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Updated: Nov 28, 2025
Pooled CRISPR-Based Genetic Screens in Mammalian Cells
Published on: September 4, 2019
Identification of regulators of poly-ADP-ribose polymerase inhibitor response through complementary CRISPR knockout
Kristen E Clements1, Emily M Schleicher1, Tanay Thakar1
1Department of Biochemistry and Molecular Biology, The Pennsylvania State University College of Medicine, Hershey, PA, 17033, USA.
Abstract:
Inhibitors of poly-ADP-ribose polymerase 1 (PARPi) are highly effective in killing cells deficient in homologous recombination (HR); thus, PARPi have been clinically utilized to successfully treat BRCA2-mutant tumors. However, positive response to PARPi is not universal, even among patients with HR-deficiency. Here, we present the results of genome-wide CRISPR knockout and activation screens which reveal genetic determinants of PARPi response in wildtype or BRCA2-knockout cells. Strikingly, we report that depletion of the ubiquitin ligase HUWE1, or the histone acetyltransferase KAT5, top hits from our screens, robustly reverses the PARPi sensitivity caused by BRCA2-deficiency. We identify distinct mechanisms of resistance, in which HUWE1 loss increases RAD51 levels to partially restore HR, whereas KAT5 depletion rewires double strand break repair by promoting 53BP1 binding to double-strand breaks. Our work provides a comprehensive set of putative biomarkers that advance understanding of PARPi response, and identifies novel pathways of PARPi resistance in BRCA2-deficient cells.
Insights
Poly (ADP-ribose) polymerase inhibitors (PARPi) are effective against BRCA2-mutant tumors. However, resistance occurs. This study identifies HUWE1 and KAT5 as key factors reversing PARPi sensitivity in BRCA2-deficient cells.
Area of Science:
- Genetics
- Cancer Biology
- Molecular Oncology
Background:
- Poly (ADP-ribose) polymerase inhibitors (PARPi) target cancer cells with homologous recombination (HR) deficiencies, such as those with BRCA2 mutations.
- Despite PARPi efficacy, not all HR-deficient tumors respond, indicating mechanisms of resistance.
- Understanding genetic determinants of PARPi response is crucial for improving cancer therapy.
Purpose of the Study:
- To identify genetic factors influencing response to PARPi in both wildtype and BRCA2-deficient cells.
- To elucidate mechanisms by which specific genes confer resistance to PARPi.
- To discover potential biomarkers for predicting PARPi treatment outcomes.
Main Methods:
- Genome-wide CRISPR knockout and activation screens were performed to identify genes affecting PARPi sensitivity.
- Functional assays were used to investigate the roles of identified genes in DNA repair pathways.
- Analysis of protein levels and interactions to understand resistance mechanisms.
Main Results:
- Depletion of ubiquitin ligase HUWE1 or histone acetyltransferase KAT5 reversed PARPi sensitivity in BRCA2-deficient cells.
- HUWE1 loss enhanced RAD51 levels, partially restoring HR and conferring resistance.
- KAT5 depletion promoted 53BP1 binding to double-strand breaks, altering DNA repair and causing resistance.
Conclusions:
- HUWE1 and KAT5 are significant genetic determinants of PARPi response and resistance.
- Distinct molecular mechanisms underlie HUWE1- and KAT5-mediated PARPi resistance.
- These findings identify novel pathways for overcoming PARPi resistance and suggest potential biomarkers.
