Reducing competition between msd and genomic DNA improves retron editing efficiency

Retrons, found in bacteria and used for defense against phages, generate a unique molecule known as multicopy single-stranded DNA (msDNA). This msDNA mimics Okazaki fragments during DNA replication, making it a promising tool for targeted gene editing in prokaryotes. However, existing retron systems often exhibit suboptimal editing efficiency. In Escherichia coli, we identify a critical bottleneck in the msd gene, which encodes the noncoding RNA template for msDNA synthesis and carries the homologous sequence of the target gene to be edited. This sequence homology causes the msDNA to bind to the msd gene, thereby reducing its efficiency in editing the target gene. To address this issue, we engineer a retron system that tailors msDNA to the leading strand of the plasmid containing the msd gene. This strategy minimizes msd gene editing and reduces competition with target genes, significantly increasing msDNA availability. Our optimized system achieves the highest retron editing efficiency reported to date, enhancing performance and expanding the potential for in vivo techniques that rely on homologous DNA synthesis.