RNA-Guided Gene Targeting in Human Cells

项目摘要/摘要 拟议的第一阶段研究的目标是开发一套商业套件，用于 无足迹基因组工程。作为这些技术基础的核心技术 试剂盒基于CRISPR/cas系统，该系统最初在细菌和 最近发现在哺乳动物细胞中也有功能。所提出的基因的主要优点是 靶向系统是靶DNA序列的特异度由“引导”RNA决定， 它可以通过形成R环与一条双链DNA碱基配对。 目前，锌指核酸酶(ZFN)和转录激活物样效应核酸酶(TALEN) 为特定和永久地修改人类基因组提供了可能性。确实有 可用于这两种技术的商业试剂和服务。然而，这两个人 技术是基于蛋白质-DNA识别的，这需要设计和创造新的 每次一个独特的DNA序列被作为靶点时，酶。新ZFN的组装或 TALEN既繁琐又昂贵，目前只有一小部分 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充分测试、可供市场使用的系统 对哺乳动物的染色体进行任何想要的改变。建议的试剂盒可以很容易地 被任何实验室采用，与ZFN和TALEN服务或 自组装过程。与最近发布的载体相反，这些载体要么提供 CAS核酸酶或引导RNA，所建议的系统是“全RNA”形式。信使核糖核酸 用于通过固有地表达所需酶作品的系统 不受基因重组影响的短寿命载体和存在问题的方面 避免了载体复制，因为只需通过培养重新补充mRNA转录本 用于实现所需水平的染色体修改所需的时间的介质。这些 特性，再加上出色的表达效率和动力学 在人类成纤维细胞的mRNA重新编程中演示，使该系统成为一个很好的 一款成功的商用套件的候选人。

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.