RNA-Guided Gene Targeting in Human Cells

RNA 引导的人类细胞基因靶向

• 批准号: 8647089
• 负责人: Jiwu Wang
• 金额: $ 15.31万
• 依托单位: ALLELE BIOTECHNOLOGY AND PHARMACEUTICALS
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-01 至 2014-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8647089
• 关键词: Adopted; Alleles; Archaea; Area; Bacteria; Base Pairing; Benchmarking; Cells; Chromosomes; Clustered Regularly Interspaced Short Palindromic Repeats; Codon Nucleotides; Complex; Culture Media; Custom; DNA; DNA Sequence; Deoxyribonuclease I; Development; Engineering; Enzymes; Eukaryotic Cell; Fibroblasts; Foundations; Gene Expression; Gene Targeting; Gene-Modified; Genes; Genetic Recombination; Genome; Genome engineering; Goals; Guide RNA; Human; Human Genome; Kinetics; Laboratories; Life; Malignant Neoplasms; Mammalian Cell; Mammalian Chromosomes; Marketing; Measures; Mediating; Messenger RNA; Modification; Molecular and Cellular Biology; Mutate; Nucleotides; Phase; Plasmids; Polynucleotides; Process; Property; Proteins; Publications; Publishing; RNA; Reagent; Reporter Genes; Research; Research Personnel; Services; Specificity; Stem cells; System; Technology; Test Result; Testing; Therapeutic; Time; Transcript; Transcription Coactivator; Variant; Virus; Work; Zinc Fingers; adaptive immunity; base; clinical application; cost; design; ds-DNA; endonuclease; innovation; nuclease; phase 2 study; prototype; public health relevance; success; tool; trafficking; vector

Project Summary/Abstract The goal of the proposed Phase I research is to develop a set of commercial kits for footprint-free genome engineering. The core technologies that serve as the foundation of these kits are based on the CRISPR/cas system, which was originally identified in bacteria and recently shown to also function in mammalian cells. The main advantage of the proposed gene targeting system is that the specificity of the target DNA sequence is dictated by a "guide" RNA, which can base-pair with one strand of double-stranded DNA by forming an "R-loop". Currently, zinc-finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN) provide possibilities to specifically and permanently modify the human genome. There are commercial reagents and services available for these two technologies. However, these two technologies are based on protein-DNA recognition, which requires designing and creating new enzymes every time a unique DNA sequence is to be targeted. The assembly of new ZFNs or TALENs is cumbersome and costly, and is presently only performed by a small number of laboratories or through expensive commercial services. Successful execution of the proposed project will establish a fully tested, market-ready system for making any desired changes to mammalian chromosomes. The proposed kits can be easily adopted by any lab at significantly lower costs compare to the ZFN and TALEN services or self-assembled processes. Opposite to the recently published vectors that delivery either the cas nuclease or the guide RNA, the proposed system is in an "all-RNA" format. The mRNA system for expression of the required enzyme works by expressing through an inherently short-lived vector which is impervious to genetic recombination and the problematic aspects of vector replication are sidestepped because mRNA transcripts are simply resupplied via culture media for the time required to achieve desired level of chromosome modifications. These properties, in conjunction with the excellent expression efficiency and kinetics which have been demonstrated in mRNA reprogramming of human fibroblasts, make the system a good candidate for a successful commercial kit.

项目摘要/摘要 拟议的第一阶段研究的目的是开发一套商业套件 无足迹基因组工程。作为这些基础的核心技术 套件基于CRISPR/CAS系统，该系统最初在细菌和 最近显示在哺乳动物细胞中也起作用。提出的基因的主要优点 靶向系统是靶DNA序列的特异性由“指南” RNA，RNA指示。 通过形成“ R环”，它可以用一条双链DNA基础对。 目前，锌指核酸酶（ZFN）和转录激活剂样效应核酸酶（TALEN） 提供特定，永久修改人类基因组的可能性。有 这两种技术可用于商业试剂和服务。但是，这两个 技术基于蛋白-DNA识别，它需要设计和创建新的 每当要靶向唯一的DNA序列时，酶。新ZFN或 塔伦斯繁琐且昂贵，目前仅由少数 实验室或通过昂贵的商业服务。 拟议项目的成功执行将建立一个经过全面测试的，可以进行市场准备就绪的系统 用于对哺乳动物染色体进行任何理想的更改。提议的套件很容易 与ZFN和TALEN Services相比，任何实验室通过的成本明显降低或 自组装过程。与最近发表的向量相反，交付 CAS核酸酶或引导RNA，提出的系统为“全RNA”格式。 mRNA 通过固有地表达所需酶表达所需酶起作用的系统 短暂的矢量不受遗传重组和有问题的方面 矢量复制之所以避免，是因为mRNA转录本简单地通过培养 培养基在达到所需的染色体修饰水平所需的时间内。这些 属性，结合出色的表达效率和动力学 在人类成纤维细胞的mRNA重编程中证明，使系统成为良好 成功商业套件的候选人。