Microbial Gene Discovery and Community Editing

The ability to manipulate microbial communities at the genomic level, coupled with the discovery of novel microbial genes, holds transformative potential for biotechnology and ecological understanding. By harnessing tools like CRISPR-Cas, we aim to develop the capacity for genetic manipulation of microbial communities, with the ultimate goal of tackling critical issues in human health and climate.. Concurrently, our deep genomic analyses spotlight previously unidentified genes from lesser-studied bacteria and archaea, emphasizing their impending biotechnological contributions. Combined, these research strands forge new paths in genome editing and unlock vast microbial genetic potential.

Genome editing of microorganisms within the context of their natural communities

Research to enable the use of CRISPR-Cas editing tools to edit the genomes of microbes without requiring that they be available as pure cultures was initiated by a multi-institution collaborative mCAFEs project funded by the Department of Energy via LBNL. Recently, an Innovative Genomics Institute team (led by Jill Banfield and Jennifer Dounda, but involving many early career investigators and their labs) secured a large 7-year award from the TED Audacious program to dramatically expand this effort, with long term goals of using manipulation of complex microbial communities to address challenges in human health and climate.

The collaborative component with LBNL aims to understand the interactions, localization, and dynamics of grass rhizosphere communities at the molecular level (genes, proteins, and 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 (called EcoFABs) 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.

We are part of a team working to pioneer microbial community editing tools for dissecting the function of genes, pathways, and microbes within complex communities. Progress with the following objectives (a), (b) was demonstrated in a Nature Microbiology publication (Rubin et al. 2021) co-led with the Dounda lab (the Doudna lab leads ongoing work in these two areas).

a) ET-Seq: Environmental Transformation Sequencing (ET-Seq) is a technology developed to assess the ability of individual species within a microbial community to acquire and integrate exogenous DNA.

b) DART: The DNA-editing all-in-one RNA-guided CRISPR–Cas transposase (DART) system was developed for locus-specific insertion of DNA into organisms within a microbial community. These systems also include barcodes that make them compatible with ET-Seq to allow for the detection and tracking of uniquely edited cells within communities.

In ongoing work, our lab is pursuing the following goals:

i) Development and refinement of methods for a genome-resolved metagenomics-based definition of microbiomes, enabling organism-specific targeting via CRISPR-Cas systems and generation of scientific hypotheses to be tested via editing.

ii) Establishing representative, lab-based microbial communities in which editing tools and hypotheses related to organism capacities and interactions can be tested. See the infant gut microbiome cultivation component of our lab website for more information.

iii) Identification of microbiome-sourced methods to deliver editing tools into targeted (and often historically not genetically tractable) organisms. Of interest is the possibility to harness plasmids and viruses that we predict associate with the organism to be edited.

iv) Microbial interaction prediction: We aim to demonstrate our community-editing tools by probing microbial interactions within microbial communities. To identify interactions in communities, we have developed a metagenomics-informed abundance correlation network analysis method for the identification of metabolite exchange, particularly vitamins, in microbial communities and have verified these predicted interactions in co-cultures and microcosms. The SFA has similarly developed metabolic models which are able to predict interactions and co-culture growth dynamics between two organisms based on genetic information. These computational models are then iteratively refined through simulations and experimentation.

Together, these efforts lay a critical foundation for understanding the role and function of genes and organisms within mixed microbial communities. These technologies will allow us to manipulate and harness beneficial microbiomes to support sustainable bioenergy, improving our understanding of nutrient cycling in the rhizosphere.

Discovery of new microbial genes for biotech

Figure 1 from Burstein, Harrington, Strutt et al. (Nature, 2017) reporting new CRISPR systems uncovered from metagenomic data. Also see Harrington et al. (Science, 2018), in which we report new miniature CRISPR systems (Cas14-based).

We are acquiring huge genomic and transcriptomic datasets from entire microbial communities from across Earth’s environments, targeting systems that often include microbes unrelated to laboratory-studied species. The datasets are searched to identify new enzymes that may be involved in DNA manipulation. Of particular interest are those sourced from extrachromosomal elements, including huge phages, plasmids and novel elements we refer to as Borgs. We are establishing assays for predicting functionalities for these enzymes such as DNA and RNA binding, unwinding, and cleavage, via various methods, including via structure modeling (using AlphaFold2).

Genomic exploration of bacteria and archaea that are only distantly related to known species is uncovering a vast diversity of novel genes and gene clusters with potential value in biotechnology and medicine. Of specific interest are genes that could be developed into genome editing tools. The existing toolkit for human genome editing has already been expanded by efforts in this area, in collaboration with the Doudna lab (Burstein et al. 2017, Harrington et al. 2018, Wright et al. 2019, Pausch et al. 2020, Al-Shayeb et al. 2022).

A new view of the Tree of Life (Hug et al. Nature Microbiology, 2016) in which red dots indicate phylum-level groups without even a single cultivated representative.

Currently, our focus is on newly described CRISPR-Cas systems (e.g., Cas14) and DNA-interacting proteins that occur in conserved genomic context. The research will lead to new insights into the functions of known proteins within CRISPR-Cas systems, and will likely uncover new kinds of proteins, enzymes, and transcripts that contribute to genome surveillance and manipulation across the microbial world. This will lead to a much larger toolbox for genome editing than is currently available, enabling researchers and clinicians to optimize technologies according to their needs.

Relevant publications

Al-Shayeb, B., Skopintsev, P., Soczek, K.K., Stahl, E.C., Li, Z., Groover, E., Smock, D., Eggers, A.R., Pausch, P., Cress, B.F., Huang, C.J., Staskawicz, B., Savage, D.F., Jacobsen, S.E., Banfield, J.F. and Doudna, J.A. (2022) Diverse virus-encoded CRISPR-Cas systems include streamlined genome editors. Cell Press, 4574-4586. e16,

Burstein, D., Harrington, L.B., Strutt, S.C., Probst, A.J., Anantharaman, K., Thomas, B.C., Doudna, J.A., and Banfield, J.F. (2017) New CRISPR-Cas systems from uncultivated microbes. Nature, doi:10.1038/nature21059

Harrington, L.B., Burstein, D., Chen, J.S., Paez-Espino, D. Ma, E., Witte, I.P., Cofsky, J.C., Kyrpides, N.C., Banfield, J.F., and Doudna, J.A. (2018) Programmed DNA destruction by miniature CRISPR-Cas14 enzymes. Science, 362, 839-842

Pausch, P. Al-Shayeb, B., Bisom-Rapp, E., Tsuchida, C.A., Li, Z., Cress, B.F., Knott, G.J., Jacobsen, S.E., Banfield, J.F., and Doudna, J.A. (2020) CRISPR-CasΦ from huge phages is a hypercompact genome editor. Science, 369, 333-337

Rubin B., Diamond S., Cress B.F., Crits-Christoph A., Lou Y.C., Borges A.L., Shivram, H., He C., Xu M., Zhou Z., Smith S.J,, Rovinsky R., Smock D.C.J., Tang K., Owens T.K., Krishnappa N., Sachdeva R., Barrangou R., Deutschbauer A.M., Banfield J.F., Dounda J.A. (2021) Species- and site-specific genome editing in complex bacterial communities. Nature Microbiology, 7: 34-47. doi: 10.1038/s41564-021-01014-7

Wright, A.W., Wang, J.Y., Burstein, D., Harrington, L.B., Paez-Espino, D., Kyrpides, N.C., Iavarone, A.T., Banfield, J.F., and Doudna, J.A. (2019) A functional mini-integrase in a two-protein-type V-C CRISPR System. Molecular Cell, 73, 727-737.e3.