Insights into the molecular mechanisms of CRISPR/Cas9-mediated gene targeting at multiple loci in Arabidopsis

Zhang, Zhengjing1; Zeng, Wenjie1,2; Zhang, Wenxin1,2,7; Li, Jing1,2; Kong, Dali1,2; Zhang, Lei1,2; Wang, Rui1,2; Peng, Fangnan1,2; Kong, Zhe1,2; Ke, Yongping1,2; Zhang, Heng1; Kim, Chanhong1; Zhang, Huiming1; Botella, Jose Ramon3; Zhu, Jian-Kang1,4,5,6; Miki, Daisuke1

2022-09-01

10.1093/plphys/kiac431

PLANT PHYSIOLOGY   Impact Factor & Quartile

0032-0889

1532-2548

["Homologous recombination-mediated gene targeting (GT) enables precise sequence knockin or sequence replacement, and thus is a powerful tool for heritable precision genome engineering. We recently established a clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9)-mediated approach for heritable GT in Arabidopsis (Arabidopsis thaliana), but its broad utility was not tested, and the underlying molecular mechanism was unclear. Here, we achieved precise GT at 14 out of 27 tested endogenous target loci using the sequential transformation approach and obtained vector-free GT plants by backcrossing. Thus, the sequential transformation GT method provides a broadly applicable technology for precise genome manipulation. We show that our approach generates heritable GT in the egg cell or early embryo of T1 Arabidopsis plants. Analysis of imprecise GT events suggested that single-stranded transfer DNA (T-DNA)/VirD2 complexes produced during the Agrobacterium (Agrobacterium tumefaciens) transformation process may serve as the donor templates for homologous recombination-mediated repair in the GT process. This study provides new insights into the molecular mechanisms of CRISPR/Cas9-mediated GT in Arabidopsis.","The sequential transformation gene targeting method provides a broadly applicable technology for precise genome manipulation in Arabidopsis."]

SCI

English

Corresponding

Shanghai Science and Technology Innovation Plan[20ZR1467000]

Plant Sciences

Plant Sciences

WOS:000864219700001

OXFORD UNIV PRESS INC

PLANT & ANIMAL SCIENCE

Web of Science

1.Chinese Acad Sci, Shanghai Ctr Plant Stress Biol, Ctr Excellence Mol Plant Sci, Shanghai 200032, Peoples R China
2.Univ Chinese Acad Sci, Beijing 100049, Peoples R China
3.Univ Queensland, Sch Agr & Food Sci, Brisbane, Qld 4072, Australia
4.Southern Univ Sci & Technol, Inst Adv Biotechnol, Shenzhen 518055, Peoples R China
5.Southern Univ Sci & Technol, Sch Life Sci, Shenzhen 518055, Peoples R China
6.Chinese Acad Agr Sci, Ctr Adv Bioind Technol, Beijing 100081, Peoples R China
7.Peking Univ, Acad Adv Interdisciplinary Studies, Peking Tsinghua Ctr Life Sci, 5 Yiheyuan Rd, Beijing 100871, Peoples R China

Zhang, Zhengjing,Zeng, Wenjie,Zhang, Wenxin,et al. Insights into the molecular mechanisms of CRISPR/Cas9-mediated gene targeting at multiple loci in Arabidopsis[J]. PLANT PHYSIOLOGY,2022.

Zhang, Zhengjing.,Zeng, Wenjie.,Zhang, Wenxin.,Li, Jing.,Kong, Dali.,...&Miki, Daisuke.(2022).Insights into the molecular mechanisms of CRISPR/Cas9-mediated gene targeting at multiple loci in Arabidopsis.PLANT PHYSIOLOGY.

Zhang, Zhengjing,et al."Insights into the molecular mechanisms of CRISPR/Cas9-mediated gene targeting at multiple loci in Arabidopsis".PLANT PHYSIOLOGY (2022).