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

编号 040037901

推送时间 20230123

研究领域 森林培育 

年份 2022 

类型 期刊 

语种 英语

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

来源期刊 Plant physiology

期 第379期

发表时间 20220915

关键词 Arabidopsis thaliana;  CRISPR/Cas9;  gene targeting;  molecular mechanisms;  genome engineering;  Agrobacterium tumefaciens;  DNA; 

摘要 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.

服务人员 孙小满

服务院士 尹伟伦

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