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核心技术专利:CN118964589B侵权必究
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使用 Prime Editing 技术高效生成同基因多能干细胞模型。

Highly efficient generation of isogenic pluripotent stem cell models using prime editing.

机构信息

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States.

Innovative Genomics Institute, University of California, Berkeley, Berkeley, United States.

出版信息

Elife. 2022 Sep 7;11:e79208. doi: 10.7554/eLife.79208.


DOI:10.7554/eLife.79208
PMID:36069759
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC9584603/
Abstract

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.

摘要

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。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/02de/9584603/619f1bc3f270/elife-79208-fig4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/02de/9584603/bc68087c3a9d/elife-79208-fig1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/02de/9584603/d4b61cfde06f/elife-79208-fig1-figsupp1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/02de/9584603/4308fd04099d/elife-79208-fig1-figsupp2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/02de/9584603/253c3a713ce1/elife-79208-fig1-figsupp3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/02de/9584603/d80e0eecaf69/elife-79208-fig2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/02de/9584603/2b58b16f5f7b/elife-79208-fig2-figsupp1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/02de/9584603/14fd6b7cf9ff/elife-79208-fig2-figsupp2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/02de/9584603/902229d15d9e/elife-79208-fig3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/02de/9584603/254542818a4a/elife-79208-fig3-figsupp1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/02de/9584603/619f1bc3f270/elife-79208-fig4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/02de/9584603/bc68087c3a9d/elife-79208-fig1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/02de/9584603/d4b61cfde06f/elife-79208-fig1-figsupp1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/02de/9584603/4308fd04099d/elife-79208-fig1-figsupp2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/02de/9584603/253c3a713ce1/elife-79208-fig1-figsupp3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/02de/9584603/d80e0eecaf69/elife-79208-fig2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/02de/9584603/2b58b16f5f7b/elife-79208-fig2-figsupp1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/02de/9584603/14fd6b7cf9ff/elife-79208-fig2-figsupp2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/02de/9584603/902229d15d9e/elife-79208-fig3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/02de/9584603/254542818a4a/elife-79208-fig3-figsupp1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/02de/9584603/619f1bc3f270/elife-79208-fig4.jpg

相似文献

[1]
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[2]
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[3]
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引用本文的文献

[1]
Precise correction of G6PD Viangchan mutation in iPSCs by prime editing strategy.

Sci Rep. 2025-8-18

[2]
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bioRxiv. 2025-3-18

[3]
Prime Editing: A Revolutionary Technology for Precise Treatment of Genetic Disorders.

Cell Prolif. 2025-4

[4]
Optimized Prime Editing of Human Induced Pluripotent Stem Cells to Efficiently Generate Isogenic Models of Mendelian Diseases.

Int J Mol Sci. 2024-12-26

[5]
Delivery of Prime editing in human stem cells using pseudoviral NanoScribes particles.

Nat Commun. 2025-1-4

[6]
Robust and inducible genome editing via an all-in-one prime editor in human pluripotent stem cells.

Nat Commun. 2024-12-30

[7]
From bench to bedside: cutting-edge applications of base editing and prime editing in precision medicine.

J Transl Med. 2024-12-20

[8]
Optimized prime editing of the Alzheimer's disease-associated APOE4 mutation.

Stem Cell Reports. 2025-1-14

[9]
Making gene editing accessible in resource limited environments: recommendations to guide a first-time user.

Front Genome Ed. 2024-9-25

[10]
Intercellular transmission of alpha-synuclein.

Front Mol Neurosci. 2024-9-11

本文引用的文献

[1]
Comprehensive analysis of prime editing outcomes in human embryonic stem cells.

Nucleic Acids Res. 2022-1-25

[2]
Generation of AAVS1 integrated doxycycline-inducible CRISPR-Prime Editor human induced pluripotent stem cell line.

Stem Cell Res. 2021-12

[3]
Enhanced prime editing systems by manipulating cellular determinants of editing outcomes.

Cell. 2021-10-28

[4]
Engineered pegRNAs improve prime editing efficiency.

Nat Biotechnol. 2022-3

[5]
Precise plant genome editing using base editors and prime editors.

Nat Plants. 2021-9

[6]
Evaluating CRISPR-based prime editing for cancer modeling and CFTR repair in organoids.

Life Sci Alliance. 2021-10

[7]
Precise correction of Duchenne muscular dystrophy exon deletion mutations by base and prime editing.

Sci Adv. 2021-4

[8]
CRISPR prime editing with ribonucleoprotein complexes in zebrafish and primary human cells.

Nat Biotechnol. 2022-2

[9]
Genome-wide specificity of prime editors in plants.

Nat Biotechnol. 2021-10

[10]
Prime editing in mice reveals the essentiality of a single base in driving tissue-specific gene expression.

Genome Biol. 2021-3-16

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