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


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.
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.
Parwiz
P
Abrahimi
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.
William G
WG
Chang
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.
Martin S
MS
Kluger
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.
Yibing
Y
Qyang
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.
George
G
Tellides
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.
W Mark
WM
Saltzman
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.
Jordan S
JS
Pober
D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D064113:CRISPR-Cas Systems / Q000235:genetics; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D066026:Endothelial Progenitor Cells / Q000166:cytology* / Q000378:metabolism / Q000637:transplantation; D005260:Female; D005312:Fetal Blood / Q000166:cytology*; D017353:Gene Deletion; D055786:Gene Knockout Techniques; D005802:Genes, MHC Class II; D005822:Genetic Vectors / Q000187:drug effects / Q000494:pharmacology*; D006684:HLA-DR Antigens / Q000096:biosynthesis / Q000276:immunology; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D016086:Lentivirus / Q000235:genetics*; D008213:Lymphocyte Activation; D007959:Lymphocyte Culture Test, Mixed; D051379:Mice; D016513:Mice, SCID; D009687:Nuclear Proteins / Q000235:genetics*; D061251:Primary Cell Culture / Q000379:methods; D011506:Proteins / Q000235:genetics; D013752:Tetracycline / Q000494:pharmacology; D015534:Trans-Activators / Q000235:genetics*; D033921:Vesicular Transport Proteins
D006684:HLA-DR Antigens; D047908:Intracellular Signaling Peptides and Proteins; C491133:Lyst protein, mouse; C083728:MHC class II transactivator protein; D009687:Nuclear Proteins; D011506:Proteins; D015534:Trans-Activators; D033921:Vesicular Transport Proteins; D013752:Tetracycline
clustered regularly interspaced short palindromic repeats; endothelial cells; genetic engineering; genetic techniques; immunologic techniques