Designing Epigenome Editors: Considerations of Biochemical and Locus Specificities

Abstract

The advent of locus-specific protein recruitment technologies has enabled a new class of studies in chromatin biology. Epigenome editors enable biochemical modifications of chromatin at almost any specific endogenous locus. Their locus-specificity unlocks unique information including the functional roles of distinct modifications at specific genomic loci. Given the growing interest in using these tools for biological and translational studies, there are many specific design considerations depending on the scientific question or clinical need. Here we present and discuss important design considerations and challenges regarding the biochemical- and locus-specificities of epigenome editors. These include how to: account for the complex biochemical diversity of chromatin; control for potential interdependency of epigenome editors and their resultant modifications; avoid sequestration effects; quantify the locus specificity of epigenome editors; and improve locus-specificity by considering concentration, affinity, avidity, and sequestration effects.

Engineering and Design of Programmable Genome Editors

“…A CRISPR-free, TALE based base editor has been developed for mitochondrial and nuclear genome editing . Epigenome editors have been developed by fusion of chromatin-modifying enzymes to ZFs, TALEs, and dCas9. , …”

“…38 Epigenome editors have been developed by fusion of chromatinmodifying enzymes to ZFs, TALEs, and dCas9. 39,10 ■ MEGANUCLEASES A genome editor was engineered by fusion of a mutant of the homing endonuclease I-SceI that lacks catalytic activity to the catalytic domain of FokI. 40 The mutant I-SceI domain was responsible for recognition of an 18 bp sequence of nucleotides, whereas the FokI nuclease introduced cuts at 2 and 6 nucleotides from the recognition site.…”

“…Most of the increases in the functional efficiency of the current genome editors have occurred by utilization of experimental molecular protein engineering approaches such as directed evolution and selection with random mutagenesis, combinatorial mutagenesis, and phage display; structural knowledge and molecular dynamics simulations have played a significant but relatively minor role. , Molecular methods for overcoming the limitations of the classical genome editors and for enhancement of the precision of genomic and epigenomic editing have been reviewed recently. − Further increases in specificity and efficiency of genome editing may be possible by leveraging the accumulated biophysical knowledge and the recent advances in protein stability, folding, and dynamics. − This review emphasizes the experimental and computational studies that have provided valuable insights regarding the relevant structures of protein–DNA complexes, conformational dynamics involved in the scanning and binding events, DNA unwinding, protein folding, and unfolding. − Computational design and engineering of sequence-specific DNA binding proteins is complicated by the structural plasticity of DNA and the altered conformations of histone-bound DNA in vivo . , Biophysical characterization of the structure and dynamics of the on-target and off-target activities of native and engineered genome editors reveal an intricate interplay of conformational changes involving both the DNA and the protein, which may involve partial protein unfolding and folding and partial unwinding of DNA, which produce a choreographed sequence of events resulting in the scanning of millions of base pairs, selection of target DNA sequence, and enzyme catalyzed site-specific DNA modifications. , Choreography of the “dance” involving the concerted conformational changes in the target DNA and the designed proteins is the key to the rational engineering and design of genome editors with improved efficiency…”

Unfolding the pathogenesis of systemic sclerosis through epigenomics

“…Without causing alterations in the DNA sequence, epigenetics can cause heritable phenotypic changes (6,7), which is vital in the regulation of gene expression and development. Major mechanisms of epigenetic gene regulation, such as chromatin remodeling, histone modification, DNA methylation, transcriptional regulation by non-coding RNA, and gene imprinting, can provide plausible links between environmental factors and disease predisposition and perpetuation.…”