title: Reversion of FMR1 Methylation and Silencing by Editing the Triplet Repeats in Fragile X iPSC-Derived Neurons.
abstract: Fragile X syndrome (FXS) is the most common form of inherited intellectual disability, resulting from a CGG repeat expansion in the fragile X mental retardation 1 (FMR1) gene. Here, we report a strategy for CGG repeat correction using CRISPR/Cas9 for targeted deletion in both embryonic stem cells and induced pluripotent stem cells derived from FXS patients. Following gene correction in FXS induced pluripotent stem cells, FMR1 expression was restored and sustained in neural precursor cells and mature neurons. Strikingly, after removal of the CGG repeats, the upstream CpG island of the FMR1 promoter showed extensive demethylation, an open chromatin state, and transcription initiation. These results suggest a silencing maintenance mechanism for the FMR1 promoter that is dependent on the existence of the CGG repeat expansion. Our strategy for deletion of trinucleotide repeats provides further insights into the molecular mechanisms of FXS and future therapies of trinucleotide repeat disorders.
authors.affiliation: Department of Physiology and Brain Korea 21 Plus Project for Medical Science, Yonsei University College of Medicine, Seoul 120-752, Korea.
authors.forename: Chul-Yong
authors.initials: CY
authors.lastname: Park
authors.affiliation: The Azrieli Center for Stem Cells and Genetic Research, Department of Genetics, Institute of Life Sciences, the Hebrew University, Givat-Ram, Jerusalem 91904, Israel.
authors.forename: Tomer
authors.initials: T
authors.lastname: Halevy
authors.affiliation: Department of Physiology and Brain Korea 21 Plus Project for Medical Science, Yonsei University College of Medicine, Seoul 120-752, Korea.
authors.forename: Dongjin R
authors.initials: DR
authors.lastname: Lee
authors.affiliation: Department of Physiology and Brain Korea 21 Plus Project for Medical Science, Yonsei University College of Medicine, Seoul 120-752, Korea.
authors.forename: Jin Jea
authors.initials: JJ
authors.lastname: Sung
authors.affiliation: Department of Physiology and Brain Korea 21 Plus Project for Medical Science, Yonsei University College of Medicine, Seoul 120-752, Korea.
authors.forename: Jae Souk
authors.initials: JS
authors.lastname: Lee
authors.affiliation: The Azrieli Center for Stem Cells and Genetic Research, Department of Genetics, Institute of Life Sciences, the Hebrew University, Givat-Ram, Jerusalem 91904, Israel.
authors.forename: Ofra
authors.initials: O
authors.lastname: Yanuka
authors.affiliation: The Azrieli Center for Stem Cells and Genetic Research, Department of Genetics, Institute of Life Sciences, the Hebrew University, Givat-Ram, Jerusalem 91904, Israel. Electronic address: nissimb@cc.huji.ac.il.
authors.forename: Nissim
authors.initials: N
authors.lastname: Benvenisty
authors.affiliation: Department of Physiology and Brain Korea 21 Plus Project for Medical Science, Yonsei University College of Medicine, Seoul 120-752, Korea. Electronic address: dwkim2@yuhs.ac.
authors.forename: Dong-Wook
authors.initials: DW
authors.lastname: Kim
mesh_terms: D064113:CRISPR-Cas Systems; D002478:Cells, Cultured; D018899:CpG Islands; D019175:DNA Methylation; D051860:Fragile X Messenger Ribonucleoprotein 1 / Q000235:genetics* / Q000378:metabolism; D005600:Fragile X Syndrome / Q000235:genetics*; D020868:Gene Silencing; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D018911:Trinucleotide Repeats
chemical_list: D051860:Fragile X Messenger Ribonucleoprotein 1; C497683:FMR1 protein, human