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Int. J. Mol. Sci. 2018, 19(12), 3925; https://doi.org/10.3390/ijms19123925

A Highly Efficient Cell Division-Specific CRISPR/Cas9 System Generates Homozygous Mutants for Multiple Genes in Arabidopsis

1
Shanghai Center for Plant Stress Biology, CAS Center of Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai 200032, China
2
University of Chinese Academy of Sciences (CAS), Beijing, 100049, China
3
School of Agriculture and Food Sciences, University of Queensland, Brisbane, QLD 4072, Australia
4
Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907, USA
*
Author to whom correspondence should be addressed.
Received: 11 October 2018 / Revised: 26 October 2018 / Accepted: 29 October 2018 / Published: 7 December 2018
(This article belongs to the Section Molecular Plant Sciences)
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Abstract

The 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
Keywords: Arabidopsis; cell division-specific Cas9 system; CRISPR/Cas9; multiplex gene editing; Pol III promoter Arabidopsis; cell division-specific Cas9 system; CRISPR/Cas9; multiplex gene editing; Pol III promoter
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).

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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.

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