title: Genome engineering of isogenic human ES cells to model autism disorders.
abstract: Isogenic pluripotent stem cells are critical tools for studying human neurological diseases by allowing one to study the effects of a mutation in a fixed genetic background. Of particular interest are the spectrum of autism disorders, some of which are monogenic such as Timothy syndrome (TS); others are multigenic such as the microdeletion and microduplication syndromes of the 16p11.2 chromosomal locus. Here, we report engineered human embryonic stem cell (hESC) lines for modeling these two disorders using locus-specific endonucleases to increase the efficiency of homology-directed repair (HDR). We developed a system to: (1) computationally identify unique transcription activator-like effector nuclease (TALEN) binding sites in the genome using a new software program, TALENSeek, (2) assemble the TALEN genes by combining golden gate cloning with modified constructs from the FLASH protocol, and (3) test the TALEN pairs in an amplification-based HDR assay that is more sensitive than the typical non-homologous end joining assay. We applied these methods to identify, construct, and test TALENs that were used with HDR donors in hESCs to generate an isogenic TS cell line in a scarless manner and to model the 16p11.2 copy number disorder without modifying genomic loci with high sequence similarity.
authors.affiliation: Allen Institute for Brain Science, Seattle, WA 98103, USA.
authors.forename: Refugio A
authors.initials: RA
authors.lastname: Martinez
authors.affiliation: Neurogenetics Program, Department of Neurology, Center for Autism Research and Treatment, Semel Institute, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.
authors.forename: Jason L
authors.initials: JL
authors.lastname: Stein
authors.affiliation: Allen Institute for Brain Science, Seattle, WA 98103, USA.
authors.forename: Anne-Rachel F
authors.initials: AR
authors.lastname: Krostag
authors.affiliation: Allen Institute for Brain Science, Seattle, WA 98103, USA.
authors.forename: Angelique M
authors.initials: AM
authors.lastname: Nelson
authors.affiliation: Allen Institute for Brain Science, Seattle, WA 98103, USA.
authors.forename: John S
authors.initials: JS
authors.lastname: Marken
authors.affiliation: Allen Institute for Brain Science, Seattle, WA 98103, USA.
authors.forename: Vilas
authors.initials: V
authors.lastname: Menon
authors.affiliation: Allen Institute for Brain Science, Seattle, WA 98103, USA.
authors.forename: Ryan C
authors.initials: RC
authors.lastname: May
authors.affiliation: Allen Institute for Brain Science, Seattle, WA 98103, USA.
authors.forename: Zizhen
authors.initials: Z
authors.lastname: Yao
authors.affiliation: Allen Institute for Brain Science, Seattle, WA 98103, USA.
authors.forename: Ajamete
authors.initials: A
authors.lastname: Kaykas
authors.affiliation: Neurogenetics Program, Department of Neurology, Center for Autism Research and Treatment, Semel Institute, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA dhg@mednet.ucla.edu.
authors.forename: Daniel H
authors.initials: DH
authors.lastname: Geschwind
authors.affiliation: Allen Institute for Brain Science, Seattle, WA 98103, USA joshuag@alleninstitute.org.
authors.forename: Joshua S
authors.initials: JS
authors.lastname: Grimley
mesh_terms: D001321:Autistic Disorder; D001665:Binding Sites; D060846:Cell Engineering; D002460:Cell Line; D002659:Child Development Disorders, Pervasive / Q000235:genetics*; D003851:Deoxyribonucleases / Q000378:metabolism; D053595:Embryonic Stem Cells; D018390:Gene Targeting; D015894:Genome, Human; D006801:Humans; D008133:Long QT Syndrome / Q000235:genetics; D008957:Models, Genetic; D059767:Recombinational DNA Repair; D012984:Software; D013576:Syndactyly / Q000235:genetics
chemical_list: D003851:Deoxyribonucleases