Pooled single-cell CRISPRa/i screens for functional genomics in bacteria at scale

Abstract

Pooled CRISPR screens using single-cell RNA sequencing (scRNA-seq) have emerged as powerful tools to uncover gene function, map regulatory networks, and identify genetic interactions. However, the inherent sparsity of bacterial scRNA-seq data has posed a major challenge toward applying these approaches to bacteria. Here, we present mapSPLiT, a pooled bacterial CRISPR activation and interference (CRISPRa/i) screening platform that enables large-scale experiments that simultaneously link hundreds of perturbations to their corresponding single-cell transcriptomes. By targeting 52 known or putative transcription factors with 118 perturbations in a pooled experiment, we expanded the E. coli regulatory network map, determined the function of putative regulators, and identified emergent global phenotypes. By targeting combinations of transcription factors simultaneously, we uncovered genetic interactions and regulatory logic between them. Mapping regulatory networks for carbon utilization in P. putida revealed control points that could expand metabolic flexibility and improve biomanufacturing. Together, these results establish mapSPLiT as a generalizable platform for single-cell functional genomics in bacteria.