CRISPR/Cas9-Mediated Multiplex Genome Editing of the BnWRKY11 and BnWRKY70 Genes in Brassica napus L.
Abstract
:1. Introduction
2. Results
2.1. Sequence Identification and Expression Analysis of BnWRKY11 and BnWRKY70 Genes in B. napus
2.2. CRISPR/Cas9 Binary Vector Construction, Rapeseed Transformation and Screening of Positive Transformants
2.3. Confirmation of Cas9-Induced Mutagenesis in Transgenic Plants of B. napus
2.4. Variety and Frequency of Mutations
2.5. Mutagenesis in T1 Plants
2.6. BnWRKY70 Mutants Enhance Resistance to S. sclerotiorum
3. Discussion
4. Materials and Methods
4.1. Target Design and Vector Construction for Targeted Gene Mutation
4.2. Genetic Transformation of B. napus
4.3. Mutation Analysis
4.4. S. sclerotiorum Infection Assay
Supplementary Materials
Author Contributions
Funding
Acknowledgements
Conflicts of Interest
Abbreviations
CRISPR | Clustered, regularly interspaced, palindromic repeat |
sgRNA | Single guide RNA |
PAM | Protospacer adjacent motif |
ZFN | Zinc finger nucleases |
TALEN | Transcription activator-like effector nucleases |
TFs | Transcription factors |
hpi | hours post-inoculation |
AS | Acetosyringone |
Cef | Cefotaxime |
CTAB | Hexadecyl trimethyl ammonium brom |
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Target Gene (Number of Transformants) | Copies | Target | Amplification Primer | No. of Plants with Mutations | Mutation Frequency (%) |
---|---|---|---|---|---|
BnWRKY1122 | BnaA.WRKY11.a | WRKY11-Tgt1, WRKY11-Tgt3 | 11subF/11subR→11F/11R | 0, 7 | 0, 31.8% |
BnaC.WRKY11.a | WRKY11-Tgt2, WRKY11-Tgt3 | 11F/11R | 12, 9 | 54.5%, 40.9% | |
BnWRKY708 | BnaC.WRKY70.b | WRKY70-Tgt1 | 70F3/70R3 | 0 | 0 |
BnaA.WRKY70.b | WRKY70-Tgt1 | 70F3/70R3 | 3 | 37.5% | |
BnaC.WRKY70.a | WRKY70-Tgt2 | 70F1/70R2 | 0 | 0 | |
BnaA.WRKY70.a | WRKY70-Tgt3 | 70F1/70R1 | 3 | 37.5% |
Line | Number of Examined Plants | Cas9:sgRNA Constructs a | Number of Edited Plants b | |||
---|---|---|---|---|---|---|
BnWRKY11 | ||||||
BnaA.WRKY11.a (WRKY11-Tgt1) | BnaA.WRKY11.a (WRKY11-Tgt3) | BnaC.WRKY11.a (WRKY11-Tgt2) | BnaC.WRKY11.a (WRKY11-Tgt3) | |||
CRI-W11-6 | 10 | 10 | 10 | 10 | 0 | 10 |
CRI-W11-10 | 10 | 10 | 10 | 10 | 0 | 10 |
BnWRKY70 | ||||||
BnaA.WRKY70.b | BnaA.WRKY70.a | BnaC.WRKY70.a | BnaC.WRKY70.b | |||
CRI-W70-6 | 10 | 8 | 8 | 8 | 0 | 8 |
CRI-W70-7 | 10 | 8 | 8 | 8 | 0 | 8 |
CRI-W70-9 | 10 | 10 | 10 | 10 | 0 | 10 |
CRI-W70-10 | 10 | 8 | 10 | 10 | 0 | 10 |
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Sun, Q.; Lin, L.; Liu, D.; Wu, D.; Fang, Y.; Wu, J.; Wang, Y. CRISPR/Cas9-Mediated Multiplex Genome Editing of the BnWRKY11 and BnWRKY70 Genes in Brassica napus L. Int. J. Mol. Sci. 2018, 19, 2716. https://doi.org/10.3390/ijms19092716
Sun Q, Lin L, Liu D, Wu D, Fang Y, Wu J, Wang Y. CRISPR/Cas9-Mediated Multiplex Genome Editing of the BnWRKY11 and BnWRKY70 Genes in Brassica napus L. International Journal of Molecular Sciences. 2018; 19(9):2716. https://doi.org/10.3390/ijms19092716
Chicago/Turabian StyleSun, Qinfu, Li Lin, Dongxiao Liu, Dewei Wu, Yujie Fang, Jian Wu, and Youping Wang. 2018. "CRISPR/Cas9-Mediated Multiplex Genome Editing of the BnWRKY11 and BnWRKY70 Genes in Brassica napus L." International Journal of Molecular Sciences 19, no. 9: 2716. https://doi.org/10.3390/ijms19092716