title: A modified TALEN-based system for robust generation of knock-out human pluripotent stem cell lines and disease models.
abstract: BACKGROUND
  Transcription activator-like effector nucleases (TALENs) have emerged as a tool for enabling targeted gene editing and disruption in difficult systems, such as human pluripotent stem cells (hPSCs). The modular architecture of TAL effectors theoretically enables targeting of any genomic locus and several cloning systems for custom TALEN assembly have recently been established. However, there is a lack of versatile TALEN expression systems applicable to hPSCs.
  
  
  RESULTS
  Here, we extend an existing TALE assembly system by a dual set of expression vectors for efficient application of TALEN technology in hPSCs. This is characterized by improved TALEN architecture as well as antibiotic resistance and fluorescent reporter cassettes, thus enabling enrichment for transfected cells. Improved functionality of the combined system was demonstrated by targeted disruption of the HPRT1 gene to create isogenic disease models of Lesch-Nyhan-Syndrome. Using female hPSCs, homozygous disruption of HPRT1 occurred at efficiencies of up to 15%. Differentiating isogenic knock-out cells both into central nervous system (CNS) as well as into sensory-like neurons recapitulated previously described phenotypes based on patient-specific induced PSCs and extended these findings to non-CNS neurons, respectively.
  
  
  CONCLUSIONS
  The combined vector system allows for flexible and affordable generation of knock-out hPSCs lines, thus enabling investigation of developmental processes as well as the generation of isogenic disease models without the need for patient material.
authors.forename: Stefan
authors.initials: S
authors.lastname: Frank
authors.forename: Boris V
authors.initials: BV
authors.lastname: Skryabin
authors.affiliation: Chemical Genomics Centre of the Max Planck Society, Dortmund, Germany. boris.greber@mpi-muenster.mpg.de.
authors.forename: Boris
authors.initials: B
authors.lastname: Greber
mesh_terms: D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics*; D004720:Endonucleases / Q000235:genetics* / Q000378:metabolism; D005260:Female; D055786:Gene Knockout Techniques; D018390:Gene Targeting; D023281:Genomics; D006720:Homozygote; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000096:biosynthesis* / Q000235:genetics; D007926:Lesch-Nyhan Syndrome / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D011984:Sensory Receptor Cells / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics*
chemical_list: D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D007041:Hypoxanthine Phosphoribosyltransferase; D004720:Endonucleases; D015252:Deoxyribonucleases, Type II Site-Specific