Selectable non-mosaic embryo editing

项目摘要 转基因动物模型对于生物医学科学的研究至关重要，并使我们能够阐明和治疗 人类多种疾病的根源。该项目的总体目标是开发健壮且易于 实施方法以在单个细胞中产生具有均匀、非嵌合基因型的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修饰的动物品系。虽然基因组编辑技术在很大程度上 简化了位点特异性基因组操作的过程，当应用于胚胎时， 通常不能统一编辑发育中胚胎的所有细胞，从而导致基因嵌合的小鼠。 此外，即使这些技术成功地编辑了胚胎的所有细胞， 哪些胚胎是非镶嵌编辑的。因此，从基因编辑的胚胎发育的小鼠必须 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， 更长的时间来产生所需的鼠标线，使得其耗时、昂贵且劳动密集。使用 结合CRISPR-Cas9系统、实时成像和腺相关病毒血清型6（AAV 6），我们 已经开发出一种方法，对小鼠基因组进行位点特异性修饰， 非嵌合体、单等位基因靶向胚胎在真实的时间。在本提案中，我们将进一步发展这一平台， 单代转基因小鼠的生产，通过（1）优化它，以实现最高的非嵌合体率 靶向可能，（2）开发它，以允许选择具有非嵌合双等位基因敲除的胚胎， ins，（3）应用它在小鼠中进行特定的核苷酸改变，以及（4）使其适应以产生遗传报告基因 老鼠.由于我们能够追踪在胚胎中经历过同源定向修复（HDR）的胚胎细胞， 真实的时间，在完成这些目标的过程中，我们也将获得对DNA双链的重大洞察 早期哺乳动物胚胎的断裂修复动力学。这项研究的成功完成将产生一套 在小鼠胚胎中高效产生各种类型的遗传修饰的工具。通过减少 时间，成本和小鼠数量需要产生新的转基因小鼠品系，这项新技术 将改善研究获得新的转基因研究动物，并有助于了解人类 疾病，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.