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

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分配每个基因的功能。虽然研究人员通常分析与基因功能丧失相关的表型变化，但用于在哺乳动物生物体中进行基因敲除的经典技术缓慢、费力且昂贵。最近，DNA序列特异性核酸酶（SSN），如TALEN和CRISPR/Cas9，已成为有望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高精度遗传改变哺乳动物生物的强大技术。通过分子进化和蛋白质组学方法的结合，该计划将为哺乳动物开发一个基因编辑工具箱，以实现这一可能性。 最初，我们将专注于改进目前最先进的基因编辑工具CRISPR/Cas9，该工具已成功用于多种生物体。Cas9采用用户定义的RNA靶序列来指导基因内互补DNA序列的切割。在DNA切割后，细胞修复过程可以导致基因失活，或者可以利用修复模板添加新的遗传物质。然而，虽然Cas9对其靶序列具有相当特异性，但它确实切割了基因组中的脱靶位点，这可能会混淆使用该技术创建的突变体的表型分析。此外，Cas9蛋白结合并影响哺乳动物细胞中蛋白质的可能性也需要研究。 为了解决这些问题，我们将在加拿大建立第一个DNA结合蛋白持续定向进化的平台，并利用它来提高Cas9的DNA结合特异性。此外，我们将鉴定与细胞中Cas9结合的哺乳动物蛋白，并研究这些相互作用对细胞生物学的影响。然后，我们将设计无法与这些细胞因子结合的Cas9变体。 从长远来看，我们将扩展该计划，以包括具有不同DNA结合特异性和DNA切割机制的CRISPR-Cas家族的其他成员，并探索工程化高度特异性RNA引导的RNA切割酶作为调节基因表达的非遗传替代品的可能性。 我们预计，他的研究计划将通过开发下一代基因编辑和基因调控工具，减少与哺乳动物和其他生物体功能基因组学研究相关的时间和研究成本，使众多领域的研究人员受益。此外，我们开发的任何新工具的商业化都可能为加拿大带来经济利益。最后，该提案将为加拿大带来尖端的蛋白质进化技术，并为高素质的人员提供使用该技术的培训。

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.