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Circ Res. 2015 Jul 3;117(2):121-8. doi: 10.1161/CIRCRESAHA.117.306290. Epub 2015 May 4.

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

Author information

1
From the Department of Immunobiology (P.A., M.S.K., J.S.P.), Department of Internal Medicine (W.G.C., Y.Q.), Department of Surgery (G.T.), and Department of Biomedical Engineering (W.M.S.), Yale University, New Haven, CT.
2
From the Department of Immunobiology (P.A., M.S.K., J.S.P.), Department of Internal Medicine (W.G.C., Y.Q.), Department of Surgery (G.T.), and Department of Biomedical Engineering (W.M.S.), Yale University, New Haven, CT. jordan.pober@yale.edu.

Abstract

RATIONALE:

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.

OBJECTIVE:

To create a simple but highly efficient method using CRISPR/Cas9 to generate biallelic gene disruption in untransformed human EC.

METHODS AND RESULTS:

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.

CONCLUSIONS:

CRISPR/Cas9-modified human EC provides a powerful platform for vascular research and for regenerative medicine/tissue engineering.

KEYWORDS:

clustered regularly interspaced short palindromic repeats; endothelial cells; genetic engineering; genetic techniques; immunologic techniques

PMID:
25940550
PMCID:
PMC4490936
DOI:
10.1161/CIRCRESAHA.117.306290
[Indexed for MEDLINE]
Free PMC Article

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