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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融化dsDNA供体分子可增强秀丽隐杆线虫的精确基因组编辑

CRISPR基因组编辑彻底改变了许多生物的遗传学。在线虫中，秀丽隐杆线虫一次注射到成年雌雄同体的两个性腺臂中，使成百上千的减数分裂生殖细胞暴露于可编辑的混合物中，从而允许从每只成功注射的动物中恢复多个插入缺失或少量精确的编辑。不幸的是，尤其是对于长插入而言，编辑效率可能有很大差异，因此需要多次注入，并且通常需要共同选择策略。在这里，我们显示在注射前融化双链DNA（dsDNA）供体分子会使精确的同源性定向修复（HDR）的频率增加几倍，以进行更长的编辑。我们描述了故障排除策略，这些策略可实现始终如一的高编辑效率，例如，最多可从单个注射动物中获得100个独立的GFP敲入。这些效率使秀丽隐杆线虫成为最容易进行基因组编辑的后生动物，从而消除了使用和采用这种简便系统作为理解动物生物学模型的障碍。