Restored glyoxylate metabolism in induced-hepatocytes after CRISPR/Cas-mediated AGXT gene correction and direct cell reprogramming of Primary Hyperoxaluria Type 1 fibroblasts
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA938377 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR23609800.fastq.gz | Fastqsanger.gz | |||
| SRR23609800_1.fastq.gz | Fastqsanger.gz | |||
| SRR23609800_2.fastq.gz | Fastqsanger.gz | |||
| SRR23609801.fastq.gz | Fastqsanger.gz | |||
| SRR23609801_1.fastq.gz | Fastqsanger.gz |
Items per page: 5 1 - 5 of 117 |
Similar Datasets
Project description:Primary Hyperoxaluria Type 1 (PH1) is a rare inherited metabolic disorder characterized by oxalate overproduction in the liver, resulting in renal damage. It is caused by mutations in the AGXT gene. Combined liver and kidney transplantation is currently the only permanent curative treatment. We combined locus-specific gene correction and hepatic direct cell reprogramming to generate autologous healthy induced hepatocytes (iHeps) from PH1 patient-derived fibroblasts. First, site-specific AGXT corrected cells were obtained by homology directed repair (HDR) assisted by CRISPR/Cas9, following two different strategies: accurate point mutation (c.853T>C) correction or knock-in of an enhanced version of AGXT cDNA. Then, iHeps were generated, by overexpression of hepatic transcription factors. Generated AGXT-corrected iHeps showed hepatic gene expression profile and exhibited in vitro reversion of oxalate accumulation compared to non-edited PH1-derived iHeps. This strategy set up a potential alternative cellular source for liver cell replacement therapy and a personalized PH1 in vitro disease model.
2024-03-27 | GSE226019 | GEO
Project description:Cornelia de Lange syndrome (CdLS) is an autosomal dominant disease mainly caused by mutations in the Nipped-B-like protein (NIPBL) gene resulting in the alteration of the cohesin pathway. Here, we generated human induced pluripotent stem cells (hiPSCs) from a CdLS patient carrying a mutation in the NIPBL gene, c.5483G>A, and tested CRISPR-Cas based approaches to repair the genetic defect. We applied an efficient and precise method of gene correction through CRISPR-Cas induced homology directed repair (HDR), which allowed the generation of hiPSC clones with regular karyotype and preserved stemness. The efficient and precise gene replacement strategy developed in this study can be extended to the modification of other genomic loci in hiPSCs. Isogenic wild-type and mutated hiPSCs produced with the CRISPR-Cas technology are fundamental CdLS cellular models to study the disease molecular determinants and identifying therapeutic targets.
2023-03-17 | E-MTAB-11604 | biostudies-arrayexpress
Project description:Single cell RNA sequencing of AATD iPSC-derived hepatocytes following Z mutation correction by base-editing
| PRJNA690576 | ENA
Project description:Obesity is becoming increasingly widespread in developed countries, and is often associated with heart diseases and diabetes. Elevated levels of plasma free fatty acids are a biochemical hallmark of obesity. Unlike plants and bacteria, mammals cannot utilize fatty acids to generate glucose because of the lack of glyoxylate shunt enzymes. Instead, fatty acids are used for energy storage, and their utilization is regulated at multiple levels ranging from hormonal to metabolic ensuring that glucose is preferentially oxidized before fatty acids. Here we designed a synthetic gene-metabolic circuit to alter the intracellular signaling and metabolic pathways that control fatty acid metabolism. By introducing the Escherichia coli glyoxylate shunt enzymes, isocitrate lyase (AceA) and malate synthase (AceB), into the mitochondria of human hepatocytes, we demonstrated that fatty acid utilization is preferentially increased while glucose uptake is significantly reduced. This bacterial pathway diverts signal metabolites (citrate, acetyl-CoA, and malonyl-CoA) that inhibits fatty acid uptake and provides an additional channel for fatty acid utilization. Remarkably, the hepatocytes readily adapted to the synthetic circuit by a series of global transcriptional and post-translational changes in metabolic and signal transduction pathways that further advanced our design objective. This systems approach illustrates the logic of the synergistic interaction between metabolism and signal transduction. The ready acceptance of this non-native pathway by hepatocytes suggests the plasticity of liver metabolism and opens a possibility for the synthetic approach in regulating human metabolism. Keywords: Fatty acid response, synthetic circuit, carbohydrate metabolism, stably transfected HepG2 cell line, glyoxylate shunt
2009-06-05 | GSE5903 | GEO
Project description:Mycobacterium tuberculosis CAS Genome sequencing
| PRJNA445659 | ENA
Project description:CRISPR-Cas immune systems function to defend prokaryotes against potentially harmful mobile genetic elements including viruses and plasmids. The multiple CRISPR-Cas systems (Types I, II, III) each recognize and target destruction of foreign invader nucleic acids via structurally and functionally diverse effector complexes (crRNPs). CRISPR-Cas effector complexes are comprised of CRISPR RNAs (crRNAs) that contain sequences homologous to the invading nucleic acids and Cas proteins specific to each immune system type. We have previously characterized a crRNP in Pyrococcus furiosus (Pfu) that contains Cmr proteins (Type III-B) associated with one of two primary size forms of crRNAs and functions through homology-dependent cleavage of target RNAs. In the current study, we have isolated and characterized two additional native Pfu CRISPR-Cas complexes containing either Csa (Type I-A) or Cst (Type I-G) proteins and distinct profiles of associated crRNAs. For each complex, the Cas proteins were identified by tandem mass spectrometry and immunoblotting and the crRNAs by RNA deep sequencing and Northern blot analysis. The crRNAs associated with both the Csa and Cst complexes originate from each of seven total CRISPR loci and contain identical 5’ ends (8-nt CRISPR RNA repeat-derived 5’ tag sequences) but heterogeneous 3’ ends (containing variable amounts of downstream repeat sequences). These crRNA forms are distinct from Cmr-associated crRNAs, indicating different 3’ end processing pathways following primary cleavage of common pre-crRNAs. We predict that the newly identified Pfu Type I-A (Csa) and Type I-G (Cst)-containing crRNPs, like other previously characterized Type I CRISPR-Cas effector complexes, each function by carrying out crRNA-guided DNA targeting of invading mobile genetic elements. Taken together, our in-depth characterization of the three isolated native complexes provides clear evidence for three compositionally distinct crRNPs containing either Cmr, Csa, or Cst Cas proteins that together make up an impressive arsenal of CRISPR-Cas defense for a single organism. 4 Samples: Protein-associated small RNAs
2015-07-10 | E-GEOD-65781 | biostudies-arrayexpress
Project description:In this study we report a comparative, transcriptome-wide analysis of CRISPR/Cas-based RNA-editing technologies in HEK293T cells. Targeted RNA editing technologies hold great process in the area of post-transcriptional genomic engineering and impermanent therapeutic correction of disease-causing variants, though off-target editing events still remain a concern. Here, we perform paired-end poly(A)+ purified RNA-seq to characterize transcriptome-wide in HEK293T cells transfected with catalytically-dead Cas9-, Cas13b, or Cas13d fused to human ADAR2 deaminase domain containing a hyperactive editing mutation (E488Q) along with either CYFIP2¬-targeting or non-targeting gRNA, in duplicate. We find that all technologies tested introduced widespread but distinct off-target mutations in the transcriptome, with the majority of edits occurring in 3’UTR and CDS regions of genes. Many of these edits, however, were in transcripts with low coverage, with the majority of editing rates being below 20%. This study is the first to characterize Cas-based RNA editing technologies in a direct manner, and reveals the relative similarity of currently available methods both in terms of on-target and off-target efficacy.
2020-11-01 | GSE152684 | GEO
Project description:Bacteria protect themselves from infection by bacteriophages (phages) using different defence systems, such as CRISPR-Cas. Although CRISPR-Cas provides phage resistance, fitness costs are incurred, such as through autoimmunity. CRISPR-Cas regulation can optimise defence and minimise these costs. We recently developed a genome-wide functional genomics approach (SorTn-seq) for high-throughput discovery of regulators of bacterial gene expression. Here, we applied SorTn-seq to identify loci influencing expression of the two type III-A Serratia CRISPR arrays. Multiple genes affected CRISPR expression, including those involved in outer membrane and lipopolysaccharide synthesis. By comparing loci affecting type III CRISPR arrays and cas operon expression, we identified PigU (LrhA) as a repressor that co-ordinately controls both arrays and cas genes. By repressing type III-A CRISPR-Cas expression, PigU shuts off CRISPR-Cas interference against plasmids and phages. PigU also represses interference and CRISPR adaptation by the type I-F system, which is also present in Serratia. RNA sequencing demonstrated that PigU is a global regulator that controls secondary metabolite production and motility, in addition to CRISPR-Cas immunity. Increased PigU also resulted in elevated expression of three Serratia prophages, indicating their likely induction upon sensing PigU-induced cellular changes. In summary, PigU is a major regulator of CRISPR-Cas immunity in Serratia.
2023-11-20 | GSE243790 | GEO
Project description:Despite advances in nuclease-based genome editing technologies, correcting human disease-causing genomic inversions remains a challenge. Here, we describe the potential use of a recombinase-based system to correct a 140 kb int1h inversion frequently found in patients diagnosed with Hemophilia A. With the use of directed molecular evolution, we developed a linked heterodimeric recombinase system (RecF8) achieving 30% inversion of the target sequence in human tissue culture cells. Transient RecF8 treatment of endothelial cells, differentiated from int1h patient derived iPSCs, resulted in prominent correction of the inversion and restored Factor VIII mRNA expression. Our data suggest that the development of designer-recombinases represent an efficient and specific mean towards treatment of large gene inversions causing monogenic diseases.
2021-12-31 | GSE159492 | GEO
Project description:Mycobacterium tuberculosis CAS/NITR204 Genome sequencing
| PRJNA193386 | ENA