Natural killer (NK) cell is one of the potential candidates to adapt Chimeric Antigen Receptor (CAR) modification owing to their potent efficacy against tumor cells, ability to transfer allogeneically, and safety profile. However, genetic modification of human primary natural killer (PNK) cells has been a major challenge in NK cell research since they are not permissive to conventional VSVG pseudotyped lentivirus. Several reported retroviral-pseudotyped lentiviruses have improved transduction efficiency on PNK but suffer from low functional viral titer during packaging. We used CRISPR-Cas9 RNP nucleofection to generate a modified HEK293T that can package high titer PNK permissive lentiviral vector (BaEVRless-LV), and a small molecule that can further boost viral titer. Also, we optimized the PNK transduction protocol, and we further demonstrated this platform could deliver and express large cargo gene (e.g., CAR). Finally, we were able to efficiently package large scale lentiviral-based CRISPR knockout library with decent sgRNA recovery. By adapting Cas9 RNP electroporation and lentiviral delivery of sgRNA, we were able to conduct the first Genome-wide CRISPR screen in primary NK cells and identify regulatory genes that are essential to maintain NK cell fitness under harsh immunosuppressive microenvironment.