期刊
ACS SYNTHETIC BIOLOGY
卷 7, 期 4, 页码 978-985出版社
AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.7b00404
关键词
CRISPR/Cas9; gene activation; AAV; VPR
资金
- National Key Basic Research Program of China [2014CB745200]
- National Natural Science Foundation of China [31771483, 81772737]
- Shenzhen Municipal Government of China [JCYJ20170413161749433]
- Tsinghua National Lab for Information Science and Technology
Nuclease dead Cas9 (dCas9) has been widely used for modulating gene expression by fusing with different activation or repression domains. However, delivery of the CRISPR/Cas system fused with various effector domains in a single adeno-associated virus (AAV) remains challenging due to the payload limit. Here, we engineered a set of downsized variants of Cas9 including Staphylococcus aureus Cas9 (SaCas9) that retained DNA binding activity by deleting conserved functional domains. We demonstrated that fusing FokI nuclease domain to the N-terminal of the minimal SaCas9 (mini-SaCas9) or to the middle of the split mini-SaCas9 can trigger efficient DNA cleavage. In addition, we constructed a set of compact transactivation domains based on the tripartite VPR activation domain and self-assembled arrays of split SpyTag:SpyCatch peptides, which are suitable for fusing to the mini-SaCas9. Lastly, we produced a single AAV containing the mini-SaCas9 fused with a downsized transactivation domain along with an optimized gRNA expression cassette, which showed efficient transactivation activity. Our results highlighted a practical approach to generate down-sized CRISPR/Cas9 and gene activation systems for in vivo applications.
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