title: Low-Dose Irradiation Enhances Gene Targeting in Human Pluripotent Stem Cells.
abstract: Human pluripotent stem cells (hPSCs) are now being used for both disease modeling and cell therapy; however, efficient homologous recombination (HR) is often crucial to develop isogenic control or reporter lines. We showed that limited low-dose irradiation (LDI) using either γ-ray or x-ray exposure (0.4 Gy) significantly enhanced HR frequency, possibly through induction of DNA repair/recombination machinery including ataxia-telangiectasia mutated, histone H2A.X and RAD51 proteins. LDI could also increase HR efficiency by more than 30-fold when combined with the targeting tools zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats. Whole-exome sequencing confirmed that the LDI administered to hPSCs did not induce gross genomic alterations or affect cellular viability. Irradiated and targeted lines were karyotypically normal and made all differentiated lineages that continued to express green fluorescent protein targeted at the AAVS1 locus. This simple method allows higher throughput of new, targeted hPSC lines that are crucial to expand the use of disease modeling and to develop novel avenues of cell therapy.
  
  
  CONCLUSIONS
  The simple and relevant technique described in this report uses a low level of radiation to increase desired gene modifications in human pluripotent stem cells by an order of magnitude. This higher efficiency permits greater throughput with reduced time and cost. The low level of radiation also greatly increased the recombination frequency when combined with developed engineered nucleases. Critically, the radiation did not lead to increases in DNA mutations or to reductions in overall cellular viability. This novel technique enables not only the rapid production of disease models using human stem cells but also the possibility of treating genetically based diseases by correcting patient-derived cells.
authors.affiliation: Board of Governors, Regenerative Medicine Institute, Department of Biomedical Sciences, iPSC Core, The David and Janet Polak Foundation Stem Cell Core Laboratory, Genomics Core Facility, Department of Surgery, and Department of Neurology, Cedars-Sinai Medical Center, Los Angeles, California, USA.
authors.forename: Seigo
authors.initials: S
authors.lastname: Hatada
authors.affiliation: Board of Governors, Regenerative Medicine Institute, Department of Biomedical Sciences, iPSC Core, The David and Janet Polak Foundation Stem Cell Core Laboratory, Genomics Core Facility, Department of Surgery, and Department of Neurology, Cedars-Sinai Medical Center, Los Angeles, California, USA.
authors.forename: Aparna
authors.initials: A
authors.lastname: Subramanian
authors.affiliation: Board of Governors, Regenerative Medicine Institute, Department of Biomedical Sciences, iPSC Core, The David and Janet Polak Foundation Stem Cell Core Laboratory, Genomics Core Facility, Department of Surgery, and Department of Neurology, Cedars-Sinai Medical Center, Los Angeles, California, USA.
authors.forename: Berhan
authors.initials: B
authors.lastname: Mandefro
authors.affiliation: Board of Governors, Regenerative Medicine Institute, Department of Biomedical Sciences, iPSC Core, The David and Janet Polak Foundation Stem Cell Core Laboratory, Genomics Core Facility, Department of Surgery, and Department of Neurology, Cedars-Sinai Medical Center, Los Angeles, California, USA.
authors.forename: Songyang
authors.initials: S
authors.lastname: Ren
authors.affiliation: Board of Governors, Regenerative Medicine Institute, Department of Biomedical Sciences, iPSC Core, The David and Janet Polak Foundation Stem Cell Core Laboratory, Genomics Core Facility, Department of Surgery, and Department of Neurology, Cedars-Sinai Medical Center, Los Angeles, California, USA.
authors.forename: Ho Won
authors.initials: HW
authors.lastname: Kim
authors.affiliation: Board of Governors, Regenerative Medicine Institute, Department of Biomedical Sciences, iPSC Core, The David and Janet Polak Foundation Stem Cell Core Laboratory, Genomics Core Facility, Department of Surgery, and Department of Neurology, Cedars-Sinai Medical Center, Los Angeles, California, USA.
authors.forename: Jie
authors.initials: J
authors.lastname: Tang
authors.affiliation: Board of Governors, Regenerative Medicine Institute, Department of Biomedical Sciences, iPSC Core, The David and Janet Polak Foundation Stem Cell Core Laboratory, Genomics Core Facility, Department of Surgery, and Department of Neurology, Cedars-Sinai Medical Center, Los Angeles, California, USA.
authors.forename: Vincent
authors.initials: V
authors.lastname: Funari
authors.affiliation: Board of Governors, Regenerative Medicine Institute, Department of Biomedical Sciences, iPSC Core, The David and Janet Polak Foundation Stem Cell Core Laboratory, Genomics Core Facility, Department of Surgery, and Department of Neurology, Cedars-Sinai Medical Center, Los Angeles, California, USA.
authors.forename: Robert H
authors.initials: RH
authors.lastname: Baloh
authors.affiliation: Board of Governors, Regenerative Medicine Institute, Department of Biomedical Sciences, iPSC Core, The David and Janet Polak Foundation Stem Cell Core Laboratory, Genomics Core Facility, Department of Surgery, and Department of Neurology, Cedars-Sinai Medical Center, Los Angeles, California, USA.
authors.forename: Dhruv
authors.initials: D
authors.lastname: Sareen
authors.affiliation: Board of Governors, Regenerative Medicine Institute, Department of Biomedical Sciences, iPSC Core, The David and Janet Polak Foundation Stem Cell Core Laboratory, Genomics Core Facility, Department of Surgery, and Department of Neurology, Cedars-Sinai Medical Center, Los Angeles, California, USA Clive.Svendsen@cshs.org Vaithi.Arumugaswami@cshs.org.
authors.forename: Vaithilingaraja
authors.initials: V
authors.lastname: Arumugaswami
authors.affiliation: Board of Governors, Regenerative Medicine Institute, Department of Biomedical Sciences, iPSC Core, The David and Janet Polak Foundation Stem Cell Core Laboratory, Genomics Core Facility, Department of Surgery, and Department of Neurology, Cedars-Sinai Medical Center, Los Angeles, California, USA Clive.Svendsen@cshs.org Vaithi.Arumugaswami@cshs.org.
authors.forename: Clive N
authors.initials: CN
authors.lastname: Svendsen
mesh_terms: D064007:Ataxia Telangiectasia Mutated Proteins / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation / Q000528:radiation effects; D002470:Cell Survival / Q000528:radiation effects; D004249:DNA Damage; D003851:Deoxyribonucleases / Q000235:genetics / Q000378:metabolism; D059472:Exome; D005720:Gamma Rays; D005786:Gene Expression Regulation / Q000528:radiation effects*; D018390:Gene Targeting / Q000379:methods*; D056426:Genetic Loci; D006657:Histones / Q000235:genetics / Q000378:metabolism; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism / Q000528:radiation effects*; D055029:Inverted Repeat Sequences; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism / Q000528:radiation effects*; D051135:Rad51 Recombinase / Q000235:genetics / Q000378:metabolism; D011829:Radiation Dosage; D059767:Recombinational DNA Repair; D015398:Signal Transduction; D014965:X-Rays; D016335:Zinc Fingers / Q000235:genetics
chemical_list: C468783:H2AX protein, human; D006657:Histones; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C495153:RAD51 protein, human; D051135:Rad51 Recombinase; D003851:Deoxyribonucleases
keywords: Clustered regularly interspaced short palindromic repeats; Gene targeting; Induced pluripotent stem cell; Irradiation; Transcription activator-like effector nuclease; Zinc finger nuclease