Engineered prime editors with minimal genomic errors.

Unlabelled: 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 Summary: Prime editors designed with repositioned DNA breaks nearly eliminate undesired genome editing errors.