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Genetic modification of NK cells to enhance cancer immunotherapy has potential application to treat a wide range of cancers. Recently, we developed a new strategy in which CRISPR/Cas9 elements are introduced into NK cells as ribonucleoproteins (RNPs) via electroporation, followed by expansion on feeder cells expressing 4-1BBL and membrane-bound IL-21 to generate large numbers of genetically modified NK cells. This method was used to genetically modify several genes in primary and expanded NK cells including suppressor of cytokine signaling 3 (SOCS3). SOCS3 negatively regulates cytokine signaling through the JAK/STAT pathway. We hypothesized that disruption of SOCS3 in primary NK cells using Cas9/RNPs could potentially maintain STAT3 signaling levels and subsequently increase their proliferation and cytotoxic function. We designed gRNAs to target exon 2 of the SOCS3 gene and electroporated them along with Cas9 protein as Cas9/RNPs into primary NK cells using the Lonza 4D electroporator. We tested 6 different conditions of gRNAs alone or in combination. The NK cells in the control group were electroporated with no Cas9/RNPs. After electroporation, the cells were rested in culture media supplemented with 100 IU of human IL-2 for 48 hours and then expanded using irradiated feeder cells. At day 7, an equal number of cells were restimulated with irradiated feeder cells to test the effect of SOCS3 KO on proliferation. Western blot was used to assay the knock out efficacy at the protein level. Calcein assay and IncuCyte Zoom (Essen) were performed to measure cytotoxicity against two cancer cell lines, K562 and Daoy. Our results showed a significant reduction in SOCS3 protein levels in 3 conditions (gRNA1, gRNA3 and gRNA1+ gRNA3) in comparison to the control group. The calcein assay and IncuCyte zoom showed the modified SOCS3 KO NK cells could kill the tumors more efficiently in comparison to the control. Proliferation data showed the SOCS3 KO cells can grow faster than the control group. In conclusion, our data demonstrate the role of SOCS3 and JAK/STAT pathway in NK cell function and suggests that SOCS3 may be a good target for genetic modification to improve cancer immunotherapy using NK cells. Disclosures Lee: CytoSen Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck, Sharp, and Dohme: Consultancy; Courier Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.