Generation of CRISPR-engineered CD38^KO/CD38-CAR human primary NK cells using Cas9/RNP and AAV. (A) Schemata of steps for CRISPR/RNP knockout of the CD38 gene and directed insertion of a CD38-CAR encoding DNA delivered by AAV6 vector with homology arms for CD38 targeting site. (B) CD38 (PE) and CAR (APC) expression levels measured by flow cytometry for binding of CD38 antigen, 7 days after stimulation. Constructs contain a 41BB signaling domain, a CD8α transmembrane domain/hinge, a CD3ζ stimulatory domain, and reversed orderings of light and heavy chain orientations. (C) Relative percentage and intensity of CD38-CAR expression (n = 10; mean ± standard deviation [SD]). (D) Fold expansion of WT and CD38-CAR NK cells over 12 days after activation with irradiated, modified mbIL21-K562 cells and IL-2 show no significant change from WT human NK cells (n = 10; mean ± SD). P values were calculated using a 2-way analysis of variance (ANOVA); ∗P = .0332; ∗∗P = .0021; ∗∗∗P = .0002; ∗∗∗∗P < .0001. (E) Cytotoxicity observed for V3 and V4 CD38^KO/CD38-CAR NK cells against high CD38-expressing MM (H929), BL (Raji), and AML (MV-11) (n = 5; mean ± SD). P values were calculated using a 2-way ANOVA; ∗P < .05; ∗∗P < .01; ∗∗∗P = .001; ∗∗∗∗P < .0001.