Effects of Runx1 or GATA3 perturbations on Spi1 expression in postcommitment pro–T stages. (A) To test Cas9-mediated disruption, Scid.adh.2c2 cells already expressing Cas9 from a GFP⁺ vector were transduced with sgRunx1 or control in an hNGFR⁺ vector or with sgGata3 or control in a CFP⁺ vector. 3 d after sgRNA transduction, lysates from the retrovirus-infected Cas9-GFP⁺CFP⁺ or Cas9-GFP⁺hNGFR⁺ Scid.adh.2c2 cells were subjected to immunoblotting (IB) for Runx1 (top) and GATA3 (bottom), respectively. Two independent experiments were performed with similar results. (B) Flow cytometric analyses of retrovirus-infected Lin⁻CD45⁺CFP⁺hNGFR⁺ postcommitment primary cells were performed at 4 d after transduction using protocol B. Gating strategies for Lin⁻CD45⁺GFP⁺hNGFR⁺ cells are shown (top). Representative profiles of CD44/CD25 in Lin⁻CD45⁺GFP⁺hNGFR⁺ cells are shown (bottom). Gates to define CD44^lo cells for sorting are labeled with red rectangles. Results are representative of three independent experiments. (C) Lin⁻CD45⁺CFP⁺hNGFR⁺ cells that had been transduced with sgRNAs before commitment were subjected to flow cytometric analysis at 4 d after transduction using protocol C. Gating strategies for Lin⁻CD45⁺GFP⁺hNGFR⁺ cells are shown (top). Representative profiles of CD44/CD25 in Lin⁻CD45⁺GFP⁺hNGFR⁺ cells are shown (bottom). CD25⁺ cells for sorting were labeled with red rectangles. The percentages of CD25⁺CD44^lo cells are indicated with SD. **, P < 0.01 by two-sided Student’s t test for the indicated sample pairs. Data are representative of two independent experiments and based on three biological replicates in an experiment.