In an experimental study using the CRISPR/Cas9 system, "enhanced" NK cell lines with knockout of CISH, the gene for the CIS protein (a negative regulator of NK cytotoxicity), as well as two lines with a knocked-out β2-microglobulin gene, which provides membrane exposure of MHC class I, were obtained from two parental lines of human natural killers (YT wild type and YT-VAV1⁺ overexpressing the VAV1 cytotoxicity enhancing protein). The knockout efficiency was determined by real-time PCR as well as by flow cytometry with specific antibodies. The resulting CISH^-/- or B2M^-/- knockout lines were tested for cytotoxicity in primary monolayer cultures of human glioblastoma multiforme. The cytotoxicity of the lines was assessed using a cell analyzer that records the cell index based on cell impedance. YT-CISH^-/- has been shown to be significantly more effective than wild-type YT in eliminating primary glioblastoma cells in an in vitro cell monolayer experiment. The cytotoxicity of the YT-VAV1⁺-CISH^-/- and YT-VAV1⁺B2M^-/- lines against glioblastoma cells was the highest, but overall, it did not significantly differ from the initially increased cytotoxicity of the YT-VAV1⁺ line. The lines of NK-like cells obtained may serve as a prototype for the creation of "enhanced" allogeneic and autologous NK- and CAR-NK cells for the immunotherapy of glioblastoma multiforme.