Retroelement-Based Genome Editing and Evolution.

While several genome editing methods exist, few are suitable for the continuous evolution of targeted sequences.  Here we develop bacterial retroelements known as "retrons" for the dynamic,  in vivo editing and mutagenesis of targeted genes. We first optimized retrons' ability to introduce preprogrammed mutations, optimizing both their expression and the host machinery that interacts with them to increase the incorporation frequency of mutations 78-fold over rates previously reported in synthetic systems. The optimized system is capable of simultaneously overwriting 13 separate positions spanning  a 31-base length, and is for the first time shown to yield targeted deletions and insertions. To engineer retrons as a tool to introduce novel, unprogrammed mutations in specific targeted regions, we expressed them under a mutagenic T7 RNA polymerase. This coupled mutagenic T7 RNA polymerase-retron system enabled the evolution of diverse variants of environmentally selected antibiotic resistance genes, producing mutation rates in the targeted region 190-fold higher than  background cellular mutation rates, potentially enabling the dynamic, continuous self-evolution of selected phenotypes.