Development of versatile non-homologous end joining-based knock-in module for genome editing.
Development of versatile non-homologous end joining-based knock-in module for genome editing.
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DOI：
10.1038/s41598-017-18911-9
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发表时间：
2018-01-12
影响因子：
4.6
通讯作者：
Sugano SS
中科院分区：
文献类型：
--
作者：
Sawatsubashi S;Joko Y;Fukumoto S;Matsumoto T;Sugano SS
基于CRISPR/Cas9的基因组编辑极大地加速了基因组工程。基因组工程的一个重要方面是有效的敲入技术。为了提高敲入效率，非同源末端连接（NHEJ）修复途径已经超过同源依赖性修复途径而被使用，但是仍然需要降低供体载体制备的复杂性。我们开发了用于基因组编辑的通用的基于NHEJ的敲入模块（维京海盗）。使用历史悠久的pUC载体的共有序列来切割供体载体，具有pUC骨架的任何载体都可以用作供体载体而无需定制。还研究了使供体载体的随机整合率最小化所需的条件。我们尝试使用选择标记盒的敲入/敲除分离人HaCaT角质形成细胞中VDR基因的空株，并且发现分离的克隆的75%被成功敲入。虽然HaCaT细胞具有亚四倍体基因组组成，但结果表明多个克隆具有VDR无效表型。维京海盗模块使得能够高效敲入任何携带pUC载体的载体。用户现在可以将各种现有载体插入基因组中的任意位点。维京海盗将有助于低成本的基因组工程。
CRISPR/Cas9-based genome editing has dramatically accelerated genome engineering. An important aspect of genome engineering is efficient knock-in technology. For improved knock-in efficiency, the non-homologous end joining (NHEJ) repair pathway has been used over the homology-dependent repair pathway, but there remains a need to reduce the complexity of the preparation of donor vectors. We developed the versatile NHEJ-based knock-in module for genome editing (VIKING). Using the consensus sequence of the time-honored pUC vector to cut donor vectors, any vector with a pUC backbone could be used as the donor vector without customization. Conditions required to minimize random integration rates of the donor vector were also investigated. We attempted to isolate null lines of the VDR gene in human HaCaT keratinocytes using knock-in/knock-out with a selection marker cassette, and found 75% of clones isolated were successfully knocked-in. Although HaCaT cells have hypotetraploid genome composition, the results suggest multiple clones have VDR null phenotypes. VIKING modules enabled highly efficient knock-in of any vectors harboring pUC vectors. Users now can insert various existing vectors into an arbitrary locus in the genome. VIKING will contribute to low-cost genome engineering.
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