优化的 Nickase 和核酸酶基的人源和鼠源细胞的 Prime 编辑。
Optimized nickase- and nuclease-based prime editing in human and mouse cells.
机构信息
School of Biomedicine and Robinson Research Institute, University of Adelaide, Adelaide, SA, Australia.
Genome Editing Program, South Australian Health and Medical Research Institute (SAHMRI), Adelaide, SA, Australia.
出版信息
Nucleic Acids Res. 2021 Oct 11;49(18):10785-10795. doi: 10.1093/nar/gkab792.
Precise genomic modification using prime editing (PE) holds enormous potential for research and clinical applications. In this study, we generated all-in-one prime editing (PEA1) constructs that carry all the components required for PE, along with a selection marker. We tested these constructs (with selection) in HEK293T, K562, HeLa and mouse embryonic stem (ES) cells. We discovered that PE efficiency in HEK293T cells was much higher than previously observed, reaching up to 95% (mean 67%). The efficiency in K562 and HeLa cells, however, remained low. To improve PE efficiency in K562 and HeLa, we generated a nuclease prime editor and tested this system in these cell lines as well as mouse ES cells. PE-nuclease greatly increased prime editing initiation, however, installation of the intended edits was often accompanied by extra insertions derived from the repair template. Finally, we show that zygotic injection of the nuclease prime editor can generate correct modifications in mouse fetuses with up to 100% efficiency.
使用精确基因组编辑（Prime Editing，PE）进行精准基因组修饰在研究和临床应用中具有巨大潜力。在本研究中，我们构建了携带 Prime Editing 所需所有组件和选择标记的一体化 Prime Editing（Prime Editing Assembly，PEA1）构建体。我们在 HEK293T、K562、HeLa 和小鼠胚胎干细胞（Embryonic Stem Cells，ES 细胞）中测试了这些构建体（带有选择标记）。我们发现，PE 在 HEK293T 细胞中的效率远高于之前的观察结果，最高可达 95%（平均 67%）。然而，在 K562 和 HeLa 细胞中的效率仍然较低。为了提高 K562 和 HeLa 中的 PE 效率，我们生成了一种核酸酶 Prime 编辑器，并在这些细胞系以及小鼠 ES 细胞中测试了该系统。PE-核酸酶大大增加了 Prime Editing 的起始效率，然而，预期编辑的安装常常伴随着来自修复模板的额外插入。最后，我们表明，核酸酶 Prime 编辑器的受精卵注射可以在高达 100%的效率下在小鼠胎儿中产生正确的修饰。
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