Different Effects of sgRNA Length on CRISPR-mediated Gene Knockout Efficiency.
Different Effects of sgRNA Length on CRISPR-mediated Gene Knockout Efficiency.
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sgRNA 长度对 CRISPR 介导的基因敲除效率的不同影响。
DOI：
10.1038/srep28566
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发表时间：
2016-06-24
影响因子：
4.6
通讯作者：
Zhang XB
中科院分区：
文献类型：
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作者：
Zhang JP;Li XL;Neises A;Chen W;Hu LP;Ji GZ;Yu JY;Xu J;Yuan WP;Cheng T;Zhang XB
CRISPR-Cas9是一种强大的基因组编辑技术，但具有脱靶效应。已发现截短的sgRNA（17 nt）减少293 T细胞中的脱靶切割而不影响中靶破坏。然而，尚未评估17 nt sgRNA相对于全长20 nt sgRNA在干细胞（如人间充质干细胞（MSC）和诱导多能干细胞（iPSC））中的效力。使用GFP报告系统，我们发现由慢病毒载体表达的17 nt和20 nt sgRNA在293 T细胞中诱导~95%敲除（KO），而在iPSC（60-70%）和MSC（65-75%）中KO效率显著较低。此外，我们观察到在iPSC和MSC两者中，与全长sgRNA相比，使用17 nt sgRNA的KO效率降低10-20个百分点。在具有1-2nt但不是3- 4 nt错配的17 nt sgRNA中观察到脱靶切割;而具有高达5 nt错配的20 nt sgRNA仍然可以诱导脱靶突变。有趣的是，我们偶尔观察到由17 nt而不是20 nt sgRNA诱导的脱靶效应。这些结果表明平衡靶基因切割效力与脱靶效应的重要性：当功效是主要问题时，例如干细胞中的基因组编辑，优选使用20 nt sgRNA。
CRISPR-Cas9 is a powerful genome editing technology, yet with off-target effects. Truncated sgRNAs (17nt) have been found to decrease off-target cleavage without affecting on-target disruption in 293T cells. However, the potency of 17nt sgRNAs relative to the full-length 20nt sgRNAs in stem cells, such as human mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs), has not been assessed. Using a GFP reporter system, we found that both 17nt and 20nt sgRNAs expressed by lentiviral vectors induce ~95% knockout (KO) in 293T cells, whereas the KO efficiencies are significantly lower in iPSCs (60–70%) and MSCs (65–75%). Furthermore, we observed a decrease of 10–20 percentage points in KO efficiency with 17nt sgRNAs compared to full-length sgRNAs in both iPSCs and MSCs. Off-target cleavage was observed in 17nt sgRNAs with 1-2nt but not 3-4nt mismatches; whereas 20nt sgRNAs with up to 5nt mismatches can still induce off-target mutations. Of interest, we occasionally observed off-target effects induced by the 17nt but not the 20nt sgRNAs. These results indicate the importance of balancing on-target gene cleavage potency with off-target effects: when efficacy is a major concern such as genome editing in stem cells, the use of 20nt sgRNAs is preferable.