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

描述（由申请人提供）：细胞命运和身份的基因重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，一些基因的靶向操作已被证明是诱导多能干细胞（iPS）细胞衍生，干细胞分化和t细胞重组的有用方法。虽然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非常低，并且需要许多辅助因子，限制了它们在有效的细胞重编程中控制不同水平的调控和协调大量基因的动态表达。该研究的主要目标是开发一种新颖的模块化和可编程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，并开发其在iPS细胞重编程和潜在的其他基于细胞的治疗方法中的应用。提出的平台是建立在我最近展示的CRISPR（集群规则间隔短回文重复序列）干扰（CRISPRi）系统上的，该系统源自细菌免疫途径，这是一种rna引导的方法，用于在多种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（催化无活性）和一个设计的小向导RNA，该RNA与任何感兴趣的基因具有20个碱基对的互补区域，而不需要改变目标位点。为了进一步开发该系统作为一种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，作为模型来量化rna引导的调控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，以实现高效和特异性的基因组靶向。其次，为了开发各种调控功能的可编程平台，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。该系统将与蛋白质效应物相结合，用于不同类型的基因组尺度靶向调控，包括转录激活、转录沉默和

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.