CRISPR- CAS SCREENING IN CANCER RESEARCH: METHODOLOGIES, APPLICATIONS, AND TRANSLATIONAL CHALLENGES

CRISPR screening has revolutionized cancer research by enabling systematic, genome-wide interrogation of gene function to identify therapeutic vulnerabilities, resistance mechanisms, and immunotherapy determinants. This review examines methodological approaches for CRISPR screenings using the Cas9 systems, evaluating how system and methodology selection profoundly influence which biological mechanisms are revealed across therapeutic resistance, metastasis regulation, and immunotherapy response. Cas9-based screens-from simple one-cell-type (1CT) in vitro approaches to complex autochthonous in vivo systems-excel at identifying cell-autonomous DNA-level dependencies. Two-cell-type (2CT) co-culture approaches validate immune-dependent mechanisms, while in vivo screens capture physiological complexity and have identified clinically relevant targets including PTPN2, now in Phase 1 trials. Critical challenges include incomplete gene editing, false-positive and false-negative hits, context-dependent essentiality, and system-specific technical limitations. Emerging technologies integrating artificial intelligence, prime editing, single-cell profiling, and patient-derived models promise to address limitations while enabling functional precision medicine. This review provides a framework for selecting appropriate systems, interpreting results within methodological constraints, and integrating complementary approaches to maximize biological insight and translational impact.