In this proof-of-principle study, we have demonstrated that dual intron-targeting CRISPR-Cas9-mediated disruption of RUNX1-RUNX1T1 leads to a reduction of chimeric RUNX1-RUNX1T1 fusion transcripts and a significant decrease in AML t(8;21) leukemic tumor cell proliferation and growth both in vitro and in vivo. This evidence concerns the gene RUNX1 and acute myeloid leukemia.