Molecular tools for targeting RNA

摘要 由于RNA测序的快速发展，科学家现在有能力轻松绘制转录组 细胞功能和疾病的变化，以补充功能基因组元件的强大目录， 变体。基因组编辑工具，特别是CRISPR-Cas9平台，允许快速控制和 操纵基因组序列进行遗传学研究。为了将这些工具扩展到直接和简便的 干扰RNA，拟议的工作旨在开发一套广泛的转录组工程工具 基于不同的RNA靶向CRISPR系统来补充RNAi。类似于DNA的dCas 9， 可编程的RNA结合将使模块化的功能模式包括和超越RNA敲低， 例如剪接异构体工程，通过利用生物信息学、生物化学和蛋白质组合 工程方法，以证明和优化系统的效用。使用患者源性诱导 多能干细胞分化为神经元作为模型系统，RNA错误剪接疾病将 靶向作为概念验证以调节可变剪接。这一提议预计将使一个强大的， 灵活的平台来询问基因表达，研究转录动力学，并剖析编码的功能， 和非编码转录本。

Abstract Due to rapid advances in RNA sequencing, scientists now have the ability to easily map transcriptome changes in cellular function and disease to complement a robust catalog of functional genome elements and variants. Genome editing tools, in particular the CRISPR-Cas9 platform, allow for rapid control and manipulation of genome sequences for genetic study. To extend these tools to the direct and facile perturbation of RNA, the proposed work aims to develop a broad suite of transcriptome engineering tools based on diverse RNA-targeting CRISPR systems to complement RNAi. Analogous to dCas9 for DNA, programmable RNA binding will enable modular modes of function including and beyond RNA knockdown, such as splice isoform engineering, by leveraging a combination of bioinformatic, biochemical, and protein engineering approaches to demonstrate and optimize the utility of the system. Using patient-derived induced pluripotent stem cells differentiated into neurons as a model system, RNA mis-splicing diseases will be targeted as a proof-of-concept to modulate alternative splicing. This proposal expects to enable a robust, flexible platform to interrogate gene expression, study transcript dynamics, and dissect the function of coding and noncoding transcripts.