CRISPR/Cas9-Based Engineering of the Epigenome

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Abstract

Determining causal relationships between distinct chromatin features and gene expression, and ultimately cell behavior, remains a major challenge. Recent developments in targetable epigenome-editing tools enable us to assign direct transcriptional and functional consequences to locus-specific chromatin modifications. This Protocol Review discusses the unprecedented opportunity that CRISPR/Cas9 technology offers for investigating and manipulating the epigenome to facilitate further understanding of stem cell biology and engineering of stem cells for therapeutic applications. We also provide technical considerations for standardization and further improvement of the CRISPR/Cas9-based tools to engineer the epigenome.

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Epigenome editing by a CRISPR/Cas9-based acetyltransferase activates genes from promoters and enhancers

Isaac B. Hilton, Anthony M. D’Ippolito, Christopher M. Vockley … (2015)

Technologies that facilitate the targeted manipulation of epigenetic marks could be used to precisely control cell phenotype or interrogate the relationship between the epigenome and transcriptional control. Here we have generated a programmable acetyltransferase based on the CRISPR/Cas9 gene regulation system, consisting of the nuclease-null dCas9 protein fused to the catalytic core of the human acetyltransferase p300. This fusion protein catalyzes acetylation of histone H3 lysine 27 at its target sites, corresponding with robust transcriptional activation of target genes from promoters, proximal enhancers, and distal enhancers. Gene activation by the targeted acetyltransferase is highly specific across the genome. In contrast to conventional dCas9-based activators, the acetyltransferase effectively activates genes from enhancer regions and with individual guide RNAs. The core p300 domain is also portable to other programmable DNA-binding proteins. These results support targeted acetylation as a causal mechanism of transactivation and provide a new robust tool for manipulating gene regulation.

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Epigenome-wide association studies for common human diseases.

Vardhman Rakyan, Thomas A. Down, David Balding … (2011)

Despite the success of genome-wide association studies (GWASs) in identifying loci associated with common diseases, a substantial proportion of the causality remains unexplained. Recent advances in genomic technologies have placed us in a position to initiate large-scale studies of human disease-associated epigenetic variation, specifically variation in DNA methylation. Such epigenome-wide association studies (EWASs) present novel opportunities but also create new challenges that are not encountered in GWASs. We discuss EWAS design, cohort and sample selections, statistical significance and power, confounding factors and follow-up studies. We also discuss how integration of EWASs with GWASs can help to dissect complex GWAS haplotypes for functional analysis.

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CRISPR RNA-guided activation of endogenous human genes

Morgan Maeder, Samantha Linder, Vincent Cascio … (2013)

Catalytically inactive CRISPR-associated 9 nuclease (dCas9) can be directed by short guide RNAs (gRNAs) to repress endogenous genes in bacteria and human cells. Here we show that a dCas9-VP64 transcriptional activation domain fusion protein can be directed by single or multiple gRNAs to increase expression of specific endogenous human genes. These results provide an important proof-of-principle that CRISPR-Cas systems can be used to target heterologous effector domains in human cells.