Making ends meet: targeted integration of DNA fragments by genome editing.
Making ends meet: targeted integration of DNA fragments by genome editing.
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DOI：
10.1007/s00412-018-0677-6
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发表时间：
2018-12
期刊：
影响因子：
1.6
通讯作者：
Gerbi SA
中科院分区：
文献类型：
--
作者：
Yamamoto Y;Gerbi SA
大片段DNA的定向插入是基因工程的一个重要目标。然而，这个目标一直难以实现，因为同源定向修复的经典方法效率低下，并且在许多系统中通常不可行。这里描述的最新进展使得相对较大的 DNA 能够在位点特异性基因组插入，效率大大提高。利用细胞中非同源末端连接的首选修复途径，通过 HITI 和 ObLiGaRe 方法可以非常精确地引入长达数 kb 的 DNA，效率高达 30-40%。利用同源定向修复（PITCH 方法；短同源臂，包括 ssODN；2H2OP）的最新进展显着提高了 DNA 插入效率，通常提高到 40-50% 甚至更高，具体取决于 DNA 的方法和长度。总结了集成精度和脱靶位点插入的剩余挑战。总体而言，当前的进展为将大DNA定点插入来自广泛细胞和生物体的基因组中迈出了重要的一步。
Targeted insertion of large pieces of DNA is an important goal of genetic engineering. However, this goal has been elusive since classical methods for homology-directed repair are inefficient and often not feasible in many systems. Recent advances are described here that enable site-specific genomic insertion of relatively large DNA with much improved efficiency. Using the preferred repair pathway in the cell of nonhomologous end-joining, DNA of up to several kb could be introduced with remarkably good precision by the methods of HITI and ObLiGaRe with an efficiency up to 30-40%. Recent advances utilizing homology-directed repair (methods of PITCh; short homology arms including ssODN; 2H2OP) have significantly increased the efficiency for DNA insertion, often to 40-50% or even more depending on the method and length of DNA. The remaining challenges of integration precision and off-target site insertions are summarized. Overall, current advances provide major steps forward for site-specific insertion of large DNA into genomes from a broad range of cells and organisms.
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