In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9

Probing gene function in the mammalian brain can be greatly assisted with methods to manipulate the genome of neurons in vivo. The clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated endonuclease (Cas)9 from Streptococcus pyogenes (SpCas9) can be used to edit single or multiple genes in replicating eukaryotic cells, resulting in frame-shifting insertion/deletion (indel) mutations and subsequent protein depletion. Here, we delivered SpCas9 and guide RNAs using adeno-associated viral (AAV) vectors to target single (Mecp2) as well as multiple genes (Dnmt1, Dnmt3a and Dnmt3b) in the adult mouse brain in vivo. We characterized the effects of genome modifications in postmitotic neurons using biochemical, genetic, electrophysiological and behavioral readouts. Our results demonstrate that AAV-mediated SpCas9 genome editing can enable reverse genetic studies of gene function in the brain.Foundation for Polish ScienceHuman Frontier Science Program (Strasbourg, France)Massachusetts Institute of Technology. Simons Center for the Social BrainEuropean Molecular Biology Organization (Fellowship)McGovern Institute for Brain Research at MIT (Friends of McGovern Institute Fellowship)National Institutes of Health (U.S.) (Grant R01EY007023)National Institutes of Health (U.S.) (Grant R01MH085802)Simons FoundationNational Institute of Mental Health (U.S.) (NIH Director's Pioneer Award 5DP1-MH100706)National Institute of Neurological Disorders and Stroke (U.S.) (NIH Transformative R01 Grant 5R01-NS073124)W. M. Keck FoundationMerkin FoundationVallee FoundationDamon Runyon Cancer Research FoundationKinship Foundation. Searle Scholars ProgramKlarman Family FoundationKlingenstein FoundationPoitras Foundatio