Viral Production and CRISPR Engineering

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ABSTRACT Viral Production and CRISPR Engineering Core The NEI UCSD Center Core Grant for Vision Research Viral Production and CRISPR Engineering Core is focused on the production of adeno-associated virus (AAV) and lentivirus (LV) vectors to allow for genome engineering in vivo. Over the last several years, Shiley Eye Institute faculty have been heavily invested in using genetic tools like high-throughput functional genomic screening (e.g., Wahlin and Welsbie) and human genetics (e.g., Weinreb, Ayyagari, Borooah) to identify genes involved in various ophthalmic diseases, including glaucoma, age-related macular degeneration (AMD) and inherited retinal degenerations (IRDs). To be able to study the function of these genes in rodent models of disease, it is necessary to produce gain-of-function (GOF) and loss-of-function (LOF) conditions. By fusing Cas9 to transcriptional activators and repressors, technologies like CRISPR activation (CRISPRa) and inhibition (CRISPRi), as well as conventional Cas9-mediated mutagenesis, can be used to produce the necessary GOF and LOF conditions. Moreover, novel CRISPR technologies are being developed therapeutically to edit specific base pairs or rewrite larger sections of genome using homology-directed repair (HDR). The key to all of these is the viral delivery of genes encoding these large Cas9 proteins as well as optimized guide RNAs (gRNAs) that direct Cas9. Finally, AAV can be used to express cDNAs (independent of CRISPRs). In all cases, faculty are using a variety of outside cores for AAV production, resulting in high costs, variable quality and only a limited selection of reagents. The Viral Production and CRISPR Engineering Core has access to compact regulatory elements that can be used to create all-in-one AAV vectors that deliver Cas9 as well as guide RNAs. The Core has a suspension cell culture setup, AAV and LV HEK293F producer cells, Cytiva AKTA Pure fast protein liquid chromatography (FPLC) system and BioRad digital droplet PCR (ddPCR) system for AAV/LV production, purification and titering. There is a dedicated part-time technician and extensive know-how regarding AAV and CRISPR engineering that will be available to faculty.