Rationally designed Campylobacter jejuni Cas9 enables efficient gene activation and base editing
Compact and adaptable CRISPR-Cas systems enable genome engineering applications in various contexts via high-efficiency delivery. The adeno-associated virus (AAV) is a widely used delivery system. One of the most compact type II-C Cas9 orthologs-CjCas9, derived from <i>Campylobacter jejuni,</i> is particularly appealing for AAV delivery. However, the editing efficiency of CjCas9 limits its applications. In this study, we used structure-guided protein engineering to improve the editing efficiency of CjCas9. Subsequently, we developed a miniature transcriptional activator (LDE-CjCas9-VPR) and base editors engineered from CjCas9 (LDE-CjABE and LDE-CjCBE). LDE-CjABE effectively induced genome editing in human and mouse cells. Through AAV delivery, LDE-CjABE enhanced the on-target editing efficiency, and off-target editing was not detected in the mouse retina. Therefore, the compact size and high editing efficiency of LDE-CjCas9 broadens the target scope of transcription activation and base editing toolsets for therapeutic applications.
Authors
- Sihui Hu
- Chengxiang Peng
- Kaixin Cui
- Junjiu Huang (ORCID: https://orcid.org/0000-0002-3417-1196)
- Xiao Luo (ORCID: https://orcid.org/0000-0001-5864-0279)
- Zhou Songyang
- Guifang Wu
- Puping Liang (ORCID: https://orcid.org/0000-0003-3593-2599)
- Yuanling Jiang
- Yue Feng Yang (ORCID: https://orcid.org/0000-0002-0080-0353)
- Guanglan Wu (ORCID: https://orcid.org/0000-0003-1569-7923)
- Rui Kang (ORCID: https://orcid.org/0000-0003-2725-1574)
- Zhan Li (ORCID: https://orcid.org/0000-0003-1658-2378)
- Jiaqi Liu (ORCID: https://orcid.org/0000-0003-0009-3457)
- Pengfei Zhang (ORCID: https://orcid.org/0000-0003-3342-0872)
- Yongming Wang (ORCID: https://orcid.org/0000-0001-8269-5296)
- Peng Fei (ORCID: https://orcid.org/0000-0001-7895-4603)
- Yuxi Chen (ORCID: https://orcid.org/0000-0001-9450-8793)
- Weijun Zhao
- Qianyi Liu (ORCID: https://orcid.org/0000-0003-1347-8954)
- Qi Zheng
Institutions
- Sun Yat-sen University (CN)
- Fudan University (CN)
- Shanghai Micro Satellite Engineering Center (CN)
- Sun Yat-sen Memorial Hospital (CN)
- Zhongshan Hospital (CN)
Publication Details
- Journal
- Molecular Therapy — Nucleic Acids
- Published
- 2024-10-18
- DOI
- https://doi.org/10.1016/j.omtn.2024.102366
- Citations
- 6
- Primary Topic
- CRISPR and Genetic Engineering
- Type
- article
- Field-Weighted Citation Impact
- 1.40
Funders
- National Natural Science Foundation of China
- Sun Yat-sen University
- Ministry of Science and Technology of the People's Republic of China
- Guangdong Science and Technology Department
- Fundamental Research Funds for the Central Universities
- Basic and Applied Basic Research Foundation of Guangdong Province