Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, MA, USA; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA; Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA.
Department of Pediatrics, University of Minnesota Medical School, Minneapolis, MN, USA.
Cell. 2023 Aug 31;186(18):3983-4002.e26. doi: 10.1016/j.cell.2023.07.039.
Prime editing enables a wide variety of precise genome edits in living cells. Here we use protein evolution and engineering to generate prime editors with reduced size and improved efficiency. Using phage-assisted evolution, we improved editing efficiencies of compact reverse transcriptases by up to 22-fold and generated prime editors that are 516-810 base pairs smaller than the current-generation editor PEmax. We discovered that different reverse transcriptases specialize in different types of edits and used this insight to generate reverse transcriptases that outperform PEmax and PEmaxΔRNaseH, the truncated editor used in dual-AAV delivery systems. Finally, we generated Cas9 domains that improve prime editing. These resulting editors (PE6a-g) enhance therapeutically relevant editing in patient-derived fibroblasts and primary human T-cells. PE6 variants also enable longer insertions to be installed in vivo following dual-AAV delivery, achieving 40% loxP insertion in the cortex of the murine brain, a 24-fold improvement compared to previous state-of-the-art prime editors.
引发编辑能够在活细胞中实现各种各样的精确基因组编辑。在这里，我们使用蛋白质进化和工程技术来生成尺寸更小、效率更高的引发编辑器。通过噬菌体辅助进化，我们将紧凑型逆转录酶的编辑效率提高了 22 倍，并生成了比当前一代编辑器 PEmax 小 516-810 个碱基对的引发编辑器。我们发现不同的逆转录酶专门用于不同类型的编辑，并利用这一见解生成了逆转录酶，其性能优于 PEmax 和 PEmaxΔRNaseH，后者是双 AAV 递送系统中使用的截断编辑器。最后，我们生成了 Cas9 结构域，以提高引发编辑的效率。这些新的编辑器（PE6a-g）增强了患者来源成纤维细胞和原代人 T 细胞中的治疗相关编辑。PE6 变体还能够在双 AAV 递送后在体内安装更长的插入物，在小鼠大脑皮层中实现 40%loxP 的插入，与之前最先进的引发编辑器相比，这一效率提高了 24 倍。
