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

• Published in: Nature microbiology Vol. 7; no. 1; pp. 34 - 47
• Main Authors: 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.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.01.2022; Nature Publishing Group
• Subjects: 42; 42/41; 45/70; 631/114; 631/1647/1511; 631/326/2565; 631/61/2300; Archaea - genetics; Bacteria; Bacteria - classification; BASIC BIOLOGICAL SCIENCES; Biomedical and Life Sciences; computational biology and bioinformatics; CRISPR; CRISPR-Cas Systems; Deoxyribonucleic acid; DNA; Gastrointestinal Microbiome - genetics; gene delivery; Gene Editing - methods; genetic engineering; Genome, Bacterial; Genomes; Humans; Infant; Infectious Diseases; Insertion; Intestinal microflora; Life Sciences; Medical Microbiology; microbial communities; Microbial Consortia - genetics; Microbiology; Microbiota; Organisms; Parasitology; RNA editing; RNA, Guide, CRISPR-Cas Systems; Soil Microbiology; Species; Transposase; Virology
• ISSN: 2058-5276; 2058-5276
• DOI: 10.1038/s41564-021-01014-7

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. A suite of methods enables programmable species- and locus-specific editing of bacteria in communities.