Rapid and precise engineering of the Caenorhabditis elegans genome with lethal mutation co-conversion and inactivation of NHEJ repair.
Rapid and precise engineering of the Caenorhabditis elegans genome with lethal mutation co-conversion and inactivation of NHEJ repair.
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DOI：
10.1534/genetics.114.172361
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发表时间：
2015-02
期刊：
影响因子：
3.3
通讯作者：
Ward JD
中科院分区：
文献类型：
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作者：
Ward JD
与其他生物体一样，CRISPR/Cas9 方法为线虫秀丽隐杆线虫的基因组编辑提供了强大的方法。寡核苷酸是引入取代和短插入的优秀修复模板，因为它们具有成本效益，不需要克隆，并且在其他生物体中出现，通过 DNA 双链断裂 (DSB) 处的同源重组来靶向变化。在这里，我描述了一种在线虫中有效敲入表位标签的方法，使用 pha-1 基因中的温度敏感致死突变作为共转化标记，在 8-9 天内有效敲入表位标签。我证明，具有 29 bp 同源性的 60mer 寡核苷酸可驱动点突变的有效敲入，并且通过 RNAi 失活 cku-80 基因来禁用非同源末端连接可显着提高敲入效率。 35-80 bp 的同源臂足以进行高效编辑，距离插入位点最多 54 bp 的 DSB 会产生敲入。这些发现可能适用于秀丽隐杆线虫的一系列基因组编辑方法，这将提高编辑效率并最大限度地减少筛选工作。
As in other organisms, CRISPR/Cas9 methods provide a powerful approach for genome editing in the nematode Caenorhabditis elegans. Oligonucleotides are excellent repair templates for introducing substitutions and short insertions, as they are cost effective, require no cloning, and appear in other organisms to target changes by homologous recombination at DNA double-strand breaks (DSBs). Here, I describe a methodology in C. elegans to efficiently knock in epitope tags in 8–9 days, using a temperature-sensitive lethal mutation in the pha-1 gene as a co-conversion marker. I demonstrate that 60mer oligos with 29 bp of homology drive efficient knock-in of point mutations, and that disabling nonhomologous end joining by RNAi inactivation of the cku-80 gene significantly improves knock-in efficiency. Homology arms of 35–80 bp are sufficient for efficient editing and DSBs up to 54 bp away from the insertion site produced knock-ins. These findings will likely be applicable for a range of genome editing approaches in C. elegans, which will improve editing efficiency and minimize screening efforts.