Molecular Mechanisms of Invader Silencing in Type III CRISPR-Cas Systems

项目摘要 簇状规则间隔短回文重复序列 相关系统是基于RNA的免疫系统，其功能是在原核生物中保护 对抗病毒和其他入侵的移动遗传因素。我们对这些的理解 适应性免疫系统的功能在过去十年中呈指数级增长。 重要的是，这些信息已经被利用和利用成了异常强大的， 用于基因组编辑和可预测的基因表达控制的新研究工具 广泛的工业和医疗应用。有多种不同的CRISPR-CA 通过不同的机制和不同的机制对抗病毒的系统，每个系统 提供独特的生物学和潜在的应用来探索。CRISPR-CAS系统雇用 入侵防御的三个一般步骤：适应、crRNA、生物发生和入侵沉默。 第一步是将入侵者DNA序列片段整合到CRISPR中 提供入侵和目标信息来源的可遗传记录的位置 (改编)。CRISPR基因座被转录，转录本被处理以产生 成熟的crRNA物种(crRNA生物发生)。CrRNA和Cas蛋白形成效应器 一种通过碱基配对寻找并破坏入侵的核酸的复合体 CrRNA和相关Cas蛋白核酸酶的破坏(入侵者沉默)。翻译 CRISPR-Cas效应复合体的研究产生了基因编辑的动力 CRISPR-CAS9来自类型II-A系统。我的研究重点是了解入侵者 由特征较差的III-A型或CSM效应器复合体沉默。第三类系统为 它们的独特之处在于它们既针对RNA，也针对DNA。这项提议的总体目标是获得 对核糖核酸和脱氧核糖核酸靶向活性的详细机制的理解 II-A CSM系统是通过相关的crRNA和 六种CaS蛋白。目的1阐明转录依赖的DNA的分子基础 被CSM系统瞄准。目标2是描述Csm6与Csm6的功能关系 CSM复合体及其在消灭入侵者中的作用一种强大的组合，主要建立在 体外和体内方法将被用来解决拟议的特定目标。这个 研究将有助于我们对CRISPR-CAS生物学的基本理解。此外, 所获得的知识将为开发新的III型CRISPR提供机会- 基于CAS的具有深远潜力的生物技术和生物医学技术 申请。

Project Summary CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated) systems are RNA-based immune systems that function in prokaryotes to protect against viruses and other invasive mobile genetic elements. Our understanding of these adaptive immune systems function has increased exponentially in the last ten years. Importantly, this information has been exploited and harnessed into exceptionally powerful, novel research tools for genome editing and predictable control of gene expression with widespread industrial and medical applications. There are multiple diverse CRISPR-Cas systems that combat viruses by distinct mechanisms and with distinct machinery, each providing unique biology and potential applications to explore. CRISPR-Cas systems employ three general steps in invader defense: adaptation, crRNA, biogenesis, and invader silencing. The initial step results in integration of a fragment of invader DNA sequence into the CRISPR locus to provide a heritable record of the invasion and source of targeting information (adaptation). The CRISPR locus is transcribed and the transcript is processed to produce mature crRNA species (crRNA biogenesis). The crRNA and Cas proteins form an effector complex that seeks out and destroys the invading nucleic acid through base-pairing of the crRNA and destruction by an associated Cas protein nuclease (invader silencing). Translational research on CRISPR-Cas effector complexes has yielded the gene editing powerhouse CRISPR-Cas9 from the Type II-A system. The focus of my research is to understand invader silencing by the less well characterized Type III-A or Csm effector complex. Type III systems are unique in that they target RNA as well as DNA. The overall objective of this proposal is to obtain a detailed mechanistic understanding of how the RNA and DNA targeting activities of the Type II-A Csm system are achieved through the concerted interactions of the associated crRNA and six Cas proteins. Aim 1 is to elucidate the molecular basis of transcription-dependent DNA targeting by the Csm system. Aim 2 is to delineate the functional relationship of Csm6 with the Csm complex and its role in invader elimination. A powerful combination of mostly established in vitro and in vivo approaches will be employed to address the proposed specific aims. The studies will contribute to our fundamental understanding of CRISPR-Cas biology. In addition, the knowledge obtained will afford opportunities for the development of novel Type III CRISPR- Cas-based technologies with far reaching potential biotechnological and biomedical applications.