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Targeted Genome Editing of Bacteria Within Microbial Communities

View ORCID ProfileBenjamin E. Rubin, View ORCID ProfileSpencer Diamond, View ORCID ProfileBrady F. Cress, Alexander Crits-Christoph, View ORCID ProfileChristine He, Michael Xu, Zeyi Zhou, Dylan C. Smock, Kimberly Tang, Trenton K. Owens, Netravathi Krishnappa, View ORCID ProfileRohan Sachdeva, Adam M. Deutschbauer, Jillian F. Banfield, View ORCID ProfileJennifer A. Doudna
doi: https://doi.org/10.1101/2020.07.17.209189
Benjamin E. Rubin
1Innovative Genomics Institute, University of California, Berkeley, CA, USA
2Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA
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  • ORCID record for Benjamin E. Rubin
Spencer Diamond
1Innovative Genomics Institute, University of California, Berkeley, CA, USA
3Department of Earth and Planetary Science, University of California, Berkeley, CA, USA
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  • ORCID record for Spencer Diamond
Brady F. Cress
1Innovative Genomics Institute, University of California, Berkeley, CA, USA
2Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA
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Alexander Crits-Christoph
4Department of Plant and Microbial Biology, University of California, Berkeley, CA, USA
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Christine He
1Innovative Genomics Institute, University of California, Berkeley, CA, USA
2Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA
3Department of Earth and Planetary Science, University of California, Berkeley, CA, USA
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Michael Xu
1Innovative Genomics Institute, University of California, Berkeley, CA, USA
2Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA
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Zeyi Zhou
1Innovative Genomics Institute, University of California, Berkeley, CA, USA
2Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA
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Dylan C. Smock
1Innovative Genomics Institute, University of California, Berkeley, CA, USA
2Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA
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Kimberly Tang
1Innovative Genomics Institute, University of California, Berkeley, CA, USA
2Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA
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Trenton K. Owens
5Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA
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Netravathi Krishnappa
1Innovative Genomics Institute, University of California, Berkeley, CA, USA
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Rohan Sachdeva
1Innovative Genomics Institute, University of California, Berkeley, CA, USA
3Department of Earth and Planetary Science, University of California, Berkeley, CA, USA
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Adam M. Deutschbauer
4Department of Plant and Microbial Biology, University of California, Berkeley, CA, USA
5Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA
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Jillian F. Banfield
1Innovative Genomics Institute, University of California, Berkeley, CA, USA
3Department of Earth and Planetary Science, University of California, Berkeley, CA, USA
6Environmental Science, Policy and Management, University of California, Berkeley, CA, USA
7School of Earth Sciences, University of Melbourne, Melbourne, Victoria, Australia
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  • For correspondence: doudna@berkeley.edu jbanfield@berkeley.edu
Jennifer A. Doudna
1Innovative Genomics Institute, University of California, Berkeley, CA, USA
2Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA
8California Institute for Quantitative Biosciences, University of California, Berkeley, CA, USA
9Department of Chemistry, University of California, Berkeley, CA, USA
10Howard Hughes Medical Institute, University of California, Berkeley, CA, USA
11Molecular Biophysics & Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA
12Gladstone Institutes, University of California, San Francisco, CA, USA
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  • ORCID record for Jennifer A. Doudna
  • For correspondence: doudna@berkeley.edu jbanfield@berkeley.edu
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Abstract

Knowledge of microbial gene functions comes from manipulating the DNA of individual species in isolation from their natural communities. While this approach to microbial genetics has been foundational, its requirement for culturable microorganisms has left the majority of microbes and their interactions genetically unexplored. Here we describe a generalizable methodology for editing the genomes of specific organisms within a complex microbial community. First, we identified genetically tractable bacteria within a community using a new approach, Environmental Transformation Sequencing (ET-Seq), in which non-targeted transposon integrations were mapped and quantified following community delivery. ET-Seq was repeated with multiple delivery strategies for both a nine-member synthetic bacterial community and a ~200-member microbial bioremediation community. We achieved insertions in 10 species not previously isolated and identified natural competence for foreign DNA integration that depends on the presence of the community. Second, we developed and used DNA-editing All-in-one RNA-guided CRISPR-Cas Transposase (DART) systems for targeted DNA insertion into organisms identified as tractable by ET-Seq, enabling organism- and locus-specific genetic manipulation within the community context. These results demonstrate a strategy for targeted genome editing of specific organisms within microbial communities, establishing a new paradigm for microbial manipulation relevant to research and applications in human, environmental, and industrial microbiomes.

Competing Interest Statement

The Regents of the University of California have patents pending related to this work on which B.E.R., S.D., B.F.C., A.M.D., J.F.B., and J.A.D. are inventors. J.A.D. is a co-founder of Caribou Biosciences, Editas Medicine, Intellia Therapeutics, Scribe Therapeutics and Mammoth Biosciences, a scientific advisory board member of Caribou Biosciences, Intellia Therapeutics, eFFECTOR Therapeutics, Scribe Therapeutics, Synthego, Mammoth Biosciences and Inari, and is a Director at Johnson & Johnson and has sponsored research projects by Biogen, Roche and Pfizer. J.F.B. is a founder of Metagenomi.

Footnotes

  • (1) Small errors fixed throughout the manuscript; (2) All supplementary tables now added; (3) Figure legends and methods revised for more explicit description of statistics and experimental procedures; (4) All raw data and additional code uploaded to GitHub

  • https://github.com/SDmetagenomics/ETsuite

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC 4.0 International license.
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Posted July 21, 2020.
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Targeted Genome Editing of Bacteria Within Microbial Communities
Benjamin E. Rubin, Spencer Diamond, Brady F. Cress, Alexander Crits-Christoph, Christine He, Michael Xu, Zeyi Zhou, Dylan C. Smock, Kimberly Tang, Trenton K. Owens, Netravathi Krishnappa, Rohan Sachdeva, Adam M. Deutschbauer, Jillian F. Banfield, Jennifer A. Doudna
bioRxiv 2020.07.17.209189; doi: https://doi.org/10.1101/2020.07.17.209189
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Targeted Genome Editing of Bacteria Within Microbial Communities
Benjamin E. Rubin, Spencer Diamond, Brady F. Cress, Alexander Crits-Christoph, Christine He, Michael Xu, Zeyi Zhou, Dylan C. Smock, Kimberly Tang, Trenton K. Owens, Netravathi Krishnappa, Rohan Sachdeva, Adam M. Deutschbauer, Jillian F. Banfield, Jennifer A. Doudna
bioRxiv 2020.07.17.209189; doi: https://doi.org/10.1101/2020.07.17.209189

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