Fig. 6: Functional interrogation of HOXA9-bound targets by genome editing.

A Competitive proliferation assay was conducted in Cas9-expressing SEM cells targeted with sgRNAs against HOXA9-bound sites close to the genes as PEBP4, ZCCNC7, AHI1, RUNX1, DCAF11, CDK6, NDUFS8, and MAN1C1. The sgRPS19 and sgFLT3-DE-1 sgRNA served as positive controls. Disruption of the loci targeted by these selected sgRNAs led to retarded cell growth of SEM cells in a time-dependent manner. The percentage of cell numbers was normalized to CFP⁺ control cells infected with non-target sgRNA (sgNT). B Competitive proliferation assay was conducted in Cas9-expressing MOLM13 cells targeted with sgRNAs against HOXA9-bound sites close to the genes as PEBP4, ZCCHC7, AHI1, RUNX1, DCAF11, CDK6, NDUFS8 and MAN1C1. The sgRPS19 and sgFLT3-DE-1 sgRNA served as positive controls. Disruption of the loci targeted by these sgRNAs led to retarded cell growth of MOLM13 cells in a time-dependent manner. The percentage of cell numbers was normalized to CFP⁺ control cells infected with non-target sgRNA (sgNT). C Competitive proliferation assay was conducted in dCas9-KRAB-expressing SEM cells targeted with sgRNAs against HOXA9-bound sites close to the genes RUNX1, DCAF11, and CDK6. The sgFLT3-DE-1 sgRNA served as positive control. D Competitive proliferation assay was conducted in dCas9-KRAB-expressing MOLM13 cells targeted with sgRNAs against HOXA9-bound sites close to the genes RUNX1, DCAF11, and CDK6. The sgFLT3-DE-1 sgRNA served as positive control. E Characterization of the chromatin conformation change upon dCas9-KRAB targeting against the HOXA9-bound site in the intron of CDK6. HiC (GEO: GSE138862), HOXA9 ChIP-seq, H3K27ac ChIP-seq and BRD4 ChIP-seq (GEO: GSE117864)⁷⁰ tracks were shown to characterize the epigenetic status of the HOXA9-bound site. F Q-PCR was conducted to quantify the transcription decrease of CDK6 when CRISPRi targeted the HOXA9-bound site in the intron of CDK6 in SEM cells. G Q-PCR was conducted to quantify the transcription decrease of CDK6 when CRISPRi targeted the HOXA9-bound site in the intron of CDK6 in MOLM13 cells. H Total RNA-seq was performed using SEM cells targeted with sgCDK6 against the HOXA9-bound site in the CDK6 intron. Differential gene expression was defined by FDR < 0.01. The CDK6 expression is the top hit. Data are shown as mean values ± SEM of three biological replicates. P values were estimated using a two-tail un-paired t-test. Source data are provided as a Source Data File.