Optical control of mammalian endogenous transcription and epigenetic states

Abstract

The dynamic nature of gene expression enables cellular programming, homeostasis, and environmental adaptation in living systems. Dissection of causal gene functions in cellular and organismal processes therefore necessitates approaches that enable spatially and temporally precise modulation of gene expression. Recently, a variety of microbial and plant-derived light-sensitive proteins have been engineered as optogenetic actuators, enabling high precision spatiotemporal control of many cellular functions1-11. However, versatile and robust technologies that enable optical modulation of transcription in the mammalian endogenous genome remain elusive. Here, we describe the development of Light-Inducible Transcriptional Effectors (LITEs), an optogenetic two-hybrid system integrating the customizable TALE DNA-binding domain12-14 with the light-sensitive cryptochrome 2 protein and its interacting partner CIB1 from Arabidopsis thaliana. LITEs do not require additional exogenous chemical co-factors, are easily customized to target many endogenous genomic loci, and can be activated within minutes with reversibility3,4,6,7,15. LITEs can be packaged into viral vectors and genetically targeted to probe specific cell populations. We have applied this system in primary mouse neurons, as well as in the brain of awake mice in vivo to mediate reversible modulation of mammalian endogenous gene expression as well as targeted epigenetic chromatin modifications. The LITE system establishes a novel mode of optogenetic control of endogenous cellular processes and enables direct testing of the causal roles of genetic and epigenetic regulation in normal biological processes and disease states.

Rational design of a split-Cas9 enzyme complex

“…For example, DNA-binding and effector domains of modified TALEs have been fused to CRY2 and CIB1 to enable light-inducible control of gene expression (15). Alternatively, genes may be split such that a single functional polypeptide is the final product of mRNA transsplicing or intein-based protein splicing (16).…”

Translating Lung Function Genome-Wide Association Study (GWAS) Findings

“…Variable levels of increases in mRNA expression were observed (from little to large) and this was accompanied by an increase in protein level. The construct allows for reversible modulation of gene transcription and epigenetic marks in spatially and temporally sensitive manners via the exposure to light (Konermann et al, 2013). This study is essentially a proof of concept that overexpression using this unique technique may be possible and it was shown to be applicable both in vitro and in vivo.…”

“…A new avenue in genome editing technology has recently emerged with lightinducible transcriptional effectors (LITEs) (Konermann et al, 2013). LITE modules consist of the light-sensitive photoreceptor cryptochrome 2 (CRY2) that is fused to TALEN DNA-binding domain, however, theoretically the concept can be applied to the CRISPR-Cas9 system as well (Figure 16).…”

A solid triple point

“…But can metabolism also steer and control cellular processes? Writing in Cell, De Bock et al 1 suggest that the answer is yes, at least in the context of glucose metabolism and angiogenesis -the formation of new blood vessels*.…”

“…The transitions between competing phases can reveal information about the underlying states that is otherwise difficult to obtain. On page 431 of this issue, Park et al 1 use a micromechanical device and single-crystal nanobeams to determine with high precision the tensile stress-temperature phase diagram of vanadium dioxide (VO 2 ), an archetypal correlated oxide. Their experiment reveals a surprising and interesting fact: the metal-insulator phase transition for which VO 2 is famous is in fact a triple point, a rare circumstance in which three phases (here two insulators and a metal) can coexist.…”