Perturb-Seq: Dissecting Molecular Circuits with Scalable Single-Cell RNA Profiling of Pooled Genetic Screens

Area of Science:

• Genomics
• Molecular Biology
• Immunology

Background:

• Assaying complex phenotypes like transcriptional profiles at scale in mammalian cells is challenging.
• Genetic screens are crucial for inferring gene function but limited in scope for complex readouts.

Purpose of the Study:

• To develop a scalable method for assaying complex phenotypes using pooled genetic perturbations.
• To enable high-throughput analysis of gene function and interactions in mammalian cells.

Main Methods:

• Development of Perturb-seq, integrating single-cell RNA sequencing (RNA-seq) with CRISPR-based perturbations.
• Application of Perturb-seq to analyze 200,000 cells, including immune cells and cell lines.
• Focus on transcription factors regulating dendritic cell response to lipopolysaccharide (LPS).

Main Results:

• Perturb-seq accurately identifies gene targets, signatures, and cell states affected by perturbations.
• Revealed genetic interactions between perturbed genes.
• Identified novel functions for regulators of differentiation, antiviral response, and mitochondrial function.

Conclusions:

• Perturb-seq significantly expands the scope of pooled genomic assays.
• The method allows decomposition of high-content measurements into perturbation effects, interactions, and cell metadata.
• Enables deeper understanding of gene function in complex biological processes like immune activation.