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Pooled CRISPR-Based Genetic Screens in Mammalian Cells
Published on: September 4, 2019
Epigenome-Wide CRISPR-Cas9-Based Knockout Screens on Chemoresistant Cells
Ozlem Yedier-Bayram1, Elif Ayca Guvener1, Tugba Bagci-Onder2
1Koç University School of Medicine; Koç University Research Center for Translational Medicine.
Abstract:
Chemotherapy resistance remains a major challenge in cancer treatment, driven by cancer cells' ability to acquire adaptive properties, rewire signaling pathways, and alter chromatin structure to evade drug-induced cytotoxicity. Because these processes rely heavily on epigenetic mechanisms that regulate chromatin organization and transcriptional plasticity, epigenetic regulators have emerged as key contributors to chemotherapy resistance. To investigate resistance to paclitaxel, one of the most widely used chemotherapeutic agents in triple-negative breast cancer (TNBC), we employed an epigenome-focused knockout library (EPIKOL), a CRISPR-Cas9-based library, designed to systematically disrupt genes involved in chromatin regulation. Chemoresistant cell lines were generated through a stepwise dose-escalation protocol that recapitulates clinically relevant drug adaptation. However, these resistant cells exhibit a multidrug-resistant (MDR) phenotype, posing significant challenges for efficient viral transduction and the selection of stable cell populations. In this study, we describe key methodological steps for achieving high-efficiency lentiviral transduction and selection, enabling the successful application of EPIKOL CRISPR screens in chemoresistant TNBC models. Following the described protocol, an epigenome-wide CRISPR screen was conducted on chemoresistant TNBC cells, and novel epigenetic regulators of chemoresistance were identified. This protocol provides a robust framework for identifying epigenetic regulators that contribute to acquired paclitaxel resistance using a CRISPR-based loss-of-function approach.
Insights
This study introduces a new CRISPR screening method to identify epigenetic regulators driving chemotherapy resistance in triple-negative breast cancer. The developed protocol enhances lentiviral transduction for discovering novel targets to overcome paclitaxel resistance.
Area of Science:
- Cancer Biology
- Epigenetics
- Genomics
Background:
- Chemotherapy resistance is a significant obstacle in cancer treatment, often involving epigenetic alterations.
- Triple-negative breast cancer (TNBC) exhibits resistance to paclitaxel, a common chemotherapeutic agent.
- Epigenetic regulators play a crucial role in the adaptive mechanisms cancer cells use to evade chemotherapy.
Purpose of the Study:
- To develop and validate a robust methodological framework for identifying epigenetic regulators of paclitaxel resistance in TNBC.
- To enable the application of epigenome-focused CRISPR screens in chemoresistant cancer models.
- To discover novel epigenetic targets that contribute to acquired chemotherapy resistance.
Main Methods:
- Generation of chemoresistant TNBC cell lines using a stepwise dose-escalation protocol.
- Development of high-efficiency lentiviral transduction and selection methods for multidrug-resistant (MDR) cells.
- Application of an epigenome-focused knockout library (EPIKOL) with CRISPR-Cas9 for loss-of-function screening.
Main Results:
- Successful implementation of a protocol for high-efficiency lentiviral transduction and selection in chemoresistant TNBC cells.
- Identification of novel epigenetic regulators contributing to acquired paclitaxel resistance through an epigenome-wide CRISPR screen.
- Demonstration of the protocol's robustness for investigating epigenetic mechanisms of drug resistance.
Conclusions:
- The developed protocol facilitates the systematic identification of epigenetic regulators involved in chemotherapy resistance.
- This approach provides a powerful tool for discovering new therapeutic targets to overcome paclitaxel resistance in TNBC.
- The findings highlight the critical role of epigenetics in cancer drug adaptation and resistance.
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