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Updated: Sep 26, 2025
Genome-Wide CRISPR Screen for Unveiling Radiosensitive and Radioresistant Genes
Published on: May 23, 2025
Genome-wide CRISPR screen identified Rad18 as a determinant of doxorubicin sensitivity in osteosarcoma
Mingrui Du1,2, Jintao Gu2, Chenlin Liu2
1Department of Orthopedics, The Second Affiliated Hospital, The Fourth Military Medical University, Xi'an, China.
Background:
Osteosarcoma (OS) is a malignant bone tumor mostly occurring in children and adolescents, while chemotherapy resistance often develops and the mechanisms involved remain challenging to be fully investigated.
Methods:
Genome-wide CRISPR screening combined with transcriptomic sequencing were used to identify the critical genes of doxorubicin resistance. Analysis of clinical samples and datasets, and in vitro and in vivo experiments (including CCK-8, apoptosis, western blot, qRT-PCR and mouse models) were applied to confirm the function of these genes. The bioinformatics and IP-MS assays were utilized to further verify the downstream pathway. RGD peptide-directed and exosome-delivered siRNA were developed for the novel therapy strategy.
Results:
We identified that E3 ubiquitin-protein ligase Rad18 (Rad18) contributed to doxorubicin-resistance in OS. Further exploration revealed that Rad18 interact with meiotic recombination 11 (MRE11) to promote the formation of the MRE11-RAD50-NBS1 (MRN) complex, facilitating the activation of the homologous recombination (HR) pathway, which ultimately mediated DNA damage tolerance and leaded to a poor prognosis and chemotherapy response in patients with OS. Rad18-knockout effectively restored the chemotherapy response in vitro and in vivo. Also, RGD-exosome loading chemically modified siRad18 combined with doxorubicin, where exosome and chemical modification guaranteed the stability of siRad18 and the RGD peptide provided prominent targetability, had significantly improved antitumor activity of doxorubicin.
Conclusions:
Collectively, our study identifies Rad18 as a driver of OS doxorubicin resistance that promotes the HR pathway and indicates that targeting Rad18 is an effective approach to overcome chemotherapy resistance in OS.
Insights
Osteosarcoma (OS) resistance to doxorubicin is driven by Rad18, which activates homologous recombination (HR). Targeting Rad18 with RGD-exosomes and siRNA offers a promising strategy to improve chemotherapy response in OS patients.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Osteosarcoma (OS) is a prevalent bone cancer in children and adolescents.
- Chemotherapy resistance is a significant challenge in OS treatment, with underlying mechanisms requiring further investigation.
Purpose of the Study:
- To identify key genes contributing to doxorubicin resistance in osteosarcoma.
- To elucidate the molecular mechanisms by which these genes confer resistance.
- To develop a novel therapeutic strategy targeting identified resistance pathways.
Main Methods:
- Genome-wide CRISPR screening and transcriptomic sequencing to identify resistance genes.
- In vitro and in vivo experiments, including cell viability assays, apoptosis studies, western blotting, qRT-PCR, and mouse models.
- Bioinformatics and IP-MS assays to determine downstream pathways.
- Development of RGD peptide-directed, exosome-delivered siRNA for targeted therapy.
Main Results:
- E3 ubiquitin-protein ligase Rad18 (Rad18) was identified as a critical factor in doxorubicin resistance in OS.
- Rad18 promotes homologous recombination (HR) pathway activation by interacting with MRE11, leading to DNA damage tolerance and poor prognosis.
- Rad18 knockout restored chemotherapy sensitivity in vitro and in vivo.
- A novel therapy using RGD-exosome loaded siRad18 combined with doxorubicin demonstrated enhanced antitumor activity.
Conclusions:
- Rad18 is a key driver of doxorubicin resistance in osteosarcoma by promoting the HR pathway.
- Targeting Rad18 presents a viable strategy to overcome chemotherapy resistance in OS.
- The developed RGD-exosome/siRad18 therapy shows potential for improved clinical outcomes in OS patients.
