Due to anatomical and physiological similarities to humans, the common marmoset (Callithrix jacchus) is an ideal organism for the study human diseases. Researchers are currently leveraging genome-editing technologies such as CRISPR/Cas9 to genetically engineer marmosets for the in vivo biomedical modeling of human neuropsychiatric and neurodegenerative diseases. The genome characterization of these cell lines greatly reinforces these transgenic efforts. It also provides the genomic contexts required for the accurate interpretation of functional genomics data. We performed haplotype-resolved whole-genome characterization for marmoset ESC line cj367 from the Wisconsin National Primate Research Center. This is the first haplotype-resolved analysis of a marmoset genome and the first whole-genome characterization of any marmoset ESC line. We identified and phased single-nucleotide variants (SNVs) and Indels across the genome. By leveraging this haplotype information, we then compiled a list of cj367 ESC allele-specific CRISPR targeting sites. Furthermore, we demonstrated successful Cas9 Endonuclease Dead (dCas9) expression and targeted localization in cj367 as well as sustained pluripotency after dCas9 transfection by teratoma assay. Lastly, we show that these ESCs can be directly induced into functional neurons in a rapid, single-step process. Our study provides a valuable set of genomic resources for primate transgenics in this post-genome era.

中文翻译：

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单倍型阶段的普通mar猴胚胎干细胞用于使用CRISPR / Cas9进行基因组编辑

由于与人类在解剖学和生理上的相似性，普通mar猴（Callithrix jacchus）是研究人类疾病的理想生物。研究人员目前正在利用CRISPR / Cas9等基因组编辑技术对edit猴进行基因工程改造，以进行人类神经精神疾病和神经退行性疾病的体内生物医学建模。这些细胞系的基因组表征大大加强了这些转基因努力。它还提供了准确解释功能基因组学数据所需的基因组环境。我们对来自威斯康星州国家灵长类动物研究中心的mar猴ESC系cj367进行了单倍型解析的全基因组鉴定。这是mar猴基因组的第一个单倍型解析分析，也是任何mar猴ESC系的第一个全基因组表征。我们在整个基因组中鉴定并定相了单核苷酸变体（SNV）和Indel。通过利用该单倍型信息，我们然后汇编了cj367 ESC等位基因特异性CRISPR靶向位点列表。此外，我们证明了成功的Cas9内切核酸酶Dead（dCas9）表达和cj367中的靶向定位以及dCas9转染后通过畸胎瘤测定法获得的持续多能性。最后，我们证明了这些ESC可以在快速，一步一步的过程中直接诱导为功能性神经元。我们的研究为后基因组时代的灵长类动物转基因提供了宝贵的基因组资源。我们通过畸胎瘤分析证实了成功的Cas9内切核酸酶死（dCas9）表达和cj367中的靶向定位以及dCas9转染后的持续多能性。最后，我们证明了这些ESC可以在快速，一步一步的过程中直接诱导为功能性神经元。我们的研究为后基因组时代的灵长类动物转基因提供了宝贵的基因组资源。我们通过畸胎瘤分析证实了成功的Cas9内切核酸酶死（dCas9）表达和cj367中的靶向定位以及dCas9转染后的持续多能性。最后，我们证明了这些ESC可以在快速，一步一步的过程中直接诱导为功能性神经元。我们的研究为后基因组时代的灵长类动物转基因提供了宝贵的基因组资源。