Chromatin Manipulation and Editing: Challenges, New Technologies and Their Use in Plants

Abstract

An ongoing challenge in functional epigenomics is to develop tools for precise manipulation of epigenetic marks. These tools would allow moving from correlation-based to causal-based findings, a necessary step to reach conclusions on mechanistic principles. In this review, we describe and discuss the advantages and limits of tools and technologies developed to impact epigenetic marks, and which could be employed to study their direct effect on nuclear and chromatin structure, on transcription, and their further genuine role in plant cell fate and development. On one hand, epigenome-wide approaches include drug inhibitors for chromatin modifiers or readers, nanobodies against histone marks or lines expressing modified histones or mutant chromatin effectors. On the other hand, locus-specific approaches consist in targeting precise regions on the chromatin, with engineered proteins able to modify epigenetic marks. Early systems use effectors in fusion with protein domains that recognize a specific DNA sequence (Zinc Finger or TALEs), while the more recent dCas9 approach operates through RNA-DNA interaction, thereby providing more flexibility and modularity for tool designs. Current developments of "second generation", chimeric dCas9 systems, aiming at better targeting efficiency and modifier capacity have recently been tested in plants and provided promising results. Finally, recent proof-of-concept studies forecast even finer tools, such as inducible/switchable systems, that will allow temporal analyses of the molecular events that follow a change in a specific chromatin mark.

Keywords: CRISPR-dCas9; chemical inhibitors; epigenome editing; histone marks.

Figure 1

Overview of epigenetic engineering approaches to study gene function and chromatin modifications at specific loci. (A) ZF-based editing tool. In this approach, the fusion of several Zn finger domains forms a polydactyl system that targets a specific DNA sequence. Direct fusion to a chromatin modifier (CM) can then trigger chromatin modifications such as methylation/demethylation of either DNA or histones in nucleosomes, or acetylation/deacetylation of histones nearby the target site. (B) TALE-based editing tool. The TALE (Transcription Activator-Like Effectors) approach also uses customizable DBDs to target a specific DNA sequence as well as a direct fusion to a CM to induce chromatin modifications nearby the target site. (C) First generation of dCas9 tools to modify chromatin at specific loci. This approach is based on the property of a guide RNA (gRNA) to target a complementary DNA region of interest. The gRNA recruits the dead Cas9 (dCas9) protein which is directly fused to a CM. (D) Second generation of dCas9 tools with the MS2 strategy. The MS2 scaffold RNAs are recognized by MCP proteins fused to CMs, thus enhancing effector capacity. (E) Second generation of dCas9 tools with the SunTag strategy. The dCas9 is fused to a multicopy antigen which is recognized by an antibody fused to TMs or CMs, thus amplifying effector capacity.