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Int J Mol Sci. 2018 Dec 7;19(12). pii: E3925. doi: 10.3390/ijms19123925.

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

Author information

1
Shanghai Center for Plant Stress Biology, CAS Center of Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai 200032, China. fengzhengyan@sibs.ac.cn.
2
Shanghai Center for Plant Stress Biology, CAS Center of Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai 200032, China. zhangzhengjing@sibs.ac.cn.
3
Shanghai Center for Plant Stress Biology, CAS Center of Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai 200032, China. huakai@sibs.ac.cn.
4
University of Chinese Academy of Sciences (CAS), Beijing, 100049, China. huakai@sibs.ac.cn.
5
Shanghai Center for Plant Stress Biology, CAS Center of Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai 200032, China. xfgao@psc.ac.cn.
6
University of Chinese Academy of Sciences (CAS), Beijing, 100049, China. xfgao@psc.ac.cn.
7
Shanghai Center for Plant Stress Biology, CAS Center of Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai 200032, China. yfmao@sibs.ac.cn.
8
School of Agriculture and Food Sciences, University of Queensland, Brisbane, QLD 4072, Australia. j.botella@uq.edu.au.
9
Shanghai Center for Plant Stress Biology, CAS Center of Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai 200032, China. jkzhu@purdue.edu.
10
Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907, USA. jkzhu@purdue.edu.

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.

KEYWORDS:

Arabidopsis; CRISPR/Cas9; Pol III promoter; cell division-specific Cas9 system; multiplex gene editing

PMID:
30544514
PMCID:
PMC6321140
DOI:
10.3390/ijms19123925
[Indexed for MEDLINE]
Free PMC Article

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