Live-Animal Epigenome Editing: Convergence of Novel Techniques

Abstract

Epigenome editing refers to the generation of precise chromatin alterations and their effects on gene expression and cell biology. Until recently, much of the efforts in epigenome editing were limited to tissue culture models of disease. However, the convergence of techniques from different fields including mammalian genetics, virology, and CRISPR engineering is advancing epigenome editing into a new era. Researchers are increasingly embracing the use of multicellular model organisms to test the role of specific chromatin alterations in development and disease. The challenge of successful live-animal epigenomic editing will depend on a well-informed foundation of the current methodologies for cell-specific delivery and editing accuracy. Here we review the opportunities for basic research and therapeutic applications.

Keywords: CRISPR; animal models; cancer; delivery; epigenomic editing; neurodevelopment.

Figure 1. Classes of CRISPR/dCas9 Epigenome Modifications.

CRISPR-based epigenome editors mediate four distinct classes of modifications on chromatin based the domains utilized. Long-range interactions between different loci and nuclear compartments are facilitated by proximity interacting domains. Nucleosome remodeling is regulate enzymatic domains that mediate arrangement of nucleosomes on DNA. Covalent histone modifications is catalyzed by enzymatic domains that target amino acids on histones. Covalent DNA modifications is catalyzed by enzymatic domains that add or remove methyl groups from cytosine.

Figure 2. Enzymatic domains for covalent histone and DNA modifications.

Several catalytic domains associated with gene activation (left) or silencing (right) have been paired with CRISPR-based epigenome editing. The enzymes and their DNA or amino acid targets on a nucleosome are shown in each panel. Open circles denote the targeted lysine and their position on the histone protein. The induced post-translational modification is denoted by brackets: me indicate methylation, ac indicates acetylation, and ub indicates ubiquitination. Arrows indicate deposition of the modification, and blunt-ended arrows indicate removal.

Figure 3. Sequences used to construct transgenic and knock-in mouse strains.

A. Strain with Cre-inducible expression of a FLAG-tagged dCas9 protein with FLP-controlled eGFP expression. B. Strain with constitutive expression of a GFP-tagged dCas9 protein. C. Strain with Cre-inducible expression of a dCas9-GCN4-epitope recombinant protein, followed by a detached single-chain antibody fused to P65 and HSF1 domains, and a detached eGFP protein. D. Strain with Cre-inducible expression of a dCas9-GCN4-epitope recombinant protein. Rosa26 denotes the locus on mouse chromosome 6. White arrow indicates a chimeric CAG promoter system. loxP-STOP-loxP denotes presence of stop codons and a poly-adenylation sequence flanked by loxP sites. P2A and T2A denote the location of exclusive intramolecular cleaving proteases for processing of the polypeptide. All mouse strains were constructed with strep pyogenes derived Cas9.

Figure 4. Summary of Methods for Live Animal Epigenome Editing.

A. Epigenomic edited human or murnie cells can be implantation into model organisms and their differentiation, growth, localization monitored over time. B. To alter endogenous mouse cells, the epigenomic editing machinery can be packaged into AAV with mutant capsid variants for specific troprim. C. Alternatively, the epigenomic edited machinery can purified and ribonucleic-protein complexes can be delivered via different routes for targeted organ distribution. D. Genetically encoding dCas9 and epigenomic editing domains in the mouse genome by transgenesis can greatly facilitate the study of epigenomic editing in live animals.