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Sci Rep. 2015 May 29;5:10342. doi: 10.1038/srep10342.

Targeted mutagenesis in soybean using the CRISPR-Cas9 system.

Author information

1
1] College of Agronomy, Northwest A&F University, Yangling, Shaanxi 712100, China [2] National Key Facilities for Crop Genetic Resources and Improvement, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
2
National Key Facilities for Crop Genetic Resources and Improvement, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
3
Tianjin Institute of Agricultural Quality Standard and Testing Technology, Tianjin Academy of Agricultural Sciences, Tianjin 300381, China.
4
College of Agronomy, Northwest A&F University, Yangling, Shaanxi 712100, China.

Abstract

Genome editing is a valuable technique for gene function analysis and crop improvement. Over the past two years, the CRISPR-Cas9 system has emerged as a powerful tool for precisely targeted gene editing. In this study, we predicted 11 U6 genes in soybean (Glycine max L.). We then constructed two vectors (pCas9-GmU6-sgRNA and pCas9-AtU6-sgRNA) using the soybean U6-10 and Arabidopsis U6-26 promoters, respectively, to produce synthetic guide RNAs (sgRNAs) for targeted gene mutagenesis. Three genes, Glyma06g14180, Glyma08g02290 and Glyma12g37050, were selected as targets. Mutations of these three genes were detected in soybean protoplasts. The vectors were then transformed into soybean hairy roots by Agrobacterium rhizogenes infection, resulting in efficient target gene editing. Mutation efficiencies ranged from 3.2-9.7% using the pCas9-AtU6-sgRNA vector and 14.7-20.2% with the pCas9-GmU6-sgRNA vector. Biallelic mutations in Glyma06g14180 and Glyma08g02290 were detected in transgenic hairy roots. Off-target activities associated with Glyma06g14180 and Glyma12g37050 were also detected. Off-target activity would improve mutation efficiency for the construction of a saturated gene mutation library in soybean. Targeted mutagenesis using the CRISPR-Cas9 system should advance soybean functional genomic research, especially that of genes involved in the roots and nodules.

PMID:
26022141
PMCID:
PMC4448504
DOI:
10.1038/srep10342
[Indexed for MEDLINE]
Free PMC Article

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