Naert Thomas, Yamamoto Taiyo, Han Shuting, Röck Ruth, Horn Melanie, Bethge Philipp, Vladimirov Nikita, Voigt Fabian F, Figueiro-Silva Joana, Bachmann-Gagescu Ruxandra, Vleminckx Kris, Helmchen Fritjof, Lienkamp Soeren S
Institute of Anatomy, University of Zurich, Zurich, Switzerland.
Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium.
Nat Biotechnol. 2025 Aug 12. doi: 10.1038/s41587-025-02771-0.
Precise CRISPR-based DNA integration and editing remain challenging, largely because of insufficient control of the repair process. We find that repair at the genome-cargo interface is predictable by deep learning models and adheres to sequence-context-specific rules. On the basis of in silico predictions, we devised a strategy of base-pair tandem repeat repair arms matching microhomologies at double-strand breaks. These repeat homology arms promote frame-retentive cassette integration and reduce deletions both at the target site and within the transgene. We demonstrate precise integrations at 32 loci in HEK293T cells. Germline-transmissible transgene integration and endogenous protein tagging in Xenopus and adult mouse brains demonstrated precise integration during early embryonic cleavage and in nondividing, differentiated cells. Optimized repair arms also facilitated small edits for scarless single-nucleotide or double-nucleotide changes using oligonucleotide templates in vitro and in vivo. We provide the design tool Pythia to facilitate precise genomic integration and editing for experimental and therapeutic purposes for a wide range of target cell types and applications.
基于CRISPR的精确DNA整合和编辑仍然具有挑战性，这主要是因为对修复过程的控制不足。我们发现，基因组-载体界面处的修复可以通过深度学习模型进行预测，并且遵循序列上下文特定的规则。基于计算机模拟预测，我们设计了一种在双链断裂处匹配微同源性的碱基对串联重复修复臂策略。这些重复同源臂促进了框架保留盒式整合，并减少了靶位点和转基因内的缺失。我们在HEK293T细胞的32个位点展示了精确整合。在非洲爪蟾和成年小鼠大脑中的种系可传递转基因整合和内源性蛋白质标记证明了在早期胚胎分裂期间以及在不分裂的分化细胞中实现了精确整合。优化后的修复臂还利用体外和体内的寡核苷酸模板促进了无痕单核苷酸或双核苷酸变化的小编辑。我们提供了设计工具Pythia，以促进针对广泛的靶细胞类型和应用，出于实验和治疗目的进行精确的基因组整合和编辑。
