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Circ Res. 2017 May 12;120(10):1561-1571. doi: 10.1161/CIRCRESAHA.116.309948. Epub 2017 Feb 28.

A Comprehensive TALEN-Based Knockout Library for Generating Human-Induced Pluripotent Stem Cell-Based Models for Cardiovascular Diseases.

Author information

1
From the Stanford Cardiovascular Institute (I.K., V.T., J.L., S.D., I.I., M.A., R.S., H.W., N.M., N.-Y.S., T.S., N.W., K.D.W., E.M., J.C.W.), Department of Cardiothoracic Surgery (I.K.), Division of Cardiovascular Medicine, Department of Medicine (V.T., J.C.W.), CA; Institute of Stem Cell Biology and Regenerative Medicine (D.A.C., V.S., J.C.W.), Departments of Pediatrics (A.H., M.H.P.), Pathology (K.D.W.), and Obstetrics and Gynecology (V.S.), Stanford University School of Medicine, CA; Berlin Institute of Health, Germany (S.D.); and Max Delbrueck Center, Berlin, Germany (S.D.).
2
From the Stanford Cardiovascular Institute (I.K., V.T., J.L., S.D., I.I., M.A., R.S., H.W., N.M., N.-Y.S., T.S., N.W., K.D.W., E.M., J.C.W.), Department of Cardiothoracic Surgery (I.K.), Division of Cardiovascular Medicine, Department of Medicine (V.T., J.C.W.), CA; Institute of Stem Cell Biology and Regenerative Medicine (D.A.C., V.S., J.C.W.), Departments of Pediatrics (A.H., M.H.P.), Pathology (K.D.W.), and Obstetrics and Gynecology (V.S.), Stanford University School of Medicine, CA; Berlin Institute of Health, Germany (S.D.); and Max Delbrueck Center, Berlin, Germany (S.D.). joewu@stanford.edu vsebast@stanford.edu.

Abstract

RATIONALE:

Targeted genetic engineering using programmable nucleases such as transcription activator-like effector nucleases (TALENs) is a valuable tool for precise, site-specific genetic modification in the human genome.

OBJECTIVE:

The emergence of novel technologies such as human induced pluripotent stem cells (iPSCs) and nuclease-mediated genome editing represent a unique opportunity for studying cardiovascular diseases in vitro.

METHODS AND RESULTS:

By incorporating extensive literature and database searches, we designed a collection of TALEN constructs to knockout 88 human genes that are associated with cardiomyopathies and congenital heart diseases. The TALEN pairs were designed to induce double-strand DNA break near the starting codon of each gene that either disrupted the start codon or introduced a frameshift mutation in the early coding region, ensuring faithful gene knockout. We observed that all the constructs were active and disrupted the target locus at high frequencies. To illustrate the utility of the TALEN-mediated knockout technique, 6 individual genes (TNNT2, LMNA/C, TBX5, MYH7, ANKRD1, and NKX2.5) were knocked out with high efficiency and specificity in human iPSCs. By selectively targeting a pathogenic mutation (TNNT2 p.R173W) in patient-specific iPSC-derived cardiac myocytes, we demonstrated that the knockout strategy ameliorates the dilated cardiomyopathy phenotype in vitro. In addition, we modeled the Holt-Oram syndrome in iPSC-cardiac myocytes in vitro and uncovered novel pathways regulated by TBX5 in human cardiac myocyte development.

CONCLUSIONS:

Collectively, our study illustrates the powerful combination of iPSCs and genome editing technologies for understanding the biological function of genes, and the pathological significance of genetic variants in human cardiovascular diseases. The methods, strategies, constructs, and iPSC lines developed in this study provide a validated, readily available resource for cardiovascular research.

KEYWORDS:

dilated cardiomyopathy; gene knockout; gene targeting; genome editing; stem cell

Comment in

PMID:
28246128
PMCID:
PMC5429194
DOI:
10.1161/CIRCRESAHA.116.309948
[Indexed for MEDLINE]
Free PMC Article

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