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

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

doi:10.1038/s41564-021-01014-7

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

Journal Article · Mon Dec 06 00:00:00 UTC 2021 · Nature Microbiology

DOI: https://doi.org/10.1038/s41564-021-01014-7 · OSTI ID:1835956

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^[1]; Crits-Christoph, Alexander ^[1]; Lou, Yue Clare ^[1];

^[1]; Shivram, Haridha ^[1];

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^[1]; Smith, Sara J. ^[1]; Rovinsky, Rachel ^[1]; Smock, Dylan C. J. ^[1];

^[1]; Owens, Trenton K. ^[2]; Krishnappa, Netravathi ^[1];

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Univ. of California, Berkeley, CA (United States)
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
North Carolina State Univ., Raleigh, NC (United States)
Univ. of California, Berkeley, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Univ. of California, Berkeley, CA (United States); Univ. of Melbourne (Australia)
Univ. of California, Berkeley, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Univ. of California, San Francisco, CA (United States)

Knowledge of microbial gene functions comes from manipulating the DNA of individual strains 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 strategy for editing the genomes of specific organisms within microbial communities. We identified genetically tractable bacteria within a community using Environmental Transformation Sequencing (ET-Seq), an approach in which non-targeted transposon integrations are mapped and quantified following community delivery. We next 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. To illustrate the utility of our approach, we selectively edited closely related strains, measured gene fitness, and enriched targeted members within soil and infant gut microbiota. These results establish a new paradigm for targeted community editing relevant to research and applications on medical, agricultural, and industrial microbiomes.

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Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: Defense Advanced Research Projects Agency; National Institute of General Medical Sciences (NIGMS); National Institutes of Health (NIH); National Science Foundation (NSF); USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 1835956

Journal Information:: Nature Microbiology, Journal Name: Nature Microbiology Journal Issue: 1 Vol. 7; ISSN 2058-5276

Publisher:: Nature Publishing GroupCopyright Statement

Country of Publication:: United States

Language:: English

References (46)

Non-Coding RNA Analysis Using the Rfam Database Kalvari, Ioanna; Nawrocki, Eric P.; Argasinska, Joanna Current Protocols in Bioinformatics, Vol. 62, Issue 1 https://doi.org/10.1002/cpbi.51	journal	June 2018
Guide RNA Categorization Enables Target Site Choice in Tn7-CRISPR-Cas Transposons Petassi, Michael T.; Hsieh, Shan-Chi; Peters, Joseph E. Cell, Vol. 183, Issue 7 https://doi.org/10.1016/j.cell.2020.11.005	journal	December 2020
Functional genetics of human gut commensal Bacteroides thetaiotaomicron reveals metabolic requirements for growth across environments Liu, Hualan; Shiver, Anthony L.; Price, Morgan N. Cell Reports, Vol. 34, Issue 9 https://doi.org/10.1016/j.celrep.2021.108789	journal	March 2021
Efficient retroelement-mediated DNA writing in bacteria Farzadfard, Fahim; Gharaei, Nava; Citorik, Robert J. Cell Systems, Vol. 12, Issue 9 https://doi.org/10.1016/j.cels.2021.07.001	journal	September 2021
Adherent-invasive E. coli metabolism of propanediol in Crohn’s disease regulates phagocytes to drive intestinal inflammation Viladomiu, Monica; Metz, Maeva L.; Lima, Svetlana F. Cell Host & Microbe, Vol. 29, Issue 4 https://doi.org/10.1016/j.chom.2021.01.002	journal	April 2021
Manipulating Bacterial Communities by in situ Microbiome Engineering Sheth, Ravi U.; Cabral, Vitor; Chen, Sway P. Trends in Genetics, Vol. 32, Issue 4 https://doi.org/10.1016/j.tig.2016.01.005	journal	April 2016
Can laboratory reference strains mirror ‘real-world’ pathogenesis? Fux, C. A.; Shirtliff, M.; Stoodley, P. Trends in Microbiology, Vol. 13, Issue 2 https://doi.org/10.1016/j.tim.2004.11.001	journal	February 2005
Infant gut strain persistence is associated with maternal origin, phylogeny, and traits including surface adhesion and iron acquisition Lou, Yue Clare; Olm, Matthew R.; Diamond, Spencer Cell Reports Medicine, Vol. 2, Issue 9 https://doi.org/10.1016/j.xcrm.2021.100393	journal	September 2021
Broad host range plasmids can invade an unexpectedly diverse fraction of a soil bacterial community Klümper, Uli; Riber, Leise; Dechesne, Arnaud The ISME Journal, Vol. 9, Issue 4 https://doi.org/10.1038/ismej.2014.191	journal	October 2014
dRep: a tool for fast and accurate genomic comparisons that enables improved genome recovery from metagenomes through de-replication Olm, Matthew R.; Brown, Christopher T.; Brooks, Brandon The ISME Journal, Vol. 11, Issue 12 https://doi.org/10.1038/ismej.2017.126	journal	July 2017
Fast gapped-read alignment with Bowtie 2 Langmead, Ben; Salzberg, Steven L. Nature Methods, Vol. 9, Issue 4 https://doi.org/10.1038/nmeth.1923	journal	March 2012
High proportions of bacteria and archaea across most biomes remain uncultured Steen, Andrew D.; Crits-Christoph, Alexander; Carini, Paul The ISME Journal, Vol. 13, Issue 12 https://doi.org/10.1038/s41396-019-0484-y	journal	August 2019
In situ reprogramming of gut bacteria by oral delivery Hsu, Bryan B.; Plant, Isaac N.; Lyon, Lorena Nature Communications, Vol. 11, Issue 1 https://doi.org/10.1038/s41467-020-18614-2	journal	October 2020
Multiplexed enrichment of rare DNA variants via sequence-selective and temperature-robust amplification Wu, Lucia R.; Chen, Sherry X.; Wu, Yalei Nature Biomedical Engineering, Vol. 1, Issue 9 https://doi.org/10.1038/s41551-017-0126-5	journal	September 2017
Engineered integrative and conjugative elements for efficient and inducible DNA transfer to undomesticated bacteria Brophy, Jennifer A. N.; Triassi, Alexander J.; Adams, Bryn L. Nature Microbiology, Vol. 3, Issue 9 https://doi.org/10.1038/s41564-018-0216-5	journal	August 2018
Mediterranean grassland soil C–N compound turnover is dependent on rainfall and depth, and is mediated by genomically divergent microorganisms Diamond, Spencer; Andeer, Peter F.; Li, Zhou Nature Microbiology, Vol. 4, Issue 8 https://doi.org/10.1038/s41564-019-0449-y	journal	May 2019
Genome-resolved metagenomics reveals site-specific diversity of episymbiotic CPR bacteria and DPANN archaea in groundwater ecosystems He, Christine; Keren, Ray; Whittaker, Michael L. Nature Microbiology, Vol. 6, Issue 3 https://doi.org/10.1038/s41564-020-00840-5	journal	January 2021
Mutant phenotypes for thousands of bacterial genes of unknown function Price, Morgan N.; Wetmore, Kelly M.; Waters, R. Jordan Nature, Vol. 557, Issue 7706 https://doi.org/10.1038/s41586-018-0124-0	journal	May 2018
Transposon-encoded CRISPR–Cas systems direct RNA-guided DNA integration Klompe, Sanne E.; Vo, Phuc L. H.; Halpin-Healy, Tyler S. Nature, Vol. 571, Issue 7764 https://doi.org/10.1038/s41586-019-1323-z	journal	June 2019
CRISPR RNA-guided integrases for high-efficiency, multiplexed bacterial genome engineering Vo, Phuc Leo H.; Ronda, Carlotta; Klompe, Sanne E. Nature Biotechnology, Vol. 39, Issue 4 https://doi.org/10.1038/s41587-020-00745-y	journal	November 2020
inStrain profiles population microdiversity from metagenomic data and sensitively detects shared microbial strains Olm, Matthew R.; Crits-Christoph, Alexander; Bouma-Gregson, Keith Nature Biotechnology, Vol. 39, Issue 6 https://doi.org/10.1038/s41587-020-00797-0	journal	January 2021
Metagenomic engineering of the mammalian gut microbiome in situ Ronda, Carlotta; Chen, Sway P.; Cabral, Vitor Nature Methods, Vol. 16, Issue 2 https://doi.org/10.1038/s41592-018-0301-y	journal	January 2019
BEDTools: a flexible suite of utilities for comparing genomic features Quinlan, Aaron R.; Hall, Ira M. Bioinformatics, Vol. 26, Issue 6 https://doi.org/10.1093/bioinformatics/btq033	journal	January 2010
IDBA-UD: a de novo assembler for single-cell and metagenomic sequencing data with highly uneven depth Peng, Y.; Leung, H. C. M.; Yiu, S. M. Bioinformatics, Vol. 28, Issue 11 https://doi.org/10.1093/bioinformatics/bts174	journal	April 2012
The TraDIS toolkit: sequencing and analysis for dense transposon mutant libraries Barquist, Lars; Mayho, Matthew; Cummins, Carla Bioinformatics, Vol. 32, Issue 7 https://doi.org/10.1093/bioinformatics/btw022	journal	January 2016
Bartender: a fast and accurate clustering algorithm to count barcode reads Zhao, Lu; Liu, Zhimin; Levy, Sasha F. Bioinformatics, Vol. 34, Issue 5 https://doi.org/10.1093/bioinformatics/btx655	journal	October 2017
Splinkerettes—improved vectorettes for greater efficiency in PCR walking Devon, Rebecca S.; Porteous, David J.; Brookes, Anthony J. Nucleic Acids Research, Vol. 23, Issue 9 https://doi.org/10.1093/nar/23.9.1644	journal	January 1995
tRNAscan-SE: A Program for Improved Detection of Transfer RNA Genes in Genomic Sequence Lowe, Todd M.; Eddy, Sean R. Nucleic Acids Research, Vol. 25, Issue 5 https://doi.org/10.1093/nar/25.5.955	journal	March 1997
Rfam 14: expanded coverage of metagenomic, viral and microRNA families Kalvari, Ioanna; Nawrocki, Eric P.; Ontiveros-Palacios, Nancy Nucleic Acids Research, Vol. 49, Issue D1 https://doi.org/10.1093/nar/gkaa1047	journal	November 2020
A versatile platform strain for high-fidelity multiplex genome editing Egbert, Robert G.; Rishi, Harneet S.; Adler, Benjamin A. Nucleic Acids Research, Vol. 47, Issue 6 https://doi.org/10.1093/nar/gkz085	journal	February 2019
Inflammation-associated Adherent-invasive Escherichia coli Are Enriched in Pathways for Use of Propanediol and Iron and M-cell Translocation Dogan, Belgin; Suzuki, Haruo; Herlekar, Deepali Inflammatory Bowel Diseases, Vol. 20, Issue 11 https://doi.org/10.1097/MIB.0000000000000183	journal	January 2014
Metabolically cohesive microbial consortia and ecosystem functioning Pascual-García, Alberto; Bonhoeffer, Sebastian; Bell, Thomas Philosophical Transactions of the Royal Society B: Biological Sciences, Vol. 375, Issue 1798 https://doi.org/10.1098/rstb.2019.0245	journal	March 2020
Toward a genetic system in the marine cyanobacterium Prochlorococcus Laurenceau, Raphaël; Bliem, Christina; Osburne, Marcia S. Access Microbiology, Vol. 2, Issue 4 https://doi.org/10.1099/acmi.0.000107	journal	April 2020
Accurate and complete genomes from metagenomes Chen, Lin-Xing; Anantharaman, Karthik; Shaiber, Alon Genome Research, Vol. 30, Issue 3 https://doi.org/10.1101/gr.258640.119	journal	March 2020
Long-term manure exposure increases soil bacterial community potential for plasmid uptake: Manure increases community plasmid uptake Musovic, Sanin; Klümper, Uli; Dechesne, Arnaud Environmental Microbiology Reports, Vol. 6, Issue 2 https://doi.org/10.1111/1758-2229.12138	journal	January 2014
Monitoring the spread of broad host and narrow host range plasmids in soil microcosms Pukall, R.; TschÃ¤pe, H.; Smalla, K. FEMS Microbiology Ecology, Vol. 20, Issue 1 https://doi.org/10.1111/j.1574-6941.1996.tb00304.x	journal	May 1996
RNA-guided DNA insertion with CRISPR-associated transposases Strecker, Jonathan; Ladha, Alim; Gardner, Zachary Science, Vol. 365, Issue 6448 https://doi.org/10.1126/science.aax9181	journal	June 2019
Cultivation-Independent Examination of Horizontal Transfer and Host Range of an IncP-1 Plasmid among Gram-Positive and Gram-Negative Bacteria Indigenous to the Barley Rhizosphere Musovic, Sanin; Oregaard, Gunnar; Kroer, Niels Applied and Environmental Microbiology, Vol. 72, Issue 10 https://doi.org/10.1128/AEM.00013-06	journal	October 2006
Plasmid Donor Affects Host Range of Promiscuous IncP-1β Plasmid pB10 in an Activated-Sludge Microbial Community De Gelder, Leen; Vandecasteele, Frederik P. J.; Brown, Celeste J. Applied and Environmental Microbiology, Vol. 71, Issue 9 https://doi.org/10.1128/AEM.71.9.5309-5317.2005	journal	September 2005
The Link between Phylogeny and Virulence in Escherichia coli Extraintestinal Infection Picard, Bertrand; Garcia, José Sevali; Gouriou, Stéphanie Infection and Immunity, Vol. 67, Issue 2 https://doi.org/10.1128/IAI.67.2.546-553.1999	journal	February 1999
Stable Neutralization of a Virulence Factor in Bacteria Using Temperate Phage in the Mammalian Gut Hsu, Bryan B.; Way, Jeffrey C.; Silver, Pamela A. mSystems, Vol. 5, Issue 1 https://doi.org/10.1128/mSystems.00013-20	journal	February 2020
Magic Pools: Parallel Assessment of Transposon Delivery Vectors in Bacteria Liu, Hualan; Price, Morgan N.; Waters, Robert Jordan mSystems, Vol. 3, Issue 1 https://doi.org/10.1128/mSystems.00143-17	journal	January 2018
Prodigal: prokaryotic gene recognition and translation initiation site identification Hyatt, Doug; Chen, Gwo-Liang; LoCascio, Philip F. BMC Bioinformatics, Vol. 11, Issue 1 https://doi.org/10.1186/1471-2105-11-119	journal	March 2010
Characterization and remediation of sample index swaps by non-redundant dual indexing on massively parallel sequencing platforms Costello, Maura; Fleharty, Mark; Abreu, Justin BMC Genomics, Vol. 19, Issue 1 https://doi.org/10.1186/s12864-018-4703-0	journal	May 2018
Cutadapt removes adapter sequences from high-throughput sequencing reads Martin, Marcel EMBnet.journal, Vol. 17, Issue 1 https://doi.org/10.14806/ej.17.1.200	journal	May 2011
Characterization and remediation of sample index swaps by non-redundant dual indexing on massively parallel sequencing platforms Costello, Maura; Fleharty, Mark; Abreu, Justin figshare https://doi.org/10.6084/m9.figshare.c.4093520	collection	January 2018

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