title: Method for Dual Viral Vector Mediated CRISPR-Cas9 Gene Disruption in Primary Human Endothelial Cells.
abstract: Human endothelial cells (ECs) are widely used to study mechanisms of angiogenesis, inflammation, and endothelial permeability. Targeted gene disruption induced by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-Associated Protein 9 (Cas9) nuclease gene editing is potentially an important tool for definitively establishing the functional roles of individual genes in ECs. We showed that co-delivery of adenovirus encoding EGFP-tagged Cas9 and lentivirus encoding a single guide RNA (sgRNA) in primary human lung microvascular ECs (HLMVECs) disrupted the expression of the Tie2 gene and protein. Tie2 disruption increased basal endothelial permeability and prevented permeability recovery following injury induced by the inflammatory stimulus thrombin. Thus, gene deletion via viral co-delivery of CRISPR-Cas9 in primary human ECs provides a novel platform to investigate signaling mechanisms of normal and perturbed EC function without the need for clonal expansion.
authors.affiliation: Department of Pharmacology, University of Illinois College of Medicine, Chicago, IL 60612, USA.|The Center for Lung and Vascular Biology, University of Illinois College of Medicine, Chicago, IL 60612, USA.
authors.forename: Haixia
authors.initials: H
authors.lastname: Gong
authors.affiliation: Department of Pharmacology, University of Illinois College of Medicine, Chicago, IL 60612, USA.|The Center for Lung and Vascular Biology, University of Illinois College of Medicine, Chicago, IL 60612, USA.
authors.forename: Menglin
authors.initials: M
authors.lastname: Liu
authors.affiliation: Department of Pharmacology, University of Illinois College of Medicine, Chicago, IL 60612, USA.|The Center for Lung and Vascular Biology, University of Illinois College of Medicine, Chicago, IL 60612, USA.
authors.forename: Jeff
authors.initials: J
authors.lastname: Klomp
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Illinois College of Medicine, Chicago, IL 60612, USA.|Genome Editing Core, University of Illinois College of Medicine, Chicago, IL 60612, USA.
authors.forename: Bradley J
authors.initials: BJ
authors.lastname: Merrill
authors.affiliation: Department of Pharmacology, University of Illinois College of Medicine, Chicago, IL 60612, USA.|The Center for Lung and Vascular Biology, University of Illinois College of Medicine, Chicago, IL 60612, USA.|Division of Cardiology, Department of Medicine, University of Illinois College of Medicine, Chicago, IL 60612, USA.
authors.forename: Jalees
authors.initials: J
authors.lastname: Rehman
authors.affiliation: Department of Pharmacology, University of Illinois College of Medicine, Chicago, IL 60612, USA.|The Center for Lung and Vascular Biology, University of Illinois College of Medicine, Chicago, IL 60612, USA.
authors.forename: Asrar B
authors.initials: AB
authors.lastname: Malik
mesh_terms: D000256:Adenoviridae / Q000235:genetics* / Q000378:metabolism; D064113:CRISPR-Cas Systems; D002463:Cell Membrane Permeability; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D000072669:Gene Editing / Q000379:methods*; D015870:Gene Expression; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D016086:Lentivirus / Q000235:genetics* / Q000378:metabolism; D008168:Lung / Q000166:cytology / Q000378:metabolism; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation; D061251:Primary Cell Culture; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics / Q000378:metabolism; D042787:Receptor, TIE-2 / Q000172:deficiency / Q000235:genetics*; D015398:Signal Transduction; D013917:Thrombin / Q000494:pharmacology
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D042787:Receptor, TIE-2; D004720:Endonucleases; D013917:Thrombin