Gene expression profile at single cell level of Min6 cells (GCs) from CRISPR-Cas9 edited cells
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE222145 | GEO | 2024/02/14
REPOSITORIES: GEO
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Project description:Gene expression profile at single cell level of Min6 cells (GCs) from CRISPR-Cas9 edited cells
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Project description:ATP-sensitive potassium (K-ATP) channels composed of a pore-forming Kir6.2 potassium channel and a regulatory ABC transporter sulfonylurea receptor 1 (SUR1) regulate insulin secretion in pancreatic beta-cells to maintain glucose homeostasis. Mutations that impair channel folding or assembly prevent cell surface expression and cause congenital hyperinsulinism. Structurally diverse K-ATP inhibitors have been shown to act as pharmacochaperones to correct mutant channel expression, but the mechanism is unknown. Here, we compare cryoEM structures of K-ATP channels bound to pharmacochaperones glibenclamide, repaglinide, and carbamazepine. DSP cross-linking mass spectrometry was used to partially confirm cryoEM structures.
2019-11-12 | PXD013873 | Pride
Project description:ATP-sensitive potassium (K-ATP) channels composed of a pore-forming Kir6.2 potassium channel and a regulatory ABC transporter sulfonylurea receptor 1 (SUR1) regulate insulin secretion in pancreatic beta-cells to maintain glucose homeostasis. Mutations that impair channel folding or assembly prevent cell surface expression and cause congenital hyperinsulinism. Structurally diverse K-ATP inhibitors have been shown to act as pharmacochaperones to correct mutant channel expression, but the mechanism is unknown. Here, we compare cryoEM structures of K-ATP channels bound to pharmacochaperones glibenclamide, repaglinide, and carbamazepine. CyanurBiotinDimercaptoPropionylSuccinimide (CBDPS) cross-linking mass spectrometry was used to partially confirm cryoEM structures.
2019-08-12 | PXD014498 | Pride
Project description:Screening for successful CRISPR/Cas9 editing events remains a time consuming technical bottleneck in the field of Drosophila genome editing. This step can be particularly laborious for events that do not cause a visible phenotype, or those which occur at relatively low frequency. A promising strategy to enrich for desired CRISPR events is to co-select for an independent CRISPR event that produces an easily detectable phenotype. Here, we describe a simple negative co-selection strategy involving CRISPR-editing of a dominant female sterile allele, ovoD1 In this system ("ovoD co-selection"), the only functional germ cells in injected females are those that have been edited at the ovoD1 locus, and thus all offspring of these flies have undergone editing of at least one locus. We demonstrate that ovoD co-selection can be used to enrich for knock-out mutagenesis via nonhomologous end-joining (NHEJ), and for knock-in alleles via homology-directed repair (HDR). Altogether, our results demonstrate that ovoD co-selection reduces the amount of screening necessary to isolate desired CRISPR events in Drosophila.
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Project description:CRISPR-Cas9 genome editing is a promising technique for clinical applications, such as the correction of disease-associated alleles in somatic cells. The use of this approach has also been discussed in the context of heritable editing of the human germ line. However, studies assessing gene correction in early human embryos report low efficiency of mutation repair, high rates of mosaicism, and the possibility of unintended editing outcomes that may have pathologic consequences. We developed computational pipelines to assess single-cell genomics and transcriptomics datasets from OCT4 (POU5F1) CRISPR-Cas9-targeted and control human preimplantation embryos. This allowed us to evaluate on-target mutations that would be missed by more conventional genotyping techniques. We observed loss of heterozygosity in edited cells that spanned regions beyond the POU5F1 on-target locus, as well as segmental loss and gain of chromosome 6, on which the POU5F1 gene is located. Unintended genome editing outcomes were present in ∼16% of the human embryo cells analyzed and spanned 4-20 kb. Our observations are consistent with recent findings indicating complexity at on-target sites following CRISPR-Cas9 genome editing. Our work underscores the importance of further basic research to assess the safety of genome editing techniques in human embryos, which will inform debates about the potential clinical use of this technology.
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Project description:A small number of HIV-1-infected individuals develop broadly neutralizing antibodies to the virus (bNAbs). These antibodies are protective against infection in animal models. However, they only emerge 1-3 yr after infection, and show a number of highly unusual features including exceedingly high levels of somatic mutations. It is therefore not surprising that elicitation of protective immunity to HIV-1 has not yet been possible. Here we show that mature, primary mouse and human B cells can be edited in vitro using CRISPR/Cas9 to express mature bNAbs from the endogenous Igh locus. Moreover, edited B cells retain the ability to participate in humoral immune responses. Immunization with cognate antigen in wild-type mouse recipients of edited B cells elicits bNAb titers that neutralize HIV-1 at levels associated with protection against infection. This approach enables humoral immune responses that may be difficult to elicit by traditional immunization.
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Project description:TAR DNA-binding protein 43 kDa (TDP-43), encoded by TARDBP, is an RNA-binding protein, the nuclear depletion of which is the histopathological hallmark of amyotrophic lateral sclerosis (ALS). Here, we show that ALS subjects have reduced early-phase insulin secretion and that the nuclear localization of TDP-43 is lost in the islets of autopsied ALS pancreas. Loss of TDP-43 inhibits exocytosis, thereby reducing early-phase insulin secretion in a cultured β cell line (MIN6). Using microarray analysis, we identified the genes causing insulin impaired in TDP knocked down MIN6 cells.
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Project description:CRISPR-Cas9 gene editing of human cells and tissues holds much promise to advance medicine and biology, but standard editing methods require weeks to months of reagent preparation and selection where much or all of the initial edited samples are destroyed during analysis. ArrayEdit, a simple approach utilizing surface-modified multiwell plates containing one-pot transcribed single-guide RNAs, separates thousands of edited cell populations for automated, live, high-content imaging and analysis. The approach lowers the time and cost of gene editing and produces edited human embryonic stem cells at high efficiencies. Edited genes can be expressed in both pluripotent stem cells and differentiated cells. This preclinical platform adds important capabilities to observe editing and selection in situ within complex structures generated by human cells, ultimately enabling optical and other molecular perturbations in the editing workflow that could refine the specificity and versatility of gene editing.
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Project description:Selenium is an essential trace element that has gained interest for its potential role in glucose homeostasis. The purpose of the present study was to investigate the impact of selenium supplementation as selenomethionine (SeMet) on insulin secretion in MIN6-K8 cells, a model of pancreatic -cells. We found that 40 μM and 80 μM SeMet significantly enhanced percent insulin secretion at low (2.8 mM), intermediate (11.7 mM), and high (16.7 mM) glucose stimulation. SeMet also increased tolbutamide- or KCl-induced percent insulin secretion at 40 μM and 80 μM. Ca2+ influx was only reduced with 80 μM SeMet. RNA-sequencing showed that 40 μM SeMet supplementation upregulated expression of selenoprotein H (SelH) and thioredoxin reductase 1 (Txnrd 1) and downregulated expression of glutathione peroxidase 3 (Gpx3), selenoprotein O (SelO), and selenoprotein P (SelP). Gpx3 knockdown increased both percent and total insulin release with high glucose stimulation. SelP knockdown increased insulin release at high glucose. In conclusion, high SeMet supplementation augments insulin secretion, while reductions in Gpx3 and SelP expression, from SeMet supplementation or via silencing, augment -cell function in the MIN6-K8 cell line. These studies support a putative role for selenium and selenoproteins in the regulation of glucose homeostasis and diabetes risk.
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Project description:In the human hematopoietic system, rare self-renewing multipotent long-term hematopoietic stem cells (LT-HSCs) are responsible for the lifelong production of mature blood cells and are the rational target for clinical regenerative therapies. However, the heterogeneity in the hematopoietic stem cell compartment and variable outcomes of CRISPR/Cas9 editing make functional interrogation of rare LT-HSCs challenging. Here, we report high efficiency LT-HSC editing at single-cell resolution using electroporation of modified synthetic gRNAs and Cas9 protein. Targeted short isoform expression of the GATA1 transcription factor elicit distinct differentiation and proliferation effects in single highly purified LT-HSC when analyzed with functional in vitro differentiation and long-term repopulation xenotransplantation assays. Our method represents a blueprint for systematic genetic analysis of complex tissue hierarchies at single-cell resolution.
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