利用CRISPR-Cas9对哺乳动物大脑中的基因功能进行体内研究。
In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9.
作者信息
Swiech Lukasz, Heidenreich Matthias, Banerjee Abhishek, Habib Naomi, Li Yinqing, Trombetta John, Sur Mriganka, Zhang Feng
机构信息
1] Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA. [2] McGovern Institute for Brain Research, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA. [3] McGovern Institute for Brain Research, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
Picower Institute for Learning and Memory, Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
出版信息
Nat Biotechnol. 2015 Jan;33(1):102-6. doi: 10.1038/nbt.3055. Epub 2014 Oct 19.
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.
体内操纵神经元基因组的方法可极大地辅助探究哺乳动物大脑中的基因功能。来自化脓性链球菌的成簇规律间隔短回文重复序列（CRISPR）相关核酸内切酶（Cas）9（SpCas9）可用于编辑复制中的真核细胞中的单个或多个基因，导致移码插入/缺失（indel）突变及随后的蛋白质缺失。在此，我们使用腺相关病毒（AAV）载体递送SpCas9和引导RNA，在成年小鼠大脑中体内靶向单个基因（Mecp2）以及多个基因（Dnmt1、Dnmt3a和Dnmt3b）。我们使用生化、遗传、电生理和行为读数来表征有丝分裂后神经元中基因组修饰的影响。我们的结果表明，AAV介导的SpCas9基因组编辑能够实现对大脑中基因功能的反向遗传学研究。
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