Improving the Efficiency of Gene Targeting Using TAL Effector Nucleases

描述（由申请人提供）：本提案的长期目标是开发广泛适用的方法，利用工程核酸酶促进人体细胞和多能细胞的基因靶向。具体目标是：1）增强我们在人体细胞和多能细胞中使用工程化核酸酶（TAL效应物核酸酶或TALEN）通过同源重组对所有类型的遗传修饰进行遗传工程化的能力，和2）提高我们使用TALEN衍生的切口酶进行同源重组的能力。该提案将利用高通量组装方法来创建工程TALEN，并将其应用于人类细胞系的基因工程。成功地开发所提出的方法，引入小插入和单碱基修饰的人类基因组将提高系统地研究人类基因变异和功能之间的关系，并使遗传操作，以前仅限于小鼠模型系统的能力。

DESCRIPTION (provided by applicant): The long-term objective of this proposal is to develop broadly applicable methods to utilize engineered nucleases to facilitate gene targeting in human somatic and pluripotent cells. The specific aims are: 1) to enhance our ability to genetically engineer all types of genetic modifications by homologous recombination using engineered nucleases (TAL effector nucleases or TALENs) in human somatic and pluripotent cells and 2) to improve our ability to use TALEN-derived nicking enzymes for homologous recombination. This proposal will leverage a high-throughput assembly method for creating engineered TALENs and apply it to the genetic engineering of human cell lines. Successful development of the proposed methods for introducing small insertions and single-base modifications to the human genome will enhance the ability to systematically investigate the relationship between human gene variation and function and enable genetic manipulations that have been previously limited to mouse model systems.