Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States.
Innovative Genomics Institute, University of California, Berkeley, Berkeley, United States.
Elife. 2022 Sep 7;11:e79208. doi: 10.7554/eLife.79208.
The recent development of prime editing (PE) genome engineering technologies has the potential to significantly simplify the generation of human pluripotent stem cell (hPSC)-based disease models. PE is a multicomponent editing system that uses a Cas9-nickase fused to a reverse transcriptase (nCas9-RT) and an extended PE guide RNA (pegRNA). Once reverse transcribed, the pegRNA extension functions as a repair template to introduce precise designer mutations at the target site. Here, we systematically compared the editing efficiencies of PE to conventional gene editing methods in hPSCs. This analysis revealed that PE is overall more efficient and precise than homology-directed repair of site-specific nuclease-induced double-strand breaks. Specifically, PE is more effective in generating heterozygous editing events to create autosomal dominant disease-associated mutations. By stably integrating the nCas9-RT into hPSCs we achieved editing efficiencies equal to those reported for cancer cells, suggesting that the expression of the PE components, rather than cell-intrinsic features, limit PE in hPSCs. To improve the efficiency of PE in hPSCs, we optimized the delivery modalities for the PE components. Delivery of the nCas9-RT as mRNA combined with synthetically generated, chemically-modified pegRNAs and nicking guide RNAs improved editing efficiencies up to 13-fold compared with transfecting the PE components as plasmids or ribonucleoprotein particles. Finally, we demonstrated that this mRNA-based delivery approach can be used repeatedly to yield editing efficiencies exceeding 60% and to correct or introduce familial mutations causing Parkinson's disease in hPSCs.
近年来,prime editing (PE) 基因组编辑技术的发展有潜力显著简化基于人类多能干细胞 (hPSC) 的疾病模型的生成。PE 是一种多组分编辑系统,它使用融合了逆转录酶 (nCas9-RT) 和扩展的 PE 指导 RNA (pegRNA) 的 Cas9 核酸酶。一旦逆转录,pegRNA 延伸就作为修复模板,在目标位点引入精确的设计突变。在这里,我们系统地比较了 PE 与 hPSC 中常规基因编辑方法的编辑效率。该分析表明,PE 在总体上比靶向核酸酶诱导的双链断裂的同源定向修复更有效和精确。具体来说,PE 更有效地产生杂合编辑事件,以创建常染色体显性疾病相关突变。通过将 nCas9-RT 稳定整合到 hPSCs 中,我们实现了与报道的癌细胞相当的编辑效率,这表明 PE 组件的表达而不是细胞内在特征限制了 hPSCs 中的 PE。为了提高 PE 在 hPSCs 中的效率,我们优化了 PE 组件的递送方式。与转染 PE 组件作为质粒或核糖核蛋白颗粒相比,将 nCas9-RT 作为 mRNA 与合成生成的、化学修饰的 pegRNA 和 nicking 向导 RNA 一起递送可将编辑效率提高高达 13 倍。最后,我们证明这种基于 mRNA 的递送方法可重复使用,以产生超过 60%的编辑效率,并纠正或引入导致 hPSCs 中帕金森病的家族突变。
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