Figure 1.

Efficient Itga2b knockout in bona fide HSCs

(A) A schematic diagram of serial transplantation of mouse bone marrow Lin⁻ cells (i.e., an HSC-containing fraction), which were electroporated with Cas9/RNP-a2b^ex1 to lethally irradiated C57BL/6J mice. (B) Surveyor assays of Itga2b exon 1 in Lin⁻ cells after transplantation. Asterisks indicate digestion products. (C, E, and G) Representative flow cytometric results showing gating of platelets (positive for CD42B) and successful knockout of ItgαIIb in those platelets isolated from primary (C), secondary (E), and tertiary recipients (G) after serial transplantation of Cas9/RNP-a2b^ex1-treated Lin⁻ cells (open histogram, transplantation of mock Lin⁻ cells as negative controls; filled-purple histogram, transplantation of Lin⁻ cells electroporated with Cas9/RNP-a2b^ex1). (D, F, and H) Flow cytometric results of ItgαIIb⁻ platelets in all mice at 4 to 12 weeks after primary to tertiary transplantation showing the knockout efficiency was around 15% even after tertiary transplantation (black circle, transplantation of mock Lin⁻ cells as negative controls; open circle, transplantation of Lin⁻ cells electroporated with Cas9/RNP-a2b^ex1). Mann-Whitney one-tailed U test: ∗p < 0.05; ∗∗p < 0.01.