Combination of Cas9 and adeno-associated vectors (AAVs) enables efficient in vivo knockdown of precise miRNAs in the rodent brain
Although the advent of Cas9 technology has expanded our ability to precisely edit the genome, manipulating microRNAs 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 downregulating defined microRNAs species in a cell-specific manner so that their function in discrete neuronal networks could be investigated. Focusing on miR-124, a microRNA highly expressed in the mammalian brain and transcribed from three 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 effect on miR-124 silencing. Similarly, when packed into AAV vectors and injected into the mouse cortex, miR-124-Cas9 vectors strongly downregulate 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 subventricular zone altered migration of newly generated neurons into the olfactory bulb. Finally, we also showed that our vectors altered the expression of Gria 2, a canonical target of miR-124, and modified the Ca2+ permeability of AMPA receptors. These tools are expected to help elucidating miRNA function in complex experimental settings such as brain networks in vivo. Overall design: A set of seven animals received an injection of AAV-Cas9-miR-124-g4 and AAV-Cas9-miR-124-empty in each motor cortex. RNA sequencing was preformed on FANS sorted Cas9+ and Cas9- nuclei.