Ramani Biswarathan, Rose Indigo V L, Teyssier Noam, Pan Andrew, Danner-Bocks Spencer, Sanghal Tanya, Yadanar Lin, Tian Ruilin, Ma Keran, Palop Jorge J, Kampmann Martin
Institute for Neurodegenerative Diseases; Weill Institute for Neurosciences, University of California, San Francisco, San Francisco, CA, USA.
Department of Pathology, University of California, San Francisco, San Francisco, CA, USA.
Nat Neurosci. 2025 Aug 22. doi: 10.1038/s41593-025-02043-9.
There is a substantial need for scalable CRISPR-based genetic screening methods that can be applied in mammalian tissues in vivo while enabling cell-type-specific analysis. Here 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 mouse tissues. We used this approach to screen two large sgRNA libraries, which collectively target over 5,000 genes, in mouse brains and uncovered genes essential for neuronal survival, of which we validated Rabggta and Hspa5. We highlight the reproducibility and scalability of the platform and show that it is sufficiently sensitive for screening in a restricted subset of neurons. We systematically characterize the impact of sgRNA library size, mouse cohort size, the size of the targeted cell population, viral titer, and coinfection rate on screen performance to establish general guidelines for large-scale in vivo screens.
迫切需要可扩展的基于CRISPR的基因筛选方法，该方法能够在哺乳动物组织中进行体内应用，同时实现细胞类型特异性分析。在此，我们开发了一种基于腺相关病毒（AAV）的CRISPR筛选平台CrAAVe-seq，该平台包含一个对Cre敏感的sgRNA构建体，用于在小鼠组织的靶向细胞群体中进行汇集筛选。我们使用这种方法在小鼠大脑中筛选了两个大型sgRNA文库，这些文库共同靶向超过5000个基因，并发现了神经元存活所必需的基因，其中我们验证了Rabggta和Hspa5。我们强调了该平台的可重复性和可扩展性，并表明它对于在有限的神经元子集中进行筛选具有足够的敏感性。我们系统地表征了sgRNA文库大小、小鼠队列大小、靶向细胞群体大小、病毒滴度和共感染率对筛选性能的影响，以建立大规模体内筛选的一般指导原则。
