Genetic manipulation of gut microbes enables single-gene interrogation in a complex microbiome

Jin, Wenbing; Li, Ting-Ting; Huo, Da; Qu, Sophia; Li, Xin V.; Arifuzzaman, Mohammad; Lima, Svetlana; Shi, Huiqing; Wang, Aolin; Putzel, Gregory G.; Longman, Randy S.; Artis, David; Guo, Chun-Jun

doi:10.1016/j.cell.2021.12.035

Cited by 82 publications

(70 citation statements)

References 83 publications

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“…A complex defined community offers a setting in which a ‘clean’ dropout can be constructed in a physiologically relevant background. This approach, in combination with recent advances in genetic systems for the microbiome (García-Bayona and Comstock, 2019; Guo et al, 2019; Jin et al, 2022), will greatly improve our understanding of strain-strain and strain-host interactions.…”

Section: Discussionmentioning

confidence: 99%

“…Together, these data suggest Cs and Ch are the only strains in the 7a-dehydroxylation niche and that dropping them out results in the complete elimination of secondary bile acids. Previous attempts to knock out secondary bile acid production were conducted in the context of simple defined communities (Marion et al, 2019(Marion et al, , 2020Streidl et al, 2021;Studer et al, 2016) or by antibiotic knockdown of resident colonists followed by colonization with an antibiotic-resistant mutant, which depletes a variety of other strains in the community (Jin et al, 2022). In contrast, our system is a defined knockout of 7a-dehydroxylation in the setting of a complex community.…”

Section: Determining the Occupancy Of The 7a-dehydroxylation Niche In...mentioning

confidence: 99%

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Strain dropouts reveal interactions that govern the metabolic output of the gut microbiome

Wang

Osborn

Jain

et al. 2022

Preprint

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The gut microbiome is complex, raising questions about the role of individual strains in the community. Here, we address this question by focusing on a functional unit within the community, the metabolic niche that controls bile acid 7-alpha-dehydroxylation. By constructing variants of a complex defined community in which we drop out strains that occupy this niche, we explore how interactions within and between niches shape community-level metabolism. Omitting both members of the niche, Clostridium scindens (Cs) and Clostridium hylemonae (Ch), eliminates secondary bile acid production and reshapes the community in a highly specific manner: eight strains go up or down in relative abundance by >100-fold, while the remaining strains are largely unaffected. In single-strain dropout communities (i.e., a strain swap within the niche), Cs and Ch reach the same relative abundance and dehydroxylate bile acids to a similar extent. However, the effect on strains in other niches differs markedly: Clostridium sporogenes increases >1000-fold in the delta-Cs but not delta-Ch dropout, reshaping the pool of microbiome-derived phenylalanine metabolites. Thus, strains that are functionally redundant within a niche can have widely varying impacts outside the niche, and a strain swap can ripple through the community in an unpredictable manner, resulting in a large impact on an unrelated community-level phenotype. Mice colonized by the delta-Cs/delta-Ch community show decreased liver steatosis relative to those colonized by the delta-Ch community, demonstrating that a single strain from the microbiome can have a substantive impact on host physiology. Our work opens the door to the mechanistic studies of the role of an individual strain on community ecology and host physiology.

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Section: Discussionmentioning

confidence: 99%

Section: Determining the Occupancy Of The 7a-dehydroxylation Niche In...mentioning

confidence: 99%

Strain dropouts reveal interactions that govern the metabolic output of the gut microbiome

Wang

Osborn

Jain

et al. 2022

Preprint

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“…For novel or fastidious species, however, trial and error and patience may be required to determine appropriate culture conditions for growth and genetic manipulation, such as the obligate intracellular pathogen Chlamydia trachomatis ( Ouellette, 2018 ). Additionally, introducing foreign DNA is often challenging for a non-model bacterium, as many are genetically recalcitrant, especially pathogens ( Fernandes et al, 2021b ) and novel strains ( Zhao et al, 2020 ; Jin et al, 2022 ), and establishing a sufficient genetic transformation method can require significant effort. Additionally, care must be taken when introducing synthetic DNA to circumvent the bacterial host’s native immunity that may degrade foreign DNA, including restriction-modification and CRISPR systems ( Marraffini and Sontheimer, 2008 ; Jin et al, 2022 ), such as by mimicking the recipient strain’s methylation patterns ( Monk et al, 2015 ; Zhao et al, 2020 ).…”

Section: Requirements and Challenges To Establish Crispri/a In Non-mo...mentioning

confidence: 99%

“…Additionally, introducing foreign DNA is often challenging for a non-model bacterium, as many are genetically recalcitrant, especially pathogens ( Fernandes et al, 2021b ) and novel strains ( Zhao et al, 2020 ; Jin et al, 2022 ), and establishing a sufficient genetic transformation method can require significant effort. Additionally, care must be taken when introducing synthetic DNA to circumvent the bacterial host’s native immunity that may degrade foreign DNA, including restriction-modification and CRISPR systems ( Marraffini and Sontheimer, 2008 ; Jin et al, 2022 ), such as by mimicking the recipient strain’s methylation patterns ( Monk et al, 2015 ; Zhao et al, 2020 ). More discussion on the isolation and domestication of non-model bacteria can be found in other reviews ( Vartoukian et al, 2010 ; Lewis et al, 2021 ; Riley and Guss, 2021 ).…”

Section: Requirements and Challenges To Establish Crispri/a In Non-mo...mentioning

confidence: 99%

“…Combined, these are the most common use of CRISPRi/a tools in non-model bacteria, with 80 reports across six phyla ( Table 1 ) ( Behler et al, 2018 ; Stamsås et al, 2018 ; Ke et al, 2021 ). The recent development of Mobile-CRISPRi ( Peters et al, 2019 ), CRAGE-CRISPR ( Ke et al, 2021 ), and a workflow for introducing genetic manipulation tools into non-model gut bacteria ( Jin et al, 2022 ) will facilitate the expansion of CRISPRi/a tools into new species and strains, including recalcitrate pathogens and novel species without sequenced genomes.…”

Section: Applications Of Crispri/a In Non-model Bacteriamentioning

confidence: 99%

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CRISPR-Based Approaches for Gene Regulation in Non-Model Bacteria

Call

Andrews

2022

Front. Genome Ed.

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CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) have become ubiquitous approaches to control gene expression in bacteria due to their simple design and effectiveness. By regulating transcription of a target gene(s), CRISPRi/a can dynamically engineer cellular metabolism, implement transcriptional regulation circuitry, or elucidate genotype-phenotype relationships from smaller targeted libraries up to whole genome-wide libraries. While CRISPRi/a has been primarily established in the model bacteria Escherichia coli and Bacillus subtilis, a growing numbering of studies have demonstrated the extension of these tools to other species of bacteria (here broadly referred to as non-model bacteria). In this mini-review, we discuss the challenges that contribute to the slower creation of CRISPRi/a tools in diverse, non-model bacteria and summarize the current state of these approaches across bacterial phyla. We find that despite the potential difficulties in establishing novel CRISPRi/a in non-model microbes, over 190 recent examples across eight bacterial phyla have been reported in the literature. Most studies have focused on tool development or used these CRISPRi/a approaches to interrogate gene function, with fewer examples applying CRISPRi/a gene regulation for metabolic engineering or high-throughput screens and selections. To date, most CRISPRi/a reports have been developed for common strains of non-model bacterial species, suggesting barriers remain to establish these genetic tools in undomesticated bacteria. More efficient and generalizable methods will help realize the immense potential of programmable CRISPR-based transcriptional control in diverse bacteria.

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Development of an integrated and project‐based laboratory course in upper‐level biochemistry and molecular biology

Liu,

Tu,

et al. 2024

Biochem Molecular Bio Educ

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An integrated and projected‐based laboratory course was described, integrating interconnected knowledge points and biochemistry and molecular biology techniques on a research project‐based system. The program, which served as an essential extension of theoretical courses to practice, was conducted with a sophomore of basic medical science who had completed the course in medical biochemistry and molecular biology. This course engaged students in learning “genetic manipulation” and “recombinant DNA technology” to understand the target gene's role in disease mechanics, thus altering evaluation and treatment for clinical disease. Students could master applied and advanced techniques, such as cell culture, transfection, inducing exogenous fusion protein expression, purifying protein and its concentration assay, quantitative polymerase chain reaction, and western bot analysis. This laboratory exercise links laboratory practices with the methods of current basic research. Students need to complete the experimental design report and laboratory report, which could be advantageous for improving their ability to write lab summaries and scientific papers in the future. The reliability and validity analyses were conducted on the questionnaire, and we examined students' satisfaction with the course and their gains from the course. The student feedback was generally positive, indicating that the exercise helped consolidate theoretical knowledge, increase scientific research enthusiasm, and provide a powerful tool to be a better person and make informed decisions.

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Genetic manipulation of gut microbes enables single-gene interrogation in a complex microbiome

Cited by 82 publications

References 83 publications

Strain dropouts reveal interactions that govern the metabolic output of the gut microbiome

Strain dropouts reveal interactions that govern the metabolic output of the gut microbiome

CRISPR-Based Approaches for Gene Regulation in Non-Model Bacteria

Development of an integrated and project‐based laboratory course in upper‐level biochemistry and molecular biology

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