Scientific Core: Perturb-seq library generation, sequencing, and data analysis

Stanford University, Stanford CA

Investigators

Abstract

PROJECT SUMMARY: Perturb-seq Core Perturb-seq is a powerful tool to map gene programs in health and disease. Perturb-seq begins with a high- throughput assay to perturb 10-10,000 genes with CRISPRi and read out their effects on cell state using single- cell RNA-seq. Computational analysis of Perturb-seq data enables the definition of gene programs — sets of genes that work together in a biological pathway. The overarching goal of this Scientific Core is to apply Perturb- seq to map gene programs in a consistent, coordinated fashion across all 3 Projects. This will enable the team to investigate the shared Program hypothesis that gene programs in endothelial cells and smooth muscle cells have context-specific activities that vary across disease states, tissue beds, and biological sex to mediate genetic risk for different vascular diseases. The Perturb-seq Core, directed by Assistant Professor Jesse Engreitz, will build on recent work and new innovations to design, collect, sequence, and analyze Perturb-seq datasets. In a recent study in collaboration with Project 1 Lead Dr. Rajat Gupta, we demonstrated the power of Perturb-seq to map gene programs and identify causal pathways for vascular disease (Schnitzler*, Kang* et al. Nature 2024). To expand this approach to map gene programs in vascular cells across contexts, our Perturb-seq Core team has collaborated with 14 other trainees and staff scientists in the Gupta, Quertermous, Rabinovitch, and Red-Horse laboratories. Together, we have conducted successful pilots in endothelial cells and smooth muscle cells in vitro, and endothelial cells in the aorta, lung, and brain in vivo. We have also optimized Perturb-seq for scalability, robustness, cost, and efficacy, and implemented the method as a core workflow in the lab. With innovative Perturb-seq technologies and analysis pipelines and validated cellular and animal models, we are now ready to conduct systematic studies of gene programs in vascular cells. The activities of the Perturb-seq Core are to: (i) Consult on experimental design and power for Perturb-seq; (ii) Design and clone gRNA libraries; (iii) Prepare and sequence Perturb-seq libraries; (iv) Conduct computational analyses to infer gene programs; (v) Manage samples and data; and (vi) Develop and share optimized methods. The work of this Scientific Core will be deeply intertwined with the 3 Projects and the Mouse Core, and each of these 6 activities will be extensively utilized by each of the 3 Projects. These activities do not duplicate existing institutional resources at Stanford or Havard/BWH. These activities will ultimately reveal causal genes for vascular diseases and advance the development of precise therapies.