Effect of targeting the +24 kb enhancer region of RUNX1 by CRIPSR/Cas9. (A) sgRNAs were developed by using the CRISPR/Cas9 target online predictor (CCTOP) algorithm and cloned into a lentiviral vector (pLenti-sgRNA). Locations and orientation of sgRNAs used are marked with a blue arrow. Pooled lentiviral particles were transduced into OCI-AML5 cells, and sgRNAs were stably selected with puromycin. Adenoviral-expressed Cas9 was transduced into stable selected sgRNA-expressing OCI-AML5 cells, and the cells were grown for 10 passages. (A) Signal density plot of a region within the +24 kb enhancer in OCI-AML5 cells with sgRNA alone and sgRNA+ Cas9 showing indels in the surviving clones, OCI-AML5 control cell ATAC-Seq, MOLM14 BRD4 ChIP-Seq, and ENCODE K562 transcription factor (TF) ChIP-Seq data sets. (B) Immunoblot analysis of RUNX1 in OCI-AML5 cells transduced with sgRNAs and/or Cas9 after 10 passages. The expression levels of β-actin in the lysates served as the loading control. (C) Cell cycle status of OCI-AML5 cells stably transduced with sgRNAs vs those transduced with sgRNAs and Cas9. *Indicates G0/G1 and S phase values that are significantly altered in the sgRNA- and Cas9-transduced cells vs cells transduced with only sgRNAs (P < .05). (D) Cell proliferation of OCI-AML5 cells with sgRNA and sgRNA+ Cas9 cells over 72 hours. *Indicates values that are significantly less in OCI-AML5 cells after Cas9 transduction (P < .05).