Metagenomic engineering of the mammalian gut microbiome in situ

Ronda, Carlotta; Chen, Sway P.; Cabral, Vitor; Yaung, Stephanie J.; Wang, Harris H.

doi:10.1038/s41592-018-0301-y

Cited by 191 publications

(165 citation statements)

References 30 publications

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“…In addition, increasing the portability of bacterial CRISPRa systems can greatly improve our ability to manipulate nonmodel organisms. In combination with in situ microbiome engineering tools (Brophy et al, 2018;Ronda et al, 2019), we envision that CasTA and similar technologies (Peters et al, 2019) could modulate gene expression in a precise and programmable fashion across diverse bacterial communities to elucidate fundamental processes underlying microbial ecology and provide useful applications in the emerging field of microbiome engineering.…”

Section: Discussionmentioning

confidence: 99%

Programmable and portable CRISPR-Cas transcriptional activation in bacteria

Fang

Cheung

et al. 2020

Preprint

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Programmable gene activation enables fine-tuned regulation of endogenous and synthetic gene circuits to control cellular behavior. While CRISPR-Cas-mediated gene activation have been extensively developed for eukaryotic systems, similar strategies have been difficult to implement in bacteria. Here, we present a generalizable platform for screening and selection of functional bacterial CRISPR-Cas transcription activators. Using this platform, we identified a novel CRISPR activator, dCas9-AsiA, that could activate gene expression by up to 200-fold across genomic and plasmid targets with diverse promoters after directed evolution. The evolved dCas9-AsiA can simultaneously mediate activation and repression of bacterial regulons in E. coli. We further identified hundreds of promoters with varying basal expression that could be induced by dCas9-AsiA, which provides a rich resource of genetic parts for inducible gene activation. Finally, we show that dCas9-AsiA can be ported to other bacteria of clinical and bioindustrial relevance, thus enabling bacterial CRISPRa in more application areas. This work expands the toolbox for programmable gene regulation in bacteria and provides a useful resource for future engineering of other bacterial CRISPR-based gene regulators.

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Section: Discussionmentioning

confidence: 99%

Programmable and portable CRISPR-Cas transcriptional activation in bacteria

Fang

Cheung

et al. 2020

Preprint

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“…Engineering complex bacterial communities in useful ways remains challenging due to the lack of understanding of the ecological principles and intercellular metabolic interactions 58 . Mobile genetic elements are potentially powerful tools for function-oriented microbiota engineering 59 . Our modeling framework and the MGE persistence potential can guide such efforts.…”

Section: Discussionmentioning

confidence: 99%

The Persistence Potential of Mobile Genetic Elements

Wang¹,

2020

Preprint

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Mobile genetic elements (MGEs), such as plasmids, phages, and transposons, play a critical role in mediating the transfer and maintenance of diverse traits and functions in microbial communities. This role depends on the ability of MGEs to persist. For a community consisting of multiple populations transferring multiple MGEs, however, the conditions underlying the persistence of these MGEs are poorly understood. Computationally, this difficulty arises from the combinatorial explosion associated with describing the gene flow in a complex community using the conventional modeling framework. Here, we describe an MGE-centric framework that makes it computationally feasible to analyze such transfer dynamics. Using this framework, we derive the persistence potential: a general, heuristic metric that predicts the persistence and abundance of any MGEs. We validate the metric with engineered microbial consortia transferring mobilizable plasmids and quantitative data available in the literature. Our modeling framework and the resulting metric have implications for developing a quantitative understanding of natural microbial communities and guiding the engineering of microbial consortia. Main text:

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“…Tet-inducible expression vectors (pHdCas9-gRNA1, pHdCas9-gRNA4, pHdCas9-gRNA5, pHdCas9 for negative control) were used to express Himar-dCas9 along with a GFP-targeting gRNA in S17 with pTarget. auxotrophic donor strain EcGT2 (S17 asd::mCherry-specR) 41 containing transposon donor plasmid pHimar6, which has a 1.4 kb Himar1 transposon with a chlor resistance cassette and the R6K origin of replication, which does not replicate in MG1655.…”

Section: Methodsmentioning

confidence: 99%

An Engineered Cas-Transposon System for Programmable and Precise DNA Transpositions

Chen

2019

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Efficient targeted insertion of heterologous DNA into a genome remains a challenge in genome engineering. Recombinases that can introduce kilobase-sized DNA constructs require pre-existing recombination sites to be present in the genome and are difficult to reprogram to other loci. Genome insertion using current CRISPR-Cas methods relies on host DNA repair machinery, which is generally inefficient. Here, we describe a Cas-Transposon (CasTn) system for genomic insertions that uses a transposase fused to a catalytically-dead dCas9 nuclease to mediate programmable, site-specific transposition. CasTn combines the power of the Himar1 transposase, which inserts multi-kb DNA transposons into TA dinucleotides by a cut-and-paste mechanism, and the targeting capability of Cas9, which uses guide-RNAs to bind to specific DNA sequences. Using in vitro assays, we demonstrated that Himar-dCas9 proteins increased the frequency of transposon insertions at a single targeted TA dinucleotide by >300-fold compared to an untargeted transposase, and that site-specific transposition is dependent on target choice while robust to log-fold variations in protein and DNA concentrations. We then showed that Himar-dCas9 mediates site-specific transposition into a target plasmid in E. coli. This work provides CasTn as a new method for host-independent, programmable, targeted DNA insertions to expand the genomic engineering toolbox.

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Metagenomic engineering of the mammalian gut microbiome in situ

Cited by 191 publications

References 30 publications

Programmable and portable CRISPR-Cas transcriptional activation in bacteria

Programmable and portable CRISPR-Cas transcriptional activation in bacteria

The Persistence Potential of Mobile Genetic Elements

An Engineered Cas-Transposon System for Programmable and Precise DNA Transpositions

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