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. 2023 Apr 13;72(8):2573–2583. doi: 10.1007/s00262-023-03443-1

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© The Author(s) 2023

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — Knockout CD19-CAR-NK92 cell clones. A Graphic abstract of the generation of knockout cell clones. CD19-CAR-NK92 cells were initially transduced with Cas9-coding plasmid (day 0). After Blasticidin selection (day 3–14), gRNA-coding plasmids (for FasL- or Prf1-binding or empty plasmid (reference)) were delivered in Cas9-equipped cells (day 14), followed by Neomycin selection. Creation of the single-cell clones begun after Blasticidin and Neomycin selection for 5 weeks (week 7). Single-cell clones were analyzed for success of knockouts after expanding for at least 4 weeks (week 11). The gRNA-complementary DNA sequences are shown in the box (underlined), the start codon is marked green. B Western blotting of perforin-, FasL-, TRAIL- and α-Tubulin-expression of the created knockout cell clones (3–10). For each knockout experiment, two single-cell clones were analyzed. Corresponding pairs of the cell clones are pictured one below the other (3/7; 4/8; 5/9; 6/10). Wildtype NK92 (1) and CD19-CAR-NK92 cells (2) were used as relation cells. C Histograms of CD19-CAR expression of the knockout cell clones. The corresponding pairs are indicated in the same color (green: reference clones, red: Prf1-ko clones, blue: FasL-ko clones, yellow: double-ko clones)