Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Gene editing of colocalized NCF1 loci is associated with homologous recombination and chromosomal rearrangements - CAST-seq

ABSTRACT: Despite lingering genotoxicity concerns, CRISPR-based genome editing has progressively entered the gene therapy field. Here we report that interactions between nucleotide sequences that are localized on the same chromosome and exhibit a high degree of homology contribute substantially to post-editing chromosomal rearrangements. We successfully employed corrective editing strategies at the NCF1 gene, as well as at the colocalized pseudogenes, NCF1B and NCF1C, in a human cell line model of p47phox-deficient chronic granulomatous disease and in patient-derived human hematopoietic stem cells. Upon genetic manipulation, an improved droplet digital PCR-based method identified cells with altered copy numbers, spanning megabases from the edited loci. We attributed the high aberration frequency to the interaction between repetitive sequences and their predisposition to recombination events. Our findings emphasize the need for careful evaluation of the target-specific genomic context, such as the presence of homologous regions, whose genome instability can constitute a risk factor for chromosomal rearrangements upon genome editing.

ORGANISM(S): Homo sapiens

PROVIDER: GSE263692 | GEO | 2024/10/16

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

Quantitative evaluation of chromosomal rearrangements in primary gene-edited human stem cells by preclinical CAST-Seq

Project description:Genome editing with programmable nucleases has shown great promise for clinical translation but also revealed the risk of genotoxicity caused by chromosomal translocations or the insertion of mutations at off-target sites. Here, we describe CAST-Seq, an innovative assay to identify and quantify chromosomal aberrations derived from on- and off-target activities of CRISPR-Cas nucleases or TALENs. CAST-Seq also detected novel types of chromosomal rearrangements, including homology-mediated translocations that are mediated by homologous recombination. Depending on the employed designer nuclease, translocations occurred in 0–0.5% of gene-edited human stem cells and some 20% of target loci harbored gross aberrations. In conclusion, CAST-Seq analyses are particularly relevant for therapeutic editing of stem cells to enable a thorough risk assessment before clinical application of gene editing products.

2021-02-14 | GSE164389 | GEO

NCF1-dependent low production of reactive oxygen species in plasmacytoid dendritic cells exacerbates lupus

Project description:Low capacity to produce reactive oxygen species (ROS) due to mutations in neutrophil cytosolic factor 1 (NCF1/p47phox) is strongly associated with lupus development both in humans and mouse models. Here, we aim to identify the major mechanisms of the Ncf1-disease association. We found that plasmacytoid dendritic cells (pDCs), the most potent producers of type I IFNs, exacerbate pristane-induced lupus in ROS-defective Ncf1-mutant and human NCF1-339 variant carrying mice. ROS deficiency in mouse models with Ncf1 mutation or human NCF1-339 variant leads to enhanced pDC generation via the TLR7/AKT/mTOR pathway and accumulation at sites of inflammation, resulting in an increased IFNα secretion. The produced IFNα further stimulates the JAK1/STAT1 pathway, which we found is hyperreactive in ROS-deficient pDCs. This, in turn, leads to increased type I IFN signature and enhanced proinflammatory responses. Our discoveries explain the causative effect of dysfunctional Ncf1 and pathogenicity of pDCs in lupus.

2023-03-12 | PXD035964 | Pride

Expanding the editable genome and CRISPR-Cas9 versatility using DNA cutting-free gene targeting based on in trans paired nicking

Project description:Genome editing typically involves recombination between donor nucleic acids and genomic sequences subjected to double-stranded DNA breaks (DSBs) made by programmable nucleases (e.g. CRISPR-Cas9). Yet, amongst other deleterious by-products, DSBs yield translocations, off-target mutations and, most pervasively, unpredictable on-target allelic disruptions. Remarkably, hitherto, the untoward phenotypic consequences of on-target disruptions at allelic and non-allelic (e.g. pseudogene) sequences have received scant scrutiny and, crucially, remain to be addressed. Here, we demonstrate that gene-edited cells can lose fitness due to on-target DSBs and report that simultaneous single-stranded DNA break formation at donor and target DNA by CRISPR-Cas9 “nickases” overcomes, to a great extent, such genotype-phenotype disrupting events. Moreover, in trans paired nicking gene editing can efficiently and precisely add large DNA segments (i.e. live-cell reporter tags) into essential and multiple-copy genomic sequences while circumventing most of the allelic and non-allelic collateral mutations and chromosomal rearrangements characteristic of nuclease-dependent gene editing procedures.

2019-12-04 | GSE135064 | GEO

On- and off-target effects of paired CRISPR-Cas nickase in primary human cells - Illumina

Project description:Undesired on- and off-target effects of CRISPR-Cas nucleases remain a challenge in therapeutic genome editing. While the use of Cas9 nickases has been shown to minimize off-target mutagenesis, their use in therapeutic genome editing has been hampered by a lack of efficacy. To overcome this limitation, we and others have developed double nickase-based strategies to generate staggered DNA double breaks to mediate gene disruption or gene correction with high efficiency. However, the impact of paired single-strand nicks on genome integrity has remained largely unexplored. Here, we developed a novel CAST-Seq pipeline, D-CAST, to characterize chromosomal rearrangements induced by paired CRISPR-Cas9 nickases at three different loci in primary keratinocytes derived from epidermolysis bullosa patients. While targeting COL7A1, COL17A1, or LAMA3 with Cas9 nucleases caused previously undescribed chromosomal rearrangements, no chromosomal translocations were detected following single or paired Cas9-based nickase editing. Conversely, whereas single nickase applications did not result in gross genomic aberrations, the double nicking strategy induced large deletions/inversions within a 10 kb region surrounding the target sites at all three loci, similar to the nucleases. Taken together, our data indicate that double-nickase approaches combine efficient editing with greatly reduced off-target effects, but still leave substantial chromosomal rearrangements at on-target sites.

2024-03-15 | GSE241778 | GEO

On- and off-target effects of paired CRISPR-Cas nickase in primary human cells - long read

2024-03-15 | GSE254922 | GEO

On- and off-target effects of paired CRISPR-Cas nickase in primary human cells - Amplicon sequencing

2024-03-15 | GSE242954 | GEO

On- and off-target effects of paired CRISPR-Cas nickase in primary human cells - long-read

2024-03-15 | GSE241779 | GEO

Characterization and Function of Neutrophils in Alcoholic Hepatitis in Humans and Mice: Roles for Neutrophilic p47phox in Disease Progression

Project description:Intrahepatic neutrophil infiltration has been implicated in the pathogenesis of severe alcoholic hepatitis (SAH), a disease with high short-term morality; however, how neutrophils contribute to SAH progression remain obscure. This study aimed to characterize intrahepatic neutrophil infiltration and its involvement in AH pathogenesis. We found that hepatic expression of neutrophil cytosolic factor 1 (NCF1), a key factor in controlling neutrophilic ROS production, was upregulated and correlated with neutrophil number, inflammation and ROS-associated genes in SAH patients. Ncf1 floxed mice were generated using CRISPR/Cas9 technology by inserting two LoxP sequences into intron 1 and intron 8 of the mouse Ncf1 gene, then crossed with Lyz Cre mice to generated myeloid cell-specific Ncf1 knockout (Ncf1Lyz-/-) mice. The LyzCre negative Ncf1 floxed mice were used as corresponding WT littermate control. The chronic-plus-binge ethanol feeding model was used in this study. The total RNAs were extracted from ETOH-fed 5 WT and 4 Ncf1Lyz-/- mouse livers and submitted for a Poly(A) RNA sequencing. Genetic deletion of the Ncf1 gene in neutrophils abolished hepatic ROS, inflammation, and fibrosis induced by ethanol feeding. RNA-sequencing analysis and the data from experimental models revealed that neutrophilic NCF1-dependent ROS promoted alcoholic liver injury by inhibiting AMP-activated protein kinase (a key regulator of lipid metabolism) and microRNA-223 (a key anti-inflammatory and anti-fibrotic microRNA). Our data suggest that divergent pathogeneses exist in SAH and neutrophilic NCF1-dependent ROS promotes alcoholic liver injury by inhibiting AMPK and microRNA-223.

2022-07-16 | GSE192720 | GEO

On- and off-target effects of paired CRISPR-Cas nickase in primary human cells [Amplicon]

Project description:Undesired on- and off-target effects of CRISPR-Cas nucleases remain a challenge in genome editing. While the use of Cas9 nickases has been shown to minimize off-target mutagenesis, their use in therapeutic genome editing has been hampered by a lack of efficacy. To overcome this limitation, we and others have developed double nickase-based strategies to generate staggered DNA double-strand breaks to mediate gene disruption or gene correction with high efficiency. However, the impact of paired single-strand nicks on genome integrity has remained largely unexplored. Here, we developed a novel CAST-Seq pipeline, D-CAST, to characterize chromosomal aberrations induced by paired CRISPR-Cas9 nickases at three different loci in primary keratinocytes derived from epidermolysis bullosa patients. While targeting COL7A1, COL17A1, or LAMA3 with Cas9 nucleases caused previously undescribed chromosomal rearrangements, no chromosomal translocations were detected following paired nickase editing. While the double nicking strategy induced large deletions/inversions within a 10 kb region surrounding the target sites at all three loci, similar to the nucleases, the chromosomal on-target aberrations were qualitatively different and included a high proportion of insertions. Taken together, our data indicate that double-nickase approaches combine efficient editing with greatly reduced off-target effects, but still leave substantial chromosomal aberrations at on-target sites.

2024-03-15 | GSE255081 | GEO

Non-allelic homologous recombination between retrotransposable elements is a major driver of de novo unbalanced translocations.

Project description:Large scale analysis of balanced chromosomal translocation breakpoints has shown nonhomologous end joining and microhomology-mediated repair to be the main drivers of interchromosomal structural aberrations. Breakpoint sequences of de novo unbalanced translocations have not yet been investigated systematically. We analyzed 12 de novo translocations and mapped the breakpoints in 9. Surprisingly, in contrast to balanced translocations, we identify non-allelic homologous recombination (NAHR) between (retro)transposable elements and especially long interspersed elements (LINEs) as the main mutational mechanism. This finding implicates (retro)transposons to be a major driver of genomic rearrangements and exposes a profoundly different mutational mechanism compared to balanced chromosomal translocations. Furthermore, we show the existence of compound maternal/paternal derivative chromosomes, reinforcing the hypothesis that human cleavage stage embryogenesis is a cradle of chromosomal rearrangements.

2012-07-13 | GSE39303 | GEO

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data