Mechanism, specificity, and design of CRISPR RNA-mediated gene regulation

项目摘要 规则间隔短回文重复序列（CRISPR）是最近发现的一种基于RNA的重复序列。 适应性免疫系统，保护细菌和古细菌免受外来DNA的侵害。CRISPR和相关的 (Cas)在90%的古生菌和40%的细菌中，包括许多人类病原体中，都发现了这种蛋白质。 这些免疫系统在控制毒力基因的水平转移方面也发挥着核心作用。的 CRISPR-Cas适应性免疫的中心步骤是通过可编程的RNA引导的外源DNA降解， 核酸酶。CRISPR-cas核酸酶-切割与CRISPR-cas核酸酶互补的靶DNA或RNA的酶。 指导CRISPR-RNA（crRNA）也被重新用于许多生物体中的精确基因调控。 尽管对这些酶的研究和临床应用都有浓厚的兴趣，但我们仍然缺乏一种新的方法。 完全理解其功能。我们的长期目标是了解CRISPR的机制- 介导的适应性免疫一个相关的目标是设计改进的CRISPR相关酶， 法规和其他生物技术应用。本提案中所述的目标将从机制上 剖析一个新发现的RNA引导核酸酶家族。为了实现这一目标，我们率先在高... 通量显微镜技术，可以成像多种酶，并记录其生化活动， 成千上万种不同的DNA底物。利用整合生物物理学的跨学科方法， 生物信息学和微/纳米尺度工程，我们将研究CRISPR相关的RNA引导的 核酸酶识别并切割靶DNA。首先，我们将确定核酸酶如何找到和切割一个 在染色质的背景下靶DNA。其次，我们将确定DNA的生物物理机制 在类似于在靶序列的脱靶位点处结合和切割。这些偏离目标的活动可以 导致意外突变，混淆研究实验并限制治疗应用。最后， 我们将设计和分析新型高保真酶，以减少脱靶活性并扩大基因组 工程工具包CRISPR介导的基因沉默和基因编辑为遗传学提供了一条令人兴奋的途径。 操纵真核细胞。我们预计，我们的研究结果将提供新的见解，了解 CRISPR相关的适应性免疫机制以及使用这些酶进行精确基因组 在科学和未来的治疗环境中的工程。

Project Summary Clustered regularly interspaced short palindromic repeats (CRISPRs) are a recently discovered RNA-based adaptive immune system that protects bacteria and archaea from foreign DNA. CRISPRs and the associated (Cas) proteins have been identified in 90% of archaea and 40% of bacteria, including many human pathogens. These immune systems also play a central role in controlling the horizontal transfer of virulence genes. The central step in CRISPR-Cas adaptive immunity is degradation of foreign DNA by a programmable RNA-guided nuclease. CRISPR-cas nucleases—enzymes that cleave a target DNA or RNA that is complementary to a guide CRISPR-RNA (crRNA)—have also been repurposed for precision gene regulation in many organisms. Despite the intense interest in these enzymes for both research and clinical applications, we still lack a complete understanding of their functions. Our long-term goal is to understand the mechanisms of CRISPR- mediated adaptive immunity. A related goal is to engineer improved CRISPR-associated enzymes for gene regulation and other biotechnological applications. The aims described in this proposal will mechanistically dissect a newly discovered family of RNA-guided nucleases. To achieve these aims, we pioneered high- throughput microscopy techniques that can image multiple enzymes and record their biochemical activities on tens of thousands of distinct DNA substrates. Using an interdisciplinary approach that integrates biophysics, bioinformatics, and micro-/nano-scale engineering, we will investigate how a CRISPR-associated RNA-guided nuclease recognizes and cleaves a target DNA. First, we will determine how the nuclease finds and cleaves a target DNA in the context of chromatin. Second, we will determine the biophysical mechanisms governing DNA binding and cleavage at off-target sites that resemble the on-target sequence. These off-target activities can cause unanticipated mutations that confound research experiments and limit therapeutic applications. Finally, we will engineer and profile novel high fidelity enzymes that reduce off-target activities and expand the genome engineering toolkit. CRISPR-mediated gene silencing and gene editing offers an exciting avenue for genetic manipulation of eukaryotic cells. We anticipate that our results will offer new insights in understanding the mechanisms of CRISPR-associated adaptive immunity and for using these enzymes for precision genome engineering in both scientific and future therapeutic settings.