New tools provide targeted genome editing in microbial communities. ET-Seq allows the isolation independent assessment of genetic accessibility of a microbe in community context. The DART vector system allows highly specific targeted DNA insertion.
New tools provide targeted genome editing in microbial communities. ET-Seq allows the isolation independent assessment of genetic accessibility of a microbe in community context. The DART vector system allows highly specific targeted DNA insertion.
Image courtesy of Ben Rubin

The Science

In nature, microbial communities contain multiple species of bacteria. This makes it difficult to isolate individual species of bacteria and culture them in the laboratory. Researchers have now developed new tools to help. These tools allow researchers to genetically manipulate distinct bacterial species within their communities. One of the tools, called ET-Seq, allows scientists to identify microbes that can be genetically modified directly within mixed communities. The other tool, dubbed DART, lets scientists test the function of genes from specific species inside their communities.

The Impact

Most bacteria live together in complex communities. With most current tools, scientists need to isolate individual species in order to study them. However, environmental microbes such as soil bacteria cannot be easily isolated and cultured in the laboratory. Furthermore, the behavior of microbial communities results from the combined contributions of its members. This is very different from the activity of isolated species. The developments of the ET-Seq and DART tools help scientists study microbial communities without the need to isolate different members. Combining both technologies, researchers can also track genetic modifications as the community grows and examine gene function in microorganisms that cannot be grown in the lab.

Summary

Cultivation and genetic analysis have been the primary means used to study gene function and the behavior of microbes. However, these classical approaches require isolating and culturing microorganisms in the lab. This severely limits scientists’ knowledge of microbes that cannot yet be cultivated in the lab. Moreover, the interactions that occur between microbes when they grow together in a community cannot be studied in isolated organisms. To address these challenges, this research developed two technologies to test functions and interactions directly within laboratory microbiomes. Environmental transformation sequencing (ET-seq) delivers a mobile genetic element (transposon) into a microbial community. The transposon inserts randomly into the genes of some bacterial species. By sequencing the genomes of all the microbes in the community, scientists can detect community members that are transformed by the transposon and how frequently. In that way, they identify genetically tractable species. Those species can be specifically targeted for manipulation of selected genes using DNA-editing all-in-one RNA-guided CRISPR–Cas transposase (DART). The researchers also combined both techniques to demonstrate the enrichment of targeted bacterial species, confer novel metabolic traits, and measure gene fitness of bacteria within a community context in a lab setting. These new capabilities will provide important new insights into the activities of uncultivated microbes and the functions of key genes, metabolites, and proteins, for example, in soil carbon cycling and mediating beneficial microbial interactions with plants for sustainable bioenergy.

Contact

Jennifer A. Doudna
Innovative Genomics Institute
University of California, Berkeley
doudna@berkeley.edu

 
Jillian F. Banfield
Innovative Genomics Institute
University of California, Berkeley
jbanfield@berkeley.edu

Funding

Funding was provided by the m-CAFE Microbial Community Analysis & Functional Evaluation in Soils Science Focus Area, which is led by Lawrence Berkeley National Laboratory and supported by the Department of Energy Office of Science, Office of Biological & Environmental Research. This research was also developed with funding from the Defense Advanced Research Projects Agency. This material is based on work supported by the National Science Foundation. Support was also provided by the Innovative Genomics Institute at the University of California, Berkeley.

Publications

Rubin, B.E., et al., Species- and site-specific genome editing in complex bacterial communitiesNature Microbiology 7, 34–47 (2021). [DOI:10.1038/s41564-021-01014-7]

Related Links

Berkeley Lab feature: Editing microbial genomes and communities in the laboratory

Innovative Genomics Institute feature: Metagenomics 101 With Spencer Diamond