Engineered prime editors with minimal genomic errors

Abstract

Prime editors make programmed genome modifications by writing new sequences into extensions of nicked DNA 3 ends. These edited 3 new strands must displace competing 5 strands to install edits, yet a bias toward retaining the competing 5 strands hinders efficiency and can cause indel errors. Using rational design of the constituent Cas9-nickase to reposition prime editor nicks, we discovered that competing 5 strands are destabilized to favor the edited 3 new strands. We exploit this mechanism to engineer efficient prime editors with strikingly low indel errors. Combining this error-suppressing strategy with the latest efficiency-boosting architecture, we design a next- generation prime editor (vPE). Compared with previous editors, vPE features comparable efficiency yet up to 60-fold lower indel errors, enabling edit:indel ratios as high as 465:1. One Sentence SummaryPrime editors designed with repositioned DNA breaks nearly eliminate undesired genome editing errors