State Key Laboratory of Plant Cell and Chromosome Engineering, Center for Genome Editing, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China.
University of Chinese Academy of Sciences, Beijing, China.
Plant Biotechnol J. 2018 Dec;16(12):2053-2062. doi: 10.1111/pbi.12938. Epub 2018 May 29.
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.
尽管在基因组编辑方面取得了巨大成就，但准确检测序列特异性核酸酶诱导的突变仍然是植物领域的一个挑战，尤其是在多倍体植物中。当突变频率较低或需要筛选大量群体时，高效的检测方法尤为重要。在这里，我们应用纯化的 CRISPR 核糖核蛋白复合物来切割 PCR 产物，以检测六倍体小麦和二倍体水稻中的基因组编辑突变。我们表明，与 Sanger 测序相比，这种突变检测方法更灵敏，与不需要限制酶位点的 PCR/RE 方法相比，更适用。我们还证明，这种检测方法在小麦的基因组编辑中特别有用，因为靶标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 蛋白诱导的无外源 DNA 的 tagw2 突变。最后，我们表明，使用高保真 SpCas9 变体或 FnCpf1 也可以检测部分碱基编辑突变。PCR/RNP 方法成本低廉，广泛适用于快速检测植物中的基因组编辑突变。
