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A Broad-Host-Range CRISPRi Toolkit for Silencing Gene Expression in Burkholderia
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posted on 2019-11-05, 18:05 authored by Andrew
M. Hogan, A. S. M. Zisanur Rahman, Tasia J. Lightly, Silvia T. CardonaGenetic
tools are critical to dissecting the mechanisms governing
cellular processes, from fundamental physiology to pathogenesis. Members
of the genus Burkholderia have potential for biotechnological
applications but can also cause disease in humans with a debilitated
immune system. The lack of suitable genetic tools to edit Burkholderia GC-rich genomes has hampered the exploration
of useful capacities and the understanding of pathogenic features.
To address this, we have developed CRISPR interference (CRISPRi) technology
for gene silencing in Burkholderia, testing it in B. cenocepacia, B. multivorans, and B. thailandensis. Tunable expression
was provided by placing a codon-optimized dcas9 from Streptococcus pyogenes under control of a rhamnose-inducible
promoter. As a proof of concept, the paaABCDE operon
controlling genes necessary for phenylacetic acid degradation was
targeted by plasmid-borne gRNAs, resulting in near complete inhibition
of growth on phenylacetic acid as the sole carbon source. This was
supported by reductions in paaA mRNA expression.
The utility of CRISPRi to probe other functions at the single cell
level was demonstrated by knocking down phbC and fliF, which dramatically reduces polyhydroxybutyrate granule
accumulation and motility, respectively. As a hallmark of the mini-CTX
system is the broad host-range of integration, we putatively identified
67 genera of Proteobacteria that might be amenable to modification
with our CRISPRi toolkit. Our CRISPRi toolkit provides a simple and
rapid way to silence gene expression to produce an observable phenotype.
Linking genes to functions with CRISPRi will facilitate genome editing
with the goal of enhancing biotechnological capabilities while reducing Burkholderia’s pathogenic arsenal.
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Burkholderia GC-rich genomesphenylacetic acid degradationBurkholderia Genetic toolsB . thailandensisB . cenocepaciasilence gene expressionB . multivoransSilencing Gene Expressionpolyhydroxybutyrate granule accumul...CRISPRi toolkitpaaA mRNA expressioncodon-optimized dcas 9Broad-Host-Range CRISPRi Toolkit
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