Engineering with care: safety assessment platforms for CRISPR-modified natural killer cells

CRISPR-based gene editing has become a transformative tool to enhance immune cell therapies. In particular, engineering natural killer (NK) cells with CRISPR/Cas systems has gained traction due to their ability to mediate strong anti-tumor responses in an MHC-unrestricted, non-alloreactive manner. Early trials show the feasibility and safety of allogeneic NK cells, paving the way as scalable "off-the-shelf" products. CRISPR/Cas9 edits genomes by inducing DNA double-strand breaks (DSBs), mainly repaired through non-homologous end joining (NHEJ) or homology-directed repair (HDR). While effective, CRISPR carries risks of off-target (OT) activity that may disrupt essential genes, cause chromosomal rearrangements, or trigger oncogenic changes - posing threats to product integrity and patient safety. These concerns intensify with multiplex editing, where multiple loci are modified to improve function, persistence, and immune evasion. Since unmodified NK cells are typically short-lived, many clinical-stage products are engineered to express IL-15 or related constructs, extending their half-life and amplifying risks associated with unintended changes. This underscores the urgent need for robust safety assessments. In this review, we summarize the current landscape of safety assessment platforms for evaluating gene edited NK cells. We highlight predictive in silico tools, biochemical <i>in vitro</i> assays, and emerging cell-based detection systems to identify and quantify CRISPR-induced OT events. Particular attention is given to their suitability, limitations, and practical use in primary NK cells and multiplex editing strategies. Our aim is to support the design of safe, effective editing workflows for NK cell therapies - ensuring rigor as the field advances rapidly toward clinical application.