Genomic Targeting of TET Activity for Targeted Demethylation Using CRISPR/Cas9

Research output: Chapter in Book/Conference paperChapterpeer-review

2 Citations (Scopus)

Abstract

Methylation of DNA at cytosine bases is an important DNA modification underlying normal development and disease states. Despite decades of research into the biological function of DNA methylation, most of the observations so far have relied primarily on associative data between observed changes in DNA methylation states and local changes in transcriptional activity or chromatin state processes. This is primarily due to the lack of molecular tools to precisely modify DNA methylation in the genome. Recent advances in genome editing technologies have allowed repurposing the CRISPR-Cas9 system for epigenome editing by fusing the catalytically dead Cas9 (dCas9) to epigenome modifying enzymes. Moreover, methods of recruiting multiple protein domains, including the SunTag system, have increased the efficacy of epigenome editing at target sites. Here, we describe an end-to-end protocol for efficient targeted removal of DNA methylation by recruiting multiple catalytic domain of TET1 enzymes to the target sites with the dCas9-SunTag system, including sgRNA design, molecular cloning, delivery of plasmid into mammalian cells, and targeted DNA methylation analysis.

Original languageEnglish
Title of host publicationTET Proteins and DNA Demethylation
Subtitle of host publicationMethods and Protocols
EditorsOzren Bogdanovic, Michiel Vermeulen
PublisherHumana Press
Chapter10
Pages181-194
Number of pages14
Volume2272
ISBN (Electronic)978-1-0716-1294-1
ISBN (Print)978-1-0716-1293-4
DOIs
Publication statusPublished - 2021

Publication series

NameMethods in Molecular Biology
Volume2272
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

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