A Multiplexed Single-Cell CRISPR Screening Platform Enables Systematic Dissection of the Unfolded Protein Response

Area of Science:

• Genomics
• Molecular Biology
• Cell Biology

Background:

• Functional genomics studies often face a trade-off between the number of perturbations and the complexity of measured phenotypes.
• Existing methods limit the scale and resolution of perturbation analysis.

Purpose of the Study:

• To develop and apply a novel method, Perturb-seq, that bridges the gap between high-throughput perturbation screening and detailed phenotyping.
• To dissect the mammalian unfolded protein response (UPR) using single and combinatorial CRISPR perturbations.

Main Methods:

• Perturb-seq integrates droplet-based single-cell RNA sequencing with a barcoding strategy for CRISPR perturbations, enabling pooled screening.
• Genome-scale CRISPR interference (CRISPRi) screens were performed to identify genes affecting ER homeostasis.
• Single-cell RNA sequencing was applied to profile the effects of numerous perturbations.

Main Results:

• Perturb-seq enabled high-precision functional clustering of genes involved in the UPR.
• Single-cell analysis revealed bifurcated activation of UPR branches within cells experiencing the same perturbation.
• Differential activation of UPR branches was uncovered, including a feedback loop between the translocon and IRE1α.

Conclusions:

• Perturb-seq is a powerful tool for dissecting complex cellular pathways like the UPR at single-cell resolution.
• The study provides insights into how ER homeostasis sensors monitor distinct stresses.
• The findings highlight the utility of Perturb-seq for large-scale functional genomics.