Figure 5.
Functional CRISPR screening identifies CEBPA cisRE as a key regulator of myeloid differentiation in high-risk leukemia. (A) Schematic overview of the CRISPR screens used to functionally interrogate MECOM-regulated cisREs. An sgRNA oligo library was designed against MECOM-regulated elements (up to 5 sgRNAs per element, depending on the availability of high-quality sgRNA-targeting sites) and packaged into a lentiviral vector. Two different populations of MUTZ-3 MECOM-FKBP12F36V cells were then transduced with this sgRNA library virus at an MOI of ∼0.33, in which one population expressed dCas9-KRAB (CRISPRi screen) and another expressed dCas9-VPR (CRISPRa screen). Cells in the CRISPRi screen were treated with 500 nM dTAGV-1 for the duration of the screen. After 14 days of in vitro culture, cells from the CRISPRi and CRISPRa screens were sorted for phenotypically rescued CD34+ cells (up-assay) and differentiated CD34– cells (down-assay), respectively. Genomically integrated sgRNAs were sequenced to assess relative sgRNA abundance. Both screens were performed with n = 3 independent replicates. (B-C) Volcano plots depicting sgRNA enrichment/depletion from sorted populations compared to plasmid library DNA (supplemental Table 8). The sgRNA library included sgRNAs targeting the TSSs of VHL, ELOB, and ELOC (5 sgRNAs per gene), which form the E3 ubiquitin-ligase complex recruited by dTAGV-1. (D) Genome browser tracks at the CEBPA locus encompassing the +42 kb cisRE. ATAC-seq tracks from MECOM-FKBP12F36V cell line models and MECOM ChIP-seq demonstrate increased chromatin accessibility upon dTAGV-1 treatment. Three top-scoring CEBPA cisRE-targeting sgRNAs were selected for single sgRNA validation experiments. (E) MUTZ-3 dCas9-KRAB cells were infected with sgRNA-expressing lentiviruses targeting either the CEBPA cisRE or a nontargeting sequence. At 48 hours after transduction, cells were treated with 500 nM dTAGV-1 vs DMSO. Histogram shows CD34 expression at day 9 (top). Percentage of CD34+ cells at day 9 (bottom). n = 3 independent replicates. Mean and SEM are shown. (F) qRT-PCR of CEBPA expression in dTAGV-1–treated cells 3 days posttreatment. Fold change represents ΔΔCt values compared to the sgNT condition. n = 3 independent replicates. Mean and SEM are shown. Two-sided Student t test was used for comparisons. ∗∗∗∗P < .0001. (G) MUTZ-3 dCas9-VPR cells were infected with sgRNA-expressing lentiviruses targeting either the CEBPA cisRE or a nontargeting sequence. Histogram shows CD34 expression at day 9 (top). Percentage of CD34+ cells at day 9 (bottom). n = 3 independent replicates. Mean and SEM are shown. (H) qRT-PCR of CEBPA expression in all conditions 3 days posttransduction. Fold change represents ΔΔCt values compared to the sgNT condition. n = 3 independent replicates. Mean and SEM are shown. Two-sided Student t test was used for comparisons. ∗∗∗∗P < .0001. FACS, fluorescence-activated cell sorted; FSC-A, forward scatter area; MOI, multiplicity of infection; qRT-PCR, quantitative reverse transcription polymerase chain reaction; sgNT, nontargeting sgRNA.

Functional CRISPR screening identifies CEBPA cisRE as a key regulator of myeloid differentiation in high-risk leukemia. (A) Schematic overview of the CRISPR screens used to functionally interrogate MECOM-regulated cisREs. An sgRNA oligo library was designed against MECOM-regulated elements (up to 5 sgRNAs per element, depending on the availability of high-quality sgRNA-targeting sites) and packaged into a lentiviral vector. Two different populations of MUTZ-3 MECOM-FKBP12F36V cells were then transduced with this sgRNA library virus at an MOI of ∼0.33, in which one population expressed dCas9-KRAB (CRISPRi screen) and another expressed dCas9-VPR (CRISPRa screen). Cells in the CRISPRi screen were treated with 500 nM dTAGV-1 for the duration of the screen. After 14 days of in vitro culture, cells from the CRISPRi and CRISPRa screens were sorted for phenotypically rescued CD34+ cells (up-assay) and differentiated CD34– cells (down-assay), respectively. Genomically integrated sgRNAs were sequenced to assess relative sgRNA abundance. Both screens were performed with n = 3 independent replicates. (B-C) Volcano plots depicting sgRNA enrichment/depletion from sorted populations compared to plasmid library DNA (supplemental Table 8). The sgRNA library included sgRNAs targeting the TSSs of VHL, ELOB, and ELOC (5 sgRNAs per gene), which form the E3 ubiquitin-ligase complex recruited by dTAGV-1. (D) Genome browser tracks at the CEBPA locus encompassing the +42 kb cisRE. ATAC-seq tracks from MECOM-FKBP12F36V cell line models and MECOM ChIP-seq demonstrate increased chromatin accessibility upon dTAGV-1 treatment. Three top-scoring CEBPA cisRE-targeting sgRNAs were selected for single sgRNA validation experiments. (E) MUTZ-3 dCas9-KRAB cells were infected with sgRNA-expressing lentiviruses targeting either the CEBPA cisRE or a nontargeting sequence. At 48 hours after transduction, cells were treated with 500 nM dTAGV-1 vs DMSO. Histogram shows CD34 expression at day 9 (top). Percentage of CD34+ cells at day 9 (bottom). n = 3 independent replicates. Mean and SEM are shown. (F) qRT-PCR of CEBPA expression in dTAGV-1–treated cells 3 days posttreatment. Fold change represents ΔΔCt values compared to the sgNT condition. n = 3 independent replicates. Mean and SEM are shown. Two-sided Student t test was used for comparisons. ∗∗∗∗P < .0001. (G) MUTZ-3 dCas9-VPR cells were infected with sgRNA-expressing lentiviruses targeting either the CEBPA cisRE or a nontargeting sequence. Histogram shows CD34 expression at day 9 (top). Percentage of CD34+ cells at day 9 (bottom). n = 3 independent replicates. Mean and SEM are shown. (H) qRT-PCR of CEBPA expression in all conditions 3 days posttransduction. Fold change represents ΔΔCt values compared to the sgNT condition. n = 3 independent replicates. Mean and SEM are shown. Two-sided Student t test was used for comparisons. ∗∗∗∗P < .0001. FACS, fluorescence-activated cell sorted; FSC-A, forward scatter area; MOI, multiplicity of infection; qRT-PCR, quantitative reverse transcription polymerase chain reaction; sgNT, nontargeting sgRNA.

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