CRISPR genome editing has revolutionized genetics in many organisms. In the nematode Caenorhabditis elegans one injection into each of the two gonad arms of an adult hermaphrodite exposes hundreds of meiotic germ cells to editing mixtures, permitting the recovery of multiple indels or small precision edits from each successfully injected animal. Unfortunately, particularly for long insertions, editing efficiencies can vary widely, necessitating multiple injections, and often requiring co-selection strategies. Here we show that melting double stranded DNA (dsDNA) donor molecules prior to injection increases the frequency of precise homology-directed repair (HDR) by several fold for longer edits. We describe troubleshooting strategies that enable consistently high editing efficiencies resulting, for example, in up to 100 independent GFP knock-ins from a single injected animal. These efficiencies make C. elegans by far the easiest metazoan to genome edit, removing barriers to the use and adoption of this facile system as a model for understanding animal biology.

中文翻译：

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熔化 dsDNA 供体分子极大地提高了秀丽隐杆线虫基因组编辑的精确度。


CRISPR 基因组编辑彻底改变了许多生物体的遗传学。在线虫秀丽隐杆线虫中，对成年雌雄同体的两个性腺臂进行一次注射，使数百个减数分裂生殖细胞暴露于编辑混合物中，从而允许从每只成功注射的动物中恢复多个插入缺失或小的精确编辑。不幸的是，特别是对于长插入，编辑效率可能差异很大，需要多次注射，并且通常需要共同选择策略。在这里，我们表明，在注射前熔化双链 DNA (dsDNA) 供体分子可将精确同源定向修复 (HDR) 的频率提高数倍，以实现更长的编辑。我们描述了能够实现持续高编辑效率的故障排除策略，例如，从单个注射动物中获得多达 100 个独立的 GFP 敲入。这些效率使秀丽隐杆线虫成为迄今为止最容易进行基因组编辑的后生动物，消除了使用和采用这种简单系统作为理解动物生物学模型的障碍。