Functional inactivation of Cadm1 in mice. (A) Experimental procedure to study the effect of Cadm1 edition on murine hematopoietic development (left panel). LSK cells from Cas9-expressing mice were purified by cell sorting (right panel) and transduced with either sgCadm1#2 or control sgRNA (sgCTL) lentiviral vectors. Transduced Cas9-expressing LSK cells (CD45.2⁺) were either plated in clonogenic assay or transplanted into syngenic recipient mice (CD45.1⁺, n = 3 for each condition). (B) Number of total CFU-GEMM, CFU-GM, and BFU-E colonies in clonogenic assay. (C) BM chimerism, analyzed by FACS 2 months after transplantation, illustrated by the percentage of transduced donor cells (% CD45.2⁺GFP⁺) found in the recipient BM (upper panel). Immunophenotype of engrafted donor-derived cells (CD45.2⁺GFP⁺) was analyzed using the Gr1 (Myeloid cells, My) and CD19 (B-cells) markers. FACS profiles and gating strategy were shown for 1 representative mouse of each condition (lower panel). (D) Proportion of myeloid and B cells (%) within total donor-derived CD45.2⁺GFP⁺ cells from the BM of recipient mice. (E-F) Donor-derived cells (CD45.2⁺GFP⁺) from each sgCTL and sgCadm1 transplanted mouse purified by cell sorting. Genome editing efficiency of targeted Cadm1 region performed on purified donor-derived cells (CD45.2⁺GFP⁺) for each condition (E). Cytological analysis was performed on purified CD45.2⁺GFP⁺ cells from each condition, and the proportion of myeloid and lymphoid cells was calculated (F).