A Comprehensive TALEN-Based Knockout Library for Generating Human Induced Pluripotent Stem Cell-Based Models for Cardiovascular Diseases
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 (KO) eighty-eight 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 KO. We observed that all the constructs were active and disrupted the target locus at high frequencies. To illustrate the general utility of the TALEN-mediated KO technique, six 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 dilated cardiomyopathy (DCM)-causing mutation (TNNT2 p.R173W) in patient-specific iPSC-derived cardiac myocytes (iPSC-CMs), we demonstrated that the KO strategy ameliorates the DCM phenotype in vitro. In addition, we modeled the Holt-Oram syndrome (HOS) in iPSC-CMs 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 technology 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.
- genome editing
- induced pluripotent stem cells
- disease modeling
- holt-roam syndrome
- dilated cardiomyopathy
- stem cell
- gene targeting
- Received September 9, 2016.
- Revision received February 23, 2017.
- Accepted February 28, 2017.