Journal
PLANT BIOTECHNOLOGY JOURNAL
Volume 16, Issue 12, Pages 2053-2062Publisher
WILEY
DOI: 10.1111/pbi.12938
Keywords
PCR/RNP; SpCas9; FnCpf1; TALEN protein; hexaploid wheat; high-fidelity SpCas9 variants
Funding
- National Key Research and Development Program of China [2016YFD0101804]
- Scientific Program of Beijing Municipal Commission of Science and Technology [Z171100001517001]
- Chinese Academy of Sciences [QYZDY-SSW-SMC030, GJHZ1602]
- National Natural Science Foundation of China [31788103, 31420103912, 31570369]
- Beijing Municipal Science and Technology [D171100007717001]
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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.
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