A Genetic Engineering Toolbox for Marmosets (GETMarm): Development and optimization of genome editing and assisted reproduction techniques for marmoset models

项目摘要 这项拨款提案的主要目标是开发和完善生殖系基因操作技术， 绒猴胚胎，以促进产生用于大脑研究的基因工程绒猴模型。到 提高产生具有所需DNA变化的基因工程绒猴模型的效率， 我们在补助金中提议开发的技术是植入前基因检测（PGT）。简单地说，在 在绒猴胚胎中进行CRISPR编辑，我们在体外培养胚胎， (from 6-8细胞期胚胎）或5-10个支持细胞（来自100-200个细胞胚泡）。我们会放大DNA 区域，并对DNA片段进行测序，以鉴定具有正确编辑的胚胎， 还在培养中。我们需要在24小时内获得测序数据，以确保良好的胚胎存活。一旦我们 识别具有正确编辑的胚胎，然后我们将正确的胚胎植入受体狨猴。 目前，最快的商业测序服务是48小时，甚至不能保证。我们有 我也检查了广泛的测序平台服务，他们的周转时间约为两周，他们 只做大批量测序。幸运的是，Thermo-Fisher最近发布了一款台式测序仪， 很容易使用，我们可以在几小时内完成测序。这些设备将极大地方便我们 PGT并大大提高了我们在绒猴中进行基因组编辑的成功率。

Project Summary The major goal of this grant proposal is to develop and refine germline genetic manipulation technologies for marmoset embryos to facilitate the generation of genetic engineered marmoset models for brain research. To increase the efficiency of generating genetic engineered marmoset models with desired DNA changes, one of the technologies we proposed to develop in the grant is preimplantation genetic testing (PGT). Briefly, after CRISPR editing in marmoset embryos, we culture the embryos in vitro and take either one embryonic cell (from 6-8 cell stage embryos) or 5-10 supporting cells (from 100-200 cell blastocysts). We will amplify the DNA region by PCR and sequence the DNA fragment to identify embryos with correct editing while the embryos are still in culture. We need to get the sequencing data withing 24 hours to ensure good embryo survival. Once we identify the embryos with correct editing, we will then implant the correct embryos to recipient marmosets. Currently, the fastest commercial sequencing service is 48 hours and even that is not guaranteed. We have also checked with Broad sequencing platform service and their turnaround time is about two weeks and they only do large batch sequencing. Fortunately, Thermo-Fisher has recently released a bench-top sequencer that is easy to use and we could get the sequencing done within hours. This equipment will greatly facilitate our PGT and dramatically increase our successful rate of genome editing in marmosets.