Development and validation of a precision genome editing platform

 描述（由申请人提供）：摘要“精确基因组编辑平台的开发和验证”实现精确基因编辑的可编程方式的开发将代表强大的新研究工具，以及基于基因编辑的人类治疗的潜在新方法。目前的基因组工程工具受到适度的基因编辑效率以及可能与所需改变竞争的不需要的基因改变的影响。该提案寻求工程化融合蛋白，使其具有易于编程的位点特异性C>T DNA编辑能力。融合将利用Cas9系统，这是一种由Cas蛋白组成的效应复合物，该蛋白通过RNA分子（称为sgRNA）靶向特定的DNA序列。Cas9和胞苷脱氨酶之间的融合复合物将被工程化，以便将它们将胞苷脱氨基为尿苷的能力引导至基因组DNA中的特定位点。各种简单融合物的酶活性和选择性都将使用基于内切酶的体外测定来表征。这些结果将被彻底分析和检查，因为它们可能提供有关Cas9结构与其进入靶DNA的能力之间关系的基本信息。如果工程化融合体显示很少或没有活性，或显著的脱靶效应，则将使用噬菌体辅助连续进化（PACE）（一种最先进的蛋白质进化方法）来优化融合体的活性和特异性。这种方法在过去已经成功地用于从不可检测的活性开始进化突变聚合酶。然后将通过校正人癌细胞系中PIK3CA基因中的癌症相关H1047R（A3140G）突变来验证所得融合酶。这些融合酶的成功开发将代表可用于研究各种生物学问题的工具开发的初始阶段。该技术可用于以高效率随意敲除蛋白质并观察结果，研究位点特异性蛋白质突变对信号通路和细胞功能的影响，并研究突变基因校正后的癌症进展。

 DESCRIPTION (provided by applicant): Abstract "Development and validation of a precision genome editing platform" The development of a programmable way to achieve precision gene editing would represent both a powerful new research tool, as well as a potential new approach to gene editing-based human therapeutics. Current genome engineering tools suffer from modest gene editing efficiencies as well as unwanted gene alterations that can compete with the desired alteration. This proposal seeks to engineer fusion proteins with readily programmable, site specific C>T DNA editing capabilities. The fusions will take advantage of the Cas9 system, an effector complex comprised of a Cas protein that is targeted to a specific DNA sequence by an RNA molecule (termed sgRNA). Fusion complexes between Cas9 and cytidine deaminase enzymes will be engineered in order to direct their ability to deaminate cytidine to uridine to a specific site in genomic DNA. Both the enzymatic activities and selectivities of the various simple fusions will be characterized using a luciferase-based assay in vitro. These results will be thoroughly analyzed and examined, as they may provide fundamental information about the relationship between the structure of Cas9 and its ability to access its target DNA. If the engineered fusions display little to no activity, or significant off-target effects, phage-assited continuous evolution (PACE), a state-of-the-art protein evolution method, will then be used to optimize the activities and specificities of the fusions. This method has been successfully used in the past to evolve mutant polymerases starting from undetectable activities. The resulting fusion enzymes will then be validated by correcting the cancer relevant H1047R (A3140G) mutation in the PIK3CA gene in a human cancer cell line. The successful development of these fusion enzymes would represent the initial phase of the development of a tool that can be used to investigate various biological problems. This technology could be used to knock out proteins at will with high efficiencies and observe the outcomes, study the effect of site specific protein mutations on signaling pathways and cell function, and investigate cancer progression following correction of a mutated gene.