Combination of Cas9 and adeno-associated vectors enables efficient in vivo knockdown of precise miRNAs in the rodent and primate brain #MMPMID41359835
Roura-Martinez D; Popa N; Jaouen F; Rombaut C; Lepolard C; Bachar D; Borges A; Cazorla M; Villet M; Moreno S; Marie H; Gascon E
Proc Natl Acad Sci U S A 2025[Dec]; 122 (50): e2513076122 PMID41359835show ga
microRNAs (miRNAs) are key regulators of multiple biological functions. Although intensively studied, inactivating miRNAs in vivo is particularly challenging, especially in the brain. Here, we designed cell-specific tools aiming at downregulating defined miRNA species in vivo and investigating their function in discrete neuronal networks. Focusing on miR-124, a miRNA highly expressed in the mammalian brain and transcribed from three independent chromosomal loci, we designed and validated different guide RNAs. In vivo, our CRISPR-Cas9 designs strongly downregulate miR-124 levels without affecting the expression of other miRNAs. As a result, levels of endogenous miR-124 targets exhibit a significant increase supporting the release of its silencing activity. We provide evidence that specific deletion of miR-124 in neural stem cells of the subventricular zone altered migration of newly generated neurons into the olfactory bulb. We also showed that our vectors modified the Ca(2+) permeability of AMPA receptors, a robust functional output downstream of miR-124. We also extended our approach to other miRNAs, mammalian species, and Cas9 proteins, confirming the versatility of CRISPR-Cas9. These tool properties support their potential for elucidating miRNA functions in complex experimental in vivo settings such as brain networks.
Proc+Natl+Acad+Sci+U+S+A 2025 ; 122 (50): e2513076122
