T cell developmental analysis in Cas9-LPs. (A) Experimental scheme (upper panel). Cas9-LPs were cocultured with OP9-DLL4, and the T cell developmental status was scored using the T-progenitor markers CD44 and CD25 among CD45⁺ cells at different time points after Notch stimulation (lower panel). (B) Cas9-LPs (5 × 10⁶, CD45.2) were mixed with BM cells from wild-type C57BL/6 congenic mice (2 × 10⁵, CD45.1) and transplanted into lethally irradiated (9 Gy) Rag2- and Cg-deficient lymphopenic hosts. Recipient mice were analyzed 8 wk after transplantation. Flow cytometric analysis of the thymocytes and splenocytes was performed. The representative CD4 and CD8 profiles of CD45.2⁺ thymocytes (left) and splenocytes (right) are shown. Results are representative of three independent biological replicates. (C) LP and DN subsets derived from Cas9-LPs were subjected to transcriptome analysis. TPM values for Spi1, Tcf7, and Bcl11b in DN subsets from Cas9-LP (upper) and in vivo thymic DNs (lower) are shown with SD (https://www.immgen.org; GSE100738) (Yoshida et al., 2019). (D) LP and DN subsets derived from Cas9-LPs were subjected to untargeted proteome analysis. Relative protein levels for PU.1, TCF1, and Bcl11b are shown, including their SDs. SD, standard deviation.