Viral Production and CRISPR Engineering

摘要 病毒生产与CRISPR工程核心 NEI UCSD中心用于视觉研究病毒生产和CRISPR工程核心的核心赠款是 专注于腺相关病毒(AAV)和慢病毒(LV)载体的生产，以允许基因组 活体工程学。在过去的几年里，Shiley Eye Institute的教职员工投入了大量资金 使用遗传工具，如高通量功能基因组筛选(例如，Wahlin和Welsbie)和 人类遗传学(如Weinreb、Ayagari、Borooah)以识别与各种眼科疾病有关的基因 疾病，包括青光眼、老年性黄斑变性(AMD)和遗传性视网膜变性 (IRDS)。为了能够研究这些基因在啮齿动物疾病模型中的功能，有必要 产生功能增益(GOF)和功能损失(LOF)条件。通过将Cas9与转录因子融合 激活剂和抑制物，以及CRISPR激活(CRISPRa)和抑制(CRISPRi)等技术 与传统的Cas9介导的诱变一样，可以用来产生必要的GOF和LOF 条件。此外，正在开发新的CRISPR技术来治疗编辑特定的碱基 使用同源定向修复(HDR)配对或重写更大的基因组片段。所有这些的关键是 编码这些大的Cas9蛋白的基因的病毒传递以及优化的引导RNA(GRNAs) 执导《九号赌场》。最后，AAV可用于表达cDNA(独立于CRISPR)。在所有情况下， 教职员工正在使用各种外部核心来生产AAV，导致成本高、质量不稳定和 只有有限的试剂可供选择。病毒制作和CRISPR工程核心可以访问 紧凑的监管元素，可用于创建一体化AAV载体，从而提供CAS9和 引导RNA。核心有悬浮细胞培养装置，AAV和LV HEK293F产生细胞，Cytiva AKTA纯快速蛋白质液相色谱(FPLC)系统和BioRad数字液滴聚合酶链式反应(DdPCR) 用于AAV/LV生产、纯化和滴定的系统。有专门的兼职技术员和 关于AAV和CRISPR工程的广泛知识将提供给教职员工。

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.