In vivo Perturb-map: scalable genetic screens with single-cell and spatial resolution in intact tissues

人类遗传学研究已经提供了与神经精神疾病风险相关的基因和基因座清单 障碍包括自闭症谱系障碍和神经发育迟缓(ASD/ND)，但系统性 评估它们的功能和机制仍然是一项艰巨的任务。哪些脑区、细胞类型和神经 电路是否参与了病理状态？不同的风险基因和遗传变异是否会影响同一组 大脑区域、细胞类型、神经元投射网络和回路？为了解决这些问题，我们将 开发可扩展的基因组技术，以实现高通量、体内单细胞和 从完整组织中读出的中尺度空间分辨率。首先，我们的目标是建立一种方法，称为扰动映射， 从完整的、完整的大脑中读出每个受扰细胞的空间位置、细胞结构和项目组 不带截面(目标1)。我们将在体内使用CRISPR-Cas9系统执行稀疏微扰 用汇集的基因扰动来收获大脑。接下来，我们将使用基于杂交的检测和整体- 脑组织清除方法，允许反复进行光片成像以推断gRNA扰动 完整的组织中的身份。我们将进一步将荧光记者的成像与内源性 标记物询问扰动如何改变细胞类型的特性、解剖分布、形态和 来自完整组织的神经元投影组。同时，这一方法将与改进的 Vivo扰动--具有前所未有的可扩展性(目标2a-b)。我们将建立一个基于AAV的扰动-SEQ系统 以实现高可伸缩性和灵活的组织特异性。然后我们将用它来研究一组ASD/ND风险基因 横跨两个大脑区域。在胚胎发育过程中会引入汇集的稀疏扰动，单细胞 多组分析和原位扰动图谱分析将揭示每一种扰动如何影响基因表达， 每种特定细胞类型跨脑区的细胞迁移、细胞结构和神经元投射组 包括与这些疾病有关的新皮质和纹状体。这将允许第一次活体筛查 具有高空间分辨率的3维中尺度光学读出。私家侦探有丰富的经验， 她是体内扰动序列技术的先驱，她将不同的团队和专业知识聚集在一起 在组织清除和高通量成像领域的长期关系和合作， 计算基因组学和人类遗传学(包括非高加索人口)，以实现公平的生物医学 (来自Pejman Mohammadi博士、Joshua Levin博士和Mireille Kamariza博士的LOS)总而言之，我们 将建立扰动图谱作为一种可扩展和推广的方法来研究复杂组织中的基因功能。 我们期望确定受不同ASD/ND风险基因影响的细胞类型、分子网络和突起 具有前所未有的规模和分辨率的变种。扰动序列和扰动映射将是模块化和宽泛的 适用的基因组工具不仅有助于神经科学，而且还有助于许多其他组织类型和领域，如我们 从基因变异转移到它们的机制和功能。

Human genetic studies have provided lists of genes and loci associated with risk for neuropsychiatric disorders including autism spectrum disorder and neurodevelopmental delay (ASD/ND), but to systematically evaluate their functions and mechanisms remains a dauting task. What brain regions, cell types, and neural circuits are involved in the pathological state? Do different risk genes and genetic variants affect the same sets of brain regions, cell types, neuronal projection networks and circuits? To address these questions, we will develop scalable genomic technologies to allow high-throughput, in vivo genetic screening with single-cell and meso-scale spatial resolution readout from intact tissues. First, we aim establish a method, named Perturb-map, to readout the spatial location, cytoarchitecture, and projectome of each perturbed cell from intact, whole brains without sectioning (Aim 1). We will perform sparse perturbation using the CRISPR-Cas9 system in vivo and harvest the brain with pooled genetic perturbations. Next, we will use hybridization-based detection and whole- brain tissue clearing methods, to allow iterative rounds of light-sheet imaging to deduce gRNA perturbation identities within an intact tissue. We will further incorporate imaging of fluorescent reporters and endogenous markers to ask how the perturbation changes the cell type identity, anatomical distribution, morphology and neuronal projectome from intact tissues. In parallel, this approach will be complemented with an improved in vivo Perturb-seq with unprecedented scalability (Aim 2a-b). We will establish an AAV-based Perturb-seq system to allow high scalability and flexible tissue-specificity. We will then apply it to study a panel of ASD/ND risk genes across two brain regions. Pooled, sparse perturbation will be introduced during embryogenesis, and single-cell multi-omic analysis and in situ Perturb-map analysis will reveal how each perturbation affect gene expression, cellular migration, cytoarchitecture, and neuronal projectome for each specific cell type across brain regions including neocortex and striatum, which are implicated in the disorders. This will allow the first in vivo screen with 3-dimensional, meso-scale optical readout with high spatial resolution. The PI has extensive experience and has pioneered the in vivo Perturb-seq technology, and she has brought together a diverse team and expertise with long-standing relationships and collaborations across the fields of tissue clearing and high-throughput imaging, computational genomics, and human genetics (including non-Caucasian populations) for equitable biomedicine (co-I Dr. Zhuhao Wu, LoS from Drs. Pejman Mohammadi, Joshua Levin, and Mireille Kamariza). Altogether, we will establish Perturb-Map as a scalable and generalizable approach to study gene function in complex tissues. We expect to identify the cell types, molecular networks, and projectomes affected by diverse ASD/ND risk gene variants with unprecedented scale and resolution. Perturb-Seq and Perturb-Map will be modular and broadly applicable genomic tools contributing to not only neuroscience but also many other tissue types and fields as we move from gene variant to their mechanisms and functions.