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

Certain phages and microbes use Diversity-Generating Retroelements (DGRs) for targeted adaptation via a process called mutagenic retrohoming. This process relies on an error-prone reverse transcriptase that specifically misincorporates bases opposite adenines in the RNA template. The resulting mutagenized complementary DNA then integrates into a homologous target gene to introduce mutations at adenine residues. Although the functions of core DGR components are well-defined, we still dont know how other cellular factors influence their activity. To address this, we reconstituted the DGR from the Bordetella phage BPP-1 in Escherichia coli. Our systematic analysis uncovered several cellular factors that influence its activity. By sequencing the cDNA, we discovered the reverse transcriptase is particularly mutagenic in the 5-AAC-3 and 5-ACC-3 contexts, which explains their abundance in natural DGR templates. A transposon sequencing screen identified both positive and negative regulators, including the single-stranded DNA exonuclease ExoI, which unexpectedly inhibits DGR activity through a nuclease-independent mechanism. Using a massively parallel reporter assay, we found that DGR preferentially edits targets located near the replication origin and co-directionally oriented with replication. In the preferred orientation, the target sequence is located on the lagging-strand template, where the extended single-stranded DNA gap likely facilitates base pairing with the incoming complementary DNA. Collectively, these findings advance our understanding of how DGRs function within the cellular context and enabled a 1000-fold improvement in editing efficiency, establishing a foundation for future targeted-mutagenesis applications.