Broadening prime editing toolkits using RNA-Pol-II-driven engineered pegRNA

The prime editor is a versatile tool for targeted precise editing to generate point mutations, small insertions, or small deletions in eukaryotes. However, canonical PE3 system is less efficient, notably in primary cells or pluripotent stem cells. Here, we employed RNA polymerase II promoter instead of RNA polymerase III promoter, whose application is limited by specific DNA contexts, to produce Csy4-processed intronic prime editing guide RNAs (pegRNAs) and, together with other optimizations, achieved efficient targeting with poly(T)-containing pegRNAs, as well as combinatorial and conditional genetic editing. We also found simultaneous suppression of both DNA mismatch repair and DNA damage response could achieve efficient and accurate editing in human embryonic stem cells. These findings relieve the restrictions of RNA polymerase III (RNA-Pol-III)-based base editors and broadened the applications of prime editing.

Automated summary

In this study, we used an RNA polymerase II promoter instead of an RNA polymerase III promoter to produce Csy4-processed intronic prime editing guide RNAs (pegRNAs), and achieved efficient genetic editing through other optimizations. We also found that simultaneous suppression of both DNA mismatch repair and DNA damage response can achieve efficient and accurate editing in human embryonic stem cells.