Genomic Science Program
U.S. Department of Energy | Office of Science | Biological and Environmental Research Program

2023 Abstracts

Pooled Microbial CRISPR Screens using Single-Cell RNA Sequencing


Jacob R. Brandner1* ([email protected]), Quoc Tran1, Jesse G. Zalatan1, Georg Seelig1, James M. Carothers1, and Anna Kuchina2


1University of Washington; and 2Institute For Systems Biology



The goal is to design genome-wide CRISPRa/i programs for carbon-conserving bioproduction. To achieve this goal, researchers will develop new approaches for high-throughput analysis powered by single-cell RNA sequencing.


CRISPR activation and interference tools have transformed the ability to reprogram microbial hosts for bioproduction. However, building large genetic programs is a time-consuming and iterative process due to the limited understanding of host metabolic and transcriptional regulatory networks. The goal is to develop a custom bacterial single-cell RNA sequencing platform to profile the impact of multi-gene CRISPR gene regulatory programs on thousands of transcriptomes. This approach will provide a high-throughput, low-cost, and information-rich technology to investigate design rules for CRISPR activation and interference (CRISPRa/i), identify heterogeneity in engineered strains, and rapidly assess both intended and unintended transcriptional responses from CRISPR programs. In eukaryotic systems, similar approaches have transformed the ability to interrogate gene function and delineate regulatory networks, but these methods have not yet been implemented in bacteria.

Here, researchers have applied a custom microbial single-cell RNA sequencing platform (microSPLiT) to profile the impact of CRISPRa perturbations on transcriptomic states in engineered E. coli. To validate the platform, researchers targeted genes involved in aromatic amino acid biosynthesis. Single-cell analysis revealed distinct gene expression signatures and variable stress responses for the CRISPRa targets despite belonging to the same metabolic pathway. These results demonstrate that the platform can provide information-rich readouts from CRISPRa programs for high-throughput metabolic engineering in bacteria.

Funding Information

This research was supported by the U.S. Department of Energy Bioenergy Technologies Office (BETO), grant no. DE-EE0008927 and DOE Office of Science, Office of Biological and Environmental Research (BER), grant no. DE-SC0023091.