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

快速探索组合多样性的方法及其在 CRISPR-Cas9 系统中的应用

• 批准号: 10472843
• 负责人: Alejandro Chavez
• 金额: --
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-15 至 2022-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10472843
• 关键词: Adopted; Animal Model; Bar Codes; Behavior; Biological Process; CRISPR screen; CRISPR/Cas technology; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Data; Disease model; Engineering; Environment; Generations; Genes; Genetic Screening; Genetic Transcription; Goals; Interdisciplinary Study; Methods; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Parkinson Disease; Pathway interactions; Property; Proteins; Research; Series; System; Technology; Testing; Tissues; Variant; Work; base; cell type; chimeric antigen receptor; combinatorial; gene function; in vivo; in vivo Model; innovation; insight; mouse model; neuronal survival; novel; rapid technique; screening; stressor; success; tool

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.

项目总结/摘要： 几十年来，生物学家从不同的蛋白质中提取部分，并将它们融合成各种蛋白质。 组合以创建具有用户定义特性的工程变体。尽管成功的 许多生成的工具（例如嵌合抗原受体和增强的CRISPR变体） 发现这些蛋白质的方法是缓慢和劳动密集型的，限制了我们的研究。 只探索了一小部分潜在的蛋白质空间。在这里，我们介绍BArcoded 组合工程和筛选（BaCES），一种能够同时 数以万计的组合蛋白变体的组装和平行测试。客观 该提案的一个重要目的是使用BaCES创建新一代增强的基于Cas9的 转录调节因子，这将与一种新的实验范式相结合，以探测 基因在体内的功能。这一建议的基本理由是，如果成功， 我们将创造几种变革性的技术，并深入了解 神经元耐受神经退行性损伤。在这里，我们证明了我们的BaCES的可行性 平台，并提供证据支持我们独特的方法，在体内筛选。进一步 我们的研究目标，我们将：1）使用BaCES生成和量化27，000 Cas9的行为 激活子和抑制子; 2）彻底验证新一代靶点和细胞类型 高效力Cas9转录调节剂;和3）应用这些工具来执行一组在 体内遗传筛选，以揭示小鼠模型中神经元存活的调节因子， 帕金森氏症从技术角度来看，这项建议是创新的，因为它创造了一个 一种新的快速搜索组合蛋白质空间的方法，并实现了一种新的 这是在复杂的细胞环境中进行体内CRISPR筛选的范例。也是 创新的方法，因为它利用高通量平台来深入了解基因， 在体内疾病模型中调节神经元存活的途径。这项工作是 其意义在于它将创造一种进行组合蛋白筛选的新方法， 鉴定一组增强的Cas9激活子和阻遏子，以实现全球研究努力， 并揭示神经元用来耐受神经退行性疾病的生物学过程- 相关压力源。我们生产广泛采用的CRISPR工具的记录， 我们的初步数据证明了拟议工作的可行性和一组长期- 长期致力于合作者，使我们的团队特别适合执行概述的 跨学科研究