SBIR Phase I: HiUGE Powered CRISPR Knock-In Library for Protein Tagging Across the Human Genome

这个小企业创新研究（SBIR）第一阶段项目的广泛影响/商业潜力影响了药物开发人员、转化研究人员和基础科学家追踪活细胞中蛋白质的能力。目前的方法耗时长、劳动密集、产量低。因此，对大多数研究人员来说，投资于这些方法是相对高风险的，而且通常只用于功能明确的蛋白质。该技术代表了一种快速和可扩展的蛋白质标记方法，允许研究人员在给定的研究中包括更广泛的感兴趣蛋白质（poi）。通过减少所需的初始投资，学术界和工业界的研究人员都有可能扩大特定研究中包含的poi。我们的期望是，这种方法的可扩展性和灵活性将加速药物筛选和开发活动，揭示更多有希望的主要候选药物。该项目将推进同源非依赖性通用基因组工程（HiUGE）方法的翻译，该方法已发展成为评估神经功能和发育中广泛蛋白质的手段。该项目推进了开发许多功能敲入载体的研究，允许荧光蛋白标记（FluorTag），功能破坏（DisrupTag）和下游蛋白质纯化（MassTag）。将这些载体与该项目中正在开发的全基因组文库配对，将使该公司能够快速提供表达感兴趣的蛋白质的细胞系，并进行定制修饰，所有这些都在内源性启动子控制下。该项目的具体目标是开发一个可扩展的高通量筛选平台，使用该技术标记人类基因组编码的每种蛋白质。技术任务包括：识别所有编码区域的基因特异性grna序列，使用定义的标准在所有蛋白质的N端和c端区域搜索和优先排序插入位点；建立一个中试规模的筛选文库，包括96个感兴趣基因中含有HiUGE插入位点的细胞；将新的自动化资源应用于所有20,000个蛋白质编码基因的规模。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

The broader impact/commercial potential of this Small Business Innovation Research (SBIR) Phase I project impacts the ability of drug developers, translational investigators, and basic scientists to track proteins in living cells. Current approaches are time-consuming, labor-intensive and low-throughput. Investing in these approaches is therefore relatively high-risk for most investigators and are most often employed only for proteins with well-defined functions. This technology represents a rapid and scalable method for protein labeling, allowing investigators to include a broader range of proteins of interest (POIs) in a given study. By reducing the initial investment required, both academic and industry investigators are likely to expand the POIs included in a given study. Our expectation is that the scalability and flexibility of this method will accelerate drug screening and development activities, revealing more promising lead candidates. The proposed project will advance translation of the homology-independent universal genome engineering (HiUGE) method, developed as a means of evaluating a broad range of proteins in neural function and development. This project advances research in developing a number of functional knock-in vectors, allowing fluorescent proteins labeling (FluorTag), functional disruption (DisrupTag), and downstream protein purification (MassTag). Pairing these vectors with the genome-wide library under development during this project will allow the company to rapidly provide cell lines expressing proteins of interest with customized modifications, all under endogenous promoter control. The specific goal of this project is to develop a scalable, high-throughput screening platform using the technology to label every protein encoded by the human genome. Technical tasks include: identify gene specific-gRNA sequences for all coding regions, searching and prioritizing insertion sites both at the N- and C-terminal regions of all proteins using defined criteria; develop a pilot scale screening library, comprising cells with HiUGE insertion sites in 96 genes of interest; apply new automation resources to scale for all 20,000 protein-encoding genes.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.