Biotechnology development

Functional genomics by pooled CRISPRa/i screens in bacteria

Single-cell CRISPR screens are powerful tools for characterization of gene function and mapping of regulatory networks. By pairing each CRISPR perturbation to a transcriptomic readout, thousands of responses to individual perturbations can be measured in a single pooled experiment. In a collaboration with Carothers, Zalatan, and Seelig groups at University of Washington, we developed a novel method, mapSPLiT (microbial analysis of perturbations using split-pool ligation transcriptomics), that makes it possible to simultaneously profile single-cell transcriptomes and link them to perturbations in bacteria at scale.

We applied mapSPLiT to profile over a hundred transcription factor perturbations across 76,000 E. coli cells in a single experiment, allowing us to map gene regulatory networks for known and putative transcription factors.  We also mapped transcriptome-wide genetic interactions in E. coli bytargeting two or three genes simultaneously with a multi-guide CRISPR cassette. Finally, we applied mapSPLiT technology to the non-model microbe Pseudomonas putida. Overall, mapSPLiT is a technological breakthrough that enables high-content CRISPR screens for mapping of transcriptional networks in multiple microbial species. Paired with computational models of bacterial metabolism or cell regulation, mapSPLiT will promote faster and more accurate engineering of microbes for bioproduction or medicine.

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

Led by: Jacob Brandner, UW