Development of CXCR4 Gene Editing in Human Umbilical Vein Endothelial Cells Using the CRISPR/CAS9 System

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Number of pages: 44-49
Year-Number: 2021-2

Abstract

Infection with the Human Immunodeficiency Virus (HIV) is associated with the progressive loss of CD4+ T cells, either through destruction or decreased production. When an individual is infected, HIV is propagated in CD4+ T cells using only the blood pathway or the transmission site. In this regard, gene targeting by the CRISPR system, which allows for genetic editing via gene inactivation, is preferred because it appears to produce significantly lasting effect than existing methods. Thus, the CRISPR-Cas9 system was tested to induce knock-out of CXCR4 gene in HUVECs, which could make the inner surface of blood vessels resistant to HIV infection and forms a separating surface. We have tested four different gRNAs and demonstrated their efficacy at genome level by T7 endonuclaase assay and protein level by CXCR4 flow cytometer. Three out of four gRNAs against CXCR4 demonstrated the efficacy in gene modification as well downregulation of CXCR4 proteins in HUVEC plasma membrane. This study demonstrates the use of the CRISPR-Cas9 system to generate gene-edited endothelial cells resistant to HIV infections by silencing the CXCR4 gene on human umbilical vein endothelial cells.

Keywords

Abstract

Infection with the Human Immunodeficiency Virus (HIV) is associated with the progressive loss of CD4+ T cells, either through destruction or decreased production. When an individual is infected, HIV is propagated in CD4+ T cells using only the blood pathway or the transmission site. In this regard, gene targeting by the CRISPR system, which allows for genetic editing via gene inactivation, is preferred because it appears to produce significantly lasting effect than existing methods. Thus, the CRISPR-Cas9 system was tested to induce knock-out of CXCR4 gene in HUVECs, which could make the inner surface of blood vessels resistant to HIV infection and forms a separating surface. We have tested four different gRNAs and demonstrated their efficacy at genome level by T7 endonuclaase assay and protein level by CXCR4 flow cytometer. Three out of four gRNAs against CXCR4 demonstrated the efficacy in gene modification as well downregulation of CXCR4 proteins in HUVEC plasma membrane. This study demonstrates the use of the CRISPR-Cas9 system to generate gene-edited endothelial cells resistant to HIV infections by silencing the CXCR4 gene on human umbilical vein endothelial cells.

Keywords