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

• Main Authors: Zhengyan Feng, Zhengjing Zhang, Kai Hua, Xifeng Gao, Yanfei Mao, Jose Ramon Botella, Jian-Kang Zhu
• Format: Article
• Language: English
• Published: MDPI AG 2018-12-01
• Series: International Journal of Molecular Sciences
• Subjects: <i>Arabidopsis</i>; cell division-specific Cas9 system; CRISPR/Cas9; multiplex gene editing; Pol III promoter
• Online Access: https://www.mdpi.com/1422-0067/19/12/3925

The CRISPR/Cas9 system has been widely used for targeted genome editing in numerous plant species. In <i>Arabidopsis</i>, 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 <i>Cas9</i> expression were evaluated. Expression of <i>Cas9</i> 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 <i>Cas9</i> under two cell division-specific promoters, <i>YAO</i> and <i>CDC45</i>, produced mutation rates of 80.9% to 100% in the T1 generation with nonchimeric mutations in the T1 (4.4&#8315;10%) and T2 (32.5&#8315;46.1%) generations. The <i>pCDC45</i> 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 <i>Arabidopsis</i>, especially in multiplex applications.