A Highly Efficient Cell Division-Specific CRISPR/Cas9 System Generates Homozygous Mutants for Multiple Genes in Arabidopsis
AbstractThe CRISPR/Cas9 system has been widely used for targeted genome editing in numerous plant species. In Arabidopsis, constitutive promoters usually result in a low efficiency of heritable mutation in the T1 generation. In this work, CRISPR/Cas9 gene editing efficiencies using different promoters to drive Cas9 expression were evaluated. Expression of Cas9 under the constitutive CaMV 35S promoter resulted in a 2.3% mutation rate in T1 plants and failed to produce homozygous mutations in the T1 and T2 generations. In contrast, expression of Cas9 under two cell division-specific promoters, YAO and CDC45, produced mutation rates of 80.9% to 100% in the T1 generation with nonchimeric mutations in the T1 (4.4–10%) and T2 (32.5–46.1%) generations. The pCDC45 promoter was used to modify a previously reported multiplex CRISPR/Cas9 system, replacing the original constitutive ubiquitin promoter. The multi-pCDC45-Cas9 system produced higher mutation efficiencies than the multi-pUBQ-Cas9 system in the T1 generation (60.17% vs. 43.71%) as well as higher efficiency of heritable mutations (11.30% vs. 4.31%). Sextuple T2 homozygous mutants were identified from a construct targeting seven individual loci. Our results demonstrate the advantage of using cell division promoters for CRISPR/Cas9 gene editing applications in Arabidopsis, especially in multiplex applications. View Full-Text
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Feng, Z.; Zhang, Z.; Hua, K.; Gao, X.; Mao, Y.; Botella, J.R.; Zhu, J.-K. A Highly Efficient Cell Division-Specific CRISPR/Cas9 System Generates Homozygous Mutants for Multiple Genes in Arabidopsis. Int. J. Mol. Sci. 2018, 19, 3925.
Feng Z, Zhang Z, Hua K, Gao X, Mao Y, Botella JR, Zhu J-K. A Highly Efficient Cell Division-Specific CRISPR/Cas9 System Generates Homozygous Mutants for Multiple Genes in Arabidopsis. International Journal of Molecular Sciences. 2018; 19(12):3925.Chicago/Turabian Style
Feng, Zhengyan; Zhang, Zhengjing; Hua, Kai; Gao, Xifeng; Mao, Yanfei; Botella, Jose R.; Zhu, Jian-Kang. 2018. "A Highly Efficient Cell Division-Specific CRISPR/Cas9 System Generates Homozygous Mutants for Multiple Genes in Arabidopsis." Int. J. Mol. Sci. 19, no. 12: 3925.
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