Efficient CRISPR/Cas9 deletions of platelet function genes are maintained in CD34⁺-derived MKs. (A) Overview of strategy. CD34⁺ cells isolated from cord blood were transfected on day 5 (D5) (unless otherwise noted) with RNP complexes containing tracrRNA, gene targeting crRNA, and Cas9 protein. MK assays, including assays normally used to measure platelet (PLT) functional responses, were performed on day 13 of culture. (B) CD34⁺ cells were transfected on different days of culture with negative (Neg) control or ITGA2B crRNA. Surface expression of αIIb (% CD41⁺ cells; see Figure 2B for gating) was measured by flow cytometry on day 13 MKs. (C-F) Western blot and densitometry analysis of proteins on day 13 MKs after targeting by negative control or the indicated gene specific CRISPR on day 5. Data are presented as mean ± standard error of the mean (SEM) (3 independent cords per group). Unpaired Student t test: *P < .05; **P < .01; ***P < .001. CalDAG, calcium diacylglycerol; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.