Scalable and multimodal brain angiogenesis and blood-brain barrier genetics by somatic mutagenesis

The endothelial blood-brain barrier (BBB) is a complex neuroprotective structure whose maturation begins during brain angiogenesis and is completed postnatally. Despite its clinical importance, our understanding of BBB ontogeny and regulation remains limited. This shortcoming results from the difficulty of modeling the BBB in vitro and the current limitation of in vivo BBB genetics to low-throughput Mendelian genetic approaches. In addition, a single animal model facilitating the study of the neurovascular events occurring during both embryogenesis and adulthood is lacking. Here, leveraging the complementary anatomical and experimental attributes of mice and zebrafish, we present an integrated somatic CRISPR-Cas9 gene disruption strategy that allows us to assess candidate genes for their role in brain angiogenesis and BBB permeability within two and six weeks, respectively. Phenotypic assessment of brain angiogenesis is conducted in the transparent embryonic zebrafish hindbrain, while BBB maintenance is evaluated at both the behavioral and the functional level by combining endothelial-specific Cas9 transgenic mice and BBB-targeting adeno-associated viruses. The quick turnaround time and multiplexing capacity of our multi-species approach enable multimodal BBB functional genomics.