A comprehensive TALEN-based knockout library for generating human-induced pluripotent stem cell-based models for cardiovascular diseases

Ioannis Karakikes, Vittavat Termglinchan, Diana A. Cepeda, Jaecheol Lee, Sebastian Diecke, Ayal Hendel, Ilanit Itzhaki, Mohamed Ameen, Rajani Shrestha, Haodi Wu, Ning Ma, Ning Yi Shao, Timon Seeger, Nicole Woo, Kitchener D. Wilson, Elena Matsa, Matthew H. Porteus, Vittorio Sebastiano, Joseph C. Wu

Research output: Contribution to journalArticlepeer-review

48 Scopus citations

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.

Original languageEnglish
Pages (from-to)1561-1571
Number of pages11
JournalCirculation Research
Volume120
Issue number10
Early online date28 Feb 2017
DOIs
StatePublished - 12 May 2017
Externally publishedYes

Bibliographical note

Publisher Copyright:
© 2017 American Heart Association, Inc.

Funding

FundersFunder number
National Heart, Lung, and Blood InstituteK08HL119251

    Keywords

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

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