Viral Production and CRISPR Engineering

摘要 病毒生产和CRISPR工程核心 NEI UCSD中心核心拨款用于视觉研究病毒生产和CRISPR工程核心， 专注于生产腺相关病毒（AV）和慢病毒（LV）载体，以实现基因组 体内工程在过去的几年里，Shiley眼科研究所的教师投入了大量资金， 使用遗传工具如高通量功能基因组筛选（例如，Wahlin和Welsbie）， 人类遗传学（例如，Weinreb，Ayyagari，Borooah），以鉴定参与各种眼科疾病的基因。 疾病，包括青光眼、年龄相关性黄斑变性（AMD）和遗传性视网膜变性 （IRD）。为了能够在啮齿动物疾病模型中研究这些基因的功能，有必要 产生功能获得（GOF）和功能丧失（LOF）条件。通过将Cas9融合到转录 激活剂和抑制剂，以及CRISPR激活（CRISPRa）和抑制（CRISPRi）等技术 作为常规的Cas9介导的诱变，可用于产生必需的GOF和LOF 条件此外，正在开发新的CRISPR技术来治疗编辑特定碱基 使用同源定向修复（HDR）配对或重写较大的基因组片段。所有这些的关键是 编码这些大Cas9蛋白的基因以及优化的指导RNA（gRNA）的病毒递送 这是Cas9的指导。最后，AAV可用于表达cDNA（独立于CRISPR）。在所有情况下， 教师正在使用各种外部核心进行AAV生产，导致成本高，质量不一， 只有有限的试剂选择。病毒生产和CRISPR工程核心可以访问 紧凑的调控元件，其可用于产生递送Cas9以及 向导RNA。Core具有悬浮细胞培养装置，AAV和LV HEK 293 F生产细胞，Cytiva AKTA Pure快速蛋白液相色谱（FPLC）系统和BioRad数字液滴PCR（ddPCR） 用于AAV/LV生产、纯化和滴定的系统。有专门的兼职技术人员， 关于AAV和CRISPR工程的广泛专业知识将提供给教师。

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.