Phage Engineering for Targeted Editing of Microbial Communities
Benjamin A. Adler1* (email@example.com), Tomas Hessler1, Avery Roberts2, Matthew Nethery2, John Beckley2, Brady F. Cress1, Arushi Lahiri1, Jillian Banfield2, Rodolphe Barrangou2, Jennifer A. Doudna1, Adam M. Deutschbauer3, and Trent R. Northern3*
1University of California–Berkeley; 2North Carolina State University; and 3Lawrence Berkeley National Laboratory
Understanding the interactions, localization, and dynamics of grass rhizosphere communities at the molecular level (genes, proteins, metabolites) to predict responses to perturbations and understand the persistence and fate of engineered genes and microbes for secure biosystems design. To do this, advanced fabricated ecosystems are used in combination with gene editing technologies such as CRISPR-Cas and bacterial virus (phage)-based approaches for interrogating gene and microbial functions in situ—addressing key challenges highlighted in recent DOE reports. This work is integrated with the development of predictive computational models that are iteratively refined through simulations and experimentation to gain critical insights into the functions of engineered genes and interactions of microbes within soil microbiomes as well as the biology and ecology of uncultivated microbes. Together, these efforts lay a critical foundation for developing secure biosystems design strategies, harnessing beneficial microbiomes to support sustainable bioenergy, and improving the understanding of nutrient cycling in the rhizosphere.
Bacteriophages are estimated to be the most abundant biological entities on Earth, outnumbering bacteria by ten to one. Owing to their natural ecological abundance, genetic diversity, and ability to transduce DNA, they represent attractive gene delivery vehicles to edit microbial communities in situ. However, the ability to broadly edit phages themselves has been limited by a diversity of mechanisms for phages to protect DNA genomes. In order to edit the diversity of phages, we establish a generalizable editing for phage genetic manipulation based off RNA-guided, RNA-targeting endonuclease, LbuCas13a. Researchers find LbuCas13a to be a remarkably potent phage inhibitor, suggesting that phage RNA is generally vulnerable during viral infection. When challenged against Escherichia coli phage phylogeny, researchers find no apparent phage-encoded limits to LbuCas13a antiviral activity. Further, researchers highlight how leveraging this potent anti-phage activity can be used to flexibly edit diverse phages with edits as small as a single codon or as large as multi-gene deletions. Researchers further discuss opportunities for engineered phages to edit microbial within fabricated ecosystems using phage-derived base editing technology and novel phages infecting members of synthetic microbial consortia. The ability to robustly edit bacteriophages will not only lead to a deeper understanding of phage genetic diversity but also facilitate meaningful genetic changes to microbial communities.
Adler, B. A., et al. 2022. Broad-Spectrum CRISPR-Cas13a Enables Efficient Phage Genome Editing. Nature Microbiology 7, 1967–79.
Nethery, M. A., et al. 2022. “CRISPR-Based Engineering of Phages for in situ Bacterial Base Editing.” The Proceedings of the National Academy of Sciences U.S. 119, e2206744119.
This material by m-CAFEs Microbial Community Analysis and Functional Evaluation in Soils, (m-CAFEs@lbl.gov) an Science Focus Area led by Lawrence Berkeley National Laboratory is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research under contract number DE-AC02-05CH11231.