Engineered prime editors with minimal genomic errors

Prime editors make programmed genome modifications by writing new sequences into extensions of nicked DNA 3' ends¹. These edited 3' new strands must displace competing 5' strands to install edits, yet a bias towards retaining the competing 5' strands hinders efficiency and can cause indel errors². Here we discover that nicked end degradation, consistent with competing 5' strand destabilization, can be promoted by Cas9-nickase mutations that relax nick positioning. 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 543:1.