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
Innovative Genomics Institute, University of California, Berkeley, CA, USA.
Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA.
Nat Microbiol. 2022 Jan;7(1):34-47. doi: 10.1038/s41564-021-01014-7. Epub 2021 Dec 6.
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.
了解微生物基因功能依赖于在培养微生物中应用实验遗传学。然而，绝大多数细菌和古菌仍无法培养，这使得传统遗传方法无法应用于这些生物体及其相互作用。在此，我们表征并验证了一种可推广的策略，用于编辑微生物群落中特定生物体的基因组。我们应用环境转化测序（ET-seq），即在将非靶向转座子插入物递送至微生物群落后对其进行定位和定量，以识别具有遗传易处理性的成分。接下来，使用DNA编辑一体化RNA引导的CRISPR-Cas转座酶（DART）系统将靶向DNA插入经ET-seq鉴定为易处理的生物体中，从而在群落背景下实现生物体和位点特异性的基因操作。通过在土壤和婴儿肠道微生物群中结合使用ET-seq和DART，我们对几种细菌进行了物种和位点特异性编辑，测量了一种非模式细菌中的基因适应性，并富集了靶向物种。这些工具能够对微生物群落进行编辑，以实现理解和控制。
