title: Reprogrammable CRISPR/Cas9-based system for inducing site-specific DNA methylation.
abstract: Advances in sequencing technology allow researchers to map genome-wide changes in DNA methylation in development and disease. However, there is a lack of experimental tools to site-specifically manipulate DNA methylation to discern the functional consequences. We developed a CRISPR/Cas9 DNA methyltransferase 3A (DNMT3A) fusion to induce DNA methylation at specific loci in the genome. We induced DNA methylation at up to 50% of alleles for targeted CpG dinucleotides. DNA methylation levels peaked within 50 bp of the short guide RNA (sgRNA) binding site and between pairs of sgRNAs. We used our approach to target methylation across the entire CpG island at the CDKN2A promoter, three CpG dinucleotides at the ARF promoter, and the CpG island within the Cdkn1a promoter to decrease expression of the target gene. These tools permit mechanistic studies of DNA methylation and its role in guiding molecular processes that determine cellular fate.
authors.affiliation: Center for Pharmacogenomics, Department of Medicine, Washington University in St. Louis School of Medicine, St. Louis, MO, USA.
authors.forename: James I
authors.initials: JI
authors.lastname: McDonald
authors.affiliation: Section of Stem Cell Biology, Division of Oncology, Department of Medicine, Washington University in St. Louis School of Medicine, St. Louis, MO 63110, USA.
authors.forename: Hamza
authors.initials: H
authors.lastname: Celik
authors.affiliation: Center for Pharmacogenomics, Department of Medicine, Washington University in St. Louis School of Medicine, St. Louis, MO, USA.
authors.forename: Lisa E
authors.initials: LE
authors.lastname: Rois
authors.affiliation: College of Arts and Science, Washington University in St. Louis, St. Louis, MO 63130, USA.
authors.forename: Gregory
authors.initials: G
authors.lastname: Fishberger
authors.affiliation: Center for Pharmacogenomics, Department of Medicine, Washington University in St. Louis School of Medicine, St. Louis, MO, USA.
authors.forename: Tolison
authors.initials: T
authors.lastname: Fowler
authors.affiliation: College of Arts and Science, Washington University in St. Louis, St. Louis, MO 63130, USA.
authors.forename: Ryan
authors.initials: R
authors.lastname: Rees
authors.affiliation: Section of Stem Cell Biology, Division of Oncology, Department of Medicine, Washington University in St. Louis School of Medicine, St. Louis, MO 63110, USA.
authors.forename: Ashley
authors.initials: A
authors.lastname: Kramer
authors.affiliation: Section of Stem Cell Biology, Division of Oncology, Department of Medicine, Washington University in St. Louis School of Medicine, St. Louis, MO 63110, USA.
authors.forename: Andrew
authors.initials: A
authors.lastname: Martens
authors.affiliation: Center for Pharmacogenomics, Department of Medicine, Washington University in St. Louis School of Medicine, St. Louis, MO, USA jedwards@dom.wustl.edu gchallen@dom.wustl.edu.
authors.forename: John R
authors.initials: JR
authors.lastname: Edwards
authors.affiliation: Section of Stem Cell Biology, Division of Oncology, Department of Medicine, Washington University in St. Louis School of Medicine, St. Louis, MO 63110, USA Developmental, Regenerative and Stem Cell Biology Program, Division of Biology and Biomedical Sciences, Washington University in St. Louis School of Medicine, St. Louis, MO 63110, USA jedwards@dom.wustl.edu gchallen@dom.wustl.edu.
authors.forename: Grant A
authors.identifier: http://orcid.org/0000-0003-4669-8814
authors.initials: GA
authors.lastname: Challen
keywords: CRISPR/Cas9-based system; CpG dinucleotides; DNA methylation