Development and validation of a precision genome editing platform

精准基因组编辑平台的开发和验证

• 批准号: 9557863
• 负责人: Alexis C. Komor
• 金额: $ 0.08万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9557863
• 关键词: ART protein; BCAR1 gene; Back; Bacteriophages; Base Pairing; Biological; Biological Assay; Cancer cell line; Candidate Disease Gene; Carcinoma; Cell physiology; Chimeric Proteins; Cleaved cell; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Cytidine; Cytidine Deaminase; DNA; DNA Sequence; Deoxyribonuclease I; Detection; Development; Directed Molecular Evolution; Engineering; Enzymes; Event; Evolution; Gene Mutation; Genes; Genome; Genome engineering; Genomic DNA; Guide RNA; HCT116 Cells; Hereditary Disease; Human; Human Genetics; In Vitro; Initiator Codon; Knock-out; Link; Location; Luciferases; Malignant Neoplasms; Mediating; Methods; Modification; Mutate; Mutation; Nonhomologous DNA End Joining; Nonsense Codon; Nucleic Acids; Nucleotides; Oncogenes; Outcome Study; PIK3CA gene; Phase; Polymerase; Proteins; RNA; RNA Sequences; Research; SKOV3 cells; Signal Pathway; Site; Specificity; Structure; System; T47D; Technology; Testing; Therapeutic; Thymidine; Uridine; Validation; Work; base; cancer cell; endonuclease; gene correction; gene function; genome editing; human disease; improved; in vitro Assay; innovation; mutant; novel strategies; novel therapeutics; programs; public health relevance; repaired; restoration; success; tool; transcription activator-like effector nucleases; tumor progression; zinc finger nuclease

 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.

 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”开发一种实现精确基因编辑的可编程方法将代表着一种强大的新研究工具，以及一种潜在的基于基因编辑的人类疗法的新方法。目前的基因组工程工具受到适度的基因编辑效率的影响，以及可能与所需的改变竞争的不必要的基因改变。这项提议寻求设计具有易于编程的、特定于C&gT；T DNA编辑能力的融合蛋白质。融合将利用Cas9系统，这是一个由RNA分子(称为sgRNA)靶向特定DNA序列的Cas蛋白组成的效应复合体。将对Cas9和胞苷脱氨酶之间的融合复合体进行改造，以将它们将胞苷脱氨为尿苷的能力定向到基因组DNA中的特定位置。各种简单融合的酶活性和选择性都将使用基于荧光素酶的体外分析来表征。这些结果将被彻底分析和检验，因为它们可能提供关于Cas9结构与其访问其靶DNA的能力之间关系的基本信息。如果工程融合显示很少或没有活性，或显著的非靶标效应，噬菌体相关的持续进化(PACE)，一种最先进的蛋白质进化方法，将被用于优化融合的活性和特异性。这种方法在过去已经成功地用于从检测不到的活性开始进化突变聚合酶。然后，将通过纠正人类癌细胞系中PIK3CA基因中与癌症相关的H1047R(A3140G)突变来验证所产生的融合酶。这些融合酶的成功开发将代表着一种可用于研究各种生物问题的工具的开发的初始阶段。这项技术可以用来高效地随意敲除蛋白质并观察结果，研究特定部位蛋白质突变对信号通路和细胞功能的影响，以及研究突变基因纠正后的癌症进展。