Diamond-Blackfan anemia (DBA) is a rare genetic disorder affecting the bone marrow's ability to produce red blood cells, leading to severe anemia and various physical abnormalities. Approximately 75% of DBA cases involve heterozygous mutations in ribosomal protein (RP) genes, classifying it as a ribosomopathy, with RPS19 being the most frequently mutated gene. Non-RP mutations, such as in GATA1, have also been identified. Current treatments include glucocorticosteroids, blood transfusions, and hematopoietic stem cell transplantation (HSCT), with HSCT being the only curative option, albeit with challenges like donor availability and immunological complications. Gene therapy, particularly using lentiviral vectors and CRISPR/Cas9 technology, emerges as a promising alternative. This review explores the potential of gene therapy, focusing on lentiviral vectors and CRISPR/Cas9 technology in combination with non-integrating lentiviral vectors, as a curative solution for DBA. It highlights the transformative advancements in the treatment landscape of DBA, offering hope for individuals affected by this condition.
- MeSH
- CRISPR-Cas Systems genetics MeSH
- Anemia, Diamond-Blackfan * genetics therapy MeSH
- Gene Editing methods MeSH
- Genetic Therapy * methods MeSH
- Genetic Vectors MeSH
- Lentivirus genetics MeSH
- Humans MeSH
- Mutation genetics MeSH
- Ribosomal Proteins genetics MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
Micronutrient deficiency conditions, such as anemia, are the most prevalent global health problem due to inadequate iron and folate in dietary sources. Biofortification advancements can propel the rapid amelioration of nutritionally beneficial components in crops that are required to combat the adverse effects of micronutrient deficiencies on human health. To date, several strategies have been proposed to increase micronutrients in plants to improve food quality, but very few approaches have intrigued `clustered regularly interspaced short palindromic repeats' (CRISPR) modules for the enhancement of iron and folate concentration in the edible parts of plants. In this review, we discuss two important approaches to simultaneously enhance the bioavailability of iron and folate concentrations in rice endosperms by utilizing advanced CRISPR-Cas9-based technology. This includes the 'tuning of cis-elements' and 'enhancer re-shuffling' in the regulatory components of genes that play a vital role in iron and folate biosynthesis/transportation pathways. In particular, base-editing and enhancer re-installation in native promoters of selected genes can lead to enhanced accumulation of iron and folate levels in the rice endosperm. The re-distribution of micronutrients in specific plant organs can be made possible using the above-mentioned contemporary approaches. Overall, the present review discusses the possible approaches for synchronized iron and folate biofortification through modification in regulatory gene circuits employing CRISPR-Cas9 technology.
- MeSH
- Biofortification * MeSH
- CRISPR-Cas Systems * MeSH
- Gene Editing methods MeSH
- Plants, Genetically Modified * metabolism genetics MeSH
- Folic Acid * metabolism MeSH
- Humans MeSH
- Oryza metabolism genetics MeSH
- Iron * metabolism MeSH
- Crops, Agricultural * metabolism genetics MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
CRISPR/Cas9-mediated genome editing has become an extremely powerful technique used to modify gene expression in many organisms, including parasitic protists. Giardia intestinalis, a protist parasite that infects approximately 280 million people around the world each year, has been eluding the use of CRISPR/Cas9 to generate knockout cell lines due to its tetraploid genome. In this work, we show the ability of the in vitro assembled CRISPR/Cas9 components to successfully edit the genome of G. intestinalis. The cell line that stably expresses Cas9 in both nuclei of G. intestinalis showed effective recombination of the cassette containing the transcription units for the gRNA and the resistance marker. This highly efficient process led to the removal of all gene copies at once for three independent experimental genes, mem, cwp1 and mlf1. The method was also applicable to incomplete disruption of the essential gene, as evidenced by significantly reduced expression of tom40. Finally, testing the efficiency of Cas9-induced recombination revealed that homologous arms as short as 150 bp can be sufficient to establish a complete knockout cell line in G. intestinalis.
- MeSH
- CRISPR-Cas Systems * MeSH
- Gene Editing methods MeSH
- Giardia lamblia * genetics MeSH
- RNA, Guide, Kinetoplastida MeSH
- Humans MeSH
- Tetraploidy MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Obesity and type 2 diabetes are associated with disturbances in insulin-regulated glucose and lipid fluxes and severe comorbidities including cardiovascular disease and steatohepatitis. Whole body metabolism is regulated by lipid-storing white adipocytes as well as "brown" and "brite/beige" adipocytes that express thermogenic uncoupling protein 1 (UCP1) and secrete factors favorable to metabolic health. Implantation of brown fat into obese mice improves glucose tolerance, but translation to humans has been stymied by low abundance of primary human beige adipocytes. Here we apply methods to greatly expand human adipocyte progenitors from small samples of human subcutaneous adipose tissue and then disrupt the thermogenic suppressor gene NRIP1 by CRISPR. Ribonucleoprotein consisting of Cas9 and sgRNA delivered ex vivo are fully degraded by the human cells following high efficiency NRIP1 depletion without detectable off-target editing. Implantation of such CRISPR-enhanced human or mouse brown-like adipocytes into high fat diet fed mice decreases adiposity and liver triglycerides while enhancing glucose tolerance compared to implantation with unmodified adipocytes. These findings advance a therapeutic strategy to improve metabolic homeostasis through CRISPR-based genetic enhancement of human adipocytes without exposing the recipient to immunogenic Cas9 or delivery vectors.
- MeSH
- Adipocytes, White metabolism MeSH
- Cell Differentiation MeSH
- Cell Culture Techniques methods MeSH
- CRISPR-Cas Systems genetics MeSH
- Diet, High-Fat adverse effects MeSH
- Adult Stem Cells physiology MeSH
- Gene Editing methods MeSH
- RNA, Guide, Kinetoplastida genetics MeSH
- Adipocytes, Brown metabolism transplantation MeSH
- Humans MeSH
- Lipid Metabolism genetics MeSH
- Disease Models, Animal MeSH
- Mice MeSH
- Nuclear Receptor Interacting Protein 1 genetics metabolism MeSH
- Obesity complications metabolism therapy MeSH
- Subcutaneous Fat cytology MeSH
- Glucose Intolerance etiology metabolism therapy MeSH
- Thermogenesis genetics MeSH
- Fatty Liver etiology metabolism prevention & control MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, N.I.H., Extramural MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
Lysosome-associated membrane glycoprotein 3 (LAMP3) is a type I transmembrane protein of the LAMP protein family with a cell-type-specific expression in alveolar type II cells in mice and hitherto unknown function. In type II pneumocytes, LAMP3 is localized in lamellar bodies, secretory organelles releasing pulmonary surfactant into the extracellular space to lower surface tension at the air/liquid interface. The physiological function of LAMP3, however, remains enigmatic. We generated Lamp3 knockout mice by CRISPR/Cas9. LAMP3 deficient mice are viable with an average life span and display regular lung function under basal conditions. The levels of a major hydrophobic protein component of pulmonary surfactant, SP-C, are strongly increased in the lung of Lamp3 knockout mice, and the lipid composition of the bronchoalveolar lavage shows mild but significant changes, resulting in alterations in surfactant functionality. In ovalbumin-induced experimental allergic asthma, the changes in lipid composition are aggravated, and LAMP3-deficient mice exert an increased airway resistance. Our data suggest a critical role of LAMP3 in the regulation of pulmonary surfactant homeostasis and normal lung function.
- MeSH
- Asthma chemically induced genetics metabolism pathology MeSH
- Bronchoalveolar Lavage Fluid MeSH
- Gene Editing methods MeSH
- Homeostasis genetics MeSH
- Lipidomics MeSH
- Disease Models, Animal MeSH
- Mice, Knockout MeSH
- Mice MeSH
- Ovalbumin administration & dosage MeSH
- Lung metabolism pathology MeSH
- Pulmonary Alveoli metabolism pathology MeSH
- Pulmonary Surfactants metabolism MeSH
- Alveolar Epithelial Cells metabolism pathology MeSH
- Protein Isoforms genetics metabolism MeSH
- Pulmonary Surfactant-Associated Protein C genetics metabolism MeSH
- Lysosomal-Associated Membrane Protein 3 deficiency genetics MeSH
- Gene Expression Regulation MeSH
- Respiratory Function Tests MeSH
- Airway Resistance MeSH
- Signal Transduction MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Genome-editing (GE) is having a tremendous influence around the globe in the life science community. Among its versatile uses, the desired modifications of genes, and more importantly the transgene (DNA)-free approach to develop genetically modified organism (GMO), are of special interest. The recent and rapid developments in genome-editing technology have given rise to hopes to achieve global food security in a sustainable manner. We here discuss recent developments in CRISPR-based genome-editing tools for crop improvement concerning adaptation, opportunities, and challenges. Some of the notable advances highlighted here include the development of transgene (DNA)-free genome plants, the availability of compatible nucleases, and the development of safe and effective CRISPR delivery vehicles for plant genome editing, multi-gene targeting and complex genome editing, base editing and prime editing to achieve more complex genetic engineering. Additionally, new avenues that facilitate fine-tuning plant gene regulation have also been addressed. In spite of the tremendous potential of CRISPR and other gene editing tools, major challenges remain. Some of the challenges are related to the practical advances required for the efficient delivery of CRISPR reagents and for precision genome editing, while others come from government policies and public acceptance. This review will therefore be helpful to gain insights into technological advances, its applications, and future challenges for crop improvement.
According to Darwin's theory, endless evolution leads to a revolution. One such example is the Clustered Regularly Interspaced Palindromic Repeats (CRISPR)-Cas system, an adaptive immunity system in most archaea and many bacteria. Gene editing technology possesses a crucial potential to dramatically impact miscellaneous areas of life, and CRISPR-Cas represents the most suitable strategy. The system has ignited a revolution in the field of genetic engineering. The ease, precision, affordability of this system is akin to a Midas touch for researchers editing genomes. Undoubtedly, the applications of this system are endless. The CRISPR-Cas system is extensively employed in the treatment of infectious and genetic diseases, in metabolic disorders, in curing cancer, in developing sustainable methods for fuel production and chemicals, in improving the quality and quantity of food crops, and thus in catering to global food demands. Future applications of CRISPR-Cas will provide benefits for everyone and will save countless lives. The technology is evolving rapidly; therefore, an overview of continuous improvement is important. In this review, we aim to elucidate the current state of the CRISPR-Cas revolution in a tailor-made format from its discovery to exciting breakthroughs at the application level and further upcoming trends related to opportunities and challenges including ethical concerns.
- MeSH
- Archaea metabolism MeSH
- Bacteria metabolism MeSH
- CRISPR-Cas Systems * MeSH
- History, 20th Century MeSH
- History, 21st Century MeSH
- Livestock MeSH
- Gene Editing methods MeSH
- Genetic Engineering history methods MeSH
- Genome MeSH
- Humans MeSH
- Clustered Regularly Interspaced Short Palindromic Repeats MeSH
- Crops, Agricultural genetics MeSH
- Animals MeSH
- Check Tag
- History, 20th Century MeSH
- History, 21st Century MeSH
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Historical Article MeSH
- Review MeSH
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.
- MeSH
- CRISPR-Cas Systems genetics MeSH
- Gene Editing methods MeSH
- Phenotype MeSH
- Mice, Knockout MeSH
- Mice MeSH
- Zinc Finger Nucleases genetics MeSH
- Transcription Activator-Like Effector Nucleases genetics MeSH
- Embryo Loss genetics prevention & control MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
Východiska: Genová terapie je cílená změna genomu v cílových buňkách s léčebným záměrem. Genetický materiál se do buňky přenáší pomocí virových nebo nevirových vektorů. V současnosti již existují metody pro vysoce přesnou, specifickou editaci genomu. Klinická aplikace genové terapie u pacientů léčených radioterapií se zaměřuje na zvýšení účinku radioterapie (tedy usmrcování nádorových buněk) a na minimalizaci poškození normálních tkání. Radiace a genová terapie mohou mít synergický účinek - radioterapie určuje čas a místo aktivace genového konstruktu a může zvýšit efektivitu genového přenosu. Existují rovněž strategie s přenosem genů zvyšující radiosenzibilitu cílové tkáně. Základní strategie genové terapie v onkologii zahrnují přenos genů pro zvýšení citlivosti nádoru na farmakoterapii nebo radioterapii, kompenzaci ztraceného nebo deregulovaného onkogenu/antionkogenu, blokování exprese onkogenu za použití antisense oligonukleotidů a zvýšení imunogenicity nádoru za účelem navození nebo stimulace protinádorové imunity (genová imunoterapie). Cíle: Cílem krátkého přehledového článku je poskytnout informace o strategiích a možnostech kombinací genové terapie a radioterapie. Závěr: Různé typy genové terapie a radioterapii lze kombinovat za účelem zvýšení lokoregionálního protinádorového účinku a snížení toxicity. Všechny aplikace genové terapie v kombinaci s radioterapií však zatím zůstávají experimentální.
Background: Gene therapy is a targeted alteration of the genome with therapeutic intent. Genetic material is transferred to the target cell by viral or non-viral vectors. Several methods for highly accurate and specific genome editing have been developed. Clinical applications of gene therapy in patients receiving radiotherapy aim at enhancing the effect of radiotherapy and minimizing damage to normal tissues. The effects of radiation and gene therapy may be synergistic. Radiotherapy determines the time and place of gene construct activation and can increase the efficiency of gene transfer. Some gene therapies increase the radiosensibility of the target tissue. Basic strategies for gene therapy in oncology include gene transfer to increase tumor sensitivity to pharmacotherapy or radiotherapy, compensate/control the lost or deregulated oncogenes, block oncogene expression using antisense oligonucleotides, and increase tumor immunogenicity in order to induce or stimulate anti-tumor immunity. Objectives: The aim of this short review is to provide the information on strategies and possibilities of combinations of gene therapy and radiotherapy. Conclusions: Various types of gene therapy and radiotherapy can be combined to increase the locoregional antitumor effect and reduce toxicity. However, all applications of gene therapy in combination with radiotherapy currently remain experimental.
- MeSH
- Targeted Gene Repair methods MeSH
- Gene Editing methods MeSH
- Genetic Therapy * methods MeSH
- Humans MeSH
- Neoplasms therapy MeSH
- Antineoplastic Protocols MeSH
- Radiotherapy * methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Review MeSH
