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.

Prime 编辑器是一种多功能工具，用于进行靶向精确编辑，以在真核生物中产生点突变、小插入或小删除。然而，规范的 PE3 系统效率较低，特别是在原代细胞或多能干细胞中。在这里，我们使用RNA聚合酶II启动子代替RNA聚合酶III启动子（其应用受到特定DNA背景的限制）来产生Csy4处理的内含子引物编辑引导RNA（pegRNA），并与其他优化一起，实现了聚（ T) 含有 pegRNA，以及组合和条件基因编辑。我们还发现同时抑制DNA错配修复和DNA损伤反应可以在人类胚胎干细胞中实现高效、准确的编辑。这些发现缓解了基于RNA聚合酶III（RNA-Pol-III）的碱基编辑器的限制，并拓宽了prime编辑的应用。