1. Genome Editing with AAV-BR1-CRISPR in Postnatal Mouse Brain Endothelial Cells
- Author
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Song, Xiaopeng, Cui, Yaxiong, Wang, Yanxiao, Zhang, Yizhe, He, Qi, Yu, Zhenyang, Xu, Chengfang, Ning, Huimin, Han, Yuying, Cai, Yunting, Cheng, Xuan, Wang, Jian, Teng, Yan, Yang, Xiao, and Wang, Jun
- Subjects
Male ,Mice, Transgenic ,Applied Microbiology and Biotechnology ,Gene Knockout Techniques ,Mice ,genome editing ,Animals ,CRISPR/Cas9 ,Molecular Biology ,beta Catenin ,Ecology, Evolution, Behavior and Systematics ,Gene Editing ,AAV-BR1 ,Endothelial Cells ,High-Throughput Nucleotide Sequencing ,Cell Biology ,blood-brain barrier ,Dependovirus ,brain endothelial cell ,Disease Models, Animal ,Luminescent Proteins ,NIH 3T3 Cells ,CRISPR-Cas Systems ,Research Paper ,RNA, Guide, Kinetoplastida ,Developmental Biology - Abstract
Brain endothelial cells (ECs) are an important component of the blood-brain barrier (BBB) and play key roles in restricting entrance of possible toxic components and pathogens into the brain. However, identifying endothelial genes that regulate BBB homeostasis remains a time-consuming process. Although somatic genome editing has emerged as a powerful tool for discovery of essential genes regulating tissue homeostasis, its application in brain ECs is yet to be demonstrated in vivo. Here, we used an adeno-associated virus targeting brain endothelium (AAV-BR1) combined with the CRISPR/Cas9 system (AAV-BR1-CRISPR) to specifically knock out genes of interest in brain ECs of adult mice. We first generated a mouse model expressing Cas9 in ECs (Tie2Cas9). We selected endothelial β-catenin (Ctnnb1) gene, which is essential for maintaining adult BBB integrity, as the target gene. After intravenous injection of AAV-BR1-sgCtnnb1-tdTomato in 4-week-old Tie2Cas9 transgenic mice resulted in mutation of 36.1% of the Ctnnb1 alleles, thereby leading to a dramatic decrease in the level of CTNNB1 in brain ECs. Consequently, Ctnnb1 gene editing in brain ECs resulted in BBB breakdown. Taken together, these results demonstrate that the AAV-BR1-CRISPR system is a useful tool for rapid identification of endothelial genes that regulate BBB integrity in vivo.
- Published
- 2022