A genome editing approach to study cancer stem cells in human tumors
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PROVIDER: PRJNA359182 | ENA |
REPOSITORIES: ENA
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Project description:A genome editing approach to study cancer stem cells in human tumors
| PRJEB18738 | ENA
Project description:The analysis of stem cell hierarchies in human cancers has been hampered by the impossibility of identifying or tracking tumor cell populations in an intact environment. To overcome this limitation, we devised a strategy based on editing the genomes of patient-derived tumor organoids using CRISPR/Cas9 technology to integrate reporter cassettes at desired marker genes. As proof of concept, we engineered human colorectal cancer (CRC) organoids that carry EGFP and lineage-tracing cassettes knocked in the LGR5 locus. Analysis of LGR5-EGFP+ cells isolated from organoid-derived xenografts demonstrated that these cells express a gene program similar to that of normal intestinal stem cells and that they propagate the disease to recipient mice very efficiently. Lineage-tracing experiments showed that LGR5+ CRC cells self-renew and generate progeny over long time periods that undergo differentiation toward mucosecreting- and absorptive-like phenotypes. These genetic experiments confirm that human CRCs adopt a hierarchical organization reminiscent of that of the normal colonic epithelium. The strategy described herein may have broad applications to study cell heterogeneity in human tumors.
| S-EPMC5494503 | biostudies-literature
Project description:This SuperSeries is composed of the SubSeries listed below.
2017-04-28 | GSE92962 | GEO
Project description:Background: Beta-thalassemia is among the commonest monogenic disorders, posing a major global health challenge. Editing of genetic modifiers of β-thalassemia, such as BCL11A erythroid enhancer and HBG promoters, enhances fetal hemoglobin (HbF) expression and confers major therapeutic potential. Double-strand-break (DSB)-independent genome editing tools, such as base editors, are potentially safer and better suited for multiplexed application than traditional DSB-dependent CRISPR/Cas technology. However, harmful inadvertent on- and off-target events remain a concern and must be excluded before clinical application, including chromosomal rearrangements, which are invisible to standard detection technologies. Results: Using primary patient-derived CD34+ cells from three donors, we investigated simplex and duplex BE-based disruption of the BCL11A erythroid enhancer and the BCL11A binding site (-115bp) on the HBG promoter for DNA-level events and functional studies at the RNA, protein, and morphological level. Analyses included direct comparison to DSB-based editing, as the current clinically applied standard, and analysis of DNA recombination events by CAST-seq to allow wider inferences for relative safety of DSB-, BE- and duplex BE-based editing. Our study reveals the effectiveness of duplex base editing, with robust γ-globin and HbF induction and significantly improved functional correction over simplex editing. Moreover, duplex editing resulted in low incidence of simple and complex genomic alterations in both therapeutically relevant target loci. Conclusions: Here we display simultaneous duplex base editing by targeting both BCL11A erythroid enhancer and HBG promoter for functional correction and genome integrity. Our study highlights the efficacy, safety, and therapeutic potential of the present duplex BE approach
2025-12-22 | GSE307482 | GEO
Project description:Genome-Wide Association Study of Testicular Germ Cell Tumors
| PRJNA376225 | ENA
Project description:Virus-induced genome editing (VIGE) using compact RNA-guided endonucleases is a transformational new approach in plant biotechnology, enabling tissue-culture-independent and transgene-free genome editing. We recently established a transgene-free VIGE approach for heritable editing at single loci in Arabidopsis by delivering ISYmu1 TnpB (Ymu1) and its guide RNA (gRNA) via Tobacco Rattle Virus (TRV). Here, we greatly improved this system by devising a multiple gRNA expression system and by utilizing an engineered high-activity Ymu1 variant (Ymu1-WFR) to develop an efficient multiplexed genome editing approach.
2026-01-15 | GSE316183 | GEO
Project description:The CRISPR/Cas genome editing approach in non-model organisms poses challenges that remain to be resolved.Here, we demonstrated a generalized roadmap for a de-novo genome-annotation approach applied to the non-model organism Macrobrachium rosenbergii. We also addressed typical genome editing challenges arising from genetic variations, such as a high frequency of single nucleotide polymorphisms, differences in sex chromosomes, and repetitive sequences that can lead to off-target events. For genome editing of M. rosenbergii, our laboratory recently adapted the CRISPR/Cas genome-editing approach to embryos and embryonic primary cell culture. In this continuation study, an annotation pipeline was trained to predict gene models by leveraging available genomic, transcriptomic, and proteomic data, enabling accurate gene prediction and guide design for knock-outs. Next-generation sequencing analysis demonstrated a high frequency of genetic variations in genes on both autosomal and sex chromosomes, which have been shown to affect the accuracy of editing analyses. To enable future applications based on the CRISPR/Cas tool in non-model organisms, we also verified the reliability of editing efficiency and tracked off-target frequencies. Despite the lack of comprehensive information on non-model organisms, this study provides an example of the feasibility of selecting and editing specific genes with a high degree of certainty.
2024-11-22 | GSE281095 | GEO
Project description:While CRISPR/Cas9 holds therapeutic promise, broader application demands understanding complications in vast non-coding regions. We found that CRISPR/Cas9 can cause premature differentiation of neural stem cells in vivo and mouse embryonic stem cells in vitro, even when cleavage occurred at distant sites tens of kilobases away from the nearest regulatory elements. To investigate this, we employed an integrated ATAC/RNA approach (AR-seq) and identified editing-induced chromatin accessibility change, with its scale varying by cell types. Cells with stemness are most affected, experiencing perturbations that extend over a hundred kilobases. Furthermore, even local DNA perturbations can disrupt CTCF- and condensate-associated chromatin architecture, causing distal transcriptional rewiring and ultimately loss of stemness identity. To minimize chromatin perturbations and preserve cell identity we refined gene editing strategies, including distance-aware sgRNA design, pharmacological attenuation of DNA resection, and alternative editing systems. This work paves the way for safer and broader application of genome editing technologies.
2026-02-26 | GSE320164 | GEO
Project description:While CRISPR/Cas9 holds therapeutic promise, broader application demands understanding complications in vast non-coding regions. We found that CRISPR/Cas9 can cause premature differentiation of neural stem cells in vivo and mouse embryonic stem cells in vitro, even when cleavage occurred at distant sites tens of kilobases away from the nearest regulatory elements. To investigate this, we employed an integrated ATAC/RNA approach (AR-seq) and identified editing-induced chromatin accessibility change, with its scale varying by cell types. Cells with stemness are most affected, experiencing perturbations that extend over a hundred kilobases. Furthermore, even local DNA perturbations can disrupt CTCF- and condensate-associated chromatin architecture, causing distal transcriptional rewiring and ultimately loss of stemness identity. To minimize chromatin perturbations and preserve cell identity we refined gene editing strategies, including distance-aware sgRNA design, pharmacological attenuation of DNA resection, and alternative editing systems. This work paves the way for safer and broader application of genome editing technologies.
2026-02-26 | GSE320163 | GEO
Project description:While CRISPR/Cas9 holds therapeutic promise, broader application demands understanding complications in vast non-coding regions. We found that CRISPR/Cas9 can cause premature differentiation of neural stem cells in vivo and mouse embryonic stem cells in vitro, even when cleavage occurred at distant sites tens of kilobases away from the nearest regulatory elements. To investigate this, we employed an integrated ATAC/RNA approach (AR-seq) and identified editing-induced chromatin accessibility change, with its scale varying by cell types. Cells with stemness are most affected, experiencing perturbations that extend over a hundred kilobases. Furthermore, even local DNA perturbations can disrupt CTCF- and condensate-associated chromatin architecture, causing distal transcriptional rewiring and ultimately loss of stemness identity. To minimize chromatin perturbations and preserve cell identity we refined gene editing strategies, including distance-aware sgRNA design, pharmacological attenuation of DNA resection, and alternative editing systems. This work paves the way for safer and broader application of genome editing technologies.
2026-02-26 | GSE320165 | GEO