Methods to Rapidly Explore Combinatorial Diversity and Their Application to CRISPR-Cas9 Systems

项目摘要/摘要： 几十年来，生物学家从不同的蛋白质中提取部分，并将它们融合在不同的 用于创建具有用户定义特性的工程变体的组合。尽管取得了成功， 产生的许多工具(例如嵌合抗原受体和增强型CRISPR变体) 发现这些蛋白质的方法是缓慢和劳动密集型的，限制了我们的 仅对潜在蛋白质空间的一小部分进行探索。在这里，我们介绍条形码 组合工程和筛选(BaCES)，一种能够同时 组装和并行测试数以万计的组合蛋白质变体。目标是 这项提议的目的是使用BaCES创建新一代基于Cas9的增强型 转录调节因子，这将与一种新的实验范式相结合来探索 基因在活体环境中发挥作用。这一提议背后的理由是，如果成功， 我们将创造几种变革性的技术，并深入了解 神经元可以忍受神经退化的侮辱。在这里，我们论证了我们的BaCES的可行性 平台，并提供支持我们体内筛查的独特方法的证据。为了进一步 我们的研究目标是：1)使用BaCES生成并量化27,000个Cas9的行为 激活物和抑制物；2)彻底验证新一代的靶点和细胞类型 高效的Cas9转录调节子；以及3)应用这些工具执行一组 活体基因筛查揭示小鼠模型中神经元存活的调节因素 帕金森氏症。这项建议从技术角度来看是创新的，因为它创建了一个 一种快速搜索组合蛋白质空间的新方法及其实现 在复杂的细胞环境中进行活体CRISPR筛选的范例。它也是 创新的方法，因为它利用高通量平台来洞察基因和 在体内疾病模型中调节神经元存活的途径。这项工作是 重要的是它将创建一种用于进行组合蛋白质筛选的新方法， 确定一组增强的Cas9激活子和抑制子，以支持全球研究努力， 并揭示神经元用来耐受神经退行性疾病的生物过程- 相关的压力源。我们生产被广泛采用的CRISPR工具的跟踪记录，结合 我们的初步数据证明了拟议工作的可行性，以及一组长期的- 坚定的合作伙伴，使我们的团队独一无二地适合执行概述的 跨学科研究。

PROJECT SUMMARY/ABSTRACT: For decades, biologists have taken parts from disparate proteins and fused them in various combinations to create engineered variants with user defined properties. Despite the success of many of the generated tools (e.g. chimeric antigen receptors and enhanced CRISPR variants) the methods by which these proteins are discovered are slow and labor intensive, limiting our exploration to only a tiny fraction of potential protein space. Here, we introduce BArcoded Combinatorial Engineering and Screening (BaCES), a method that enables the simultaneous assembly and parallel testing of tens of thousands of combinatorial protein variants. The objective of this proposal is to use BaCES to create a new generation of enhanced Cas9-based transcriptional regulators, which will be combined with a novel experimental paradigm to probe gene function within in vivo contexts. The rationale underlying this proposal is that, if successful, we will create several transformative technologies and gain insight into the mechanism by which neurons tolerate neurodegenerative insults. Herein we demonstrate the feasibility of our BaCES platform and provide evidence supporting our unique approach to in vivo screening. To further our research goals, we will: 1) use BaCES to generate and quantify the behavior of 27,000 Cas9 activators and repressors; 2) thoroughly validate across targets and cell types a new generation of highly-potent Cas9 transcriptional modulators; and 3) apply these tools to perform a set of in vivo genetic screens to uncover regulators of neuronal survival within a mouse model of Parkinson’s Disease. This proposal is innovative from a technical perspective in that it creates a new method for rapidly searching through combinatorial protein space and implements a new paradigm for performing in vivo CRISPR screens within a complex cellular environment. It is also innovative in approach as it utilizes a high-throughput platform to gain insight into the genes and pathways that regulate neuronal survival within an in vivo model of disease. This work is significant in that it will create a novel method for performing combinatorial protein screens, identify a set of enhanced Cas9 activators and repressors to enable global research endeavors, and uncover the biological processes that neurons use to tolerate neurodegenerative disease- associated stressors. Our track record of producing widely adopted CRISPR tools, combined with our preliminary data demonstrating the feasibility of the proposed work and a group of long- standing committed collaborators, makes our team uniquely suited to carry out the outlined interdisciplinary research.