四倍pegRNA使可编程和高效的大基因组插入，这一成果由武汉大学张楹团队经过不懈努力而取得。这一研究成果发表在2026年4月22日出版的国际学术期刊《自然》上。

在这里，课题组研究人员提出了一种合理设计的四倍pegRNA策略（QuadPE），用于高效和可编程的大DNA片段插入。通过筛选不同的设计，课题组研究人员发现两个基因组靶向pegRNA在PAM-out或PAM-in方向的组合，当与两个供体靶向pegRNA以线性或圆形形式配对时，产生最佳效率。使用QuadPE，该团队实现了稳定的DNA片段整合效率，范围在1.6到26在多个位点上的效率约为40%，并且脱靶插入活动最小。QuadPE明显优于重组酶介导的（PASSIGE和PASTE）和转座酶介导的（CAST）插入系统，特别是对于较大的有效载荷，在9.5分别插入KB。值得注意的是，QuadPE在分裂和非分裂原代细胞（如人类原代T细胞和有丝分裂后神经元）中都有效，这使QuadPE成为一个强大而精确的平台，无需双链断裂或重组酶即可插入大片段基因。

据悉，精确的、特定位点的大基因序列插入为治疗各种遗传疾病带来了巨大的希望。尽管启动编辑主题配对向导RNA（pegRNAs）可以介导靶向整合，但对于超过300个碱基对的有效载荷，插入效率会急剧下降。

附：英文原文

Title: Quadruple pegRNA enables programmable and efficient large genomic insertion

Author: Shi, Ya-jing, Ding, Zi-yi, Wu, Ying, He, Zhou, Zhang, Yi-zhou, Zhang, Yu-lu, Zhang, Yu-ming, Huang, Xiang-rui, Yin, Hao, Zhang, Ying

Issue&Volume: 2026-04-22

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 300base pairs1,2,3. 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 26kb, 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 9.5kb 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.

DOI: 10.1038/s41586-026-10395-w

Source: https://www.nature.com/articles/s41586-026-10395-w