NatureApr 22, 2026

Using four pegRNAs to efficiently insert large DNA sequences into the genome

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Jun 27, 2026

Abstract

Essence

QuadPE enabled programmable large DNA insertions with high efficiency across multiple loci and primary cell types without double-stranded breaks or recombinases.

Evidence

This genome-editing platform experiment screened quadruple pegRNA designs and tested 1.6 to 26 kb insertions, reporting around 40% stable integration at multiple loci and improved 9.5 kb insertion versus PASSIGE, PASTE, and CAST systems.

Caveat

The evidence is platform and cell-model performance data, so therapeutic efficacy, delivery, and safety in living organisms or patients remain outside the abstract's scope.

Simplified

Precise, site-specific insertion of large gene sequences holds great promise for the treatment of diverse genetic disorders. Although prime editing using paired guide RNAs (pegRNAs) can mediate targeted integration, insertion efficiency drops sharply for payloads exceeding 300 base pairs. Here we present a rationally designed quadruple pegRNA strategy (QuadPE) for efficient and programmable insertion of large DNA fragments. Through screening different designs, we identified that combinations of two genome-targeting pegRNAs in a PAM-out or PAM-in orientation, when paired with two donor-targeting pegRNAs in linear or circular form, yield optimal efficiency. Using QuadPE, we achieved stable integration efficiency of DNA fragments ranging from 1.6 to 26 kb, with efficiencies of around 40% at multiple loci with minimal off-target insertion activity. QuadPE substantially outperformed recombinase-mediated (PASSIGE and PASTE)and transposase-mediated (CAST)insertion systems, particularly for larger payloads, showing a 11-fold, 61-fold and 12-fold improvement for a 9.5 kb insertion, respectively. Notably, QuadPE was effective in both dividing and non-dividing primary cells such as human primary T cells and post-mitotic neurons, establishing QuadPE as a powerful and precise platform for large-fragment gene insertion without the need for double-stranded breaks or recombinases. 1-3 4,5 6