Selectable non-mosaic embryo editing

项目总结 转基因动物模型对生物医学科学的研究至关重要，并使我们能够阐明和治疗 人类许多疾病的根本原因。该项目的总体目标是开发健壮且易于实现的 实现方法来生成具有统一的、非镶嵌的基因类型的经过遗传编辑的小鼠品系 一代。随着基因组编辑技术的出现，我们现在可以对小鼠胚胎进行基因操作 为生物医学研究制造精确改良的动物品系。虽然基因组编辑技术在很大程度上 简化了特定部位基因组操作的过程，当应用于胚胎时，目前的方法 通常不能统一编辑发育中的胚胎的所有细胞，导致小鼠的基因嵌合体。 此外，即使这些技术成功地编辑了胚胎的所有细胞，也无法检测到 哪些胚胎是非镶嵌编辑的。因此，从经过基因编辑的胚胎发育出来的小鼠必须是 交叉以在下一代中生成所需的非马赛克编辑的鼠标线。可能需要六个月或者 生产所需的鼠标品系的时间更长，这使得它耗时、成本和劳动密集型。使用 CRISPR-Cas9系统、活体成像和腺相关病毒血清6型(AAV6)的组合，WE 已经开发出一种方法，可以对小鼠基因组进行非镶嵌的定点修改，并检测到 非镶嵌、单等位基因的实时靶向胚胎。在这份提案中，我们将进一步开发这个平台，以 通过(1)优化单代转基因小鼠以获得最高的非嵌合率 可能的目标，(2)发展它，以允许选择具有非镶嵌双等位基因敲击的胚胎。 INS，(3)将其应用于在小鼠身上进行特定的核苷酸改变，以及(4)使其适应以产生遗传报告 老鼠。由于我们能够追踪经过同源定向修复(HDR)的胚胎细胞 实时，在实现这些目标的过程中，我们还将对DNA双链有重要的了解 哺乳动物早期胚胎的断裂修复动力学。这项研究的成功完成将产生一套 高效率地在小鼠胚胎中产生各种类型的遗传修饰的工具。通过减少 生产新的转基因小鼠品系所需的时间、成本和小鼠数量，这项新技术 将改善获得新的转基因研究动物的机会，并为了解人类的努力做出贡献 并开发新的治疗方法。

PROJECT SUMMARY Transgenic animal models are vital for research in biomedical science and have allowed us to elucidate and treat the root causes of numerous human diseases. The overall goal of this project is to develop robust and easy to implement methods to generate genetically edited mouse lines with uniform, non-mosaic genotypes in a single generation. With the advent of genome editing technologies we can now genetically manipulate mouse embryos to make precisely modified animal lines for biomedical research. While genome editing technologies have greatly simplified the process of site-specific genomic manipulation, when applied to embryos, current methods generally fail to uniformly edit all the cells of a developing embryo resulting in a mouse that is genetically mosaic. Furthermore, even when these technologies successfully edit all the cells of an embryo there is no way to detect which embryos are non-mosaically edited. As a result, mice that develop from the gene-edited embryos must be crossed to generate the desired non-mosaic edited mouse line in the next generation. It can take six months or longer to generate a desired mouse line making it time consuming, costly, and labor intensive. Using a combination of the CRISPR-Cas9 system, live imaging, and Adeno Associated Virus Serotype 6 (AAV6), we have developed a method to make site-specific modifications to the mouse genome non-mosaically and to detect non-mosaic, mono-allelic targeted embryos in real time. In this proposal, we will further develop this platform for single-generation transgenic mouse production by (1) optimizing it to achieve the highest rates of non-mosaic targeting possible, (2) developing it to allow for the selection of embryos that have non-mosaic bi-allelic knock- ins, (3) applying it to make specific nucleotide changes in mice, and (4) adapting it to generate a genetic reporter mouse. Due to our ability to track cells of an embryo that have undergone homology-directed repair (HDR) in real time, in the process of completing these aims we will also gain significant insight into DNA double strand break repair dynamics in early mammalian embryos. Successful completion of this research will yield a suite of tools to produce various types of genetic modifications in mouse embryos at high efficiency. By reducing the time, cost, and numbers of mice required to produce new genetically modified mouse lines, this new technology will improve research access to new transgenic research animals and contribute to efforts to understand human diseases and to develop new therapies.