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：
7466640
负责人：
RAYMOND J MONNAT
金额：
$ 38.72万
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/7466640
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 Galvanic Skin Response Gene Mutation Gene Silencing Gene Targeting Genes Genetic Recombination Genome Genome engineering Goals Hematopoietic Hemolytic Anemia Homing Human Immune Inborn Errors of Metabolism Indium Lateral Lead Life Link Methods Mus Object Attachment 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 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.

拟议的研究的长期目标是设计新的，高度序列特异性的DMA结合具有靶向和裂解特定人类基因的能力的蛋白质。我们的目标是生成和使用新的基因特异性试剂（GSR），以促进特定基因的有效重组修复或消融在活细胞中。新颖的GSR将是由先前存在的归巢核酸内切酶蛋白而产生的拉格里达（Laglidadg）家庭。这些归巢核酸酶催化寄生DNA的位点特异性侧向转移生活中所有王国的元素，并且已经拥有许多理想的GSR工程特性。为了。例如，laglidadg归核核酸内切酶的长DNA靶位点为18-24 bp，高度的 DNA序列特异性以及DNA位点识别与DNA链裂解的紧密耦合。最后属性允许归巢核酸酶准确地靶向DNA链裂解至单磷酸酯由于脱靶或虚假的乳沟，复杂基因组中的键很少或没有“附带损害” 事件。在拟议的研究中，我们将产生laglidadg Housing内核酸酶变体，该变体针对9人基因及其犬或鼠。这些基因靶标是因为它们在可遗传中的作用而选择的人造血疾病；在可遗传的免疫缺陷综合症中；或I型酪氨酸血症，其中之一最常见的遗传天生新陈代谢错误。我们的实验目的是： AIM 1：生成优化的laglidadg Housing内核酸酶蛋白用于哺乳动物基因组工程 AIM 2：开发针对针对的laglidadg Housing noclelases的基因特异性靶向变体人类疾病基因靶标 AIM 3：确定工程化的基因特异性laglidadg变体蛋白催化的能力重组基因修复或体内基因失活。