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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）

基本信息

批准号：
8128487
负责人：
RAYMOND J MONNAT
金额：
$ 37.48万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
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/8128487
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

项目摘要

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.

拟议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) 可促进特定基因的有效重组修复或消融在活细胞中。新型 GSR 将从先前存在的归巢核酸内切酶蛋白中产生拉格利达家族。这些归巢核酸内切酶催化寄生 DNA 的位点特异性横向转移生命王国中的元素，并且已经拥有 GSR 工程的许多理想特性。为了。例如，LAGLIDADG 归巢核酸内切酶具有 18-24 bp 的长 DNA 靶位点，高度 DNA 序列特异性以及 DNA 位点识别与 DNA 链切割的紧密耦合。最后这个该特性允许归巢核酸内切酶将 DNA 链切割精确地靶向单个磷酸二酯复杂基因组中的键合很少或没有因脱靶或虚假切割而造成的“附带损害” 事件。在拟议的研究中，我们将生成针对 9 种人类基因的 LAGLIDADG 归巢核酸内切酶变体基因及其犬或鼠的对应物。这些基因靶标因其在遗传中的作用而被选择人类造血系统疾病；遗传性免疫缺陷综合症；或在 I 型酪氨酸血症中，其中之一最常见的遗传性先天性代谢缺陷。我们的实验目标是：目标 1：生成用于哺乳动物基因组工程的优化 LAGLIDADG 归巢核酸内切酶蛋白目标 2：开发 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标 3：确定工程基因特异性 LAGLIDADG 变体蛋白的催化能力体内重组基因修复或基因失活。