CAGE-Enabled Single-Lineage RNA-Seq for Bacterial PERTURB-Seq and Host-Pathogen Interaction Analysis

About This Grant

Project Summary This R21 pilot grant aims to benchmark single-lineage RNA-Seq (slRNA-Seq), an innovative technology that enables transcriptome analysis of microbial lineages with sensitivity far exceeding current single-microbe methods. Current approaches for bacterial RNA-seq are severely limited by technical challenges including short mRNA half-lives, absence of poly-A tails, and small transcript numbers. We established an approach using “Capsules with Amphiphilic Gel Envelopes” (CAGEs) that allows clonal expansion of individual cells followed by multi-step in-capsule library preparation, dramatically improving transcript detection to thousands of genes per lineage while efficiently capturing CRISPR interference perturbations. Building on this, this pilot grant will evaluate the broad utility of slRNA-Seq through two specific aims: (1) evaluate bacterial PERTURB-Seq by optimizing library preparation protocols and generating a benchmark dataset of all transcription factor perturbations in E. coli; and (2) develop slRNA-Seq as a tool for host- pathogen interaction profiling through co-encapsulation of human immune cells with microbial populations. Our specific focus will be on detecting immune cell transcriptional responses alongside pathogen identity in each CAGE and evaluating that CAGEs are not perturbative to immune cells or their response. We propose multiple strategies to optimize CAGEs and slRNA-Seq if we identify challenges during the pilot. This technology directly addresses NIAID priorities for tools to systematically dissect host-pathogen responses. If successful, this project will establish slRNA-Seq as a robust platform for bacterial functional genomics and microbiome research, providing the scientific community with transformative tools to investigate previously intractable questions about microbial gene function and host-pathogen interactions.