title: A Comprehensive TALEN-Based Knockout Library for Generating Human-Induced Pluripotent Stem Cell-Based Models for Cardiovascular Diseases.
abstract: BACKGROUND
  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.
  
  
  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, andNKX2.5) 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 byTBX5in 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.
authors.affiliation: 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.).
authors.forename: Ioannis
authors.initials: I
authors.lastname: Karakikes
authors.affiliation: 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.).
authors.forename: Vittavat
authors.initials: V
authors.lastname: Termglinchan
authors.affiliation: 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.).
authors.forename: Diana A
authors.initials: DA
authors.lastname: Cepeda
authors.affiliation: 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.).
authors.forename: Jaecheol
authors.initials: J
authors.lastname: Lee
authors.affiliation: 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.).
authors.forename: Sebastian
authors.initials: S
authors.lastname: Diecke
authors.affiliation: 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.).
authors.forename: Ayal
authors.initials: A
authors.lastname: Hendel
authors.affiliation: 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.).
authors.forename: Ilanit
authors.initials: I
authors.lastname: Itzhaki
authors.affiliation: 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.).
authors.forename: Mohamed
authors.initials: M
authors.lastname: Ameen
authors.affiliation: 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.).
authors.forename: Rajani
authors.initials: R
authors.lastname: Shrestha
authors.affiliation: 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.).
authors.forename: Haodi
authors.initials: H
authors.lastname: Wu
authors.affiliation: 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.).
authors.forename: Ning
authors.initials: N
authors.lastname: Ma
authors.affiliation: 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.).
authors.forename: Ning-Yi
authors.initials: NY
authors.lastname: Shao
authors.affiliation: 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.).
authors.forename: Timon
authors.initials: T
authors.lastname: Seeger
authors.affiliation: 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.).
authors.forename: Nicole
authors.initials: N
authors.lastname: Woo
authors.affiliation: 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.).
authors.forename: Kitchener D
authors.initials: KD
authors.lastname: Wilson
authors.affiliation: 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.).
authors.forename: Elena
authors.initials: E
authors.lastname: Matsa
authors.affiliation: 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.).
authors.forename: Matthew H
authors.initials: MH
authors.lastname: Porteus
authors.affiliation: 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.
authors.forename: Vittorio
authors.initials: V
authors.lastname: Sebastiano
authors.affiliation: 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.
authors.forename: Joseph C
authors.initials: JC
authors.lastname: Wu
mesh_terms: D001483:Base Sequence; D002318:Cardiovascular Diseases / Q000235:genetics* / Q000628:therapy; D002478:Cells, Cultured; D055786:Gene Knockout Techniques / Q000379:methods*; D015723:Gene Library; D018390:Gene Targeting / Q000379:methods; D005818:Genetic Engineering / Q000379:methods*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000502:physiology* / Q000637:transplantation
keywords: dilated cardiomyopathy; gene knockout; gene targeting; genome editing; stem cell