Zinc Finger Nucleases Research Articles

A narrative review of in utero gene therapy: advances, challenges, and future considerations.

The field of in utero gene therapy (IUGT) represents a crossroad of technologic advancements and medical ethical boundaries. Several strategies have been developed for IUGT focusing on either modifying endogenous genes, replacing missing genes, or modifying gene transcription products. The list of candidate diseases such as hemoglobinopathies, cystic fibrosis, lysosomal storage disorders continues to grow with new strategies being developed as our understanding of their respective underlying molecular pathogenesis increases. Treatment in utero has several distinct advantages to postnatal treatment. Biologic and physiologic phenomena enable the delivery of a higher effective dose, generation of immune tolerance, and the prevention of phenotypic onset for genetic diseases. Therapeutic technology for IUGT including CRISPR-Cas9 systems, zinc finger nucleases (ZFN), and peptide nucleic acids (PNAs) has already shown promise in animal models and early postnatal clinical trials. While the ability to detect fetal diagnoses has dramatically improved with developments in ultrasound and next-generation sequencing, treatment options remain experimental, with several translational gaps remaining prior to implementation in the clinical realm. Complicating this issue, the potential diseases targeted by this approach are often debilitating and would otherwise prove fatal if not treated in some manner. The leap from small animals to large animals, and subsequently, to humans will require further vigorous testing of safety and efficacy.

Time course of ventilatory failure in a novel rat model of Pompe Disease

Pompe disease (PD) is caused by mutations in the gene encoding the enzyme acid-α-glucosidase (GAA). Since GAA acts to degrade lysosomal glycogen, reduced or loss of enzyme activity leads to glycogen accumulation and neuromuscular impairments. Respiratory dysfunction and ventilator-dependence is common in both early- and late-onset PD. Our group has created a rat model of PD using Zinc Finger Nuclease (ZFN) technology. The goal of the current study was to characterize ventilation and metabolism in unrestrained ZFN Pompe rats using whole body plethysmography. Data were collected during a normoxic baseline period, acute exposures to hypoxia (FiO2=10.5%), hypercapnia (FiCO2=7%), and combined hypercapnic-hypoxic challenge intended to produce a maximal ventilatory drive. ZFN Pompe rats and age-matched, wild type (WT) controls were studied at age 3, 6, and 10-11 months to assess ventilation throughout disease progression. Both male and female 3 month old ZFN Pompe rats hyperventilated (greater VE/VCO2) due to greater tidal volume compared to WT rats. However, as the ZFN Pompe rats aged, the ventilatory phenotype changed such that by 10-11 months they showed significant hypoventilation as well as a blunted ability to increase ventilation in response to respiratory challenge relative to WT controls. Histological data from these rats, as well as prior data from the Gaa-/- mouse model of PD, show that respiratory neuromuscular pathology is present at a very young age. Accordingly, the current ventilatory data suggest that animals with PD initially increase breathing in response to the onset of pathology, possibly representing a period of “overcompensation”. However, this is followed by respiratory failure as the respiratory neuromuscular pathology progresses towards disease end-stage.

CRISPR/Cas9 technology in disease research and therapy: a review

Genome editing is a genetic manipulation technique that can modify DNA sequences at the genome level, including insertion, knockout, replacement and point mutation of specific DNA fragments. The ultimate principle of genome editing technology relying on engineered nucleases is to generate double-stranded DNA breaks at specific locations in genome and then repair them through non-homologous end joining or homologous recombination. With the intensive study of these nucleases, genome editing technology develops rapidly. The most used nucleases include meganucleases, zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats associated Cas proteins. Based on introducing the development and principles of above mentioned genome editing technologies, we review the research progress of CRISPR/Cas9 system in the application fields of identification of gene function, establishment of disease model, gene therapy, immunotherapy and its prospect.

Molecular Switch Engineering for Precise Genome Editing.

Genome editing technology commenced in 1996 with the discovery of the first zinc-finger nuclease. Application of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) associated protein 9 (Cas9) technology to genome editing of mammalian cells allowed researchers to use genome editing more easily and cost-effectively. However, one of the technological problems that remains to be solved is "off-target effects", which are unexpected mutations in nontarget DNA. One significant improvement in genome editing technology has been achieved with molecular/protein engineering. The key to this engineering is a "switch" to control function. In this review, we discuss recent efforts to design novel "switching" systems for precise editing using genome editing tools.

Compound heterozygous KCTD7 variants in progressive myoclonus epilepsy

KCTD7 is a member of the potassium channel tetramerization domain-containing protein family and has been associated with progressive myoclonic epilepsy (PME), characterized by myoclonus, epilepsy, and neurological deterioration. Here we report four affected individuals from two unrelated families in which we identified KCTD7 compound heterozygous single nucleotide variants through exome sequencing. RNAseq was used to detect a non-annotated splicing junction created by a synonymous variant in the second family. Whole-cell patch-clamp analysis of neuroblastoma cells overexpressing the patients’ variant alleles demonstrated aberrant potassium regulation. While all four patients experienced many of the common clinical features of PME, they also showed variable phenotypes not previously reported, including dysautonomia, brain pathology findings including a significantly reduced thalamus, and the lack of myoclonic seizures. To gain further insight into the pathogenesis of the disorder, zinc finger nucleases were used to generate kctd7 knockout zebrafish. Kctd7 homozygous mutants showed global dysregulation of gene expression and increased transcription of c-fos, which has previously been correlated with seizure activity in animal models. Together these findings expand the known phenotypic spectrum of KCTD7-associated PME, report a new animal model for future studies, and contribute valuable insights into the disease.

Progress in Gene Therapy of Sickle Cell Disease Based on Hemoglobin F--Review

Sickle cell disease (SCD) is a single gene genetic disease, which seriously threatens the life span and quality of patients. On the basis of the pathogenesis of SCD and the alternative therapy based on fetal hemoglobin F (HbF), the research progress of transcription factors involved in the regulation of HbF gene expression, such as BCL11A, ZBTB7A, KLF-1, c-MYB and SOX6, as well as the application of CRISPR / Cas9, TALEN, zinc finger nuclease and other gene editing technologies in this field has been made, providing a solid theoretical basis for the exploration of new treatment schemes for β- like hemoglobin diseases, such as sickle cell disease and β- thalassemia.

Overview of sixteen scientific opinions on genetically modified plants obtained by new genomic techniques

RIVM was asked by EFSA in the frame of a procurement (PO/EFSA/GMO/2020/01) to producean overview of 16 scientific opinions published by European competent authorities and national institutions since 2012 ongenetically modified plants obtained by new genomic techniques (NGTs). The 16 opinions were provided by the European Commission in the frame of a mandate asking EFSA to provide an overview on the risk assessment of plants developed through NGTsbased on its own previous and current work and on work carried out at national level. NGTs are defined as techniques capable to change the genetic material of an organism and that have emerged or have been developed since the adoption of the GMO legislation in 2001. Based on this definition for NGTs provided by EC, the following NGTs as described in a report of the Joint Research Council on new plant breeding techniques, published in 2011, were considered: (1) Zinc finger nuclease technology, (defined more broadly as site-directed nuclease technology); (2) oligonucleotide directed mutagenesis; (3) cisgenesis and intragenesis; (4) RNA-dependent DNA methylation; (5) grafting (on genetically modified rootstock); (6) reverse breeding; (7) agro-infiltration and (8) synthetic genomics.The current report presents the information on the defined NGTsmethod descriptionand on the risk assessment of plants developed through these NGTs, as extracted from the 16opinions. In order to extract the relevant information a baseline descriptionfor each NGT was set and inclusion and exclusion criteria for information extraction were defined. Most (14 out of 16) opinions discuss the SDN technology, whereas no opinion contains information on synthetic genomics. A new NGT, base-editing, is described in four opinions and therefore information on this technique is also presented.As specified by the procurement, no critical appraisal onthe information extracted from the scientific opinions was performed.

Recent Advances in Genome Editing Tools in Medical Mycology Research.

Manipulating fungal genomes is an important tool to understand the function of target genes, pathobiology of fungal infections, virulence potential, and pathogenicity of medically important fungi, and to develop novel diagnostics and therapeutic targets. Here, we provide an overview of recent advances in genetic manipulation techniques used in the field of medical mycology. Fungi use several strategies to cope with stress and adapt themselves against environmental effectors. For instance, mutations in the 14 alpha-demethylase gene may result in azole resistance in Aspergillus fumigatus strains and shield them against fungicide’s effects. Over the past few decades, several genome editing methods have been introduced for genetic manipulations in pathogenic fungi. Application of restriction enzymes to target and cut a double-stranded DNA in a pre-defined sequence was the first technique used for cloning in Aspergillus and Candida. Genome editing technologies, including zinc-finger nucleases (ZFNs) and transcriptional activator-like effector nucleases (TALENs), have been also used to engineer a double-stranded DNA molecule. As a result, TALENs were considered more practical to identify single nucleotide polymorphisms. Recently, Class 2 type II Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 technology has emerged as a more useful tool for genome manipulation in fungal research.

ZFN-mediated in vivo gene editing in hepatocytes leads to supraphysiologic α-Gal A activity and effective substrate reduction in Fabry mice

Fabry disease, a lysosomal storage disorder resulting from thedeficient activity of α-galactosidase A (α-Gal A), is characterized by cardiac, renal, and/or cerebrovascular disease due to progressive accumulation of the enzyme's substrates, globotriaosylceramide (Gb3) and globotriaosylsphingosine (Lyso-Gb3). We report here the preclinical evaluation of liver-targeted invivo genome editing using zinc-finger nuclease (ZFN) technology to insert the human α-galactosidase A (hGLA) cDNA into the albumin "safe harbor" locus of Fabry mice, thereby generating an albumin-α-Gal A fusion protein. The mature α-Gal A protein is secreted into the circulation for subsequent mannose-6-phosphate receptor-mediated tissue uptake. Donor vector optimization studies showed that replacing the hGLA cDNA signal peptide sequence with that of human iduronate 2-sulfatase (IDS) achieved higher transgene expression. Intravenous adeno-associated virus (AAV) 2/8-mediated co-delivery of the IDS-hGLA donor and ZFNs targeting the albumin locus resulted in continuous, supraphysiological plasma and tissue α-Gal A activities, which essentially normalized Gb3 and Lyso-Gb3 levels in key tissues of pathology. Notably, this was achieved with <10% of hepatocytes being edited to express hGLA, occurring mostly via non-homologous end joining (NHEJ) rather than homology-directed repair (HDR). These studies indicate that ZFN-mediated invivo genome editing has the potential to be an effective one-time therapy for Fabry disease.

CRISPR/Cas9-Mediated Gene Editing Revolutionizes the Improvement of Horticulture Food Crops

Horticultural food crops are important sources of nutrients for humans. With the increase of the global population, enhanced horticulture food crop production has become a new challenge, and enriching their nutritional content has also been required. Gene editing systems, such as zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9), have accelerated crop improvement through the modification of targeted genomes precisely. Here, we review the development of various gene editors and compare their advantages and shortcomings, especially the newly emerging CRISPR/Cas systems, such as base editing and prime editing. We also summarize their practical applications in crop trait improvement, including yield, nutritional quality, and other consumer traits.

Advanced genome editing strategies for manipulation of plant specialized metabolites pertaining to biofortification

Emerging trends in molecular biology have progressed the knowledge of plant specialized metabolites with respect to diversity in structure, function and biosynthetic pathways. Being powerful genome-editing tools, Zinc Finger Nuclease, Transcription Activator-Like Effector Nuclease, and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems have found potential application in genome and epigenome engineering. CRISPR/Cas9 is being used for studying the functions of multiple genes and gene families in plants. Our analysis suggests that although a rapid progress has occurred for utilization of CRISPR/Cas9 tool in crop improvement, limited studies are available for its application in manipulation of gene clusters of useful specialized metabolites in plants. In this review, after describing briefly about the recent advancements in genome editing techniques, we have further discussed their applicability in the modulation of metabolite production and biofortification of food crops. We have also emphasized the importance of CRISPR/Cas9-based targeted deletion of larger chromosomal fragments or gene clusters towards value addition of crop plants. The current policies for CRISPR/Cas9-edited crop plants in different countries and their acceptability in market place is also discussed. We propose that advanced genome editing techniques, including a multiplex CRISPR/Cas9 system could serve as a versatile tool for rewiring of metabolite gene clusters and improving the levels of useful metabolites in plants.

The Current Status of Gene Therapy for the Treatment of Cancer.

Gene therapy is the administration of foreign genomic material into the host tissue to modify the expression of a gene product or to change the biological properties of cells for therapeutic use. Initially, the major objective of gene therapy was to manage genetic diseases, but now different disorders with several patterns of acquired and inherited disorders are targets of gene therapy. Over three decades, the advancement of Genome engineering technologies facilitated gene therapy for the prevention and management of intractable diseases. Researchers are advancing with cautious optimism that safe and effective treatment will give to patients with single-gene disorders and complex acquired disorders. To date, over 3000 genes associates with disease-causing mutations, and about 2600 gene therapy trials are undergoing for the management of various disorders. This review summarizes the principles of genome-editing approaches, such as zinc finger nucleases, transcription activator-like effector nucleases, meganucleases, and the CRISPR/Cas9 system with the underlying mechanisms. This review also explains the types of gene delivery systems as viral [adenoviral, adeno association, herpes simplex virus] and nonviral delivery systems (physical: DNA bombardment, electroporation) and (chemical: Cationic lipids, cationic polymers). Finally, this review summarizes gene therapy medicines approved to treat cancer in detail, including names, indications, vectors, and mode of gene therapy. Gene therapy becomes an alternative to an existing management for different diseases. Therefore, gene products with safe vectors and better biotechnologies play a significant role in the prophylaxis and management of various disorders in the future.

Rescuing lethal phenotypes induced by disruption of genes in mice: a review of novel strategies.

Approximately 35 % of the mouse genes are indispensable for life, thus, global knock-out (KO) of those genes may result in embryonic or early postnatal lethality due to developmental abnormalities. Several KO mouse lines are valuable human disease models, but viable homozygous mutant mice are frequently required to mirror most symptoms of a human disease. The site-specific gene editing systems, the transcription activator-like effector nucleases (TALENs), Zinc-finger nucleases (ZFNs) and the clustered regularly interspaced short palindrome repeat-associated Cas9 nuclease (CRISPR/Cas9) made the generation of KO mice more efficient than before, but the homozygous lethality is still an undesired side-effect in case of many genes. The literature search was conducted using PubMed and Web of Science databases until June 30th, 2020. The following terms were combined to find relevant studies: "lethality", "mice", "knock-out", "deficient", "embryonic", "perinatal", "rescue". Additional manual search was also performed to find the related human diseases in the Online Mendelian Inheritance in Man (OMIM) database and to check the citations of the selected studies for rescuing methods. In this review, the possible solutions for rescuing human disease-relevant homozygous KO mice lethal phenotypes were summarized.

Potential Application of CRISPR/Cas9 System to Engineer Abiotic Stress Tolerance in Plants.

Abiotic stresses in plants such as salinity, drought, heavy metal toxicity, heat, and nutrients limitations significantly reduce agricultural production worldwide. The genome editing techniques such as transcriptional activator-like effector nucleases (TALENs) and zinc finger nucleases (ZFNs) have been used for genome manipulations in plants. However, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technique has recently emerged as a promising tool for genome editing in plants to acquire desirable traits. The CRISPR/Cas9 system has a great potential to develop crop varieties with improved tolerance against abiotic stresses. This review is centered on the biology and potential application of the CRISPR/Cas9 system to improve abiotic stress tolerance in plants. Furthermore, this review highlighted the recent advancements of CRISPR/Cas9-mediated genome editing for sustainable agriculture.

CRISPR/Cas9-mediated grna gene knockout leads to neurodevelopmental defects and motor behavior changes in zebrafish.

Progranulin (PGRN) is a secreted glycoprotein with multiple biological functions in early embryogenesis, anti-inflammation, and neurodegeneration. A good model for the functional study of PGRN is the zebrafish with knockdown or knockout of grn, the gene encoding PGRN. Morpholino oligonucleotides (MOs) and zinc finger nucleases have been used to generate zebrafish grn models, yet they have shown inconsistent phenotypes due to either the neurotoxicity of the MOs or possible genetic compensation responses during gene editing. In this study, we generated stable grna (one of the major grn homologues of zebrafish) knockout zebrafish by using CRISPR/Cas9-mediated genome editing. A grna sgRNA was designed to target the similar repeated sequence shared by exon 13, exon 15, and exon 19 in zebrafish. The F1 generation with the frameshift mutation of+4bp (the addition of 4bp to exon15), which causes a premature termination, was obtained and subjected to morphological and behavioral evaluation. The grna knockout zebrafish showed neurodevelopmental defects, including spinal motor neurons with shorter axons, decreased sensory hair cells, thinning of the outer nuclear layer and thickening of the inner nuclear layer of the retina, decreased expression of rhodopsin in the cone cells, and motor behavior changes. Moreover, the phenotypes of grna knockout zebrafish could be rescued with the Tol2 system carrying the grna gene. The grna knockout zebrafish model generated in this study provides a useful tool to study PGRN function and has potential for high-throughput drug screening for disease therapy.

Mini-Review Regarding the Applicability of Genome Editing Techniques Developed for Studying Infertility.

Infertility is a highly debated topic today. It has been long hypothesized that infertility has an idiopathic cause, but recent studies demonstrated the existence of a genetic substrate. Fortunately, the methods of editing the human genome proven to be revolutionary. Following research conducted, we identified a total of 21 relevant studies; 14 were performed on mice, 5 on zebrafish and 2 on rats. We concluded that over forty-four genes in total are dispensable for fertility in both sexes without affecting host homeostasis. However, there are genes whose loss-of-function induces moderate to severe phenotypic changes in both sexes. There were situations in which the authors reported infertility, exhibited by the experimental model, or other pathologies such as cryptorchidism, cataracts, or reduced motor activity. Overall, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 are techniques that offer a wide range of possibilities for studying infertility, even to create mutant variants. It can be concluded that ZFNs, TALENs, and CRISPR/Cas9 are crucial tools in biomedical research.

Microbial community changes in a female rat model of Rett syndrome

Rett syndrome (RTT) is an X-linked neurodevelopmental disorder that is predominantly caused by alterations of the methyl-CpG-binding protein 2 (MECP2) gene. Disease severity and the presence of comorbidities such as gastrointestinal distress vary widely across affected individuals. The gut microbiome has been implicated in neurodevelopmental disorders such as Autism Spectrum Disorder (ASD) as a regulator of disease severity and gastrointestinal comorbidities. Although the gut microbiome has been previously characterized in humans with RTT compared to healthy controls, the impact of MECP2 mutation on the composition of the gut microbiome in animal models where the host and diet can be experimentally controlled remains to be elucidated. By evaluating the microbial community across postnatal development as behavioral symptoms appear and progress, we have identified microbial taxa that are differentially abundant across developmental timepoints in a zinc-finger nuclease rat model of RTT compared to WT. We have additionally identified p105 as a key translational timepoint. Lastly, we have demonstrated that fecal SCFA levels are not altered in RTT rats compared to WT rats across development. Overall, these results represent an important step in translational RTT research.

Combinatorial Strategies for Long-term Control of HIV Infection.

AIDS is a disease caused by a chronic infection of HIV. Recently, long-term control of HIV infection has been demonstrated through the bone marrow transplantation of hematopoietic stem cells (HSC), in which the C-C chemokine receptor type 5 (CCR5) gene is mutated innately. However, it is very difficult to obtain CCR5 mutant HSC that match human leukocyte antigen between donor and recipient. To solve this problem, this review will summarize and discuss various reports related to the generation of patient-specific CCR5 geneedited HSC. The fusion of current gene editing (zinc-finger nuclease, transcription activator-like effector nuclease, and clustered regulatory interspaced short palindromic repeats) and cellular reprogramming technology (somatic cell nuclear transfer, induced pluripotent stem cells technology, and direct phenotypic conversion) enables the generation of patient-specific CCR5 edited HSC. These cells can be useful as valuable therapeutic agents for long-term control of HIV-infected patients in the future.

Empowering of reproductive health of farm animals through genome editing technology

To cater the exponential growth of human population, need to improve food production and quality through modern biotechnology with limited recourses in a way that has minimal impact on the environment. The selective breeding and genomic selection have attended the momentum gain in livestock productivity. Recent advancement in genome-editing technologies offers exciting prospects for the production of healthy and prolific livestock. Genome editing involves altering genetic material by manipulation, addition, or removal of certain deoxyribonucleic acid (DNA) sequences at a specific locus in a way that does not occur naturally. The major genome editors are zinc finger nucleases, transcription-activator-like endonucleases, and clustered regularly interspaced short palindromic repeats associated protein nine systems which are proficient of cutting the nuclear DNA precisely at a predetermined position. This review provides an update on the use of genome editing systems to modify the genes related to reproduction of farm animal vis-à-vis human, update knowledge on the underlying mechanism and discusses new opportunities to produce genetically modified farm animals.

Advances in Genome Editing With CRISPR Systems and Transformation Technologies for Plant DNA Manipulation

The year 2020 marks a decade since the first gene-edited plants were generated using homing endonucleases and zinc finger nucleases. The advent of CRISPR/Cas9 for gene-editing in 2012 was a major science breakthrough that revolutionized both basic and applied research in various organisms including plants and consequently honored with “The Nobel Prize in Chemistry, 2020.” CRISPR technology is a rapidly evolving field and multiple CRISPR-Cas derived reagents collectively offer a wide range of applications for gene-editing and beyond. While most of these technological advances are successfully adopted in plants to advance functional genomics research and development of innovative crops, others await optimization. One of the biggest bottlenecks in plant gene-editing has been the delivery of gene-editing reagents, since genetic transformation methods are only established in a limited number of species. Recently, alternative methods of delivering CRISPR reagents to plants are being explored. This review mainly focuses on the most recent advances in plant gene-editing including (1) the current Cas effectors and Cas variants with a wide target range, reduced size and increased specificity along with tissue specific genome editing tool kit (2) cytosine, adenine, and glycosylase base editors that can precisely install all possible transition and transversion mutations in target sites (3) prime editing that can directly copy the desired edit into target DNA by search and replace method and (4) CRISPR delivery mechanisms for plant gene-editing that bypass tissue culture and regeneration procedures including de novo meristem induction, delivery using viral vectors and prospects of nanotechnology-based approaches.