Expanding the CRISPR/Cas toolbox for RNA modulation

规则间隔短回文重复序列（CRISPR）/Cas系统已经广泛应用于临床。 用于DNA识别，实现基因组编辑和效应子招募等应用 蛋白质的特定位点，以调节转录或使直接成像。最近我们发现， 根据特定的修饰，Cas9能够结合活的哺乳动物细胞中的特定mRNA， 跟踪它们的运动，或促进靶RNA的降解，为许多RNA Cas蛋白的应用在这项提案中，我们寻求进一步扩展RNA的CRISPR/Cas工具箱 调变为了实现我们的目标，Corbett和Yeo实验室将合作使用蛋白质工程， 生物化学和细胞生物学技术，以（1）合理设计，然后验证和优化RNA靶向 （2）开发RNA靶向Cas9（RCas 9），以可视化 并在活细胞中以单分子分辨率追踪特定RNA;（3）开发RCas 9作为一种系统， 可编程编辑和/或靶向破坏人细胞中含有重复序列的mRNA，以及（4）适应 RCas 9用于使用化学诱导蛋白二聚化的动态RNA控制。完成工作 该提案中概述的内容将导致扩展的RNA靶向Cas蛋白工具箱， 通过直接编辑靶向RNA碱基，可编程切割疾病， 将含有重复序列的转录物以及其它选择的RNA与通用RNA内切核酸酶结合， 以及改变RNA代谢和翻译的动态控制手段。这些工具将提供一个 功能性转录组工程的基础，并在未来，使治疗的发展， 强直性肌营养不良、C9 ALS、亨廷顿氏病和其它由含重复序列的RNA引起的病症。

The Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system has become widely adopted for DNA recognition, enabling applications such as genome-editing and recruitment of effector proteins to specific loci, to modulate transcription or enable direct imaging. Recently we showed that depending on specific modifications, Cas9 is able to bind specific mRNAs in living mammalian cells allowing tracking of their movement, or promotes degradation of target RNAs, opening up the potential for many RNA applications of Cas proteins. In this proposal, we seek to further expand the CRISPR/Cas toolbox for RNA modulation. To achieve our goal, the Corbett and Yeo labs will team up to use protein engineering, biochemistry, and cell biology techniques to (1) rationally design, then validate and optimize RNA-targeting activity of minimized Cas9 proteins from multiple species; (2) develop RNA-targeting Cas9 (RCas9) to visualize and track specific RNAs at single-molecule resolution in live cells; (3) develop RCas9 as a system for programmable editing and/or targeted destruction of repeat-containing mRNAs in human cells, and (4) adapt RCas9 for dynamic RNA control using chemically-inducible protein dimerization. Completion of the efforts outlined in this proposal will result in an expanded RNA-targeting Cas protein toolbox that will allow multiplex engineering of the transcriptome via direct editing of targeted RNA bases, programmable cleavage of disease- associated repeat-containing transcripts as well as other RNAs-of-choice with a universal RNA endonuclease, as well as a dynamically-controlled means to alter RNA metabolism and translation. These tools will provide a foundation for functional transcriptome engineering and in the future, enable development of therapeutics for myotonic dystrophy, C9ALS, Huntington’s disease and other conditions caused by repeat-containing RNAs.