Single Nucleotide Genome Modifications in Oocytes

描述（由申请人提供）：我们结合了多种新技术，并将其应用于卵母细胞和胚胎，目标是快速、高效、有效地改变动物基因组。我们以非转基因方法利用卵母细胞和早期胚胎来改变特定的基因功能。卵母细胞将用于构建 F0 代的稳定转基因系，并使用胚胎进行纯合基因改变以进行快速基因筛选。这项技术将为研究新生物体以及修改基因进行功能分析或纠正原本功能不全的基因打开大门。由于能够引入外源 DNA 构建体，基因组操作方法主要使用培养细胞和遗传易处理的胚胎进行。然而，为了创造稳定的转基因动物，这些方法需要回交以减少遗传嵌合或整合到种系中，从而增加时间和资源的成本。这些限制最大限度地降低了在生物研究关键生物体中广泛利用的有效性，特别是那些遗传易处理性较低的生物体。在卵母细胞中使用与 DNA 脱氨酶活性的酶促结构域相关的转录激活因子样效应器 (TALE) 功能是新颖的，该项目的成功将使研究人员能够在 F0 代构建基因改变。该项目利用 ADAR 的催化结构域在预定位点构建单核苷酸基因组靶向系统。我们最近的初步结果证明了该结构域的 DNA 脱氨基活性，并与可证明的 TALE 靶向机制相结合，增加了该项目的可行性。该项目的结果将对所有研究人员有用，特别是那些研究遗传上不易处理的生物体的研究人员，以及那些希望在不使用转基因方法的情况下快速筛选基因功能的研究人员。我们相信这项技术将从基因研究术语中删除“非模式生物”一词，并在未来通过纠正与先天性疾病状态相关的点突变而应用于临床。

DESCRIPTION (provided by applicant): We combine several new technologies and apply them to oocytes and embryos with the goal of quickly, efficiently, and effectively altering genomes of animals. We utilize oocytes and early embryos in a non-transgenic approach to alter specific gene function. Oocytes will be used for construction of stable transgenic lines at the F0 generation, and embryos for homozygous gene alteration for quick gene screening. This technology will open the door for the study of new organisms and to either modify a gene for functional analysis or correct a gene that is otherwise rendered sub-functional. Genome manipulation approaches have been performed mostly with cultured cells and genetically tractable embryos because of the capabilities to introduce exogenous DNA constructs. To create stable transgenic animals, however, these approaches require back-crosses to reduce genetic mosaicism or integration into the germ line and thus increase the cost of time and resources. These limitations minimize the effectiveness for broad utilization in organisms key for biological research, especially those whose genetic tractability is low. The use transcriptional activator like effector (TALE) functions linked to an enzymatic domain of DNA deaminase activity into oocytes is novel, and success of this project will allow researchers to construct gen alterations at the F0 generation. This project crafts a single nucleotide genome targeting system at a predetermined site by use of the catalytic domains of ADAR. Our recent preliminary results demonstrate DNA-deaminating activity of this domain and coupled with a demonstrable TALE-targeting mechanism increases feasibility of this project. The outcome of this project will be useful to all researchers, especially those working in genetically less-tractable organisms, and those wishing to rapidly screen gene functions without transgenic approaches. We believe this technology will remove the wording non-model organism from the genetic research vernacular, and in the future may be applicable for clinical use by correcting a point mutation related to a congenital disease state.