CRISPR/Cas technology is a powerful tool for genome engineering in Aspergillus oryzae as an industrially important filamentous fungus. Previous study has reported the application of the CRISPR/Cpf1 system based on the Cpf1 (LbCpf1) from Lachnospiraceae bacterium in A. oryzae. However, multiplex gene editing have not been investigated using this system. Here, we presented a new CRISPR/Cpf1 multiplex gene editing system in A. oryzae, which contains the Cpf1 nuclease (FnCpf1) from Francisella tularensis subsp. novicida U112 and CRISPR-RNA expression cassette. The crRNA cassette consisted of direct repeats and guide sequences driven by the A. oryzae U6 promoter and U6 terminator. Using the constructed FnCpf1 gene editing system, the wA and pyrG genes were mutated successfully. Furthermore, simultaneous editing of wA and pyrG genes in A. oryzae was performed using two guide sequences targeting these gene loci in a single crRNA array. This promising CRISPR/Cpf1 genome-editing system provides a powerful tool for genetically engineering A. oryzae.
- MeSH
- Aspergillus oryzae * genetics MeSH
- Gene Editing MeSH
- Francisella * MeSH
- RNA, Guide, CRISPR-Cas Systems MeSH
- Publication type
- Journal Article MeSH
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
Rozvoj možností ovlivnění koncentrací iipoproteinů krevní plazmy jde velmi rychle kupředu. Kromě zcela nových léčiv se rozšiřuje spektrum kombinací déie užívaných moiekui, stejně jako praktické možnosti preskripce a dostupnost léčiv, která v našich podmínkách nebyla k dispozici (iomitapid). Dostáváme k dispozici nová léčiva ovlivňující koncentrace LDL (iipoproteiny s nízkou hustotou, iow-density lipoproteins) cholesterolu (např. bempedoová kyselina, RNA terapeutika či monokionáiní protilátky - evinacumab), klinickým testováním procházejí malé molekuly (obicetrapib) i peroráině podávané inhibitory proprotein konvertázy subtiiisinu/kexinu typu 9 (proprotein convertase subtiiisin/kexin type 9, PCSK9). Koncentrace trigiyceridů budeme moci cíleně ovlivňovat léčivy s účinky na iipoiytické schopnosti iipoproteinové iipázy (inhibitory apoiipoproteinu CIII [apo-CIII] či angiopoetinu podobný protein 3 [angiopoietin-iike 3 protein, ANGPTL3]). Veiká pozornost je věnována novým možnostem ovlivnění koncentrací iipoproteinu (a) pomocí RNA terapeutik (antisense [protisměrné] oiigonukieotidy i maié interferující RNA fragmenty). Mimořádně zajímavý je rozvoj technik genové editace, který se v první fázi rovněž obrací k iipidovému metabolismu. Aktuálně probíhá první studie u pacientů s heterozygotní familiární hyperchoiesteroiemií, u nichž byia provedena jednorázová editace genu pro systémem editace genů segmenty nahromaděných pravidelně rozmístěných krátkých paiindromických repetic (clustered regularly interspaced palindromic repeats associated genes, CRISPR/Cas9) následovaná poklesem koncentrací LDL cholesterolu o 50 %.
The deveiopment of options for influencing biood piasma iipoprotein ieveis is progressing rapidiy. In addition to compieteiy new drugs, the spectrum of combinations of ionger used moiecuies is expanding, as weii as the practicai possibiiities of prescription and avaiiabiiity of drugs that were not avaiiabie in our conditions (iomitapide). New drugs affecting LDL (iow-density iipoproteins) choiesteroi ieveis are becoming avaiiabie (e.g., bempedoic acid, RNA therapeutics or monocionai antibodies - evinacumab), smaii moiecuies (obicetrapib) and new oraiiy administered PCSK9 (proprotein convertase subtiiisin/kexin type 9) inhibitors are undergoing ciinicai triais. We wiii be abie to target trigiyceride ieveis with drugs affecting the iipoiytic capacity of iipoprotein iipase (inhibitors of apoiipoprotein CIII [apo-CIII] or angiopoietin-iike 3 protein [ANGPTL3]). Much attention is paid to new possibiiities of influencing iipoprotein (a) concentrations by RNA therapeutics (antisense oiigonucieotides and smaii interfering RNA fragments). Of particuiar interest is the deveiopment of gene editing techniques, which in the first phase aiso turn to iipid metaboiism. Currentiy, the first study is underway in patients with heterozygous famiiiai hyperchoiesteroiemia, in whom a singie gene editing of the PCSK9 gene by the ciustered reguiariy interspaced paiindromic repeats associated genes (CRISPR/Cas9) system foiiowed by a 50% decrease in LDL-choiesteroi ieveis.
- Keywords
- studie VERDE 101, kyselina bempedoová, inhibitory CETP,
- MeSH
- Apolipoprotein C-III pharmacology MeSH
- Dyslipidemias * drug therapy blood MeSH
- Gene Editing MeSH
- Clinical Trials as Topic MeSH
- Cholesterol, LDL pharmacology blood MeSH
- Humans MeSH
- Antibodies, Monoclonal pharmacology therapeutic use MeSH
- PCSK9 Inhibitors pharmacology MeSH
- Triglycerides pharmacology blood MeSH
- Check Tag
- Humans MeSH
- Publication type
- Research Support, Non-U.S. Gov't 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
The incidence and the burden of cardiovascular disease (CVD), coronary heart disease (CHD), type 2 diabetes mellitus (T2DM), and the metabolic syndrome are greatly increasing in our societies. Together, they account for 31% of all deaths worldwide. This chapter focuses on the role of two revolutionary discoveries that are changing the future of medicine, induced pluripotent stem cells (iPSCs) and CRISPR/Cas9 technology, in the study, and the cure of cardiovascular and metabolic diseases.We summarize the state-of-the-art knowledge about the possibility of editing iPSC genome for therapeutic applications without hampering their pluripotency and differentiation, using CRISPR/Cas technology, in the field of cardiovascular and metabolic diseases.
- MeSH
- Diabetes Mellitus, Type 2 * genetics therapy MeSH
- Gene Editing MeSH
- Induced Pluripotent Stem Cells * MeSH
- Cardiovascular System * MeSH
- Humans MeSH
- Metabolic Diseases * genetics therapy MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article 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
The article explores whether the broader regulatory framework applicable to the member states of the EU contains suitable tools to react to the rapid advances in science, especially as to the question of germline editing technologies. From the perspective of EU member states, the regulatory framework is fragmented between norms of international law, secondary EU law and national legislation. The rules and their interpretation are strongly influenced by the concept of precaution, which reflects the concern that there is not enough knowledge to assess the impact of genome editing technology on individuals, society and future populations. However, the argument of precaution loses its strength with every new scientific discovery. The expanding knowledge in the field creates the need to replace regulation, which is based on the lack of knowledge (such as precautionary moratoriums) by the regulation that is based on the actual knowledge. The article reaches a conclusion that the EU framework for advanced treatments and medicinal products is in a state where it can, in principle, address the questions associated with the safety and efficacy of germline editing technologies. The EU framework is, however, not suitable to assess the moral and societal impacts of new technology, which should be left for member states.
- MeSH
- Gene Editing * MeSH
- Humans MeSH
- Technology * MeSH
- Germ Cells MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
