Farboud Behnom
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA.
Wiley Interdiscip Rev Dev Biol. 2017 Nov;6(6). doi: 10.1002/wdev.287. Epub 2017 Aug 15.
Utilization of programmable nucleases to generate DNA lesions at precise endogenous sequences has transformed the ability to edit genomes from microbes to plants and animals. This is especially true in organisms that previously lacked the means to engineer precise genomic changes, like Caenorhabditis elegans. C. elegans is a 1 mm long free-living, nonparasitic, nematode worm, which is easily cultivated in a laboratory. Its detailed genetic map and relatively compact genome (~100 megabases) helped make it the first metazoan to have its entire genome sequenced. With detailed sequence information came development of numerous molecular tools to dissect gene function. Initially absent from this toolbox, however, were methods to make precise edits at chosen endogenous loci. Adapting site-specific nucleases for use in C. elegans, revolutionized studies of C. elegans biology. Zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and then CRISPR-associated protein 9 (Cas9) were used to target specific endogenous DNA sequences to make double-strand DNA breaks (DSBs). Precise changes could be engineered by providing repair templates targeting the DSB in trans. The ease of programming Cas9 to bind and cleave DNA sequences with few limitations has led to its widespread use in C. elegans research and sped the development of strategies to facilitate mutant recovery. Numerous innovative CRISPR/Cas9 methodologies are now primed for use in C. elegans. WIREs Dev Biol 2017, 6:e287. doi: 10.1002/wdev.287 For further resources related to this article, please visit the WIREs website.
利用可编程核酸酶在精确的内源性序列处产生DNA损伤，已经改变了从微生物到植物和动物的基因组编辑能力。在以前缺乏精确基因组工程手段的生物体中，情况尤其如此，比如秀丽隐杆线虫。秀丽隐杆线虫是一种1毫米长的自由生活、非寄生性线虫，易于在实验室培养。其详细的遗传图谱和相对紧凑的基因组（约100兆碱基）有助于使其成为第一个完成全基因组测序的后生动物。随着详细的序列信息而来的是众多用于剖析基因功能的分子工具的开发。然而，在这个工具包中最初缺少的是在选定的内源性位点进行精确编辑的方法。将位点特异性核酸酶应用于秀丽隐杆线虫，彻底改变了秀丽隐杆线虫生物学的研究。锌指核酸酶（ZFN）、转录激活样效应因子核酸酶（TALEN），然后是CRISPR相关蛋白9（Cas9）被用于靶向特定的内源性DNA序列以产生双链DNA断裂（DSB）。通过提供反式靶向DSB的修复模板，可以设计精确的变化。Cas9易于编程以结合和切割DNA序列且限制很少，这导致了它在秀丽隐杆线虫研究中的广泛应用，并加速了促进突变体恢复策略的发展。现在，许多创新的CRISPR/Cas9方法已准备好在秀丽隐杆线虫中使用。WIREs发育生物学2017年，6：e287。doi：10.1002/wdev.287 有关本文的更多资源，请访问WIREs网站。
