Genome Editing With AAV-BR1-CRISPR In Postnatal Mouse Brain Endothelial Cells

The blood-brain barrier（BBB）is a unique and complex multicellular barrier structure in the central nervous system（CNS）.BBB is mainly composed of cerebral microvascular endothelial cells（ECs）,pericytes,basement membrane,and the end-feet of astrocytes.As a barrier structure between blood and brain,BBB provides a stable environment for the development and homeostasis of CNS.The loss of BBB integrity promotes the influx of neurotoxins,plasma proteins and immune cells from the blood,and then causes or aggravates a series of cerebrovascular and neurological diseases,such as ischemic stroke,Alzheimer’s disease（AD）and Parkinson’s disease（PD）.The core component of the BBB is specialized ECs,which are closely connected to each other to form a monolayer endothelial structure and act as the inner layer of cerebral vessels.Brain vascular ECs have several special characteristics to ensure the unique function of BBB.Under physiological conditions,the junctions between brain vascular ECs are tightly closed,allowing only small molecules（<400Da）to passively cross the BBB.In addition,very few vesicle transport and highly active efflux transporters on the surface of brain vascular ECs promote only a small but necessary substance exchange.Compared with peripheral organ-derived ECs,brain vascular ECs are rich in the expression of tight junctions and transporters.These characteristics play an extremely important role in maintaining the BBB homeostasis.Using genetically modified animals,researchers revealed important functional molecules and signal pathways involved in the development and maintenance of BBB homeostasis.Even so,our understanding of the mechanism of maintaining the adult BBB homeostasis is still very limited.In recent years,high throughput single cell sequencing enables to understand the heterogeneity and complexity of brain vascular ECs at the whole transcriptome level,and provides data resources for understanding the genetic and molecular basis of BBB function.However,only a small number of genes essential for BBB development and homeostasis maintenance have been verified by experiments.On the one hand,to develop an in vitro BBB model that accurately mimics the in vivo BBB,a very complex culture system must be employed,and some BBB characteristics will be lost in long-term culture of brain vascular ECs in vitro.On the other hand,the development of genetically modified mice is a time-consuming process.Overall,there is no technical method to quickly identify the essential brain endothelial genes essential for BBB homeostasis.Clustered regularly interspaced short palindromic repeats（CRISPR）and CRISPR-associated endonuclease（Cas）is an immune defense system found in most bacteria and all archaea,which is used to recognize and eliminate invading viruses and nucleic acids.The principle of CRISPR-Cas9 system as a genome editing tool is that Cas9 endonuclease recognizes and cuts the target DNA locus under the guidance of artificially designed short guide RNA（sg RNA）.Because the CRISPR-Cas9 gene editing tool has many advantages such as simplicity,time saving,high efficiency and low off-target effect,it is used for somatic gene editing and rapid identification of functional genes in various tissues,and construct disease animal models.AAV vector is the main vector for gene therapy in vivo.AAV is safe and capable of delivering its single stranded DNA vector genome to various tissues and different cell types,which causes mild immune response.As a CRISPR element delivery vector,AAV is widely used in somatic gene editing and disease animal model development.Recent studies have developed an AAV capsid variant,AAV-BR1,which has high specificity and long-term transduction efficiency for mouse brain vascular ECs,without infecting other tissues or cell types.In this study,we explored the feasibility and effectiveness of using AAV-BR1mediated CRISPR-Cas9 technology to edit adult mouse brain endothelial genes to construct BBB breakdown mouse model.We select major facilitator super family domain containing 2a（Mfsd2a）,which is critical to the integrity of BBB,as the target gene.We constructed the recombinant AAV2（r AAV2）vector containing sg RNA for Mfsd2a gene and Cre recombinase gene,and packaged it into AAV-BR1 virus（AAV-BR1-sg Mfsd2a-Cre）.We infected Cas9 gene knock-in mice（R26-lox P-STOP-lox P-3x FLAG-h Sp Cas9-e GFP）with AAV-BR1-sg Mfsd2a-Cre virus at a dose of 1.8×10¹¹ vg（viral genomes）.The brain vascular ECs expressed Cas9 gene under the action of Cre recombinase and Mfsd2a gene in brain vascular ECs was edited under the guidance of sg RNA for Mfsd2a,and we determined 11%were efficiently edited by in-depth DNA-sequencing analysis.Immunofluorescence results showed that the MFSD2A protein level was decreased,and electron microscope results showed that the vesicle density in the brain vascular ECs was increased.Sulfo-NHS-LC-biotin dye perfusion test showed that Mfsd2a gene editing caused BBB leakage in Cas9 gene knock-in mice.The above results suggested that AAV-BR1-sg Mfsd2a-Cre successfully induced Mfsd2a gene editing in brain vascular ECs of Cas9 gene knock-in mice,resulting in BBB breakdown.Given that the limitation of AAV packaging capacity and AAV-BR1 is able to infect a few scattered neurons,we obtained EC-specific Cas9 gene knock-in mice(Tie2^Cas9)by breeding Tie2-Cre transgenic mice with Cas9 gene knock-in mice to improve the transduction efficiency and specificity of AAV-BR1-CRISPR system to the brain vascular ECs.We selected cadherin-associated proteinβ1（Ctnnb1）,which plays an important role in maintaining BBB integrity,as the target gene.We packaged AAV-BR1 virus with sg RNA for Ctnnb1 and red fluorescent reporter gene td Tomato（AAV-BR1-sg Ctnnb1-td Tomato）and infected Tie2^Cas9 mice with a dose of 1.8×10¹¹vg.The results of flow sorting showed that the optimized system improved the transduction efficiency of cerebrovascular EC by about 40%.After infection,～36%of the target locus was mutated by in-depth DNA-sequencing analysis.Real-time PCR and Western Blot results confirmed that the m RNA and protein levels of Ctnnb1 were down-regulated.The results of immunofluorescence staining also showed that the protein level of CTNNB1 in cerebral ECs was decreased.Sulfo-NHS-LC-biotin dye perfusion results revealed that Ctnnb1 gene editing caused BBB leakage in Tie2^Cas9mice.The above results indicated that AAV-BR1-sg Ctnnb1-td Tomato virus targeting brain vascular ECs can effectively induce the editing of Ctnnb1 gene in brain vascular ECs,and lead to the destruction of BBB integrity in multiple brain regions of Tie2^Cas9mice.Taken together,we showed that the AAV-BR1-CRISPR system can effectively and specifically achieve gene editing in brain ECs of adult mice,demonstrating that the AAV-BR1-CRISPR system is a useful tool for screening genes essential for BBB integrity and generating BBB breakdown mouse models.