Induction of t(9;11) chromosomal translocations encoding MLL-AF9 fusion genes by CRISPR/Cas9 gene editing. (A) PCR amplification products for MLL-AF9 (upper panel) and AF9-MLL (lower panel) junction regions in genomic DNA harvested from genome-edited cells at day 31 of posttreatment culture. (B) Genomic DNA sequences of MLL-AF9 and AF9-MLL junctions in edited cells. sgRNA and PAM sequences are denoted by bold type and gray shading, respectively. ▼, Cas9 cutting position. (C) Karyotype analysis at day 41 of culture shows balanced t(9;11) translocation (red arrowheads). (D) FISH analysis of cells at day 41 of culture for MLL translocation using MLL break-apart probes. Red and green arrowheads indicate the split signals of the break-apart probe indicating MLL translocation. Yellow arrowheads represent nontranslocated MLL gene. (E) Expression of MLL-AF9 and reciprocal AF9-MLL transcripts (black arrowheads) were detected by RT-PCR on cDNA from 2 independent MLLr cell cultures (day 59). TALEN-induced MLLr cells⁹  and Cas9-treated human HSPCs are positive and negative controls, respectively. Exon skipping at MLL exon 11 in MLL-AF9 and at MLL exon 12 in AF9-MLL are indicated (white arrowheads). (F) cDNA sequences of MLL-AF9 junctions (upper left panel) and AF9-MLL junctions (upper right panel) in gene-edited cells. Exon-skipping at MLL exon 11 and exon 12 in MLL-AF9 and AF9-MLL, respectively (lower panels). (G) MLL-AF9 fusion proteins are visualized by western blot using anti-MLL antibody. Positive and negative controls are the same as in panel E. Wild-type (wt) MLL and MLL-AF9 proteins are indicated. GAPDH was used for loading control. *, nonspecific band. (H) Representative growth curves for MLLr cells compared with Cas9-alone nucleofection or HSPCs lacking CRISPR treatment. The average cell counts of 6 MLLr cell cultures are displayed. Error bars indicate 95% confidence intervals.