基于非分裂神经元系统的CRISPR interference作用机制及应用研究

CRISPR/Cas9 can be directed to specific genomic loci by single-guide RNA and introduce double strand breaks within the target site, which is a powerful genome editing tool to generate transgenic cell lines and animal strains. In addition, CRISPRi is also emerging as a powerful system for targeted genome repression as well..Currently, thoughtful insights regarding the mechanism of the Cas9-sgRNA complex have been gained based upon in vitro reconstitution systems or fast-dividing cell lines. However, the Cas9-involved events detected in proliferating cells are so transient that a nuclease-null dCas9 mutant and high-cost sequencing analysis were often used for the study of Cas9-sgRNA-DNA interactions. On the one hand, it has remained largely unknown whether the dCas9 mutant has identical biophysical characteristics to the wild-type Cas9 protein; on the other hand, the cells could proliferate in an imbalanced pattern, which would consequently affect the data interpretation. Hence, a non-dividing cell model such as postmitotic neuron, by which the Cas9-chromatin interacting events can faithfully display and be conveniently monitored, would be ideal for investigation of the in vivo Cas9 mechanism..At present, the guidelines designing optimized sgRNA with maximal activity and minimal off-target effects are yet-to-be established to guarantee the knockout efficiency. However, in diploid cells, incomplete loss of function often compensates the phenotype at the single cell levels. For neurons, it is non-dividing cells and unlikely to screen homozygote cell line to characterize phenotype. Moreover, neuronal diversity requires neuron subtype-specific gene disrupting tools. The application of CRISPR/Cas9 in neurobiological research, especially at single neuron levels, has been limited..In this project, utilizing the postmitotic neuron as a model to faithfully retain the bona fide interaction between Cas9 and dCas9 with chromatin in living cells, we will firstly employ the ChIP assay to quantitatively study the interaction of Cas9 and dCas9 with chromatin. In addition, we will address the effects of sgRNA mutation on Cas9 and dCas9 interrogation. Secondly, using the accurate electrophysiology as an assessment, we will systematically evaluate the efficiency of disrupting gene function by CRISPR/Cas9 based gene knockout and CRISPRi/dCas9 based gene inhibition at single neuron levels. Furthermore, we will develop CRISPR based multiplexed and neuron subtype-specific gene disrupting.toolkits, and apply these technology to validate gene function in animals. Collectively, our study will not only provide with a novel postmitotic neuron model harboring the intact in situ interaction between Cas9 and dCas9 with chromatin for CRISPR mechanism research, but also develop versatile CRISPR toolkits to facilitate neurobiology research.

目前，CRISPR/Cas9基因敲除技术已被广泛应用于细胞系和动物模型的构建。由于染色体复制会破坏Cas9/dCas9与染色质原位相互作用，基于dCas9的研究并不能忠实地预测Cas9在细胞中的行为。非分裂神经元是研究Cas9寻靶机制的理想细胞模型。但是，鉴于神经元的高度异质性和缺乏高效sgRNA的设计原则，CRISPR在神经元中的效率、均一性和普遍适用性仍不理想。CRISPRi 基因敲低技术可能是另一选择。本课题将系统地比较基于CRISPR的基因敲除和敲低技术在神经元水平沉默特定基因的适用性。在此基础上，完善靶向多重基因和针对神经元亚型的条件性基因干扰技术，将其推广到动物在体基因研究，推进CRISPRi在神经生物学研究中的应用。另一方面，本课题将利用非分裂神经元为模型，结合ChIP-qPCR和免疫印迹技术解析Cas9和dCas9在活细胞中寻靶的异同，为CRISPR的机制研究提供新思路。

CRISPR/Cas9基因敲除技术已被广泛应用于细胞系和动物模型的构建。由于染色体复制会破坏Cas9/dCas9与染色质原位相互作用，基于dCas9的研究并不能忠实地预测Cas9在细胞中的行为。非分裂神经元是研究Cas9寻靶机制的理想细胞模型。但是，鉴于神经元的高度异质性和缺乏高效sgRNA的设计原则，CRISPR在神经元中的效率、均一性和普遍适用性仍不理想。CRISPRi 基因敲低技术可能是另一选择。本项目系统地比较基于CRISPR的基因敲除和敲低技术在神经元水平沉默特定基因的适用性。在此基础上，完善靶向多重基因和针对神经元亚型的条件性基因干扰技术，将其应用于解决神经生物学领域长期悬而未决的难题，Synaptotagmin-1和SNARE 蛋白质复合物在突触小泡docking中的作用机制研究。本课题的研究目标已经圆满完成，针对神经系统改良了基于dCas9的CRISPR interference技术，研究成果发表于Nature Neuroscience和Cell Reports杂志。