Dissecting Immune Circuits by Linking CRISPR-Pooled Screens with Single-Cell RNA-Seq

Area of Science:

• Immunology
• Genomics
• Cell Biology

Background:

• Multicellular organisms rely on complex regulatory circuits for cell diversity and responses.
• Cellular heterogeneity and circuit crosstalk present significant research challenges.
• Understanding these regulatory networks is crucial for deciphering biological processes.

Purpose of the Study:

• To develop an integrated method for simultaneously profiling genomic perturbations and transcriptomes in single cells.
• To elucidate the function of multiple factors and their interactions within regulatory circuits.
• To investigate the regulatory circuits of innate immunity.

Main Methods:

• CRISP-seq: an integrated method combining massively parallel single-cell RNA sequencing (RNA-seq) and clustered regularly interspaced short palindromic repeats (CRISPR)-pooled screens.
• Application of CRISP-seq to tens of thousands of perturbed cells in vitro and in vivo (mice).
• Analysis of genomic perturbation and transcriptome data from individual cells.

Main Results:

• Identified interactions and redundancies between developmental and signaling-dependent factors in innate immunity.
• Revealed opposing effects of Cebpb and Irf8 in regulating monocyte/macrophage versus dendritic cell lineages.
• Discovered differential functions for Rela and Stat1/2 in monocyte versus dendritic cell responses to pathogens.

Conclusions:

• CRISP-seq is a broadly applicable, comprehensive, and unbiased approach for elucidating mammalian regulatory circuits.
• The method enables simultaneous elucidation of multiple factor functions and interactions.
• CRISP-seq provides novel insights into the complexities of innate immune regulation.