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Genome-Wide CRISPR Screen for Unveiling Radiosensitive and Radioresistant Genes
Published on: May 23, 2025
Genome-wide CRISPR screens identify the EXO1-CAF-1 pathway suppressing R-loop-associated DNA damage
Alexandra Nusawardhana1, Anastasia Hale1, Joshua Straka1
1Department of Molecular and Precision Medicine, The Pennsylvania State University College of Medicine, Hershey, PA 17033, United States.
Abstract:
DNA repair is critical for cellular homeostasis under both normal conditions as well as in response to genotoxic agents such as chemotherapeutics. EXO1 is a 5'-3' exonuclease with multiple roles in DNA biology. To better understand these roles, we employed CRISPR loss-of-function genome-wide screening to identify genes required for proliferation and cisplatin sensitivity in EXO1-deficient cells. We uncovered differential regulators of cisplatin sensitivity between wildtype (WT) and EXO1-deficient cells. By analyzing the genetic networks that these regulators belong to, we found that DNA repair was the main biological process suppressing cisplatin sensitivity in WT cells, but this was not the case in EXO1-deficient cells, indicating that EXO1 is critical for the repair of cisplatin-induced DNA damage. Moreover, synthetic lethality screens identified a genetic interaction between EXO1 and the histone chaperone CAF-1. Mechanistically, we show that EXO1 and CAF-1 are independently recruited to R-loops and participate in separate, synergistic pathway of R-loop suppression. Even in the absence of DNA damage treatment, concomitant loss of EXO1 and CAF-1 causes R-loop accumulation and increased R-loop-associated DNA damage. Our work sheds light on the critical roles of EXO1 in genomic stability.
Insights
The exonuclease EXO1 is crucial for repairing DNA damage from chemotherapy drugs like cisplatin. Its absence, along with the histone chaperone CAF-1, leads to R-loop accumulation and DNA damage, impacting genomic stability.
Area of Science:
- Molecular Biology
- Genetics
- Cellular Biology
Background:
- DNA repair mechanisms are essential for maintaining cellular homeostasis and responding to genotoxic stress.
- EXO1 is a 5'-3' exonuclease involved in various DNA biological processes.
- Understanding EXO1's function is critical, especially in the context of chemotherapeutic treatments.
Purpose of the Study:
- To identify genes affecting proliferation and cisplatin sensitivity in EXO1-deficient cells using CRISPR screening.
- To elucidate the specific roles of EXO1 in repairing DNA damage induced by genotoxic agents.
- To investigate the functional relationship between EXO1 and other DNA maintenance pathways, including R-loop suppression.
Main Methods:
- CRISPR loss-of-function genome-wide screening to identify essential genes in EXO1-deficient cells.
- Analysis of genetic networks to understand differential regulation of cisplatin sensitivity.
- Synthetic lethality screens to uncover genetic interactions, specifically with histone chaperone CAF-1.
- Mechanistic studies investigating the recruitment and function of EXO1 and CAF-1 in R-loop suppression.
Main Results:
- EXO1 is critical for the repair of cisplatin-induced DNA damage, a role not observed in wildtype cells.
- A synthetic lethal interaction was identified between EXO1 and the histone chaperone CAF-1.
- EXO1 and CAF-1 are independently recruited to R-loops and function synergistically to suppress them.
- Concomitant loss of EXO1 and CAF-1 leads to R-loop accumulation and DNA damage, even without exogenous genotoxic treatment.
Conclusions:
- EXO1 plays a vital role in the DNA damage response pathway, particularly in repairing chemotherapy-induced damage.
- The combined action of EXO1 and CAF-1 is crucial for preventing R-loop accumulation and maintaining genomic stability.
- Disruptions in the EXO1-CAF-1 pathway can lead to significant R-loop-associated DNA damage, highlighting a novel mechanism of genomic instability.
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