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

体内扰动图：在完整组织中具有单细胞和空间分辨率的可扩展遗传筛选

• 批准号: 10578616
• 负责人: Xin Jin
• 金额: $ 77.33万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-02 至 2027-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10578616
• 关键词: 3-Dimensional; Address; Affect; Anatomy; Artificial nanoparticles; Biological; Biological Process; Biology; Brain; Brain region; CRISPR/Cas technology; Cells; Cellular Morphology; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Complement; Complex; Corpus striatum structure; Data; Detection; Development; Disease; Elements; Embryonic Development; Equity; Gene Delivery; Gene Expression; Genes; Genetic; Genetic Risk; Genetic Screening; Genetic screening method; Genetic study; Genome; Genomics; Goals; Guide RNA; Harvest; Human Genetics; Image; In Situ; Investigation; Light; Location; Maps; Methodology; Methods; Molecular; Morphology; Names; Nature; Neocortex; Neurons; Neurosciences; Optics; Pathologic; Phenotype; Population; Reaction; Reporter; Resolution; Risk; Science; Screening procedure; Series; Serotyping; Spatial Distribution; Specificity; System; Technology; Thick; Tissues; Transgenes; Transposase; Work; autism spectrum disorder; brain research; brain tissue; cell motility; cell type; delivery vehicle; experience; flexibility; fluorescence imaging; functional genomics; gene function; genetic risk factor; genetic variant; genomic tools; human disease; improved; in vivo; interdisciplinary approach; multiple omics; neocortical; neural circuit; neuropsychiatric disorder; novel; risk variant; screening; tool; transgene delivery

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.

人类遗传学研究提供了与神经精神疾病风险相关的基因和基因座列表 包括自闭症谱系障碍和神经发育迟缓 (ASD/ND) 在内的疾病，但要系统地 评估其功能和机制仍然是一项艰巨的任务。大脑区域、细胞类型和神经元有哪些 电路是否参与病理状态？不同的风险基因和遗传变异会影响同一组吗 大脑区域、细胞类型、神经元投射网络和电路？为了解决这些问题，我们将 开发可扩展的基因组技术，以实现单细胞和高通量体内遗传筛选 从完整组织中读取中尺度空间分辨率。首先，我们的目标是建立一种方法，名为 Perturb-map， 从完整的全脑中读出每个受干扰细胞的空间位置、细胞结构和投影组 无需切片（目标 1）。我们将使用 CRISPR-Cas9 系统在体内进行稀疏扰动， 通过汇集遗传扰动收获大脑。接下来，我们将使用基于杂交的检测和整体检测 脑组织透明化方法，允许迭代轮次光片成像来推断 gRNA 扰动 完整组织内的身份。我们将进一步整合荧光报告基因和内源性的成像 标记物询问扰动如何改变细胞类型身份、解剖分布、形态和 来自完整组织的神经元投射组。与此同时，这种方法将得到改进的补充 Vivo Perturb-seq 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Perturb-seq系统 以实现高可扩展性和灵活的组织特异性。然后我们将应用它来研究一组 ASD/ND 风险基因 跨越两个大脑区域。在胚胎发生过程中将引入汇集的、稀疏的扰动，并且单细胞 多组学分析和原位扰动图分析将揭示每个扰动如何影响基因表达， 跨大脑区域的每种特定细胞类型的细胞迁移、细胞结构和神经元投射组 包括与疾病有关的新皮质和纹状体。这将使第一个体内屏幕 具有高空间分辨率的 3 维介观尺度光学读数。 PI拥有丰富的经验并拥有 开创了体内 Perturb-seq 技术，她汇集了多元化的团队和专业知识 组织透明化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（包括非白人群体）以实现公平的生物医学 （联合作者 Zhuhao Wu 博士，Pejman Mohammadi 博士、Joshua Levin 博士和 Mireille Kamariza 博士的 LoS）。总而言之，我们 将建立 Perturb-Map 作为一种可扩展和通用的方法来研究复杂组织中的基因功能。 我们期望识别受不同 ASD/ND 风险基因影响的细胞类型、分子网络和投影组 具有前所未有的规模和分辨率的变体。 Perturb-Seq 和 Perturb-Map 将是模块化且广泛的 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，因为我们 从基因变异转向其机制和功能。