Current advances and future directions of CRISPR/Cas9 screening for therapeutic target discovery in breast cancer

Abstract

Breast cancer continues to be a major contributor to global cancer mortality, driven by pronounced molecular diversity and the frequent emergence of drug resistance, which together highlight the pressing demand for innovative treatment approaches. This review offers a thorough appraisal of CRISPR-Cas9-based functional genomic screening technologies—encompassing genome-wide knockout, transcriptional activation, and epigenome-modifying platforms—and their utility in uncovering genetic dependencies and exploitable therapeutic targets across various breast cancer subtypes. High-throughput CRISPR approaches have facilitated the large-scale identification of tumor suppressors, oncogenes, immune-regulatory factors, and synthetic lethal gene pairs, while also shedding light on resistance pathways to established treatments such as tyrosine kinase inhibitors and antibody–drug conjugates. Within the context of cancer immunotherapy, CRISPR-mediated disruption of immune checkpoint molecules has been shown to bolster T-cell resilience and cytotoxic function, and pooled in vivo screening strategies have further revealed novel enhancers of anti-tumor immune activity. Beyond target discovery, CRISPR-engineered cellular immunotherapies—specifically CAR-T and TCR-T products directed against HER2, MUC1-C, and PRAME—have progressed to clinical evaluation, with multiple Phase I/II trials currently underway in breast cancer patients. Nevertheless, several obstacles remain, including unintended genomic edits, inefficient in vivo delivery, intratumoral heterogeneity, and the necessity for robust orthogonal validation using physiologically relevant disease models. The integration of CRISPR screening outputs with computational modeling, single-cell transcriptomics, and multi-omic datasets has improved the accuracy of target selection and deepened mechanistic insights. Overall, this review underscores that the synergistic application of functional genomics and CRISPR-based interrogation offers a potent foundation for devising rational combination regimens and precision medicine approaches in breast cancer, while also delineating key hurdles that must be overcome to enable effective clinical implementation.