Modulated efficacy CRISPRi reveals evolutionary conservation of essential gene expression-fitness relationships in bacteria

Js, Hawkins; Mr, Silvis; B, Koo; Jm, Peters; Jost, Marco; Cc, Hearne; Weissman, Jonathan S.; Todor, Horia; Ca, Gross

doi:10.1101/805333

Cited by 7 publications

(11 citation statements)

References 40 publications

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“…Large data sets have been produced to understand the effect of mismatches between the guide and the target (63)(64)(65)(66)(67). A particularly creative strategy was to tether many targets to a sequencing flow cell, locate the different targets by next-generation sequencing, then measure the binding of a fluorescent-labeled CRISPR effectors directly on the same flow cell (68,69).…”

Section: Taming Crispr Guides With Imperfect Complementaritymentioning

confidence: 99%

“…The tx/tl in vitro translation system was also used to measure DNA cleavage and repression for many targets (70). Finally, recent studies employed libraries of CRISPR guides combined with fluorescent-activated cell sorting (FACS) to measure how the target sequence and the presence of mismatches affect the repression strength (65,67).…”

Section: Taming Crispr Guides With Imperfect Complementaritymentioning

confidence: 99%

“…The applications of these data include discovery of drug targets, searching for synthetic lethal pairs, or genome minimization. Partial repression can also be useful to find phenotypes for essential genes (43,67). CRISPR screens are also more versatile than TnSeq, as one can target only a subset of genes of interest, for example by targeting only genes implicated in a function of interest.…”

Section: Genome-wide Crispr Screens In Bacteriamentioning

confidence: 99%

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CRISPR Tools To Control Gene Expression in Bacteria

Vigouroux

Bikard

2020

Microbiol Mol Biol Rev

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SUMMARY CRISPR-Cas systems have been engineered as powerful tools to control gene expression in bacteria. The most common strategy relies on the use of Cas effectors modified to bind target DNA without introducing DNA breaks. These effectors can either block the RNA polymerase or recruit it through activation domains. Here, we discuss the mechanistic details of how Cas effectors can modulate gene expression by blocking transcription initiation or acting as transcription roadblocks. CRISPR-Cas tools can be further engineered to obtain fine-tuned control of gene expression or target multiple genes simultaneously. Several caveats in using these tools have also been revealed, including off-target effects and toxicity, making it important to understand the design rules of engineered CRISPR-Cas effectors in bacteria. Alternatively, some types of CRISPR-Cas systems target RNA and could be used to block gene expression at the posttranscriptional level. Finally, we review applications of these tools in high-throughput screens and the progress and challenges in introducing CRISPR knockdown to other species, including nonmodel bacteria with industrial or clinical relevance. A deep understanding of how CRISPR-Cas systems can be harnessed to control gene expression in bacteria and build powerful tools will certainly open novel research directions.

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Section: Taming Crispr Guides With Imperfect Complementaritymentioning

confidence: 99%

Section: Taming Crispr Guides With Imperfect Complementaritymentioning

confidence: 99%

Section: Genome-wide Crispr Screens In Bacteriamentioning

confidence: 99%

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CRISPR Tools To Control Gene Expression in Bacteria

Vigouroux

Bikard

2020

Microbiol Mol Biol Rev

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“…Predicting the shape of expression-fitness landscapes requires quantitative characterization of the cellular state at multiple levels. Although numerous expression-fitness landscapes have been previously mapped (Chou et al, 2014; Dekel and Alon, 2005; Duveau et al, 2017; Hawkins et al, 2019; Jost et al, 2020; Kavčič et al, 2019; Keren et al, 2016; Knöppel et al, 2016; Li et al, 2014; Palmer et al, 2018; Parker et al, 2020; Schober et al, 2019; Tubulekas and Hughes, 1993), these measurements rarely include concomitant assessment of the internal cell state following perturbations (but see (Jost et al, 2020; Parker et al, 2020)). With limited information bridging the molecular to cellular scales, the root causes of observed fitness defects are challenging to pinpoint (Fig.…”

Section: Introductionmentioning

confidence: 99%

Spurious regulatory connections dictate the expression-fitness landscape of translation termination factors

Lalanne

Parker

2020

Preprint

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During steady-state cell growth, individual enzymatic fluxes can be directly inferred from growth rate by mass conservation, but the inverse problem remains unsolved. Perturbing the flux and expression of a single enzyme could have pleiotropic effects that may or may not dominate the impact on cell fitness. Here we quantitatively dissect the molecular and global responses to varied expression of translation termination factors (peptide release factors, RFs) in bacterium Bacillus subtilis. While endogenous RF expression maximizes proliferation, deviations in expression lead to unexpected distal regulatory responses that dictate fitness reduction. Molecularly, RF depletion causes expression imbalance at specific operons, which activates master regulators and detrimentally overrides the transcriptome. Through these spurious connections, RF abundances are thus entrenched by focal points within the regulatory network, in one case located at a single stop codon. Such regulatory entrenchment suggests that predictive bottom-up models of expression-fitness landscapes will require near-exhaustive characterization of parts.HighlightsPrecision measurements enable multiscale expression-to-fitness mapping.RF depletion leads to imbalanced translation for co-transcribed gene pairs.Imbalanced translation induces unintended regulons to the detriment of cell fitness.Swapping a single stop codon rewires global susceptibility to RF perturbation.

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“…fitness and gene expression varies by gene and is generally unknown(49,50). This relationship is especially important to consider for essential genes, which have a fitness of zeroat full knockdown but a large range of possible fitness values at intermediate levels of knockdown depending on the function of the gene product.…”

mentioning

confidence: 99%

A High-efficacy CRISPRi System for Gene Function Discovery inZymomonas mobilis

Banta

Enright

Siletti³

et al. 2020

Preprint

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ABSTRACTZymomonas mobilis is a promising biofuel producer due to its high alcohol tolerance and streamlined metabolism that efficiently converts sugar to ethanol. Z. mobilis genes are poorly characterized relative to model bacteria, hampering our ability to rationally engineer the genome with pathways capable of converting sugars from plant hydrolysates into valuable biofuels and bioproducts. Many of the unique properties that make Z. mobilis an attractive biofuel producer are controlled by essential genes; however, these genes cannot be manipulated using traditional genetic approaches (e.g., deletion or transposon insertion) because they are required for viability. CRISPR interference (CRISPRi) is a programmable gene knockdown system that can precisely control the timing and extent of gene repression, thus enabling targeting of essential genes. Here, we establish a stable, high-efficacy CRISPRi system in Z. mobilis that is capable of perturbing all genes—including essentials. We show that Z. mobilis CRISPRi causes either strong knockdowns (>100-fold) using single guide RNA (sgRNA) spacers that perfectly match target genes, or partial knockdowns using spacers with mismatches. We demonstrate the efficacy of Z. mobilis CRISPRi by targeting essential genes that are universally conserved in bacteria, key to the efficient metabolism of Z. mobilis, or underlie alcohol tolerance. Our Z. mobilis CRISPRi system will enable comprehensive gene function discovery, opening a path to rational design of biofuel production strains with improved yields.IMPORTANCEBiofuels produced by microbial fermentation of plant feedstocks provide renewable and sustainable energy sources that have the potential to mitigate climate change and improve energy security. Engineered strains of the bacterium Z. mobilis can convert sugars extracted from plant feedstocks into next generation biofuels such as isobutanol; however, conversion by these strains remains inefficient due to key gaps in our knowledge about genes involved in metabolism and stress responses such as alcohol tolerance. Here, we develop CRISPRi as a tool to characterize gene function in Z. mobilis. We identify genes that are essential for growth, required to ferment sugar to ethanol, and involved in resistance to alcohol. Our Z. mobilis CRISPRi system makes it straightforward to define gene function and can be applied to improve strain engineering and increase biofuel yields.

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Modulated efficacy CRISPRi reveals evolutionary conservation of essential gene expression-fitness relationships in bacteria

Cited by 7 publications

References 40 publications

CRISPR Tools To Control Gene Expression in Bacteria

CRISPR Tools To Control Gene Expression in Bacteria

Spurious regulatory connections dictate the expression-fitness landscape of translation termination factors

A High-efficacy CRISPRi System for Gene Function Discovery inZymomonas mobilis

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