College of Plant Protection, China Agricultural University, Beijing 100193, China.
Institute of Plant Quarantine, Chinese Academy of Inspection and Quarantine, Beijing 100176, China.
Int J Mol Sci. 2019 Aug 23;20(17):4125. doi: 10.3390/ijms20174125.
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system has been widely applied in functional genomics research and plant breeding. In contrast to the off-target studies of mammalian cells, there is little evidence for the common occurrence of off-target sites in plants and a great need exists for accurate detection of editing sites. Here, we summarized the precision of CRISPR/Cas9-mediated mutations for 281 targets and found that there is a preference for single nucleotide deletions/insertions and longer deletions starting from 40 nt upstream or ending at 30 nt downstream of the cleavage site, which suggested the candidate sequences for editing sites detection by whole-genome sequencing (WGS). We analyzed the on-/off-target sites of 6 CRISPR/Cas9-mediated plants by the optimized method. The results showed that the on-target editing frequency ranged from 38.1% to 100%, and one off target at a frequency of 9.8%-97.3% cannot be prevented by increasing the specificity or reducing the expression level of the Cas9 enzyme. These results indicated that designing guide RNA with high specificity may be the preferred factor to avoid the off-target events, and it is necessary to predict or detect off-target sites by WGS-based methods for preventing off targets caused by genome differences in different individuals.
簇状规律间隔短回文重复 (CRISPR)/CRISPR 相关蛋白 (Cas) 系统已广泛应用于功能基因组学研究和植物育种。与哺乳动物细胞的脱靶研究相比，在植物中很少有证据表明普遍存在脱靶位点，因此非常需要准确检测编辑位点。在这里，我们总结了 281 个靶点的 CRISPR/Cas9 介导的突变的精确性，发现对于切割位点上游 40nt 或下游 30nt 起始的单核苷酸缺失/插入和更长的缺失有偏好，这表明候选序列可用于通过全基因组测序 (WGS) 检测编辑位点。我们通过优化方法分析了 6 种 CRISPR/Cas9 介导的植物的靶上/靶外位点。结果表明，靶上编辑频率范围为 38.1%-100%，不能通过提高 Cas9 酶的特异性或降低表达水平来防止一个频率为 9.8%-97.3%的靶外位点。这些结果表明，设计具有高特异性的向导 RNA 可能是避免脱靶事件的首选因素，有必要通过基于 WGS 的方法预测或检测脱靶位点，以防止由于不同个体的基因组差异而导致的脱靶。
