title: Variant-aware saturating mutagenesis using multiple Cas9 nucleases identifies regulatory elements at trait-associated loci.
abstract: Cas9-mediated, high-throughput, saturating in situ mutagenesis permits fine-mapping of function across genomic segments. Disease- and trait-associated variants identified in genome-wide association studies largely cluster at regulatory loci. Here we demonstrate the use of multiple designer nucleases and variant-aware library design to interrogate trait-associated regulatory DNA at high resolution. We developed a computational tool for the creation of saturating-mutagenesis libraries with single or multiple nucleases with incorporation of variants. We applied this methodology to the HBS1L-MYB intergenic region, which is associated with red-blood-cell traits, including fetal hemoglobin levels. This approach identified putative regulatory elements that control MYB expression. Analysis of genomic copy number highlighted potential false-positive regions, thus emphasizing the importance of off-target analysis in the design of saturating-mutagenesis experiments. Together, these data establish a widely applicable high-throughput and high-resolution methodology to identify minimal functional sequences within large disease- and trait-associated regions.
authors.affiliation: Division of Hematology/Oncology, Boston Children's Hospital; Department of Pediatric Oncology, Dana-Farber Cancer Institute; Harvard Stem Cell Institute; and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Matthew C
authors.initials: MC
authors.lastname: Canver
authors.affiliation: Montreal Heart Institute, Université de Montréal, Montréal, Québec, Canada.
authors.forename: Samuel
authors.initials: S
authors.lastname: Lessard
authors.affiliation: Department of Molecular Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Luca
authors.initials: L
authors.lastname: Pinello
authors.affiliation: Division of Hematology/Oncology, Boston Children's Hospital; Department of Pediatric Oncology, Dana-Farber Cancer Institute; Harvard Stem Cell Institute; and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Yuxuan
authors.initials: Y
authors.lastname: Wu
authors.affiliation: Montreal Heart Institute, Université de Montréal, Montréal, Québec, Canada.
authors.forename: Yann
authors.initials: Y
authors.lastname: Ilboudo
authors.affiliation: Division of Hematology/Oncology, Boston Children's Hospital; Department of Pediatric Oncology, Dana-Farber Cancer Institute; Harvard Stem Cell Institute; and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Emily N
authors.initials: EN
authors.lastname: Stern
authors.affiliation: Division of Hematology/Oncology, Boston Children's Hospital; Department of Pediatric Oncology, Dana-Farber Cancer Institute; Harvard Stem Cell Institute; and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Austen J
authors.initials: AJ
authors.lastname: Needleman
authors.affiliation: Red Cell Genetic Disease Unit, Hôpital Henri-Mondor, Assistance Publique-Hôpitaux de Paris (AP-HP), UPeC, IMRB U955 Equipe no. 2, Créteil, France.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Galactéros
authors.affiliation: Department of Laboratory Medicine, Boston Children's Hospital, Boston, Massachusetts, USA.
authors.forename: Carlo
authors.initials: C
authors.lastname: Brugnara
authors.affiliation: Department of Medicine, Sickle Cell Center, Augusta University, Augusta, Georgia, USA.
authors.forename: Abdullah
authors.initials: A
authors.lastname: Kutlar
authors.affiliation: The Caribbean Institute for Health Research, University of the West Indies, Mona, Kingston, Jamaica.
authors.forename: Colin
authors.initials: C
authors.lastname: McKenzie
authors.affiliation: The Caribbean Institute for Health Research, University of the West Indies, Mona, Kingston, Jamaica.
authors.forename: Marvin
authors.initials: M
authors.lastname: Reid
authors.affiliation: Division of Hematology/Oncology, Boston Children's Hospital; Department of Pediatric Oncology, Dana-Farber Cancer Institute; Harvard Stem Cell Institute; and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Diane D
authors.initials: DD
authors.lastname: Chen
authors.affiliation: Division of Hematology/Oncology, Boston Children's Hospital; Department of Pediatric Oncology, Dana-Farber Cancer Institute; Harvard Stem Cell Institute; and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Partha Pratim
authors.initials: PP
authors.lastname: Das
authors.affiliation: Division of Hematology/Oncology, Boston Children's Hospital; Department of Pediatric Oncology, Dana-Farber Cancer Institute; Harvard Stem Cell Institute; and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Mitchel
authors.initials: M
authors.lastname: A Cole
authors.affiliation: Division of Hematology/Oncology, Boston Children's Hospital; Department of Pediatric Oncology, Dana-Farber Cancer Institute; Harvard Stem Cell Institute; and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Jing
authors.initials: J
authors.lastname: Zeng
authors.affiliation: Department of Research and Development, Central Blood Institute, Japanese Red Cross Society, Tokyo, Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Kurita
authors.affiliation: Cell Engineering Division, RIKEN BioResource Center, Tsukuba, Japan.|Faculty of Medicine, University of Tsukuba, Tsukuba, Japan.
authors.forename: Yukio
authors.initials: Y
authors.lastname: Nakamura
authors.affiliation: Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute and Harvard T.H. Chan School of Public Health, Boston, Massachusetts, USA.
authors.forename: Guo-Cheng
authors.initials: GC
authors.lastname: Yuan
authors.affiliation: Montreal Heart Institute, Université de Montréal, Montréal, Québec, Canada.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Lettre
authors.affiliation: Division of Hematology/Oncology, Boston Children's Hospital; Department of Pediatric Oncology, Dana-Farber Cancer Institute; Harvard Stem Cell Institute; and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Daniel E
authors.initials: DE
authors.lastname: Bauer
authors.affiliation: Division of Hematology/Oncology, Boston Children's Hospital; Department of Pediatric Oncology, Dana-Farber Cancer Institute; Harvard Stem Cell Institute; and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts, USA.|Howard Hughes Medical Institute, Boston, Massachusetts, USA.
authors.forename: Stuart H
authors.initials: SH
authors.lastname: Orkin
mesh_terms: D002478:Cells, Cultured; D021901:DNA, Intergenic / Q000235:genetics; D018628:Gene Dosage / Q000235:genetics; D014644:Genetic Variation / Q000235:genetics*; D055106:Genome-Wide Association Study / Q000379:methods; D057809:HEK293 Cells; D006801:Humans; D016296:Mutagenesis / Q000235:genetics*; D040641:Quantitative Trait Loci / Q000235:genetics*; D050436:Regulatory Elements, Transcriptional / Q000235:genetics*
chemical_list: D021901:DNA, Intergenic