Genome Editing with AAV-BR1-CRISPR in Postnatal Mouse Brain Endothelial Cells

• Published in: International journal of biological sciences Vol. 18; no. 2; pp. 652 - 660
• Main Authors: 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, Wang, Jun
• Format: Journal Article
• Language: English
• Published: Australia Ivyspring International Publisher Pty Ltd 2022; Ivyspring International Publisher
• Subjects: Animals; beta Catenin - genetics; Biocompatibility; Blood-brain barrier; Blood-Brain Barrier - metabolism; Cloning; CRISPR; CRISPR-Cas Systems; CTNNB1 gene; Dependovirus; Disease Models, Animal; Efficiency; Endothelial cells; Endothelial Cells - metabolism; Endothelium; Gene Editing; Gene Knockout Techniques; Gene loci; Genes; Genetic modification; Genomes; High-Throughput Nucleotide Sequencing; Homeostasis; Integrity; Intravenous administration; Kinases; Luminescent Proteins - genetics; Male; Mice; Mice, Transgenic; Molecular weight; Mutation; NIH 3T3 Cells; Physiology; Proteins; Red Fluorescent Protein; Research Paper; RNA, Guide, CRISPR-Cas Systems - genetics; Transgenic mice; Vascular endothelial growth factor; Viruses; β-Catenin; blood-brain barrier; genome editing; AAV-BR1; CRISPR/Cas9; brain endothelial cell
• ISSN: 1449-2288; 1449-2288
• DOI: 10.7150/ijbs.64188

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 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 ( ). We selected endothelial β-catenin ( ) gene, which is essential for maintaining adult BBB integrity, as the target gene. After intravenous injection of AAV-BR1-sg -tdTomato in 4-week-old transgenic mice resulted in mutation of 36.1% of the alleles, thereby leading to a dramatic decrease in the level of CTNNB1 in brain ECs. Consequently, 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 .