The biotransformation of nanoparticles plays a crucial role in determining their biological fate and responses. Although a few engineering strategies (e.g., surface functionalization and shape control) have been employed to regulate the fate of nanoparticles, the genetic control of nanoparticle biotransformation remains an unexplored avenue. Herein, we utilized a CRISPR-based genome-scale knockout approach to identify genes involved in the biotransformation of rare earth oxide (REO) nanoparticles. We found that the biotransformation of REOs in lysosomes could be genetically controlled via SMPD1. Specifically, suppression of SMPD1 inhibited the transformation of La2O3 into sea urchin-shaped structures, thereby protecting against lysosomal damage, proinflammatory cytokine release, pyroptosis and RE-induced pneumoconiosis. Overall, our study provides insight into how to control the biological fate of nanomaterials.

• MeSH
• biotransformace genetika   MeSH
• CRISPR-Cas systémy MeSH
• ježovky metabolismus   MeSH
• kovové nanočástice * chemie   MeSH
• kovy vzácných zemin * metabolismus   chemie   MeSH
• lidé MeSH
• lyzozomy metabolismus   MeSH
• myši MeSH
• nanočástice * metabolismus   chemie   MeSH
• pyroptóza MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

CFTR is a membrane protein that functions as an ion channel. Mutations that disrupt its biosynthesis, trafficking or function cause cystic fibrosis (CF). Here, we present a novel in vitro model system prepared using CRISPR/Cas9 genome editing with endogenously expressed WT-CFTR tagged with a HiBiT peptide. To enable the detection of CFTR in the plasma membrane of live cells, we inserted the HiBiT tag in the fourth extracellular loop of WT-CFTR. The 11-amino acid HiBiT tag binds with high affinity to a large inactive subunit (LgBiT), generating a reporter luciferase with bright luminescence. Nine homozygous clones with the HiBiT knock-in were identified from the 182 screened clones; two were genetically and functionally validated. In summary, this work describes the preparation and validation of a novel reporter cell line with the potential to be used as an ultimate building block for developing unique cellular CF models by CRISPR-mediated insertion of CF-causing mutations.

• MeSH
• buněčná membrána metabolismus   MeSH
• buněčné linie MeSH
• CRISPR-Cas systémy genetika   MeSH
• cystická fibróza * genetika   metabolismus   MeSH
• lidé MeSH
• protein CFTR * genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Familiární onemocnění krvetvorby (FHD) jsou vzácnou a heterogenní skupinou onemocnění. Variabilní klinické projevy a prolínající se formy FHD zapříčiňují mnohy nesprávné stanovení diagnózy. Díky moderním genomickým přístupům (celoexomové sekvenování) dnes dokážeme vyřešit mnohé dosud neobjasněné případy. Nicméně u některých pacientů detekujeme jedinečnou a rodinně-specifickou genetickou variantu nejasného klinického významu (VUS). Cílem navrhované studie je vyhledávat zárodečné varianty u postižených rodin s FHD. Dále budeme zjišťovat funkční dopad identifikovaných VUS, abychom potvrdili jejich kauzalitu pomocí in vitro modelů (CRISPR/Cas9, mikroskopické a proteomické eseje). Na základě těchto zkušeností plánujeme zavést nový biomedicínský postup funkčního testování VUS do rutinní laboratorní praxe. Stanovení správné diagnózy u pacienta je zásadní i z důvodů určení rizika výskytu onkologických malignit, ke kterým některé zárodečné varianty predisponují. Očekáváme přímý dopad naší studie do translačního výzkumu hematopoézy, karcinogenézy a genové terapie.; Familial haematopoietic disorders (FHD) are a rare and heterogeneous group of disorders. Variable clinical expressivity and overlapping forms of FDH cause many misdiagnoses. Thanks to modern genomic approaches (whole exome sequencing), we are able to solve many previously unclear cases today. However, in some patients we detect a unique and family-specific gene variant of uncertain significance (VUS). The aim of the proposed study is to search for germline variants in affected families with FHD phenotype. Furthermore, we plan to investigate the functional impact of identified VUSs to verify their causality using in vitro models (CRISPR/Cas9, microscopic, and proteomic assays). Based on these experiences, we will introduce new biomedical pipeline for functional testing of VUSs into routine laboratory practice. Determining the correct diagnosis of a patient is also essential to define the risk of cancer, which some germline variants predispose. We expect a direct impact of our study in translational research of haematopoiesis, cancerogenesis and gene therapy.

• Klíčová slova
• variant of uncertain significance, funkční studie, functional studies, familiární poruchy krvetvorby, varianta nejasného klinického významu, whole exome sequencing, familial haematopoietic disorders, celoexomové sekvenování,

• NLK Publikační typ
• závěrečné zprávy o řešení grantu AZV MZ ČR

Navrhovaný projekt je zaměřen na studium genetického podkladu, etiologie a patogeneze nových případů talasémií, erytroenzymopatií, defektů membrány erytrocytů vedoucích k hemolytickým anémiím, vrozených dyserytropoetických anémií, vrozených poruch transportu železa v erytroidních buňkách a vrozených polycytémií. Na základě zkušeností získaných v průběhu předchozích grantových období odhadujeme, že přibližně u dvou set dětských pacientů budou potvrzeny vrozené defekty erytropoézy. V rámci projektu budeme využívat několika moderních přístupů (CRISPR/Cas9 editace, indukované pluripotentní kmenové buňky, myší a rybí modely), které nám umožní odhalit molekulárně-genetické mechanismy přispívající k defektní erytropoéze. Výsledky naší práce pomohou pochopit molekulárně genetické faktory přispívající k patologické erytropoéze, objasnit propojení procesu nevyvážené erytropoézy s metabolismem železa a úlohu proteinů signalizačních cest hypoxie, jednoho z důležitých cílů pro terapeutické intervence u nádorů.; This project aims to establish genetic background, etiology and pathogenesis of new cases of thalassemias, enzymopathies, erythrocyte membrane defects associated with hemolytic anemia, congenital dyserythropoietic anemia as well as some congenital defects of the iron transport pathway in erythroid cells and congenital polycythemias. Based on experiences gained during previous grant periods we estimate that in approximately two hundreds of pediatric patients hereditary defects of erythropoiesis will be confirmed. We propose several approaches (CRISPR/Cas9 editing, induced pluripotent stem cells, zebrafish and mouse model) to unravel the molecular-genetic mechanisms contributing to defective erythropoiesis. Results of our work will improve understanding of the molecular-genetic factors contributing to the pathogenesis of disordered erythropoiesis, and will help to elucidate the interconnections of disbalanced erythropoiesis and iron metabolism and the role of the hypoxia signaling pathway proteins, which emerge as important targets for therapeutic interventions in tumors.

• Klíčová slova
• zebrafish, hemolytická anémie, kongenitální polycytémie, HIF dráha, Danio rerio, hemolytic anemia, congenital polycythemia, HIF pathway,

• NLK Publikační typ
• závěrečné zprávy o řešení grantu AZV MZ ČR

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 * genetika   MeSH
• editace genu MeSH
• Francisella * MeSH
• vodící RNA, systémy CRISPR-Cas MeSH
• Publikační typ
• časopisecké články 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 systémy genetika   MeSH
• Diamondova-Blackfanova anemie * genetika   terapie   MeSH
• editace genu metody   MeSH
• genetická terapie * metody   MeSH
• genetické vektory MeSH
• Lentivirus genetika   MeSH
• lidé MeSH
• mutace genetika   MeSH
• ribozomální proteiny genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy 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
• biofortifikace * MeSH
• CRISPR-Cas systémy * MeSH
• editace genu metody   MeSH
• geneticky modifikované rostliny * metabolismus   genetika   MeSH
• kyselina listová * metabolismus   MeSH
• lidé MeSH
• rýže (rod) metabolismus   genetika   MeSH
• železo * metabolismus   MeSH
• zemědělské plodiny * metabolismus   genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Persistent infection with high-risk types of human papillomaviruses (HPV) is a major cause of cervical cancer, and an important factor in other malignancies, for example, head and neck cancer. Despite recent progress in screening and vaccination, the incidence and mortality are still relatively high, especially in low-income countries. The mortality and financial burden associated with the treatment could be decreased if a simple, rapid, and inexpensive technology for HPV testing becomes available, targeting individuals for further monitoring with increased risk of developing cancer. Commercial HPV tests available in the market are often relatively expensive, time-consuming, and require sophisticated instrumentation, which limits their more widespread utilization. To address these challenges, novel technologies are being implemented also for HPV diagnostics that include for example, isothermal amplification techniques, lateral flow assays, CRISPR-Cas-based systems, as well as microfluidics, paperfluidics and lab-on-a-chip devices, ideal for point-of-care testing in decentralized settings. In this review, we first evaluate current commercial HPV tests, followed by a description of advanced technologies, explanation of their principles, critical evaluation of their strengths and weaknesses, and suggestions for their possible implementation into medical diagnostics.

• MeSH
• infekce papilomavirem * komplikace   MeSH
• lidé MeSH
• lidské papilomaviry MeSH
• nádory děložního čípku * MeSH
• Papillomaviridae genetika   MeSH
• technologie MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

BACKGROUND: This paper brings new information about the genome and phenotypic characteristics of Pantoea agglomerans strain DBM 3797, isolated from fresh Czech hop (Humulus lupulus) in the Saaz hop-growing region. Although P. agglomerans strains are frequently isolated from different materials, there are not usually thoroughly characterized even if they have versatile metabolism and those isolated from plants may have a considerable potential for application in agriculture as a support culture for plant growth. METHODS: P. agglomerans DBM 3797 was cultured under aerobic and anaerobic conditions, its metabolites were analyzed by HPLC and it was tested for plant growth promotion abilities, such as phosphate solubilization, siderophore and indol-3-acetic acid productions. In addition, genomic DNA was extracted, sequenced and de novo assembly was performed. Further, genome annotation, pan-genome analysis and selected genome analyses, such as CRISPR arrays detection, antibiotic resistance and secondary metabolite genes identification were carried out. RESULTS AND DISCUSSION: The typical appearance characteristics of the strain include the formation of symplasmata in submerged liquid culture and the formation of pale yellow colonies on agar. The genetic information of the strain (in total 4.8 Mb) is divided between a chromosome and two plasmids. The strain lacks any CRISPR-Cas system but is equipped with four restriction-modification systems. The phenotypic analysis focused on growth under both aerobic and anaerobic conditions, as well as traits associated with plant growth promotion. At both levels (genomic and phenotypic), the production of siderophores, indoleacetic acid-derived growth promoters, gluconic acid, and enzyme activities related to the degradation of complex organic compounds were found. Extracellular gluconic acid production under aerobic conditions (up to 8 g/l) is probably the result of glucose oxidation by the membrane-bound pyrroloquinoline quinone-dependent enzyme glucose dehydrogenase. The strain has a number of properties potentially beneficial to the hop plant and its closest relatives include the strains also isolated from the aerial parts of plants, yet its safety profile needs to be addressed in follow-up research.

• Publikační typ
• časopisecké články MeSH

The corpora allata-corpora cardiaca (CA-CC) is an endocrine gland complex that regulates mosquito development and reproduction through the synthesis of juvenile hormone (JH). Epoxidase (Epox) is a key enzyme in the production of JH. We recently utilized CRISPR/Cas9 to establish an epoxidase-deficient (epox-/-) Aedes aegypti line. The CA from epox-/- mutants do not synthesize epoxidated JH III but methyl farneosate (MF), a weak agonist of the JH receptor, and therefore have reduced JH signalling. Illumina sequencing was used to examine the differences in gene expression between the CA-CC from wild type (WT) and epox-/- adult female mosquitoes. From 18,034 identified genes, 317 were significantly differentially expressed. These genes are involved in many biological processes, including the regulation of cell proliferation and apoptosis, energy metabolism, and nutritional uptake. In addition, the same CA-CC samples were also used to examine the microRNA (miRNA) profiles of epox-/- and WT mosquitoes. A total of 197 miRNAs were detected, 24 of which were differentially regulated in epox-/- mutants. miRNA binding sites for these particular miRNAs were identified using an in silico approach; they target a total of 101 differentially expressed genes. Our results suggest that a lack of epoxidase, besides affecting JH synthesis, results in the diminishing of JH signalling that have significant effects on Ae. aegypti CA-CC transcriptome profiles, as well as its miRNA repertoire.

• MeSH
• Aedes * genetika   metabolismus   MeSH
• corpora allata metabolismus   MeSH
• exprese genu MeSH
• juvenilní hormony metabolismus   MeSH
• mikro RNA * genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH