High‐efficiency delivery of CRISPR‐Cas9 by engineered probiotics enables precise microbiome editing

Neil, Kevin; Allard, Nancy; Roy, Patricia J; Grenier, Frédéric; Menéndez, Alfredo; Burrus, Vincent; Rodrigue, Sébastien

doi:10.15252/msb.202110335

Cited by 58 publications

(70 citation statements)

References 50 publications

(72 reference statements)

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“…A series of single amino acid replacements were performed by recombineering with a PCR product containing the appropriate point mutation in pilS along with an antibiotic resistance marker to select for transformants. The cysteine knock-in mutations were introduced in both TP114 and the previously described eB-TP114 derivative ( 44 ). The introduction of cysteine in Caulobacter crescentus major pilin (PilS) did not apparently affect the structure of the pilus fiber or the cell envelope ( 45 ).…”

Section: Resultsmentioning

confidence: 99%

“…Before the observations, E. coli BW25113Δ fliA cells harboring either TP114 or TP114 derivatives were subjected to conjugation in broth with E. coli BW25113Δ fliA recipient cells carrying pNeonGreen. Conjugations in broth were performed as described for in vitro conjugation experiments, except that cells were harvested after 5 min of mating for eB-TP114 ( 44 ) plasmid or after 70 min for TP114 wild type. Cells were then centrifuged for 1 min at 400 × g and resuspended in sterile phosphate-buffered saline (PBS) 1×.…”

Section: Methodsmentioning

confidence: 99%

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The Type IV Pilus of Plasmid TP114 Displays Adhesins Conferring Conjugation Specificity and Is Important for DNA Transfer in the Mouse Gut Microbiota

et al. 2022

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

The Type IV Pilus of Plasmid TP114 Displays Adhesins Conferring Conjugation Specificity and Is Important for DNA Transfer in the Mouse Gut Microbiota

et al. 2022

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“…Enhancements can be achieved by engineering an E. faecalis donor strain to be more competitive against wild strains. Another previously proposed avenue is to utilize gut-adapted bacteria, such as probiotic strains, which could be more resilient against other gut bacteria (46). Collectively, our results support our hypothesis that the differences observed between the two recipient groups are due to the presence (or lack thereof) of competitive factors.…”

Section: Discussionmentioning

confidence: 99%

Efficacy of plasmid-encoded CRISPR-Cas antimicrobial is affected by competitive factors found in wildEnterococcus faecalisisolates

Araya

Islam

Moni

et al. 2022

Preprint

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Enterococcus faecalis is a leading cause of hospital-acquired infections. These infections are becoming more difficult to treat due to the increasing emergence of E. faecalis strains resistant to last resort antibiotics. Over the past decade, multiple groups have engineered the naturally occurring bacterial defense system CRISPR-Cas as a sequence-specific antimicrobial to combat antibiotic-resistant bacteria. We have previously established that the type II CRISPR-Cas system of E. faecalis can be reprogrammed as a CRISPR-Cas antimicrobial and delivered to antibiotic-resistant recipients on a conjugative pheromone-responsive plasmid. Using a co-culture system, we showed sequence-specific depletion of antibiotic resistance from E. faecalis model strains, both in vitro and in vivo. Although this and other studies have demonstrated the potential use for CRISPR-Cas as an antimicrobial, most have deployed the system against model bacterial strains. Thus, there is limited knowledge on how effective these potential therapies are against recently isolated and uncharacterized strains with limited laboratory passage, which we refer to here as wild strains. Here, we compare the efficacy of our previously established CRISPR-Cas antimicrobials against both E. faecalis model strains and wild E. faecalis fecal isolates. We demonstrate that these wild isolates can antagonize the CRISPR-Cas antimicrobial donor strain via competitive factors like cytolysin. Furthermore, we show that the wild isolates can effectively prevent delivery of the CRISPR-Cas antimicrobial plasmids, consequently avoiding CRISPR-Cas targeting. Our results emphasize the requisite to study CRISPR-Cas antimicrobials against wild strains to understand limitations and develop delivery systems that can endure competitive interspecies interactions in the gut microenvironment and effectively deliver CRISPR-Cas antimicrobials to their intended targets.

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“…Microbiome engineering is one of the most promising fields of application of synthetic biology in areas as diverse as human therapeutics, crop improvement, and environmental bioremediation [1][2][3]. Communities can be designed from either scratch or the composition and functions of those already in place modified upon the introduction of one or more members into the existing partnership [4].…”

Section: Introductionmentioning

confidence: 99%

Propagation of Recombinant Genes through Complex Microbiomes with Synthetic Mini-RP4 Plasmid Vectors

2022

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The promiscuous conjugation machinery of the Gram-negative plasmid RP4 has been reassembled in a minimized, highly transmissible vector for propagating genetically encoded traits through diverse types of naturally occurring microbial communities. To this end, the whole of the RP4-encoded transfer determinants (tra, mob genes, and origin of transfer oriT) was excised from their natural context, minimized, and recreated in the form of a streamlined DNA segment borne by an autoselective replicon. The resulting constructs (the pMATING series) could be self-transferred through a variety of prokaryotic and eukaryotic recipients employing such a rationally designed conjugal delivery device. Insertion of GFP reporter into pMATING exposed the value of this genetic tool for delivering heterologous genes to both specific mating partners and complex consortia (e.g., plant/soil rhizosphere). The results accredited the effective and functional transfer of the recombinant plasmids to a diversity of hosts. Yet the inspection of factors that limit interspecies DNA transfer in such scenarios uncovered type VI secretion systems as one of the factual barriers that check otherwise high conjugal frequencies of tested RP4 derivatives. We argue that the hereby presented programming of hyperpromiscuous gene transfer can become a phenomenal asset for the propagation of beneficial traits through various scales of the environmental microbiome.

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High‐efficiency delivery of CRISPR‐Cas9 by engineered probiotics enables precise microbiome editing

Cited by 58 publications

References 50 publications

The Type IV Pilus of Plasmid TP114 Displays Adhesins Conferring Conjugation Specificity and Is Important for DNA Transfer in the Mouse Gut Microbiota

The Type IV Pilus of Plasmid TP114 Displays Adhesins Conferring Conjugation Specificity and Is Important for DNA Transfer in the Mouse Gut Microbiota

Efficacy of plasmid-encoded CRISPR-Cas antimicrobial is affected by competitive factors found in wildEnterococcus faecalisisolates

Propagation of Recombinant Genes through Complex Microbiomes with Synthetic Mini-RP4 Plasmid Vectors

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