Editing the microbiome the CRISPR way

Ramachandran, Gayetri; Bikard, David

doi:10.1098/rstb.2018.0103

Cited by 86 publications

(61 citation statements)

References 129 publications

(152 reference statements)

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“…Although this is still a very young field, the developments have moved fast and scientists now have a variety of CRISPR-based tools at hand to alter microbiome composition and function, and use them as next-generation antimicrobials. Ramachandran & Bikard [101] provide a thorough overview of these various CRISPR-based applications to alter the microbiome, including its application in editing bacteria and phage genomes, controlling their gene expression using CRISPRi, killing pathogenic bacteria using CRISPRbased antimicrobials and removing ARGs or other virulence determinants. While these CRISPR-based ecological engineering approaches have great potential to solve ecological problems, there are many technological, regulatory, societal and ethical challenges.…”

Section: (D) Implications Of Crispr-cas For Human Health and Wellbeingmentioning

confidence: 99%

The ecology and evolution of microbial CRISPR-Cas adaptive immune systems

Westra

Houte

Gandon

et al. 2019

Phil. Trans. R. Soc. B

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Section: (D) Implications Of Crispr-cas For Human Health and Wellbeingmentioning

confidence: 99%

The ecology and evolution of microbial CRISPR-Cas adaptive immune systems

Westra

Houte

Gandon

et al. 2019

Phil. Trans. R. Soc. B

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“…At least three trans-conjugant colonies were combined and grown for 4 h at 37°C in 2 ml of LB. Bacteria were then streaked on freshly prepared LB-no salt agar plates [24] containing 20% sucrose and 0.5 µg/ml AHT. Plates were incubated at 28°C protected from light for at least 24h.…”

Section: Methodsmentioning

confidence: 99%

“…Currently, λ-Red recombineering is the method of choice for introducing genetic manipulations in S. enterica and E. coli [18] but it has been difficult to implement in several other bacterial species such as Pseudomonas aeruginosa . Recently, CRISPR-Cas has revolutionized eukaryotic genome editing [19–21], but this strategy is more cumbersome for bacteria with limited recombination activities [22–24].…”

Section: Introductionmentioning

confidence: 99%

Efficient Dual-Negative Selection for Bacterial Genome Editing

Cianfanelli

Cunrath

Bumann

2020

Preprint

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11We describe a versatile method for chromosomal gene editing based on classical consecutive 12 single-crossovers. The method exploits rapid plasmid construction using Gibson assembly, a 13 convenient E. coli donor strain, and efficient dual-negative selection for improved suicide vector 14 resolution. We used this method to generate in frame deletions, insertions and point mutations in 15Salmonella enterica with limited hands-on time. Similar strategies allowed efficient gene editing 16 also in Pseudomonas aeruginosa and multi-drug-resistant (MDR) Escherichia coli clinical isolates. 17 18 [19][20][21], but this strategy is more cumbersome for bacteria with limited recombination activities 34 [22][23][24]. 35Here, we combined several improvements for establishing a time-efficient versatile 36 method for consecutive single cross-overs in multiple bacterial species. We used rapid Gibson 37 assembly of PCR products [25] to generate suicide vectors with dual negative selection with I-38SceI and SacB [26, 27] (Fig 1a), which increased counter-selection efficiency to 100% for nearly 39 all tested deletions, insertions and point mutations. We employed an E. coli donor strain that 40 simplifies donor removal after conjugation and avoids common problems with contaminating 100 mM (Sigma Aldrich C1016-500G) containing 15% of glycerol (AppliChem, A1123,1000). 57 Bacteria were resuspended in 5 ml ice-cold CaCl2 100 mM containing 15% of glycerol and 200 µl 58 aliquots were frozen and stored at -80°C. Super-Optimal broth with Catabolite repression (SOC) 59 (tryptone 20 g/L, yeast extract 5 g/L, NaCl 0.5 g/L, KCl 0.186 g/L, MgSO4 4.8 g/L and glucose 3.6 60 g/L) medium was used after heat shock. Kanamycin (Roth T832.4) at a final concentration of 50 61 µg/ml and gentamicin (Gibco 15750-037) at a final concentration of 15 µg/ml were used to select 62 E. coli transformants. For positive selection, kanamycin (Roth T832.4) at a final concentration of 63 50 µg/ml, gentamicin (Gibco 15750-037) at a final concentration of 30 µg/ml, or potassium tellurite 64 (Sigma P0677) at a final concentration of 10 µg/ml, were used to select for S. typhimurium, P.

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“…The drastically reduced complexity of new therapies based on genetic engineering technologies but incomplete knowledge of the microbiota may hamper the translation to an effective modulation of an extremely complex system. On the other hand, highly synthetic strategies reduce or avoid the risk of transferring potentially unwanted bacteria or other components of the microbiota 66 . The challenges of microbiota-modifying therapies are even more obvious when targeting bacterial communities beyond the gut microbiota.…”

Section: Compassionate Usementioning

confidence: 99%

The global preclinical antibacterial pipeline

Theuretzbacher¹,

et al. 2019

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| Antibacterial resistance is a great concern and requires global action. A critical question is whether enough new antibacterial drugs are being discovered and developed. A review of the clinical antibacterial drug pipeline was recently published, but comprehensive information about the global preclinical pipeline is unavailable. This Review focuses on discovery and preclinical development projects and has found, as of 1 May 2019, 407 antibacterial projects from 314 institutions. The focus is on Gram-negative pathogens, particularly bacteria on the WHO priority bacteria list. The preclinical pipeline is characterized by high levels of diversity and interesting scientific concepts, with 135 projects on direct-acting small molecules that represent new classes, new targets or new mechanisms of action. There is also a strong trend towards non-traditional approaches, including diverse antivirulence approaches, microbiome-modifying strategies, and engineered phages and probiotics. The high number of pathogen-specific and adjunctive approaches is unprecedented in antibiotic history. Translational hurdles are not adequately addressed yet, especially development pathways to show clinical impact of nontraditional approaches. The innovative potential of the preclinical pipeline compared with the clinical pipeline is encouraging but fragile. Much more work , focus and funding are needed for the novel approaches to result in effective antibacterial therapies to sustainably combat antibacterial resistance.

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Editing the microbiome the CRISPR way

Cited by 86 publications

References 129 publications

The ecology and evolution of microbial CRISPR-Cas adaptive immune systems

The ecology and evolution of microbial CRISPR-Cas adaptive immune systems

Efficient Dual-Negative Selection for Bacterial Genome Editing

The global preclinical antibacterial pipeline

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