Performance of the Cas9 Nickase System in Drosophila melanogaster
Performance of the Cas9 Nickase System in Drosophila melanogaster
复制标题
Cas9 切口酶系统在果蝇中的性能
DOI：
10.1534/g3.114.013821
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发表时间：
2014-10-01
影响因子：
2.6
通讯作者：
Ni, Jian-Quan
中科院分区：
文献类型：
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作者：
Ren, Xingjie;Yang, Zhihao;Ni, Jian-Quan
最近对黑腹果蝇基因组编辑中Cas9/sgRNA系统的研究为以高通量方式产生位点特异性突变体集合开辟了新的机会。然而，在分析突变表型时，系统的脱靶效应仍然是一个主要问题。将Cas9转化为DNA切口酶的突变具有在体外减少脱靶效应的巨大潜力。在这里，我们证明了将编码相邻偏移sgRNA的两个质粒注射到转基因Cas 9D 10A切口酶果蝇中有效地产生可遗传的indel突变体。然后，我们确定了两个sgRNA靶标之间的有效距离和它们的取向，其影响了sgRNA对在转基因切口酶果蝇中表达时产生突变的能力。有趣的是，Cas9切口酶大大降低了用一种sgRNA产生突变体的能力，这表明Cas9切口酶和sgRNA对的应用在产生indel突变体时几乎可以避免脱靶效应。最后，一个定义的piwi突变体等位基因产生与此系统通过同源定向修复。然而，Cas 9D 10A在用两个sgRNA替换piwi的整个编码序列方面不如Cas9有效。
Recent studies of the Cas9/sgRNA system in Drosophila melanogaster genome editing have opened new opportunities to generate site-specific mutant collections in a high-throughput manner. However, off-target effects of the system are still a major concern when analyzing mutant phenotypes. Mutations converting Cas9 to a DNA nickase have great potential for reducing off-target effects in vitro. Here, we demonstrated that injection of two plasmids encoding neighboring offset sgRNAs into transgenic Cas9D10A nickase flies efficiently produces heritable indel mutants. We then determined the effective distance between the two sgRNA targets and their orientations that affected the ability of the sgRNA pairs to generate mutations when expressed in the transgenic nickase flies. Interestingly, Cas9 nickase greatly reduces the ability to generate mutants with one sgRNA, suggesting that the application of Cas9 nickase and sgRNA pairs can almost avoid off-target effects when generating indel mutants. Finally, a defined piwi mutant allele is generated with this system through homology-directed repair. However, Cas9D10A is not as effective as Cas9 in replacing the entire coding sequence of piwi with two sgRNAs.