Background The oncogenic protein HOXA9 plays a critical role in leukemia transformation and maintenance, and its aberrant expression is a hallmark of most aggressive acute leukemia. Although inhibiting the upstream regulators of HOXA9 has been proven as a significant therapeutic intervention, the comprehensive regulation network controlling HOXA9 expression in leukemia has not been systematically investigated. Results Here, we perform genome-wide CRISPR/Cas9 screening in the HOXA9-driven reporter acute leukemia cells. We identify a poorly characterized RNA-binding protein, RBM5, as the top candidate gene required to maintain leukemia cell fitness. RBM5 is highly overexpressed in acute myeloid leukemia (AML) patients compared to healthy individuals. RBM5 loss triggered by CRISPR knockout and shRNA knockdown significantly impairs leukemia maintenance in vitro and in vivo. Through domain CRISPR screening, we reveal that RBM5 functions through a noncanonical transcriptional regulation circuitry rather than RNA splicing, such an effect depending on DNA-binding domains. By integrative analysis and functional assays, we identify HOXA9 as the downstream target of RBM5. Ectopic expression of HOXA9 rescues impaired leukemia cell proliferation upon RBM5 loss. Importantly, acute protein degradation of RBM5 through auxin-inducible degron system immediately reduces HOXA9 transcription. Conclusions We identify RBM5 as a new upstream regulator of HOXA9 and reveal its essential role in controlling the survival of AML. These functional and molecular mechanisms further support RBM5 as a promising therapeutic target for myeloid leukemia treatment.

Accumulating evidence indicates that HOXA9 dysregulation is necessary and sufficient for leukemic transformation and maintenance. However, it remains largely unknown how HOXA9, as a homeobox transcriptional factor, binds to noncoding regulatory sequences and controls the downstream genes. Here, we conduct dropout CRISPR screens against 229 HOXA9-bound peaks identified by ChIP-seq. Integrative data analysis identifies reproducible noncoding hits, including those located in the distal enhancer of FLT3 and intron of CDK6. The Cas9-editing and dCas9-KRAB silencing of the HOXA9-bound sites significantly reduce corresponding gene transcription and impair cell proliferation in vitro, and in vivo by transplantation into NSG female mice. In addition, RNA-seq, Q-PCR analysis, chromatin accessibility change, and chromatin conformation evaluation uncover the noncoding regulation mechanism of HOXA9 and its functional downstream genes. In summary, our work improves our understanding of how HOXA9-associated transcription programs reconstruct the regulatory network specifying MLL-r dependency.

MLL-rearranged (MLL-r) leukemias count for more than 80% of infant leukemia, ~5-10% of B-cell acute lymphoblastic leukemia (B-ALL), and ~10% of acute myeloid leukemia (AML) cases, where they confer a particularly poor outcome. Despite treatment with intensive multi-agent chemotherapy, most MLL-r patients ultimately relapsed after an initial remission. So far, the molecular mechanism by which MLL-r leukemia maintains progression and prevents differentiation remains largely unclear. Observations in the clinic support aberrant expression and concomitant activating driver mutations in the gene encoding the tyrosine kinase FLT3 that occur in leukemia, including the MLL-r subtype. To interrogate the novel MLL-r vulnerable genes regulating FLT3, we developed an algorithm so-called “context-dependent association analysis” (CDAA) to explore potential MLL-r dependent FLT3 regulators using the DepMap dataset. The RNA-binding protein MBNL1 was identified as the top candidate. MBNL1 has been recognized as an RNA-binding protein (RBP) involved in splicing, RNA export, and stability. However, our study suggested its non-canonical transcriptional activator function by uniquely controlling FLT3 expression in MLL-r leukemias. CRISPR/Cas9 disruption of the coding region of MBNL1 or CRISPR-interference (CRISPRi) against the MBNL1-bound site in the FLT3 distal enhancer notably decreased FLT3 mRNA expression in MLL-r leukemia cell lines. However, the FLT3 gene splicing defect was not observed upon MBNL1 loss, suggesting MBNL1 could be affecting FLT3 expression through a transcriptional regulation mechanism. Genome-wide ChIP-seq assays performed in MLL-r SEM and MOLM13 cells identified only two reproducible MBNL1-bound peaks at a genome-wide scale, one of which was located in the distal enhancer of FLT3, ~170kb away from the FLT3 promoter. CRISPR-interference (CRISPRi) against the MBNL1-bound site in the FLT3 distal enhancer notably decreased FLT3 mRNA expression in MLL-r leukemia cell lines without affecting enhancer-promoter looping. Using in vivo assay, SEM cells targeted with sgRNA against the MBNL1-bound site in the distal enhancer of FLT3 transplanted into NSG mice exhibited significant growth retardation. Furthermore, the impaired cell fitness crisis phenotype could be entirely rescued by overexpression of FLT3 cDNA. To identify the protein domains of MBNL1 required for its oncogenic function in MLL-r leukemia cells, an MBNL1 domain CRISPR screen was performed on the SEM cell line, stably expressing Cas9 and a pooled sgRNA library targeting the coding region of MBNL1. Essential domains observed from the screen included MBNL1's zinc finger domains. A biochemistry electrophoretic mobility shift assay (EMSA) was conducted using purified recombinant MBNL1 protein and a 207 bp DNA oligo synthesized based on the MBNL1 ChIP-seq peak at the FLT3 enhancer. The result clearly demonstrated that MBNL1 could directly bind to the enhancer DNA of FLT3 through its zinc fingers 1 and 2 domains, supporting its role as a novel transcriptional regulator of FLT3. Using state-of-art research tools, including genome editing, ChIP-seq, and EMSA assays, we have systematically interrogated an undocumented transcriptional regulation axis of MBNL1/FLT3 in MLL-r leukemias. This work will significantly advance our understanding of how transcription programs and novel factors reconstruct the regulatory network specifying MLL-r dependency, promoting the future development of alternative therapeutic targets for MLL-r leukemia in patients.

Bromo-and extra-terminal domain inhibitors (BETi) have exhibited therapeutic activ-ities in many cancers. However, the mechanisms controlling BETi response and resist-ance are not well understood. We conducted genome-wide loss-of-function CRISPR screens using BETi-treated KMT2A-rearranged (KMT2A-r) cell lines. We revealed that Speckle-type POZ protein (SPOP) gene (Speckle Type BTB/POZ Protein) deficiency caused significant BETi resistance, which was further validated in cell lines and xenograft models. Proteomics analysis and a kinase-vulnerability CRISPR screen indicated that cells treated with BETi are sensitive to GSK3 perturbation. Pharmaceutical inhibition of GSK3 reversed the BETi-resistance phenotype. Based on this observation, a com-bination therapy regimen inhibiting both BET and GSK3 was developed to impede KMT2A-r leukemia progression in patient-derived xenografts in vivo. Our results revealed molecular mechanisms underlying BETi resistance and a promising combi-nation treatment regimen of ABBV-744 and CHIR-98014 by utilizing unique ex vivo and in vivo KMT2A-r PDX models.

Background CTCF is a well-established chromatin architectural protein that also plays various roles in transcriptional regulation. While CTCF biology has been extensively studied, how the domains of CTCF function to regulate transcription remains unknown. Additionally, the original auxin-inducible degron 1 (AID1) system has limitations in investigating the function of CTCF. Results We employ an improved auxin-inducible degron technology, AID2, to facilitate the study of acute depletion of CTCF while overcoming the limitations of the previous AID system. As previously observed through the AID1 system and steady-state RNA analysis, the new AID2 system combined with SLAM-seq confirms that CTCF depletion leads to modest nascent and steady-state transcript changes. A CTCF domain sgRNA library screening identifies the zinc finger (ZF) domain as the region within CTCF with the most functional relevance, including ZFs 1 and 10. Removal of ZFs 1 and 10 reveals genomic regions that independently require these ZFs for DNA binding and transcriptional regulation. Notably, loci regulated by either ZF1 or ZF10 exhibit unique CTCF binding motifs specific to each ZF. Conclusions By extensively comparing the AID1 and AID2 systems for CTCF degradation in SEM cells, we confirm that AID2 degradation is superior for achieving miniAID-tagged protein degradation without the limitations of the AID1 system. The model we create that combines AID2 depletion of CTCF with exogenous overexpression of CTCF mutants allows us to demonstrate how peripheral ZFs intricately orchestrate transcriptional regulation in a cellular context for the first time.

HOXA9 overexpression is observed in 50-70% of human acute myeloid leukemia (AML) and a subset of acute lymphoblastic leukemia (ALL) and positively correlates with poor patient outcomes. Leukemia subtypes with hallmark overexpression of HOXA9 include those carrying MLL gene rearrangements (MLL-r), NPM1c mutations, and other genetic alterations. Frequent genetic translation of NUP98-HOXA9 retained the DNA binding domain of HOXA9, suggesting the transcription factor function is essential for leukemia initiation and maintenance. Accumulating evidence indicates that HOXA9 dysregulation is sufficient and necessary for leukemic transformation. However, HOXA9 protein itself is a poor therapeutic target as it lacks targetable binding domains. Therefore, understanding HOXA9's functional downstream targets will provide alternative therapeutic targets. However, it remains largely unknown how HOXA9, as a homeobox transcriptional factor, binds to noncoding regulatory sequences and controls the downstream genes in MLL-r leukemia and across leukemias of other genetic subtypes. In this study, we have successfully established a HOXA9-miniAID expressing MLL-r B-ALL cell line to execute acute HOXA9 degradation in the same isogenic cell line background upon auxin treatment. Compared with conventional loss-of-function strategies, our AID cellular model allows the investigation of immediate downstream targets controlled by HOXA9. We have therefore identified about 1,800 reproducible peaks bound by HOXA9 at a genome-wide scale. About 80% of HOXA9 binding peaks were co-bound by HOXA9's binding partner MEIS1, and motif enrichment confirmed that more than 75% of peaks contained a typical HOXA9 consensus motif. Interestingly, >80% binding peaks are located in non-promoter cis-regulator elements. Given the essential role of HOXA9 in regulating MLL-r leukemia cell survival and differentiation, we reasoned that targeting specific HOXA9-bound peaks will affect downstream gene expression leading to a similar survival crisis seen in HOXA9 knockout cells. Based on this rationale, combinatorial CRISPR screens were performed in three biological replicates on the MLL-r SEM cell line stably expressing Cas9 and base editor (ABE8.0), followed by time-course dropout selection on days 7, 14, and 21. Integrative CRISPR screen analysis identified six reproducible noncoding hits, including a positive control located in the distal enhancer of FLT3. Functional validation by targeting these six noncoding segments with Cas9, dCas9-KRAB in additional MLL-r leukemia cell lines MOLM13, MV4,11 confirmed the survival essential of these HOXA9-bound regions. No cellular phenotype was observed in non-MLL-r leukemia cell lines, including NALM6, JURKAT, or OCI-AML2. Transplanting SEM cells targeted with sgHOXA9-in-FLT3 into NSG mice, significant growth retardation was monitored by flow analysis of peripheral blood. In addition, RNA-seq and Q-PCR analysis further identified the functional relevant downstream genes upon CRISPR editing of these HOXA9-bound noncoding segments, including FLT3, XBP1, JUN, BAHCC1, and CCDC200. Chromatin conformation characterization by Capture-C further confirmed the long-range interaction between HOXA9 bound enhancers and corresponding gene promoters in MLL-r SEM cells. In summary, using state-of-art research tools and newly established cell models, we have conducted a systematic functional screen and mechanism study of an undocumented downstream transcriptional network of HOXA9 in MLL-r leukemia cells. The work is significant as it will advance our understanding of how HOXA9-associated transcription programs reconstruct the regulatory network specifying MLL-r dependency. Moreover, our study will promote the future development of alternative therapeutic targets in HOXA9-driven (including HOXA9 and possibly NUP98-HOXA9 subtype) leukemia in patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract MLL-rearranged (MLLr) leukemias count for more than 80% of infant leukemia, ~5-10% of B-cell acute lymphoblastic leukemia (B-ALL), and ~10% of acute myeloid leukemia (AML) cases, where they confer a particularly poor outcome. Despite treatment with intensive multi-agent chemotherapy, most MLLr patients achieved an initial remission but ultimately relapsed. Bromo- and Extra-Terminal domain inhibitors (BETi) prevent the progression of many cancer types in preclinical studies, including MLLr leukemia. However, the mechanisms controlling drug response and resistance of BET inhibitors are not well understood. We have addressed this timely, crucial scientific question by completing genetic screens to explore potential BETi resistance mechanisms. By conducting genome-wide and targeted loss-of-function CRISPR screens using MLLr AML cell lines upon BETi treatment including ABBV-744, JQ1, and dBET1, we discovered that Speckle Type POZ (SPOP) gene deficiency leads to significant BETi resistance in in vitro cell culture systems (SEM, OCI-AMl2 and MV4,11), and by in vivo transplantation of human MLLr leukemia SEM cells into immune-deficient mice. However, no BETi resistance phenotype was seen in non-MLLr SPOP-deficient cells. SPOP was previously reported as an adaptor protein to bridge the E3 ubiquitination complex component CUL3 to the substrate proteins BRD4 and MYC in prostate and many other solid cancers. However, in SPOP knockout MLLr leukemia cells, TRIM24, not BRD4 and MYC, was identified as a substrate likely responsible for SPOP's role in drug resistance. Genetically blocking TRIM24 via CRISPR knockout in SPOP-knockout cells reversed the BETi resistance phenotype. Transcriptomic analysis of TRIM24-deficient cells identified the GSK3A signature as the top influenced pathway. Additionally, proteomics expression analysis and a kinase vulnerability CRISPR screen also indicated that resistant cells are sensitive to GSK3B inhibition. Further validation by CRISPR knockout and pharmaceutical blockage of GSK3A/3B (by ChIR-98014) sensitized the SPOP-deficient resistant cells to BETi treatment in vitro. In SEM xenograft models in NSG mice, ABBV-744 or CHIR-98014 minimally impacted human CD45 + leukemia cell proliferation while synergistic treatment significantly reduced the tumor progression. In summary, our data suggest the novel SPOP/TRIM24/GSK3A/3B axis plays an essential role in BETi therapy-resistant leukemia cells. Targeting GSK3A/3B pathways by ChIR-98014 can overcome SPOP-associated BETi resistance in in vivo preclinical models of MLLr leukemia. Successful outcomes following combination therapy using ChIR-98014 and BETi in PDX models would translate to a clinical application that holds the promise to cure MLLr leukemia. Disclosures No relevant conflicts of interest to declare.

Defining the mechanisms that control the perinatal switch from γ-globin (HBG1 and HBG2) to β-globin (HBB) gene expression in human red blood cells (RBCs) has informed novel approaches to reactivate fetal hemoglobin (HbF, α2γ2) therapeutically for sickle cell disease and β-thalassemia. However, one longstanding unsolved problem is to explain how HbF becomes elevated in conditions such as blood loss, hypoxia and hemolysis. These conditions are associated with accelerated RBC production, also referred to as stress erythropoiesis, driven by activation of hypoxia-inducible factor (HIF) via a canonical O 2 sensing pathway. At high O 2 levels (“normoxia”), O 2-dependent prolyl hydroxylase domain (PHD) enzymes hydroxylate HIFα, thereby targeting it for ubiquitination by the von Hippel-Lindau (VHL) E3 ubiquitin ligase complex, followed by proteasomal degradation. At low O 2 tension (hypoxia), PHD activity is reduced, causing HIF1α to accumulate, dimerize with constitutively expressed HIF1β, and bind hypoxia response elements (HREs) to activate a broad array of genes that facilitate hypoxic adaptation. We identified VHL and HIF1α, as negative and positive regulators of HbF expression, respectively.

T reg cells bearing a diverse antigen receptor repertoire suppress pathogenic T cells and maintain immune homeostasis during their long lifespan. How their robust function is determined genetically remains elusive. Here, we investigate the regulatory space of the cis-regulatory elements of T reg lineage-specifying factor Foxp3. Foxp3 enhancers are known as distinct readers of environmental cues controlling T reg cell induction or lineage stability. However, their single deficiencies cause mild, if any, immune dysregulation, leaving the key transcriptional mechanisms determining Foxp3 expression and thereby T reg cell suppressive capacity uncertain. We examined the collective activities of Foxp3 enhancers and found that they coordinate to maximize T reg cell induction, Foxp3 expression level, or lineage stability through distinct modes and that ablation of synergistic enhancers leads to lethal autoimmunity in young mice. Thus, the induction and maintenance of a diverse, stable T reg cell repertoire rely on combinatorial Foxp3 enhancers, suggesting broad, stage-specific, synergistic activities of cellintrinsic factors and cell-extrinsic cues in determining T reg cell suppressive capacity.

Additional file 5: Supplementary Tables-Tables S4. Insulator targets.

BACKGROUND:The transcription factor CTCF appears indispensable in defining topologically associated domain boundaries and maintaining chromatin loop structures within these domains, supported by numerous functional studies. However, acute depletion of CTCF globally reduces chromatin interactions but does not significantly alter transcription.RESULTS:Here, we systematically integrate multi-omics data including ATAC-seq, RNA-seq, WGBS, Hi-C, Cut&Run, and CRISPR-Cas9 survival dropout screens, and time-solved deep proteomic and phosphoproteomic analyses in cells carrying auxin-induced degron at endogenous CTCF locus. Acute CTCF protein degradation markedly rewires genome-wide chromatin accessibility. Increased accessible chromatin regions are frequently located adjacent to CTCF-binding sites at promoter regions and insulator sites associated with enhanced transcription of nearby genes. In addition, we use CTCF-associated multi-omics data to establish a combinatorial data analysis pipeline to discover CTCF co-regulatory partners. We successfully identify 40 candidates, including multiple established partners. Interestingly, many CTCF co-regulators that have alterations of their respective downstream gene expression do not show changes of their own expression levels across the multi-omics measurements upon acute CTCF loss, highlighting the strength of our system to discover hidden co-regulatory partners associated with CTCF-mediated transcription.CONCLUSIONS:This study highlights that CTCF loss rewires genome-wide chromatin accessibility, which plays a critical role in transcriptional regulation.

Aberrant HOXA9 expression is a hallmark of most aggressive acute leukemias, notably those with KMT2A (MLL) gene rearrangements. HOXA9 overexpression not only predicts poor diagnosis and outcome but also plays a critical role in leukemia transformation and maintenance. However, our current understanding of HOXA9 regulation in leukemia is limited, hindering development of therapeutic strategies. Here, we generated the HOXA9-mCherry knock-in reporter cell lines to dissect HOXA9 regulation. By utilizing the reporter and CRISPR/Cas9 screens, we identified transcription factors controlling HOXA9 expression, including a novel regulator, USF2, whose depletion significantly down-regulated HOXA9 expression and impaired MLLr leukemia cell proliferation. Ectopic expression of Hoxa9 rescued impaired leukemia cell proliferation upon USF2 loss. Cut and Run analysis revealed the direct occupancy of USF2 at HOXA9 promoter in MLLr leukemia cells. Collectively, the HOXA9 reporter facilitated the functional interrogation of the HOXA9 regulome and has advanced our understanding of the molecular regulation network in HOXA9 -driven leukemia.

We developed cis-X, a computational method for discovering regulatory noncoding variants in cancer by integrating whole-genome and transcriptome sequencing data from a single cancer sample. cis-X first finds aberrantly cis-activated genes that exhibit allele-specific expression accompanied by an elevated outlier expression. It then searches for causal noncoding variants that may introduce aberrant transcription factor binding motifs or enhancer hijacking by structural variations. Analysis of 13 T-lineage acute lymphoblastic leukemias identified a recurrent intronic variant predicted to cis-activate the TAL1 oncogene, a finding validated in vivo by chromatin immunoprecipitation sequencing of a patient-derived xenograft. Candidate oncogenes include the prolactin receptor PRLR activated by a focal deletion that removes a CTCF-insulated neighborhood boundary. cis-X may be applied to pediatric and adult solid tumors that are aneuploid and heterogeneous. In contrast to existing approaches, which require large sample cohorts, cis-X enables the discovery of regulatory noncoding variants in individual cancer genomes. A new computational method integrates whole-genome sequencing and transcriptomic data to identify regulatory noncoding variants in an individual cancer genome.

Loss of function of CDKN2A/B, also known as INK4/ARF [encoding p16INK4A, p15INK4B, and p14ARF (mouse p19Arf)], confers susceptibility to cancers, whereas its up-regulation during organismal aging provokes cellular senescence and tissue degenerative disorders. To better understand the transcriptional regulation of p16INK4A, a CRISPR screen targeting open, noncoding chromatin regions adjacent to p16INK4A was performed in a human p16INK4A-P2A-mCherry reporter cell line. We identified a repressive element located in the 3' region adjacent to the ARF promoter that controls p16INK4A expression via long-distance chromatin interactions. Coinfection of lentiviral dCas9-KRAB with selected single-guide RNAs against the repressive element abrogated the ARF/p16INK4A chromatin contacts, thus reactivating p16INK4A expression. Genetic CRISPR screening identified candidate transcription factors inhibiting p16INK4A regulation, including ZNF217, which was confirmed to bind the ARF/p16INK4A interaction loop. In summary, direct physical interactions between p16INK4A and ARF genes provide mechanistic insights into their cross-regulation.

Numerous pieces of evidence support the complex, 3D spatial organization of the genome dictates gene expression. CTCF is essential to define topologically associated domain boundaries and to facilitate the formation of insulated chromatin loop structures. To understand CTCF's direct role in global transcriptional regulation, we integrated the miniAID-mClover3 cassette to the endogenous CTCF locus in a human pediatric B-ALL cell line, SEM, and an immortal erythroid precursor cell line, HUDEP-2, to allow for acute depletion of CTCF protein by the auxin-inducible degron system. In SEM cells, CTCF loss notably disrupted intra-TAD loops and TAD integrity in concurrence with a reduction in CTCF-binding affinity, while showing no perturbation to nuclear compartment integrity. Strikingly, the overall effect of CTCF's loss on transcription was minimal. Whole transcriptome analysis showed hundreds of genes differentially expressed in CTCF-depleted cells, among which MYC and a number of MYC target genes were specifically downregulated. Mechanically, acute depletion of CTCF disrupted the direct interaction between the MYC promoter and its distal enhancer cluster residing ∼1.8 Mb downstream. Notably, MYC expression was not profoundly affected upon CTCF loss in HUDEP-2 cells suggesting that CTCF could play a B-ALL cell line specific role in maintaining MYC expression.

The most aggressive of four medulloblastoma (MB) subgroups are cMyc-driven group 3 (G3) tumors, some of which overexpress EZH2, the histone H3K27 mono-, di-, and trimethylase of polycomb-repressive complex 2. Ezh2 has a context-dependent role in different cancers as an oncogene or tumor suppressor and retards tumor progression in a mouse model of G3 MB. Engineered deletions of Ezh2 in G3 MBs by gene editing nucleases accelerated tumorigenesis, whereas Ezh2 re-expression reversed attendant histone modifications and slowed tumor progression. Candidate oncogenic drivers suppressed by Ezh2 included Gfi1, a proto-oncogene frequently activated in human G3 MBs. Gfi1 disruption antagonized the tumor-promoting effects of Ezh2 loss; conversely, Gfi1 overexpression collaborated with Myc to bypass effects of Trp53 inactivation in driving MB progression in primary cerebellar neuronal progenitors. Although negative regulation of Gfi1 by Ezh2 may restrain MB development, Gfi1 activation can bypass these effects.

Manipulating the developmental switch from γ- to β-globin expression that occurs after birth has been intensively investigated as therapeutic strategy for sickle cell anemia and β-thalassemia. Rare individuals with a benign condition termed hereditary persistence of fetal hemoglobin (HPFH) exhibit an attenuated or absent γ-to-β switch, resulting in high levels of fetal hemoglobin (α2γ2) in all red blood cells (RBCs) throughout life. Moreover, individuals with HPFH and homozygosity for sickle cell disease (SCD) mutations exhibit few or no clinical manifestations of the latter. We used genome editing to induce a naturally occurring 13-nucleotide (-102 to -114) deletional HPFH mutation in the γ-globin (HBG1) gene promoter. Heterozygosity for this mutation is associated with HbF levels > 30% in adults. We used the clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) system to create small deletions around -102 to -114 in the γ-globin genes in peripheral blood CD34+ cells from healthy donors. We delivered guide RNA (gRNA) and Cas9 using lentiviruses, sorted transduced hematopoietic progenitors by FACS, and cultured them using a 3-phase erythroid differentiation protocol. Real time PCR showed that γ-globin mRNA increased more than 10-fold in Cas9/gRNA transduced cells compared to controls. HbF flow cytometry and high-performance liquid chromatography (HPLC) demonstrated that induced γ-globin chains were effectively incorporated into hemoglobin tetramers. HPLC revealed 1-3% HbF in negative controls and an increase to 15% in cells transduced with gRNA and Cas9. Expression of erythroid differentiation markers CD235 and CD71 were unaffected, suggesting that the γ-globin increase is not due to impaired erythroid maturation. Next generation sequencing demonstrated that a single gRNA created one predominant mutation that co-segregated with high HbF expression and represented over 50% of the sequencing coverage. Interestingly, this mutation is identical to the 13-nucleotide HPFH deletion. We also tested the gRNA mutation efficiency after transient expression of gRNA and Cas9 in human CD34+ cells by electroporation followed by analysis of single burst-forming unit-erythroid (BFU-E) colonies formed in methylcellulose. Genomic DNA analysis revealed that one gRNA targeted 50% of HBG1 alleles, and cells that received two overlapping gRNAs demonstrated 80% mutation frequency. Real-time PCR of mRNA from edited BFU-Es showed that mutations stimulated γ-globin mRNA expression to 19-55% total globin synthesis, whereas control colonies contained 1-5% γ-globin. Together, our data demonstrate that the CRISPR-Cas9 system can generate precisely the -102 to -114 HPFH mutation at high efficiency in primary human progenitor cells and thereby induce the expression of HbF to potentially therapeutic levels. This work provides proof of concept for targeted genome editing for γ-globin activation as a therapy for patients with β hemoglobinopathies.