Strategy for detecting off-target sites in genome-edited rice
Strategy for detecting off-target sites in genome-edited rice
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DOI：
10.1101/2021.05.28.446070
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发表时间：
2021-05
期刊：
影响因子：
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通讯作者：
Jumpei Narushima;Shinya Kimata;Yuh Shiwa;T. Gondo;Satoru Akimoto;K. Soga;Satoko Yoshiba;Kosuke Nakamura;N. Shibata;K. Kondo
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文献类型：
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作者：
Jumpei Narushima;Shinya Kimata;Yuh Shiwa;T. Gondo;Satoru Akimoto;K. Soga;Satoko Yoshiba;Kosuke Nakamura;N. Shibata;K. Kondo
使用CRISPR-Cas9系统进行基因组编辑可以大大加快作物育种。由于脱靶编辑是该系统的主要问题，因此需要一种可靠的方法来全面检测脱靶位点，以便对粮食作物基因组进行编辑。然而，目前还没有一种准确预测脱靶部位的方法。在这项研究中，我们进行了SITE-Seq分析来预测潜在的脱靶位点。SITE-Seq是一种适用于体外检测双链断裂（dsb）的无偏方法。为了分析SITE-Seq数据，我们开发了一种新颖的Galaxy系统，该系统无需命令行操作即可进行简单且可重复的分析。我们对OsFH15 gRNA-Cas9修饰的水稻基因组进行了SITE-Seq分析，并在注释区域鉴定了41个DSB位点。扩增子测序揭示了基因组编辑水稻中一个脱靶位点的突变。一个不常见的原间隔邻近基序（NTG PAM）的存在可能使这种脱靶位点难以用计算机方法识别。在六个测试的程序中，只有CRISPRdirect预测了这个脱靶位点，但它也预测了总共6080个脱靶位点。这些结果表明，本文提出的SITE-Seq方法可以有效地预测脱靶位点，并可用于评估基因组编辑食品的安全性。
Genome-editing using the CRISPR-Cas9 system can substantially accelerate crop breeding. Because off-target editing is the main problem with this system, a reliable method for comprehensively detecting off-target sites is required for the editing of food crop genomes. However, a method that accurately predicts off-target sites has not been established. In this study, we performed a SITE-Seq analysis to predict potential off-target sites. SITE-Seq is an unbiased method applicable for the in vitro detection of double-strand breaks (DSBs). To analyze SITE-Seq data, we developed a novel Galaxy system, which can perform simple and reproducible analyses without a command line operation. We conducted a SITE-Seq analysis of a rice genome modified by OsFH15 gRNA-Cas9, and identified 41 DSB sites in the annotated regions. Amplicon-sequencing revealed mutations at one off-target site in the genome-edited rice. The presence of an uncommon protospacer adjacent motif (NTG PAM) likely makes this off-target site difficult to identify using in silico methods. Of the six tested programs, only CRISPRdirect predicted this off-target site, but it also predicted 6,080 off-target sites in total. These results suggest the SITE-Seq method presented herein can efficiently predict off-target sites and is useful for assessing the safety of genome-edited food.