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

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 (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. Conclusion 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.

Polycaprolactone electrospun fiber scaffold loaded with iPSCs-NSCs and ASCs as a novel tissue engineering scaffold for the treatment of spinal cord injury

“…50 IPSCs are gradually applied to disease models, drug screening, regenerative medicine, and other fields. 51,52 Führmann et al have reported a peptide-modified, minimally invasive, injectable hydrogel containing hyaluronan and methylcellulose. 53 They used the biomaterial to enhance the survival of human iPSCs-derived oligodendrocyte progenitor cells and cell differentiation.…”

Inherited cardiac diseases, pluripotent stem cells, and genome editing combined—the past, present, and future

“…For example, this can be achieved with viruses, RNA interference or transgenic approaches to either overexpress or repress the gene or specific variant implicated as being disease causative. Indeed these approaches have all been used to examine the pathogenicity of both channelopathy-and cardiomyopathycausing cardiac variants [66][67][68][69] (see Supplemental Table S1 for complete list). Although the genetically modified hiPSC-CMs exhibited the expected phenotypic changes, as the introduced constructs are heterologously expressed, the resulting lines might not accurately reflect the molecular mechanisms underlying the disease pathology.…”

Genome Editing for the Understanding and Treatment of Cardiomyopathies

“…TALENs have been used to generate a wide variety of human embryonic stem cell (hESC)-based models to study cardiomyopathies. Karakikes and colleagues (2017) used TALEN constructs to knockout 88 genes associated with cardiomyopathies and congenital heart diseases in hiPSCs [65]. All constructs were validated and found to disrupt the target locus at high frequencies.…”