Nature biomedical engineeringApr 7, 2026

Improving prime editing using specially designed guide RNAs

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Jun 27, 2026

Abstract

Essence

Non-canonical pegRNAs substantially boosted prime-editing RNP efficiency across cells and a mouse disease model.

Evidence

This genome-editing engineering study tested loop-integrated npegRNAs across genomic sites and cell types, a tyrosinaemia mouse model, and human cell lines, with average RNP editing yields 26.8-fold above canonical pegRNAs and 5.9-fold above epegRNAs.

Caveat

The work reports editing efficiency and gene-correction performance, but therapeutic application remains preclinical and dependent on tested sites, cells, and delivery context.

Simplified

Prime editing (PE) enables precise genetic modifications using canonical prime editing guide RNA (pegRNA), with the reverse transcription template and primer binding site (RTT-PBS) attached to the 3' ends of CRISPR-Cas guide RNAs. Although PE ribonucleoprotein (RNP) delivery holds great therapeutic potential, its weak genomic editing capability limits therapeutic applications. Here we present structure-guided engineering of the PE complex using non-canonical pegRNAs (npegRNAs), with the RTT-PBS integrated within the single guide RNA loops, to improve PE efficiency. This approach demonstrates enhanced precise editing rates across various genomic sites and cell types, and improves therapeutic gene correction in a tyrosinaemia mouse model. Cas9-associated npegRNAs are more resistant to exonuclease degradation, probably enhancing the PE complex's targeting efficiency in living cells. Using PE RNP delivery, npegRNAs achieve increased average editing yields of 26.8-fold over canonical pegRNAs and 5.9-fold over engineered pegRNAs (epegRNAs). Furthermore, npegRNA-mediated RNPs increased the efficiency of installing disease-relevant mutations up to 123-fold in human cell lines, including Jurkat T cells and induced pluripotent stem cells. Collectively, our findings demonstrate a robust PE strategy and highlight the potential of npegRNAs for therapeutic PE applications.