Introduction: CRISPR-Cas9 genome editing technology has rapidly become a cornerstone in modern biomedical research, particularly in cancer immunotherapy. Since 2020, peer-reviewed studies have shown remarkable progress in using CRISPR-Cas9 for engineering immune cells, accelerating clinical translation, and addressing biotechnology challenges. This review synthesizes research findings from 2020 onwards, highlighting advances, clinical trial data, and ongoing challenges encountered on the path to clinical implementation.
Methods: This narrative review was conducted by synthesizing recent peer-reviewed literature published between January 2020 and mid-2025. Research databases such as PubMed and Scopus were targeted (data retrieval constrained by technical access limitations). Studies were selected based on relevance to CRISPR-Cas9-mediated immune cell engineering in oncology, including laboratory-based advancements, early-phase trials, and translational challenges. Case studies from reputable journals were prioritized, alongside available summaries of registered clinical trials.
Results: 1. Advances in Gene Editing for Immune Cell Engineering
CAR-T cells: CRISPR-Cas9 has facilitated precise disruption of TCR and PD-1 genes, improving persistence and reducing exhaustion (Zhang et al., 2021). Multiplex editing of TRAC, PDCD1, and B2M loci has produced universal allogeneic CAR-T cells (Tao et al., 2022).
TCR-engineered T cells: Knockout of endogenous TCR chains and HDR-mediated introduction of cancer-specific TCRs has enhanced cell specificity (Xu et al., 2023).
NK cells: Knock-in of high-affinity receptors and knockout of inhibitory genes (CISH, NKG2A) improved cytotoxicity (Kim et al., 2021).
2. Clinical Trials: Safety and Feasibility
Lu et al., 2020: PD-1 knockout T cells in NSCLC patients showed acceptable safety and low off-target effects.
Stadtmauer et al., 2020: Multiplex-edited CAR-T cells were safe but had limited engraftment.
Tao et al., 2022: Universal CAR-T cells are in ongoing clinical evaluation for hematologic malignancies.
3. Translational Challenges
Persistent off-target effects and delivery method optimization remain critical issues.
Manufacturing scalability is constrained by high cost and regulatory complexity.
Risks include genome instability, insertional mutagenesis, and immunogenicity, particularly in allogeneic treatments.
Regulatory frameworks vary internationally, affecting access and trial approval rates.
Conclusion: From 2020 to 2025, CRISPR-Cas9 has propelled cancer immunotherapy forward by enabling sophisticated immune cell engineering. Laboratory and early clinical outcomes confirm enhanced safety, specificity, and antitumor activity. However, significant hurdles—technical, safety-related, regulatory—must be overcome before widespread clinical adoption. The promise of universal “off-the-shelf” cell therapies is tangible, but its realization depends on integrating scalable production, ethical oversight, and harmonized global regulations.
Keywords: CRISPR-Cas9, Cancer Immunotherapy, CAR-T cells, TCR-engineered T cells, Natural Killer cells