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.
Widespread use of Stylosanthes scabra cv. Seca to improve native pastures in northern Australia makes it necessary to monitor changes in the anthracnose pathogen, Colletotrichum gloeosporioides, because new damaging races have arisen to devastate many agronomically promising cultivars in the past. A total of 103 isolates collected during the past 20 years were analyzed by using virulence and molecular markers to determine whether aggressive strains have evolved in the field. Data on severity for eight host differentials were obtained from a greenhouse assay and analyzed by linear discriminant functions developed from existing data on 182 isolates of known races. A molecular analysis of a subset of 21 isolates by random amplified polymorphic DNA (RAPD) and electrophoretic karyotyping gave comparable results for both markers, although electrophoretic karyotyping detected a higher level of polymorphism. The majority of the 103 isolates were placed into three groups on the basis of RAPD analysis. Virulence analysis detected isolates that were highly aggressive toward Seca. During the past 20 years, the frequency of aggressive isolates has increased. Many of the aggressive isolates were collected during the 1990s, and one had a distinct RAPD genotype. The lack of a strong association between RAPD genotypes and pathogen races suggests that races have arisen independently within each genetic group. This work highlights the need for regular monitoring of the pathogen population.
In the absence of target cleavage, 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, referred to 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 by Rho-dependent transcription termination were overcome by combining a strong processive promoter with a boxA element upstream of a repeat/spacer array.
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