Puchta Holger, Fauser Friedrich
Botanical Institute II, Karlsruhe Institute of Technology, PO Box 6980, Karlsruhe, 76049, Germany.
Plant J. 2014 Jun;78(5):727-41. doi: 10.1111/tpj.12338. Epub 2013 Nov 5.
By inducing double-strand breaks (DSB), it is possible to initiate DNA recombination. For a long time, it was not possible to use DSB induction for efficient genome engineering due to the lack of a means to target DSBs to specific sites. This limitation was overcome by development of modified meganucleases and synthetic DNA-binding domains. Domains derived from zinc-finger transcription factors or transcription activator-like effectors may be designed to recognize almost any DNA sequence. By fusing these domains to the endonuclease domains of a class II restriction enzyme, an active endonuclease dimer may be formed that introduces a site-specific DSB. Recent studies demonstrate that gene knockouts via non-homologous end joining or gene modification via homologous recombination are becoming routine in many plant species. By creating a single genomic DSB, complete knockout of a gene, sequence-specific integration of foreign DNA or subtle modification of individual amino acids in a specific protein domain may be achieved. The induction of two or more DSBs allows complex genomic rearrangements such as deletions, inversions or the exchange of chromosome arms. The potential for controlled genome engineering in plants is tremendous. The recently discovered RNA-based CRISPR/Cas system, a new tool to induce multiple DSBs, and sophisticated technical applications, such as the in planta gene targeting system, are further steps in this development. At present, the focus remains on engineering of single genes; in the future, engineering of whole genomes will become an option.
通过诱导双链断裂（DSB），可以启动DNA重组。长期以来，由于缺乏将DSB靶向特定位点的方法，无法利用DSB诱导进行高效的基因组工程。修饰的大范围核酸酶和合成DNA结合结构域的开发克服了这一限制。源自锌指转录因子或转录激活因子样效应子的结构域可被设计用于识别几乎任何DNA序列。通过将这些结构域与II类限制性内切酶的内切核酸酶结构域融合，可形成一种活性内切核酸酶二聚体，其可引入位点特异性DSB。最近的研究表明，通过非同源末端连接进行基因敲除或通过同源重组进行基因修饰在许多植物物种中已成为常规操作。通过产生单个基因组DSB，可以实现基因的完全敲除、外源DNA的序列特异性整合或特定蛋白质结构域中单个氨基酸的精细修饰。诱导两个或更多个DSB可实现复杂的基因组重排，如缺失、倒位或染色体臂的交换。植物中可控基因组工程的潜力巨大。最近发现的基于RNA的CRISPR/Cas系统是一种诱导多个DSB的新工具，以及复杂的技术应用，如植物体内基因靶向系统，都是这一发展过程中的进一步举措。目前，重点仍然是单个基因的工程改造；未来，全基因组工程将成为一种选择。
