Massively Parallel Screens Reveal Factors Influencing the Activity of a Diversity-Generating Retroelement Reconstituted in Escherichia coli [Tn-Seq]

ABSTRACT: Some phages and microbes employ Diversity-Generating Retroelements (DGRs) to achieve rapid targeted adaptation. DGRs mutagenize a specific gene without harming the rest of the genome. Mutagenesis relies on the DGR reverse transcriptase (dRT), which is highly error-prone when transcribing adenines of an RNA template. The resulting mutagenized cDNA then alters the target gene through a poorly understood mechanism. Transplanting DGRs into a tractable organism, like Escherichia coli, holds promise for creating a powerful system for gene-targeted editing. Here, we successfully reconstituted the archetypal BPP DGR in E. coli, demonstrating that DGRs are "plug-and-play" systems adaptable to non-native hosts. Systematic analysis of reconstituted DGR established principles for target mutagenesis. Our results revealed that cytosine misincorporation is the most common error when dRT transcribes adenines. However, error rates peak in 5'-AAC-3' and 5'-ACC-3' contexts, driven by elevated adenine and guanosine misincorporations, respectively. Using high-throughput genetics, we discovered that cells lacking the single-stranded DNA exonuclease ExoI exhibit 15-fold higher DGR activity. Our evidence indicates that ExoI inhibits DGRs by binding a limited cellular factor, rather than through direct cDNA degradation. Finally, by profiling DGR targets across thousands of chromosomal loci, we discovered that DGRs preferentially edit targets near the replication origin and oriented in the same direction as replication. We showed that the directionality of replication underlies this bias, possibly through unwinding the target to facilitate base-pairing with incoming cDNA. These findings establish a reconstituted DGR system in E. coli and provide a foundation for optimizing targeted gene-editing applications in the future.

ORGANISM(S): Escherichia coli

PROVIDER: GSE304216 | GEO | 2025/08/06

REPOSITORIES: GEO