1. Genome Editing with AAV-BR1-CRISPR in Postnatal Mouse Brain Endothelial Cells.
- Author
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Song X, Cui Y, Wang Y, Zhang Y, He Q, Yu Z, Xu C, Ning H, Han Y, Cai Y, Cheng X, Wang J, Teng Y, Yang X, and Wang J
- Subjects
- Animals, Blood-Brain Barrier metabolism, CRISPR-Cas Systems, Disease Models, Animal, Gene Knockout Techniques, High-Throughput Nucleotide Sequencing, Male, Mice, Mice, Transgenic, NIH 3T3 Cells, RNA, Guide, CRISPR-Cas Systems genetics, Red Fluorescent Protein, Dependovirus, Endothelial Cells metabolism, Gene Editing, Luminescent Proteins genetics, beta Catenin genetics
- 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 ( Tie2
Cas9 ). 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-sg Ctnnb1 -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 ., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)- Published
- 2022
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