Dimeric Cys2His2 Zinc Finger Proteins for Gene Targeting

用于基因打靶的二聚 Cys2His2 锌指蛋白

• 批准号: 7783350
• 负责人: SCOT A WOLFE
• 金额: $ 32.84万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-02-01 至 2013-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7783350
• 关键词: Address; Affinity; Agriculture; Alleles; Animal Model; Architecture; Biochemical; Biomedical Engineering; Cell Line; Characteristics; Communities; Complex; Conserved Sequence; DNA; DNA Binding; DNA Restriction Enzymes; DNA Sequence; Development; Disease; Elements; Engineering; Enzymes; Equilibrium; Event; Family; Future; Gene Family; Gene Targeting; Generations; Genes; Genetic Transcription; Genome; Genomics; Human; Knock-out; Laboratories; Location; Mus; Play; Property; Proteins; Research; Role; Site; Specificity; System; Technology; Testing; Therapeutic; Work; Zebrafish; Zinc Fingers; design; dimer; fly; gene therapy; homeodomain; homologous recombination; human disease; improved; in vivo; infancy; innovation; member; novel; nuclease; positional cloning; public health relevance; repaired; tool; transcription factor; vertebrate genome

DESCRIPTION (provided by applicant): Novel DNA-binding specificity can be engineered into Cys2His2 zinc finger proteins by either selection or design, and they can be used to deliver a DNA cleavage domain to a specific "address" in a genome. These tailor-made restriction endonucleases ("Zinc Finger Nucleases" or ZFNs), which function as (hetero)dimers, can introduce double stranded breaks at a specific genomic location to inactivate a target gene through imprecise repair or recode a target gene through homologous recombination with an exogenously supplied donor DNA. ZFN technology can potentially be used to apply simple or sophisticated reverse genetic approaches to a broad range of metazoan systems that were previously only accessible in the mouse and fly. This technology, which should also find application in bioengineering and human gene therapy, is still in its infancy. The current generation of ZFNs has not been thoroughly characterized nor completely optimized. We propose to develop a new generation of artificial nucleases with improved properties (activity, precision, range of targetable sequences) that will fully realize the potential of this technology for tailored genome editing. In Aims 1 & 2 we will define the optimal assembly of ZFPs and the optimal fusion of the nuclease domain to create heterodimeric ZFNs that are both efficient and precise. We will also develop new nuclease architectures to expand the types of DNA sequences that can be targeted. In Aim 3 we will explore the potential of ZFNs to perform knockouts of highly related gene families, which would allow complex knockout combinations to be rapidly created. PUBLIC HEALTH RELEVANCE: This research focuses on the development of artificial proteins that can be targeted to a unique site in a vertebrate genome to alter its content. This technology has potential application in human gene therapy, but much work remains to define versions of these proteins that will be highly active, yet will not damage other parts of the genome. Understanding how to improve the characteristics of these artificial systems will be the focus of this study.

描述（由申请人提供）：新颖的DNA结合特异性可以通过选择或设计将其设计到CYS2HIS2锌指蛋白中，并且可以用于将DNA裂解域传递到基因组中的特定“地址”。这些量身定制的限制性核酸内切酶（“锌指核酸酶”或ZFNs）起作用（异伴）二聚体，可以在特定的基因组位置引入双链断裂，以通过不准确的修复或通过与本质上补充的donor donor dna的同源性重组来灭活靶基因。 ZFN技术可以可能用于将简单或复杂的反向遗传方法应用于以前仅在鼠标和飞行中访问的广泛的后生系统。该技术还应在生物工程和人类基因疗法中找到应用，但仍处于起步阶段。当前的ZFN一代尚未得到彻底表征或完全优化。我们建议开发新一代的人造核酸酶，具有改进的性质（活动，精度，可靶向序列范围），这些核酸酶将充分实现这项技术对量身定制的基因组编辑的潜力。在AIMS 1和2中，我们将定义ZFP的最佳组装和核酸酶域的最佳融合，以创建有效且精确的异二聚体ZFN。我们还将开发新的核酸酶体系结构，以扩展可以靶向的DNA序列的类型。在AIM 3中，我们将探讨ZFN执行高度相关基因家族的敲除的潜力，这将使复杂的敲除组合能够迅速产生。 公共卫生相关性：这项研究的重点是人造蛋白质的开发，这些蛋白质可以针对脊椎动物基因组中的独特地点以改变其含量。该技术在人类基因疗法中具有潜在的应用，但是仍有许多工作要定义这些蛋白质的版本，这些蛋白质将是高度活跃的，但不会损害基因组的其他部分。了解如何改善这些人工系统的特征将成为这项研究的重点。