Viral Production and CRISPR Engineering

病毒生产和 CRISPR 工程

基本信息

批准号：
10709406
负责人：
Derek Stuart Welsbie
金额：
$ 8.86万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-01 至 2028-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10709406
关键词：
Age related macular degeneration Base Pairing CRISPR-mediated transcriptional activation CRISPR/Cas technology Cell Culture Techniques Cells Center Core Grants Clustered Regularly Interspaced Short Palindromic Repeats Communities Complementary DNA Culture Setup Dedications Dependovirus Development Disease Disease model Engineering Equipment Eye Eye diseases Faculty Genes Genetic Genome Genome engineering Glaucoma Guide RNA Human Genetics Human Resources Investments Lentivirus Lentivirus Vector Mediating Mutagenesis Production Proteins RNA Sequences Reagent Regulatory Element Rodent Model Services Suspensions System Technology Therapeutic Time Transcription Coactivator Transcription Repressor Viral Vision research Writing adeno-associated viral vector cost digital fast protein liquid chromatography functional genomics gain of function gene function in vivo inherited retinal degeneration loss of function novel repaired screening tool vector viral gene delivery

项目摘要

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.

抽象的病毒生产和 CRISPR 工程核心 NEI UCSD 中心视觉研究病毒生产和 CRISPR 工程核心核心资助是专注于生产腺相关病毒（AAV）和慢病毒（LV）载体以允许基因组体内工程。在过去的几年里，希利眼科研究所的教职员工在以下方面投入了大量资金：使用高通量功能基因组筛选等遗传工具（例如 Wahlin 和 Welsbie）以及人类遗传学（例如 Weinreb、Ayyagari、Borooah）来识别与各种眼科相关的基因疾病，包括青光眼、年龄相关性黄斑变性 (AMD) 和遗传性视网膜变性（税务局）。为了能够研究这些基因在啮齿动物疾病模型中的功能，有必要产生功能获得（GOF）和功能丧失（LOF）条件。通过将 Cas9 与转录融合激活剂和阻遏物，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，可用于产生必要的 GOF 和 LOF 状况。此外，正在开发新的 CRISPR 技术来编辑特定碱基使用同源定向修复（HDR）配对或重写基因组的较大部分。所有这些的关键是编码这些大 Cas9 蛋白以及优化引导 RNA (gRNA) 的基因的病毒传递直接Cas9。最后，AAV 可用于表达 cDNA（独立于 CRISPR）。在所有情况下，教师使用各种外部核心来生产 AAV，导致成本高、质量参差不齐仅有有限的试剂选择。病毒生产和 CRISPR 工程核心可以访问紧凑的调控元件，可用于创建一体化 AAV 载体，可传递 Cas9 以及引导RNA。 Core 具有悬浮细胞培养装置、AAV 和 LV HEK293F 生产细胞、Cytiva AKTA Pure 快速蛋白质液相色谱 (FPLC) 系统和 BioRad 数字微滴 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和有关 AAV 和 CRISPR 工程的丰富专业知识可供教师使用。