The Circadian Clock

Our lives are governed by repeated cycles, including those of day and night, wake and sleep, and breakfast and dinner. In response to external cues (particularly visible light), animals have evolved endogenous timekeeping machinery to optimize survival in a predictable environment. The 'master clock', which sets time for the rest of the cells in the body, is located in a hypothalamic structure called the suprachiasmatic nucleus (SCN; pictured below; credit: JCB). As this nucleus receives direct input from the retina, it can adjust the phase of the clock in accordance with the light/dark cycle. 

Outside of the SCN, the role so called 'local clocks' play in behavior is largely unknown. To investigate this, I use CRISPR/Cas9 and Cre/lox technology to disrupt core clock genes in specific sets of neurons throughout the brain. Uncovering the roles of the circadian clock in discrete sets of neurons will likely provide new targets for drug development for psychiatric and sleep disorders.

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CRISPR/Cas9 is a powerful gene editing platform that uses a single guide RNA (sgRNA) complementary to a target gene of interest. This RNA 'guides' the molecular scissors 'Cas9' to specific sites on the gene, where precise edits can be made, depending on the repair pathway the DNA takes. (image credit: Redman et al., 2016)

For example, to examine the role of local clocks in ventral tegmental area (VTA) dopamine neurons one can use use the dopamine transporter (DAT) to drive Cre expression in tandem with Cas9 in these cells. Injecting mice with multiple sgRNAs targeting the core clock gene 'bmal1', and equipping them with EEG/EMG electrodes, we are able to investigate how midbrain dopaminergic clocks influence natural sleep and contribute to motivated behavior and anxiety. 

Banner image: The ventral tegmental area of a DAT-Cre/Cas9 mouse injected with Cre-dependent sgRNAs targeting BMAL1, visualized by mCherry signal. (credit: JCB)

Relevant Reading

Transcriptional architecture of the mammalian circadian clock - Joseph Takahashi

What is CRISPR/Cas9? - Redman et al., 2016

All rights reserved: Jeremy Borniger, PhD