Editing the epigenome: technologies for programmable transcription and epigenetic modulation

Abstract

Gene regulation is a complex and tightly controlled process that defines cell identity, health and disease, and response to pharmacologic and environmental signals. Recently developed DNA-targeting platforms, including zinc finger proteins, transcription activator-like effectors (TALEs) and the clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 system, have enabled the recruitment of transcriptional modulators and epigenome-modifying factors to any genomic site, leading to new insights into the function of epigenetic marks in gene expression. Additionally, custom transcriptional and epigenetic regulation is facilitating refined control over cell function and decision making. The unique properties of the CRISPR-Cas9 system have created new opportunities for high-throughput genetic screens and multiplexing targets to manipulate complex gene expression patterns. This Review summarizes recent technological developments in this area and their application to biomedical challenges. We also discuss remaining limitations and necessary future directions for this field.

Figure 1. Applications of epigenome editing

Targeted control over epigenetic regulation is achieved by fusing programmable DNA-binding domains (DBDs) to epigenome editing effectors. Engineered epigenome editing proteins can be used to study mechanisms of epigenetic regulation and the contributions of gene regulation to cellular function and disease. Novel gene regulation relationships can be discovered through high-throughput screens performed with gRNA libraries, and new gene regulatory networks can be constructed with orthogonal epigenome editing proteins. Therapeutic applications of epigenome editing include cellular reprogramming and gene therapies that correct aberrant gene expression.

Figure 2. Programmable DNA-binding domains

A) Zinc fingers and B) TALEs are DNA-targeting platforms consisting of protein modules that bind within the major groove of DNA to recognize specific DNA base pairs. Zinc fingers domains recognize 3–4 nucleotide sequences, whereas TALE modules recognize single nucleotides according to a specific code. C) Cas9 is directed to the target site by an engineered guide RNA (gRNA). The gRNA consists of a 20 base pair targeting sequence, which recognizes its complementary genomic sequence via Watson-Crick base-pairing, and a constant region that interacts with the Cas9 protein. In order to bind target DNA, Cas9 also requires the presence of a protospacer-adjacent motif (PAM) immediately following the target sequence. Cas9 derived from S. pyogenes recognizes a 5’-NGG-3’ PAM. PDB files 2I13, 3UGM, and 4OO8 for the zinc finger protein, TALE, and CRISPR/Cas9 structures, respectively

Figure 3. Orthogonal CRISPR/dCas9 systems for complex regulation of distinct genomic targets

A) dCas9 orthologs with distinct PAM requirements can be adapted from different host species or engineered via directed evolution. Through unique gRNAs and PAM recognition sites, dCas9 ortholog-fusions can be used to effect distinct gene regulation events at multiple targets in a single host genome simultaneously. B) Alternatively, complex gene regulation events can be coordinated by recruiting epigenetic effectors directly to the gRNA molecule. Protein-binding motifs and long RNAs can be incorporated directly in the stem-loop structure of the gRNA.