BackgroundA point mutation in sickle cell disease (SCD) alters one amino acid in the β-globin subunit of hemoglobin, with resultant anemia and multiorgan damage that typically shortens lifespan by decades. Because SCD is caused by a single mutation, and hematopoietic stem cells (HSCs) can be harvested, manipulated, and returned to an individual, it is an attractive target for gene correction.ResultsAn optimized Cas9 ribonucleoprotein (RNP) with an ssDNA oligonucleotide donor together generated correction of at least one β-globin allele in more than 30% of long-term engrafting human HSCs. After adopting a high-fidelity Cas9 variant, efficient correction with minimal off-target events also was observed. In vivo erythroid differentiation markedly enriches for corrected β-globin alleles, indicating that erythroblasts carrying one or more corrected alleles have a survival advantage.SignificanceThese findings indicate that the sickle mutation can be corrected in autologous HSCs with an optimized protocol suitable for clinical translation.

Naturally occurring point mutations in the HBG promoter switch hemoglobin synthesis from defective adult beta-globin to fetal gamma-globin in sickle cell patients with hereditary persistence of fetal hemoglobin (HPFH) and ameliorate the clinical severity. Inspired by this natural phenomenon, we tiled the highly homologous HBG proximal promoters using adenine and cytosine base editors that avoid the generation of large deletions and identified novel regulatory regions including a cluster at the –123 region. Base editing at –123 and –124 bp of HBG promoter induced fetal hemoglobin (HbF) to a higher level than disruption of well-known BCL11A binding site in erythroblasts derived from human CD34+ hematopoietic stem and progenitor cells (HSPC). We further demonstrated in vitro that the introduction of –123T > C and –124T > C HPFH-like mutations drives gamma-globin expression by creating a de novo binding site for KLF1. Overall, our findings shed light on so far unknown regulatory elements within the HBG promoter and identified additional targets for therapeutic upregulation of fetal hemoglobin.

Fetal development and anemias such as β-hemoglobinopathies trigger rapid production of red blood cells in a process known as stress erythropoiesis. Cellular stress prompts differentiating erythroid precursors to express high levels of fetal γ-globin, which has suggested strategies to treat hemoglobinopathies such as thalassemia and sickle cell disease. However, the mechanisms underlying γ-globin production during cellular stress are still poorly defined. Here we use CRISPR-Cas genome editing and CRISPRi transcriptional repression to model the stress caused by reduced levels of adult β-globin. We find that loss of β-globin is sufficient to induce widespread globin compensation, including robust re-expression of γ-globin. Time-course RNA-seq of differentiating isogenic erythroid precursors identified the ATF4 transcription factor as a causal regulator of this response. ChIP-seq of multiple erythroid precursor genotypes and differentiation states revealed that β-globin knockout leads to reduced engagement of ATF4 targets involved in the unfolded protein response. This ATF4 program indirectly regulates the levels of BCL11A, a key repressor of γ-globin. Identification of ATF4 as a key regulator of globin compensation adds mechanistic insight to the poorly understood phenomenon of stress-induced globin compensation and could be relevant for proposed gene editing strategies to treat hemoglobinopathies.

β-Hemoglobinopathies can trigger rapid production of red blood cells in a process known as stress erythropoiesis. Cellular stress prompts differentiating erythroid precursors to express high levels of fetal γ-globin. However, the mechanisms underlying γ-globin production during cellular stress are still poorly defined. Here, we use CRISPR-Cas genome editing to model the stress caused by reduced levels of adult β-globin. We find that decreased β-globin is sufficient to induce robust re-expression of γ-globin, and RNA sequencing (RNA-seq) of differentiating isogenic erythroid precursors implicates ATF4 as a causal regulator of this response. ATF4 binds within the HBS1L-MYB intergenic enhancer and regulates expression of MYB, a known γ-globin regulator. Overall, the reduction of ATF4 upon β-globin knockout decreases the levels of MYB and BCL11A. Identification of ATF4 as a key regulator of globin compensation adds mechanistic insight to the poorly understood phenomenon of stress-induced globin compensation and could inform strategies to treat hemoglobinopathies.

The decoy exon model has been proposed to regulate a subset of intron retention (IR) events involving predominantly larger introns (>1kb). Splicing reporter studies have shown that decoy splice sites are essential for activity, suggesting that decoys act by engaging intron-terminal splice sites and competing with cross-intron interactions required for intron excision. The decoy model predicts that antisense oligonucleotides blocking decoy splice sites in endogenous pre-mRNA should increase productive gene expression by reducing IR. Indeed, we now demonstrate that targeting a decoy 5′ splice site in the O-GlcNAc transferase (OGT) gene reduced IR from ∼80% to ∼20% in primary human erythroblasts, accompanied by increases in spliced OGT RNA and OGT protein expression. The remaining OGT IR was refractory to antisense treatment and might be mediated by independent mechanism(s). In contrast, other retained introns were strongly dependent on decoy function, since IR was nearly eliminated by antisense targeting of 5′ splice sites. Genes in the latter group encode the widely expressed splicing factor (SF3B1), and the erythroid-specific structural protein, alpha-spectrin (SPTA1). These results show that modulating decoy exon function can dramatically alter IR, and suggest that dynamic regulation of decoy exons could be a mechanism to fine tune gene expression post-transcriptionally in many cell types.

Genome editing often takes the form of either error-prone sequence disruption by non-homologous end joining (NHEJ) or sequence replacement by homology-directed repair (HDR). Although NHEJ is generally effective, HDR is often difficult in primary cells. Here, we use a combination of immunophenotyping, next-generation sequencing, and single-cell RNA sequencing to investigate and reprogram genome editing outcomes in subpopulations of adult hematopoietic stem and progenitor cells. We find that although quiescent stem-enriched cells mostly use NHEJ, non-quiescent cells with the same immunophenotype use both NHEJ and HDR. Inducing quiescence before editing results in a loss of HDR in all cell subtypes. We develop a strategy of controlled cycling and quiescence that yields a 6-fold increase in the HDR/NHEJ ratio in quiescent stem cells ex vivo and in vivo. Our results highlight the tension between editing and cellular physiology and suggest strategies to manipulate quiescent cells for research and therapeutic genome editing.

Background:Sickle Cell Disease (SCD) is a devastating disorder caused by a single base change in the ß‐globin gene (HBB) that is one component of the adult hemoglobin tetramer (HbA, which contains 2 a‐globin and 2 ß‐globin proteins). SCD is a recessive disorder – if one of the 2 alleles of HBB is normal, the disease is not present; thus gene correction needs to correct only one of the two HBB alleles in any given stem cell.Aims:To correct the sickle allele in human hematopoietic stem cellsMethods:To correct the sickle mutation, we developed a process that directly introduces a preassembled Cas9 ribonucleoprotein (RNP) complex (composed of recombinant Cas9 protein and single guide RNA) along with a single‐stranded DNA oligonucleotide donor template (ssODN) for homology‐directed repair (HDR). We studied gene correction after long‐term xenografting of human sickle HSCs. After electroporation with RNP and ssODN, sickle human HSCs were injected into immunodeficient NBSGW mice, which permit engraftment of human HSCs and erythroid differentiation in the bone marrow.Results:Sixteen to 20 weeks after injection, we analyzed engrafted human cells in the bone marrow of 43 mice in 4 cohorts. We quantified editing in xenografted cells, and found an average of 23.4% of HBB alleles with the corrected genotype, and 65.2% with insertion/deletion mutations (indels). To assess the distribution of HBB alleles within populations of xenografted cells, we inferred HBB genotypes using RNA‐Seq data from 359 individual clonal BFU‐E colonies derived from marrow CD34+ cells (figure 1A). This reveals that a substantial proportion of corrected (HDR) alleles are heterozygous with indel alleles equivalent to ß‐thalassemia mutations, confirming that corrected (wild type) HBB alleles in edited HSCs are distributed such that many erythrocytes have only a single wild type HBB allele. Data from the colony analysis, extrapolated to the entire experimental group, indicates that the 23.4% of corrected HBB alleles are distributed among >30% of HSCs, producing a comparable proportion of functional erythrocytes. SCD is a recessive disorder; based upon observations of mixed chimerism after allogeneic transplantation, this proportion of normal cells is consistent with cure.From engrafted NBSGW mice, we immunoselected human erythroid cells with anti‐CD235a (GlycophorinA), carried out RNA‐Seq, and inferred HBB genotypes in the erythroblast population. For each individual mouse, we compared the proportion of corrected HBB alleles in the xenografted bone marrow, in CD34+ cells immunoselected from marrow, and in CD235a+ erythroblasts. To control for enrichment of alleles corrected by HDR, we distinguished two types of HDR: “PAM‐only” HDR events mutate the Cas9 PAM without the conversion tract reaching the sickle mutation, whereas “sickle‐corrected” HDR events mutate the Cas9 PAM and also correct the sickle mutation. We observe a marked enrichment of “sickle‐corrected” HDR alleles in erythroblasts when compared to marrow or CD34+ cells from the same mouse, but no enrichment of “PAM‐only” HDR alleles (figure 1B).Summary/Conclusion:In summary, we have developed a Cas9‐mediated gene editing protocol that reproducibly yields a cell product in which the sickle mutation is corrected to wild type at a level that is sufficient to cure SCD. Evidence we have presented indicates that cells carrying the corrected ß‐globin gene are enriched during in vivo erythroid differentiation. These results support moving this therapeutic approach to the clinic.image

Sickle Cell Disease and ß-thalassemia, which are caused by defective or deficient adult ß-globin (HBB) respectively, are the most common serious genetic blood diseases in the world. Persistent expression of the fetal ß-like globin, also known as 𝛾-globin, can ameliorate both disorders by serving in place of the adult ß-globin as a part of the fetal hemoglobin tetramer (HbF). Here we use CRISPR-Cas9 gene editing to explore a potential 𝛾-globin silencer region upstream of the δ-globin gene identified by comparison of naturally-occurring deletion mutations associated with up-regulated 𝛾-globin. We find that deletion of a 1.7 kb consensus element or select 350 bp sub-regions from bulk populations of cells increases levels of HbF. Screening of individual sgRNAs in one sub-region revealed three single guides that caused increases in 𝛾-globin expression. Deletion of the 1.7 kb region in HUDEP-2 clonal sublines, and in colonies derived from CD34+ hematopoietic stem/progenitor cells (HSPCs), does not cause significant up-regulation of 𝛾-globin. These data suggest that the 1.7 kb region is not an autonomous 𝛾-globin silencer, and thus by itself is not a suitable therapeutic target for gene editing treatment of ß-hemoglobinopathies.

The erythroid transcriptome is extensively remodeled during terminal erythropoiesis by dynamic changes in RNA splicing of cassette exons and retained introns. Mechanistic studies of these RNA processing networks will provide new insight into pathways that impact structure and function of the erythroid proteome during erythroblast differentiation. We previously showed that up-regulation of EPB41 exon 16 splicing imparts new functionality to the encoded protein, enhancing protein-protein interactions that mechanically strengthen the red cell membrane. More recently, RNA-seq analysis revealed that numerous erythroid transcripts exhibit up-regulation of intron retention (IR) events, some of which are controlled by a decoy exon-mediated mechanism that can reduce the output of translated mRNA so as to limit protein expression. Here we demonstrate that modulation of decoy-mediated IR quantitatively affects protein expression in primary human erythroid cells. We first studied the OGT gene (O-GlcNAc transferase), a key regulator of O-GlcNAC homeostasis. OGT expression responds to pharmacological inhibitors by regulating intron 4 retention, by a mechanism requiring a intronic splicing silencer (1) that functions as a decoy exon (2) to nonproductively engage annotated splice sites at the ends of the intron, thereby blocking excision and enforcing its retention. In erythroid CD34+ progenitors at day 7 of culture, we found that treatment with an OGT inhibitor (OSMI-1) reduced OGT IR and increased spliced OGT RNA and OGT protein. Conversely, an inhibitor of the antagonistic OGA enzyme (thiamet-G) induced greater OGT IR and reduced OGT protein expression. These results are similar to what was reported previously in established cell lines (1), and suggest that modulation of IR can vary mRNA and protein expression in primary cells >5-fold. To further explore the model, we independently blocked OGT IR in erythroid cultures by electroporation of an antisense morpholino directed against the OGT decoy exon 5' splice site. RT-qPCR and western blot analysis confirmed substantial reduction in IR, coupled with an increase in spliced RNA and elevated OGT protein expression, compared to control cells or cells treated with an irrelevant morpholino. The OGT-specific MO also substantially blocked IR induced by thiamet-G. These results show that pharmacological- or antisense-mediated alteration in IR can significantly change protein expression in primary erythroblast cultures. We propose that the abundance of IR transcripts in late erythropoiesis represents a widespread modulation of protein output by post-transcriptional pathways operating at the level of intron retention. Park SK, et al. (2017) Cell Rep. 20: 1088-99.Parra M et al. (2018) RNA 24: 1255-65. Disclosures No relevant conflicts of interest to declare.

Sickle Cell Disease (SCD), one of the world9s most common genetic disorders, causes anemia and progressive multiorgan damage that typically shortens lifespan by decades; currently there is no broadly applicable curative therapy. Here we show that Cas9 RNP-mediated gene editing with an ssDNA oligonucleotide donor yields more than 20% correction of the sickle mutation in long-term engrafting human HSCs. Using RNA-seq, we further find that in vivo erythroid differentiation markedly enriches for cells carrying corrected β-globin alleles. Adoption of a high-fidelity Cas9 variant demonstrates that this approach can yield efficient editing with almost no off-target events. These findings indicate that the sickle mutation can be corrected in human HSCs at levels that are likely to be curative if translated into a therapy.

INTRODUCTION:Sickle cell anemia (SCA) is a hereditary blood disease caused by a single-gene mutation that affects millions of individuals world-wide. In this review, we focus on techniques to treat SCA by ex vivo genetic manipulation of hematopoietic stem/progenitor cells (HSPC), emphasizing replacement gene therapy and gene editing. AREAS COVERED:Viral transduction of an anti-sickling β-like globin gene has been tested in pre-clinical and early-phase clinical studies, and shows promising preliminary results. Targeted editing of endogenous genes by site-directed nucleases has been developed more recently, and several approaches also are nearing clinical translation. EXPERT OPINION:The indications and timing of gene therapy for SCA in lieu of supportive care treatment and allogeneic hematopoietic cell transplantation are still undefined. In addition, ensuring access to the treatment where the disease is endemic will present important challenges that must be addressed. Nonetheless, gene therapy and gene editing techniques have transformative potential as a universal curative option in SCA.

The beta hemoglobinopathies, beta thalassemia and sickle cell disease, are the most common serious genetic diseases in the world. Both are caused by defect or absence of the adult beta globin gene (HBB). Symptoms of both diseases can be ameliorated by increased re-expression of fetal globin (HBG) in the adult, and upregulation of HBG is a recognized therapeutic approach. Here, we evaluate a novel gene editing strategy for fetal globin de-repression using the Cas9 RNP gene editing reagent as a key tool. We focus on a 1.7 kb region, just upstream of delta globin, whose absence in naturally-occurring mutations appears to predict fetal globin de-repression. We found that deletion of the entire region is not sufficient to effectively de-repress fetal globin. Interrogation of sub-regions identify 2 sub-regions whose deletion may increase fetal globin expression. This phenotype is not consistent across isogenic clones in a model cell lines, and is below clinical significance in primary hematopoietic stem/progenitor cells. We conclude that this region is likely not a valid therapeutic target for these diseases.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated system (Cas9)-mediated gene editing of human hematopoietic stem cells (hHSCs) is a promising strategy for the treatment of genetic blood diseases through site-specific correction of identified causal mutations. However, clinical translation is hindered by low ratio of precise gene modification using the corrective donor template (homology-directed repair, HDR) to gene disruption (nonhomologous end joining, NHEJ) in hHSCs. By using a modified version of Cas9 with reduced nuclease activity in G1 phase of cell cycle when HDR cannot occur, and transiently increasing the proportion of cells in HDR-preferred phases (S/G2), we achieved a four-fold improvement in HDR/NHEJ ratio over the control condition in vitro, and a significant improvement after xenotransplantation of edited hHSCs into immunodeficient mice. This strategy for improving gene editing outcomes in hHSCs has important implications for the field of gene therapy, and can be applied to diseases where increased HDR/NHEJ ratio is critical for therapeutic success. Stem Cells 2019;37:284-294.

Semiconductor quantum dots (QDs) have proven to be superior probes for single-molecule imaging compared to organic or genetically encoded fluorophores, but they are limited by difficulties in protein targeting, their larger size, and on-off blinking. Here, we report compact aqueous CdSe/CdS QDs with significantly improved bioconjugation efficiency and superior single-molecule optical properties. We have synthesized covalent protein labeling ligands (i.e., SNAP tags) that are optimized for nanoparticle use, and QDs functionalized with these ligands label SNAP-tagged proteins ∼10-fold more efficiently than existing SNAP ligands. Single-molecule analysis of these QDs shows 99% of time spent in the fluorescent on-state, ∼4-fold higher quantum efficiency than standard CdSe/ZnS QDs, and 350 million photons detected before photobleaching. Bright signals of these QDs enable us to track the stepping movement of a kinesin motor in vitro, and the improved labeling efficiency enables tracking of single kinesins in live cells.

CRISPR/Cas9-based therapeutics, especially those that can correct gene mutations via homology directed repair (HDR), have the potential to revolutionize the treatment of genetic diseases. However, HDR-based therapeutics are challenging to develop because they require simultaneous in vivo delivery of Cas9 protein, guide RNA and donor DNA. Here, we demonstrate that a delivery vehicle composed of gold nanoparticles conjugated to DNA and complexed with cationic endosomal disruptive polymers can deliver Cas9 ribonucleoprotein and donor DNA into a wide variety of cell types, and efficiently correct the DNA mutation that causes Duchenne muscular dystrophy in mice via local injection, with minimal off-target DNA damage.

The CRISPR-Cas genome editing system is very powerful. The format of the CRISPR reagents and the means of delivery are often important factors in targeting efficiency. Delivery of recombinant Cas9 protein and guide RNA (gRNA) as a preformed ribonucleoprotein (RNP) complex has recently emerged as a powerful and general approach to genome editing. Here we outline methods to produce and deliver Cas9 RNPs. A donor DNA carrying desired sequence changes can also be included to program precise sequence introduction or replacement. RNP delivery limits exposure to genome editing reagents, reduces off-target events, drives high rates of homology-dependent repair, and can be applied to embryos to rapidly generate animal models. RNP delivery thus minimizes some of the pitfalls of alternative editing modalities and is rapidly being adopted by the genome editing community.

Chemical modification of the gRNA and donor DNA has great potential for improving the gene editing efficiency of Cas9 and Cpf1, but has not been investigated extensively. In this report, we demonstrate that the gRNAs of Cas9 and Cpf1, and donor DNA can be chemically modified at their terminal positions without losing activity. Moreover, we show that 5’ fluorescently labeled donor DNA can be used as a marker to enrich HDR edited cells by a factor of two through cell sorting. In addition, we demonstrate that the gRNA and donor DNA can be directly conjugated together into one molecule, and show that this gRNA-donor DNA conjugate is three times better at transfecting cells and inducing HDR, with cationic polymers, than unconjugated gRNA and donor DNA. The tolerance of the gRNA and donor DNA to chemical modifications has the potential to enable new strategies for genome engineering.