文献类型：期刊文献

英文题名：Dual-single-guide RNA strategy improves CRISPR-mediated homology-directed repair in Aspergillus

作者：Fu, Mingxin[1];Wang, Jing[1];Li, Jingyi[1];Zhou, Yao[1];Huang, Xiaofei[1];Jia, Zehan[1];Luo, Yiqing[1];Tan, Xinyu[1];Gao, Yan[1];Yu, Bingzi[1];Duan, Yuting[1];Bu, Qianyun[1];Li, Xiaoying[1];Wang, Yifan[1];Takaya, Naoki[2];Zhou, Shengmin[1,3]

机构：[1]East China Univ Sci & Technol, Sch Biotechnol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China;[2]Univ Tsukuba, Tsukuba Inst Adv Res, Fac Life & Environm Sci, Microbiol Res Ctr Sustainabil, Tsukuba, Ibaraki 3058572, Japan;[3]Peking Univ, State Key Lab Nat & Biomimet Drugs, Beijing 100191, Peoples R China

年份：2026

卷号：54

期号：4

外文期刊名：NUCLEIC ACIDS RESEARCH

收录：;WOS:【SCI-EXPANDED(收录号:WOS:001680166600001)】；

基金：This study was supported by the National Natural Science Foundation of China (22077032 and 21672065), the State Key Laboratory of Natural and Biomimetic Drugs (K202415).

语种：英文

摘要：CRISPR-Cas9 knock-in efficiency is often limited by geometric misalignment between donor DNA and the endogenous strand-invasion path. In Aspergillus nidulans, we found that integration drops sharply when the insertion site is offset from the invasion entry point, producing premature annealing or unsupported 3 ' ends that stall DNA synthesis. Chromatin immunoprecipitation-based profiling shows directional loading of the RAD51 homolog UvsC around Cas9-induced double-strand breaks, thereby defining the spatial origin of strand invasion. Guided by this insight, we introduce a dual-single-guide RNA design that places two cuts flanking the insertion site to create a geometry-matched strand-invasion window. This alignment consistently and markedly increases homology-directed-repair-mediated integration across insert sizes and editing tasks-including C-terminal tagging, bidirectional promoter rewiring, and long-distance dual-site mutagenesis-and generalizes across multiple fungal species. We propose a structural-docking model in which pairing fidelity between the resected chromosomal strand and donor homology arms governs knock-in outcomes, providing a practical design principle for efficient and precise genome engineering at structurally constrained loci.