Tailor-made variants of site-specific recombinases as tools for genome engineerin

作为基因组工程工具的位点特异性重组酶的定制变体

• 批准号: 7846108
• 负责人: Yuri Voziyanov
• 金额: $ 23.23万
• 依托单位: LOUISIANA TECH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7846108
• 关键词: Amino Acids; Antibiotic Resistance; Antibiotics; Bacteria; Base Pairing; Binding; Cell Line; Cells; Code; Competence; Cystic Fibrosis; Cytosine deaminase; DNA; DNA Sequence; DNA Sequence Rearrangement; Data; Development; Drops; Elements; Enzymes; Escherichia coli; Event; Excision; Exhibits; Genes; Genetic; Genetic Recombination; Genetic Screening; Genome; Genome engineering; Genomics; Goals; Hand; Hereditary Disease; Human; Human Cell Line; Human Genome; Hybrids; In Vitro; Individual; Institutes; Length; Lesch-Nyhan Syndrome; Mammalian Cell; Mediating; Medical Research; Methods; Modeling; Mutagenesis; Mutation; Patients; Pattern; Pilot Projects; Plasmids; Probability; Prodrugs; Property; Reaction; Reporter; Research; Scheme; Screening procedure; Sickle Cell Anemia; Site; Specificity; Structure; Syndrome; System; Technology; Testing; Thymidine Kinase; Time; Transfection; Variant; Work; base; cell type; cystic fibrosis patients; directed evolution; disease-causing mutation; established cell line; gene replacement; homologous recombination; interest; programs; recombinase; research study; sex; suicide gene; tool

DESCRIPTION (provided by applicant): Main goal of the proposed research is development of efficient and universal genome manipulation technology based on the tailor-made variants of site-specific recombinase Flp. We aim to (1) identify and classify all DNA sequences in human genome that resemble native recombination target for Flp, FRT; (2) evolve Flp variants that can recombine the corresponding classes of FRT-like sequences; (3) develop an efficient selection system to identify cells in which the desired site-specific recombination took place; and (4) correct genetic disease-causing mutations in model human cell lines using the evolved Flp variants. To identify and classify all FRT-like sites in human genome, we will enhance our existing search program TargetFinder. The modified program will overcome limitations of the current program on the length of DNA to be searched and will be able to screen the longest contigs. After all individual DNA sequences that make up human genome are screened for potential FRT-like sites based on their resemblance to FRT, these sites will be grouped into classes depending on particular sequence patterns of these sites. We will employ structure-based amino acid alterations and directed evolution strategies to evolve Flp variants that can recombine the corresponding classes of FRT-like sequences. All of the evolutionary steps will be carried out by in vitro mutagenesis and gene shuffling, followed by efficient genetic screens in Escherichia coli. Selected representatives of Flp variants will be expressed in bacteria, isolated and analyzed in in vitro recombination reactions. After tests in bacterial system, Flp variants will be assessed in CHO and human cells for mediating integration and gene replacement reactions. To develop efficient selection system for identifying cells in which desired gene replacement reaction took place, we will modify the well-established approach used to identify cells, in which gene replacement has occurred by homologous recombination. Our selection scheme will have two steps. In the first step, we will transfect cells with a construct that contains an antibiotic resistance gene and a suicide gene flanked by the FRT-like sites that correspond to the ones that flank a genomic region to be replaced. This construct will also contain another suicide gene located outside the region flanked by the FRT-like sites. Upon site-specific recombination in some cells, the genomic region of interest will be replaced with the antibiotic/suicide genes cassette. In the second step, the cells selected for antibiotic resistance will be transfected with a construct that contains a DNA fragment for replacement flanked by the corresponding FRT-like sites. The transfected cells will be then treated with prodrugs, which will be converted into toxic products by enzymes coded by the suicide genes. The enzymes will be active in the cells in which second gene replacement did not occur or in which a construct used in the first step integrated into genome randomly by non-homologous recombination. We will experiment with different suicide genes to find their optimal combination that maximizes the yield of cells, in which perfect gene replacement has occurred. We also aim to correct genetic disease-causing mutations in model cell lines that represent (1) sickle cell disease, (2) cystic fibrosis, (3) Tay-Sachs syndrome and (4) Lesch-Nyhan syndrome. To achieve this goal, we will locate FRT-like sites in the vicinity of the disease-causing mutations of the corresponding genes and, using the data obtained under Aim 2, identify Flp variants able to recombine the corresponding FRT-like sites. After testing activity of Flp variants on the corresponding sites in bacterial and mammalian systems, we will accordingly modify the constructs of the gene replacement selection system developed under Aim 3 and replace the disease-causing mutations in the cell lines.

描述（由申请人提供）：拟议研究的主要目标是基于现场特异性重组酶FLP的量身定制变体的高效和通用基因组操纵技术的开发。我们的目标是（1）识别和分类人类基因组中类似于FLP的天然重组靶标的所有DNA序列； （2）进化可以重组类似FRT的序列的FLP变体； （3）开发一个有效的选择系统，以识别发生所需位点特异性重组的细胞； （4）使用进化的FLP变体中的模型人细胞系中正确引起遗传疾病的突变。为了识别和分类人类基因组中的所有类似FRT的站点，我们将增强现有的搜索程序目标捕获器。修改后的程序将克服当前程序对要搜索的DNA长度的局限性，并能够筛选最长的重叠群。在所有构成人类基因组的单个DNA序列都基于与FRT相似的潜在frt样位点筛选出来的所有DNA序列，这些位点将根据这些位点的特定序列模式分为类。我们将采用基于结构的氨基酸改变和定向演化策略来进化可以重组相应的FRT样序列的FLP变体。所有进化步骤将通过体外诱变和基因改组进行，然后在大肠杆菌中有效的遗传筛选。选定的FLP变体代表将在细菌中表达，并在体外重组反应中分离和分析。在细菌系统中进行测试后，将在CHO和人类细胞中评估FLP变体，以介导整合和基因替代反应。为了开发有效的选择系统，以识别发生所需的基因替代反应的细胞，我们将修改用于识别细胞的公认方法，其中通过同源重组发生了基因置换。我们的选择方案将有两个步骤。在第一步中，我们将使用包含抗生素耐药性基因和自杀基因的构建体转染细胞，该基因的侧面是FRT样位点的侧面，这些基因与侧面基因组区域的基因组区域相对应。该构建体还将包含另一个自杀基因，该基因位于侧面是FRT样地点的区域外。在某些细胞中位点特异性重组后，感兴趣的基因组区域将被抗生素/自杀基因盒取代。在第二步中，选择用于抗生素耐药性的细胞将用包含DNA片段的构造转染，用于替换为相应的FRT样位点的替换。然后将转染的细胞用前药处理，该前药将通过自杀基因编码的酶转化为有毒产物。这些酶将活跃在未发生第二个基因置换的细胞中，或者在第一步中使用的构建体通过非同学重组随机整合到基因组中。我们将尝试不同的自杀基因，以找到最佳组合，以最大化细胞的产量，其中发生了完美的基因置换。我们还旨在纠正代表（1）镰状细胞疾病，（2）囊性纤维化，（3）Tay-Sachs综合征和（4）Lesch-Nyhan综合征的模型细胞系中引起遗传疾病的突变。为了实现这一目标，我们将在相应基因的引起疾病的突变附近找到类似FRT的位点，并使用AIM 2下的数据确定能够重新组合相应的FRT样位点的FLP变体。在测试了细菌和哺乳动物系统中相应位点上FLP变体的活性之后，我们将相应地修改基因置换选择系统在AIM 3开发的基因替代选择系统的构建体，并替换细胞系中引起疾病​​的突变。