Quadruple pegRNA enables programmable and efficient large genomic insertion

  • Nature. 2026 Jun;654(8117):272-281. doi: 10.1038/s41586-026-10395-w.
Ya-Jing Shi  #  1 ,  Zi-Yi Ding  #  1 ,  Ying Wu  #  1 ,  Zhou He  2 ,  Yi-Zhou Zhang  1 ,  Yu-Lu Zhang  1 ,  Yu-Ming Zhang  1 ,  Xiang-Rui Huang  1 ,  Hao Yin  3  4  5 ,  Ying Zhang  6  7
Affiliations
  • 1. State Key Laboratory of Virology and Biosafety, Departments of Rheumatology and Immunology, Clinical Laboratory and Urology, Medical Research Institute, Frontier Science Center for Immunology and Metabolism, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, China.
  • 2. TaiKang Centre for Life and Medical Sciences, School of Artificial Intelligence, RNA Institute, Hubei Key Laboratory of Cell Homeostasis, Wuhan University, Wuhan, China.
  • 3. State Key Laboratory of Virology and Biosafety, Departments of Rheumatology and Immunology, Clinical Laboratory and Urology, Medical Research Institute, Frontier Science Center for Immunology and Metabolism, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, China. [email protected].
  • 4. TaiKang Centre for Life and Medical Sciences, School of Artificial Intelligence, RNA Institute, Hubei Key Laboratory of Cell Homeostasis, Wuhan University, Wuhan, China. [email protected].
  • 5. Shanghai Key Laboratory of Gene Editing and Cell Therapy for Rare Diseases, Shanghai, China. [email protected].
  • 6. State Key Laboratory of Virology and Biosafety, Departments of Rheumatology and Immunology, Clinical Laboratory and Urology, Medical Research Institute, Frontier Science Center for Immunology and Metabolism, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan, China. [email protected].
  • 7. TaiKang Centre for Life and Medical Sciences, School of Artificial Intelligence, RNA Institute, Hubei Key Laboratory of Cell Homeostasis, Wuhan University, Wuhan, China. [email protected].
  • # Contributed equally.
PMID: 42020736 DOI: 10.1038/s41586-026-10395-w
Abstract

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 pairs1-3. 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)4,5 and transposase-mediated (CAST)6 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.

Products