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 是当今生物学面临的最大挑战之一。基因的数量乘以 表达它们的细胞状态的数量强调了对可扩展的，全面的， 无偏的，和强大的技术扰动基因，以确定其功能。CRISPR-Cas9技术 是迄今为止描述的用于基因组实验操作的最有前途的工具， 核心C的重点是开发、压力测试和大规模部署这项技术。核心C将 支持U19提案中的研究项目1和2，“定义急性感染免疫调节剂 使用CRISPR筛选。” 具体来说，我们将与计算核心（核心B）和每个研究中的个体研究者一起工作。 两个研究项目，以策划一组靶基因，用于在小鼠模型中进行筛选（目标1）。我们将产生 使用优化的设计算法为每个项目汇集gRNA文库， 试剂，使得每个基因能够使用少量的gRNA，从而筛选更多的基因。这些 然后将文库作为慢病毒产生以递送到细胞中。然后我们将进行小鼠筛选 核心（核心D），以确定每个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样品提供给核心C进行处理和分析。芯C 将对gRNA插入物进行PCR，通过Illumina对产物进行测序，并使用定制的软件来解卷积 测序读数。最后，核心C将联合收割机结合来自靶向每个基因的多个gRNA的信息， 每个屏幕的命中列表，使用STARS算法。这份名单是系统网络的起点 核心B分析。在主屏幕之后，Core C将支持个性化创建 重点跟踪所需的试剂（目标3）。这种支持包括设计和生产 单个gRNA试剂、定制载体的创建以及替代微扰的实施 CRISPRa、CRISPRi、ORF和RNAi等技术。这三个目标将共同加速 研究两个研究项目，并实现跨多个模型的深入功能知识， 免疫力

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.