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Plant Cell Rep. 2017 Mar;36(3):399-406. doi: 10.1007/s00299-016-2089-5. Epub 2016 Dec 19.

Efficient CRISPR/Cas9-based gene knockout in watermelon.

Author information

1
National Engineering Research Center for Vegetables, Beijing Academy of Agriculture and Forestry Sciences, Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (North China), Beijing Key Laboratory of Vegetable Germplasm Improvement, Beijing, 100097, China.
2
Department of Plant Pathology, China Agricultural University, Beijing, 100193, China.
3
Beijing University of Agriculture, Beijing, 102206, China.
4
National Engineering Research Center for Vegetables, Beijing Academy of Agriculture and Forestry Sciences, Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (North China), Beijing Key Laboratory of Vegetable Germplasm Improvement, Beijing, 100097, China. xuyong@nercv.org.

Abstract

CRISPR/Cas9 system can precisely edit genomic sequence and effectively create knockout mutations in T0 generation watermelon plants. Genome editing offers great advantage to reveal gene function and generate agronomically important mutations to crops. Recently, RNA-guided genome editing system using the type II clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) has been applied to several plant species, achieving successful targeted mutagenesis. Here, we report the genome of watermelon, an important fruit crop, can also be precisely edited by CRISPR/Cas9 system. ClPDS, phytoene desaturase in watermelon, was selected as the target gene because its mutant bears evident albino phenotype. CRISPR/Cas9 system performed genome editing, such as insertions or deletions at the expected position, in transfected watermelon protoplast cells. More importantly, all transgenic watermelon plants harbored ClPDS mutations and showed clear or mosaic albino phenotype, indicating that CRISPR/Cas9 system has technically 100% of genome editing efficiency in transgenic watermelon lines. Furthermore, there were very likely no off-target mutations, indicated by examining regions that were highly homologous to sgRNA sequences. Our results show that CRISPR/Cas9 system is a powerful tool to effectively create knockout mutations in watermelon.

KEYWORDS:

CRISPR/Cas9; Genome editing; PDS; Watermelon

PMID:
27995308
DOI:
10.1007/s00299-016-2089-5
[Indexed for MEDLINE]

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