title: Simultaneous Reprogramming and Gene Correction of Patient Fibroblasts.
abstract: The derivation of genetically modified induced pluripotent stem (iPS) cells typically involves multiple steps, requiring lengthy cell culture periods, drug selection, and several clonal events. We report the generation of gene-targeted iPS cell lines following a single electroporation of patient-specific fibroblasts using episomal-based reprogramming vectors and the Cas9/CRISPR system. Simultaneous reprogramming and gene targeting was tested and achieved in two independent fibroblast lines with targeting efficiencies of up to 8% of the total iPS cell population. We have successfully targeted the DNMT3B and OCT4 genes with a fluorescent reporter and corrected the disease-causing mutation in both patient fibroblast lines: one derived from an adult with retinitis pigmentosa, the other from an infant with severe combined immunodeficiency. This procedure allows the generation of gene-targeted iPS cell lines with only a single clonal event in as little as 2 weeks and without the need for drug selection, thereby facilitating "seamless" single base-pair changes.
authors.affiliation: Morgridge Institute for Research, 330 North Orchard Street, Madison, WI 53715, USA. Electronic address: sara.howden@mcri.edu.au.
authors.forename: Sara E
authors.initials: SE
authors.lastname: Howden
authors.affiliation: Morgridge Institute for Research, 330 North Orchard Street, Madison, WI 53715, USA.
authors.forename: John P
authors.initials: JP
authors.lastname: Maufort
authors.affiliation: Morgridge Institute for Research, 330 North Orchard Street, Madison, WI 53715, USA.
authors.forename: Bret M
authors.initials: BM
authors.lastname: Duffin
authors.affiliation: Department of Anatomy and Developmental Biology, Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton, Victoria 3800, Australia; Department of Paediatrics, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, Victoria 3052, Australia.
authors.forename: Andrew G
authors.initials: AG
authors.lastname: Elefanty
authors.affiliation: Department of Anatomy and Developmental Biology, Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton, Victoria 3800, Australia; Department of Paediatrics, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, Victoria 3052, Australia.
authors.forename: Edouard G
authors.initials: EG
authors.lastname: Stanley
authors.affiliation: Morgridge Institute for Research, 330 North Orchard Street, Madison, WI 53715, USA; Cell and Regenerative Biology, University of Wisconsin School of Medicine and Public Health, Madison, WI 53707-7365, USA; Department of Molecular, Cellular, and Developmental Biology, University of California Santa Barbara, Santa Barbara, CA 93106, USA.
authors.forename: James A
authors.initials: JA
authors.lastname: Thomson
mesh_terms: D000328:Adult; D001483:Base Sequence; D064113:CRISPR-Cas Systems; D002460:Cell Line; D002478:Cells, Cultured; D065150:Cellular Reprogramming; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics; D018274:Electroporation / Q000379:methods; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D008969:Molecular Sequence Data; D050814:Octamer Transcription Factor-3 / Q000235:genetics; D000097584:DNA Methyltransferase 3B
chemical_list: D004248:DNA (Cytosine-5-)-Methyltransferases; D050814:Octamer Transcription Factor-3; D000097584:DNA Methyltransferase 3B; C494173:POU5F1 protein, human