Dataset Information

Precise CCM1 gene correction and inactivation in patient-derived endothelial cells: Modeling Knudson's two-hit hypothesis in vitro.

ABSTRACT: BACKGROUND:The CRISPR/Cas9 system has opened new perspectives to study the molecular basis of cerebral cavernous malformations (CCMs) in personalized disease models. However, precise genome editing in endothelial and other hard-to-transfect cells remains challenging. METHODS:In a proof-of-principle study, we first isolated blood outgrowth endothelial cells (BOECs) from a CCM1 mutation carrier with multiple CCMs. In a CRISPR/Cas9 gene correction approach, a high-fidelity Cas9 variant was then transfected into patient-derived BOECs using a ribonucleoprotein complex and a single-strand DNA oligonucleotide. In addition, patient-specific CCM1 knockout clones were expanded after CRISPR/Cas9 gene inactivation. RESULTS:Deep sequencing demonstrated correction of the mutant allele in nearly 33% of all cells whereas no CRISPR/Cas9-induced mutations in predicted off-target loci were identified. Corrected BOECs could be cultured in cell mixtures but demonstrated impaired clonal survival. In contrast, CCM1-deficient BOECs displayed increased resistance to stress-induced apoptotic cell death and could be clonally expanded to high passages. When cultured together, CCM1-deficient BOECs largely replaced corrected as well as heterozygous BOECs. CONCLUSION:We here demonstrate that a non-viral CRISPR/Cas9 approach can not only be used for gene knockout but also for precise gene correction in hard-to-transfect endothelial cells (ECs). Comparing patient-derived isogenic CCM1+/+ , CCM1+/- , and CCM1-/- ECs, we show that the inactivation of the second allele results in clonal evolution of ECs lacking CCM1 which likely reflects the initiation phase of CCM genesis.

SUBMITTER: Spiegler S

PROVIDER: S-EPMC6625102 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Precise CCM1 gene correction and inactivation in patient-derived endothelial cells: Modeling Knudson's two-hit hypothesis in vitro.

Spiegler Stefanie S Rath Matthias M Much Christiane D CD Sendtner Barbara S BS Felbor Ute U

Molecular genetics & genomic medicine 20190523 7

<h4>Background</h4>The CRISPR/Cas9 system has opened new perspectives to study the molecular basis of cerebral cavernous malformations (CCMs) in personalized disease models. However, precise genome editing in endothelial and other hard-to-transfect cells remains challenging.<h4>Methods</h4>In a proof-of-principle study, we first isolated blood outgrowth endothelial cells (BOECs) from a CCM1 mutation carrier with multiple CCMs. In a CRISPR/Cas9 gene correction approach, a high-fidelity Cas9 varia ...[more]

PMID: 31124307

Similar Datasets

Project description:BackgroundPreterm infants are at high risk of diffuse white matter injury and adverse neurodevelopmental outcome. The multiple hit hypothesis suggests that the risk of white matter injury increases with cumulative exposure to multiple perinatal risk factors. Our aim was to test this hypothesis in a large cohort of preterm infants using diffusion weighted magnetic resonance imaging (dMRI).MethodsWe studied 491 infants (52% male) without focal destructive brain lesions born at < 34 weeks, who underwent structural and dMRI at a specialist Neonatal Imaging Centre. The median (range) gestational age (GA) at birth was 30+ 1 (23+ 2-33+ 5) weeks and median postmenstrual age at scan was 42+ 1 (38-45) weeks. dMRI data were analyzed using tract based spatial statistics and the relationship between dMRI measures in white matter and individual perinatal risk factors was assessed. We tested the hypothesis that increased exposure to perinatal risk factors was associated with lower fractional anisotropy (FA), and higher radial, axial and mean diffusivity (RD, AD, MD) in white matter. Neurodevelopmental performance was investigated using the Bayley Scales of Infant and Toddler Development, Third Edition (BSITD-III) in a subset of 381 infants at 20 months corrected age. We tested the hypothesis that lower FA and higher RD, AD and MD in white matter were associated with poorer neurodevelopmental performance.ResultsIdentified risk factors for diffuse white matter injury were lower GA at birth, fetal growth restriction, increased number of days requiring ventilation and parenteral nutrition, necrotizing enterocolitis and male sex. Clinical chorioamnionitis and patent ductus arteriosus were not associated with white matter injury. Multivariate analysis demonstrated that fetal growth restriction, increased number of days requiring ventilation and parenteral nutrition were independently associated with lower FA values. Exposure to cumulative risk factors was associated with reduced white matter FA and FA values at term equivalent age were associated with subsequent neurodevelopmental performance.ConclusionThis study suggests multiple perinatal risk factors have an independent association with diffuse white matter injury at term equivalent age and exposure to multiple perinatal risk factors exacerbates dMRI defined, clinically significant white matter injury. Our findings support the multiple hit hypothesis for preterm white matter injury.

Project description:The dual-hit hypothesis of neurodegeneration states that severe stress sensitizes vulnerable cells to subsequent challenges so that the two hits are synergistic in their toxic effects. Although the hippocampus is vulnerable to a number of neurodegenerative disorders, there are no models of synergistic cell death in hippocampal neurons in response to combined proteotoxic and oxidative stressors, the two major characteristics of these diseases. Therefore, a relatively high-throughput dual-hit model of stress synergy was developed in primary hippocampal neurons. In order to increase the rigor of the study and strengthen the interpretations, three independent, unbiased viability assays were employed at multiple timepoints. Stress synergy was elicited when hippocampal neurons were treated with the proteasome inhibitor MG132 followed by exposure to the oxidative toxicant paraquat, but only after 48 h. MG132 and paraquat only elicited additive effects 24 h after the final hit and even loss of heat shock protein 70 activity and glutathione did not promote stress synergy at this early timepoint. Dual hits of MG132 elicited modest glutathione loss and slightly synergistic toxic effects 48 h after the second hit, but only at some concentrations and only according to two viability assays (metabolic fitness and cytoskeletal integrity). The thiol N-acetyl cysteine protected hippocampal neurons against dual MG132/MG132 hits but not dual MG132/paraquat hits. These findings support the view that proteotoxic and oxidative stress propel and propagate each other in hippocampal neurons, leading to synergistically toxic effects, but not as the default response and only after a delay. The neuronal stress synergy observed here lies in contrast to astrocytic responses to dual hits, because astrocytes that survive severe proteotoxic stress resist additional cell loss following second hits. In conclusion, a new model of hippocampal vulnerability was developed for the testing of therapies, because neuroprotective treatments that are effective against severe, synergistic stress are more likely to succeed in the clinic. © 2016 Wiley Periodicals, Inc.

Dataset Information

Precise CCM1 gene correction and inactivation in patient-derived endothelial cells: Modeling Knudson's two-hit hypothesis in vitro.

Publications

Precise CCM1 gene correction and inactivation in patient-derived endothelial cells: Modeling Knudson's two-hit hypothesis in vitro.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure