MMEJ in living animals through
(i) Profiling and dynamics of DSB repair genes expression
(ii) Impact of loss-of-function mutations in DSB repair genes on embryonic development

- Determination of cellular, developmental and physiological contexts that favour/disfavour MMEJ over other DSB repair pathways by
(i) Visualization at cellular resolution of DSB repair pathways using fluorescent reporters
(ii) Characterization of repair products including frequency of MMEJ-induced deletions

- Identification of novel genes involved in MMEJ and the balance with other DSB repair pathways, using
(i) A CRISPR-based screen in cultured cells by developing appropriate reporters for the DSB repair pathways
(i) Validation of candidate genes in cultured cells and in the two animal models

Our project combines the expertise of the three partners in developmental biology and genetics of animal models as well as in genome editing and DSB repair. Our project will allow to better understand how cells in living animals deal with DSB, how different pathways are switched on depending on the cellular context and finally to better characterize the MMEJ pathway. In addition, we will also exploit our data to improve precise genome modification in genome editing approaches.