Efficient high-precision transgene knock-in by Recombinases (Redα/β)-enhanced DNA integration-CRISPR-Cas9 (RED-CRISPR)
CRISPR-Cas9 tools have revolutionized genetic engineering, yet the efficient precise integration of DNA cargos, particularly for large DNA payloads (>1 kilobase, kb), remains a technical bottleneck. Herein, we develop a Recombinases (Redα/β)-enhanced DNA integration-CRISPR-Cas9 approach, referred to as RED-CRISPR, which offers a versatile yet robust homology-directed repair (HDR) strategy enabling efficient and precise kb-scale DNA insertion across various cell types, including immortalized and primary cells of variable origins. RED-CRISPR significantly enhances HDR efficiencies by 2- to 5-fold change across diverse loci and further elevates HDR rates by 1.5- to 2.5-fold when synergizing with other HDR-enhancing strategies. We achieved up to 45% knock-in efficiency for CAR-T cell manufacturing, and attained 43% knock-in rate for generation of genetically modified mice using an 8-kb DNA cargo. Through a head-to-head comparison, RED-CRISPR profoundly mitigates off-target mutational burden and chromosomal translocations. We envision RED-CRISPR as a powerful genome-editing tool with broad biomedical and therapeutic applications.
Authors
- Chengkun Wang
- Fudong Li (ORCID: https://orcid.org/0000-0002-9434-3762)
- Xiaoyu Fang (ORCID: https://orcid.org/0000-0002-4168-3130)
- Jiaqi Zou
- Jianqiang Bao (ORCID: https://orcid.org/0000-0003-1248-2687)
- Qin Jiang
- Senquan Liu (ORCID: https://orcid.org/0000-0003-1444-5994)
- Wenqing Li (ORCID: https://orcid.org/0009-0000-6591-9059)
- Xiaoxiao Gao (ORCID: https://orcid.org/0000-0002-3153-9370)
- Wenjie Han (ORCID: https://orcid.org/0000-0002-2779-9196)
- Hongbin Liu (ORCID: https://orcid.org/0000-0003-2550-7492)
- Linzhao Cheng (ORCID: https://orcid.org/0009-0003-2128-8525)
- Xuemei Xing
- Xiaoli Zhu
- Yuzhu Cao (ORCID: https://orcid.org/0000-0001-5175-0643)
- Xiaolin Min
- Muhammad Azhar
- Youming Zhang (ORCID: https://orcid.org/0009-0004-1702-6335)
Institutions
- University of Science and Technology of China (CN)
- Shandong University (CN)
- Emmanuel College - Massachusetts (US)
- Hefei National Center for Physical Sciences at Nanoscale (CN)
- Cell Technology (China) (HK)
- Shandong University of Science and Technology (CN)
- Nanjing Medical University (CN)
Publication Details
- Journal
- Nature Communications
- Published
- 2025-12-10
- DOI
- https://doi.org/10.1038/s41467-025-67239-w
- Primary Topic
- CRISPR and Genetic Engineering
- Type
- article
- Field-Weighted Citation Impact
- 0.00
Funders
- National Natural Science Foundation of China
- University of Science and Technology of China
- Natural Science Foundation of Anhui Province
- Fundamental Research Funds for the Central Universities