PT - JOURNAL ARTICLE AU - Benjamin E. Rubin AU - Spencer Diamond AU - Brady F. Cress AU - Alexander Crits-Christoph AU - Christine He AU - Michael Xu AU - Zeyi Zhou AU - Dylan C. Smock AU - Kimberly Tang AU - Trenton K. Owens AU - Netravathi Krishnappa AU - Rohan Sachdeva AU - Adam M. Deutschbauer AU - Jillian F. Banfield AU - Jennifer A. Doudna TI - Targeted Genome Editing of Bacteria Within Microbial Communities AID - 10.1101/2020.07.17.209189 DP - 2020 Jan 01 TA - bioRxiv PG - 2020.07.17.209189 4099 - http://biorxiv.org/content/early/2020/07/21/2020.07.17.209189.short 4100 - http://biorxiv.org/content/early/2020/07/21/2020.07.17.209189.full AB - 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 StatementThe 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.