CRISPR interference-based specific and efficient gene inactivation in the brain.
CRISPR interference-based specific and efficient gene inactivation in the brain.
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基于 CRISPR 干扰的大脑中特定且高效的基因失活。
DOI：
10.1038/s41593-018-0077-5
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发表时间：
2018
期刊：
影响因子：
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通讯作者：
Yao Jun
中科院分区：
文献类型：
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作者：
Zheng Yi;Shen Wei;Zhang Jian;Yang Bo;Liu Yao-Nan;Qi Huihui;Yu Xia;Lu Si-Yao;Chen Yun;Xu Yu-Zhou;Li Yun;Gage Fred H;Mi Shuangli;Yao Jun
CRISPR-Cas9已被证明可以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高度同质性的基因编辑结果更具挑战性。在这里，我们证明了基于dCas9的CRISPR干扰(CRISPRi)可以有效地沉默神经元中的基因。使用假目标钓鱼策略，我们证明了CRISPRi在没有检测到目标外活动的情况下表现出更好的目标特异性。此外，CRISPRi可以高效地实现神经递质释放基础基因的多重失活。通过开发针对突触素I(Syt1)的条件性CRISPRi工具，我们将小鼠海马齿状回的兴奋性平衡修改为抑制性平衡，发现齿状回在小鼠的学习和情感过程中具有明显的调节作用。因此，我们推荐CRISPRi作为一种有用的工具来更快地研究哺乳动物大脑中的基因功能。
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.