Nature biotechnologySep 30, 2024

Fast creation of long, chemically changed guide RNAs for prime editing

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Mar 25, 2026

Abstract

Approximately 90% production efficiency was achieved for chemically modified pegRNA and engineered pegRNA.

An efficient method for producing pegRNA and epegRNA has been developed.
L-epegRNA shows enhanced editing efficiencies, with improvements of over tenfold in RNP delivery and several hundredfold in RNA delivery compared to epegRNA from in vitro transcription.
L-epegRNA-mediated RNP delivery surpasses plasmid-encoded prime editing in most comparisons.
These advancements may facilitate the use of prime editing in therapeutic applications.

Simplified

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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Funding

Competing interests

Competing interests: H.Y., Ying Zhang, X.L. and X.W. have filed a patent application on ligation of pegRNA through Wuhan University (application number PCT/CN2024/078744). The other authors declare no competing interests.

PubMed

Fast creation of long, chemically changed guide RNAs for prime editing

Abstract

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Funding

Competing interests

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Abstract

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