Gene Editing Strategies Research Articles

Gene Therapy as the New Frontier for Sickle Cell Disease.

Sickle Cell Disease (SCD) is one of the most common monogenic disorders caused by a point mutation in the β-globin gene. This mutation results in polymerization of hemoglobin (Hb) under reduced oxygenation conditions, causing rigid sickle-shaped RBCs and hemolytic anemia. This clearly defined fundamental molecular mechanism makes SCD a prototypical target for precision therapy. Both the mutant β-globin protein and its downstream pathophysiology are pharmacological targets of intensive research. SCD also is a disease well-suited for biological interventions like gene therapy. Recent advances in hematopoietic stem cell (HSC) transplantation and gene therapy platforms, like Lentiviral vectors and gene editing strategies, expand the potentially curative options for patients with SCD. This review discusses the recent advances in precision therapy for SCD and the preclinical and clinical advances in autologous HSC gene therapy for SCD.

Bayesian Network Analysis of Lysine Biosynthesis Pathway in Rice

Lysine is the first limiting essential amino acid in rice because it is present in the lowest quantity compared to all the other amino acids. Amino acids are the building block of proteins and play an essential role in maintaining the human body’s healthy functioning. Rice is a staple food for more than half of the global population; thus, increasing the lysine content in rice will help improve global health. In this paper, we studied the lysine biosynthesis pathway in rice (Oryza sativa) to identify the regulators of the lysine reporter gene LYSA (LOC_Os02g24354). Genetically intervening at the regulators has the potential to increase the overall lysine content in rice. We modeled the lysine biosynthesis pathway in rice seedlings under normal and saline (NaCl) stress conditions using Bayesian networks. We estimated the model parameters using experimental data and identified the gene DAPF(LOC_Os12g37960) as a positive regulator of the lysine reporter gene LYSA under both normal and saline stress conditions. Based on this analysis, we conclude that the gene DAPF is a potent candidate for genetic intervention. Upregulating DAPF using methods such as CRISPR-Cas9 gene editing strategy has the potential to upregulate the lysine reporter gene LYSA and increase the overall lysine content in rice.

Clinically relevant gene editing in hematopoietic stem cells for the treatment of pyruvate kinase deficiency

Pyruvate kinase deficiency (PKD), an autosomal-recessive disorder, is the main cause of chronic non-spherocytic hemolytic anemia. PKD is caused by mutations in the pyruvate kinase, liver and red blood cell (PKLR) gene, which encodes for the erythroid pyruvate kinase protein (RPK). RPK is implicated in the last step of anaerobic glycolysis in red blood cells (RBCs), responsible for the maintenance of normal erythrocyte ATP levels. The only curative treatment for PKD is allogeneic hematopoietic stem and progenitor cell (HSPC) transplant, associated with a significant morbidity and mortality, especially relevant in PKD patients. Here, we address the correction of PKD through precise gene editing at the PKLR endogenous locus to keep the tight regulation of RPK enzyme during erythropoiesis. We combined CRISPR-Cas9 system and donor recombinant adeno-associated vector (rAAV) delivery to build an efficient, safe, and clinically applicable system to knock in therapeutic sequences at the translation start site of the RPK isoform in human hematopoietic progenitors. Edited human hematopoietic progenitors efficiently reconstituted human hematopoiesis in primary and secondary immunodeficient mice. Erythroid cells derived from edited PKD-HSPCs recovered normal ATP levels, demonstrating the restoration of RPK function in PKD erythropoiesis after gene editing. Our gene-editing strategy may represent a lifelong therapy to correct RPK functionality in RBCs for PKD patients.

Simple, efficient and open-source CRISPR/Cas9 strategy for multi-site genome editing in Populus tremula × alba.

Although the CRISPR/Cas9 system has been successfully used for crop breeding, its application remains limited in forest trees. Here, we describe an efficient gene editing strategy for hybrid poplar, (Populus tremula × alba INRA clone 717-1B4) based on the Golden Gate MoClo cloning. To test the system efficiency for generating single gene mutants, two single guide RNAs (sgRNAs) were designed and incorporated into the MoClo Tool Kit level 2 binary vector with the Cas9 expression cassette to mutate the SHORT ROOT (SHR) gene. Moreover, we also tested its efficiency for introducing mutations in two genes simultaneously by expressing one sgRNA targeting a single site of the YUC4 gene and the other sgRNA targeting the PLT1 gene. For a robust evaluation of the approach, we repeated the strategy to target the LBD12 and LBD4 genes simultaneously, using an independent construct. We generated hairy roots by Agrobacterium rhizogenes-mediated leaf transformation. Sequencing results confirmed the CRISPR/Cas9-mediated mutation in the targeted sites of PtaSHR. Biallelic and homozygous knockout mutations were detected. A deletion spanning both target sites and small insertions/deletions were the most common mutations. Out of the 22 SHR alleles sequenced, 21 were mutated. The phenotype’s characterization showed that transgenic roots with biallelic mutations for the SHR gene lacked a defined endodermal single cell layer, suggesting a conserved gene function similar to its homolog in Arabidopsis Arabidopsis thaliana (L.) Heynh. Sequencing results also revealed the high efficiency of the system for generating double mutants. Biallelic mutations for both genes in the yuc4/plt1 and lbd12/lbd4 roots were detected in three (yuc4/plt1) and two (lbd12/lbd4) out of four transgenic roots evaluated. A small deletion or a single nucleotide insertion at the single target site was the most common mutations. This CRISPR/Cas9 strategy arises as a rapid, simple and standardized gene-editing tool to evaluate the gene role in essential developmental programs such as radial cell differentiation of poplar roots.

CRISPR/Cas9 Genome Editing of the Human Topoisomerase IIα Intron‐19 5′ Splice Site Circumvents Etoposide Resistance in Human Leukemia K562 Cells

An essential function of DNA topoisomerase IIα (170 kDa, TOP2α/170) is to resolve DNA topologic entanglements during chromosome dysjunction by introducing transient DNA double-strand breaks (DSBs). TOP2α/170 is an important target for DNA damage-stabilizing anticancer drugs, whose clinical efficacy is compromised by drug resistance often associated with decreased TOP2α/170 expression. We recently demonstrated that an etoposide-resistant K562 clonal subline, K/VP.5, with reduced levels of TOP2α/170, expresses high levels of a novel C-terminal truncated TOP2α isoform (90 kDa, TOP2α/90). TOP2α/90, the translation product of a TOP2α mRNA that retains a processed intron 19 (I19), heterodimerizes with TOP2α/170 and is a resistance determinant through a dominant negative effect on drug activity. We hypothesized that genome editing to enhance I19 removal would provide a tractable strategy to circumvent acquired TOP2α-mediated drug resistance. To enhance I19 removal in K/VP.5 cells, CRISPR/Cas9 was utilized to make changes (GAG//GTAAACàGAG//GTAAGT) in the TOP2α gene's suboptimal exon 19/intron 19 5′ splice site (E19/I19 5′ SS). Gene edited clones were identified by qPCR and verified by sequencing. Characterization of a clone with all TOP2α alleles edited revealed improved I19 removal, decreased TOP2α/90 protein, and increased TOP2α/170 protein. Sensitivity to etoposide-induced DNA damage (γH2AX, comet assays) and growth inhibition was restored to the levels comparable to those in parental K562 cells. Together results indicate that our gene editing strategy for optimizing the TOP2α E19/I19 5′ SS in K/VP.5 cells circumvents resistance to etoposide and other TOP2-targeted drugs.

Gene Replacement Therapies for Genodermatoses: A Status Quo.

Epidermolysis bullosa (EB) is a genodermatosis, characterized by the formation of extended blisters and lesions on the skin and mucous membranes upon minimal mechanical trauma. The disease is caused by mutations in genes encoding proteins that are essential for skin stability. Functional impairment, reduction, or absence of one of these proteins results in skin fragility due to reduced connectivity between dermis and epidermis. Currently, gene therapy represents the only treatment option with the potential to cure this severe blistering skin disease. Two promising forms of gene therapy are potentially feasible for EB: gene replacement and genome editing. While genome editing for genodermatoses remains at the preclinical stage, gene replacement approaches are clinically advanced and have been applied already to a small number of patients with junctional and dystrophic forms of EB. Here, the viral transduction of the “wild-type” transgene into skin stem cells, followed by autologous grafting of corrected epidermal sheets, led to the regeneration of stable skin. Recent developments regarding designer nuclease-based gene editing strategies enable the establishment of alternative options to restore the gene function in genodermatoses. This is particularly true in cases wherein genetic constellation hinders gene therapy-based gene replacement.

Delivery technologies for T cell gene editing: Applications in cancer immunotherapy.

While initial approaches to adoptive T cell therapy relied on the identification and expansion of rare tumour-reactive T cells, genetic engineering has transformed cancer immunotherapy by enabling the modification of primary T cells to increase their therapeutic potential. Specifically, gene editing technologies have been utilized to create T cell populations with improved responses to antigens, lower rates of exhaustion, and potential for use in allogeneic applications. In this review, we provide an overview of T cell therapy gene editing strategies and the delivery technologies utilized to genetically engineer T cells. We also discuss recent investigations and clinical trials that have utilized gene editing to enhance the efficacy of T cells and broaden the application of cancer immunotherapies.

Single-strand template repair: key insights to increase the efficiency of gene editing.

DNA double-strand breaks (DSBs) pose a serious hazard for the stability of the genome. CRISPR-Cas9-mediated gene editing intentionally creates a site-specific DSB to modify the genomic sequence, typically from an introduced single-stranded DNA donor. However, unlike typical forms of homologous recombination, single-strand template repair (SSTR) is Rad51-independent. Moreover, this pathway is distinct from other previously characterized Rad51-independent processes. Here, we briefly review the work characterizing this pathway, and how these findings can be used to guide and improve current gene editing strategies.

The Strategies and Challenges of CCR5 Gene Editing in Hematopoietic Stem and Progenitor Cells for the Treatment of HIV.

HIV infection continues to be a serious health issue with an alarming global spread, owing to the fact that attempts at developing an effective vaccine or a permanent cure remains futile. So far, the only available treatment for the clinical management of HIV is the combined Anti-Retroviral Therapy (cART), but the long-term cART is associated with metabolic changes, organ damages, and development and transmission of drug resistant HIV strains. Thus, there is a need for the development of one-time curative treatment for HIV infection. The allogeneic transplantation with the Hematopoietic Stem and Progenitor cells (HSPCs) having 32bp deletion in Chemokine receptor 5 gene (CCR5 Δ32) demonstrated successful HIV remission in the Berlin and London patients, and highlighted that transplantation of CCR5 null HSPCs is a promising approach for a long- term HIV remission. The advent of gene editing technologies offers a new choice of generating ex vivo CCR5 ablated allogeneic or autologous HSPCs for stem cell transplantation into HIV patients. Many groups are attempting CCR5 disruption in HSPCs using various gene-editing strategies. At least two such studies, involving CCR5 gene editing in HSPCs have entered the clinical trials. This review aims to outline the strategies taken for CCR5 gene editing and discuss the challenges associated with the development of CCR5 manipulated HSPCs for the gene therapy of HIV infection.

Classifying molecular phenotypes of G6PC variants for pathogenic properties and to guide therapeutic development.

Due to advances in sequencing technologies, identification of genetic variants is rapid. However, the functional consequences of most genomic variants remain unknown. Consequently, variants of uncertain significance (VUS) that appear in clinical DNA diagnostic reports lack sufficient data for interpretation. Algorithms exist to aid prediction of a variant's likelihood of pathogenicity, but these predictions usually lack empiric evidence. To examine the feasibility of generating functional evidence in vitro for a given variant's role in disease, a panel of 29 coding sequence variants in the G6PC gene was assessed. G6PC encodes glucose‐6 phosphatase enzyme, and reduction in its function causes the rare metabolic disease glycogen storage disease type 1a (GSD1a). Variants were heterologously expressed as fusion proteins in a hepatocyte‐derived cell line and examined for effects on steady‐state protein levels, biosynthetic processing, and intracellular distribution. The screen revealed variant effects on protein levels, N‐linked glycosylation status, and cellular distribution. Of the eight VUS tested, seven behaved similar to wild‐type protein while one VUS, p.Cys109Tyr, exhibited features consistent with pathogenicity for all molecular phenotypes assayed, including significantly reduced protein levels, alteration in protein glycosylation status, and abnormally diffuse protein localization pattern, and has recently been reported in a patient with GSD1a. Our results show that such a screen can add empiric evidence to existing databases to aid in diagnostics, and also provides further classification for molecular phenotypes that could be used in future therapeutic screening approaches for small molecule or gene editing strategies directed at specific variants.

Gene Therapies for Transfusion-Dependent β-Thalassemia

β-Thalassemia is one of the most prevalent monogenic diseases usually caused by quantitative defects in the production of b-globin, a component of adult hemoglobin (a2b2), leading to severe anemia. Technological advances in genome sequencing, stem cell selection, viral vector development, transduction and gene-editing strategies now allow for efficient ex-vivo genetic manipulation of human hematopoietic stem cells that can lead to a meaningful clinical benefit in thalassemia patients. In this perspective, the status of the gene-therapy approaches available for transfusion-dependent thalassemia and early results of clinical trials are discussed. It is highly anticipated that gene therapies will soon become a treatment option for patients lacking compatible donors for hematopoietic stem cell transplant and will offer a suitable alternative for definitive treatment of b-thalassemia, even in young children.

MiniCAFE, a CRISPR/Cas9-based compact and potent transcriptional activator, elicits gene expression invivo.

CRISPR-mediated gene activation (CRISPRa) is a promising therapeutic gene editing strategy without inducing DNA double-strand breaks (DSBs). However, in vivo implementation of these CRISPRa systems remains a challenge. Here, we report a compact and robust miniCas9 activator (termed miniCAFE) for in vivo activation of endogenous target genes. The system relies on recruitment of an engineered minimal nuclease-null Cas9 from Campylobacter jejuni and potent transcriptional activators to a target locus by a single guide RNA. It enables robust gene activation in human cells even with a single DNA copy and is able to promote lifespan of Caenorhabditis elegans through activation of longevity-regulating genes. As proof-of-concept, delivered within an all-in-one adeno-associated virus (AAV), miniCAFE can activate Fgf21 expression in the liver and regulate energy metabolism in adult mice. Thus, miniCAFE holds great therapeutic potential against human diseases.

Efficient correction of Duchenne muscular dystrophy mutations by SpCas9 and dual gRNAs

CRISPR gene therapy is one promising approach for treatment of Duchenne muscular dystrophy (DMD), which is caused by a large spectrum of mutations in the dystrophin gene. To broaden CRISPR gene editing strategies for DMD treatment, we report the efficient restoration of dystrophin expression in induced myotubes by SpCas9 and dual guide RNAs (gRNAs). We first sequenced 32 deletion junctions generated by this editing method and revealed that non-homologous blunt-end joining represents the major indel type. Based on this predictive repair outcome, efficient in-frame deletion of a part of DMD exon 51 was achieved in HEK293T cells with plasmids expressing SpCas9 and dual gRNAs. More importantly, we further corrected a frameshift mutation in human DMD (exon45del) fibroblasts with SpCas9-dual gRNA ribonucleoproteins. The edited DMD fibroblasts were transdifferentiated into myotubes by lentiviral-mediated overexpression of a human MYOD transcription factor. Restoration of DMD expression at both the mRNA and protein levels was confirmed in the induced myotubes. With further development, the combination of SpCas9-dual gRNA-corrected DMD patient fibroblasts and transdifferentiation may provide a valuable therapeutic strategy for DMD.

PnB Designer: a web application to design prime and base editor guide RNAs for animals and plants

BackgroundThe rapid expansion of the CRISPR toolbox through tagging effector domains to either enzymatically inactive Cas9 (dCas9) or Cas9 nickase (nCas9) has led to several promising new gene editing strategies. Recent additions include CRISPR cytosine or adenine base editors (CBEs and ABEs) and the CRISPR prime editors (PEs), in which a deaminase or reverse transcriptase are fused to nCas9, respectively. These tools hold great promise to model and correct disease-causing mutations in animal and plant models. But so far, no widely-available tools exist to automate the design of both BE and PE reagents.ResultsWe developed PnB Designer, a web-based application for the design of pegRNAs for PEs and guide RNAs for BEs. PnB Designer makes it easy to design targeting guide RNAs for single or multiple targets on a variant or reference genome from organisms spanning multiple kingdoms. With PnB Designer, we designed pegRNAs to model all known disease causing mutations available in ClinVar. Additionally, PnB Designer can be used to design guide RNAs to install or revert a SNV, scanning the genome with one CBE and seven different ABE PAM variants and returning the best BE to use. PnB Designer is publicly accessible at http://fgcz-shiny.uzh.ch/PnBDesigner/ConclusionWith PnB Designer we created a user-friendly design tool for CRISPR PE and BE reagents, which should simplify choosing editing strategy and avoiding design complications.

A Small Key for a Heavy Door: Genetic Therapies for the Treatment of Hemoglobinopathies.

Throughout the past decades, the search for a treatment for severe hemoglobinopathies has gained increased interest within the scientific community. The discovery that ɤ-globin expression from intact HBG alleles complements defective HBB alleles underlying β-thalassemia and sickle cell disease, has provided a promising opening for research directed at relieving ɤ-globin repression mechanisms and, thereby, improve clinical outcomes for patients. Various gene editing strategies aim to reverse the fetal-to-adult hemoglobin switch to up-regulate ɤ-globin expression through disabling either HBG repressor genes or repressor binding sites in the HBG promoter regions. In addition to these HBB mutation-independent strategies involving fetal hemoglobin (HbF) synthesis de-repression, the expanding genome editing toolkit is providing increased accuracy to HBB mutation-specific strategies encompassing adult hemoglobin (HbA) restoration for a personalized treatment of hemoglobinopathies. Moreover, besides genome editing, more conventional gene addition strategies continue under investigation to restore HbA expression. Together, this research makes hemoglobinopathies a fertile ground for testing various innovative genetic therapies with high translational potential. Indeed, the progressive understanding of the molecular clockwork underlying the hemoglobin switch together with the ongoing optimization of genome editing tools heightens the prospect for the development of effective and safe treatments for hemoglobinopathies. In this context, clinical genetics plays an equally crucial role by shedding light on the complexity of the disease and the role of ameliorating genetic modifiers. Here, we cover the most recent insights on the molecular mechanisms underlying hemoglobin biology and hemoglobinopathies while providing an overview of state-of-the-art gene editing platforms. Additionally, current genetic therapies under development, are equally discussed.

Hematopoietic Stem Cell-Targeted Gene-Addition and Gene-Editing Strategies for β-hemoglobinopathies

Sickle cell disease (SCD) is caused by a well-defined point mutation in the β-globin gene and therefore is an optimal target for hematopoietic stem cell (HSC) gene-addition/editing therapy. In HSC gene-addition therapy, a therapeutic β-globin gene is integrated into patient HSCs via lentiviral transduction, resulting in long-term phenotypic correction. State-of-the-art gene-editing technology has made it possible to repair the β-globin mutation in patient HSCs or target genetic loci associated with reactivation of endogenous γ-globin expression. With both approaches showing signs of therapeutic efficacy in patients, we discuss current genetic treatments, challenges, and technical advances in this field.

CRISPR/Cas9 Genome Editing of the Human Topoisomerase IIα Intron 19 5' Splice Site Circumvents Etoposide Resistance in Human Leukemia K562 Cells.

An essential function of DNA topoisomerase IIα (TOP2α; 170 kDa, TOP2α/170) is to resolve DNA topologic entanglements during chromosome disjunction by introducing transient DNA double-stranded breaks. TOP2α/170 is an important target for DNA damage-stabilizing anticancer drugs, whose clinical efficacy is compromised by drug resistance often associated with decreased TOP2α/170 expression. We recently demonstrated that an etoposide-resistant K562 clonal subline, K/VP.5, with reduced levels of TOP2α/170, expresses high levels of a novel C-terminal truncated TOP2α isoform (90 kDa, TOP2α/90). TOP2α/90, the translation product of a TOP2α mRNA that retains a processed intron 19 (I19), heterodimerizes with TOP2α/170 and is a resistance determinant through a dominant-negative effect on drug activity. We hypothesized that genome editing to enhance I19 removal would provide a tractable strategy to circumvent acquired TOP2α-mediated drug resistance. To enhance I19 removal in K/VP.5 cells, CRISPR/Cas9 was used to make changes (GAG//GTAA AC →GAG//GTAA GT ) in the TOP2α gene's suboptimal exon 19/intron 19 5' splice site (E19/I19 5' SS). Gene-edited clones were identified by quantitative polymerase chain reaction and verified by sequencing. Characterization of a clone with all TOP2α alleles edited revealed improved I19 removal, decreased TOP2α/90 mRNA/protein, and increased TOP2α/170 mRNA/protein. Sensitivity to etoposide-induced DNA damage (γH2AX, Comet assays) and growth inhibition was restored to levels comparable to those in parental K562 cells. Together, the results indicate that our gene-editing strategy for optimizing the TOP2α E19/I19 5' SS in K/VP.5 cells circumvents resistance to etoposide and other TOP2α-targeted drugs. SIGNIFICANCE STATEMENT: Results presented here indicate that CRISPR/Cas9 gene editing of a suboptimal exon 19/intron 19 5' splice site in the DNA topoisomerase IIα (TOP2α) gene results in circumvention of acquired drug resistance to etoposide and other TOP2α-targeted drugs in a clonal K562 cell line by enhancing removal of intron 19 and thereby decreasing formation of a truncated TOP2α 90 kDa isoform and increasing expression of full-length TOP2α 170 kDa in these resistant cells. Results demonstrate the importance of RNA processing in acquired drug resistance to TOP2α-targeted drugs.

Advances allowing feasible pyrG gene editing by a CRISPR-Cas9 system for the edible mushroom Pleurotus eryngii

For decades, the edible mushroom Pleurotus eryngii (P. eryngii) has been cultivated as important raw materials for food and pharmaceutical industries in most of Asian countries, especially in China. Unfortunately, the generation and improvement of new cultivars are very difficult since there are many barriers which have not been solved thoroughly by gene editing tools, even though the CRISPR-Cas9 technique has been widely applied in other species. In this study, we identified the point-mutated variant of the endogenous sdhB gene (cbxr) as a more stable selection marker than hygromycin B resistance gene (hph) in P. eryngii. Furthermore, using a codon-optimized Cas9, a predicted native U6 promoter-guided sgRNA, as well as an optimized protoplast transformation system, a highly efficient pyrG gene editing system was established in P. eryngii, that incorporated varied insertions and deletions (indels) by non-homologous end joining (NHEJ) and homology-directed repair (HDR). Findings for a successful targeted gene editing strategy in the edible mushroom P. eryngii may open a new chapter for the improvement of edible mushroom cultivars.

Genome Editing of Rice by CRISPR-Cas: End-to-End Pipeline for Crop Improvement.

CRISPR-Cas resonates a revolutionary genome editing technology applicable through a horizon spreading across microbial organism to higher plant and animal. This technology can be harnessed with ease to understand the basic genetics of a living system by altering sequence of individual genes and characterizing their functions. The precision of this technology is unparallel. It allows very precise and targeted base pair level edits in the genome. Here, in the current chapter, we have provided end-to-end process outline on how to generate genome edited plants in crops like rice to evaluate for agronomic traits associated with yield, disease resistance and abiotic stress tolerance, etc. Genome editing process includes designing of gene editing strategy, vector construction, plant transformation, molecular screening, and phenotyping under control environment conditions. Furthermore, its application for development of commercial crop product may require additional processes, including field trials in the target geography for evaluation of product efficacy. Evaluation of genome edited lines in controlled greenhouse/net house or open field condition requires few generations for outcrossing with wild-type parent to eliminate and/or reduce any potential pleiotropic effect in the edited genome which may arise during the process. The genome edited plant selected for advancement shall harbor the genome with only the intended changes, which can be analyzed by various molecular techniques, advanced sequencing methods, and genomic data analysis tools. CRISPR-Cas-based genome editing has opened a plethora of opportunities in agriculture as well as human health.

Development of Gene Editing Strategies for CCR5 Gene in Endothelial Cells

Development of Gene Editing Strategies for CCR5 Gene in Endothelial Cells