Cas9-mediated in vivo miRNA silencing

Although the advent of Cas9 technology has expanded our ability to precisely edit the genome,manipulating micro RNAs in vivo has been shown to be particularly challenging, especially in the brain.Here, we sought to generate novel tools aiming at targeting and efficiently down regulating defined microRNAsspecies in a cell-specific manner so that their function in discrete neuronal networks could be investigated.Focusing on miR-124, a micro RNA highly expressed in the mammalian brain and transcribed fromthree independent chromosomal loci, we designed and validated different gRNAs directed against this miRNA.In vitro, our Cas9 designs show not only a significant reduction in miR-124 levels but also a functional effecton miR-124 silencing. Similarly, when packed into AAV vectors and injected into the mouse cortex, miR-124-Cas9vectors strongly down regulate miR-124 levels without affecting the expression of other miRNAs. In parallel,levels of endogenous miR-124 targets exhibit a significant increase supporting the release of its silencing activity.To functionally validate our tools, we provide evidences that deletion of miR-124 in the sub ventricular zone alteredmigration of newly generated neurons into the olfactory bulb. Finally, we also showed that our vectors also modifiedthe Ca2+ permeability of AMPA receptors, a robust functional output downstream of miR-124. These tools are expectedto help elucidating miRNA function in complex experimental settings such as brain networks in vivo.