Summary
DNA double-strand breaks (DSBs) occur frequently during a cell’s lifetime and constitute the most cytotoxic DNA lesion. DSBs compromise genetic integrity and have the potential to trigger cell death and generate pathological mutations. To counteract such threat, cells possess a complex protein network termed DSB response (DSBR) which senses, signals and repairs DSBs. Two predominant mechanisms repair DSBs: homologous recombination (HR) and non-homologous end joining (NHEJ).
While loss of DSBR factors such as KU and XRCC4 (i.e. NHEJ proteins) or BRCA1 and BRCA2 (i.e. HR proteins) leads to early embryonic lethality and cell death, inactivating somatic mutations in these very same genes are associated with certain immunological and neurological disorders, premature ageing and cancer. This implies the existence of a suppressive (epi)genetic background that enables diseased cells to tolerate the otherwise lethal loss of essential DSBR factors.
This project aims to systematically identify suppressor mutations which rescue viability upon loss of essential HR (BRCA1 and BRCA2) and NHEJ (KU80 and XRCC4) effectors. To reach such goal, I propose to (a.) genetically sensitize wild-type “healthy” mouse embryonic stem cells and B lymphoid cells by introducing mutations using a genome-wide CRISPR KO library. In such diversely sensitized background, I will then (b.) trigger the acute degradation of KU80, XRCC4, BRCA1 and BRCA2 proteins, using the auxin-inducible degron (AID) system. Suppressor mutations will be directly identified by sequencing sgRNA retrieved from survivor cells. (c.) The resulting suppression network maps will be integrated to clinical datasets to identify the relevance of these mutations in disease.
Together, this project will unravel the DSBR protein networks that permit cell survival under different DNA repair-deficient conditions, encountered notably in cancer; paving way for personalized disease prevention and treatment strategies.
While loss of DSBR factors such as KU and XRCC4 (i.e. NHEJ proteins) or BRCA1 and BRCA2 (i.e. HR proteins) leads to early embryonic lethality and cell death, inactivating somatic mutations in these very same genes are associated with certain immunological and neurological disorders, premature ageing and cancer. This implies the existence of a suppressive (epi)genetic background that enables diseased cells to tolerate the otherwise lethal loss of essential DSBR factors.
This project aims to systematically identify suppressor mutations which rescue viability upon loss of essential HR (BRCA1 and BRCA2) and NHEJ (KU80 and XRCC4) effectors. To reach such goal, I propose to (a.) genetically sensitize wild-type “healthy” mouse embryonic stem cells and B lymphoid cells by introducing mutations using a genome-wide CRISPR KO library. In such diversely sensitized background, I will then (b.) trigger the acute degradation of KU80, XRCC4, BRCA1 and BRCA2 proteins, using the auxin-inducible degron (AID) system. Suppressor mutations will be directly identified by sequencing sgRNA retrieved from survivor cells. (c.) The resulting suppression network maps will be integrated to clinical datasets to identify the relevance of these mutations in disease.
Together, this project will unravel the DSBR protein networks that permit cell survival under different DNA repair-deficient conditions, encountered notably in cancer; paving way for personalized disease prevention and treatment strategies.
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| Web resources: | https://cordis.europa.eu/project/id/101066732 |
| Start date: | 01-09-2023 |
| End date: | 31-08-2025 |
| Total budget - Public funding: | - 211 754,00 Euro |
Cordis data
Original description
DNA double-strand breaks (DSBs) occur frequently during a cell’s lifetime and constitute the most cytotoxic DNA lesion. DSBs compromise genetic integrity and have the potential to trigger cell death and generate pathological mutations. To counteract such threat, cells possess a complex protein network termed DSB response (DSBR) which senses, signals and repairs DSBs. Two predominant mechanisms repair DSBs: homologous recombination (HR) and non-homologous end joining (NHEJ).While loss of DSBR factors such as KU and XRCC4 (i.e. NHEJ proteins) or BRCA1 and BRCA2 (i.e. HR proteins) leads to early embryonic lethality and cell death, inactivating somatic mutations in these very same genes are associated with certain immunological and neurological disorders, premature ageing and cancer. This implies the existence of a suppressive (epi)genetic background that enables diseased cells to tolerate the otherwise lethal loss of essential DSBR factors.
This project aims to systematically identify suppressor mutations which rescue viability upon loss of essential HR (BRCA1 and BRCA2) and NHEJ (KU80 and XRCC4) effectors. To reach such goal, I propose to (a.) genetically sensitize wild-type “healthy” mouse embryonic stem cells and B lymphoid cells by introducing mutations using a genome-wide CRISPR KO library. In such diversely sensitized background, I will then (b.) trigger the acute degradation of KU80, XRCC4, BRCA1 and BRCA2 proteins, using the auxin-inducible degron (AID) system. Suppressor mutations will be directly identified by sequencing sgRNA retrieved from survivor cells. (c.) The resulting suppression network maps will be integrated to clinical datasets to identify the relevance of these mutations in disease.
Together, this project will unravel the DSBR protein networks that permit cell survival under different DNA repair-deficient conditions, encountered notably in cancer; paving way for personalized disease prevention and treatment strategies.
Status
SIGNEDCall topic
HORIZON-MSCA-2021-PF-01-01Update Date
09-02-2023
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