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

拟议研究的长期目标是设计新的、高度特定于序列的DMA绑定 具有靶向和切割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‘s)促进特定基因的高效重组修复或去除 在活细胞中。新的GSR将从先前存在的归巢内切酶蛋白中产生 拉格利达德家族。这些归巢内切酶催化寄生DNA的定点侧向转移 所有生命王国中的元素，并且已经拥有许多GSR工程所需的特性。为了。 例如，LAGLIDADG归巢内切酶具有18-24个碱基的长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。这是最后一个 特性允许定位内切酶精确地将DNA链裂解为单磷酸二酯 复杂基因组中的键，很少或没有因非靶标或虚假切割而造成的‘附带损害’ 事件。 在这项拟议的研究中，我们将产生针对9个人类的LAGLIDADG归巢内切酶变体 基因和它们的犬类或小鼠的同源基因。这些基因靶点的选择是因为它们在可遗传的 人类造血疾病；可遗传免疫缺陷综合征；或在I型酪氨酸血症中， 最常见的遗传性新陈代谢错误。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LAGLIDG归巢核酸内切酶蛋白 目的2：建立针对LAGLIDG归巢核酸内切酶的基因特异性靶向突变体 人类疾病基因靶点 目的3：确定工程的、基因特异的LAGLIDG变异蛋白的催化能力 体内重组基因修复或基因失活。

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.