Genotyping genome‐edited mutations in plants using CRISPR ribonucleoprotein complexes

Wiley - Tập 16 Số 12 - Trang 2053-2062 - 2018
Zhen Liang1,2, Kunling Chen1, Yan Yan1, Yi Zhang3, Caixia Gao1
1State Key Laboratory of Plant Cell and Chromosome Engineering, Center for Genome Editing, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China
2University of Chinese Academy of Sciences, Beijing, China
3Key Laboratory of Plant Stress Research, College of Life Science, Shandong Normal University, Jinan, China

Tóm tắt

SummaryDespite 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.

Từ khóa


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Wiley

Tập 16 Số 12

2053-2062

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