A novel RNA-guided platform for sequence-specific cell reprogramming

描述(由申请人提供)：对细胞命运和身份的遗传重新编程是基于细胞的治疗和组织再生的一种有前途的策略。特别是，靶向操作少数基因已被证明是诱导多能干细胞(IPS)来源、干细胞分化和T细胞重组的有用方法。尽管已经取得了快速的进展，但目前的靶向基因组操作方法效率很低，需要许多辅助因素，限制了它们在有效的细胞重新编程以控制不同水平的调控和协调大量基因的动态表达方面的有效性。该研究的主要目标是开发一种新型的模块化和可编程的RNA引导平台，可用于靶向基因组中的多个基因进行转录或表观遗传调节，并开发其在iPS细胞重新编程和潜在的其他基于细胞的治疗方法中的应用。建议的平台建立在我最近展示的CRISPR(集群规则间隔短回文重复)干扰系统(CRISPRi)的基础上，该系统源于细菌免疫途径，这是一种RNA引导的方法，可以在不同的宿主细胞中转录沉默任意基因。该方法只需要一个修饰的CaS蛋白(催化无效)和一个设计的具有20个碱基对的互补区域的小引导RNA，而不需要改变目标基因的基因。为了进一步将该系统开发为一个新的细胞重编程平台，我将首先在细菌中进行高通量表征实验，以量化RNA引导的调节效率和特异性的决定因素。数学模型将被开发并用于合理设计大型RNA文库，以实现高效和特定的基因组靶向。其次，为了开发一个可编程的平台来实现各种调控功能，我将把CRISPRi系统扩展为用于哺乳动物细胞的模块化DNA结合系统。该系统将与不同类型的基因组规模靶向调控的蛋白质效应器结合，包括转录激活、转录沉默和 可遗传的组蛋白修饰。我还将介绍使用以下工具来管理这些功能的功能 光，通过使用光基因控制的蛋白质相互作用。第三，作为试验台，我将重点介绍使用可编程CRISPRi平台作为生成iPS细胞的替代方法，或许更简单。我将创建光门转录电路，以精确控制内源性转录因子的表达程序，这些转录因子已知对iPS细胞重新编程非常重要。我还将使用CRISPRi平台来靶向和修改这些因素的表观遗传调控，并研究调控表观遗传标记是否可以实现更有效、更稳定和更安全的重新编程。总之，这些目标将通过提供一个新的RNA引导的平台来解决细胞重新编程的关键障碍，为各种类型的调节提供多个基因的序列特异性调节。此外，该应用将为构建协调iPS细胞产生的多个基因的基因电路提供新的技术基础，也直接适用于其他细胞重新编程应用，如干细胞分化和T细胞工程。

DESCRIPTION (provided by applicant): Genetic reprogramming of cell fate and identity is a promising strategy for cell-based therapies and tissue regeneration. In particular, targeted manipulation of a few genes has been shown as a useful method for induced pluripotent stem (iPS) cell derivation, stem cell differentiation, and T-cell reengineering. Though rapid progress has been made, current methods for targeted genome manipulation are very inefficient and require many accessory factors, limiting their utility for effective cell reprogramming to control different levels of regulation and to coordinate the kinetic expression of large numbers of genes. The major goal of the research is to develop a novel modular and programmable RNA-guided platform that can be used to target multiple genes in a genome for transcriptional or epigenetic regulation, and to exploit its applications for iPS cell reprogramming and potentially other cell-based therapeutic approaches. The proposed platform is built on my recently demonstrated CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) interference (CRISPRi) system derived from the bacterial immune pathway, which is an RNA-guided method for transcriptional silencing of arbitrary genes in diverse host cells. The method requires only a single modified Cas protein (catalytically inactive) and a designed small guide RNA with a 20-basepair complementary region to any gene of interest, without genetically altering the target locus. To further develop the system as a novel cell-reprogramming platform, I will first perform high-throughput characterization experiments in bacteria as a model to quantify the determinants of RNA-guided regulatory efficiency and specificity. Mathematical models will be developed and used for the rational design of large RNA libraries for efficient and specific genome targeting. Second, to develop a programmable platform for various regulatory functions, I will extend the CRISPRi system as a modular DNA-binding system for use in mammalian cells. The system will be combined with protein effectors for different types of genome-scale targeted regulations, including transcription activation, transcription silencing, and heritable histone modification. I will also introduce the ability to regulate these functions using light, through the use of optogenetically controlled protein interactions. Third, as a test bed, I ill focus on using the programmable CRISPRi platform as an alternative and perhaps easier way to generate iPS cells. I will create light-gated transcription circuits to precisely control the expression program of endogenous transcription factors that are known to be important for iPS cell reprogramming. I will also use the CRISPRi platform to target and modify epigenetic regulation of these factors, and study if regulating epigenetic marks could achieve more efficient, more stable, and safer reprogramming. Together, these aims will address a critical barrier for cell reprogramming by providing a novel RNA-guided platform for sequence-specific regulation of multiple genes for various types of regulation. Further, the application will providea novel technological basis for constructing gene circuits to coordinate multiple genes for iPS cell generation, which is also directly applicable to other cell reprogramming applications such as stem cell differentiation and T-cell engineering.