De novo synthesis of a conjugative system from human gut metagenomic data for targeted delivery of Cas9 antimicrobials

Metagenomic sequence represents an untapped source of genetic novelty, particularly for conjugative systems that could be used for plasmid-based delivery of Cas9-derived antimicrobial agents. However, unlocking the functional potential of conjugative systems purely from metagenomic sequence requires the identification of suitable candidate systems as starting scaffolds for de novo DNA synthesis. Here, we developed a bioinformatics approach that searches through the metagenomic trash bin for conjugative systems present on contigs that are typically excluded from common metagenomic analysis pipelines. Using a human metagenomic gut dataset representing 2805 taxonomically distinct units, we identified 1598 contigs containing conjugative systems with a differential distribution in human cohorts. We synthesized de novo an entire Citrobacter spp. conjugative system of 54 kb and containing at least 47 genes, pCitro, and found that pCitro conjugates from Escherichia coli to Citrobacter rodentium with a 30-fold higher frequency than to E. coli, and is compatible with Citrobacter resident plasmids. Mutations in the traV and traY conjugation components of pCitro inhibited conjugation. We showed that pCitro can be re-purposed as an antimicrobial delivery agent by programming it with the TevCas9 nuclease and Citrobacter -specific sgRNAs to kill C. rodentium. Our study reveals a trove of uncharacterized conjugative systems in metagenomic data and describes an experimental framework to animate these large genetic systems as novel target-adapted delivery vectors for Cas9-based editing of bacterial genomes.