High-Throughput Crispri Studies of Gene Function and Regulation in E. coli

The CRISPR-associated protein Cas9 has been adapted as a versatile tool for transcriptional regulation, genome editing, and imaging in a number of organisms. Here, we apply the catalytically inactive dCas9 to conduct high-throughput transcriptional and regulatory studies in E. coli. Using an Agilent OLS library of 32992 unique sgRNAs, we targeted 4500 genes, 5400 promoters, 640 transcription factor binding sites, and 106 sRNAs in the E. coli genome. These genomic targets cover a wide range of functionalities such as metabolism, stress response, transport, and cell division. By leveraging CRISPRi with next-generation sequencing, we were able to interrogate the fitness effect of transcriptional knockdown for each of the aforementioned genomic features in a single-pot experiment both aerobically and anaerobically. Our fitness results agreed well with current knockout databases, and our ability to induce transcriptional knockdown at any point during an experiment has allowed us to explore target essentiality under different conditions (aerobic, anaerobic, etc.) with great ease. We demonstrated this by showing that although nrdA and nrdB are essential under aerobic conditions â?? and are annotated as such in databases â?? they are dispensable anaerobically. Furthermore, our preliminary screens have identified the most effective sgRNAs for targeting E. coli features and have provided useful data on what makes different sites and sgRNA designs most effective. Overall, HT-CRISPRi enables single-pot, precise measurements of fitness for a large set of genomic features and will prove useful in genomic studies of model and non-model organisms.