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

描述（由申请人提供）：细胞命运和身份的遗传重编程是基于细胞的治疗和组织再生的有前途的策略。特别是，靶向操作的几个基因已被证明是一个有用的方法诱导多能干细胞（iPS）的衍生，干细胞分化，和T细胞再造。尽管已经取得了快速进展，但目前用于靶向基因组操作的方法非常低效，并且需要许多辅助因子，限制了它们用于有效细胞重编程以控制不同水平的调控和协调大量基因的动力学表达的效用。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控，并利用其在iPS细胞重编程和其他潜在的基于细胞的治疗方法中的应用。该平台建立在我最近展示的CRISPR（重复的规则间隔短回文重复序列）干扰（CRISPRi）系统上，该系统来自细菌免疫途径，这是一种RNA指导的方法，用于在不同宿主细胞中转录沉默任意基因。该方法仅需要单个修饰的Cas蛋白（无催化活性）和设计的小向导RNA，其具有与任何感兴趣的基因互补的20个碱基对的区域，而不会遗传地改变靶基因座。为了进一步开发该系统作为一种新型的细胞重编程平台，我将首先在细菌中进行高通量表征实验，作为量化RNA引导的调控效率和特异性的决定因素的模型。将开发数学模型，并用于合理设计大型RNA文库，以实现高效和特异的基因组靶向。其次，为了开发一个可编程的平台，用于各种调控功能，我将扩展CRISPRi系统作为一个模块化的DNA结合系统，用于哺乳动物细胞。该系统将与蛋白质效应器结合，用于不同类型的基因组规模靶向调控，包括转录激活，转录沉默， 遗传性组蛋白修饰我还将介绍使用 光，通过使用光遗传学控制的蛋白质相互作用。第三，作为一个试验平台，我将专注于使用可编程CRISPRi平台作为替代方案，也许更容易产生iPS细胞。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，这些转录因子对iPS细胞重编程至关重要。我还将使用CRISPRi平台来靶向和修改这些因子的表观遗传调控，并研究调节表观遗传标记是否可以实现更有效，更稳定，更安全的重编程。总之，这些目标将通过提供一种新的RNA指导平台来解决细胞重编程的关键障碍，该平台用于多种基因的序列特异性调控，以进行各种类型的调控。此外，该应用将为构建基因电路以协调用于iPS细胞生成的多个基因提供新的技术基础，这也可直接应用于其他细胞重编程应用，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.