NEW LAGLIDADG HOMING ENDONUCLEASES FOR GENOME ENGINEERING (Component 2 of 11)

用于基因组工程的新型 LAGLIDADG 归巢核酸内切酶（11 项中的第 2 项）

• 批准号: 7660379
• 负责人: RAYMOND J MONNAT
• 金额: $ 45.07万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-28 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7660379
• 关键词: Ablation; Agammaglobulinemia; Binding Proteins; Canis familiaris; Cells; Cleaved cell; Complex; Coupling; DNA; DNA Sequence; Disease; Elements; Engineered Gene; Engineering; Erythrocytes; Event; Family; Fumarylacetoacetase Deficiency Disease; Gene Mutation; Gene Silencing; Gene Targeting; Genes; Genetic Recombination; Genome; Genome engineering; Goals; Hematopoietic; Hemolytic Anemia; Homing; Human; Human Genome; Immune; Inborn Errors of Metabolism; Indium; Lateral; Lead; Life; Link; Methods; Mus; Positioning Attribute; Property; Proteins; Reagent; Research; Research Personnel; Role; Severe Combined Immunodeficiency; Site; Solubility; Specificity; Syndrome; Testing; Toxic effect; Variant; Work; X-Linked Severe Combined Immunodeficiency; base; disease-causing mutation; endonuclease; gene repair; human disease; in vivo; mammalian genome; mutant; novel; phosphodiester; recombinational repair; repaired

The long term goal of the proposed research is to engineer new, highly sequence-specific DMA binding proteins with the ability to target and cleave specific human genes. Our aim is to generate and use new Gene-Specific Reagents (GSR's) to promote the efficient recombinational repair or ablation of specific genes in living cells. Novel GSR's will be generated from preexisting homing endonuclease proteins of the LAGLIDADG family. These homing endonucleases catalyze the site-specific lateral transfer of parasitic DNA elements in all Kingdoms of life, and already possess many desirable properties for GSR engineering. For . example, LAGLIDADG homing endonucleases have long DNA target sites of 18-24 bp, a high degree of DNA sequence specificity, and tight coupling of DNA site recognition to DNA strand cleavage. This last property allows homing endonucleases to precisely target DNA strand cleavage to single phosphodiester bonds in complex genomes with little or no 'collateral damage' as a result of off-target or spurious cleavage events. In the proposed research we will generate LAGLIDADG homing endonuclease variants that target 9 human genes and their canine or murine counterparts. These gene targets were chosen for their role in heritable human hematopoietic disease; in heritable immune deficiency syndromes; or in tyrosinemia type I, one of the most common heritable inborn errors of metabolism. Our experimental Aims are: Aim 1: Generate optimized LAGLIDADG homing endonuclease proteins for mammalian genome engineering Aim 2: Develop gene-specific targeting variants of LAGLIDADG homing endonucleases directed against human disease gene targets Aim 3: Determine ability of engineered, gene-specific LAGLIDADG variant proteins to catalyze recombinational gene repair or gene inactivation in vivo.

该研究的长期目标是设计新的，高度序列特异性的DMA结合 具有靶向和切割特定人类基因能力的蛋白质。我们的目标是产生和使用新的 基因特异性试剂（GSR），用于促进特定基因的有效重组修复或消除 在活细胞中。新型GSR将由先前存在的归巢核酸内切酶蛋白产生 LAGLIDADG家族这些归巢核酸内切酶催化寄生虫DNA的位点特异性侧向转移 元素在所有王国的生活，并已拥有许多理想的属性GSR工程。For . 例如，LAGLIDADG归巢核酸内切酶具有18-24 bp的长DNA靶位点，高度的同源性， DNA序列特异性和DNA位点识别与DNA链切割的紧密耦合。这最后一 特性允许归巢核酸内切酶精确靶向DNA链切割至单个磷酸二酯 复杂基因组中的键，由于脱靶或假切割而几乎没有或没有“附带损害” 事件 在所提出的研究中，我们将产生靶向9个人类肿瘤的LAGLIDADG归巢核酸内切酶变体。 基因和它们的犬或鼠对应物。选择这些基因靶点是因为它们在遗传性疾病中的作用。 人类造血系统疾病;遗传性免疫缺陷综合征;或I型酪氨酸血症， 最常见的遗传性先天代谢缺陷。我们的实验目标是： 目的1：为哺乳动物基因组工程制备优化的LAGLIDADG归巢核酸内切酶蛋白 目的2：开发针对抗HLA-LIDADG的LAGLIDADG归巢核酸内切酶的基因特异性靶向变体。 人类疾病基因靶标 目的3：确定工程化的基因特异性LAGLIDADG变体蛋白催化 重组基因修复或体内基因失活。