期刊
ACS SYNTHETIC BIOLOGY
卷 11, 期 4, 页码 1397-1407出版社
AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.1c00606
关键词
epigenetics; CRISPR-Cas9; chemical induced proximity; bifunctional molecules; synthetic gene regulation; ChIP-seq
资金
- U.S. National Institutes of Health [R01GM118653, R01GM132299, R01CA242305, R61DA047023]
- Eshelman Institute for Innovation
- Mark Foundation for Cancer Research
- UNC School of Medicine Bioinformatics and Analytics Research Collaborative (BARC)
CRISPR-Cas9 systems have been developed for gene expression regulation through fusions with epigenetic regulators or chemical-mediated strategies. This chemical approach utilizes a new chemical epigenetic modifier (CEM) to optimize gene targeting by linking it to catalytically inactive Cas9 (dCas9) and FKBP.
CRISPR-Cas9 systems have been developed to regulate gene expression by using either fusions to epigenetic regulators or, more recently, through the use of chemically mediated strategies. These approaches have armed researchers with new tools to examine the function of proteins by intricately controlling expression levels of specific genes. Here we present a CRISPR-based chemical approach that uses a new chemical epigenetic modifier (CEM) to hone to a gene targeted with a catalytically inactive Cas9 (dCas9) bridged to an FKS06-binding protein (FKBP) in mammalian cells. One arm of the bifunctional CEM recruits BRD4 to the target site, and the other arm is composed of a bumped ligand that binds to a mutant FKBP with a compensatory hole at F36V. This bump-and-hole strategy allows for activation of target genes in a dose-dependent and reversible fashion with increased specificity and high efficacy, providing a new synthetic biology approach to answer important mechanistic questions in the future.
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