An in vivo analysis of autism risk genes CRISPR targeting in vivo, especially in mammals, can be difficult and time consuming when attempting to determine the effects of a single gene. However, such studies may be required to identify pathological gene variants with effects in specific cells along a developmental trajectory. To study the function of genes implicated in autism spectrum disorders (ASDs), Jin et al. applied a gene-editing and single-cell–sequencing system, Perturb-Seq, to knock out 35 ASD candidate genes in multiple mice embryos (see the Perspective by Treutlein and Camp). This method identified networks of gene expression in neuronal and glial cells that suggest new functions in ASD-related genes. Science, this issue p. eaaz6063; see also p. 1038 An in utero Perturb-Seq genetic screen reveals autism-related gene modules affected during mouse brain development. INTRODUCTION Human genetic studies have revealed long lists of genes and loci associated with risk for many diseases and disorders, but to systematically evaluate their phenotypic effects remains challenging. Without any a priori knowledge, these risk genes could affect any cellular processes in any cell type or tissue, which creates an enormous search space for identifying possible downstream effects. New high-throughput approaches are needed to functionally dissect these large gene sets across a spectrum of cell types in vivo. RATIONALE Analysis of trio-based whole-exome sequencing has implicated a large number of de novo loss-of-function variants that contribute to autism spectrum disorder and developmental delay (ASD/ND) risk. Such de novo variants often have large effect sizes, thus providing a key entry point for mechanistic studies. We have developed in vivo Perturb-Seq to allow simultaneous assessment of the individual phenotypes of a panel of such risk genes in the context of the developing mouse brain. RESULTS Using CRISPR-Cas9, we introduced frameshift mutations in 35 ASD/ND risk genes in pools, within the developing mouse neocortex in utero, followed by single-cell transcriptomic analysis of perturbed cells from the early postnatal brain. We analyzed five broad cell classes—cortical projection neurons, cortical inhibitory neurons, astrocytes, oligodendrocytes, and microglia—and selected cells that had received only single perturbations. Using weighted gene correlation network analysis, we identified 14 covarying gene modules that represent transcriptional programs expressed in different classes of cortical cells. These modules included both those affecting common biological processes across multiple cell subsets and others representing cell type–specific features restricted to certain subsets. We estimated the effect size of each perturbation on each of the 14 gene modules by fitting a joint linear regression model, estimating how module gene expression in cells from each perturbation group deviated from their expression level in internal control cells. Perturbations in nine ASD/ND genes had significant effects across five modules across four cell classes, including cortical projection neurons, cortical inhibitory neurons, astrocytes, and oligodendrocytes. Some of these results were validated by using a single-perturbation model as well as a germline-modified mutant mouse model. To establish whether the perturbation-associated gene modules identified in the mouse cerebral cortex are relevant to human biology and ASD/ND pathology, we performed co-analyses of data from ASD and control human brains and human cerebral organoids. Both gene expression and gene covariation (“modularity”) of several of the gene modules identified in the mouse Perturb-Seq analysis are conserved in human brain tissue. Comparison with single-cell data from ASD patients showed overlap in both affected cell types and transcriptomic phenotypes. CONCLUSION In vivo Perturb-Seq can serve as a scalable tool for systems genetic studies of large gene panels to reveal their cell-intrinsic functions at single-cell resolution in complex tissues. In this work, we demonstrated the application of in vivo Perturb-Seq to ASD/ND risk genes in the developing brain. This method can be applied across diverse diseases and tissues in the intact organism. In vivo Perturb-Seq identified neuron and glia-associated effects by perturbations of risk genes implicated in ASD/ND. De novo risk genes in this study were chosen from Satterstrom et al. (2018), and co-analysis with ASD patient data at bottom right is from Velmeshev et al. (2019); full citations for both are included in the full article online. The number of disease risk genes and loci identified through human genetic studies far outstrips the capacity to systematically study their functions. We applied a scalable genetic screening approach, in vivo Perturb-Seq, to functionally evaluate 35 autism spectrum disorder/neurodevelopmental delay (ASD/ND) de novo loss-of-function risk genes. Using CRISPR-Cas9, we introduced frameshift mutations in these risk genes in pools, within the developing mouse brain in utero, followed by single-cell RNA-sequencing of perturbed cells in the postnatal brain. We identified cell type–specific and evolutionarily conserved gene modules from both neuronal and glial cell classes. Recurrent gene modules and cell types are affected across this cohort of perturbations, representing key cellular effects across sets of ASD/ND risk genes. In vivo Perturb-Seq allows us to investigate how diverse mutations affect cell types and states in the developing organism.

中文翻译：

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体内 Perturb-Seq 揭示与自闭症风险基因相关的神经元和神经胶质异常

在尝试确定单个基因的影响时，对以 CRISPR 为目标的体内自闭症风险基因的体内分析，尤其是在哺乳动物中，可能既困难又耗时。然而，可能需要进行此类研究来确定对特定细胞沿发育轨迹产生影响的病理基因变异。为了研究与自闭症谱系障碍 (ASD) 相关的基因的功能，Jin 等人。应用基因编辑和单细胞测序系统 Perturb-Seq，在多个小鼠胚胎中敲除 35 个 ASD 候选基因（参见 Treutlein 和 Camp 的观点）。这种方法确定了神经元和神经胶质细胞中的基因表达网络，这些网络暗示了 ASD 相关基因的新功能。科学，这个问题 p。eaaz6063; 另见第 1038 子宫内 Perturb-Seq 遗传筛选揭示了在小鼠大脑发育过程中受影响的自闭症相关基因模块。引言 人类遗传研究已经揭示了与许多疾病和病症的风险相关的一长串基因和位点，但系统地评估它们的表型效应仍然具有挑战性。在没有任何先验知识的情况下，这些风险基因可能会影响任何细胞类型或组织中的任何细胞过程，这为识别可能的下游影响创造了巨大的搜索空间。需要新的高通量方法来在体内对一系列细胞类型中的这些大基因集进行功能剖析。基本原理 对基于三重组的全外显子组测序的分析涉及大量导致自闭症谱系障碍和发育迟缓 (ASD/ND) 风险的从头功能丧失变异。这种从头变体通常具有较大的效应量，因此为机理研究提供了一个关键的切入点。我们开发了体内 Perturb-Seq，以允许在发育中的小鼠大脑的背景下同时评估一组此类风险基因的个体表型。结果使用 CRISPR-Cas9，我们在子宫内发育中的小鼠新皮层中引入了 35 个 ASD/ND 风险基因库中的移码突变，然后对出生后早期大脑中的扰动细胞进行了单细胞转录组学分析。我们分析了五种广泛的细胞类别——皮质投射神经元、皮质抑制神经元、星形胶质细胞、少突胶质细胞、和小胶质细胞——以及只接受单一扰动的选定细胞。使用加权基因相关网络分析，我们确定了 14 个共变基因模块，它们代表在不同类别的皮质细胞中表达的转录程序。这些模块包括影响跨多个细胞子集的常见生物过程的模块和其他表示仅限于某些子集的细胞类型特定特征的模块。我们通过拟合联合线性回归模型来估计每个扰动对 14 个基因模块中每一个的影响大小，估计来自每个扰动组的细胞中的模块基因表达如何偏离它们在内部对照细胞中的表达水平。九个 ASD/ND 基因的扰动对四个细胞类别的五个模块产生了显着影响，包括皮质投射神经元、皮质抑制性神经元、星形胶质细胞和少突胶质细胞。其中一些结果通过使用单扰动模型以及种系修饰的突变小鼠模型进行了验证。为了确定在小鼠大脑皮层中鉴定的扰动相关基因模块是否与人类生物学和 ASD/ND 病理学相关，我们对来自 ASD 的数据进行了联合分析，并控制了人类大脑和人类大脑类器官。在小鼠 Perturb-Seq 分析中鉴定的几个基因模块的基因表达和基因协变（“模块化”）在人脑组织中都是保守的。与来自 ASD 患者的单细胞数据的比较显示，受影响的细胞类型和转录组表型均存在重叠。结论 体内 Perturb-Seq 可作为大型基因组系统遗传研究的可扩展工具，以在复杂组织中以单细胞分辨率揭示其细胞内在功能。在这项工作中，我们展示了体内 Perturb-Seq 对发育中大脑中 ASD/ND 风险基因的应用。这种方法可以应用于完整生物体中的各种疾病和组织。In vivo Perturb-Seq 通过扰动 ASD/ND 中涉及的风险基因来识别神经元和神经胶质相关效应。本研究中的从头风险基因选自 Satterstrom 等人。（2018），右下角与 ASD 患者数据的共同分析来自 Velmeshev 等人。(2019); 两者的完整引用都包含在在线全文中。通过人类遗传研究确定的疾病风险基因和位点的数量远远超过系统研究其功能的能力。我们应用了一种可扩展的遗传筛选方法，即体内 Perturb-Seq，对 35 种自闭症谱系障碍/神经发育迟缓 (ASD/ND) 从头功能丧失风险基因进行功能评估。使用 CRISPR-Cas9，我们在子宫内发育中的小鼠大脑内在池中的这些风险基因中引入移码突变，然后对出生后大脑中的受扰细胞进行单细胞 RNA 测序。我们从神经元和神经胶质细胞类别中鉴定了细胞类型特异性和进化上保守的基因模块。循环基因模块和细胞类型在这一系列扰动中受到影响，代表了 ASD/ND 风险基因组的关键细胞效应。