Efficient genome editing of human natural killer cells by CRISPR RNP

Abstract The ability to genetically modify CD8 T cells using viral gene delivery has facilitated the development of next generation of cancer immunotherapies such as chimeric antigen receptor (CAR) T cells engineered to specifically kill tumor cells. Development of immunotherapies targeting natural killer (NK) cells have stalled in part by their resistance to viral gene delivery. Here, we describe an efficient approach to genetically edit human NK cells by electroporation and CRISPR-Cas9 ribonucleoprotein (RNP) complexes. We detail electroporation pulse codes and buffer optimization for protein uptake by human NK cells and viability, and the efficiency of this approach over other methods. To highlight the transformative step this technique will have for NK cell immunotherapy drug discovery, we deleted NKp46 and CIS in primary human NK cells and validated murine findings on their key roles in regulating NK cell anti-tumor function.