Targeted Genome Editing of Bacteria Within Microbial Communities

Abstract

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.

Chemistry of Class 1 CRISPR-Cas effectors: Binding, editing, and regulation

“…Lastly, transposaseassociated Type I-F systems have been shown to specifically insert synthetic "cargo" DNA up to 10 kb (kilobases) in length in E. coli with high fidelity, and thus holds promise as a technique to knock-in genes without requiring homologous recombination (Fig 4A)(66). Indeed, recent preprints have reported the use of transposaseassociated Type I-F systems for targeted insertion of antibiotic resistance genes in members of a microbial community, and multiplexed gene insertion in several medically and industrially important bacterial species (133,134).…”

“…Transposase-associated Type I-F systems also integrate DNA cargo with fewer off-target events than a transposase-associated Type V (Cas12k) system (66,134,139). The unique, cOA-regulated RNases of Type III systems could also be repurposed for control of cellular growth or behavior, in response to an upstream signal generated by a cOA synthetase (Fig.…”

Experimental and computational approaches to unravel microbial community assembly

“…While this approach can only be employed with strains amenable to genetic manipulation, there is continuing work on specific tools aiming to greatly expand the range of taxa open to such studies [44] , [45] and in a high-throughput manner [46] . Furthermore, a recent study has opened the road of genome editing within a microbial community context without requiring prior isolation [47] . This technique may be useful in future studies to assess the role of particular genes on fitness, community function and community assembly.…”

Translating New Synthetic Biology Advances for Biosensing Into the Earth and Environmental Sciences

“…The conjugative plasmid in this strain contained the IncPαRP4 replication and transfer system, which is expected to be useful in diverse environmental communities beyond the gut. New technologies also enable targeting of specific members of a microbial community for genome editing (Rubin et al, 2020 ).…”

“…High-performance conjugation systems hold promise for programming diverse environmental microbes (Brophy et al, 2018 ; Ronda et al, 2019 ; Wang et al, 2019 ), as do methods for targeted genome editing in communities (Rubin et al, 2020 ). However, conjugation systems have only been used to program microbiomes from a limited number of environmental settings.…”