Efficient prime editing in two-cell mouse embryos using PEmbryo

Here, we show that prime editing, when deployed during a permissive stage of early development and in the presence of an engineered inhibitor of DNA mismatch repair, enables highly efficient editing of mouse embryos with few on-target byproducts. While temporary inhibition of mismatch repair increased nonspecific, off-target indels, as revealed by whole genome sequencing, these primarily -1 bp deletions were found in genomic regions of low sequence complexity and did not result in obvious phenotypic changes nor viability issues in mice. Moreover, on-target editing was of sufficient efficiency and purity to allow us, in proof-of-principle experiments, to directly characterize founder mice for a recessive phenotype without outcrossing. Our findings thus establish PEmbryo as a promising technique for rapidly phenotyping genetic variants and for accelerated germline engineering in mice.

本研究表明，当在早期发育的许可阶段应用先导编辑（prime editing），并辅以DNA错配修复（DNA mismatch repair）的工程化抑制剂时，可实现小鼠胚胎的高效精准编辑，且靶标副产物极少。尽管全基因组测序（whole genome sequencing）结果显示，暂时抑制错配修复会增加非特异性的脱靶插入缺失突变（indels），但这类突变主要为1碱基缺失，且仅富集于序列复杂度较低的基因组区域，不会导致小鼠出现明显的表型变化或生存能力异常。此外，靶标编辑的效率与纯度均满足实验需求，使我们能够在原理验证实验中，无需远交（outcrossing）即可直接对携带隐性表型的首建小鼠（founder mice）进行表型鉴定。综上，本研究成果确立了PEmbryo作为一种极具潜力的技术，可用于小鼠遗传变异的快速表型鉴定以及加速生殖系工程改造。