Species- and site-specific genome editing in complex bacterial communities

Benjamin E Rubin; Spencer Diamond; Brady F Cress; Alexander Crits-Christoph; Yue Clare Lou; Adair L Borges; Haridha Shivram; Christine He; Michael Xu; Zeyi Zhou; Sara J Smith; Rachel Rovinsky; Dylan C J Smock; Kimberly Tang; Trenton K Owens; Netravathi Krishnappa; Rohan Sachdeva; Rodolphe Barrangou; Adam M Deutschbauer; Jillian F Banfield; Jennifer A Doudna

doi:10.1038/s41564-021-01014-7

Species- and site-specific genome editing in complex bacterial communities

Nat Microbiol. 2022 Jan;7(1):34-47. doi: 10.1038/s41564-021-01014-7. Epub 2021 Dec 6.

Authors

Benjamin E Rubin^#^{1

2}, Spencer Diamond^#^{1

3}, Brady F Cress^#^{1

2}, Alexander Crits-Christoph⁴, Yue Clare Lou^{1

4}, Adair L Borges^{1

5}, Haridha Shivram^{1

2}, Christine He^{1

2

3}, Michael Xu^{1

2}, Zeyi Zhou^{1

2}, Sara J Smith^{1

2}, Rachel Rovinsky^{1

2}, Dylan C J Smock^{1

2}, Kimberly Tang^{1

2}, Trenton K Owens⁶, Netravathi Krishnappa¹, Rohan Sachdeva^{1

3}, Rodolphe Barrangou⁷, Adam M Deutschbauer^{4

6}, Jillian F Banfield^{8

9

10

11}, Jennifer A Doudna^{12

13

14

15

16

17

18}

Affiliations

¹ Innovative Genomics Institute, University of California, Berkeley, CA, USA.
² Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA.
³ Department of Earth and Planetary Science, University of California, Berkeley, CA, USA.
⁴ Department of Plant and Microbial Biology, University of California, Berkeley, CA, USA.
⁵ Environmental Science, Policy and Management, University of California, Berkeley, CA, USA.
⁶ Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.
⁷ Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, NC, USA.
⁸ Innovative Genomics Institute, University of California, Berkeley, CA, USA. jbanfield@berkeley.edu.
⁹ Department of Earth and Planetary Science, University of California, Berkeley, CA, USA. jbanfield@berkeley.edu.
¹⁰ Environmental Science, Policy and Management, University of California, Berkeley, CA, USA. jbanfield@berkeley.edu.
¹¹ School of Earth Sciences, University of Melbourne, Melbourne, Victoria, Australia. jbanfield@berkeley.edu.
¹² Innovative Genomics Institute, University of California, Berkeley, CA, USA. doudna@berkeley.edu.
¹³ Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA. doudna@berkeley.edu.
¹⁴ California Institute for Quantitative Biosciences, University of California, Berkeley, CA, USA. doudna@berkeley.edu.
¹⁵ Department of Chemistry, University of California, Berkeley, CA, USA. doudna@berkeley.edu.
¹⁶ Howard Hughes Medical Institute, University of California, Berkeley, CA, USA. doudna@berkeley.edu.
¹⁷ Molecular Biophysics & Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA. doudna@berkeley.edu.
¹⁸ Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA, USA. doudna@berkeley.edu.

^# Contributed equally.

Abstract

Understanding microbial gene functions relies on the application of experimental genetics in cultured microorganisms. However, the vast majority of bacteria and archaea remain uncultured, precluding the application of traditional genetic methods to these organisms and their interactions. Here, we characterize and validate a generalizable strategy for editing the genomes of specific organisms in microbial communities. We apply environmental transformation sequencing (ET-seq), in which nontargeted transposon insertions are mapped and quantified following delivery to a microbial community, to identify genetically tractable constituents. Next, DNA-editing all-in-one RNA-guided CRISPR-Cas transposase (DART) systems for targeted DNA insertion into organisms identified as tractable by ET-seq are used to enable organism- and locus-specific genetic manipulation in a community context. Using a combination of ET-seq and DART in soil and infant gut microbiota, we conduct species- and site-specific edits in several bacteria, measure gene fitness in a nonmodel bacterium and enrich targeted species. These tools enable editing of microbial communities for understanding and control.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Archaea / genetics
Bacteria / classification
CRISPR-Cas Systems
Gastrointestinal Microbiome / genetics*
Gene Editing / methods*
Genome, Bacterial*
Humans
Infant
Microbial Consortia / genetics*
RNA, Guide, CRISPR-Cas Systems
Soil Microbiology*

Substances

RNA, Guide, CRISPR-Cas Systems

Abstract

Publication types

MeSH terms

Substances

Grants and funding