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

Author:

Zhang Zhengjing1,Zeng Wenjie12ORCID,Zhang Wenxin12ORCID,Li Jing12,Kong Dali12ORCID,Zhang Lei12,Wang Rui12,Peng Fangnan12,Kong Zhe12ORCID,Ke Yongping12ORCID,Zhang Heng1ORCID,Kim Chanhong1ORCID,Zhang Huiming1ORCID,Botella Jose Ramón3ORCID,Zhu Jian-Kang145ORCID,Miki Daisuke1ORCID

Affiliation:

1. Shanghai Center for Plant Stress Biology, Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences , Shanghai 200032, China

2. University of Chinese Academy of Sciences , Beijing 100049, China

3. School of Agriculture and Food Sciences, University of Queensland , Brisbane 4072, Australia

4. Institute of Advanced Biotechnology and School of Life Sciences, Southern University of Science and Technology , Shenzhen 518055, China

5. Center for Advanced Bioindustry Technologies, Chinese Academy of Agricultural Sciences , Beijing 100081, China

Abstract

Abstract 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.

Funder

Shanghai Science and Technology Innovation Plan

Chinese Academy of Sciences

Publisher

Oxford University Press (OUP)

Subject

Plant Science,Genetics,Physiology

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