ENABLING GENETIC ANALYSIS OF DIVERSE BACTERIA WITH MOBILE-CRISPRi

ENABLING GENETIC ANALYSIS OF DIVERSE BACTERIA WITH MOBILE-CRISPRi

Researchers at UCSF, funded in part by the Chan Zuckerberg Biohub, have generated a modular and effective CRISPRi system for the genetic dissection of non-model bacteria.

The vast majority of bacteria, including human pathogens and microbiome species, lack genetic tools needed to associate genes with phenotypes. A method of gene disruption, clustered regularly interspaced short palindromic repeats interference (CRISPRi), enables the robust silencing of target genes by interfering with gene transcription. CRISPRi has demonstrated utility in species ranging from bacteria to humans. However, the difficulty of establishing robust genetic tools, such as CRISPRi, in non-model bacteria has profoundly limited our understanding of the contributions of genes and gene networks to bacterial physiology and pathogenicity.

Stage of Research

The inventors have developed Mobile-CRISPRi, a suite of modular CRISPRi systems for use in non-model bacterial systems. Mobile-CRISPRi can be stably integrated into diverse bacterial species and used in combination with organism-specific genetic libraries and tools. Mobile-CRISPRi modules achieve transfer and genomic integration by distinct mechanisms, while ensuring a specified orientation and without gene disruption. Importantly, genomic integration is stable for more than 50 generation, even in the absence of selection. The inventors demonstrate the efficacy and applicability of Mobile-CRISPRi in drug-gene synergy studies in several pathogenic bacteria.

Applications

Genetic analysis of pathogenic bacteria lacking genetic tools
Analyses of microbiome function, antibiotic resistances and sensitivities of pathogenic bacteria
Comprehensive screens for host-microorganism interactions
Mechanism of action studies in relevant human pathogens

Advantages

A suite of CRISPRi systems that combines modularity, stable genomic integration and ease of transfer to diverse bacteria by conjugation
Inducible and tunable conditional knockdown, allowing for analysis of essential and non-essential genes
Stable genetic integration without antibiotic selection, enabling studies of gene and antibiotic function, in which maintaining selection is problematic or impossible

Stage of Development

Research – in vitro

Publications

Peters JM, Koo BM, Patino R, Heussler GE, Hearne CC, Qu J, Inclan YF, Hawkins JS, Lu CHS, Silvis MR, Harden MM, Osadnik H, Peters JE, Engel JN, Dutton RJ, Grossman AD, Gross CA, Rosenberg OS. Enabling genetic analysis of diverse bacteria with Mobile-CRISPRi. Nature Microbiology 4:244-250 (2019)