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. 2024 Jun 26;32(8):2692–2710. doi: 10.1016/j.ymthe.2024.06.033

Figure 2.

Figure 2

Quantification of T cell target knockout in individual cells

(A–C) Flow cytometry histograms to show protein levels for CD52, TCRa/b, PD1, and B2M following co-delivery of Pin-point or SpCas9 mRNAs and their compatible four target sgRNAs in (A) non-activated and (C) activated T cells, analyzed 7 days post electroporation. Non-edited mock electroporated cells are used as control. In this comparison, optimal gRNAs for SpCas9 have been used and these differ in their spacer sequence from the optimal Pin-point gRNAs (further details in the materials and methods). Quantification of protein knockout reported as the percentage of (B) non-activated and (D) activated T cells expressing undetectable levels of the protein, normalized to the frequency of expression in mock electroporated cells. Data in (B) and (D) represented as mean (SEM), n = 4 independent biological T cell donors. Protein expression phenotypes within populations of (E) non-activated and (F) activated T cells either mock electroporated as controls or edited with either SpCas9 or the Pin-point platform. Columns are single cells classified as positive (colored) or negative (white) for the four target proteins with respect to the gates shown in (A) and (C). Color bar shows clusters of cells with similar patterns of expression of the protein targets (rows). k-means clustering, k = 16. Quantification of single, double, triple, and quadruple negative target protein expression in (G) non-activated and (H) activated T cells following co-delivery of Pin-point or SpCas9 mRNAs and four target gRNAs, analyzed by flow cytometry 7 days post electroporation. Control is mock electroporated T cells without RNA. Single cells are classified as either positive or negative for the four target proteins according to the gates shown in Figure S2. Data in (G) and (H) represented as mean (SEM), n = 4 independent biological T cell donors. (I) Fold expansion of T cells as measured by cell counts 3 days post co-delivery of Pin-point or SpCas9 mRNAs and three or four target gRNAs. Data represented as mean (SEM), n = 4 independent biological T cell donors. A paired student t-test was applied.