Engineered prime editors with minimal genomic errors

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. 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.