Jul 2026· Journal of Visualized Experiments· Vol 233· 0 citations
Medicine
TL;DR
Key methodological steps for achieving high-efficiency lentiviral transduction and selection are described, enabling the successful application of EPIKOL CRISPR screens in chemoresistant TNBC models.
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.
It is proposed that integrating precise editing, in vivo screening, single-cell multi-omics, and emerging artificial intelligence (AI)-assisted design may provide information and a design basis for future combined strategies that simultaneously target vulnerabilities in senescent cells and malignant populations.
Bo Fan, Aiwei Wu, Xue Pan et al.· Ageing and Cancer Research &...· 0 citations
Background: Leukemia is a cancer of hematopoietic stem cells in the bone marrow. It is classified as lymphoid or myeloid, and as acute (rapid onset) or chronic (slow progression). Advances in CRISPR technology enable deeper study of leukemia biology and therapeutic targets. While AML, CML, ALL, and CLL are distinct, comparative studies highlight shared and unique features. Identifying key pathways across subtypes may yield improved treatments. Our lab has established a unique CRISPR Activation (CRISPRa) platform for gain-of-function screening applications. We hypothesize that genome-wide CRISPRa screening will uncover subtype-specific genetic dependencies and drug resistance mechanisms, offering novel therapeutic insights.
We engineered leukemia cell lines by nucleofecting a self-selecting CRISPRa PiggyBac plasmid carrying a blasticidin resistance gene, enabling selection of CRISPRa-competent cells. Functionality was validated using lentiviral delivery of sgRNAs targeting cell surface markers. Cells were transduced with our whole-genome CRISPRa library, Sonata, at MOI 0.4, followed by puromycin selection to enrich for sgRNA-expressing cells. Post-transduction, cells were harvested at various timepoints for sequencing to track sgRNA abundance and identify growth-modulating genes.
We engineered and validated four CRISPRa-competent leukemia lines: K562 (CML), Jurkat (T-ALL), THP-1 (AML), and HL60 (AML). We have completed whole-genome screening campaigns in the CML and T-ALL backgrounds, identifying hundreds of shared and context specific growth modifiers. Functional validation, pathway analysis, and potential clinical significance is ongoing, as is expansion of our screens with the AML contexts.
Conclusions: Our screens reveal genes that, upon activation, influence leukemia cell growth. These findings support discovery of new therapeutic targets and enhance understanding of leukemia subtype biology, guiding future personalized treatment strategies.
The Cole Foundation, FHMR
Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)
Jeffrey Sullivan, Xiaozen Wen, Gabriela Flores-Vargas et al.· Journal of Immunology· 0 citations
Background CRISPR-mediated viability assays in diverse cancer cell lines have informed cancer biology and precision medicine, but cell fitness is not the only cancer-relevant phenotype. Gene expression profiling provides insight into cellular stress, inflammation, and differential state, while still identifying activation of cell-death pathways. Perturb-seq allows scalable functional genomics screening of expression phenotypes at single-cell resolution, however existing datasets cover only a small number of work-horse cell lines. Results We produced a proof-of-concept Perturb-seq dataset targeting 100 genes in 16 diverse cancer cell lines. In the process, we established methods to address single-cell technical artifacts, identified Cas9-mediated chromosomal aberrations and assessed screen quality. Even with a limited library, we observed common signatures of deleting essential genes as well as context-specific responses based on intrinsic genomic properties of the models. For example, we inferred a previously undescribed relationship between dependence on the ER-golgi transport gene immediate early response 3 interacting protein 1 (IER3IP1) and oxidative stress, demonstrating the potential of integrated Perturb-seq for hypothesis generation. Conclusions We established a framework for building a comprehensive map of post-perturbational transcriptional phenotypes using parallel Perturb-seq experiments across multiple cell lines. We demonstrated that integrated Perturb-seq experiments spanning diverse contexts enable hypotheses about gene function specific to tissue types or cancer subtypes – suggesting large-scale, genome-wide datasets would offer invaluable insight into the highly context-dependent nature of cancer biology.
Samuel Maffa, Isabella Boyle, Lie Ward et al.· bioRxiv· 0 citations
An overview of the CRISPR screen methodology, which involves introducing guide RNAs targeting specific genes into cells, followed by phenotype selection and analysis, is presented, providing valuable insights into gene function and potential therapeutic targets.
Xinyue Zhou, Rui Lu· Methods in molecular biology· 0 citations
Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies worldwide, characterized by late-stage diagnosis, profound chemoresistance, and a five-year survival rate that barely exceeds 12%. The fibrotic stromal barrier surrounding the tumor actively suppresses immune infiltration and blocks drug delivery, rendering conventional treatment options largely ineffective. CRISPR-Cas9-mediated gene knockout represents a promising strategy to overcome this stromal barrier-associated therapeutic resistance by enabling precise disruption of genes that sustain desmoplastic signaling, stromal-immune crosstalk, and drug efflux pathways within the tumor microenvironment. In this context, CRISPR-Cas9-guided gene knockout has opened a new chapter in PDAC research by enabling precise, scalable analysis of the cancer genome. Functional screens using this technology have mapped critical oncogenic dependencies, identifying mutant KRAS, TP53, SMAD4, and CDKN2A as high-value targets, while simultaneously revealing synthetic lethal interactions that were previously inaccessible through pharmacological approaches. These discoveries are now being translated into therapeutic strategies aimed at silencing driver mutations, restoring chemosensitivity, and reprogramming the immunosuppressive tumor microenvironment. Delivery platforms, including lipid nanoparticles, viral vectors, and extracellular vesicles, are being refined to navigate the physical barriers unique to PDAC. Patient-derived organoids and xenograft models are providing the translational framework needed to evaluate these interventions under clinically relevant conditions. This review examines the molecular mechanisms of CRISPR-guided knockout, the genetic vulnerabilities it has uncovered in PDAC, the therapeutic strategies emerging from this work, and the delivery systems supporting clinical translation. The remaining barriers and the steps needed to bring this technology to patients are also discussed.
Muhammad Shahid Mehmood, Maida Noor, Somal Meraj et al.· Discover Oncology· 0 citations
Triple-negative breast cancer (TNBC) is defined by profound heterogeneity, dormant metastatic reservoirs, and rapid therapy resistance. Building on our AI-Driven CRISPR Strategies in Breast Cancer framework, CRISPR–Cas9 is emerging as more than a gene-editing tool, capable of restoring circadian integrity, eliminating dormant clones, and re-programming immune surveillance. A structured PubMed, Scopus, and ClinicalTrials.gov review through 2025 integrated mechanistic, preclinical, and early clinical evidence. Beyond standard knockout, base, and prime editing, we highlight chrono-genomic repair of BMAL1/PER2, dormancy-focused synthetic-lethality screens, and genomic-collapse tactics for BRCA1-deficient tumors. Adaptive AI pipelines that iteratively refine guide RNAs and exosome-mimetic carriers, incorporating Boolean logic gates, were also evaluated for self-regulated, tumor-specific delivery. Proof-of-concept studies show that HER2 deletion, TP53 rescue, and ABCB1 silencing enhance chemosensitivity across luminal, HER2-positive, and TNBC models. Circadian restoration expands therapeutic windows and delays relapse in xenografts. Dormancy-directed CRISPR screens reveal unique vulnerabilities in disseminated tumor cells, whereas genomic collapse selectively destroys BRCA1-mutant clones. Integration with CAR-T cells and antibody–drug conjugates amplifies cytotoxicity, and transient nanoparticle or exosome systems improve solid-tumor penetration while minimizing off-target events. CRISPR–Cas9 is transitioning from a molecular scalpel to an adaptive, self-learning therapeutic ecosystem. By uniting AI-guided design, circadian reprogramming, dormancy eradication, and logic-gated delivery, the strategies detailed here define a next-generation precision-oncology paradigm capable of anticipating tumor evolution, overcoming resistance, and preventing metastatic relapse.
Anmar Ghanim, Anmar Ghanim Taki, Abdulkareem Shareef et al.· Iranian Journal of Basic Med...· 0 citations
