Pooled CRISPR-Cas9–KO library screening identified a CBS7/9 boundary critical for posterior HOXA expression in AML cells. (A) Schematic diagram representing the pooled CRISPR-Cas9–KO library screening of the entire 4 HOX gene loci for CTCF boundary function in MLL-AF9–rearranged MOLM13 AML cells. (B) One-step RT-droplet digital PCR screening of HOXA9 expression in single clones infected with lentivirus containing the sgRNA library. Shown is the screening of 528 sgRNA library–infected clones for HOXA9 expression levels. (C) RT-droplet digital PCR analysis of HOXA9 levels in WT MOLM13 cells and the 21 clones containing single targeted sgRNA. HOXA9 expression data were grouped into 5 groups in accordance with the categories of sgRNA sequences: HOXA7/9 CTCF site, nonhuman targets, other CTCF sites in the HOX loci, HOX-associated lincRNAs, and other human targets. (D) The SURVEYOR nuclease assays of mutations occurred in the CBS7/9 site from the representative clones that exhibited reduced (red line), unchanged (blue line), or increased (purple line) levels of HOXA9 expression. The HOXA9-decreased clones 5, 6, 28, and 121 exhibited mutations in the CBS7/9 boundary. (E) ChIP analysis of CTCF binding across the HOXA locus in MOLM13 cells compared with the WT control and the CBS7/9^+/− clone. Data are mean ± SD from 3 or 4 independent experiments. (F) Western blot analysis of CTCF protein levels compared with the WT control and the CBS7/9^+/− MOLM13 clone. *P < .05, **P < .01, Student t test. ns, not significant.