UCSF

Essential genes are critical for cell viability. In bacterial cells these genes are unusually connected across their genetic network, frequently direct targets of antibiotics, and disproportionately expressed to the transcriptome and proteome. Despite their centrality to bacterial biology, essential genes have been poorly studied in vivo for want of the facile and high-throughput methods needed to systematically compromise activity essential genes while still maintaining the population levels necessary for measurements and assays of their functional impact. We applied CRISPR interference (CRISPRi) to create knockdowns of every essential gene in Bacillus subtilis, then assayed those knockdowns using chemical genomics, high-throughput microscopy, and growth experiments, which collectively provided significant insights into the organization and functional relevance of the bacterial essentialome. With this work as a springboard we developed a modified CRISPRi system leveraging the predictable reduction in efficacy of imperfectly matched sgRNAs to generate specific levels of CRISPRi activity and demonstrate its broad applicability in bacteria. Using libraries of such mismatched sgRNAs, we characterized the expression fitness relationships of essential genes in Escherichia coli and Bacillus subtilis. These organisms, though separated by ~2 billion years of evolution, conserve not only the essential genes themselves but in the majority of cases the relationships connecting their individual expression to overall fitness, suggesting that the tradeoffs underlying bacterial homeostasis are deeply fundamental.