Browse
Submit Data
Databases
API
Help

Dataset Information

30 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Reduced Expression of VEGF-A in Human Retinal Pigment Epithelial Cells and Human Muller Cells Following CRISPR-Cas9 Ribonucleoprotein-Mediated Gene Disruption.

ABSTRACT:

Purpose

To evaluate the effects of vascular endothelial growth factor-A (VEGF-A) gene editing in human retinal pigment epithelial (RPE) cells and human Muller cells, which are the main VEGF-A producing cells in the eye.

Methods

CRISPR-Cas9 ribonucleoprotein was used to target exon 1 in VEGF-A gene. Lipofectamine CRISPRMAX was used as a vehicle. In vitro gene editing efficiency was assessed on oligonucleotides and genomic DNAs. Sanger sequencing was performed to detect indels. VEGF-A messenger RNA and protein expressions were assessed using quantitative polymerase chain reaction and enzyme-linked immunosorbent assay.

Results

In vitro cleavage assay on a 60-nucleotide DNA duplex showed 88% cleavage of the precursor. The cleavage efficiency was 40% in RPE cells and 32% in Muller cells. Sanger sequencing in the CRISPR-Cas9 treated RPE and Muller cells showed indels at the predicted cut site in both cells. After the VEGF-A gene disruption, VEGF-A protein levels decreased 43% in RPE cells (P < 0.0001) and 38% in Muller cells (P < 0.0001).

Conclusions

CRISPR-Cas9-mediated gene disruption resulted in a significant decrease in the VEGF-A gene protein expression in human RPE and Muller cells. CRISPR-Cas9 ribonucleoprotein may allow simultaneous targeting of multiple VEGF-A producing cells.

Translational relevance

VEGF-A gene disruption using CRISPR-Cas9 ribonucleoprotein has a potential in treating retinal vascular diseases.

SUBMITTER: Ameri H

PROVIDER: S-EPMC7422915 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Genomic Disruption of VEGF-A Expression in Human Retinal Pigment Epithelial Cells Using CRISPR-Cas9 Endonuclease.

Project description:PurposeTo employ type II clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonuclease to suppress ocular angiogenesis by genomic disruption of VEGF-A in human RPE cells.MethodsCRISPR sequences targeting exon 1 of human VEGF-A were computationally identified based on predicted Cas9 on- and off-target probabilities. Single guide RNA (gRNA) cassettes with these target sequences were cloned into lentiviral vectors encoding the Streptococcuspyogenes Cas9 endonuclease (SpCas9) gene. The lentiviral vectors were used to infect ARPE-19 cells, a human RPE cell line. Frequency of insertion or deletion (indel) mutations was assessed by T7 endonuclease 1 mismatch detection assay; mRNA levels were assessed with quantitative real-time PCR; and VEGF-A protein levels were determined by ELISA. In vitro angiogenesis was measured using an endothelial cell tube formation assay.ResultsFive gRNAs targeting VEGF-A were selected based on the highest predicted on-target probabilities, lowest off-target probabilities, or combined average of both scores. Lentiviral delivery of the top-scoring gRNAs with SpCas9 resulted in indel formation in the VEGF-A gene at frequencies up to 37.0% ± 4.0% with corresponding decreases in secreted VEGF-A protein up to 41.2% ± 7.4% (P < 0.001), and reduction of endothelial tube formation up to 39.4% ± 9.8% (P = 0.02). No significant indel formation in the top three putative off-target sites tested was detected.ConclusionsThe CRISPR-Cas9 endonuclease system may reduce VEGF-A secretion from human RPE cells and suppress angiogenesis, supporting the possibility of employing gene editing for antiangiogenesis therapy in ocular diseases.

| S-EPMC5072539 | biostudies-literature

Functional Assessment of Patient-Derived Retinal Pigment Epithelial Cells Edited by CRISPR/Cas9.

Project description:Retinitis pigmentosa is the most common form of inherited blindness and can be caused by a multitude of different genetic mutations that lead to similar phenotypes. Specifically, mutations in ubiquitously expressed splicing factor proteins are known to cause an autosomal dominant form of the disease, but the retina-specific pathology of these mutations is not well understood. Fibroblasts from a patient with splicing factor retinitis pigmentosa caused by a missense mutation in the PRPF8 splicing factor were used to produce three diseased and three CRISPR/Cas9-corrected induced pluripotent stem cell (iPSC) clones. We differentiated each of these clones into retinal pigment epithelial (RPE) cells via directed differentiation and analyzed the RPE cells in terms of gene and protein expression, apicobasal polarity, and phagocytic ability. We demonstrate that RPE cells can be produced from patient-derived and corrected cells and they exhibit morphology and functionality similar but not identical to wild-type RPE cells in vitro. Functionally, the RPE cells were able to establish apicobasal polarity and phagocytose photoreceptor outer segments at the same capacity as wild-type cells. These data suggest that patient-derived iPSCs, both diseased and corrected, are able to differentiate into RPE cells with a near normal phenotype and without differences in phagocytosis, a result that differs from previous mouse models. These RPE cells can now be studied to establish a disease-in-a-dish system relevant to retinitis pigmentosa.

| S-EPMC6321630 | biostudies-literature

Multiplexed CRISPR gene editing in primary human islet cells with Cas9 ribonucleoprotein.

Project description:Successful genome editing in primary human islets could reveal features of the genetic regulatory landscape underlying β cell function and diabetes risk. Here, we describe a CRISPR-based strategy to interrogate functions of predicted regulatory DNA elements using electroporation of a complex of Cas9 ribonucleoprotein (Cas9 RNP) and guide RNAs into primary human islet cells. We successfully targeted coding regions including the PDX1 exon 1, and non-coding DNA linked to diabetes susceptibility. CRISPR/Cas9 RNP approaches revealed genetic targets of regulation by DNA elements containing candidate diabetes risk SNPs, including an in vivo enhancer of the MPHOSPH9 gene. CRISPR/Cas9 RNP multiplexed targeting of two cis-regulatory elements linked to diabetes risk in PCSK1, which encodes an endoprotease crucial for insulin processing, also demonstrated efficient simultaneous editing of PCSK1 regulatory elements, resulting in impaired β cell PCSK1 regulation and insulin secretion. Multiplex CRISPR/Cas9 RNP provides powerful approaches to investigate and elucidate human islet cell gene regulation in health and diabetes.

| S-EPMC10516051 | biostudies-literature

Ascorbate Suppresses VEGF Expression in Retinal Pigment Epithelial Cells.

Project description:PurposeTo investigate the impact of ascorbate, via DNA hydroxymethylation, on VEGF expression in retinal pigment epithelial (RPE) cells.MethodsDot-blot and hydroxymethylated DNA immunoprecipitation sequencing were applied to evaluate the impact of ascorbate on DNA hydroxymethylation in ARPE-19 cells. RNA sequencing (RNA-seq) was carried out to analyze the transcriptome. Quantitative RT-PCR and ELISA were conducted to examine the transcription and secretion of VEGF from cultured cells. Primary human fetal RPE cells and RPE-J cells were used to verify the effect of ascorbate on VEGF expression. ELISA was used to measure VEGF in the vitreous humor of Gulo-/- mice, which, like humans, cannot synthesize ascorbate de novo.ResultsTreatment with ascorbate (50 μM) promoted 5-hydroxymethycytosine (5hmC) generation and changed the genome-wide profiles of 5hmC in ARPE-19 cells. Ascorbate also caused a dramatic shift in the transcriptome-3186 differential transcripts, of which 69.3% are correlated with altered 5hmC in promoters or gene bodies. One of the most downregulated genes was VEGFA, which encodes the VEGF protein. The suppression of VEGF by ascorbate is independent of hypoxia-inducible factor 1-alpha (HIF-1α) but correlates with increased 5hmC in the gene body. The decreased transcription and secretion of VEGF by ascorbate were verified in primary human fetal RPE cells. Furthermore, adding ascorbate in the diet for Gulo-/- mice resulted in decreased levels of VEGF in the RPE/choroid and vitreous humor.ConclusionsAscorbate inhibits VEGF expression in RPE cells likely via DNA hydroxymethylation. Thus, ascorbate could be implicated in the prevention or treatment of diseases such as age-related macular degeneration (AMD).

| S-EPMC6049987 | biostudies-literature

Efficient gene disruption in cultured primary human endothelial cells by CRISPR/Cas9.

Project description:The participation of endothelial cells (EC) in many physiological and pathological processes is widely modeled using human EC cultures, but genetic manipulation of these untransformed cells has been technically challenging. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) technology offers a promising new approach. However, mutagenized cultured cells require cloning to yield homogeneous populations, and the limited replicative lifespan of well-differentiated human EC presents a barrier for doing so.To create a simple but highly efficient method using CRISPR/Cas9 to generate biallelic gene disruption in untransformed human EC.To demonstrate proof-of-principle, we used CRISPR/Cas9 to disrupt the gene for the class II transactivator. We used endothelial colony forming cell-derived EC and lentiviral vectors to deliver CRISPR/Cas9 elements to ablate EC expression of class II major histocompatibility complex molecules and with it, the capacity to activate allogeneic CD4(+) T cells. We show the observed loss-of-function arises from biallelic gene disruption in class II transactivator that leaves other essential properties of the cells intact, including self-assembly into blood vessels in vivo, and that the altered phenotype can be rescued by reintroduction of class II transactivator expression.CRISPR/Cas9-modified human EC provides a powerful platform for vascular research and for regenerative medicine/tissue engineering.

| S-EPMC4490936 | biostudies-literature

CRISPR/Cas9-Mediated TERT Disruption in Cancer Cells.

Project description:Mammalian telomere lengths are primarily regulated by telomerase, a ribonucleoprotein consisting of a reverse transcriptase (TERT) and an RNA subunit (TERC). TERC is constitutively expressed in all cells, whereas TERT expression is temporally and spatially regulated, such that in most adult somatic cells, TERT is inactivated and telomerase activity is undetectable. Most tumor cells activate TERT as a mechanism for preventing progressive telomere attrition to achieve proliferative immortality. Therefore, inactivating TERT has been considered to be a promising means of cancer therapy. Here we applied the CRISPR/Cas9 gene editing system to target the TERT gene in cancer cells. We report that disruption of TERT severely compromises cancer cell survival in vitro and in vivo. Haploinsufficiency of TERT in tumor cells is sufficient to result in telomere attrition and growth retardation in vitro. In vivo, TERT haploinsufficient tumor cells failed to form xenograft after transplantation to nude mice. Our work demonstrates that gene editing-mediated TERT knockout is a potential therapeutic option for treating cancer.

| S-EPMC7014288 | biostudies-literature

CRISPR-Cas9 Ribonucleoprotein-Mediated Genomic Editing in Primary Innate Immune Cells.

Project description:CRISPR-Cas9 genome engineering can be used to functionally investigate the complex mechanisms of immune system regulation. Decades of work have aimed to genetically reprogram innate immunity, but current approaches are inefficient or nonspecific, limiting their use. Here, we detail an optimized strategy for non-viral CRISPR-Cas9 ribonucleoprotein (cRNP) genomic editing of primary innate lymphocytes (ILCs) and myeloid lineage cells, resulting in high-efficiency editing of target gene expression from a single electroporation. For complete details on the use and execution of this protocol, please refer to Riggan et al. (2020).

| S-EPMC7756923 | biostudies-literature

CRISPR/Cas9 ribonucleoprotein-mediated genome and epigenome editing in mammalian cells.

Project description:The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system has revolutionized the ability to edit the mammalian genome, providing a platform for the correction of pathogenic mutations and further investigation into gene function. CRISPR reagents can be delivered into the cell as DNA, RNA, or pre-formed ribonucleoproteins (RNPs). RNPs offer numerous advantages over other delivery approaches due to their ability to rapidly target genomic sites and quickly degrade thereafter. Here, we review the production steps and delivery methods for Cas9 RNPs. Additionally, we discuss how RNPs enhance genome and epigenome editing efficiencies, reduce off-target editing activity, and minimize cellular toxicity in clinically relevant mammalian cell types. We include details on a broad range of editing approaches, including novel base and prime editing techniques. Finally, we summarize key challenges for the use of RNPs, and propose future perspectives on the field.

| S-EPMC8844242 | biostudies-literature

CRISPR-Cas9 Ribonucleoprotein-Mediated Genomic Editing in Mature Primary Innate Immune Cells.

Project description:CRISPR genome engineering has become a powerful tool to functionally investigate the complex mechanisms of immune system regulation. While decades of work have aimed to genetically reprogram innate immunity, the utility of current approaches is restricted by poor knockout efficiencies or limited specificity for mature cell lineages in vivo. Here, we describe an optimized strategy for non-viral CRISPR-Cas9 ribonucleoprotein (cRNP) genomic editing of mature primary mouse innate lymphocyte cells (ILCs) and myeloid lineage cells that results in an almost complete loss of single or double target gene expression from a single electroporation. Furthermore, we describe in vivo adoptive transfer mouse models that can be utilized to screen for gene function during viral infection using cRNP-edited naive natural killer (NK) cells and bone-marrow-derived conventional dendritic cell precursors (cDCPs). This resource will enhance target gene discovery and offer a specific and simplified approach to gene editing in the mouse innate immune system.

| S-EPMC7294876 | biostudies-literature

Highly efficient DNA-free gene disruption in the agricultural pest Ceratitis capitata by CRISPR-Cas9 ribonucleoprotein complexes.

Project description:The Mediterranean fruitfly Ceratitis capitata (medfly) is an invasive agricultural pest of high economic impact and has become an emerging model for developing new genetic control strategies as an alternative to insecticides. Here, we report the successful adaptation of CRISPR-Cas9-based gene disruption in the medfly by injecting in vitro pre-assembled, solubilized Cas9 ribonucleoprotein complexes (RNPs) loaded with gene-specific single guide RNAs (sgRNA) into early embryos. When targeting the eye pigmentation gene white eye (we), a high rate of somatic mosaicism in surviving G0 adults was observed. Germline transmission rate of mutated we alleles by G0 animals was on average above 52%, with individual cases achieving nearly 100%. We further recovered large deletions in the we gene when two sites were simultaneously targeted by two sgRNAs. CRISPR-Cas9 targeting of the Ceratitis ortholog of the Drosophila segmentation paired gene (Ccprd) caused segmental malformations in late embryos and in hatched larvae. Mutant phenotypes correlate with repair by non-homologous end-joining (NHEJ) lesions in the two targeted genes. This simple and highly effective Cas9 RNP-based gene editing to introduce mutations in C. capitata will significantly advance the design and development of new effective strategies for pest control management.

| S-EPMC5577161 | biostudies-literature

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