Method for Dual Viral Vector Mediated CRISPR-Cas9 Gene Disruption in Primary Human Endothelial Cells

Haixia Gong,Asrar B Malik,Menglin Liu,Jalees Rehman,Bradley J Merrill,Jeff Klomp

doi:10.1038/srep42127

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.

Highlights

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas[9] when applied to a cell population generates a mosaic of cells with varying insertions and deletions[12] which necessitates clonal expansion of individual cells to obtain a cell line with only the desired gene disruption[13,14]
Passage 3 HMLVECs were transduced with adenovirus-mediated expression of EGFP-Cas[9] at an Multiplicity of infection (MOI) of 10 as well as a lentivirus expressing a single guide RNA (sgRNA) targeting the endothelial cells (ECs) receptor kinase Tie[2] at an MOI of 10 or a vector control
T7E1 assay was used to confirm the presence of CRISPR-Cas9induced mutations in human lung microvascular ECs (HLMVECs) following Tie-2 gene deletion (Fig. 1D)

Summary

Introduction

CRISPR-Cas[9] when applied to a cell population generates a mosaic of cells with varying insertions and deletions[12] which necessitates clonal expansion of individual cells to obtain a cell line with only the desired gene disruption[13,14]. The feasibility of using CRISPR-Cas[9] for gene deletion in primary ECs, which cannot be clonally expanded[15,16], has not yet been established. A recent method using CRISPR-Cas[9] achieved gene deletion following antibiotic selection of clones in cord blood derived endothelial colony forming cells (ECFCs)[17], which exhibit an endothelial cell-like phenotype but represent a specialized progenitor population of highly proliferative ECs found in umbilical cord blood and which can undergo clonal expansion[18]

Methods

Results

Conclusion