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

• DOI: 10.3390/ijms19123925
• Publication Date: 2018-12-07T08:46:14Z
• Authors: Zhengyan Feng; Zhengjing Zhang; Kai Hua; Xifeng Gao; Yanfei Mao; Jose Ramon Botella; Jian-Kang Zhu
• Keywords: Multiplex gene editing; 1503 Catalysis; Arabidopsis; 1607 Spectroscopy; <i>Arabidopsis</i>; Article; 03 medical and health sciences; 1312 Molecular Biology; 1706 Computer Science Applications; Cell division-specific Cas9 system; Promoter Regions, Genetic; CRISPR/Cas9; Gene Editing; 0303 health sciences; cell division-specific Cas9 system; Pol III promoter; 1604 Inorganic Chemistry; Arabidopsis Proteins; Plants, Genetically Modified; multiplex gene editing; Mutation; CRISPR-Cas Systems; 1606 Physical and Theoretical Chemistry; Cell Division; Genome, Plant; 1605 Organic Chemistry

The CRISPR/Cas9 system has been widely used for targeted genome editing in numerous plant species. In Arabidopsis, constitutive promoters usually result a low efficiency of heritable mutation the T1 generation. this work, gene efficiencies using different to drive Cas9 expression were evaluated. Expression under CaMV 35S promoter resulted 2.3% rate plants and failed produce homozygous mutations T2 generations. contrast, two cell division-specific promoters, YAO CDC45, produced rates 80.9% 100% generation with nonchimeric (4.4–10%) (32.5–46.1%) pCDC45 was modify previously reported multiplex system, replacing original ubiquitin promoter. multi-pCDC45-Cas9 higher than multi-pUBQ-Cas9 (60.17% vs. 43.71%) as well (11.30% 4.31%). Sextuple mutants identified from construct targeting seven individual loci. Our results demonstrate advantage division applications especially applications.