Structure, function and engineering of nuclease catalysts

核酸酶催化剂的结构、功能和工程

• 批准号: 8038697
• 负责人: BARRY L. STODDARD
• 金额: $ 36.92万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-01 至 2014-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8038697
• 关键词: Affinity; Amino Acid Substitution; Anopheles Genus; Architecture; Area; Bacteriophages; Base Sequence; Behavior; Binding; Bioinformatics; Biological Assay; Boxing; Catalysis; Cells; Chloride Ion; Chlorides; Cleaved cell; Cystic Fibrosis; DNA; DNA Binding; DNA Sequence; Data; Development; Endonuclease I; Engineered Gene; Engineering; Enzymes; Excision; Exhibits; Family; Gene Conversion; Gene Targeting; Genes; Genetic Recombination; Genome; Genome Mappings; Genome engineering; Genomics; Homing; Human; In Vitro; Length; Life; Maps; Measures; Methods; Modification; Monitor; Monoamine Oxidase; Monoamine Oxidase B; Nucleic acid sequencing; Plasmids; Property; Protein Family; Proteins; Publishing; Reagent; Relative (related person); Reporter; Reporting; Scaffolding Protein; Scheme; Series; Single Strand Break Repair; Site; Specificity; Structure; System; Technology; Tertiary Protein Structure; Testing; Toxic effect; Transgenes; Transplantation; Variant; Work; X Chromosome; base; catalyst; design; directed evolution; endonuclease; gene therapy; homologous recombination; improved; in vivo; member; metagenomic sequencing; novel; nuclease; repaired; research study; scaffold

DESCRIPTION (provided by applicant): Homing endonucleases (also termed meganucleases) are highly specific enzymes that are under intense study for the purpose of targeted genome engineering and gene therapy. We have previously determined the structures of representatives from many known homing endonuclease families, characterized their mechanisms of DNA recognition and catalysis, and created variants that cleave noncognate DNA targets. We will now pursue two specific aims that build upon these results and that pursue new areas of enquiry: Aim 1: Engineering and characterization. We will characterize our successfully reengineered, gene-targeted endonucleases in living cells. Using different experimental strategies, we have recently completed the successful selection and redesign of two different endonuclease scaffolds that specifically cleave target sites in (i) the human cystic fibrosis-associated chloride transporter (CFTR) gene and (ii) the human monoamine oxidase (MOAB) gene. Starting with these two enzymes, we will (a) establish the relationship of their in vitro recognition specificity and cleavage activity to their in vivo ability to induce homologous recombination versus nonhomologous end-joining, while (b) simultaneously measuring their toxicity profiles. We will also (c) compare their relative efficiencies of gene conversion when they introduce double strand breaks, versus when engineered "nickase" versions of the same enzymes introduce single-strand breaks. The constructs mentioned above were produced using two very different methods, each developed for a specific target. In subaim (d), we will continue to improve and combine methods for homing endonuclease redesign. This work involves the iterative application of bioinformatics (to identify new endonuclease scaffolds), computational structure-based design, and directed evolution of targeted cleavage activity. Aim 2: Determination of new endonuclease structures and functions. We will determine the structure of the gp29 endonuclease from bacteriophage 0305f8-36. This protein family was discovered while examining metagenomic sequence data. Its members are shown to display a novel combination of protein domain organization and DNA recognition that is appropriate for several important genomic applications. PUBLIC HEALTH RELEVANCE: The development of highly specific, nontoxic gene targeting proteins that are capable of inducing the site-specific modification of a DNA sequence (either the insertion of novel genes or the disruption or alteration of existing genes) is a critical technology for genome engineering and for corrective gene therapy. Newly discovered endonucleases also provide raw material for the creation of new genome mapping reagents.

描述（由申请人提供）：归巢核酸内切酶（也称为大范围核酸酶）是高度特异性的酶，正在进行深入研究，用于靶向基因组工程和基因治疗。我们先前已经确定了许多已知归巢核酸内切酶家族的代表的结构，表征了它们的DNA识别和催化机制，并创建了切割非同源DNA靶标的变体。我们现在将追求两个具体目标，建立在这些结果的基础上，并追求新的调查领域：目标1：工程和表征。我们将在活细胞中描述我们成功重组的基因靶向核酸内切酶。使用不同的实验策略，我们最近完成了两种不同的核酸内切酶支架的成功选择和重新设计，这些核酸内切酶支架特异性切割（i）人类囊性纤维化相关氯转运蛋白（CFTR）基因和（ii）人类单胺氧化酶（MOAB）基因中的靶位点。从这两种酶开始，我们将（a）建立它们的体外识别特异性和切割活性与它们诱导同源重组与非同源末端连接的体内能力之间的关系，同时（B）同时测量它们的毒性特征。我们还将（c）比较它们在引入双链断裂时与在相同酶的工程化“切口酶”版本引入单链断裂时的基因转换的相对效率。上面提到的构建体是使用两种非常不同的方法产生的，每种方法都是针对特定靶标开发的。在子目标（d）中，我们将继续改进和联合收割机用于归巢核酸内切酶重新设计的方法。这项工作涉及生物信息学的迭代应用（以识别新的核酸内切酶支架），基于计算结构的设计，以及靶向切割活性的定向进化。目的2：确定新的核酸内切酶的结构和功能。我们将确定来自噬菌体0305 f8 -36的gp 29核酸内切酶的结构。这个蛋白质家族是在检查宏基因组序列数据时发现的。它的成员显示出一种新的蛋白质结构域组织和DNA识别的组合，适用于几个重要的基因组应用。 公共卫生相关性：开发能够诱导DNA序列的位点特异性修饰（插入新基因或破坏或改变现有基因）的高度特异性、无毒的基因靶向蛋白是基因组工程和校正基因治疗的关键技术。新发现的核酸内切酶也为创造新的基因组作图试剂提供了原料。