Genome editing is a powerful technique for delineating complex signaling circuitry and enhancing the functionality of immune cells for immunotherapy. Natural killer (NK) cells are potent immune effectors against cell malignancy, but they are challenging to modify genetically by conventional methods due to the toxicity of DNA when introduced into cells coupled with limited transfection and transduction efficiency. Here, we describe an integrated platform that streamlines feeder-free ex vivo expansion of cryopreserved primary human NK cells and nonviral genome editing by the nucleofection of CRISPR-Cas9 ribonucleoproteins (Cas9 RNPs). The optimized Cas9 nucleofection protocol allows efficient and multiplex gene knockout in NK cells while preserving high cell viability and negligible off-target effects. Cointroduction of a DNA template also enables in-frame gene knock-in of an HA affinity tag and a gfp reporter across multiple loci. This work demonstrates the advantages and flexibility of working with cryopreserved NK cells as potential off-the-shelf engineered therapeutic agents.

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