Identifying virulence-critical Legionella pneumophila effectors using CRISPR-dCas9 mediated gene interrogation

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Authors

• B Kim
• MP Machner

Abstract

The bacterium L. pneumophila is the causative agent of a severe pneumonia called Legionnaires’ disease. During infection, L. pneumophila modifies host cell processes by injecting almost 300 effector proteins into the host cytosol. One question that has puzzled the field is which of the 300 effectors contribute to the infection of human cells as opposed to effectors that only play a role in amoeba, the natural host of Legionella? Randomized screening approaches such as transposon or chemical mutagenesis were mostly unsuccessful in addressing this issue due to a high level of functional redundancy among Legionella effectors. We now established a novel genetic tool for the simultaneous deactivation of several effector-encoding genes by adapting the Streptococcus pyogenes CRISPR/dCas9 gene silencing (CRISPRi) technology in L. pneumophila. We showed that CRISPRi can specifically silence individual L. pneumophila genes without off-target effects, validating that the CRISPRi system is an effective gene editing tool in L. pneumophila. We also confirmed simultaneous silencing of two genes or even up to ten effector-encoding genes in L. pneumophila by CRISPRi. To our knowledge, this is the first time that such a large number of genes have been simultaneously silenced in any laboratory organism using the CRISPRi system, and it paves the way for a systematic interrogation of Legionella’s effector-encoding genes.

Scientific Focus Area: Microbiology and Infectious Diseases

This page was last updated on Friday, March 26, 2021