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.
Document Details
- Document Type
- Pub Defense Publication
- Publication Date
- May 11, 2016
- Source ID
- 10.1242/bio.019067
Entities
People
- Andrew Martens
- Ashley Kramer
- Grant A Challen
- Gregory Fishberger
- Hamza Celik
- James I. Mcdonald
- John R. Edwards
- Lisa E. Rois
- Ryan Rees
- Tolison Fowler
Organizations
- Alex's Lemonade Stand Foundation
- American Society of Hematology
- Congressionally Directed Medical Research Programs
- Edward Mallinckrodt Jr. Foundation
- National Institutes of Health
- V Foundation for Cancer Research
- Washington University in St. Louis