Melting dsDNA Donor Molecules Greatly Improves Precision Genome Editing in Caenorhabditis elegans.
Melting dsDNA Donor Molecules Greatly Improves Precision Genome Editing in Caenorhabditis elegans.
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DOI：
10.1534/genetics.120.303564
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发表时间：
2020-11
期刊：
影响因子：
3.3
通讯作者：
Mello CC
中科院分区：
文献类型：
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作者：
Ghanta KS;Mello CC
在注射前熔化和快速冷却双链DNA供体分子显著增加了编辑的同源性定向修复的频率，例如在秀丽隐杆线虫中插入荧光蛋白标记。这里所描述的策略能够始终如一地...... CRISPR基因组编辑已经彻底改变了许多生物体的遗传学。在线虫秀丽隐杆线虫中，向成年雌雄同体的两个性腺臂中的每一个注射一次，将数百个减数分裂生殖细胞暴露于编辑混合物，允许从每个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，对于更长的编辑，精确同源定向修复（HDR）的频率增加了数倍。我们描述了故障排除策略，使一致的高编辑效率的结果，例如，在多达100个独立的GFP基因敲入从一个单一的注射动物。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。
Melting and fast cooling double stranded DNA donor molecules prior to injection dramatically increases the frequency of homology-directed repair for edits such as insertions of fluorescent protein markers in Caenorhabditis elegans. Strategies described here enable consistently ..... 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 coselection 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.