A randomized multiplex CRISPRi-Seq approach for the identification of critical combinations of genes

Ellis, N.; Myers, Kevin S.; Tung, Jessica; Ward, Anne Davidson; Johnston, Kathryn V.; Bonnington, Katherine E.; Donohue, Timothy J.; Machner, Matthias P.

doi:10.7554/elife.86903

Cited by 4 publications

(1 citation statement)

References 47 publications

(57 reference statements)

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“…This enables straight-forward and cost-efficient investigation of gene function across many bacterial species through the design and construction of sgRNAs or sgRNA libraries for high-throughput studies, replacing the time-consuming construction of gene knockouts or transposon mutant libraires (Peters et al, 2019). Also, expression of more than one sgRNA enables multiplex approaches to manipulate several genetic targets at once, to explore combinatorial effects (Ellis et al, 2023), and to engineer metabolic pathways and flux (Kim et al, 2017). Other engineering avenues have explored using CRISPRi-dCas9 in bacteria to activate transcription by fusing transcriptional activation domains to dCas9 (Dong et al, 2018), or to construct more complex regulatory and repressilator-like circuits (Kuo et al, 2020; Rueff et al, 2023).…”

Section: Introductionmentioning

confidence: 99%

Programming CRISPRi-dCas9 to control the lifecycle of bacteriophage T7

Bergmiller

2024

Preprint

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CRISPR interference (CRISPRi) based on catalytically dead Cas9 nuclease of Streptococcus pyogenes is a programmable and highly flexible tool to interrogate gene function and essentiality in bacteria due to its ability to block transcription elongation at nearly any desired DNA target. Here, I assess how CRISPRi-dCas9 can be programmed to control the life cycle and infectivity of Escherichia coli bacteriophage T7, a highly virulent and obligatory lytic phage, by blocking critical host-dependent promoters that are required for host cell entry and life cycle execution. Using fluorescent reporters demonstrates that the efficacy of CRISPRi-dCas9 towards E. coli RNAP promoters depends on target promoter strength, and that the phage's own T7 RNAP can be downregulated very efficiently. Effects on the time to lysis were partially dictated by the chronological order of phage DNA and dCas9 target appearance in the cell, suggesting an accessory role of E. coli RNAP during the early stages of T7 genome ejection. The stringency of the CRISPRi-dCas9 approach was greatly improved when using multiplex sgRNAs to target multiple phage regions simultaneously, resulting in a 25% increase in the time to lysis and up to 8-fold reduction in plaque size. Overall, this work expands CRISPRi-dCas9 as a flexible tool to non-invasively manipulate and probe the lifecycle and infectivity of otherwise native T7 phage.

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Section: Introductionmentioning

confidence: 99%

Programming CRISPRi-dCas9 to control the lifecycle of bacteriophage T7

Bergmiller

2024

Preprint

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Integrating bacterial molecular genetics with chemical biology for renewed antibacterial drug discovery

Parkhill,

Johnson

2024

Biochemical Journal

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The application of dyes to understanding the aetiology of infection inspired antimicrobial chemotherapy and the first wave of antibacterial drugs. The second wave of antibacterial drug discovery was driven by rapid discovery of natural products, now making up 69% of current antibacterial drugs. But now with the most prevalent natural products already discovered, ∼107 new soil-dwelling bacterial species must be screened to discover one new class of natural product. Therefore, instead of a third wave of antibacterial drug discovery, there is now a discovery bottleneck. Unlike natural products which are curated by billions of years of microbial antagonism, the vast synthetic chemical space still requires artificial curation through the therapeutics science of antibacterial drugs — a systematic understanding of how small molecules interact with bacterial physiology, effect desired phenotypes, and benefit the host. Bacterial molecular genetics can elucidate pathogen biology relevant to therapeutics development, but it can also be applied directly to understanding mechanisms and liabilities of new chemical agents with new mechanisms of action. Therefore, the next phase of antibacterial drug discovery could be enabled by integrating chemical expertise with systematic dissection of bacterial infection biology. Facing the ambitious endeavour to find new molecules from nature or new-to-nature which cure bacterial infections, the capabilities furnished by modern chemical biology and molecular genetics can be applied to prospecting for chemical modulators of new targets which circumvent prevalent resistance mechanisms.

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The rise and future of CRISPR-based approaches for high-throughput genomics

Vercauteren,

Fiesack,

Maroc

et al. 2024

FEMS Microbiology Reviews

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Clustered regularly interspaced short palindromic repeats (CRISPR) has revolutionized the field of genome editing. To circumvent the permanent modifications made by traditional CRISPR techniques and facilitate the study of both essential and non-essential genes, CRISPR interference (CRISPRi) was developed. This gene silencing technique employs a deactivated Cas effector protein and a guide RNA to block transcription initiation or elongation. Continuous improvements and a better understanding of the mechanism of CRISPRi have expanded its scope, facilitating genome-wide high-throughput screens to investigate the genetic basis of phenotypes. Additionally, emerging CRISPR-based alternatives have further expanded the possibilities for genetic screening. This review delves into the mechanism of CRISPRi, compares it with other high-throughput gene-perturbation techniques and highlights its superior capacities for studying complex microbial traits. We also explore the evolution of CRISPRi, emphasizing enhancements that have increased its capabilities, including multiplexing, inducibility, titratability, predictable knockdown efficacy, and adaptability to non-model microorganisms. Beyond CRISPRi, we discuss CRISPR activation, RNA-targeting CRISPR systems and single-nucleotide resolution perturbation techniques for their potential in genome-wide high-throughput screens in microorganisms. Collectively, this review gives a comprehensive overview of the general workflow of a genome-wide CRISPRi screen, with an extensive discussion of strengths and weaknesses, future directions and potential alternatives.

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A randomized multiplex CRISPRi-Seq approach for the identification of critical combinations of genes

Cited by 4 publications

References 47 publications

Programming CRISPRi-dCas9 to control the lifecycle of bacteriophage T7

Programming CRISPRi-dCas9 to control the lifecycle of bacteriophage T7

Integrating bacterial molecular genetics with chemical biology for renewed antibacterial drug discovery

The rise and future of CRISPR-based approaches for high-throughput genomics

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