Reprogrammable CRISPR/Cas9-based system for inducing sitespecific DNA methylation

James I. McDonald, Hamza Celik, Lisa E. Rois, Gregory Fishberger, Tolison Fowler, Ryan Rees, Ashley Kramer, Andrew Martens, John R. Edwardsand, Grant A. Challen

Research output: Contribution to journalArticlepeer-review

169 Scopus citations

Abstract

Advances in sequencing technology allow researchers to map genomewide 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.

Original languageEnglish
Pages (from-to)866-874
Number of pages9
JournalBiology Open
Volume5
Issue number6
DOIs
StatePublished - Jun 15 2016

Keywords

  • CRISPR/Cas9-based system
  • CpG dinucleotides
  • DNA methylation

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