State Key Laboratory of Membrane Biology, Tsinghua-Peking Joint Center for Life Sciences, IDG/McGovern Institute for Brain Research, School of Life Sciences, Tsinghua University, Beijing, China.
Key Laboratory of Genomics and Precision Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China.
Nat Neurosci. 2018 Mar;21(3):447-454. doi: 10.1038/s41593-018-0077-5. Epub 2018 Feb 5.
CRISPR-Cas9 has been demonstrated to delete genes in postmitotic neurons. Compared to the establishment of proliferative cell lines or animal strains, it is more challenging to acquire a highly homogeneous consequence of gene editing in a stable neural network. Here we show that dCas9-based CRISPR interference (CRISPRi) can efficiently silence genes in neurons. Using a pseudotarget fishing strategy, we demonstrate that CRISPRi shows superior targeting specificity without detectable off-target activity. Furthermore, CRISPRi can achieve multiplex inactivation of genes fundamental for neurotransmitter release with high efficiency. By developing conditional CRISPRi tools targeting synaptotagmin I (Syt1), we modified the excitatory to inhibitory balance in the dentate gyrus of the mouse hippocampus and found that the dentate gyrus has distinct regulatory roles in learning and affective processes in mice. We therefore recommend CRISPRi as a useful tool for more rapid investigation of gene function in the mammalian brain.
CRISPR-Cas9 已被证明可删除有丝分裂后神经元中的基因。与建立增殖细胞系或动物品系相比，在稳定的神经网络中获得基因编辑的高度均一后果更具挑战性。在这里，我们展示了基于 dCas9 的 CRISPR 干扰（CRISPRi）可有效沉默神经元中的基因。我们使用伪靶标捕捞策略证明，CRISPRi 具有优越的靶向特异性，而无明显的脱靶活性。此外，CRISPRi 可以高效地实现对神经递质释放至关重要的基因的多重失活。通过开发针对突触结合蛋白 I（Syt1）的条件性 CRISPRi 工具，我们改变了小鼠海马齿状回中的兴奋性到抑制性平衡，发现齿状回在小鼠的学习和情感过程中具有独特的调节作用。因此，我们推荐 CRISPRi 作为一种有用的工具，可更快速地研究哺乳动物大脑中的基因功能。
