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Sci Rep. 2016 Feb 19;6:21451. doi: 10.1038/srep21451.

Selection of highly efficient sgRNAs for CRISPR/Cas9-based plant genome editing.

Author information

1
Key Laboratory of Tropical Plant Resources and Sustainable Use, Xishuangbanna Tropical Botanical Garden, Kunming, Yunnan 650223, China.
2
School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, China.
3
University of Chinese Academy of Sciences, Beijing 100049, China.

Abstract

The CRISPR/Cas9-sgRNA system has been developed to mediate genome editing and become a powerful tool for biological research. Employing the CRISPR/Cas9-sgRNA system for genome editing and manipulation has accelerated research and expanded researchers' ability to generate genetic models. However, the method evaluating the efficiency of sgRNAs is lacking in plants. Based on the nucleotide compositions and secondary structures of sgRNAs which have been experimentally validated in plants, we instituted criteria to design efficient sgRNAs. To facilitate the assembly of multiple sgRNA cassettes, we also developed a new strategy to rapidly construct CRISPR/Cas9-sgRNA system for multiplex editing in plants. In theory, up to ten single guide RNA (sgRNA) cassettes can be simultaneously assembled into the final binary vectors. As a proof of concept, 21 sgRNAs complying with the criteria were designed and the corresponding Cas9/sgRNAs expression vectors were constructed. Sequencing analysis of transgenic rice plants suggested that 82% of the desired target sites were edited with deletion, insertion, substitution, and inversion, displaying high editing efficiency. This work provides a convenient approach to select efficient sgRNAs for target editing.

PMID:
26891616
PMCID:
PMC4759811
DOI:
10.1038/srep21451
[Indexed for MEDLINE]
Free PMC Article

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