Modulating pathogenesis with Mobile-CRISPRi

Qu, Jiuxin; Prasad, Neha; Yu, Michelle; Looney, Mark R.; Chen, Shuyan; Lyden, Amy; Crawford, Emily; Silvis, Melanie R.; Peters, Joseph E.; Rosenberg, Oren S.

doi:10.1101/618637

Cited by 3 publications

(1 citation statement)

References 49 publications

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“…Both systems were demonstrated to be functional for repression of genes encoding fluorescent proteins, and Kim et al (2020) also employed CRISPRi for metabolic engineering via gene repression by depleting the GlpR regulator to enhance the glycerol-dependent synthesis of mevalonate. Other examples on the development of CRISPRi systems have been reported for P. aeruginosa (Peters et al, 2019;Qu et al, 2019) and P. fluorescens (Noirot-Gros et al, 2019). While the CRISPRi toolbox for Pseudomonas species offers alternatives depending on the intended application (ranging from fundamental studies to simple metabolic engineering manipulations), the techniques applied so far are afflicted by either leaky expression of the components or limited ability to titrate repression levelsthus restricting the applicability of the tool in complex engineering approaches.…”

Section: Introductionmentioning

confidence: 99%

An expanded CRISPRi toolbox for tunable control of gene expression in Pseudomonas putida

Batianis

Kozaeva

Damalas

et al. 2020

Microbial Biotechnology

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Summary Owing to its wide metabolic versatility and physiological robustness, together with amenability to genetic manipulations and high resistance to stressful conditions, Pseudomonas putida is increasingly becoming the organism of choice for a range of applications in both industrial and environmental applications. However, a range of applied synthetic biology and metabolic engineering approaches are still limited by the lack of specific genetic tools to effectively and efficiently regulate the expression of target genes. Here, we present a single‐plasmid CRISPR‐interference (CRISPRi) system expressing a nuclease‐deficient cas9 gene under the control of the inducible XylS/Pm expression system, along with the option of adopting constitutively expressed guide RNAs (either sgRNA or crRNA and tracrRNA). We showed that the system enables tunable, tightly controlled gene repression (up to 90%) of chromosomally expressed genes encoding fluorescent proteins, either individually or simultaneously. In addition, we demonstrate that this method allows for suppressing the expression of the essential genes pyrF and ftsZ, resulting in significantly low growth rates or morphological changes respectively. This versatile system expands the capabilities of the current CRISPRi toolbox for efficient, targeted and controllable manipulation of gene expression in P. putida.

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Section: Introductionmentioning

confidence: 99%

An expanded CRISPRi toolbox for tunable control of gene expression in Pseudomonas putida

Batianis

Kozaeva

Damalas

et al. 2020

Microbial Biotechnology

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A High-efficacy CRISPRi System for Gene Function Discovery in Zymomonas 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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Essential Gene Knockdowns Reveal Genetic Vulnerabilities and Antibiotic Sensitivities in Acinetobacter baumannii

Ward

Tran

Banta

et al. 2023

Preprint

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The emergence of multidrug-resistant Gram-negative bacteria underscores the need to define genetic vulnerabilities that can be therapeutically exploited. The Gram-negative pathogen,Acinetobacter baumannii, is considered an urgent threat due to its propensity to evade antibiotic treatments. Essential cellular processes are the target of existing antibiotics and a likely source of new vulnerabilities. AlthoughA. baumanniiessential genes have been identified by transposon sequencing (Tn-seq), they have not been prioritized by sensitivity to knockdown or antibiotics. Here, we take a systems biology approach to comprehensively characterizeA. baumanniiessential genes using CRISPR interference (CRISPRi). We show that certain essential genes and pathways are acutely sensitive to knockdown, providing a set of vulnerable targets for future therapeutic investigation. Screening our CRISPRi library against last-resort antibiotics uncovered genes and pathways that modulate beta-lactam sensitivity, an unexpected link between NADH dehydrogenase activity and growth inhibition by polymyxins, and anticorrelated phenotypes that underpin synergy between polymyxins and rifamycins. Our study demonstrates the power of systematic genetic approaches to identify vulnerabilities in Gram-negative pathogens and uncovers antibiotic-essential gene interactions that better inform combination therapies.ImportanceAcinetobacter baumanniiis a hospital-acquired pathogen that is resistant to many common antibiotic treatments. To combat resistantA. baumanniiinfections, we need to identify promising therapeutic targets and effective antibiotic combinations. In this study, we comprehensively characterize the genes and pathways that are critical forA. baumanniiviability. We show that genes involved in aerobic metabolism are central toA. baumanniiphysiology and may represent appealing drug targets. We also find antibiotic-gene interactions that may impact the efficacy of carbapenems, rifamycins, and polymyxins, providing a new window into how these antibiotics function in mono- and combination therapies. Our studies offer a useful approach for characterizing interactions between drugs and essential genes in pathogens to inform future therapies.

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Modulating pathogenesis with Mobile-CRISPRi

Cited by 3 publications

References 49 publications

An expanded CRISPRi toolbox for tunable control of gene expression in Pseudomonas putida

An expanded CRISPRi toolbox for tunable control of gene expression in Pseudomonas putida

A High-efficacy CRISPRi System for Gene Function Discovery in Zymomonas mobilis

Essential Gene Knockdowns Reveal Genetic Vulnerabilities and Antibiotic Sensitivities in Acinetobacter baumannii

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