PDF(Pubmed)
Abstract：
Despite the great achievements in genome editing, accurately detecting mutations induced by sequence-specific nucleases is still a challenge in plants, especially in polyploidy plants. An efficient detection method is particularly vital when the mutation frequency is low or when a large population needs to be screened. Here, we applied purified CRISPR ribonucleoprotein complexes to cleave PCR products for genome-edited mutation detection in hexaploid wheat and diploid rice. We show that this mutation detection method is more sensitive than Sanger sequencing and more applicable than PCR/RE method without the requirement for restriction enzyme site. We also demonstrate that this detection method is especially useful for genome editing in wheat, because target sites are often surrounded by single nucleotide polymorphisms. Using this screening method, we were also able to detect foreign DNA-free tagw2 mutations induced by purified TALEN protein. Finally, we show that partial base editing mutations can also be detected using high-fidelity SpCas9 variants or FnCpf1. The PCR/RNP method is low-cost and widely applicable for rapid detection of genome-edited mutation in plants.
摘要：
尽管在基因组编辑方面取得了巨大的成就，准确检测序列特异性核酸酶诱导的突变在植物中仍然是一个挑战，尤其是在多倍体植物中。当突变频率低或需要筛查大量人群时，有效的检测方法尤为重要。这里,我们应用纯化的CRISPR核糖核蛋白复合物切割PCR产物,对六倍体小麦和二倍体水稻进行基因组编辑突变检测.我们表明，这种突变检测方法比Sanger测序更灵敏，并且比PCR/RE方法更适用，而不需要限制酶位点。我们还证明了这种检测方法对于小麦的基因组编辑特别有用，因为靶位点通常被单核苷酸多态性包围。使用这种筛选方法，我们还能够检测到由纯化的TALEN蛋白诱导的外源无DNA的tagw2突变.最后,我们表明,部分碱基编辑突变也可以使用高保真SpCas9变体或FnCpf1检测到.PCR/RNP方法成本低，可广泛用于植物基因组编辑突变的快速检测。
