Towards a toolbox for mammalian gene editing: Removing off-target activity and protein interactions of Cas9

打造哺乳动物基因编辑工具箱：消除 Cas9 的脱靶活性和蛋白质相互作用

• 批准号: RGPIN-2016-06381
• 负责人: Hubbard, Basil
• 金额: $ 3.13万
• 依托单位: University of Alberta
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=616268
• 关键词: Towards; toolbox; mammalian; gene; editing

In the past 30 years, molecular genetics research has resulted in unprecedented breakthroughs in sequencing and annotating the mammalian genome. One of the main challenges now facing researchers is how to efficiently assign function to each gene. While researchers typically analyze phenotypic changes associated with gene loss-of-function, classical techniques for performing gene knockout in mammalian organisms are slow, laborious, and costly. Recently, DNA sequence-specific nucleases (SSNs) such as TALENs and CRISPR/Cas9 have emerged as tools that are poised to revolutionize this process. An over-arching goal of my lab is to develop robust technology for genetically altering mammalian organisms with high precision. Through a combination of molecular evolution and proteomics approaches, this program will develop a gene editing toolbox for mammals that will realize this possibility. Initially, we will focus on improving the current state-of-the-art gene editing tool, CRISPR/Cas9, which has been used successfully in diverse organisms. Cas9 employs a user-defined RNA target sequence to guide cleavage of a complementary DNA sequence within a gene. After DNA cleavage, cellular repair processes can result in gene inactivation, or can be exploited to add in new genetic material by using a repair template. However, while Cas9 is quite specific for its target sequence, it does cleave off-target sites in the genome, which could confound phenotypic analysis of mutants created using this technology. In addition, the possibility that the Cas9 protein binds to and influences proteins in mammalian cells, needs to be examined. To address these issues, we will set up the first platform for the continuous directed evolution of DNA-binding proteins in Canada, and use it to improve the DNA-binding specificity of Cas9. In addition, we will identify mammalian proteins that bind to Cas9 in cells and study the effects of these interactions on cell biology. We will then engineer Cas9 variants that are incapable of binding to these cellular factors. In the longer term we will extend this program to include other members of the CRISPR-Cas family with different DNA-binding specificities and mechanisms of DNA-cleavage, and explore the possibility of engineering highly specific RNA-guided RNA cleaving enzymes as a non-genetic alternative for regulating gene expression. We anticipate that his research program will benefit researchers in numerous fields by leading to the development of next-generation gene editing and gene regulating tools that will cut down on time and research costs associated with functional genomics studies in mammals and other organisms. In addition, commercialization of any new tools that we develop could lead to economic benefits for Canada. Finally, this proposal will bring cutting-edge protein evolution technology to Canada, and provide training for highly qualified personnel in its use.

在过去的30年里，分子遗传学研究在哺乳动物基因组测序和注释方面取得了前所未有的突破。研究人员目前面临的主要挑战之一是如何有效地为每个基因分配功能。虽然研究人员通常分析与基因功能丧失相关的表型变化，但在哺乳动物生物体中进行基因敲除的传统技术是缓慢、费力和昂贵的。最近，DNA序列特异性核酸酶（ssn）如TALENs和CRISPR/Cas9已经成为准备彻底改变这一过程的工具。我的实验室的一个首要目标是开发强大的技术，以高精度地改变哺乳动物有机体的基因。通过结合分子进化和蛋白质组学方法，该项目将为哺乳动物开发一个基因编辑工具箱，实现这一可能性。