The study of gene regulation is dominated by a focus on the control of gene activation or increase in the level of expression. Just as critical is the process of gene repression or silencing. Chromatin signatures have identified enhancers, however, genome-wide identification of silencers by computational or experimental approaches are lacking. Here, we first define uncharacterized cis-regulatory elements likely containing silencers and find that 41.5% of ~7500 tested elements show silencer activity using massively parallel reporter assay (MPRA). We trained a support vector machine classifier based on MPRA data to predict candidate silencers in over 100 human and mouse cell or tissue types. The predicted candidate silencers exhibit characteristics expected of silencers. Leveraging promoter-capture HiC data, we find that over 50% of silencers are interacting with gene promoters having very low to no expression. Our results suggest a general strategy for genome-wide identification and characterization of silencer elements.

Genome editing using the CRISPR/Cas9 system has been used to make precise heritable changes in the DNA of organisms. Although the widely used Streptococcus pyogenes Cas9 (SpCas9) and its engineered variants have been efficiently harnessed for numerous gene-editing applications across different platforms, concerns remain regarding their putative off-targeting at multiple loci across the genome. Here we report that Francisella novicida Cas9 (FnCas9) shows a very high specificity of binding to its intended targets and negligible binding to off-target loci. The specificity is determined by its minimal binding affinity with DNA when mismatches to the target single-guide RNA (sgRNA) are present in the sgRNA:DNA heteroduplex. FnCas9 produces staggered cleavage, higher homology-directed repair rates, and very low nonspecific genome editing compared to SpCas9. We demonstrate FnCas9-mediated correction of the sickle cell mutation in patient-derived induced pluripotent stem cells and propose that it can be used for precise therapeutic genome editing for a wide variety of genetic disorders.

Genome compaction is a universal feature of cells and has emerged as a global regulator of gene expression. Compaction is maintained by a multitude of architectural proteins, long non-coding RNAs (lncRNAs), and regulatory DNA. Each component comprises interlinked regulatory circuits that organize the genome in three-dimensional (3D) space to manage gene expression. In this review, we update the current state of 3D genome catalogues and focus on how recent technological advances in 3D genomics are leading to an enhanced understanding of disease mechanisms. We highlight the use of genome-wide chromatin conformation capture (Hi-C) coupled with oligonucleotide capture technology (capture Hi-C) to map interactions between gene promoters and distal regulatory elements such as enhancers that are enriched for disease variants from genome-wide association studies (GWASs). We discuss how aberrations in architectural units are associated with various pathological outcomes, and explore how recent advances in genome and epigenome editing show great promise for a systematic understanding of complex genetic disorders. Our growing understanding of 3D genome architecture-coupled with the ability to engineer changes in it-may create novel therapeutic opportunities.

Ionic liquids (ILs) are salts with poor ionic coordination, resultantly remaining in liquid state below 100 °C and some may retain liquid state even at room temperature. ILs are known to provide a conducive environment for many biological enzymatic reactions, but their interaction with biomacromolecules are poorly understood. In the present study, we investigate the effect of various ionic liquids on DNA-small molecule interaction using calf thymus DNA (ctDNA)-ethidium bromide (EB) as a model system. The effect of various ionic liquids on these interactions is studied by an array of techniques such as circular dichroism (CD), UV melting, fluorescence exclusion and isothermal titration calorimetry. Interestingly, we observed that presence of IL increased the stability of ctDNA without altering its structure. The binding affinities Kbs for EB binding to ctDNA in the presence of 300 mM ILs are about half order of magnitude smaller than the Kbs in absence of ILs and correspond to a less favorable free energy. We noted that, when adjusted to corresponding buffer condition, the unfavorable shift in ΔG of ctDNA-EB interaction is attributed to decreased entropy in the case of ILs, whereas the same effect by NaCl was due to increased enthalpy.