CRISPR technologies for precise epigenome editing

Over 100 types of chemical modifications have been identified in cellular RNAs. While the 5' cap modification and the poly(A) tail of eukaryotic mRNA play key roles in regulation, internal modifications are gaining attention for their roles in mRNA metabolism. The most abundant internal mRNA modification is N6-methyladenosine (m6A), and identification of proteins that install, recognize, and remove this and other marks have revealed roles for mRNA modification in nearly every aspect of the mRNA life cycle, as well as in various cellular, developmental, and disease processes. Abundant noncoding RNAs such as tRNAs, rRNAs, and spliceosomal RNAs are also heavily modified and depend on the modifications for their biogenesis and function. Our understanding of the biological contributions of these different chemical modifications is beginning to take shape, but it's clear that in both coding and noncoding RNAs, dynamic modifications represent a new layer of control of genetic information.

Chromatin is not an inert structure, but rather an instructive DNA scaffold that can respond to external cues to regulate the many uses of DNA. A principle component of chromatin that plays a key role in this regulation is the modification of histones. There is an ever-growing list of these modifications and the complexity of their action is only just beginning to be understood. However, it is clear that histone modifications play fundamental roles in most biological processes that are involved in the manipulation and expression of DNA. Here, we describe the known histone modifications, define where they are found genomically and discuss some of their functional consequences, concentrating mostly on transcription where the majority of characterisation has taken place.

Targeted gene regulation on a genome-wide scale is a powerful strategy for interrogating, perturbing, and engineering cellular systems. Here, we develop a method for controlling gene expression based on Cas9, an RNA-guided DNA endonuclease from a type II CRISPR system. We show that a catalytically dead Cas9 lacking endonuclease activity, when coexpressed with a guide RNA, generates a DNA recognition complex that can specifically interfere with transcriptional elongation, RNA polymerase binding, or transcription factor binding. This system, which we call CRISPR interference (CRISPRi), can efficiently repress expression of targeted genes in Escherichia coli, with no detectable off-target effects. CRISPRi can be used to repress multiple target genes simultaneously, and its effects are reversible. We also show evidence that the system can be adapted for gene repression in mammalian cells. This RNA-guided DNA recognition platform provides a simple approach for selectively perturbing gene expression on a genome-wide scale. Copyright © 2013 Elsevier Inc. All rights reserved.