Genome engineering tools for functional screening of non-coding elements

描述(申请人提供)：自人类基因组计划完成以来，一个主要目标是了解人类基因组中的所有功能元件及其在正常生物过程和疾病中所起的作用。为此，已经开发了用于全基因组功能丧失筛选的大型RNA干扰试剂池(RNAi)试剂库，但受到三个问题的阻碍：1)部分敲除所固有的蛋白质耗尽的不完整性；2)种子序列的脱靶效应；以及3)不能转录的遗传元件无法操作。使用精确靶向核酸酶的基因组工程已经成为一种修改基因组并因果询问不同功能元件的作用的创新技术。最近，我开发了一种使用RNA引导的CRISPR/Cas9核酸酶进行功能基因组筛选的新技术(Shalem*，Sanjana*等人，2014年)。由于CRISPR在DNA水平上工作，因此有可能操纵RNAi无法访问的非编码元件。这项提议的研究目标是开发新的生物学工具和分析技术，用于使用汇集的CRISPR屏幕对非编码元件进行功能注释。指导阶段：首先，我计划开发和优化针对内含子、UTRs、启动子、非编码RNA和基因间隔区的高通量CRISPR非编码突变文库，以实现高分辨率和兆基覆盖的筛选。接下来，我将验证功能上的非编码元件，并使用这个大型数据集来寻找非编码元件如何调节基因表达的统一原则。独立阶段：我计划开发一种新的CRISPR架构，用于平铺删除屏幕，能够删除整个染色体甚至整个基因组的许多片段。有了这项技术及其增加的筛查能力，我将能够在几个可能的方向上开发一个长期的独立研究计划，包括进一步的基因组生物学、个性化功能基因组学以及药物与基因组相互作用的预测性诊断。帮助我实现研究目标所需的两个主要培训领域是1)CRISPR技术开发(导师：张峰博士)和2)人类遗传学和非编码变异知识(导师：David Altshuler博士)。每一位导师都是这些领域的知名专家。我的职业发展计划包括额外的实验室培训，世界专家提供的人类遗传学专业教程，我研究的地方和国家演示，以及科学写作、赠款和求职策略方面的课程。为了帮助做出与科学和职业相关的决策，我组建了一个咨询委员会，成员包括资深的基因组学专家：Eric Lander博士、Steven Hyman博士和David Root博士。远大研究所是一个理想的环境：所有的导师和顾问都位于一栋大楼里，并且在TH RNAi平台上有用于高通量功能筛选的设施(主任：Dr.Root)。

DESCRIPTION (provided by applicant): A major goal since the completion of the Human Genome Project has been to understand all functional elements in the human genome and the role they play in normal biological processes and disease. To that end, large pooled libraries of RNA interference (RNAi) reagents have been developed for genome-wide loss-of-function screens but have been hindered by 3 problems: 1) the incompleteness of protein depletion inherent in partial knock-down; 2) off-target effects from the seed sequence; and 3) genetic elements that are not transcribed are inaccessible to manipulation. Genome engineering using precisely targeted nucleases has emerged as an innovative technology to modify the genome and causally interrogate the role of different functional elements. Recently, I developed a new technology for functional genomic screening using the RNA- guided CRISPR/Cas9 nuclease (Shalem*, Sanjana*, et al., Science, 2014). Since CRISPR works on the DNA level, it is possible to manipulate non-coding elements that are inaccessible to RNAi. The research goal of this proposal is to develop new biological tools and analysis techniques for functional annotation of non-coding elements using pooled CRISPR screens. Mentored phase: First, I plan to develop and optimize high-throughput CRISPR non-coding mutagenesis libraries targeting introns, UTRs, promoters, non-coding RNAs, and intergenic regions to enable screening at high-resolution with megabase-scale coverage. Next, I will validate functional non-coding elements and use this large dataset to find unifying principles of how non-coding elements regulate gene expression. Independent phase: I plan to develop a novel CRISPR architecture for tiled deletion screens capable of deleting many segments over entire chromosomes or even entire genomes. With this technology and the increased screening throughput it enables, I will be able to develop a long-term independent research program in several possible directions, including further genome biology, personalized functional genomics, and predictive diagnostics for drug-genome interactions. The two primary areas of training needed to help me succeed in my research goals are 1) CRISPR technology development (mentor: Dr. Feng Zhang) and 2) knowledge of human genetics and non-coding variation (mentor: Dr. David Altshuler). Each mentor is an established expert in these fields. My career development plan integrates additional laboratory training, specialized tutorials in human genetics from world experts, local and national presentations of my research, and courses in scientific writing, grantsmanship and job search strategies. To assist with science- and career-related decisions, I have assembled an Advisory Committee with a team of established, senior genomics experts: Drs. Eric Lander, Steven Hyman, and David Root. The Broad Institute is an ideal environment: All Mentors and Advisors are located in one building and there are facilities for high-throughput functional screening in th RNAi Platform (Director: Dr. Root).