A multiplex CRISPR interference tool for virulence gene interrogation in Legionella pneumophila

Nicole A Ellis,Byoungkwan Kim,Matthias P Machner,Jessica Tung

doi:10.1038/s42003-021-01672-7

Abstract

Catalytically inactive dCas9 imposes transcriptional gene repression by sterically precluding RNA polymerase activity at a given gene to which it was directed by CRISPR (cr)RNAs. This gene silencing technology, known as CRISPR interference (CRISPRi), has been employed in various bacterial species to interrogate genes, mostly individually or in pairs. Here, we developed a multiplex CRISPRi platform in the pathogen Legionella pneumophila capable of silencing up to ten genes simultaneously. Constraints on precursor-crRNA expression were overcome by combining a strong promoter with a boxA element upstream of a CRISPR array. Using crRNAs directed against virulence protein-encoding genes, we demonstrated that CRISPRi is fully functional not only during growth in axenic media, but also during macrophage infection, and that gene depletion by CRISPRi recapitulated the growth defect of deletion strains. By altering the position of crRNA-encoding spacers within the CRISPR array, our platform achieved the gradual depletion of targets that was mirrored by the severity in phenotypes. Multiplex CRISPRi thus holds great promise for probing large sets of genes in bulk in order to decipher virulence strategies of L. pneumophila and other bacterial pathogens.

Highlights

Inactive dCas[9] imposes transcriptional gene repression by sterically precluding RNA polymerase activity at a given gene to which it was directed by CRISPRRNAs
We established an adaptable multiplex Clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi) platform capable of silencing up to ten genes simultaneously (10-plex). We demonstrate that this platform can be applied to study bacterial virulence genes in axenic media but, importantly, under disease-relevant conditions such as the infection of macrophages by the intracellular pathogen Legionella pneumophila, adding CRISPRi to our toolbox for efficiently studying genetically less tractable human pathogens
Using immunoblot analyses and quantitative polymerase chain reaction, we found that, while the intended target genes’ protein and mRNA levels were dramatically reduced by CRISPRi, none of the other surveyed genes chosen at random were repressed by crRNA expression

Summary

Introduction

Inactive dCas[9] imposes transcriptional gene repression by sterically precluding RNA polymerase activity at a given gene to which it was directed by CRISPR (cr)RNAs. In the simplest CRISPR-Cas system, Type II, only a single protein known as Cas[9] is required for crRNA-guided DNA cleavage, lending it to be the most developed as a genetic tool[11,12,13] In bacteria, this system has been adapted for gene silencing by making two simple changes: First, the gene encoding Cas[9] was replaced with a catalytically inactive variant of Cas[9], called deactivated Cas[9] (dCas9), in which the two nuclease domains, RuvClike and HNH, have been mutated, preventing DNA cleavage[14,15]; and second, by designing arrays in which the spacer sequence(s), that are typically directed against invading DNA elements, have been replaced with sequences complementary to the bacterium’s own genes. The Streptococcus pyogenes dCas9-encoding sequence was inserted into the chromosomal thyA locus (lpg2868) as this L. pneumophila gene was already disrupted by a mutation in this strain background[27], creating

Methods

Results

Conclusion