Rapid generation of long, chemically modified pegRNAs for prime editing

IF 33.1 1区生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Nature biotechnology Pub Date : 2024-09-30 DOI:10.1038/s41587-024-02394-x

Xinlin Lei, Anhui Huang, Didi Chen, Xuebin Wang, Ruijin Ji, Jinlin Wang, Yizhou Zhang, Yuming Zhang, Shuhan Lu, Kun Zhang, Qiubing Chen, Ying Zhang, Hao Yin

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Abstract

The editing efficiencies of prime editing (PE) using ribonucleoprotein (RNP) and RNA delivery are not optimal due to the challenges in solid-phase synthesis of long PE guide RNA (pegRNA) (>125 nt). Here, we develop an efficient, rapid and cost-effective method for generating chemically modified pegRNA (125–145 nt) and engineered pegRNA (epegRNA) (170–190 nt). We use an optimized splint ligation approach and achieve approximately 90% production efficiency for these RNAs, referred to as L-pegRNA and L-epegRNA. L-epegRNA demonstrates enhanced editing efficiencies across various cell lines and human primary cells with improvements of up to more than tenfold when using RNP delivery and several hundredfold with RNA delivery of PE, compared to epegRNA produced by in vitro transcription. L-epegRNA-mediated RNP delivery also outperforms plasmid-encoded PE in most comparisons. Our study provides a solution to obtaining high-quality pegRNA and epegRNA with desired chemical modifications, paving the way for the use of PE in therapeutics and various other fields.

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快速生成用于素材编辑的化学修饰长 pegRNA

由于固相合成长PE引导RNA（pegRNA）（125 nt）的难题，使用核糖核蛋白（RNP）和RNA递送的质粒编辑（prime editing，PE）的编辑效率并不理想。在此，我们开发了一种高效、快速、经济的方法，用于生成化学修饰的 pegRNA（125-145 nt）和工程化的 pegRNA（epegRNA）（170-190 nt）。我们采用优化的夹板连接方法，这些 RNA 的生产效率约为 90%，分别称为 L-pegRNA 和 L-epegRNA。与体外转录产生的 epegRNA 相比，L-epegRNA 在各种细胞系和人类原代细胞中的编辑效率都有所提高，使用 RNP 运送时可提高 10 倍以上，使用 PE 的 RNA 运送时可提高几百倍。在大多数比较中，L-epegRNA介导的RNP递送也优于质粒编码的PE。我们的研究为获得具有所需化学修饰的高质量 pegRNA 和 epegRNA 提供了一种解决方案，为 PE 在治疗学和其他各种领域的应用铺平了道路。

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来源期刊

Nature biotechnology 工程技术-生物工程与应用微生物

CiteScore

63.00

自引率

1.70%

发文量

382

审稿时长

3 months

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