CRISPR-Cas9–induced t(4;11)/MA4 targeting either MLLⁱ¹⁰ (Mⁱ¹⁰A4) or MLLⁱ¹² (Mⁱ¹²A4) in human FL and CB CD34⁺ HSPCs causes MA4-driven myeloid immortalization in vitro. (A) Scheme showing the location of the molecular regions targeted by single-guide MLLⁱ¹⁰ (sgMLLⁱ¹⁰) or sgMLLⁱ¹² and sgAF4ⁱ³ sgRNAs (top) and the resulting t(4;11)/MA chromosomal translocation (bottom). Filled light or dark gray/orange boxes depict exons and the lines between boxes depict introns. (B) Cartoon of the experimental design for in vitro studies. (C) Representative 60-day cell expansion of control and edited (Mⁱ¹⁰A4 and Mⁱ¹²A4) FL (left) and CB (right) CD34 myeloid progeny (CD45⁺CD34^–CD19^–CD33⁺). Two additional independent experiments for each cell of origin are shown in supplemental Figure 1A-B. (D) Representative G-banding karyotype confirming the presence of chromosomal translocation after 42 days of culture. Enlarged images of the translocated chromosomes are also shown. (E) MLL split-apart iFISH quantification of MLL-edited cells in control and edited cell cultures at the indicated time points, for FL (left) and CB (middle); right panels show representative images of MLLr and MLL germ line cells. The centromeric portion of the MLL gene breakpoint cluster region (bcr) is labeled in green, and the telomeric portion of the bcr is labeled in orange. Scale bars represent 10 μm.