Engineering the RNA-guided Cascade complex for genome editing

对于人类来说，花了几千代人的时间才发展出现代驯化的动植物。自托马斯摩根时代以来，科学家们一直在寻找直接操纵不同生物体基因组和修复人类基因缺陷的方法。复杂基因组的精确修饰一直是基础科学和应用科学领域科学家的主要目标之一。目前用于基因组工程的分子工具包括锌指核酸酶（ZFN）和转录激活因子样效应物核酸酶（TALEN）。这些蛋白质引导的基因组工程系统的主要缺点是，每个新靶序列的校正需要费力的努力来设计特异性识别该靶序列的新核酸酶。因此，基因工程仍然需要负担得起、易于使用和有效的新技术。 最近，微生物CRISPR/Cas系统迅速成为一种RNA引导的基因组编辑工具，用于强大且可多路复用的基因组工程。在CRISPR机制中，短CRISPR RNA（crRNA）将CRISPR相关蛋白复合物（Cascade）或核酸酶（Cas9）引导至互补DNA或RNA序列，从而促进特异性靶标结合和切割。几种Cas9核酸酶用于基因组编辑和调控的成功应用已经在细菌、酵母、植物和人类细胞中得到证实。然而，Cascade复合物用于基因组编辑的应用仍有待探索。这项研究的长期目标是了解Cascade活性的分子机制，并设计新的RNA指导系统，以实现高效的基因组编辑。使用纯化的Cascade蛋白及其复合物，我们将表征它们与合成crRNA和靶DNA的相互作用。对于序列特异性DNA切割和基因组编辑，我们将在工程化Cascade复合物中引入核酸酶结构域。这些研究将有助于优化工程化Cascade复合物的蛋白质和RNA组分，以促进它们在靶细胞中的递送和组装。将使用大肠杆菌和酵母细胞作为模型系统在体内表征工程化级联复合物的靶识别和切割的特异性。我们的工作将促进新型RNA引导的分子工具在基因组编辑，基因治疗和生物技术中的应用。

For humans, it took thousands of generations to develop modern domesticated plants and animals. Since the time of Thomas Morgan, scientists have been seeking methods to directly manipulate genomes of different organisms and fix faulty human genes. Precise modifications of complex genomes have been one of the major goals for scientists working in basic and applied science. Molecular tools that are currently used for genome engineering include zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). A major disadvantage of these protein-guided genome engineering systems is that the correction of each new target sequence requires laborious efforts to design a new nuclease that specifically recognizes this target sequence. Therefore, new technologies that are affordable, easy to use and efficient are still needed for the genetic engineering.        Recently, the microbial CRISPR/Cas system has rapidly emerged as an RNA-guided genome editing tool for robust and multiplexable genome engineering. In the CRISPR mechanism, short CRISPR RNAs (crRNAs) guide the CRISPR-associated protein complexes (Cascade) or nucleases (Cas9) to complementary DNA or RNA sequences promoting specific target binding and cleavage. Successful applications of several Cas9 nucleases for genome editing and regulation have already been demonstrated in bacteria, yeast, plants, and human cells. However, the application of the Cascade complex for genome editing remains to be explored. The long-term objective of this research is to understand the molecular mechanisms of activity of Cascade and to design novel RNA-guided systems for efficient genome editing. Using the purified Cascade proteins and their complexes, we will characterize their interaction with synthetic crRNAs and target DNAs. For sequence-specific DNA cleavage and genome editing, we will introduce a nuclease domain into the engineered Cascade complex. These studies will help to optimize the protein and RNA components of engineered Cascade complexes to facilitate their delivery and assembly in targeted cells. The specificity of target recognition and cleavage by engineered Cascade complexes will be characterized in vivo using Escherichia coli and yeast cells as model systems. Our work will promote the development of novel RNA-guided molecular tools for applications in genome editing, gene therapy and biotechnology.