PE-STAR improves bacterial efficiency and expands edit sizes in Escherichia coli.
Evidence
This platform engineering study tested exonuclease removal, RecJ overexpression, SOS-linked counterselection, and integrase coupling across E. coli loci, reaching 80%-90% short-fragment editing and up to 46 bp edits.
Caveat
The results are limited to engineered E. coli systems and screened-colony outcomes, so performance in other bacteria or broader genomic contexts remains untested.
Simplified
enables precise genome modifications without DNA double-strand breaks, yet bacterial applications are limited by low efficiency and small edit sizes. Here, we develop PE-STAR, Prime Editing with SOS-Triggered and RecJ-Augmented Repair, to enhance prime editing in Escherichia coli. Removing three inhibitory 3'→5' exonucleases (SbcB, ExoX, and XseA) improved edited-strand retention, and extending post-transformation outgrowth increased editing efficiency. RecJ overexpression strengthened 5'-directed processing during flap resolution and gap expansion, biasing repair toward incorporation of the reverse-transcribed edited strand. To enrich edited cells, we integrated an SOS-responsive counter-selection circuit that links PE3-associated dual nicking to LexA-dependent gRNA expression targeting a plasmid encoding the toxin CcdB, thereby eliminating unedited cells. PE-STAR achieved up to 80%-90% editing efficiency for short-fragment modifications, representing up to 16-fold improvement across loci. The platform supported insertions, deletions, and replacements of up to 46 bp with high efficiency. Furthermore, installing an attB site by prime editing, followed by Bxb1 integrase recombination, enabled chromosomal integration of 3.2 and 8.0 kb cassettes with 100% recombination efficiency among screened colonies, including GFP reporter and riboflavin biosynthetic pathway. PE-STAR expands both the efficiency and functional scope of bacterial prime editing for programmable genome engineering.
Key numbers
80%–90%
Achieved for short-fragment modifications in E. coli.
16×
Improvement Factor
Observed across various genomic loci.
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