Genome Editing in Mouse Spermatogonial Stem Cell Lines Using TALEN and Double-Nicking CRISPR/Cas9.
Genome Editing in Mouse Spermatogonial Stem Cell Lines Using TALEN and Double-Nicking CRISPR/Cas9.
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DOI：
10.1016/j.stemcr.2015.05.011
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发表时间：
2015-07-14
影响因子：
5.9
通讯作者：
Ogawa T
中科院分区：
文献类型：
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作者：
Sato T;Sakuma T;Yokonishi T;Katagiri K;Kamimura S;Ogonuki N;Ogura A;Yamamoto T;Ogawa T
小鼠精原干细胞（SSCs）可以在保持其生精能力的同时进行增殖培养和长时间维持。虽然培养的ssc被称为种系干细胞（GS），是基因组修饰的目标，但这一过程在技术上仍然困难。在本研究中，我们在GS细胞上测试了TALEN和双缺口CRISPR/Cas9，分别针对Rosa26和Stra8位点作为精子发生中不可缺少和不可缺少的代表基因。收获的GS细胞集落对TALEN和CRISPR/Cas9都显示出很高的靶向效率。rosa26靶向的GS细胞在移植后分化为具有生育能力的精子。另一方面，stra8靶向的GS细胞在移植后表现出精子发生缺陷，证实了其在减数分裂起始中的主要作用。TALEN和CRISPR/Cas9在GS细胞中的应用，将成为研究精子发生和揭示生精失败遗传机制的重要工具。在药物选择克隆中，靶向准确性和特异性接近100%，基因组编辑过程本身不会干扰GS细胞的生精能力，GS细胞的基因组修饰为研究精子发生提供了工具。使用TALEN和双nick CRISPR/Cas9系统，Ogawa, Sato，和同事的研究表明，在药物选择克隆中，用转基因靶向Rosa26和Stra8位点是成功的，效率很高，没有随机整合，导致每个靶向GS细胞的生精能力分别得到完全维持和适当的干扰。这种方法有助于揭示生精失败的遗传机制。
Mouse spermatogonial stem cells (SSCs) can be cultured for multiplication and maintained for long periods while preserving their spermatogenic ability. Although the cultured SSCs, named germline stem (GS) cells, are targets of genome modification, this process remains technically difficult. In the present study, we tested TALEN and double-nicking CRISPR/Cas9 on GS cells, targeting Rosa26 and Stra8 loci as representative genes dispensable and indispensable in spermatogenesis, respectively. Harvested GS cell colonies showed a high targeting efficiency with both TALEN and CRISPR/Cas9. The Rosa26-targeted GS cells differentiated into fertility-competent sperm following transplantation. On the other hand, Stra8-targeted GS cells showed defective spermatogenesis following transplantation, confirming its prime role in the initiation of meiosis. TALEN and CRISPR/Cas9, when applied in GS cells, will be valuable tools in the study of spermatogenesis and for revealing the genetic mechanism of spermatogenic failure. Genome editing in GS cells is available by TALEN and double-nicking CRISPR/Cas9 Targeting accuracy and specificity are nearly 100% in drug-selected clones The genome-editing procedure itself does not disturb the spermatogenic ability of GS cells Genome modification in GS cells provides a tool in the study of spermatogenesis Using the TALEN and double-nicking CRISPR/Cas9 systems, Ogawa, Sato, and colleagues show that targeting at Rosa26 and Stra8 loci with transgenes was successful in drug-selected clones with high efficiency, without random integrations, which resulted in the complete maintenance and proper disturbance of the spermatogenic ability, respectively, in each targeted GS cells. This methodology is useful in revealing the genetic mechanism of spermatogenic failure.