Induction of Brain Arteriovenous Malformation Through CRISPR/Cas9-Mediated Somatic Alk1 Gene Mutations in Adult Mice
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Brain arteriovenous malformation (bAVM) is an important risk factor for intracranial hemorrhage. The pathogenesis of bAVM has not been fully understood. Animal models are important tools for dissecting bAVM pathogenesis and testing new therapies. We have developed several mouse bAVM models using genetically modified mice. However, due to the body size, mouse bAVM models have some limitations. Recent studies identified somatic mutations in sporadic human bAVM. To develop a feasible tool to create sporadic bAVM in rodent and animals larger than rodent, we made tests using the CRISPR/Cas9 technique to induce somatic gene mutations in mouse brain in situ. Two sequence-specific guide RNAs (sgRNAs) targeting mouse Alk1 exons 4 and 5 were cloned into pAd-Alk1e4sgRNA + e5sgRNA-Cas9 plasmid. These sgRNAs were capable to generate mutations in Alk1 gene in mouse cell lines. After packaged into adenovirus, Ad-Alk1e4sgRNA + e5sgRNA-Cas9 was co-injected with an adeno-associated viral vector expressing vascular endothelial growth factor (AAV-VEGF) into the brains of wild-type C57BL/6J mice. Eight weeks after viral injection, bAVMs were detected in 10 of 12 mice. Compared to the control (Ad-GFP/AAV-VEGF-injected) brain, 13% of Alk1 alleles were mutated and Alk1 expression was reduced by 26% in the Ad-Alk1e4sgRNA + e5sgRNA-Cas9/AAV-VEGF-injected brains. Around the Ad-Alk1e4sgRNA + e5sgRNA-Cas9/AAV-VEGF injected site, Alk1-null endothelial cells were detected. Our data demonstrated that CRISPR/Cas9 is a feasible tool for generating bAVM model in animals.
KeywordsAlk1 CRISPR/Cas9 Brain arteriovenous malformation Somatic gene mutation
This study was supported by grants to Dr. Hua Su from the NIH (R01 NS027713 and R01 HL122774) and the Michael Ryan Zodda Foundation.
Compliance with Ethical Standards
Conflict of Interest
The authors declare that they do not have conflict of interest.
All applicable international, national, and institutional guidelines for the care and use of animals were followed. The protocol and experimental procedures for using laboratory animals were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of California, San Francisco. Animal husbandry and veterinary cares were provided by staffs in the IACUC and the Animal Core Facility.
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