CRISPR screening by AAV episome-sequencing (CrAAVe-seq) is a highly scalable cell type-specific in vivo screening platform.

Biswarathan Ramani, Indigo V L Rose, Noam Teyssier, Andrew Pan, Spencer Danner-Bocks, Tanya Sanghal, Lin Yadanar, Ruilin Tian, Keran Ma, Jorge J Palop, Martin Kampmann
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Abstract

There is a significant need for scalable CRISPR-based genetic screening methods that can be applied directly in mammalian tissues in vivo while enabling cell type-specific analysis. To address this, we developed an adeno-associated virus (AAV)-based CRISPR screening platform, CrAAVe-seq, that incorporates a Cre-sensitive sgRNA construct for pooled screening within targeted cell populations in the mouse tissues. We demonstrate the utility of this approach by screening two distinct large sgRNA libraries, together targeting over 5,000 genes, in mouse brains to create a robust profile of neuron-essential genes. We validate two genes as strongly neuron-essential in both primary mouse neurons and in vivo, confirming the predictive power of our platform. By comparing results from individual mice and across different cell populations, we highlight the reproducibility and scalability of the platform and show that it is highly sensitive even for screening smaller neuronal subpopulations. We systematically characterize the impact of sgRNA library size, mouse cohort size, the size of the targeted cell population, viral titer, and multiplicity of infection on screen performance to establish general guidelines for large-scale in vivo screens.

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在小鼠大脑中进行可扩展的、细胞类型选择性的、基于AAV的体内CRISPR筛选。
由于需要可扩展的细胞类型选择性递送和回收引导RNA文库,直接在体内哺乳动物组织中进行基于CRISPR的基因筛选具有挑战性。我们开发了一种基于腺相关病毒和Cre重组酶依赖的体内工作流程,用于小鼠组织中细胞类型选择性CRISPR干扰筛选。我们通过使用一个靶向2000多个基因的文库来鉴定小鼠大脑中的神经元必需基因,从而证明了这种方法的强大性。
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