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)1 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.
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We thank A. Trevino and C. Le for technical assistance and the entire Zhang lab for technical support and critical discussions; we thank R. Platt (Broad Institute) and H. Worman (Columbia University) for sharing plasmids, R. Rikhye for providing a template for electrophysiology analysis; and X. Yu for statistical discussions. L.S. is a European Molecular Biology Organization (EMBO) Fellow and is supported by the Foundation for Polish Science. M.H. is supported by the Human Frontiers Scientific Program. A.B. holds a postdoctoral fellowship from the Simons Center for the Social Brain. N.H. is an EMBO Fellow and Y.L. is supported by Friends of the McGovern Institute Fellowship. M.S. is supported by grants from the US National Institutes of Health (NIH) (R01EY007023 and R01MH085802) and the Simons Foundation. F.Z. is supported by the National Institute of Mental Health (NIMH) through NIH Director's Pioneer Award (5DP1-MH100706), the NINDS through a NIH Transformative R01 grant (5R01-NS073124), the Keck, Merkin, Vallee, Damon Runyon, Searle Scholars, Klarman Family Foundation, Klingenstein, Poitras and Simons Foundations, and Bob Metcalfe. The authors plan on making the reagents widely available to the academic community through Addgene and to provide software tools via the Zhang lab website (http://www.genome-engineering.org/).
F.Z. is a scientific advisor of Editas Medicine and Horizon Discovery. A patent application has been filed relating to this work.
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Swiech, L., Heidenreich, M., Banerjee, A. et al. In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9. Nat Biotechnol 33, 102–106 (2015). https://doi.org/10.1038/nbt.3055
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