Core C: Defining regulators of immunity to acute infection using CRISPR screens

基因功能的系统表征，以及对基因功能障碍如何导致 疾病状态，是当今生物学面临的最大挑战之一。基因的数量乘以 表达它们的蜂窝状态的数量强调了可扩展的、全面的 不偏不倚，强大的技术干扰基因，以确定其功能。CRISPR-CAS9技术 代表了迄今为止描述的最有希望的基因组实验操作工具，以及一个中心 核心C的重点是这项技术的开发、压力测试和大规模部署。核心C将 支持本U19提案中的研究项目1和2，定义对急性感染免疫的调节器 使用CRISPR屏幕。 具体地说，我们将与计算核心(核心B)和每个 在小鼠模型中筛选一组目标基因的两个研究项目(目标1)。我们将生成 使用优化设计算法为每个项目汇集gRNA文库，最大限度地提高 试剂，使得每个基因能够使用少量的gRNA，从而能够筛选更多的基因。这些 然后，文库将以慢病毒的形式被生产出来，以便传递到细胞中。然后我们将使用鼠标筛选 核心(核心D)，以确定每个单独项目的最佳筛选条件(目标2)。核心D将 执行体内筛选，并将基因组DNA样本提供给Core C进行处理和分析。核心C 将对插入的gRNA进行聚合酶链式反应，通过Illumina对产品进行测序，并使用定制的软件来解卷 测序结果显示。最后，核心C将结合来自多个针对每个基因的gRNA的信息来定义 每个屏幕的点击率列表，使用STARS算法。这份黑名单是系统化网络的起点 按核心B进行分析。在主屏幕之后，核心C将支持个人的定制创建 重点随访所需的试剂(目标3)。这项支持包括设计和制作 单独的gRNA试剂，定制载体的创建，以及替代扰动的实施 技术，如CRISPRa、CRISPRi、ORF和RNAi。这三个目标加在一起将加速 对两个研究项目进行研究，并在多个模型中实现深入的功能知识 豁免权。

The systematic characterization of gene function, and the understanding of how gene dysfunction leads to disease states, is one of the largest challenges in biology today. The number of genes multiplied by the number of cellular states in which they are expressed emphasizes the need for scalable, comprehensive, unbiased, and robust techniques for perturbing genes to ascertain their functions. CRISPR-Cas9 technology represents the most promising tool yet described for experimental manipulation of the genome, and a central focus of Core C is the development, pressure testing, and deployment at-scale of this technology. Core C will support Research Projects 1 and 2 in this U19 proposal, “Defining regulators of immunity to acute infection using CRISPR screens.” Specifically, we will work with the computational core (Core B) and the individual investigators in each of the two Research Projects to curate a set of target genes for screening in mouse models (Aim 1). We will generate pooled libraries of gRNA for each project using optimized design algorithms that maximize the efficiency of the reagents, enabling the use of small numbers of gRNAs per gene and thus the screening of more genes. These libraries will then be produced as lentivirus for delivery into cells. We will then work with the mouse screening core (Core D) to determine optimal screening conditions for each of the individual projects (Aim 2). Core D will execute the in vivo screens and provide genomic DNA samples to Core C for processing and analysis. Core C will PCR the gRNA inserts, sequence the product via Illumina, and use customized software to deconvolute the sequencing reads. Finally, Core C will combine information from multiple gRNAs targeting each gene to define a hit list for each screen, using the STARS algorithm. This hit list is the starting point for systematic network analysis by Core B. Following primary screens, Core C will support the customized creation of individual reagents necessary for focused follow-up (Aim 3). This support includes the design and production of individual gRNA reagents, creation of customized vectors, and implementation of alternative perturbational technologies, such as CRISPRa, CRISPRi, ORFs, and RNAi. Together, these three aims will accelerate the research of both Research Projects and enable deep functional knowledge across multiple models of immunity.