Fig 1: Successful generation of CD33CAR-expressing NK cells using combination of Cas9/RNP and AAV6(A) Schematic of anti-CD33 CAR constructs (Gen2 and Gen4v2) with HAs for AAVS1-targeting site and cloned in ssAAV.(B) Schematic of CAR protein structural design.(C) Representative flow cytometry showing the expression level of CD33CAR on NK cells 7 days after Cas9/RNP electroporation and AAV6 transduction (MOI = 3 × 105).(D) Mean fluorescence intensity (MFI) of CD33CAR expression for Gen2 versus Gen4v2 (p = 0.0014, unpaired t test).(E) CD33CAR expression level on NK cells 7 and 14 days after transduction and electroporation (n = 3, n.s. from two-way ANOVA adjusted for multiple comparisons).(F) Fold expansion of CD33CAR-expressing NK cells on feeder cells for 14 days starting from 3 × 105 cells (n = 3, n.s. from one-way ANOVA adjusted for multiple comparisons). Data are shown as mean ± SD. Significant p values indicated are from individual ratio paired t tests and are not adjusted for multiple comparisons. ∗∗p < 0.01.