Auswahl der wissenschaftlichen Literatur zum Thema „Crispr-case9“

Hypoxia is an important risk for renal disease. The mitochondrial enzyme arginase-II (Arg-II) is expressed and/or induced by hypoxia in proximal tubular epithelial cells (PTECs) and in podocytes, leading to cellular damage. Because PTECs are vulnerable to hypoxia and located in proximity to podocytes, we examined the role of Arg-II in the crosstalk of PTECs under hypoxic conditions with podocytes. A human PTEC cell line (HK2) and a human podocyte cell line (AB8/13) were cultured. Arg-ii gene was ablated by CRISPR/Case9 in both cell types. HK2 cells were exposed to normoxia (21% O2) or hypoxia (1% O2) for 48 h. Conditioned medium (CM) was collected and transferred to the podocytes. Podocyte injuries were then analyzed. Hypoxic (not normoxic) HK2-CM caused cytoskeletal derangement, cell apoptosis, and increased Arg-II levels in differentiated podocytes. These effects were absent when arg-ii in HK2 was ablated. The detrimental effects of the hypoxic HK2-CM were prevented by TGF-β1 type-I receptor blocker SB431542. Indeed, TGF-β1 levels in hypoxic HK2-CM (but not arg-ii−/−-HK2-CM) were increased. Furthermore, the detrimental effects of TGF-β1 on podocytes were prevented in arg-ii−/−-podocytes. This study demonstrates crosstalk between PTECs and podocytes through the Arg-II-TGF-β1 cascade, which may contribute to hypoxia-induced podocyte damage.

Gemini viruses account for destructive scourge wastes that endanger nourishment safety. The genomic sequence of Gemini virus comprises of a round or disk shaped, unique or isolated stranded or grounded DNA crumb, which set off a duplex DNA replica center in the parenchyma cell core and translate almost seven to four universal polypeptide Weed attribute manipulate needs effectual pick out genetic editing machinery. Clustered regularly interspaced palindromic repeats accompanying (CAS) type II organization cast off select genomic changing implementation beyond the organisms that have nucleus in their cell containing plants that cause resistance of these devastating Gemi virus. We know the evolution (TRV) tobacco rattle virus, which is, mediated during the genomic changing or during editing in tobacco plant family not long ago. We know that tobacco rattle virus also affects the new and newly growing plantlets that have possess a very small size genomic size that help in multiplication and binary fusion and also in cloning and agricultural agro infections that cause the plants to diseases. closer, constant action and distinctiveness of the tobacco rattle virus, which is mediated CRISPR Cas9 system for targeted modification of the tobacco rattle family plant Nicotiana benthamiana genome. Informational and experimental report perseverance TRV-moderate Cas-9 task for about 30 days agroinefection. In addition, our results show or demonstrate that TRV make up genomic sequence changing illustrated no in-exact work generable in exact arguing the exactness of the organization for vascular plantlets genomic sequencing scheme. Are hold of at once, the particular statics authorize reasonableness arousing chances of utilized the viral particle as a make peace CRISPER/Cas9 for chosen manipulate of the plant genomic sequence.

Abstract Previous studies by our group and others have demonstrated that platelet targeted FVIII expression can rescue the bleeding phenotype in hemophilia A (HA) mice in both the inhibitor and non-inhibitor models. The clinical efficacy has been further proven in a dog HA non-inhibitor model. While these are informative, neither the canine nor mouse model entirely mimic human disease because 1) neither HA mice nor dogs exhibit the frequent spontaneous bleeding in severe HA patients, and 2) canine platelets do not contain FVIII carrier protein VWF and therefore store and release FVIII differently, thus precluding examining the efficacy of platelet gene therapy in the presence of inhibitors. The efficacy of platelet-FVIII gene therapy, therefore, needs to be examined in an animal model more representative of human disease. Since the inversion of FVIII gene is the most common mutation that causes severe HA in patients, using a CRISPR/Case9 strategy we generated a novel HA rat model in a Dahl/Salt Sensitive (Dahl) inbred congenic background (Dahl-F8KO) with the nearly entire murine FVIII gene inverted causing a translational stop 6 amino acids after the signal sequence of FVIII. There is no detectable FVIII in plasma in Dahl-F8KO rats (LOD is 1 U/dL). Spontaneous bleeding in the soft tissue, muscles, or joints occurred in 56.5% of homozygous affected animals (n=46) by the age of 8-weeks and up to 82.6% by the age of 32-weeks. This was significantly higher (P<0.0001) than the occurrence of spontaneous bleeding in another colony in Sprague Dawley (SD) background developed by Nielsen et al. using zinc finger nuclear strategy (J Thromb Haemost 2014) with a 13-bp deletion in exon 16 of FVIII. In SD-F8KO rat model, spontaneous bleeds occur in 23.3% and 42.1% of homozygous affected animals (n=85) that were housed in our facility by the age of 8- and 32-weeks, respectively. Likewise, the percentages of homozygous affected animals with 2, 3, or 4 bleeding episodes in the Dahl-F8KO colony were significantly higher than those in the SD-F8KO colony. In contrast, none of the heterozygous (F8+/-) animals ever have a spontaneous bleed. Similar to SD-F8KO rats, Dahl-F8KO rats can develop anti-FVIII inhibitory antibodies after 2 doses of recombinant human FVIII (rhF8) treatment. Thus, our novel Dahl-F8KO rat model is a unique animal model with the severest spontaneous bleeding phenotype yet reported. To investigate if platelet-derived FVIII can rescue the spontaneous bleeding phenotype in HA rats, we generated transgenic rats (2bF8Tg) in both SD- and Dahl-background in which FVIII expression is under control of the platelet-specific αIIb promoter (2bF8) using lentivirus-mediated oocyte transduction transgenesis. Bone marrow (BM) mononuclear cells were isolated from femurs of 2bF8Tg rats and transplanted into F8-/- rats preconditioned with 950 R TBI. Animals were closely monitored after BM transplantation (BMT), and blood samples were collected for FVIII assays and whole blood thrombin generation assay (wbTGA). After BMT from SD-2bF8Tg rats, no spontaneous bleeding was observed in SD-F8KO recipients within the entire study course (1 year at the time of this report) with a sustained platelet-FVIII expression of 20.9±8.1 mU/108 platelets (n=3). Similarly, we performed BMT from Dahl-2bF8Tg rats into Dahs-F8KO and found that 1 of 3 recipients had a bruise at the early stage of BM reconstitution. However, no any other spontaneous bleeding has yet been observed (5 months). To confirm that the bleeding diathesis in F8-/- rats was ameliorated after 2bF8 gene expression, wbTGA was performed. All parameters, including Lag Time, Peak Time, Peak Thrombin, Endogenous Thrombin Potential in 2bF8Tg-BMT recipients were significantly different compared to F8-/- control rats, but were similar to those obtained in WT control animals. Of note, neither detectable levels of plasma FVIII nor anti-F8 antibodies were detected in F8-/- recipients after receiving BMT from 2bF8Tg rats. In summary, we have developed a novel HA rat model with both the pathophysiology and clinical phenotype found in severe HA patients, which could be an ideal model for evaluating the clinical efficacy of new therapeutics. Our studies demonstrated that transplantation of BM cells that are genetically modified to express FVIII only in platelets can efficiently prevent the severe spontaneous bleeding in HA rats with no anti-F8 antibody development. Disclosures Montgomery: BCW: Patents & Royalties: GPIbM assay patent to the BloodCenter of Wisconsin.

AbstractChimeric antigen receptor T (CAR-T) cells therapy has revolutionized the treatment paradigms for hematological malignancies, with multi-line therapy-refractory patients achieving durable complete remissions (CR) and relatively high objective response rate (ORR). So far, many CAR-T products, such as Kymriah, Yescarta and Tecartus, have been developed and got the unprecedented results. However, some patients may relapse afterwards, driving intense investigations into promoting the development of novel strategies to overcome resistance and mechanisms of relapse. Notable technical progress, such as nanobodies and CRISPR-Case9, has also taken place to ensure CAR-T cell therapy fully satisfies its medical potential. In this review, we outline the basic principles for the development and manufacturing processes of CAR-T cell therapy, summarize the similarities and differences in efficacy of different products as well as their corresponding clinical results, and discuss CAR-T immunotherapy combined with other clinical effects of drug therapy.

Abstract Background Strong multiple stress-tolerance is a desirable characteristic for Saccharomyces cerevisiae when different feedstocks are used for economical industrial ethanol production. Random mutagenesis or genome shuffling has been applied for improving multiple stress-tolerance, however, these techniques are generally time-consuming and labor cost-intensive and their molecular mechanisms are unclear. Genetic engineering, as an efficient technology, is poorly applied to construct multiple stress-tolerant industrial S. cerevisiae due to lack of clear genetic targets. Therefore, constructing multiple stress-tolerant industrial S. cerevisiae is challenging. In this study, some target genes were mined by comparative transcriptomics analysis and applied for the construction of multiple stress-tolerant industrial S. cerevisiae strains with prominent bioethanol production. Results Twenty-eight shared differentially expressed genes (DEGs) were identified by comparative analysis of the transcriptomes of a multiple stress-tolerant strain E-158 and its original strain KF-7 under five stress conditions (high ethanol, high temperature, high glucose, high salt, etc.). Six of the shared DEGs which may have strong relationship with multiple stresses, including functional genes (ASP3, ENA5), genes of unknown function (YOL162W, YOR012W), and transcription factors (Crz1p, Tos8p), were selected by a comprehensive strategy from multiple aspects. Through genetic editing based on the CRISPR/Case9 technology, it was demonstrated that expression regulation of each of these six DEGs improved the multiple stress-tolerance and ethanol production of strain KF-7. In particular, the overexpression of ENA5 significantly enhanced the multiple stress-tolerance of not only KF-7 but also E-158. The resulting engineered strain, E-158-ENA5, achieved higher accumulation of ethanol. The ethanol concentrations were 101.67% and 27.31% higher than those of the E-158 when YPD media and industrial feedstocks (straw, molasses, cassava) were fermented, respectively, under stress conditions. Conclusion Six genes that could be used as the gene targets to improve multiple stress-tolerance and ethanol production capacities of S. cerevisiae were identified for the first time. Compared to the other five DEGs, ENA5 has a more vital function in regulating the multiple stress-tolerance of S. cerevisiae. These findings provide novel insights into the efficient construction of multiple stress-tolerant industrial S. cerevisiae suitable for the fermentation of different raw materials. Graphical Abstract

Le poisson-zèbre est devenu un modèle d’étude du cancer de plus en plus répandu au cours des dernières décennies. Alors que la plupart de ces modèles sont générés par l'expression d'oncogènes de mammifères sous des promoteurs tissulaires spécifiques, nous décrivons ici une méthode qui permet un contrôle optique précis de l'activation ou d’inactivation de l’expression de gènes, in vivo, chez le poisson-zèbre. Ainsi, cette méthode permet l’induction de phénotypes tumoraux par l’activation de l’expression constitutive d’un oncogène humain typique, KRASG12V, dans des tissus et des cellules individuelles sélectionnées sans promoteurs tissus-spécifiques, et ce, dans les embryons de poisson-zèbre. Nous démontrons également le contrôle optique de l'expression des oncogènes KRASG12V, CMYC et BRAFV600E ainsi que le contrôle de l’expression et de l’activité du système CRISPR-Cas9. En outre, il convient de noter que la manipulation précise de l'expression des gènes est essentielle pour comprendre la plupart des processus biologiques. De ce fait, notre travail présente une nouvelle approche dont l’objectif est d’initier et d’étudier le cancer chez le poisson-zèbre. De plus, la haute résolution spatio-temporelle de cette méthode en fait un outil précieux pour étudier l'initiation du cancer à partir de cellules uniques
The zebrafish has become an increasingly popular and valuable cancer model over the past decades. While most of these models are generated by expressing mammalian oncogenes under tissue-specific promoters, here we describe a method that allows for the precise optical control of oncogene expression or inactivation in live zebrafish. Thus, this technique allows for the induction of tumor phenotypes by activating the constitutive expression of a typical human oncogene, KRASG12V, in selected tissues and single cells without tissue-specific promoters in live zebrafish. We also demonstrate the optical control of oncogene expression as KRASG12V, CMYC and BRAFV600E as well as the control of the expression and the activity of the CRISPR-Cas9 system. In addition, it should be noted that accurate manipulation of gene expression is essential to understand most biological processes. Therefore, our work presents a novel approach to initiate and study cancer in zebrafish. Finally, it is also worth noting that the high spatio-temporal resolution of this method makes it a valuable tool for studying cancer initiation from single cells