Bradley McColl

A Cas9 Variant for Efficient Generation of Indel-Free Knockin or Gene-Corrected Human Pluripotent Stem Cells

Summary While Cas9 nucleases permit rapid and efficient generation of gene-edited cell lines, the CRISPR-Cas9 system can introduce undesirable “on-target” mutations within the second allele of successfully modified cells via non-homologous end joining (NHEJ). To address this, we fused the Streptococcus pyogenes Cas9 (SpCas9) nuclease to a peptide derived from the human Geminin protein (SpCas9-Gem) to facilitate its degradation during the G1 phase of the cell cycle, when DNA repair by NHEJ predominates. We also use mRNA transfection to facilitate low and transient expression of modified and unmodified versions of Cas9. Although the frequency of homologous recombination was similar for SpCas9-Gem and SpCas9, we observed a marked reduction in the capacity for SpCas9-Gem to induce NHEJ-mediated indels at the target locus. Moreover, in contrast to native SpCas9, we demonstrate that transient SpCas9-Gem expression enables reliable generation of both knockin reporter cell lines and genetically repaired patient-specific induced pluripotent stem cell lines free of unwanted mutations at the targeted locus.

Transcriptional regulators Myb and BCL11A interplay with DNA methyltransferase 1 in developmental silencing of embryonic and fetal β-like globin genes

The clinical symptoms of hemoglobin disorders such as β‐thalassemia and sickle cell anemia are significantly ameliorated by the persistent expression of γ‐globin after birth. This knowledge has driven the discovery of important regulators that silence γ‐globin postnatally. Improved understanding of the γ‐ to β‐globin switching mechanism holds the key to devising targeted therapies for β‐hemoglobinopathies. To further investigate this mechanism, we used the murine erythroleukemic (MEL) cell line containing an intact 183‐kb human β‐globin locus, in which the Gγ‐ and β‐globin genes are replaced by DsRed and eGFP fluorescent reporters, respectively. Following RNA interference (RNAi)‐mediated knockdown of two key transcriptional regulators, Myb and BCL11A, we observed a derepression of γ‐globin, measured by DsRed fluorescence and qRT‐PCR (P < 0.001). Interestingly, double knockdown of Myb and DNA methyltransferase 1 (DNMT1) resulted in a robust induction of ε‐globin, (up to 20% of total β‐like globin species) compared to single knockdowns (P<0.001). Conversely, double knockdowns of BCL11A and DNMT1 enhanced γ‐globin expression (up to 90% of total β‐like globin species) compared to single knockdowns (P<0.001). Moreover, following RNAi treatment, expression of human β‐like globin genes mirrored the expression levels of their endogenous murine counterparts. These results demonstrate that Myb and BCL11A cooperate with DNMT1 to achieve developmental repression of embryonic and fetal β‐like globin genes in the adult erythroid environment.—Roosjen, M., McColl, B., Kao, B., Gearing, L. J., Blewitt, M. E., Vadolas, J. Transcriptional regulators Myb and BCL11A interplay with DNA methyltransferase 1 in developmental silencing of embryonic and fetal β‐like globin genes. FASEB J. 28, 28–1610 (1620). www.fasebj.org

Generation of a genomic reporter assay system for analysis of γ- and β-globin gene regulation

A greater understanding of the regulatory mechanisms that govern γ‐globin expression in humans, especially the switching from γ‐ to β‐globin, which occurs after birth, would help to identify new therapeutic targets for patients with β‐hemoglobinopathy. To further elucidate the mechanisms involved in γ‐globin expression, a novel fluorescent‐based cellular reporter assay system was developed. Using homologous recombination, two reporter genes, DsRed and EGFP, were inserted into a 183‐kb intact human β‐globin locus under the control of Gγ‐ or Aγ‐globin promoter and β‐globin promoter, respectively. The modified constructs were stably transfected into adult murine erythroleukaemic (MEL) cells and human embryonic or fetal erythroleukemic (K562) cells, allowing for rapid and simultaneous analysis of fetal and adult globin gene expression according to their developmental stage‐specific expression. To demonstrate the utility of this system, we performed RNA interference (RNAi)‐mediated knockdown of BCL11A in the presence or absence of known fetal hemoglobin inducers and demonstrated functional derepression of a γ‐globin‐linked reporter in an adult erythroid environment. Our results demonstrate that the cellular assay system represents a promising approach to perform genetic and functional genomic studies to identify and evaluate key factors associated with γ‐globin gene sup pression.—Chan, K. S. K., Xu, J., Wardan, H., McColl, B., Orkin, S., Vadolas, J. Generation of a genomic reporter assay system for analysis of y‐ and β‐globin gene regulation. FASEB J. 26, 1736‐1744 (2012). www.fasebj.org

GFP to BFP Conversion: A Versatile Assay for the Quantification of CRISPR/Cas9-mediated Genome Editing

To the Editor: Genome editing via programmable endonucleases enables us to generate site-specific double-strand breaks at virtually any position in a target genome.1–3 Exploiting cellular repair mechanisms, this can be used for targeted gene disruption via nonhomologous end joining (NHEJ) or for the precise manipulation of a target sequence through homology-directed repair (HDR) in the presence of a suitable DNA template. The latter carries great promise for the field of gene therapy as it can be utilized for the correction of disease-causing mutations. Earlier this year, De Ravin et al. reported HDR rates >50% in human hematopoietic stem and progenitor cells using zinc finger nucleases, demonstrating that therapeutic levels of gene correction can be achieved in clinically relevant cell types.4 However, the efficiency of HDR remains considerably lower than that of NHEJ in many experimental settings and a background of mutagenic NHEJ is currently limiting the usefulness of genome editing for gene therapy approaches. This limitation signifies a need to identify conditions that bias genome editing toward HDR. Strategies have been developed to encourage HDR over NHEJ, including stimulation with small molecules and inhibition or disruption of DNA ligase 4 activity, but optimal conditions still need to be established.5–7 Reliable quantification of HDR and NHEJ is essential to the identification of conditions that favor HDR over NHEJ. This was first achieved through the generation of single-cell clones,2 which is impractical for the determination of overall NHEJ and HDR frequencies. The Traffic Light Reporter system provided the first fluorescence-based assay for the simultaneous quantification of HDR and NHEJ.8 However, this system requires the generation of reporter cell lines and therefore can not be applied easily in primary cells or animal models. Sophisticated methods such as single molecule real time sequencing or sib-selection/droplet digital polymerase chain reaction allow for the quantification of HDR and NHEJ at endogenous loci without the necessity of generating individual clones.9,10 However, downstream sample processing requirements limit the use of these techniques in a high-throughput format. As an alternative, we propose a simple strategy for the simultaneous quantification of HDR and NHEJ by targeting the ubiquitous enhanced green fluorescent protein (EGFP) fluorescent reporter (Figure 1a, b). In 1994, Heim et al. discovered that a single base substitution (196T > C) in the chromophore of wild-type (wt) GFP could shift its fluorescence absorption and emission toward the blue spectrum, thus creating blue fluorescent protein (BFP).11 Here, we demonstrate that EGFP can be converted into BFP in EGFP-expressing cell lines using the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) system. HDR and NHEJ can subsequently be quantified as blue fluorescence and loss of fluorescence, respectively. K562 cells carrying an EGFP-modified human β-globin locus in the AAVS-1 site in chromosome 19,12 and HEK293T cells that were stably transduced with an integration competent lentiviral EGFP expression construct (K562-50 and HEK293T-EGFP, Figure 2a) were used in this study. Two guide RNA (gRNA) vectors based on px330-IRES mCherry were designed to target Cas9 into close proximity to the target site (Figure 1b). A double-stranded BFP PCR reaction product amplified from the vector pLMP (primers 5′-CCTGAAGTTCATCTGC ACCACC-3′ and 5′-GACGTAGCCTTCGGGCATGG-3′) was compared with two single-stranded repair templates (ssODN) (Figure 1c). The gRNA/Cas9 plasmids (5 μg) and HDR templates (100 pmol) were coelectroporated into the target cells using a BioRad Gene Pulser II electroporator. GFP and BFP fluorescence were assessed 10 days later using flow cytometry. HDR and NHEJ were quantified as the percentage of BFP+ cells and nonfluorescent cells, respectively. HDR/total editing ratios (R) were determined using the formula: R = (HDR)/(NHEJ + HDR) * 100. Our data shows that a single 196T > C substitution using ssODN1 is sufficient to convert GFP to BFP. However, low fluorescence intensity and a low HDR frequency were observed in comparison with the other templates in K562-50 cells (Figure 1d,e). An additional 194C > G substitution in ssODN2, corresponding to a reversion of the EGFP amino acid sequence back to that of wild-type GFP, was sufficient to restore BFP fluorescence intensity to that observed with the PCR template (Figure 1e). The low HDR frequency observed with ssODN1 was theorized to result from recutting of the repaired sequence by Cas9, as the sequence resulting from HDR retains the complete target sequence for gRNA1 and contains only one mismatch in the gRNA2 target site. The 194C > G substitution introduces an additional mismatch in the gRNA2 target site and eliminates the gRNA1 protospacer adjacent motif sequence. To further reduce the target sequence similarity with gRNA1 after HDR, ssODN2 was designed with an GFP to BFP Conversion: A Versatile Assay for the Quantification of CRISPR/Cas9-mediated Genome Editing LETTER TO ThE EDITOR

Animal models of β-hemoglobinopathies: utility and limitations.

The structural and functional conservation of hemoglobin throughout mammals has made the laboratory mouse an exceptionally useful organism in which to study both the protein and the individual globin genes. Early researchers looked to the globin genes as an excellent model in which to examine gene regulation – bountifully expressed and displaying a remarkably consistent pattern of developmental activation and silencing. In parallel with the growth of research into expression of the globin genes, mutations within the β-globin gene were identified as the cause of the β-hemoglobinopathies such as sickle cell disease and β-thalassemia. These lines of enquiry stimulated the development of transgenic mouse models, first carrying individual human globin genes and then substantial human genomic fragments incorporating the multigenic human β-globin locus and regulatory elements. Finally, mice were devised carrying mutant human β-globin loci on genetic backgrounds deficient in the native mouse globins, resulting in phenotypes of sickle cell disease or β-thalassemia. These years of work have generated a group of model animals that display many features of the β-hemoglobinopathies and provided enormous insight into the mechanisms of gene regulation. Substantive differences in the expression of human and mouse globins during development have also come to light, revealing the limitations of the mouse model, but also providing opportunities to further explore the mechanisms of globin gene regulation. In addition, animal models of β-hemoglobinopathies have demonstrated the feasibility of gene therapy for these conditions, now showing success in human clinical trials. Such models remain in use to dissect the molecular events of globin gene regulation and to identify novel treatments based upon the reactivation of developmentally silenced γ-globin. Here, we describe the development of animal models to investigate globin switching and the β-hemoglobinopathies, a field that has paralleled the emergence of modern molecular biology and clinical genetics.

An in vivo model for analysis of developmental erythropoiesis and globin gene regulation

Expression of fetal γ‐globin in adulthood ameliorates symptoms of β‐hemoglobinopathies by compensating for the mutant β‐globin. Reactivation of the silenced γ‐globin gene is therefore of substantial clinical interest. To study the regulation of γ‐globin expression, we created the GG mice, which carry an intact 183‐kb human β‐globin locus modified to express enhanced green fluorescent protein (eGFP) from the Gγ‐globin promoter. GG embryos express eGFP first in the yolk sac blood islands and then in the aorta–gonad mesonephros and the fetal liver, the sites of normal embryonic hematopoiesis. eGFP expression in erythroid cells peaks at E9.5 and then is rapidly silenced (>95%) and maintained at low levels into adulthood, demonstrating appropriate developmental regulation of the human β‐globin locus. In vitro knockdown of the epigenetic regulator DNA methyltransferase‐1 in GG primary erythroid cells increases the proportion of eGFP+ cells in culture from 41.9 to 74.1%. Furthermore, eGFP fluorescence is induced > 3‐fold after treatment of erythroid precursors with epigenetic drugs known to induce γ‐globin expression, demonstrating the suitability of the Gγ‐globin eGFP reporter for evaluation of γ‐globin inducers. The GG mouse model is therefore a valuable model system for genetic and pharmacologic studies of the regulation of the β‐globin locus and for discovery of novel therapies for the P‐hemoglobinopathies.—McColl, B., Kao, B. R., Lourthai, P., Chan, K., Wardan, H., Roosjen, M., Delagneau, O., Gearing, L. J., Blewitt, M. E., Svasti, S., Fucharoen, S., Vadolas, J. An in vivo model for analysis of developmental erythropoiesis and globin gene regulation. FASEB J. 28, 2306–2317 (2014). www.fasebj.org

Epigenetic interplay at the β-globin locus

During development, the α- and β-globin genes exhibit a highly conserved pattern of expression, giving rise to several developmental stage-specific hemoglobin variants. Networks of regulatory proteins interact with epigenetic complexes to regulate DNA accessibility and histone modifications, thereby determining appropriate patterns of globin gene expression. In this review, we focus on recent advances in the understanding of the molecular mechanisms that underpin globin gene expression, focusing on multi-subunit regulatory complexes that bind to specific regions of DNA to orchestrate globin gene transcription throughout development.

The therapeutic potential of genome editing for β-thalassemia.

The rapid advances in the field of genome editing using targeted endonucleases have called considerable attention to the potential of this technology for human gene therapy. Targeted correction of disease-causing mutations could ensure lifelong, tissue-specific expression of the relevant gene, thereby alleviating or resolving a specific disease phenotype. In this review, we aim to explore the potential of this technology for the therapy of β-thalassemia. This blood disorder is caused by mutations in the gene encoding the β-globin chain of hemoglobin, leading to severe anemia in affected patients. Curative allogeneic bone marrow transplantation is available only to a small subset of patients, leaving the majority of patients dependent on regular blood transfusions and iron chelation therapy. The transfer of gene-corrected autologous hematopoietic stem cells could provide a therapeutic alternative, as recent results from gene therapy trials using a lentiviral gene addition approach have demonstrated. Genome editing has the potential to further advance this approach as it eliminates the need for semi-randomly integrating viral vectors and their associated risk of insertional mutagenesis. In the following pages we will highlight the advantages and risks of genome editing compared to standard therapy for β-thalassemia and elaborate on lessons learned from recent gene therapy trials.

Reduced PU.1 expression underlies aberrant neutrophil maturation and function in β-thalassemia mice and patients

β-Thalassemia is associated with several abnormalities of the innate immune system. Neutrophils in particular are defective, predisposing patients to life-threatening bacterial infections. The molecular and cellular mechanisms involved in impaired neutrophil function remain incompletely defined. We used the Hbbth3/+ β-thalassemia mouse and hemoglobin E (HbE)/β-thalassemia patients to investigate dysregulated neutrophil activity. Mature neutrophils from Hbbth3/+ mice displayed a significant reduction in chemotaxis, opsonophagocytosis, and production of reactive oxygen species, closely mimicking the defective immune functions observed in β-thalassemia patients. In Hbbth3/+ mice, the expression of neutrophil CXCR2, CD11b, and reduced NAD phosphate oxidase components (p22phox, p67phox, and gp91phox) were significantly reduced. Morphological analysis of Hbbth3/+ neutrophils showed that a large percentage of mature phenotype neutrophils (Ly6GhiLy6Clow) appeared as band form cells, and a striking expansion of immature (Ly6GlowLy6Clow) hyposegmented neutrophils, consisting mainly of myelocytes and metamyelocytes, was noted. Intriguingly, expression of an essential mediator of neutrophil terminal differentiation, the ets transcription factor PU.1, was significantly decreased in Hbbth3/+ neutrophils. In addition, in vivo infection with Streptococcus pneumoniae failed to induce PU.1 expression or upregulate neutrophil effector functions in Hbbth3/+ mice. Similar changes to neutrophil morphology and PU.1 expression were observed in splenectomized and nonsplenectomized HbE/β-thalassemia patients. This study provides a mechanistic insight into defective neutrophil maturation in β-thalassemia patients, which contributes to deficiencies in neutrophil effector functions.

Corrigendum to GFP to BFP Conversion: A Versatile Assay for the Quantification of CRISPR/Cas9-mediated Genome Editing

Molecular Therapy–Nucleic Acids (2016) 5, e360; doi:10.1038/mtna.2016.78; published online 13 September 2016