Viral and Transgenic CRISPR Epigenomic-Editing Tools to Investigate Consequences of Arc Modulation

Abstract

Arc is an immediate early gene that acts as a direct link between neuronal activity and changes in synaptic plasticity. Its complicated regulation paired with its impact on learning and memory make it challenging yet important to investigate. Previous studies have used a variety of knockouts, knockdowns, and manipulations to study its function in the brain. Although the functions of Arc in development are well described, the role of the activity-dependent expression in the adult brain and its relationship to circuit and behavioral plasticity is not well understood. Here, we used CRISPR inhibition of Arc transcription to address this role in the context of different kinds of cellular and behavioral plasticity. Through a variety of CRISPR-dCas9 tools, we provide in vitro and in vivo methods to manipulate Arc in a temporally and spatially controlled manner. These tools allow us to use transcriptional modulators fused to dCas9 to have epigenetic control of key regulatory regions. Arc’s highly inducible expression is thought to be due in part to its single distal enhancer, which binds activity-regulated transcription factors. In this work, we first show how targeting the enhancer and promoter of the Arc gene individually with a transcriptional activator or repressor can be used to dissect the relationship between enhancer RNA, mRNA, and protein expression in cultured neurons. Next, we extensively demonstrate how a dCas9 transgenic mouse line can be used to knock down Arc expression in multiple brain regions in response to different stimuli. Finally, through calcium imaging and behavioral testing, we demonstrate that despite Arc’s purported role as a master regulator, Arc knockdown does not always lead to substantial downstream circuit and behavioral effects. These experiments will provide future studies with precise tools to control Arc expression, but the small effects observed downstream of our manipulation point to the importance of fully elucidating where, when, and how Arc is knocked down to understand what specific role it is playing in the adult brain.