Fig 2: Loss of Zbtb1 and Cbfa2t3 leads to the T-lineage differentiation arrest in LPs.A, specific depletion of targeted Lmo2, Zbtb1, and Cbfa2t3 proteins. sgRNA against Lmo2, Zbtb1, Cbfa2t2, or Cbfa2t3 was introduced into the Cas9-expressing (GFP+) LPs. Four days after sgRNA transduction, nuclear lysates from retrovirus infected GFP+hNGFR+ cells were subjected to immunoblotting for Lmo2, Zbtb1, Cbfa2t3, Bcl11a, and LaminB. Two independent experiments were performed with similar results. B, an experimental scheme for the deletion of Lmo2, Zbtb1, Cbfa2t3, and Cbfa2t2 using the CRISPR/Cas9 system in Ebf1-deficient LPs is shown. C, retroviral vectors encoding sgRNAs against luciferase (sgControl), Lmo2 (sgLmo2), Zbtb1 (sgZbtb1), Cbfa2t3 (sgCbfa2t3), or Cbfa2t2 (sgCbfa2t2) were introduced into Cas9-expressing (GFP+) LPs. Five days after sgRNA introduction, LPs were transferred onto OP9-DLL4 stromal cells and cocultured for 2 days. GFP+hNGFR+ sgRNA transduced cells were gated and analyzed for CD44 and CD25 expression. D, the percentage of CD25+ cells among GFP+hNGFR+ sgRNA transduced cells (C) is shown with standard deviation (SD). E, ten days after sgRNA introduction, LPs were transferred onto OP9-DLL4 stromal cells and cocultured for 2 days. GFP+hNGFR+ sgRNA-transduced cells were gated and analyzed for CD44 and CD25 expression. F, the percentage of CD25+ cells among GFP+hNGFR+ sgRNA transduced cells (E) is shown with SD. Data are representative of two (A) or three (C and E) independent experiments. Data represent the mean values of three independent biological replicates (D and F). **p < 0.01 by two-sided Student’s t test. LP, lymphoid progenitor; sgRNA, single-guide RNA.